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WO2022150878A1 - Compositions and methods of using targeting peptides and nanopharmaceuticals for the sterilization of cats and dogs - Google Patents

Compositions and methods of using targeting peptides and nanopharmaceuticals for the sterilization of cats and dogs Download PDF

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Publication number
WO2022150878A1
WO2022150878A1 PCT/AU2022/050011 AU2022050011W WO2022150878A1 WO 2022150878 A1 WO2022150878 A1 WO 2022150878A1 AU 2022050011 W AU2022050011 W AU 2022050011W WO 2022150878 A1 WO2022150878 A1 WO 2022150878A1
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Prior art keywords
seq
amino acids
peptide
acid sequence
peptides
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PCT/AU2022/050011
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French (fr)
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Lee Brett SMITH
Robert John Aitken
Barbara Anne FRASER
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University Of Newcastle
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Publication of WO2022150878A1 publication Critical patent/WO2022150878A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
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    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • This application relates to compositions and methods of administering nanoparticle constructs containing an apoptotic or cytotoxic protein or a polynucleotide encoding for the apoptotic or cytotoxic protein, such as Diphtheria toxin A (“DTA”), where the nanoparticle constructs comprise one or more peptides that can bind to reproductive cell receptors and cause cell death, and where the compositions and methods are useful for the sterilization of subjects, including male and female cats and dogs.
  • DTA Diphtheria toxin A
  • Dogs and cats are the most common domesticated carnivores across the world (Hughes & Macdonald, 2013). Estimates of the world dog population varies widely but is generally believed to be upwards of between 500 - 600 million (Statistics on Dogs - CAROdog, n.d.; Stray Animal Control, n.d.); approximately 75% ( ⁇ 480 million animals) of which are considered to be free-ranging or strays (Chawla et al., 2020). Additionally, the feral cat population is estimated to be at least 100 million, including 60 million in the USA and up to 12 million in Australia alone (Legge et al., 2017). These numbers highlight the magnitude of the worldwide problem posed by free-roaming domestic dogs and cats, which impacts not only human health but also imposes significant economic and environmental costs.
  • the ASPCA American Society for the Prevention of Cruelty to Animals® has indicated the pet homelessness problem results in millions of healthy dogs and cats being euthanized in the United States each year. This is a worldwide problem that is not limited to just the United States.
  • the ASPCA also indicates there are medical and behavioral benefits to spaying and neutering animals including: preventing certain infections or tumors (e.g., uterine infections and mammary tumors in females and testicular cancer and prostate problems in males); avoiding female pets going into heat; making it less likely for male pets to roam; and possibly leading to better behaved males. Sterilization procedures such as spaying and neutering are common surgeries, but there can be health risks.
  • General anesthesia for example, is relatively expensive, and requires specialized equipment, facilities and personnel making it impracticable in remote areas and in low-income countries. Additionally, it takes time for the animals to heal after the surgery. Accordingly, bathing must be avoided for, e.g., at least ten days after surgery; the animal must refrain from running or jumping post-surgery; and the incision site must be monitored to avoid infection and proper healing. Accordingly, the present disclosure provides methods and compositions for sterilizing subjects that can avoid surgery.
  • nonsurgical fertility control is increasingly advocated as both a more humane (Fagerstone et al., 2010; McUaughlin & Aitken, 2011) and cost-effective (Massei & Miller, 2013) solution for free-roaming dog and cat management and to resolve human-wildlife conflicts.
  • nonsurgical sterilization may provide a higher throughput, compared to surgical sterilization.
  • Previous attempts to generate non-surgical approaches to animal sterilization have not been successful. This is particularly true of contraceptive vaccines which have been the subject of considerable investment, for instance by governments interested in developing strategies to control feral animal populations.
  • Vaccines for example, have been used to sterilize females by attempting to permanently remove the primordial follicle population (Aitken et al., 1996). But sterilization via vaccine was somewhat ineffective as infertility takes several months to materialize because the primordial follicle pool is progressively depleted over time. Furthermore, no active immunization approach has ever been found to induce sterility in males.
  • Non-surgical sterilization technologies include: (i) hormonal methods such as implants (Driancourt & Briggs, 2020); (ii) the induction of immunocontraception that relies on antibody production against reproductive cells or signaling molecules; and (iii) reproductive senescence induced by a chemosterilant (Rafatmah et al., 2019; Silva et al., 2018). Hormonal and immunocontraception require periodic re-administration which may not always be practicable (e.g., where there is not continued access to the animal).
  • untargeted chemosterilants are often not species specific, require anesthesia and highly trained administrants (Madbouly et al., 2021; Silva et al., 2018), may have off-target effects at the doses required for sterilization (Abolaji et al., 2016), and/or may be environmental pollutants.
  • the injectable chemical agents, such as zinc gluconate, currently available for sterilization of male animals are effective, but still require specialized equipment and personnel to conduct intratesticular administration (Massei & Miller, 2013).
  • an injectable sterilizing agent will be able to be administered either intraperitoneally or intravenously to deliver an agent that will selectively target the gonads.
  • an aspect of the present invention provides a non-surgical permanent and, in certain embodiments, single dose fertility treatment that is safe and effective in male and female subjects.
  • the male gonads i.e., testes
  • the male gonads perform two distinct functions, that is, the production of spermatozoa within the seminiferous tubules and the synthesis of steroids by Leydig cells (Ye et al., 2017), which reside within the interstitial space outside the seminiferous tubules (Evans & Ganjam, 2011; Rebourcet et al., 2014).
  • testicular function is regulated via the hypothalamo-pituitary-gonadal (HPG) axis and secretion of the glycoprotein hormones (GPH), that is, Follicle-Stimulating Hormone (FSH) and Luteinising hormone (LH), by gonadotrophs in the anterior pituitary (Abel et al., 2008; Cheng & Mruk, 2010).
  • GPH glycoprotein hormones
  • FSH Follicle-Stimulating Hormone
  • LH Luteinising hormone
  • the anti -Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors.
  • AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood. Nevertheless, AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015). AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Cimino et al., 2016).
  • the present invention is directed to permanently disrupting both cell types by delivering a gene, for example, to Sertoli and/or Leydig cells, that will cause cell death.
  • the genetic payload can be delivered intravenously, for example, by a lipid sphere (nanoparticle), which protects the DNA inside until being internalized by the target cell.
  • viral vector technologies require the modification of the virus’s genome to incorporate genes of interest to be introduced.
  • the virus is then manufactured with the DNA or other polynucleotide inside.
  • Viral vectors can be limited, however, in their ability to be modified to introduce targeting peptides on their surface.
  • Nanoparticle constructs are formulated herein to deliver a gene.
  • Nanoparticles can be formulated with different components and different ratios of components that form the sphere that protects the DNA or other polynucleotide until it enters the cell.
  • Nanoparticles provide versatility of manufacturing. They can be tailored and functionalized to target specific organs by biasing the biodistribution to the site of action of the therapeutic that they deliver. This can also be achieved by grafting or linking ligands to the surface of the nanoparticles, which target a specific receptor expressed by the cells or tissue of interest.
  • Nanoparticles themselves are a relatively new drug delivery technology. They are currently used in humans, for example, in the oncology sector for delivering small molecule organic pharmaceuticals (largely chemotherapeutic agents).
  • PEGylated liposome is Doxil, indicated for the treatment of AIDS-associated Kaposi’s sarcoma.
  • nanoparticles can be designed to incorporate small peptide sequences on their surface that bind to receptors present on the target cells (e.g., FSH and LH receptors on Sertoli and/or Leydig cells), thereby adding a level of safety preventing the gene drug from being internalized by non-Sertoli and/or non-Leydig cells.
  • receptors present on the target cells e.g., FSH and LH receptors on Sertoli and/or Leydig cells
  • the inventors have developed a unique array of peptides that are capable of targeting these cells in vitro and in vivo.
  • cell -targeting peptides were generated through the use of random peptide phage display technology (Eidne et al., 2000) or by the synthesis of peptides capable of binding to receptors that are restricted to the target cell population (e.g., FSH, LH).
  • the nanoparticles can also be utilized, for example, to target Sertoli and/or Leydig cells, gonocyte cells (precursors of spermatogonia), or to target female cells such as primordial follicle cells, or primordial germ cells in utero.
  • targeting peptides are incorporated on the surface of nanoparticles that encompass an apoptotic or cytotoxic protein or recombinant viral-based (e.g., recombinant lentiviral-based) DNA construct encoding the apoptotic or cytotoxic protein.
  • Targeting peptides based on, e.g., FSH and LH expressed on the lentiviral coats can be grafted or linked to a nanoparticle to allow specific delivery to, for example, Sertoli and/or Leydig cells in males and primordial follicle cells in females. Species- and cell-specific promoters may also be incorporated to further reduce off-target effects.
  • targeting peptides are incorporated on the surface of nanoparticles that encompass a linear DNA construct that encodes an apoptotic or cytotoxic protein.
  • sterility may be achieved following a single administration of nanoparticles. Accordingly, sterilization of stray/homeless animals will no longer require a surgical process which can be expensive, time consuming and stressful for the animals. Alongside this, the numbers of animals euthanized as a result of overcrowding in shelters will also be reduced (as a result of fewer litters being produced due to sterilization). Furthermore, this technology could also be used for routine neutering of domestic pets (cats and dogs) as opposed to the surgical processes currently in practice. This technology could both reduce the costs of this procedure and result in a reduced impact on the animals due to lack of anesthetic requirements and surgery.
  • peptides, conjugated peptides, nanoparticle constructs, pharmaceutical compositions comprising the peptides and nanoparticle constructs, and methods of sterilizing a subject comprising administering to the subject an effective amount of the peptides, nanoparticle constructs or pharmaceutical compositions are provided.
  • the disclosure provides an isolated Follicle-Stimulating Hormone (FSH) peptide comprising a fragment of an FSH protein or variant thereof.
  • FSH Follicle-Stimulating Hormone
  • the FSH peptide comprises an amino acid sequence of TVX9GLGPX10Y (SEQ ID No. 95), wherein X 9 is R or Q and X10 is S or G.
  • the FSH peptide comprises an amino acid sequence of CTVX9GLGPX10Y (SEQ ID No. 96), wherein X 9 is R or Q and X10 is S or G.
  • the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4Q (SEQ ID No. 33), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
  • the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 34), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, orN; and X4 is T orN.
  • the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4Q (SEQ ID No. 35), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, orN; and X4 is T orN.
  • the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 36), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; and X4 is T or N.
  • the FSH peptide comprises an amino acid sequence of YTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 49), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
  • the FSH peptide comprises an amino acid sequence of CYTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 50), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
  • the FSH peptide comprises an amino acid sequence of CHX7GX8X7DSDSTDX7T (SEQ ID No. 63), wherein X 7 is C, S, or A and X 8 is K, R, or G.
  • the FSH peptide comprises an amino acid sequence of X8CDSDSTDX7TVX9GL (SEQ ID No. 78), wherein X 8 is K, R, or G; X 7 is C, S, or A; and X 9 is R or Q.
  • the FSH peptide comprises an amino acid sequence of CDSDSTDX7TVX9GL (SEQ ID No. 79), wherein X 8 is K, R, or G; X 7 is C, S, or A; and X 9 is R or Q.
  • the FSH peptide comprises an amino acid sequence of SDSTSX7TVX9GLGPX10Y (SEQ ID No. 89), wherein X 7 is C, S, or A; X 9 is R or Q; and X10 is S or G.
  • the FSH peptide comprises an amino acid sequence of CSDSTSX7TVX9GLGPX10Y (SEQ ID No. 90), wherein X 7 is C, S, or A; X 9 is R or Q; and X10 is S or G.
  • the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
  • the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No.
  • SEQ ID No. 46 SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No.
  • SEQ ID No. 75 SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
  • the FSH peptide comprises the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2. In certain aspects, the FSH peptide comprises an amino acid sequence consisting of SEQ ID No. 1 and/or SEQ ID No. 2.
  • the FSH peptide comprises the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No.
  • SEQ ID No. 51 SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No.
  • SEQ ID No. 76 SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
  • the FSH peptide comprises an amino acid sequence consisting of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No.
  • SEQ ID No. 51 SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No.
  • SEQ ID No. 76 SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
  • the FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and/or SEQ ID No. 18.
  • the isolated FSH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
  • LH Luteinizing Hormone
  • the LH peptide comprises an amino acid sequence of SX18SDX7GGPX8X19X20X21 (SEQ ID No. 144), wherein Xis is T, S, orN; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
  • the LH peptide comprises an amino acid sequence of CSX18SDX7GGPX8X19X20X21, (SEQ ID No. 145), wherein Xis is T, S, or N; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
  • the LH peptide comprises an amino acid sequence of X11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 112), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
  • the LH peptide comprises an amino acid sequence of CX11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 113), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
  • the LH peptide comprises an amino acid sequence of SFPVALX7RX7GPX7RX17 (SEQ ID No. 125), wherein X 7 is C, S, or A and X17 is L or R.
  • the LH peptide comprises an amino acid sequence of CSFPVALX7RX7GPX7RX17 (SEQ ID No. 126), wherein X 7 is C, S, or A and X17 is L or R.
  • the LH peptide comprises an amino acid sequence of CRX17SX18SDX7G (SEQ ID No. 133), wherein X17 is L or R; Xis is T, S, or N; and X7 is C, S, or A.
  • the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
  • the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No.
  • SEQ ID No. 124 SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
  • the LH peptide comprises the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4. In certain aspects, the LH peptide comprises an amino acid sequence consisting of SEQ ID No. 3 and/or SEQ ID No. 4.
  • the LH peptide comprises the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No.
  • SEQ ID No. 130 SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
  • the LH peptide comprises an amino acid sequence consisting of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No.
  • SEQ ID No. 130 SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
  • the LH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, and/or SEQ ID No. 104.
  • the LH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
  • the disclosure provides an isolated Anti-Mullerian Hormone (AMH) peptide comprising a fragment of an AMH protein or variant thereof.
  • AMH Anti-Mullerian Hormone
  • the AMH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
  • the AMH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
  • the AMH peptide comprises the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165. In certain aspects, the AMH peptide comprises an amino acid sequence consisting of SEQ ID No. 5 and/or SEQ ID No. 165. [0060] In certain aspects, the AMH peptide comprises the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
  • the AMH peptide comprises an amino acid sequence consisting of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
  • the AMH peptide has one substitution modification, two substitution modifications, three substitution modifications, or four substitution modifications relative to SEQ ID No. 149, SEQ ID No. 153, SEQ ID No. 157, and/or SEQ ID No. 161.
  • the AMH peptide is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
  • the isolated FSH, LH, and/or AMH is amidated.
  • the peptide comprises a cysteine residue at the N-terminus.
  • the peptide comprises a linker.
  • the linker is a maleimide group, 1,6- aminohexanoic acid (Ahx), y-aminobutanoate-mercaptopropionic acid, alanine with 3- mercaptopropionic acid (MPA), y-aminobutanoic acid with MPA, mini-polyethyleneglycol with MPA, or a cathepsin cleavable valine-citrulline(vc)-PABC linker.
  • the protein is an apoptotic protein or a cytotoxic protein.
  • the small molecule is an auristatin (Aur) molecule, such as such monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), or a menadione (Md) molecule, such as 2-methyl-l,4-naphthoquinone.
  • the label is fluorescein isothiocyanate (FITC).
  • composition comprising a peptide conjugate comprising the isolated FSH, LH, and/or AMH peptide of any of the preceding aspects.
  • the disclosure provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein.
  • the disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein.
  • the disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein; and c) a reporter protein or a polynucleotide encoding a reporter protein.
  • the one or more peptides comprises a fragment of a protein or a variant thereof that binds the receptor of the reproductive cell.
  • the one or more peptides is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
  • the nanoparticle construct comprises a polynucleotide encoding the apoptotic or cytotoxic protein.
  • the apoptotic protein is Diphtheria toxin fragment A (DTA).
  • DTA Diphtheria toxin fragment A
  • the DTA comprises the amino acid sequence of SEQ ID No: 10 or a variant thereof comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID No: 10.
  • the reporter protein is selected from red fluorescent protein (RFP), green fluorescent protein (GFP), and enhanced green fluorescent protein (EGFP).
  • RFP red fluorescent protein
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • the reproductive cell is a Sertoli cell and/or a Leydig cell.
  • the one or more peptides bind a receptor of a Sertoli cell and/or a Leydig cell.
  • the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell and/or the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.
  • the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 1 or SEQ ID No: 2.
  • the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 3 or SEQ ID No: 4.
  • the reproductive cell is a gonocyte, a primordial follicle cell, and/or a primordial germ cell.
  • the one or more peptides bind an anti-Mullerian hormone on a primordial follicle cell.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 5.
  • the one or more peptides bind a primordial germ cell.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 6.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 7.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 8.
  • the one or more peptides comprise an amino acid sequence of SEQ ID No: 9.
  • the one or more peptides contain a terminal cysteine residue.
  • the polynucleotide encoding the apoptotic or cytotoxic protein is operatively linked to a promoter.
  • the promoter is a cell-specific promoter.
  • the promoter is selected from the group consisting of ABP, Rhox5, and HSD17B3.
  • the promoter is a species-specific promoter.
  • the nanoparticle construct comprises poly(lactide-co-glycolide) (PLGA).
  • the nanoparticle construct comprises polyethylene glycol (PEG). In certain aspects, the nanoparticle construct comprises maleimide-terminated PEG. In certain aspects, the nanoparticle construct comprises DSPE-PEG.
  • the nanoparticle construct comprises cationic lipids, ionizable lipids, and/or helper lipids.
  • the one or more peptides are conjugated to lipids.
  • the mean peptide density is from 5,000 to 50,000 peptides per nanoparticle.
  • the polynucleotide is condensed with protamine, lysine, or poly lysine.
  • the apoptotic or cytotoxic protein or the polynucleotide is encapsulated within the nanoparticle construct.
  • the nanoparticle construct comprises a nanoparticle having a PLGA matrix with maleimide terminated PEG lipids; wherein the one or more peptides are conjugated to the maleimide terminated PEG lipids; and wherein the apoptotic or cytotoxic protein or the polynucleotide is encapsulated in the nanoparticle.
  • the nanoparticle construct comprises a viral vector comprising the polynucleotide encoding the apoptotic or cytotoxic protein.
  • the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector.
  • the viral vector is a lentiviral vector.
  • the nanoparticle construct further comprises a fluorescent label.
  • the disclosure also provides a pharmaceutical composition comprising the isolated FSH, LH, or AMH peptide, the composition, or the nanoparticle construct of any of the preceding aspects.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an aqueous solution.
  • the isolated FSH, LH, or AMH peptide, composition, or the nanoparticle construct is freeze-dried.
  • the disclosure also provides a method of sterilizing a subject comprising administering to the subject an effective amount of the nanoparticle construct or pharmaceutical composition of any one of the preceding aspects.
  • the isolated FSH, LH, or AMH peptide, the nanoparticle construct, or the pharmaceutical composition is freeze-dried as a powder and dispersed in an aqueous medium prior to administration.
  • the composition is administered via injection.
  • the administering comprises a one-time injection.
  • the administering is intravenous, intraperitoneal, or intratesticular administration. In certain aspects, the administering is intravenous injection. In certain aspects, the administering is a one-time intravenous injection into the cephalic vein.
  • the effective amount comprises a dosage capable of inducing sterilization of the subject.
  • sterilization or ablation occurs within 24 hours, 48 hours, or 72 hours after injection.
  • sterilization is permanent.
  • the effective amount comprises a dosage capable of inducing ablation of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of one or more reproductive cell types of the subject. In certain aspects, the effective amount comprises a dosage capable of inducing ablation of at least about 60% of one or more reproductive cell types of the subject. In certain aspects, the effective amount comprises a dosage capable of inducing ablation of at least about 80% of one or more reproductive cell types of the subject.
  • the one or more reproductive cell types of the subject is Sertoli cells and/or Leydig cells.
  • the one or more reproductive cell types of the subject is gonocytes, primordial follicle cells, and/or primordial germ cells.
  • the subject is a cat or dog. In certain aspects, the subject is a cat. In certain aspects, the subject is a dog. In certain aspects, the subject is a male cat or dog. In certain aspects, the subject is a female cat or dog.
  • the subject is pre-pubescent. In certain aspects, the subject has reached pubertal maturation.
  • the disclosure also provides a method of determining uptake efficiency of a nanoparticle construct, comprising: a. administering to a subject the nanoparticle construct of a previous embodiment (comprising a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein) or a composition comprising the nanoparticle construct; and b. assessing ligand density on the nanoparticle surface, wherein increased ligand density or transfection efficiency for the nanoparticle as compared to a control or reference value is indicative of optimal uptake efficiency.
  • Figs. 1A-F show exemplary follicle-stimulating hormone (FSH) peptides mapped to the [3-subunit of the FSH protein.
  • the synthetic FSH peptides are indicated by brackets.
  • Fig. 1A peptide F49 (residues 35-49);
  • Fig. IB peptide F53 (residues 33-53);
  • Fig. 1C peptide F95 (residues 81-95);
  • Fig. ID peptide F99 (residues 86-99);
  • Fig. IE peptide F103 (residues 89-103);
  • Fig. IF peptide FSHa (residues 95-103).
  • Figs. 2A-B show immunocytochemistry using an anti-FSH receptor antibody with an Alexa Fluor 488 secondary antibody (third column from the left) indicates that the FSH receptor is expressed at high levels by the TM4 Sertoli cell line, but not by the mEcapl8 cell line.
  • Fig. 2A TM4 Sertoli, 40x objective.
  • FIGs. 3A-H show in vitro peptide targeting and binding affinity for FSH peptides F49, F53, F95, F99, F103, and FSHa.
  • FACS fluorescence -activated cell sorting
  • Fig. 3G shows the shift in FITC median fluorescence intensity of the TM4 Sertoli cells in response to increasing concentrations of FSH- peptides after 1 hour.
  • Fig. 3H shows the shift in FITC median fluorescence intensity of the mEcap 18 cells in response to increasing concentrations of FSH-peptides after 1 hour. A greater shift in median fluorescence indicates greater binding capability.
  • FIG. 4 shows representative images of epididymis and excretory organs (kidney, liver, and spleen) 8 hours post-injection.
  • Male adult mice were treated with IP injections of 300 pL of either 1 mM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each treatment group.
  • the peptide-FITCs applied are designated in the first image of each row.
  • Each field includes tissue from a vehicle-treated animal.
  • Figs. 5A-C show representative images of peptide-FITC localized to testes 8 hours post-injection compared to the vehicle control.
  • Fig. 5A FSHa-FITC.
  • Fig. 5B F49-FITC and F103-FITC.
  • Fig. 5C F53-FITC, F95-FITC, and F99-FITC.
  • Fig. 5D shows corresponding Imagel analysis of mean fluorescence intensity/pixel in testes of peptide-FITC injected males. Mean fluorescence intensity/pixel was normalized to that of the vehicle control within the same image.
  • Figs. 6A-C show the structure and chemistry of naphthoquinones.
  • Fig. 6A shows the structure of menadione (Md).
  • Fig. 6B show redox chemistry of the naphthoquinones.
  • SOD superoxide dismutase.
  • Fig. 6C shows linker designs for peptide conjugation. Structures of all linkers attached to the N-terminal of the peptide with menadione attached through a thioether bond to the linker. Structures are drawn using software: Advanced Chemistry Development Inc. ACD/3D Viewer (Freeware) Product Version 12.01 (Build 32890, 18 May 2009).
  • Figs. 7A-B show reactive oxygen species (ROS) production by TM4 Sertoli cells in response to FSH-Md and comparison of linkers.
  • ROS reactive oxygen species
  • Figs. 8A-D show production of ROS and oxidative damage in TM4 Sertoli cells.
  • TM4 Sertoli cells were treated with 50 pM of the peptide, peptide-Md conjugate or Md alone (as positive control) or with vehicle and then incubated at 37°C for 90 minutes.
  • Figs. 9A-E show analysis of male mice treated with FSH2Md at 10 weeks post-injection. Ten males were treated with IP injections of 300 pL/30g 14.5 mM FSH2Md in 30% Kolliphor/phosphate buffer saline (PBS). Controls were age-matched, untreated males.
  • Fig. 9A average testes weights (mg).
  • Fig. 9B sperm motility.
  • Fig. 9C sperm vitality.
  • the sperm chromatin dispersion test (HALO assay) was used to count the percentage of sperm with DNA damage in the form of strand breaks. p ⁇ 0.05.
  • Fig. 9A average testes weights (mg).
  • Fig. 9B sperm motility.
  • Fig. 9C sperm vitality.
  • Fig. 9D the sperm chromatin dispersion test (HALO assay) was used to count the percentage of sperm with DNA damage in the
  • FIGs. 10A-B show representative images of H and E staining of testes sections showing histology of FSH2Md-treated or control mice at two different magnifications. Seminiferous tubules from FSH2Md-treated mice exhibited uninterrupted spermatogenesis.
  • FIG. 11 show testes section of FSH2Md-treated or control mice. Seminiferous tubules from treated mice exhibited uninterrupted spermatogenesis. The ApopTag assay showed that there was some apoptosis of germ cells.
  • Figs. 12A-D show exemplary luteinizing hormone (LH) peptides mapped to the [3-subunit of the LH protein, which targets receptors on the cells in the ovary and Leydig cells of the testes.
  • the synthetic LH peptides are indicated by brackets.
  • Fig. 12A peptide L57 (residues 38-57);
  • Fig. 12B peptide L95 (residues 81-95);
  • Fig. 12C peptide L101 (residues 93- 101);
  • Fig. 12D peptide LHa (residues 96-107).
  • Figs. 13A-B show a Leydig cell model.
  • Fig. 13A shows relative expression of LHr mRNA in the mouse Leydig cell lines, MLTC-1 and TM3 cells, whole testis, and mEcapl8 cells, as assessed by qPCR.
  • Fig. 13B shows immunocytochemistry using an anti- LHr antibody with an Alexa Fluor 488 secondary antibody, which indicates that the LH receptor is expressed at high levels in MLTC-1 cells but not in mEcapl8 cells.
  • FIGS. 14A-F show in vitro peptide targeting and binding affinity for LH peptides.
  • Fig. 14A L57; Fig. L95; Fig. 14C: L101; Fig. 14D LHa.
  • Fig. 14E-F LH peptides exhibited specificity of binding to MLTC-1 Leydig cells when compared to binding to
  • FIG. 14E shows the shift in FITC median fluorescence intensity of the MLTC-1 Leydig cells in response to increasing concentrations of LH-peptides.
  • Fig. 14F shows the shift in FITC median fluorescence intensity of the mEcapl8 cells in response to increasing concentrations of LH-peptides. A greater shift in median fluorescence indicates greater binding capacity.
  • Figs. 15A-B show representative images and mean intensity of fluorescence of peptide-FITC localized to testes 8 hours post-injection compared to the vehicle control.
  • Fig. 15A shows images of testes from LHa-FITC, L57-FITC, L101-FITC, and L95- FITC-treated animals alongside the vehicle control.
  • 16 shows representative bright field and fluorescence images of excretory organs (kidney, liver, and spleen) 8 hours post-injection.
  • Male adult mice were treated with IP injections of 300 pL of either 1 mM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each treatment group.
  • Peptide-FITCs applied are as shown in the first image of each row.
  • the fourth (unlabeled) tissue in the bottom three images were from animals treated with an unrelated peptide-FITC.
  • Fig. 17 shows lysosomal processing of vcMonomethylAuristatin E (vcMMAE), an anticancer agent that interacts with a-tubulin in a similar way to Vinca alkaloids to block a-tubulin polymerization and prevents the formation of the mitotic apparatus.
  • vcMMAE vcMonomethylAuristatin E
  • the linked maleimide allows conjugation of the peptide via the N-terminal thiol group. Between the maleimide and the Aur, there is a cleavable linker made up of valine, citrulline (circled) and para-aminobenzyl carbamate (PABC).
  • the dipeptide linker is cleaved and then Aur is released from the PABC by 1,6-elimination. Structures drawn using software: Advanced Chemistry Development Inc. ACD/3D Viewer (Freeware) Product Version 12.01 (Build 32890, 18 May 2009).
  • Figs. 18A-C show the effect of FSH2Md and LH2Auristatin (LH2Aur) in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. Males were euthanized immediately after the matin period ( ⁇ I0 weeks post-injection).
  • Fig. 18C shows the average area occupied by seminiferous tubule in the testes of each treatment group.
  • Figs. 19A-C show the effect of FSH2Md and LH2Auristatin (LH2Aur) in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. Males were euthanized immediately after the matin period ( ⁇ I0 weeks postinjection).
  • Fig. 19C shows the percentage of tubules affected (>4 stained cells) per testes section, which were counted with the number of stained cells per affected tubule. NB. Subjects were randomly selected from the treatment groups but those testes weighing less than 60 mg were excluded.
  • Figs. 20A-C show the effect of FSH2Md and LH2Aur in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Fig. 5B shows the average number of Sertoli cells per tubule.
  • NB Subjects were randomly selected from the treatment groups; subjects whose testes weighed less than 60 mg were excluded.
  • Figs. 21A-C show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Fig. 21 A Percent sperm with DNA strand breaks was accessed by probing sperm for DNA strand breaks using the HALO assay.
  • Fig. 2 IB Percent sperm with oxidative DNA damage was assessed using an anti-8-OH-dG antibody to detect oxidized DNA guanosine bases.
  • Fig. 21C morphology assessment of sperm. Sperm smears were fixed in methanol and then stained using Rapid Diff Kit (Pathtech). Sperm were categorized as either having normal or abnormal morphology.
  • Figs. 22A-D show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Figs. 22B-D mean motility score.
  • FIG. 23A-D show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Flow cytometry was used with probes to assess mitochondrial ROS (MSR; Fig. 23 A) and cytoplasmic superoxide (DHE; FIG. 23B).
  • Fig. 22C shows caudal sperm vitality as assessed by the eosin exclusion method.
  • Fig. 23D shows percent DNA fragmentation index (DFI) as a measure of susceptibility to acid-mediated DNA fragmentation assessed by the sperm chromatin structure assay (SCSA).
  • DFI percent DNA fragmentation index
  • Figs. 24A-B show the effect of FSH2Md and LH2Aur on fertility in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. At 13 days post-coitus, females were euthanized then embryos and resorption moles were counted.
  • Fig. 24A shows the number of embryos per pregnant female.
  • Fig. 24B shows the average number of resorption moles per pregnant female and the proportion of pregnant females that had at least 1 resorption mole.
  • Fig. 25A-B show the effect of FSH2Md and LH2Aur on fertility in vivo.
  • Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group.
  • Haematoxylin- and eosin-stained sections illustrate normal development of the seminiferous tubules in a vehicle control testis section (Fig. 25A) and abnormal development of the seminiferous epithelia in a testis section from a FSH2Md and LH2Aur-treated male (Fig. 25B).
  • Scale bar 100 pm.
  • Fig. 26 shows an exemplary anti-Mullerian hormone peptide (AMHa) mapped to the surface of the murine AMH protein (residues 93-102).
  • the synthetic AMH peptide is indicated by a circle and an arrow.
  • FIGs. 27A-B show expression of AMH receptor type II (AMHr) mRNA.
  • FIG. 28A shows relative expression of MLTC-1 mRNA by qPCR.
  • Fig. 28 shows in vitro binding of AMHa-FITC to MLTC-1 cells.
  • a shift in FITC median fluorescence intensity of the MLTC-1 cells is observed in response to increasing concentrations of AMHa-FITC after 1 hour.
  • Figs. 29A-I show representative images of AMHa-FITC localization following IP injection of male adult mice with 300 pl of either ImM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each group. Representative images from 8 hours post-injection are shown.
  • Fig. 29A Testis.
  • Fig. 29B Adrenal.
  • Fig. 29C Brain.
  • Fig. 29D Pituitary.
  • Fig. 29E Heart.
  • Fig. 29F Lung.
  • Fig. 29G Kidney.
  • Fig. 29H Liver.
  • Fig. 291 Spleen.
  • Fig. 29J shows ImageJ analysis of mean fluorescence intensity /pixel in testes of AMHa-FITC injected males. Mean fluorescence intensity/pixel was normalized to that of the vehicle control within the same image.
  • Figs. 30A-E show analysis of caudal sperm of male mice 10 weeks postinjection with AMH2Md.
  • Fig. 30A shows percent live positive for mitochondrial ROS.
  • Fig. 30B shows percent DNA fragmentation index (DFI) as a measure of susceptibility to acid-mediated DNA fragmentation assessed by SCSA.
  • Fig. 30C shows percent sperm vitality.
  • Fig. 30D shows percent sperm motility.
  • Fig. 30E shows percent oxidative DNA damage as determined by immunocytochemistry using anti-8-OHdG to detect DNA oxidation.
  • Figs. 31A-B show histology of the testes of male mice 10 weeks postinjection with AMH2Md or vehicle.
  • Fig. 31A shows testes section of AMH2Md-treated males.
  • Figs. 32A-D show in vitro binding of peptide-FITC.
  • Fig. 32A shows LH- FITC on a Leydig cell line;
  • Fig. 32 shows FSH-FITC on a Sertoli cell line.
  • Cells were cultured in growth specific medium and then, when confluent, incubated for 1 hour at 37°C with 50 pM peptide-FITC, washed, fixed and counterstained with DAPI nuclear stain before imaging at 40x magnification.
  • Figs. 32C-D shows binding of LH-FITC (Fig. 32C) and FSH-FITC (Fig. 32D) to Leydig and Sertoli cell lines.
  • Fig. 33A-D shows in vitro binding and specificity of peptide-targeted nanoparticles (NPs) encapsulating FITC-dextran.
  • Cultured cells were incubated with 2 mg/ml of NP- untargeted NP, nonspecific peptide (nsp)NP, or targeted NP - for 24 hours. Cells were then washed, fixed and counterstained with DAPI before imaging at 40x.
  • Fig. 33A shows LH-NP- FITC
  • Fig. 33B shows FSH-NP-FITC
  • Fig. 33C shows a Leydig cell line
  • Fig. 34 shows transfection efficiency of TM4 Sertoli cells over time.
  • the cells were treated with Lipofectamine + pDNA (first row), empty FSH-targeted lipid NPs (second row), FSH peptide -lacking lipid NPs + pDNA (third row), and FSH peptide -conjugated lipid NPs + pDNA (fourth row).
  • Table 1 provides a listing of exemplary sequences referenced herein.
  • the present disclosure further relates to nanoparticle constructs (e.g., targeting peptides conjugated to the lipids on the surface of the nanoparticle and a nucleotide sequence encapsulated in the nanoparticle) and compositions thereof.
  • the present disclosure further relates to a method of sterilizing a male or female cat or dog comprising administering to the male or female cat or dog an effective amount of the nanoparticle constructs or compositions thereof, where the nanoparticle constructs comprise one or more peptides that each bind to one or more receptors of one or more reproductive cells; and an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein.
  • the male gonads i.e., testes
  • the male gonads perform two distinct functions, that is, the production of spermatozoa within the seminiferous tubules and the synthesis of steroids by Leydig cells (Ye et al., 2017), which reside within the interstitial space outside the seminiferous tubules (Evans & Ganjam, 2011).
  • testicular function is regulated via the hypothalamic- pituitary-gonadal (HPG) axis and secretion of the glycoprotein hormones (GPH), that is, Follicle-Stimulating Hormone (FSH) and Luteinizing hormone (LH), by gonadotrophs in the anterior pituitary (Abel et al., 2008; Cheng & Mruk, 2010).
  • GPH glycoprotein hormones
  • FSH Follicle-Stimulating Hormone
  • LH Luteinizing hormone
  • Sertoli and Leydig cells are functionally interdependent and essential for normal testicular function.
  • Sertoli cells are selectively ablated in the mature animal, then germ cell development is bought to an abrupt halt (Rebourcet et al., 2017).
  • Sertoli cell function is regulated by androgens as well as by FSH (Abel et al., 2008). While androgen receptors are ubiquitously expressed, the FSH receptor is specific to Sertoli cells and the LH receptor is specific to Leydig cells.
  • GPHs are generally composed of an a- and a [3-subunit; the former is shared by several related hormones, including FSH, LH, and Thyroid-Stimulating Hormone (TSH), while the latter confers specificity on the binding of a particular GPH to its cognate receptor.
  • Anti-Mullerian hormone (AMH) also known as Mullerian Inhibiting Substance, has a sexually dimorphic expression in Sertoli cells of the testis (Alves et al., 2013) and granulosa cells of the ovary (Josso et al., 2001) and, in the embryo is critical for normal differentiation of the internal reproductive tract structures.
  • the anti-Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors.
  • AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood.
  • AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015).
  • AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Cimino et al., 2016).
  • AMH belongs to the TGF-J3 superfamily of glycoproteins, which consists of a large number of structurally related polypeptide growth factors involved in the regulation of growth and differentiation (Xu et al., 2019), including the activins and bone morphogenetic proteins (BMPs).
  • BMPs bone morphogenetic proteins
  • the TGF-J3 superfamily bind to paired receptors - a type I receptor that is shared by different members of the family and a type II receptor that endows specificity.
  • AMH binds to BMP receptors la and lb, which it shares with the BMPs and the growth/differentiation factors (GDF), however, AMH also has its own unique receptor type II (Hinck, 2012).
  • Anti-Mullerian hormone also known as Mullerian Inhibiting Substance, has a sexually dimorphic expression in Sertoli cells of the testis (Alves et al., 2013) and granulosa cells of the ovary (Josso et al., 2001) and, in the embryo is critical for normal differentiation of the internal reproductive tract structures.
  • the anti-Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors.
  • AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood. Nevertheless, AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015). AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Ohyama et al., 2015).
  • AMH belongs to the TGF-J3 superfamily of glycoproteins, which consists of many structurally related polypeptide growth factors involved in the regulation of growth and differentiation (Xu et al., 2019), including the activins and bone morphogenetic proteins (BMPs).
  • BMPs bone morphogenetic proteins
  • the TGF-J3 superfamily bind to paired receptors - a type I receptor that is shared by different members of the family and a type II receptor that endows specificity.
  • AMH binds to BMP receptors la and lb, which it shares with the BMPs and the growth/differentiation factors (GDF), however, AMH also has its own unique receptor type II (Hinck, 2012).
  • an apoptotic protein is targeted to reproductive cells or reproductive organs to cause cell death that may lead to sterilization.
  • the apoptotic protein is Diphtheria toxin fragment A (DTA).
  • DTA has been used in unrelated indications such as glioblastoma multiforme and prostate cancer.
  • Diphtheria toxin (“DTX”) is a single protein composed of two components; fragment A and fragment B.
  • Fragment B is the component responsible for binding to the HBEGF receptor (the heparin-binding EGF-like growth factor receptor), which leads to its entry into the cell.
  • Fragment B binds to the HBEGF receptor present on the surface of many cell-types, leading to internalization and delivery of fragment A into the cells. Once inside the host cell, fragment A is able to block protein synthesis of the host cell. This induces death of the specific host cell. Without fragment B, fragment A cannot enter any other non-host cell, and is thus inactive or inert without it.
  • diphtheria toxin for controlled cell ablation of a target cell is that it has not been shown to have a toxic effect on other cell types or systems in vivo (Rebourcet et al., 2017). Diphtheria toxin was previously shown to be a means of selectively ablating Sertoli cells from the testis of adult transgenic mice carrying the diphtheria toxin receptor on the surface of these cells. Use of Diphtheria toxin was shown to be an acute means to specifically induce ablation of Sertoli cells with cell death occurring within 24 hours (Rebourcet et al., 2017).
  • DTA comprises the amino acid sequence of SEQ ID No. 10 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID No: 10.
  • the variations can comprise modifications, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • polypeptide refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental, such as through mutations of hosts which produce the proteins or errors.
  • the nanoparticle construct further comprises a viral vector (i.e., a recombinant viral-based DNA construct).
  • the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector.
  • the nanoparticles comprise a lentiviral vector.
  • the lentiviral constructs are self-inactivating and/or cannot be re-activated by serendipitous viral infections to cause insertional mutagenesis and cannot activate surrounding genes due to the mutations in the 3’ viral long terminal repeat.
  • the tumorigenic WPRE common to many lentiviral vectors have been replaced with a non-tumorigenic OPRE.
  • the nanoparticles comprise a lentiviral vector containing the coding sequence for DTA.
  • the nanoparticle construct further comprises a transgene coding a reporter protein (e.g., a fluorescent reporter protein) or a fluorescent label. This labeling is useful, for example, to determine body distribution and cellular uptake (in vitro or in vivo).
  • a fluorescent reporter transgene delivered by these nanoparticles allows for analysis of tissues collected post-mortem using fluorescent imaging equipment and detection of the fluorescent protein within fixed tissues.
  • the fluorescent protein is red fluorescent protein (RFP), green fluorescent protein (GFP), or enhanced green fluorescent protein (EGFP).
  • the fluorescent label is fluorescent isothiocyanate (FITC)- dextran. FITC-dextran loaded NPs can be used to optimize targeting ligand density and confirm the targeting specificity and uptake efficacy of NP preparations.
  • the polynucleotide is operatively linked to a promoter (i.e., a specific component controlling delivered gene expression carried within the nanoparticle).
  • a promoter i.e., a specific component controlling delivered gene expression carried within the nanoparticle.
  • the promoter can be cell-specific or species-specific (e.g., specific for cats or dogs, or specific for a particular breed of cat or dog). Use of the promoter can provide an additional aspect of cell specificity, to enable the delivered gene to be ‘switched on’ when delivered to the cell types of interest.
  • the promoter is cell-specific.
  • the cell-specific promoters are selected from ABP, Rhox5, or HSD17B3.
  • a peptide used to target reproductive cells or reproductive organs can be from any vertebrate source (e.g., any species or breed of dogs or cats).
  • the peptide targets receptors on reproductive cells selected from primordial germ cells or gonocytes, and primordial follicle cells.
  • the peptides target FSH and/or LH receptors on Sertoli and/or Leydig cells.
  • the peptide is directed to both Sertoli and Leydig cells. It is known in the art that the number of Leydig cell numbers is correlated with Sertoli cell number in adults (mature subjects). It was previously shown in cell ablation studies that ablation (i.e., selective reduction or removal) of Sertoli cells also results in reduction of Leydig cell numbers by 75% (Rebourcet et al., 2014, 2017). A reduction of Leydig cells have been seen when Sertoli cell population is decreased by about 50% or more (Rebourcet et al., 2017).
  • Ablating Leydig cells along with Sertoli cell ablation may ablate testosterone production to a degree to confer infertility. Ablating Sertoli cells alone will also impact Leydig cell function.
  • the peptide is directed to Sertoli cells alone. [00169] In certain aspects of the invention, the peptide targets the AMH receptor on the primordial follicle.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of TVX9GLGPX10Y (SEQ ID No. 95), wherein X9 is R or Q and X10 is S or G.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CTVX9GLGPX10Y (SEQ ID No. 96), wherein X 9 is R or Q and X10 is S or G.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4Q (SEQ ID No. 33), wherein Xi is K or G; X 2 is A or P; X 3 is K, S, G, orN; and X4 is T orN.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 34), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4Q (SEQ ID No. 35), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, orN; and X4 is T orN.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 36), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, or N; and X4 is T or N.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of YTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 49), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CYTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 50), wherein Xi is K or G; X2 is A or P; X 3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CHX7GX8X7DSDSTDX7T (SEQ ID No.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of X8CDSDSTDX7TVX9GL (SEQ ID No. 78), wherein Xs is K, R, or G; X7 is C, S, or A; and X9 is R or Q.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CDSDSTDX7TVX9GL (SEQ ID No. 79), wherein X 8 is K, R, or G; X 7 is C, S, or A; and X 9 is R or Q.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of SDSTSX7TVX9GLGPX10Y (SEQ ID No. 89), wherein X 7 is C, S, or A; X 9 is R or Q; and X10 is S or G.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CSDSTSX7TVX9GLGPX10Y (SEQ ID No. 90), wherein X 7 is C, S, or A; X 9 is R or Q; and X10 is S or G.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No.
  • SEQ ID No. 72 SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
  • an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No.
  • SEQ ID No. 46 SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No.
  • SEQ ID No. 75 SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
  • the isolated FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and/or SEQ ID No. 18.
  • the isolated FSH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of SX18SDX7GGPX8X19X20X21 (SEQ ID No. 144), wherein Xis is T, S, orN; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CSX18SDX7GGPX8X19X20X21, (SEQ ID No. 145), wherein Xis is T, S, or N; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of X11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 112), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CX11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 113), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of SPPVALX7RX7GPX7RX17 (SEQ ID No. 125), wherein X 7 is C, S, or A and X17 is L or R.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CSPPVALX7RX7GPX7RX17 (SEQ ID No. 126), wherein X 7 is C, S, or A and X17 is L or R.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CRX17SX18SDX7G (SEQ ID No. 133), wherein X17 is L or R; Xis is T, S, orN; and X7 is C, S, or A.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No.
  • SEQ ID No. 121 SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
  • an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No.
  • SEQ ID No. 127 SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
  • the isolated FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, and/or SEQ ID No. 104.
  • the isolated LH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
  • an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
  • an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
  • the isolated AMH peptide has one substitution modification, two substitution modifications, three substitution modifications, or four substitution modifications relative to SEQ ID No. 149, SEQ ID No. 153, SEQ ID No. 157, and/or SEQ ID No. 161.
  • an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the AMH peptide comprises the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
  • an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the AMH peptide comprises the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
  • the isolated AMH peptide is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
  • the peptide sequences are modified.
  • the peptide sequences may be modified to include a terminal cysteine or terminal methionine, which may be used to attach the peptide to the nanoparticle, a protein, a peptide, a small molecule, or other moiety.
  • the terminal cysteine or terminal methionine is at the N- terminus.
  • the terminal cysteine or terminal methionine is at the C-terminus.
  • the peptide is amidated.
  • the N-terminus is amidated.
  • the peptide comprises a linker.
  • the linker is a maleimide group, 1,6-aminohexanoic acid (Ahx), a y-aminobutanoate -mercaptopropionic acid (y-aminobutanoate-MPA or y-aminobutanoic acid with MPA) linker, alanine with 3- mercaptopropionic acid (MPA), mini-polyethyleneglycol with MPA, or a cathepsin cleavable valine-citrulline(vc)-linker, such as a vc-PABC linker.
  • the peptide is conjugated to a protein, a peptide, a nanoparticle, a label, a small molecule, and/or other moiety.
  • the conjugated protein is an apoptotic protein or a cytotoxic protein.
  • the conjugated small molecule is a cytotoxic agent, an antimimotic agent, or an antineoplastic agent.
  • the conjugated small molecule is an auristatin molecule, such monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • the conjugated small molecule is a redox cycling compound.
  • the conjugated small molecule is 2-methyl-l,4-naphthoquinone (i.e., menadione (Md)).
  • the conjugated small molecule is a fluorescent label, such as fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • the peptide sequences may be modified, for example to purify protein from cell material for use in in vitro experiments.
  • a modified peptide comprises a spacer, such as 1,6- aminohexanoic acid (Ahx).
  • the spacer is at the N-terminus of the peptide. In certain aspects, the spacer is at the C-terminus of the peptide.
  • the compositions comprise a nanoparticle.
  • the nanoparticle comprises peptides on its surface that target a receptor on a reproductive cell.
  • Nanoparticles can be lipid-based, polymer-based, or be a polymer/lipid hybrid. Nanoparticles provide many unique advantages over conventional delivery systems. For example, nanoparticles can encapsulate various therapeutics, such as nucleic acids, proteins and small compounds, and enhance both drug stability and bioavailability thus leading to substantial reduction in dosage as well as dose frequency. In certain aspects, the nanoparticles encapsulate an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein.
  • Targeting ligands can also be incorporated onto the nanoparticle surface in order to enable drug delivery to specific cell populations, achieving improvements in efficacy with a corresponding reduction in unwanted effects.
  • Targeting ligands can be introduced on the surface of the nanoparticles with varied densities, which can affect uptake efficiency of the nanoparticles.
  • the nanoparticles can be polymer-based, lipid-based, a polymer/lipid hybrid, or can comprise biocompatible inorganic nanovectors based on layered- double hydroxide strategy.
  • Nanoparticle constructs e.g., nanoparticle itself together with the targeting peptides conjugated to the lipids of the nanoparticle and nucleotide sequence encapsulated in the nanoparticle
  • the activity of a nanoparticle construct can depend on, for example, the materials used in the nanoparticle, the type of peptides, peptide density, as well as length of spacer-peptide ligands as well as the testing of cell-specific promoters.
  • Nanoparticles may be prepared using a variety of methods.
  • a nanoparticle is prepared with methods comprising single or double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation.
  • Methods for making nanoparticles are available to one skilled in the art, such as those described in “Microcapsules and Nanoparticles in Medicine and Pharmacy,” which is incorporated in its entirety herein by reference ⁇ Microcapsules and Nanoparticles in Medicine and Pharmacy, n.d.).
  • Related methods are also described in and are incorporated in their entireties by reference (Mathiowitz et al., 1987; Mathiowitz & Langer, 1987; Polyanhydride Microspheres as Drug Carriers. II. Microencapsulation by Solvent Removal - Mathiowitz - 1988 - Journal of Applied Polymer Science - Wiley Online Library, n.d.).
  • peptide density or “ligand density” of targeted nanoparticles refers to the density of targeting peptide/ligand on the nanoparticle surface.
  • Ligand density relates to the ability of the nanoparticles to navigate across biological barriers and promote cell-specific uptake.
  • there is an optimal ligand density for a nanoparticle construct and targeted receptor that can be determined by a person of ordinary skill in the art.
  • the ligand density of targeted nanoparticles may be indicative of the uptake efficiency of the nanoparticles and may reflect targeted delivery.
  • the mean peptide density is from 5,000 to 50,000 peptides per nanoparticle.
  • N/P ratio refers to the ratio of positively charged amine (N) groups of the polymer to negatively charged phosphate (P) groups of DNA, which is one characteristic of nanoparticles in affecting gene transfection efficiency and cytotoxicity.
  • Various other properties characterized, such as surface chemistry, drug loading, stability in blood serum, and release profile of payloads can be indicative of cell expression or viability.
  • Modem spectroscopic NMR, MS, UV-Vis, HPLC, IR
  • other known techniques in the art dynamic light scattering, size exclusion chromatography, gel electrophoresis, electron microscopy, X-ray photoelectron spectroscopy
  • the nanoparticle is polymer-based.
  • the nanoparticle comprises components selected from poly(hydroxy acids), polyglycolic acid (PGA), polylactic acid (PLA), poly(D,L-lactic acid), poly(lactide-co-glycolide) (“PLGA”), polyamides, polycarbonates, polyalkylenes, polyethylene, polypropylene, polyethylene glycol), polyethylene oxide), poly(Ethylene terephthalate), polyvinyl compounds, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses, polysaccharides, peptides, proteins, copolymers, PEG+linear polymers, dendritic polymers, or copolymers or blends thereof.
  • the nanoparticle comprises PLGA.
  • PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions known by a person of ordinary skill in the art to effect drug release.
  • the degradation time of PLGA nanoparticles can be altered from days to years to achieve a safe and sustained action by varying the polymer molecular weight, the composition of the block copolymer and the structure of the nanoparticles.
  • the nanoparticle comprises PLGA and 1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-poly(ethylene glycol) (“DSPE-PEG”).
  • the nanoparticles further comprise cationic lipids, ionizable lipids, and other helper lipids.
  • helper lipids include DC-Cholesterol, dilinoleylmethyl-4- dimethylaminobutyrate, DLin-KC2-DMA, DLin-MC3-DMA, and Lipofectamine 3000.
  • Polymeric nanoparticles may be prepared using a variety of methods.
  • a nanoparticle is prepared with methods comprising single or double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation.
  • Methods for making nanoparticles are available to one skilled in the art, such as those described in “Microcapsules and Nanoparticles in Medicine and Pharmacy,” which is incorporated in its entirety herein by reference (Microcapsules and Nanoparticles in Medicine and Pharmacy, n.d.).
  • Related methods are also described in and are incorporated in their entireties by reference (Mathiowitz et al., 1987; Mathiowitz & Langer, 1987; Polyanhydride Microspheres as Drug Carriers. II. Microencapsulation by Solvent Removal - Mathiowitz - 1988 - Journal of Applied Polymer Science - Wiley Online Library, n.d.).
  • the nanoparticles comprise liposomes. Liposomes using different lipid components may be produced according to methods available to one skilled in the art (Kim et al., 1983; Lee et al., 1992; D. Liu et al., 1992; Wang & Huang, 1989).
  • the lipids of choice are dissolved in an organic solvent and are mixed and dried onto the bottom of a glass tube under vacuum. The lipid fdm is rehydrated using an aqueous buffered solution containing the material to be encapsulated by gentle swirling. The hydrated lipid vesicles or liposomes are washed by centrifugation and can be filtered and stored at 4°C (Gold & Dritschilo, 1992).
  • liposomes can be made using the method described in “Production of Unilamellar Vesicles Using an Inverted Emulsion” (Pautot et al., 2003).
  • the liposomes comprise streptavidin-coated lipids, e.g., DPPC, DSPC, and similar lipids.
  • the polynucleotide to be delivered by the nanoparticle construct is condensed with protamine, lysine, or polylysine to further enhance transfection efficiency prior to encapsulation by the nanoparticles.
  • transfection efficiency refers to the percentage of cells successfully transfected by nanoparticle delivery as evidenced, for example, by the polynucleotide payload delivery to the nucleus resulting in transgene expression as related to an entire population.
  • the polynucleotide is delivered inside a PLGA nanoparticle, wherein the polynucleotide is condensed with protamine, lysine, or poly lysine.
  • the nanoparticle comprises PEG (“polyethylene glycol)”). PEGylation can minimize non-specific particle uptake and clearance by immune cells.
  • the nanoparticle comprises a maleimide-terminated PEG, e.g., as a surface coating. Such a coating allows for conjugation of the targeting peptide (synthesized with a terminal cysteine) to the PEG via a covalent thiol -maleimide linkage.
  • Each peptide may be engineered to contain a terminal cysteine for coupling purposes and attachment to the nanoparticle surface may be achieved using maleimide-terminated PEG.
  • the peptide may comprise a terminal cysteine to couple with maleimide-terminated PEG via covalent thiol- maleimide linkage.
  • SEQ ID No. 2 comprises the sequence of SEQ ID No. 1 but modified to include a C-terminal cysteine (FSH receptor of Sertoli cells).
  • SEQ ID No. 4 also comprises the sequence of SEQ ID No. 3, but modified to include the C-terminal cysteine (LH receptor of Leydig cells).
  • the one or more peptides are conjugated to lipids on the surface of the nanoparticle construct.
  • a first component i.e., a protein, an apoptotic protein, a peptide (e.g., an FSH, an LH, and/or an AMH peptide), a nanoparticle, a small molecule, or other moiety
  • a second component i.e., a protein, an apoptotic protein, a peptide, a nanoparticle, a small molecule, or other moiety
  • a first or second component may be modified by reacting the peptide with a modifying agent.
  • a “modifying agent,” as used herein, refers to a suitable organic group e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group.
  • An “activating group,” as used herein, is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group, thereby forming a covalent bond between the modifying agent and the second chemical group.
  • amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.
  • Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl dilsulfides, 5- thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like.
  • An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
  • a first component is attached to a second component via a covalent linker.
  • the covalent linker comprises a 1,2, 3 -triazole linker, an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, a urea or thiourea linker, an amidine linker, an amine linker, or a sulfonamide linker.
  • the covalent linker comprises a 1,2, 3 -triazole linker formed by the 1,3-dipolar cycloaddition reaction of azido groups on the surface of a nanoparticle with a peptide containing an alkyne group.
  • the 1,2, 3 -triazole linker is formed by the 1,3- dipolar cycloaddition reaction of alkynes on the surface of a first component with a second component containing an azido group. Such cycloaddition reactions may be performed with or without a catalyst.
  • the reaction includes a Cu(I) catalyst, a suitable Cu(I) -ligand, and a reducing agent to reduce Cu(II) compound to catalytic active Cu(I) compound.
  • This Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC) can also be referred to as the click reaction.
  • the covalent linker comprises an amide linker formed via an amide bond between an amine on a first component with the carboxylic acid group of a second component.
  • the amide bond in the linker can be made using any of the conventional amide bond forming reactions with suitably protected amino acids or peptides and activated carboxylic acid such N-hydroxy succinimide-activated ester.
  • the covalent linker comprises a disulfide linker made via the formation of a disulfide (S-S) bond between two sulfur atoms of the form, for instance, of Ri-S-S-R2.
  • a disulfide bond can be formed by thiol exchange of a first component containing thiol/mercaptan group(-SH) with another activated thiol group on a second component containing thiol/mercaptan groups.
  • the covalent linker comprises a thioether linker made by the formation of a sulfur-carbon (thioether) bond in the form, for instance, of R S-R2.
  • Thioether can be made by either alkylation of a thiol/mercaptan (-SH) group on a first component with an alkylating group such as halide or epoxide on a second component.
  • Thioether linkers can also be formed by Michael addition of a thiol/mercaptan group on a first component to an electron-deficient alkene group on a second component containing a maleimide group or vinyl sulfone group as the Michael acceptor.
  • thioether linkers can be prepared by the radical thiol-ene reaction of a thiol/mercaptan group on a first component with an alkene group a second component.
  • the covalent linker comprises a hydrazone linker made by the reaction of a hydrazide group on a first component with an aldehyde/ketone group on a second component.
  • the covalent linker comprises a hydrazide linker formed by the reaction of a hydrazine group on a first component with a carboxylic acid group on a second component. Such reaction is generally performed using chemistry that is similar to the formation of amide bond where the carboxylic acid is activated with an activating reagent.
  • the covalent linker comprises an imine or oxime linker formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on a first component with an aldehyde or ketone group on a second component.
  • the covalent linker comprises a urea or thiourea linker prepared by the reaction of an amine group on a first component with an isocyanate or thioisocyanate group on a second component.
  • the covalent linker comprises an amidine linker prepared by the reaction of an amine group on a first component with an imidoester group on a second component.
  • the covalent linker comprises an amine linker made by the alkylation reaction of an amine group on a peptide with an alkylating group such as halide, epoxide, or sulfonate ester group on a nanoparticle.
  • the covalent linker comprises an amine linker made by reductive amination of an amine group on a first component with an aldehyde or ketone group on a second component with a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.
  • the covalent linker comprises a sulfonamide linker made by the reaction of an amine group on a first component with a sulfonyl halide (such as sulfonyl chloride) group on a second component.
  • a sulfonamide linker made by the reaction of an amine group on a first component with a sulfonyl halide (such as sulfonyl chloride) group on a second component.
  • the covalent linker comprises a sulfone linker made by Michael addition of a nucleophile to a vinyl sulfone.
  • the vinyl sulfone or the nucleophile may be on the surface of or attached to the first or second component.
  • a first component may be attached to a second component via non-covalent conjugation methods.
  • a negatively charged peptide can be conjugated to a positively charged nanoparticle through electrostatic adsorption.
  • a peptide containing a metal ligand can also be conjugated to a nanoparticle containing a metal complex via a metal-ligand complex.
  • a first component is attached to a second component via a (streptjavidin-biotin interaction.
  • a first or second component can be attached to a polymer.
  • a peptide prior to the assembly of a nanoparticle, can be attached to polylactic acid-block-polyethylene glycol.
  • a nanoparticle can be formed with reactive or activatable groups on its surface. In the latter case, the peptide is prepared with a group which is compatible with the attachment chemistry that is presented on the surface of the nanoparticle surface.
  • a peptide may be attached to virus-like particles (VLP) or liposomes using a suitable linker.
  • the linker is a compound or reagent that capable of coupling two molecules together.
  • the linker can be a homobifimctional or heterobifimctional reagent.
  • an VLP or liposome synthetic nanoparticle containing a carboxylic group on the surface can be treated with a homobifunctional linker, adipic dihydrazide (ADH), in the presence of l-ethyl-3-(3- dimethylamino) propyl carbodiimide, hydrochloride (EDC) to form the corresponding synthetic nanoparticle with the ADH linker.
  • ADH adipic dihydrazide
  • EDC hydrochloride
  • the resulting ADH linked synthetic nanoparticle is then conjugated with an agent containing an acid group via the other end of the ADH linker on the NC to produce the corresponding VLP or liposome peptide conjugate.
  • Suitable conjugation methods are known in the art, for example, as described in “Bioconjugate Techniques” (Hermanson, 2013), which is incorporated herein in its entirety by reference.
  • the nanoparticle constructs are formulated into compositions comprising a pharmaceutically acceptable carrier.
  • the compositions comprise compendial and widely used excipients.
  • the composition comprises at least one excipient.
  • compositions comprise a freeze-dried or lyophilized powder, or aqueous solution.
  • compositions comprise a freeze-dried powder that can be re-dispersed in aqueous medium prior to administration. This can benefit storage and shelf life.
  • a subject is administered the nanoparticle construct or compositions thereof at an effective dosage.
  • the subject is administered a composition containing the nanoparticles described above at an effective dosage.
  • the effective dosage comprises an effective amount of the nanoparticles or compositions thereof sufficient to induce ablation of cells.
  • the effective amount of nanoparticle constructs or compositions thereof described above results in partial or complete cell ablation of the reproductive cells. Even partial ablation of the cells results in significant disruption of the blood testes barrier, which has been shown to lead to infertility in males.
  • the percentage of cells ablated comprise at least about 50%, at least 60%, at least about 70%, at least about 80%, or at least about 90% of cells. In certain aspects, complete cell ablation occurs. In certain aspects, at least about 60% of cells are ablated.
  • the invention relates to administering to a male cat or dog an apoptotic or cytotoxic protein or transgene encoding an apoptotic or cytotoxic protein.
  • administration refers to intravenous, intraperitoneal, or intratesticular administration, although other methods of administration known by persons of ordinary skill in the art in delivering nanoparticle constructs can also be used.
  • the nanoparticle constructs or compositions thereof are administered by intravenous administration or injection.
  • the nanoparticle constructs or compositions thereof are administered in a single dose or multiple doses.
  • administration comprises a single dose.
  • administration comprises a single intravenous injection.
  • administration comprises a single intravenous injection into the cephalic vein.
  • the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent.
  • Pubertal maturation in males is defined as the presence of motile sperm in the ejaculate (male dogs), or presence of penile spines (male cats).
  • Pubertal maturation in females is defined as the females undergoing at least one normal estrous cycle (female dogs: presence of serosanginuous vulvar discharge, vulvar swelling and increase of the plasma progesterone concentration > 9nmol/L indicating that ovulation has occurred).
  • the subjects are mature or have reached pubertal maturation.
  • the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent cats or dogs.
  • the subject is selected from a mature male dog, mature male cat, mature female dog, or mature female cat.
  • induction of cell death occurs within 24 hours, 48 hours, or 72 hours of injection or ablation. In certain aspects, induction of cell death leads to sterilization of the subject. In certain aspects, sterilization of the subject is permanent.
  • Anti-Mullerian hormone also known as Mullerian Inhibiting Substance, is a hormone that plays a role in growth differentiation and folliculogenesis. AMH expression inhibits the development of the female reproductive tract in the male embryo and thus is critical to sex differentiation during fetal development.
  • an “apoptotic protein” is a protein that can cause cell death.
  • apoptosis of certain reproductive cells can lead to sterilization.
  • Nanoparticles relate to precursors of spermatogonia that differentiate from primordial germ cells.
  • nanoparticle construct refer to materials with overall dimensions in the nanoscale, i.e., under about 100 nm. Nanoparticles can be prepared for a variety of materials, for example, lipids and polymers.
  • nanoparticle may be interpreted to also mean “nanoparticle construct” with the proper context set forth above.
  • the “primordial follicles” are the first class of follicles formed in mammalian ovaries.
  • reproductive cell(s) refer to any cell(s), ablation (i.e., selective reduction or removal) of which could affect or result in sterilization of a subject.
  • “reproductive cells” as used herein include germ cells, as well as cells whose function effects the development or maturation of reproductive systems (“reproductive supportive cells”) during embryogenesis, including but not limited to, for example, Sertoli cells, Leydig cells, cells of the primordial follicle, or granulosa cells. Other such “reproductive cells” or “reproductive supportive cells” would be known to a person of ordinary skill in the art.
  • nucleic acid As used herein will generally refer to a molecule (i.e., strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g.
  • oligonucleotide refers to a molecule of between about 3 and about 100 nucleobases in length.
  • nucleic acid also refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double -stranded polynucleotides.
  • the term “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • a “gene” refers to coding sequence of a gene product, as well as non-coding regions of the gene product, including 5’UTR and 3’UTR regions, introns and the promoter of the gene product. These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule.
  • a nucleic acid may encompass a doublestranded molecule or a double-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a molecule.
  • the term “gene” refers to the segment of DNA involved in producing a polypeptide chain, it includes regions preceding and following the coding region as well as intervening sequences (introns) between individual coding segments (exons).
  • binding e.g., to a receptor
  • a molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc.
  • vector includes any genetic element, including, but not limited to, a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, minichromosome, expression vector, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer nucleic acid molecules to cells.
  • Vectors are well known in the art and include, but are not limited to, cloning and expression vectors, as well as viral vectors.
  • viral vector includes DNA vectors, RNA vectors, and circular or linear vectors and refers to the recombinant viral-based (e.g., recombinant lentiviral -based DNA) construct.
  • the vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid.
  • Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper-dependent adenoviral vectors (HD Ad), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, pox virus vectors, and retrovirus vectors.
  • the nanoparticle constructs and compositions thereof comprise a lentivirus.
  • the lentivirus may be integrating or non-integrating.
  • peptide refers to a polymer of amino acid residues, and are not limited to a minimum length.
  • Peptides, oligopeptides, dimers, multimers, and the like are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof are encompassed by the definition.
  • polypeptide encompasses a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity.
  • Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids.
  • the term “peptide” as used herein typically refers to a sequence of amino acids of made up of a single chain of D- or L- amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length.
  • wild-type refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof.
  • a wild-type polypeptide may be produced recombinantly.
  • variant means a biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide.
  • a variant can have, for example, at least 1, 2, 3, 4, or 5 amino acids substituted by a different amino acid.
  • a variant has at least about 50% sequence identity with the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C- terminus of the polypeptide.
  • percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINETM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.
  • amino acid substitution refers to the replacement of one amino acid in a polypeptide with another amino acid.
  • An amino acid substitution can be a non-conservative substitutions, which will entail exchanging a member of one of these classes with another class.
  • An amino acid substitution can be a conservative substitution.
  • Nonlimiting exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.
  • Amino acids may be grouped according to common side-chain properties:
  • hydrophobic Norleucine, Met, Ala, Vai, Leu, He
  • neutral hydrophilic Cys, Ser, Thr, Asn, Gin
  • to “reduce” or “inhibit” means to decrease, reduce, or arrest an activity, function, parameter, or amount as compared to a reference or control.
  • “reduce” or “inhibit” refers to the ability to cause an overall decrease of about 20%, about 50%, about 75%, about 85%, about 90%, or about 95%, or greater.
  • the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.
  • a “reference” or “control” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
  • a “subject” means a human or animal.
  • the subject is a mammal, e.g., a cat or a dog.
  • a subject can be male or female. Additionally, a subject can be an adult or can be pre-pubescent.
  • compositions and nanoparticle constructs of the present invention can be administered by the methods described herein and any appropriate route known to one of ordinary skill in the art that results in partial or complete apoptosis of reproductive cells in a subject.
  • the term “effective amount” as used herein refers to a sufficient amount of pharmacological composition to provide the desired effect. Thus, it is not possible to specify the exact “effective amount.” However, for any given case as set forth in detail herein, an appropriate “effective amount” can be determined by one of ordinary skill in the art. The efficacy of treatment can be judged by an ordinarily skilled practitioner.
  • compositions or “pharmaceutical composition” are used interchangeably herein refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells. Exemplary compositions and pharmaceutical compositions are described in detail herein.
  • the cells may be part of a subject, for example for therapeutic, diagnostic, or prophylactic purposes.
  • the cells may also be cultured, for example cells as part of an assay for screening potential pharmaceutical compositions, and the cells may be part of a transgenic animal for research purposes.
  • the composition can also be a cell culture, in which a polypeptide or polynucleotide encoding a metabolic regulator of the present invention is present in the cells and/or in the culture medium.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. The acceptability of treatment can be judged by an ordinarily skilled practitioner.
  • phrases “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid fdler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • each carrier must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Example 1 Materials and methods for Examples 2-4
  • MLTC-1 Leydig cells (CRL-2065), TM3 Leydig cells (CRL-1714), and TM4 Sertoli cells (CRL-1715) were purchased from ATCC (Manassas, Virginia, USA).
  • Mouse epididymal epithelial (mECapI8) cells were obtained as a gift from Dr. Petra Sipila (Institute of Biomedicine, Department of Physiology, University of Turku, Turku, Finland).
  • LSBio rabbit polyclonal anti-FSH receptor antibody (LS-C400751), Bioss rabbit polyclonal anti-LH receptor antibody (bs-6431R), LSBio rabbit polyclonal anti-AMH receptor II antibody (LS-C377389), and donkey serum were purchased from Sapphire Bioscience (Redfern, NSW, Australia).
  • Goat polyclonal anti-8-hydroxydeoxyguanosine (8-OH-dG) was purchased from Millipore (Temecula, CA, USA), Rabbit anti-cleaved Caspase 3 (Asp 175) from Cell Signalling Technologies (Danvers, Massachusetts, USA), and anti-4HNE antiserum from Jomar Diagnostics (Mulgrave, Vic, Australia).
  • Agarose low melting point was obtained from Applichem GMBH (Ottoweg 4, 64291 Darmstadt, Germany) and ethanol purchased from POCD Scientific (North Rocks, NSW, Australia).
  • DNA/RNA Damage Antibody (15A3) was obtained from Novus Biologicals (Littleton, Colarado, USA).
  • Maleimide-vcMMAE (Auristatin) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).
  • cysteine and methionine residues were removed or replaced, unless the cysteine or methionine occurs at the N-terminus or C-terminus of the peptide.
  • cysteine is replaced by serine, which does not have a thiol group and has a similar structure to cysteine.
  • the 3D structure of the peptide should not be altered by the substitution and the chances of peptide cyclization and peptide oligomerization (which leads to a mixed population of products) should be reduced or eliminated.
  • the reactive thiol group of a cysteine residue at the N-terminus of a peptide is useful for attaching the peptide to other moieties or to a nanoparticle, through a linker, such as a maleimide group. See Martinez-Jothar et al., 2018; Varanko et al., 2020. Moreover, removal of internal reactive thiol groups enables a single site is available for linkage, such as a maleimide-thiol mediated coupling of the peptide to the desired pay load, and avoids the potential for mixed end products to be formed.
  • the peptide may not attach to the surface of the nanoparticle in the correct orientation conducive to peptide-target binding.
  • the C-terminal may be amidated to inhibit protease degradation of the peptide in vivo. See Di, 2015; Werle & Bemkop-Schntirch, 2006.
  • synthetic peptides linked to maleimide are used.
  • synthetic peptides linked to fluorescein isothiocyanate (FITC) are used where the peptide is attached to FITC via an N-terminal spacer, 1,6-aminohexanoic acid (Ahx).
  • TM4 Sertoli cells were cultured in growth medium composed of DMEM/F12 supplemented with 5% (v/v) horse serum, 2.5% (v/v) fetal bovine serum, 10 mM HEPES, and 100 U/mL penicillin-streptomycin and subcultured at a ratio of 1:5 into culture flasks.
  • MLTC- 1 Leydig cells were cultured in growth medium composed of RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum, and 100 U/mL penicillin-streptomycin. MLTC-1 Leydig cells were subcultured at a ratio of 1:5 into cell culture flasks.
  • TM3 Leydig cells were cultured in growth medium composed of F12/Dulbecco’s modified Eagle’s medium, 2.5 mM L-glutamine, 0.5 mM sodium pyruvate, 15 mM HEPES, 2.5% of a mixture of 92.5% horse serum and 5% fetal bovine serum, and 100 U/mL penicillin-streptomycin.
  • TM3 Leydig cells were subcultured at a ratio of 1:5 into cell culture flasks.
  • Immortalized mouse proximal caput epididymal epithelial (mE-Capl8) cultures were used as a nonspecific cell control for investigating the specificity of FSH, LH, and AMH peptide interactions (Sipila et al., 2004) and were maintained in growth medium composed of DMEM supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin-streptomycin, 1 mM sodium pyruvate and 50 nM 5-a-androstan-17p-ol-3- one and sub-cultured at a ratio of 1: 10 into culture flasks.
  • Cells were grown to 80% confluency before being harvested using 0.5% trypsin-EDTA, quenched, centrifuged at 300x g for 5 min, then washed in warmed growth medium. After centrifugation, the cells were resuspended in warm medium and subjected to incubation with peptide-FITC or peptide-menadione conjugates.
  • mice were obtained from a breeding colony at the University of Newcastle Central Animal House and housed under a conventional controlled light and temperature regimen (12-h light: 12-h dark cycle, 21-22°C). All procedures involving mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Additionally, mice were handled, monitored and euthanized with the approval of the University of Newcastle’s Animal Care and Ethics Committee (approval number A-2014-417) in accordance with NSW Animal Research Act 1998, NSW Animal Research Regulation 2010 and the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Ed. Immediately prior to tissue isolation, animals were euthanized by CO2 asphyxiation.
  • RNA isolation was performed using two rounds of a modified acid guanidinium thiocyanate-phenol-chloroform protocol (Aitken et al., 1998) followed by isopropanol precipitation.
  • Total RNA was DNase treated prior to reverse transcription to remove genomic DNA.
  • Reverse transcription to cDNA was conducted using M-MLV Reverse Transcriptase (Promega, Ml 701) following the manufacturer’s instructions with subsequent confirmation of transcription using cyclophilin qPCR.
  • Taq quantitative PCR was performed using SyBr Green GoTaq qPCR master mix (Promega) according to manufacturer's instructions on a LightCycler 96 SW 1.0 (Roche Diagnostics GmbH, Sandhofer Strasse 116, Mannheim, Germany) thermocycler. Certain qPCR primer sequences (SEQ ID Nos. 173-180) are provided in Table 1. Reactions were performed on cDNA equivalent to 100 ng of total RNA and conducted for 45 amplification cycles. SYBR Green fluorescence was measured after the extension step at the end of each amplification cycle and quantified using LightCycler Analysis Software (Roche). For each sample, replicates omitting the reverse transcription step were undertaken as negative control. Each PCR was performed on at least 3 separate cell or tissue isolations, of which a representative average is shown.
  • buffer 1 phosphate buffered saline (PBS) with 3% BSA and 0.015% Triton-X-100
  • buffer 2 PBS with 10% goat serum, 3% BSA and 0.015% Triton-X-100
  • anti-FSHr, anti-LHr, or anti-AMHr antibodies diluted 1/250 in buffer 1).
  • Cells were then re-blocked in buffer 2 for 15 min at RT before incubating for 1 h at RT in 1/200 of goat anti-rabbit AF488 in buffer 2.
  • Quantitative PCR as described above, was also used to assess expression of peptide receptors relative to the house-keeping gene, GAPDH. Subsequently, based on the results obtained in the qPCR, LHr and AMHr protein expression in cultured cells were examined using anti-LHr and anti-AMHr II antibodies, respectively.
  • TM4 Sertoli cells or MLTC-1 Leydig cells were used to assess the relative binding capabilities and the specificity of targeting of the synthetic FSH, LH, or AMHa peptides.
  • mEcapl8 cells were used as a negative control. All peptides were purchased with an N-terminal spacer, 1,6-aminohexanoic acid (Ahx), attached to fluorescein isothiocyanate (FITC) and dissolved in DMSO to form a 2.5 mM stock solution. Cells were treated for analysis by fluorescence assisted cell sorting (FACS). The median fluorescence intensity was recorded for each treatment group and the shift in median fluorescence intensity was then calculated by subtracting the median fluorescence intensity of the vehicle (0 pM) from the median fluorescence intensity of each dose.
  • FACS fluorescence assisted cell sorting
  • Cells were grown to 80% confluency in 175 cm 2 cell culture flasks before they were harvested using 0.25% trypsin-EDTA, washed, counted and resuspended in growth medium. Cells were then split equally between treatments (in triplicate) of 300,000 cells/200 pL/trcatmcnt in 1.5 mb Protein Lo-bind Eppendorf tubes. Treatments included separate tubes for untreated (non-labeled) and untreated Live/dead stained for each cell type. Cells were treated with FSH-FITC, LH-FITC, or AMHa-FITC in concentrations ranging from 0 to 50 pM, with the 0 pM representing the vehicle (DMSO) control.
  • DMSO vehicle
  • Treated cells were gently mixed by inversion, and then incubated at 37°C for 1 h. Cells were then centrifuged at 300x g for 5 min, washed with warmed HBSS and resuspended in 180 pL of HBSS. Except for the unstained controls, 20 pL of 1/1000 Live/dead Far Red stain was added to all tubes. Unstained Live/dead tubes were boiled for 3 min and cooled to RT before addition of the Live/Dead stain. Samples were gently mixed and then incubated at 37°C for 20 min before washing and resuspension in HBSS. Cells were then centrifuged, as above, and washed twice with HBSS before being analyzed using FACS.
  • Flow cytometry was performed using a FACSCanto II flow cytometer (BD Biosciences) with a 488-nm argon ion laser. Emission measurements were made using 530/30 band pass (green/FL-1), 585/42 band pass (red/FL-2), and >670 long pass (far red/FL-3) fdters. Forward scatter and side scatter measurements were recorded to generate a scatter plot, which was used to gate for cells only, excluding any contaminating cell debris. All data were acquired and analyzed using BD FACSDiva software (BD Biosciences), with a total of 10,000 events collected per sample.
  • Tissues were imaged in cold PBS with a Leica MZFLIII microscope with an epifluore scent green filter. All images included the vehicle control to enable comparison of median fluorescence intensity of the treatments relative to the control using ImageJ software [ImageJ version 1.52a; Wayne Rasband, National Institutes of Health, USA. Error! Hyperlink reference not valid.imagei.nih.gov.ii, Java 1.8.0 112 (64-bit)].
  • FSH and AMHa peptides were attached, via the N-terminal thiol, to menadione (Md) as follows. Peptides were weighed and then dissolved 6 mg/mL MilliQ water. The solution was deoxygenated by bubbling through nitrogen gas and then 0.8 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) were added. The mixture was left at RT for 20 min. Meanwhile, 10 molar equivalents of menadione were dissolved in DMSO in a sealed reaction vessel, deoxygenated, and heated in a water bath to 55°C.
  • TCEP tris(2-carboxyethyl)phosphine
  • Samples were eluted from a Waters 250 x 10.0 mm Jupiter 4u Proteo 90A column using a gradient method with solvent A (0. 1 % v/v formic acid in H2O) and Solvent B (90% acetonitrile with 0.1% v/v formic acid) with a flow rate of 2 mL/min over 26 min followed by 2 min at 100% Solvent B before re-equilibration for 7 min. Samples were fdtered through 0.45 pm syringe fdter before injection onto the column. The eluted species were detected by their absorbance at 220 nm. Peak fractions were collected and peptide-quinone identity and purity was verified using MALDI-TOF/TOF mass spectrometry, as above, before the product was freeze dried under vacuum and stored as a powder at -20°C.
  • LH2 peptide (see Table 4 in Example 3) with a y-aminobutanoate- mercaptopropionic acid linker and was attached, via the N-terminal thiol, to maleimide- auristatin (Aur) (in the form of maleimide-valine-citrulline- para-aminobenzyloxycarbonyl - monomethyl-auristatin E) as follows.
  • LH2 peptide (MW 1333) was dissolved in 10 mM PBS, pH 7, at a rate of 8.5 mg/250 pL. The peptide was then reduced, using immobilized TCEP disulphide reducing gel, the solution pH adjusted to 7, and then deoxygenated.
  • maleimide-Aur (MW 1316) were dissolved in 53% dimethyl formamide/10 mM PBS, pH 7, at 10 mg/3 mb and placed in a reaction vessel.
  • the maleimide- Aur solution was then deoxygenated, and the reduced peptide solution was added dropwise, using a syringe, whilst stirring and under positive pressure with nitrogen gas.
  • the reaction was then stirred overnight at RT.
  • the success of the conjugation reaction was confirmed by mass spectrometry, using a Bruker Ultraflextreme MALDI-TOF/TOF (mode: reflectron positive; matrix: a-cyano-hydroxy-cinnamic acid).
  • the solvents were removed from the reaction by freeze drying under vacuum.
  • Chemiluminometic analysis was used to ascertain whether the Md portion of a peptide-Md conjugate retained its ability to produce hydrogen peroxide. Chemiluminometry was used to measure the production of hydrogen peroxide by a redox cycling agent in the presence of cells. Specifically, after conjugation of Md to the FSH peptides, the FSH-Md conjugates were assessed for retention of redox cycling ability.
  • TM4 Sertoli cells were grown to 80% confluency in 175 cm 2 cell culture flasks. Cells were harvested and split 500,000 cells/380 pL/treatment (in duplicate or triplicate). Cell free control tubes containing 380 pL of HBSS were also included in each analysis as negative controls. Treatments comprised 50 pM each of the unconjugated peptides and each of the peptides conjugated to Md, as well as Md alone. Luminol-peroxidase dependent assessment of extracellular hydrogen peroxide was recorded on a Berthold 953 luminometer.
  • HRP horseradish peroxidase
  • PBS final peroxidase activity 51.2 U/mL
  • Luminol 25 mM in DMSO
  • ROS Assessment reactive oxygen species
  • This example describes methods for assessing the impact of reactive oxygen species (ROS) produced by the FSH-Md conjugates on TM4 Sertoli cells.
  • ROS reactive oxygen species
  • a y-aminobutanoic acid linker (referred to as linker 2), was used to covalently link FSH to Md.
  • this peptide is referred to herein as FSH2Md.
  • TM4 Sertoli cells were grown to 8% confluency in 175 cm 2 cell culture flasks. Cells were harvested, washed, and resuspended in growth medium and split equally between four treatments (in triplicate): 50 pM of FSH2Md, FSH2 peptide, Md, or the vehicle alone (30% Kolliphor/PBS) and incubated at 37°C for 90 min. After washing in HBSS, treated cells were then either used for immunocytochemistry to detect apoptosis (anti-Caspase 3) and oxidative DNA damage (anti-8-OHdG), or prepared for FACS to assess mitochondrial ROS (MSR) or lipid peroxidation (anti-4HNE). Each experiment was conducted three times in triplicate.
  • FACS Fluorescence-activated Cell Sorting
  • ROS mitochondrial reactive oxygen species
  • TM4 Sertoli cells were resuspended in 200 pL DMEM with additional tubes of untreated cells to create the following controls: untreated cells, unstained cells, MSR untreated, and Live/Dead Green (L/D G) positive (i.e., cells boiled at 100°C for 3 min).
  • SCSA Sperm chromatin structure assay
  • FACS FACS-activated spermatozoa susceptibility to DNA fragmentation.
  • the SCSA was performed as previously described (Campbell et al., 1991; Santa Coloma & Reichert, 1990). Briefly, aliquots of spermatozoa were thawed at 37°C and stored on ice, after which 200 pL of acid detergent solution (0.08 N HC1, 0.15 M NaCl, 0.1% Triton X-100, pH of 1.2) was added to 100 pL of sperm suspension. After 30 s, 600 pL of acridine orange staining solution (0.1 M citric acid, 0.2 M NazPOi.
  • DNA fragmentation index ratio of single stranded or denatured DNA to total DNA
  • DFI DNA fragmentation index
  • mice Seven male adult mice were treated with IP injections of 100 pL 14.5 mM FSH2Md (in 30% Kolliphor/PBS) /10 g body weight (with this concentration equating to the solubility of FSH2Md); ostensibly providing a final FSH2Md concentration of 145 pM.
  • IP injections 100 pL 14.5 mM FSH2Md (in 30% Kolliphor/PBS) /10 g body weight (with this concentration equating to the solubility of FSH2Md); ostensibly providing a final FSH2Md concentration of 145 pM.
  • 6 weeks post injection 5 of the treated mice and 4 age-matched untreated males were mated with 2 untreated females each for 3 weeks. Females were monitored for mating plugs each day and 12.5 days post-mating, resorption moles and embryos were counted.
  • Spermatozoa were allowed to swim-out for 10 min before being collected into clean tubes.
  • Sperm motility was assessed using a light microscope at lOOx magnification and sperm vitality was assessed using the eosin exclusion test (WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction ⁇ Obstetrics and Gynecology, Repro, n.d.).
  • spermatozoa per sample were analyzed to determine percentage of total motility and vitality of each sample.
  • Sperm motility was assessed using computer-assisted sperm analysis (CASA). The remaining spermatozoa were aliquoted into tubes containing 10 6 sperm/mL, snap frozen in liquid nitrogen and stored at -80°C until use.
  • CASA computer-assisted sperm analysis
  • This example describes methods for assessing the impact of treating mice with an FSH-Md conjugate and a LH-Aur conjugate.
  • a y-aminobutanoic acid linker (referred to as linker 2), was used to covalently link FSH to Md and LH2 to Aur.
  • linker 2 a y-aminobutanoic acid linker
  • the dose of 1 mg/kg of conjugated Aur was based on previous work finding the maximum tolerated dose (MTD) of conjugated Aur for mice is 1.1 mg/kg (Doronina et al., 2003). Solubility of the FSH2Md was limited to 14.5 mM (25 mg/kg) in the biocompatible reagents (Kolliphor/PBS) delivering a dose much lower than menadione’s MTD of 830 mg/kg.
  • MTD maximum tolerated dose
  • Females were monitored for mating plugs each day and on the thirteenth day post-mating plug, or if weight gain indicated approximately 13 days of pregnancy, females were euthanized weighed, embryos and resorption moles were counted, ovaries were collected, weighed, and corpora lutea counted. After the mating period, at ten weeks post-injection, all males were euthanized, weighed and blood collected by cardiac puncture. Blood samples from each animal were decanted into EDTA-coated tubes and plasma was separated (10 min, 400 g). Plasma was stored at -80°C for later measurement of serum FSH levels. An AVIVA FSH ELISA Kit was purchased from Sapphire Bioscience (Redfern, NSW, Australia) and conducted as per manufacturer’s instructions.
  • Tissues salivas (seminal vesicles, epididymis, adrenals, kidneys, liver, brain, heart, spleen and testes) were collected and weighed. A sample of each tissue was fixed in Bouin’s solutionfor around 40 min per mg tissue. One testis from each male was fixed and the other snap frozen in liquid nitrogen and stored at -80°C. Fixed samples were processed for H and E staining as described in the preceding section. H and E stained sections were used to evaluate cross-sectional tubule areas as described below. To evaluate the impact on TM4 Sertoli cells numbers, an anti-SOX9 antibody was used to stain sections, while an ApopTag Kit (Merck) was used to detect apoptotic cells in accordance with the manufacturer’s instructions.
  • ApopTag Kit Merck
  • Sperm motility was assessed using a HTM-IVOS II Computer Assistedsperm Analysis and sperm vitality was assessed using the eosin exclusion test (WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction ⁇ Obstetrics and Gynecology, Repro, n.d.) and viewed using a light microscope at 40 x magnification. At least 100 spermatozoa per sample were analyzed to determine percentage vitality of each sample. Spermatozoa were stained for FACS analysis of mitochondrial and cytosolic superoxide using MSR and DHE staining (Koppers et al., 2008, 2011).
  • sperm smears were made for staining with Rapid Diff (Pathtech, Preston, Vic, Australia), as per manufacturer’s instructions, to evaluate sperm morphology.
  • Sperm were categorized as either having normal or abnormal morphology.
  • Analyses of DNA strand breaks (HALO), and detection of the oxidative DNA adducts (anti-8-OH-dG) were conducted as described in section U below.
  • At least 100 sperm/sample 10,000 sperm/sample for FACS were assessed for each assay and, where possible, all counts were conducted blind.
  • the remaining spermatozoa were aliquoted into tubes containing 10 6 sperm/mL, snap frozen in liquid nitrogen and stored at -80°C until use.
  • mice Three male adult mice were twice injected intraperitoneally with 100 pL/10 g 30 mM AMH2Md (in 30% Kolliphor/PBS), with injections spaced 16 h apart.
  • the treated mice along with three aged-matched untreated males were housed with one untreated female each for three weeks.
  • Females were monitored for mating plugs each day and 12.5 days post-mating, resorption moles and embryos were counted. At the end of the mating period, males were sacrificed and testes were collected, weighed, and fixed in Bouin’s solution.
  • sperm chromatin dispersion test (HALO assay) was used to determine DNA fragmentation in sperm. If the chromatin is intact, it will spring out and form a ‘halo’ around the sperm head, however, if there is DNA fragmentation then the chromatin is unable to spring out and ‘no halo’ effect is observed. Briefly, microscope slides were pre-coated with 0.65% standard agarose. After thawing, 1% low-melting agarose solution was added to the sperm at an agarose to sperm ratio of 7:3. 50-60 pl was then spread evenly across the pre-coated slides and immediately covered with a coverslip (24 x 50 mm).
  • the slides were then incubated for 2 min at RT in TBE (0.45 M Tris, 0.45 M boric acid, 10 mM EDTA, pH 7.5), then serially with 70%, 90%, and 100% ethanol for 2 min each at RT. Slides were then air dried and stained with DAPI for 10 min at RT. After rinsing in PBS, Mowiol was dropped onto the slides and a coverslip added before sealing with clear nail varnish and viewing using fluorescence microscopy. At least 100 cells on each slide were viewed and classified as either having a halo or no halo.
  • Testes sections were dewaxed using xylene and then rehydrated in an ethanol gradient. Heat-assisted epitope retrieval was conducted in Tris-EDTA, pH 9. Slides were then incubated in 0.3% hydrogen peroxide in Tris buffered saline (TBS) for 30 min at RT. After washing in TBS (3 x 5 min), the sections were blocked using 5% normal goat serum (NGS) for 30 min at RT in a humidified chamber. The sections were then incubated with rabbit anti-SOX9 antibodies (Merck: AB5535) in NGS (1:500) overnight at 4°C.
  • TBS Tris buffered saline
  • NGS normal goat serum
  • Slides were washed with TBS (3 x 5 min) and counterstained with haematoxylin for 8 min before being dehydrated and mounted with Eukitt. Slides were imaged using bright field microscopy and the number of TM4 Sertoli cells counted in all tubules across one section from each slide.
  • FSH peptide sequences include residues from the L2 loop or the “seatbelt” receptor binding region of FSH with varying charge and hydrophobicity
  • Each of the six FSH peptides were modeled as described in Example 1 above. The peptides were found to map to the surface of the ‘seatbelt’ region and the tip of the PL2 loop of FSH (Figs. 1A-F). Both of these regions are known to contain residues important for FSH receptor binding.
  • TM4 Sertoli model cell line was identified for use in targeting the synthetic FSH peptides to the FSHr.
  • a control cell line that was devoid of FSHr was also identified.
  • the TM4 Sertoli cell line was used, originally purchased from ATCC (derived from 11-14 day old murine testis) and which has been shown in other studies to express the FSH receptor (X. Liu et al., 2014). According to ATCC (atcc.org), the TM4 cell line is reported to respond to FSH with an increase in cAMP production. However, the FSH responsiveness was reportedly much reduced compared to primary Sertoli cell cultures.
  • FSH peptides possess binding capability and specificity in vitro
  • each of the synthetic FSH peptides (shown in Table 3) was found to be capable of binding to TM4 Sertoli cells, with the intensity of labeling increasing in accordance with increasing doses of the applied peptide. This was evidenced by a proportional shift in median fluorescence intensity of the stained TM4 Sertoli cell population across the examined peptide dose-range of 5 to 50 pM (Figs. 3A-F). Additionally, flow cytometry analyses provided evidence that all of the TM4 Sertoli cells exhibited peptide uptake, such that their labeling intensity shifted uniformly as a single population as the peptide dose was increased.
  • each of the peptides showed good specificity with 10-fold higher median fluorescence labeling intensity being detected across the dose range when peptides were applied to TM4 Sertoli cells as compared to the control mECapl8 cells.
  • the peptides with the greatest shift in median fluorescence and, by inference, the greatest binding capability, were identified as F53, F49, and FSHa (Figs. 3G-H).
  • FSH peptides target the testes in vivo and have minimal off-target localization
  • the results are expressed as a ratio of the mean fluorescence intensity/pixel of the treated tissue in any image to that of the vehicle control in the same image (Fig. 5D).
  • the testes of FSHa-FITC-treated males exhibited a significantly higher (p ⁇ 0.005) ratio of mean fluorescence intensity/pixel compared to the testes of animals treated with the alternative array of FSH synthetic peptides. Based on these findings, FSHa was selected as the focus for further studies.
  • 2-methyl-l,4-naphthoquinone is a redox cycling compound (also known as menadione, Md, or Vitamin K3) that undergoes one and two electron reduction to produce superoxide radicals.
  • the redox cycling of the naphthoquinone family is facilitated by enzymes, usually flavoenzymes, that use NAD(P)H as an electron source (Gutierrez, 2000a, 2000b).
  • NAD(P)H as an electron source
  • the superoxide radicals are catalytically converted by superoxide dismutase (SOD) to hydrogen peroxide (Melov, 2002).
  • the superoxide radicals also have a propensity to release iron from ferritin, while the production of hydrogen peroxide can facilitate the release of iron from heme proteins (Wiseman & Halliwell, 1996).
  • the free iron can then participate in Fenton reactions whereby hydrogen peroxide is converted to hydroxyl and hydroperoxyl radicals that, in turn, act as oxidizing agents.
  • ROS reactive oxygen species
  • the radicals so formed can cause loss of function through oxidative damage to cellular lipids (Halliwell, 1991), proteins, and DNA (Wiseman & Halliwell, 1996).
  • linkers were subsequently attached to the N-terminal of the peptide and thereafter modified through the addition of 3-mercaptopropionic acid (MPA) to maintain a free thiol for attachment of the Md.
  • MPA 3-mercaptopropionic acid
  • these linkers were: (1) alanine with MPA, (2) y- aminobutanoic acid with MPA, and (3) mini-polyethyleneglycol with MPA.
  • linkers 1, 2, and 3 respectively.
  • the unconjugated FSH peptides displayed only basal levels of redox activity, equivalent to the chemiluminescence generated in the untreated control samples.
  • the FSHa peptide, conjugated to Md using linkers 1, 2, or 3 exhibited favorable chemiluminescence compared to the free Md agent over the 90 min time course of this experiment (Fig. 7A).
  • FSH2-Menadione created oxidative stress and induced mitochondrial ROS, lipid peroxidation, and DNA damage in TM4 Sertoli cells in vitro
  • FSH2Menadione induces targeted oxidative damage to the male germ line in vivo
  • Example 3 builds on this concept and discusses additional targeting of the testes with Auristatin (Aur) towards improved methods for nonsurgical sterilization.
  • Aur Auristatin
  • Md was used to induce oxidative damage while Aur was used to inhibit mitosis, demonstrating that decreased fertility in male mice is possible when a dual-targeting approach is used.
  • a targeting peptide should accumulate in the target tissue but not in off-target tissues, thereby increasing efficacy and minimizing side-effects (Le Joncour & Laakkonen, 2018).
  • F53 and F49 were designed to encompass the sequence of the PL2 loop binding region of the P-subunit of FSH whereas FSHa was representative of residues residing within the “seatbelt” region of FSH P-subunit (Table 3, Fig. 1). Since both regions of FSHp had been implicated in ligand binding to the cognate FSH receptor (Q. R.
  • Md was covalently attached to the N-terminus of the FSH peptides via the reactive C3. This mode of attachment negates the alkylating properties of Md meaning that any oxidative damage observed may be attributed to ROS production rather than the adduction of Md to essential biomolecules (Klotz et al., 2014). Additionally, with the C-terminus of the FSH peptides protected by amidation and the N-terminus modified with Md, the peptides may be less susceptible to degradation by exopeptidases (Di, 2015; Diao & Meibohm, 2013) in vivo thereby prolonging the in vivo half-lives and extending the time that the peptides are exposed to the FSHr.
  • FSHr-targeting peptides were designed that binds with high capability and specificity to a Sertoli cell line in vitro as well as selectively targeting the testes in vivo. Furthermore, modifying these peptides to incorporate an appropriate linker domain and redox active payload (i.e., Md), can be exploited to propagate localized redox cycling within the Sertoli cell population in vivo.
  • an appropriate linker domain and redox active payload i.e., Md
  • Example 3 builds on the method of this Example of targeted delivery of Md to mouse testes.
  • Example 3 discusses additional targeted delivery of Aur to mouse testes to induce germ cell apoptosis, resulting in decreased fertility in male mice.
  • LH peptide sequences include residues from the L2 loop and “seatbelt” region of the LH -subunit with varying charge and hydrophobicity
  • the peptides were found to map to the (3 L2 loop and the “seatbelt” region of the LH P-subunit , which are the two binding regions on the surface of the P-subunit (Figs. 12A-D). Additionally, their position on the surface of the protein means that they are accessible for receptor binding.
  • a cell line with strong expression of the LH receptor gene (Lhcgr) was identified as an in vitro model cell line for use in targeting the synthetic LH peptides to LHr.
  • qPCR and an anti-LHr antibody were used to probe for the presence of the LH receptor in both MLTC-1 and TM3 Leydig cell lines, as well as in the control mECapl8.
  • MLTC-1 cells and mECapl8 cells were used (as described in Example 1 above) to assess the relative binding capabilities and the targeting specificity of the synthetic LH peptides.
  • Each peptide bound to the MLTC-1 Leydig cells in a dose-dependent manner. This was evidenced by a proportional shift in the median fluorescence intensity of the stained MLTC- 1 cell population across the dose range of applied peptides (Figs. 14A-D). Additionally, each LH peptide displayed suitable specificity with three-fold higher median fluorescence intensity when applied to MLTC-1 Leydig cells as compared to the mECapl8 control cells (Figs. 14E-F).
  • L95-FITC showed a lack of specificity at the highest dose tested, 50 pM, with only 1.5-fold relative binding on MLTC-1 cells above that of the background mECapl8 cell binding.
  • FITC-Ahx tag conjugated to the peptide means that important factors that determine the pharmacokinetics, such as the theoretical isoelectric point, the charge and the logP value (which is a measure of hydrophobicity that increases with increasing hydrophobicity) of a modified peptide differs from that of an unmodified peptide and, most likely, that of a peptide with cargo. Therefore, further analysis of the peptides was warranted.
  • Aqueous solubility was also considered, both in the context of conjugating with Aur and for use of biocompatible solvents for in vivo delivery.
  • the Aur employed in this study has a valine-citrulline linker (Fig. 17) with a terminal maleimide for ease of conjugation and has very poor aqueous solubility with a logP value of 6.04 (Error!
  • Aur was to be conjugated to a y-aminobutanoate-MPA linker (i.e., linker 2) on the N-terminal of the peptide via a thiol-maleimide reaction.
  • the linkers were expected to allow the peptide to bind to the receptor unhampered by the attached Aur. Therefore, a cysteine extra to the N-terminal cysteine, such as in L101 peptide, indicates that a non-ideal mixed product of mono-conjugated and bi-conjugated peptide-Aur, may result. Receptor binding by a bi-conjugated product may be sterically hindered by the extra Aur moiety.
  • linker 2 i.e., a linker comprised of y-aminobutanoic acid and mercaptopropionic acid (MPA)
  • MPA mercaptopropionic acid
  • linker 2 allows Md to produce ROS by redox cycling despite its covalent attachment to the peptide.
  • LH2 and FSH2 The LHa and FSH peptides with linker 2 are hereinafter referred to as LH2 and FSH2.
  • FSH2 was attached, via the N-terminal cysteine, to C3 of Md and the product, FSH2Md, was isolated and the powdered product was stored at -20°C.
  • LH2Auristatin localized to the testes and impacted seminiferous tubule morphology and germ cell viability
  • mice were injected IP with FSH2Md, LH2Aur, or FSH2Md and LH2Aur (FSH2Md/LH2Aur) as described in Example 1 above.
  • Treated mice maintained healthy weight gain over a ten-week post-injection surveillance period, with no difference in mean body weights detected between the treatment groups. Additionally, with the exception of the testes, there was no difference in tissue (seminal vesicles, epididymis, adrenals, kidneys, liver, brain, heart, and spleen) weights or morphology between the treatment groups, indicating that the injected peptides did not elicit pronounced off-target effects.
  • ApopTag was used to detect single-stranded and double-stranded breaks associated with this form of cell death.
  • the results of this assay confirmed that the testes sections from both the LH2Aur and the FSH2Md/LH2Aur treatment groups exhibited significantly (p ⁇ 0.05) more tubules containing germ cells undergoing apoptosis (Figs. 19A) and a significantly (p ⁇ 0.05) greater number of apoptotic cells per affected tubule (Fig. 19B) than the FSH2Md alone or vehicle treatment groups.
  • the apoptosis was mostly precipitated by damage inflicted by the Auristatin.
  • FSH2Menadione FSH2Md
  • LH2Auristatin LH2Aur
  • Sertoli cells per seminiferous tubule were determined by immunolabeling of testes sections with antibodies directed against SOX9, a protein whose testicular expression is restricted to the Sertoli cell population (Figs. 20A-C). Methods used were as described in Example 1 above. This analysis revealed a trend, albeit not statistically significant, of reduced mean numbers of Sertoli cells within the testes of the FSH2Md/LH2Aur treatment group (i.e., ⁇ 14 Sertoli cells/tubule) compared to the number of these cells in the vehicle control group (i.e., ⁇ 17 Sertoli cells/tubule) (Fig. 20B).
  • oxidative DNA damage observed in the spermatozoa was evidently not a product of ongoing ROS production as there was no difference in the levels of either mitochondrial ROS or cytoplasmic superoxide detected within these cells (Figs. 23A-B) between the four treatment groups.
  • the DNA damage in the caudal spermatozoa of treated males was not accompanied by an increase in the incidence of abnormal sperm gross morphology compared to the control males (Fig. 21C).
  • FSH2Md administration alone was not sufficient to compromise male fertility.
  • all 5 males in the FSH2Md treatment group were fertile, with each of the 10 females housed with these males becoming pregnant and carrying live embryos at 13 days postmating (Fig. 24A).
  • Only 2 of the females that had been mated with males from this treatment group exhibited one resorption mole each, with none detected in the remaining eight females.
  • LHr-targeting peptides were designed to complement the FSHr-targeting peptides described in Example 2 above. Importantly, ten weeks (i.e., two complete spermatogenic cycles) after a single administration of FSH2Md and LH2Aur, the mature epididymal sperm of treated males exhibited a higher level of DNA strand breaks and oxidative DNA damage than that of their control counterparts. Administration of a single dose of LH2Aur resulted in extensive germ cell apoptosis and a reduction in fertility. Although spermatogenesis and fertility persisted, the treatments facilitated enduring damage to the testes and confirmed that the Sertoli cell and Leydig cell populations had been targeted.
  • LH2Aur had worked synergistically with FSH2Md to impact the Sertoli cells and decrease the viability of the embryos.
  • FSH2Md FSH2Md
  • the extent of the aforementioned effects varied widely within the treatment groups.
  • a targeting peptide should accumulate in the target tissue but not in off-target tissues(Le Joncour & Laakkonen, 2018).
  • the peptide with the highest binding capability in vitro, L95, along with L101 and LHa were designed to encompass the sequence of the “seatbelt” region of LH P-subunit.
  • the L57 peptide which comprised residues residing within the L2 loop binding region of the -subunit of LH (Table 4, Fig. 12), displayed higher binding capability than either L101 or LHa in vitro. Since both the “seatbelt” and the PL2 loop regions of LHP have been implicated in ligand binding to the cognate LH receptor (Q. R. Fan & Hendrickson, 2005; Jiang et al., 2014), it is possible that differences in in vitro peptide binding efficacy are attributed to their unique physicochemical properties. L95 peptide, despite displaying a higher binding capability, also lost specificity at higher concentrations (Fig. 14E).
  • the increased attraction may act to increase the concentration of the peptide that accumulates within the vicinity of the LH receptors and thus account for the enhanced binding capability of L95 and L57.
  • this advantage did not translate directly to an in vivo setting in which all four of the peptides displayed similar localization to the testes.
  • LH2 The LHa peptide with an N-terminal linker 2 (i.e., LH2) was tested for in vivo delivery of the anticancer cell ablation agent, Auristatin (Aur) ( Fig. 17).
  • a hydrophobic form of Aur was chosen because it readily enters the cell and, once within the cell, acts as an antimitotic agent that binds to a-tubulin and prevents the formation of the mitotic spindle, leading to G2-M cell cycle arrest.
  • Aur is highly potent and may be delivered selectively by conjugation to a targeting agent (most commonly an antibody) so as to reduce adverse off-target effects.
  • Aur was attached to the LH2 peptide via a cathepsin cleavable valine- citrulline(vc)-PABC linker (Fig. 17).
  • This linker has been reported to have high stability in serum or plasma and, once the peptide-Aur conjugate binds to the LH receptor, the premise is that it will be taken into the cell where the complex will be metabolized by lysosomal proteases (Sutherland et al., 2006) followed by 1,6-elimination to liberate the active drug (Fig. 17).
  • LH2Aur was used to target the Leydig cells and a FSH2 peptide conjugated to Menadione was used to target the Sertoli cells.
  • the Aur can be freed from LH2 and released from the cell. Free Aur may then kill surrounding cells via the bystander effect (Li et al., 2020; Lucas et al., 2019). Bystander killing occurs when the drug from atargeting reagent is released either from the target cell following internalization and degradation of the peptide-drug or release of the drug within the extracellular space.
  • Sertoli cells are critical for the formation of the stem cell niche within the testis and the subsequent support of all stages of spermatogenesis (Hermo et al., 2010a, 2010b).
  • Spermatogonial germ cells are sensitive to the effects of ionizing radiation (Kangasniemi et al., 1996) that, additional to damage to the DNA in the form of strand breaks by acting directly on the ribose-phosphate backbone, mainly arise from the radiolysis of water leading to the formation of hydroxyl radicals. The hydroxyl radicals subsequently react with DNA bases and cause persistent DNA damage (Eastman & Barry, 1992).
  • Ionizing radiation also generates other ROS, such as superoxide and hydrogen peroxide, all of which can interact with DNA, cellular lipids and proteins (Tominaga et al., 2004) thus facilitating irreversible damage to essential cellular machinery.
  • Md may recapitulate these effects as the superoxide radicals produced by Md can be catalytically converted by superoxide dismutase (SOD) to hydrogen peroxide (Melov, 2002).
  • SOD superoxide dismutase
  • Hydrogen peroxide may be converted through a series of reactions to hydroxyl and hydroperoxyl radicals that, in turn, may act as oxidizing agents. Together with other ROS, the radicals so formed can cause loss of function through oxidative damage to cellular lipids (Halliwell, 1991), proteins, and DNA (Wiseman & Halliwell, 1996).
  • Challenging both the Sertoli cell and Leydig populations concurrently by the production of localized oxidative stress in the vicinity of the germ cells and in the presence of an anti-mitotic agent may deplete the Leydig cell population as well as enhance the impact of ROS within the testes.
  • Aur is a potent tubulin inhibitor. This inhibition may provide a devastating effect on Leydig cell function as it binds to microtubules, introduces structural defects, and suppresses microtubule (MT) dynamics, while reducing kinetics and extent of MT assembly. This may lead to suppression of proliferation, mitosis, and disrupted MT network (Best et al., 2021). A depleted Leydig cell population may mean that their support of Sertoli cell health is disrupted.
  • the cytoskeleton is responsible for movement of organelles and molecules involved in steroidogenesis (17-P-estradiol synthesis) (Marchlewicz et al., 2004) in the Leydig cells. If, as a result, testosterone levels are reduced, then Sertoli cell nurturing of spermatogenesis (Smith & Walker, 2014) may be disrupted and may contribute to the apoptosis of the germ cells. If Aur indeed reached the Sertoli cells through the aforementioned bystander effect, then its suppression of MT dynamics may also impact Sertoli cell function and cytoskeleton, disrupting the germ cell niche, which, in turn, may adversely affect the germ cell population (Rebourcet et al., 2017).
  • Aur may exert its effects directly on the germ cells by precipitating mitotic arrest (Bourillon et al., 2019) and lead to their apoptosis. Indeed, all stages of spermatogenesis and male fertility are reliant on the dynamic organization/reorganization of microtubules (O’Donnell & O’Bryan, 2014) and the cytoskeleton (Dunleavy et al., 2019). Despite the absence of Md, sperm from LH2Aur-treated males also displayed a higher level of oxidative damage than the control-treated males. A similar observation was made in FSH2Md-treated males.
  • OGGI 8-oxoguanine DNA glycosylase
  • the higher rate of resorption moles in the females housed with FSH2Md/LH2Aur-treated males may be the consequence of DNA damage to the germ line precipitated by the presence of ROS, produced by the menadione, further potentiated by the radiosensitization effect and disruption of microtubules precipitated by Aur. Furthermore, Aur increases the fraction of cells that are in G2/M which is the most radiosensitive phase of the cell cycle (Bourillon et al., 2019). The germ cells, as pachytene spermatocytes and round spermatids, remain adherent to Sertoli cells throughout their development (Mruk & Cheng, 2004).
  • Sertoli cells are involved in regulating the levels of oxidative stress to which developing germ cells are exposed.
  • ROS generated via the redox cycling of Md, may have contributed to a level of oxidative stress in the testes that overwhelmed the regulatory mechanisms present within the Sertoli cell population (Y oganathan et al., 1989) so that they were no longer able to protect the germ cells from oxidative stress.
  • Sertoli cell function may have been impaired by the impact of Aur on the Leydig cells, thus potentiating the effects of ROS on the germ cells without the protective support normally provided by the Sertoli cells.
  • the persistence of this damage in the DNA may be directly related to the increase in resorption moles as it has been established that the paternal genome (Barton et al., 1984) and genomic imprinting (Thamban et al., 2020) play a vital part in the normal development of the embryo.
  • the accumulation of oxidative DNA damage in the germ cells may have affected key genes within the genome so as to impact the viability of embryos sired by FSH2Md/LH2Auristatin-treated males .
  • One method of increasing in vivo stability is to increase the hydrodynamic radius of the peptide by the addition of polyethylene glycol (Werle & Bemkop-Schntirch, 2006) or fusion with albumin (Di, 2015; Diao & Meibohm, 2013) or other serum proteins (Penchala et al., 2015).
  • polyethylene glycol Werle & Bemkop-Schntirch, 2006
  • albumin Di, 2015; Diao & Meibohm, 2013
  • other serum proteins Pierchala et al., 2015.
  • such peptide modifications including others such as replacement of labile amino acids or replacement of L- amino acids with D-amino acids (Werle & Bemkop-Schntirch, 2006), may disrupt the binding capabilities of the peptide to its cognate receptor.
  • the vc-PABC linker that attaches the Aur to the peptide must be stable in vivo.
  • Extracellular cleavage of the vc-PABC linker can be catalyzed by carboxylesterase 1C in mouse semm, thereby releasing the Aur before it reaches the target cell (Dorywalska et al., 2016). Indeed, plasma studies using antibody conjugated-Aur have shown that up to 25% of the Aur is released over the first 96 hours in comparison to less than 2% in human, monkey and rat plasma . Finally, the toxicity of Aur necessitated the delivery of a very low final concentration of 1.42 pM. This dose may not have been high enough to bring the local concentration in the testes to an efficacious level. While in certain aspects, treatment may comprise the administration of more than one dose, in a preferred embodiment, a single injection that results in permanent sterilization is desired.
  • this Example describes the development of LHr-targeting peptides that demonstrated binding capability and specificity to a Leydig cell line in vitro as well as selectively targeting the testes in vivo.
  • An exemplary LHr-targeting peptide was conjugated to Aur for delivery to Leydig cells in vivo, both with and without an FSHr-targeting peptide conjugated to Md for delivery to Sertoli cells.
  • the results showed precipitating germ cell apoptosis with varying outcomes within treatment groups. This variation may have stemmed from susceptibility of enzymatic degradation of the peptide and/or the vc-PABC linker in vivo resulting in a suboptimal quantity of Aur reaching the target cell.
  • the results indicate that both the LHr and FSHr targeting peptides selectively localized to the testes and can be used as delivery agents for chemosterilization.
  • the targeting peptides may be attached to the surface of nanoparticles (NPs) encapsulating cytotoxic agents and used for targeted delivery of the cytotoxic agents to testis resulting in targeted testis cell ablation.
  • NPs nanoparticles
  • Targeted delivery of such nanosystems offers the advantage of simultaneously protecting the peptide from clearance and degradation and of increasing the amount of cargo that can be delivered.
  • AMH peptide maps to solvent accessible portions of AMH protein
  • AMH anti-Mullerian hormone receptor II targeting protein
  • BMP7 bone morphogenetic protein 7
  • AMH protein A region in the AMH protein was selected that included several of the residues important for binding of human BMP7 with its cognate receptor II.
  • the mouse AMH peptide, ERISAHHVPNM SEQ ID No. 164 was identified.
  • the C-terminal methionine was replaced with a glutamate to eliminate the reactive thiol group and to increase peptide solubility, yielding ERISAHHVPNE (AMHa; SEQ ID No. 165).
  • This AMHa peptide was mapped to the surface of the AMH protein model (as described in Example 1) indicating that the selected residues are likely accessible and involved in receptor binding (Fig. 26).
  • the predicted physicochemical properties of the AMHa peptide showed good aqueous solubility, a charge of +0.2 at pH 7, and a ratio of hydrophobic to hydrophilic amino acid residues of 1.
  • An in vitro Leydig model cell line was identified for use in targeting the AMHa peptide to the anti -Mullerian hormone receptor II (AMHr).
  • a control cell line that was devoid of AMHr was also identified.
  • the MLTC-1 Leydig cell line was originally purchased from ATCC (established from the M548OP transplantable Leydig cell tumor carried in C57BL/6 mice). According to ATCC (atcc.org/products/crl-2065), the MLTC-1 cells retain the hormonal responsiveness of the original tumor and are stimulated by treatment with human chorionic gonadotropin (hCG) and luteinizing hormone.
  • hCG human chorionic gonadotropin
  • luteinizing hormone Although ATCC does not mention the presence of the AMH receptor, both qPCR (Fig.
  • FIG. 27A and immunocytochemistry (Fig. 27B) confirmed that AMHr was expressed in MLTC- 1 Leydig cells but not in the TM4 Sertoli cell line, epididymal epithelial cell line (mECapl8), or the alternative Leydig cell line (TM3). Therefore, the MLTC- 1 cell line was used as an in vitro Leydig model cell line and mECapl8 was used as the negative control cell line.
  • mECapl8 epididymal epithelial cell line
  • C AMHa exhibited selective in vitro binding capability
  • AMHa peptide was confirmed to bind to MLTC-1 cells in vitro in a dose-dependent manner. This was evidenced by a proportional shift in median fluorescence intensity of the cell population across the dose range (Fig. 28). Furthermore, AMHa peptide exhibited selective binding at concentrations of up to 25 pM with four-fold higher median fluorescence intensity when applied to MLTC-1 Leydig cells as compared to the mECapl8 cells across the tested dose range. However, the magnitude of the shift in median fluorescence intensity dropped to two-fold that of the binding to mECapl8 cells at a concentration of 50 pM (Fig. 28), indicating reduced specificity of binding at this higher dose.
  • AMHa-FITC conjugate in the mouse testes was verified using methods described above in Example 1.Either the AMHa-FITC conjugate or the vehicle alone was injected IP into each of three adult male mice per treatment. Eight hours after peptide administration, no significant difference was observed in the weight of mice exposed to either the vehicle or the AMHa-FITC treatments. Similarly, no differences in the weight or gross morphology of the representative tissues collected were observed. AMHa-FITC localized within the testes with strong fluorescence intensity while no fluorescence signal was detected in vehicle-treated males (Fig. 29A). This contrasted with findings on the adrenals (Fig. 29B), brain (Fig. 29C), lungs (Fig.
  • AMH2Md AMH2Md conjugate with linker 2
  • the methods used were as described above in Example 1. Specifically, adult male mice were administered with two IP injections of AMH2Md at a concentration (30 mM) approaching that of the compound’s limit of solubility.
  • spermatogenesis appears to be normal
  • AMHr is expressed in the hypothalamus. If the hypothalamo-pituitary-gonadal axis is disrupted, fertility may also be impacted.
  • AMHa was used to deliver Md, a redox cycling reagent, to the testes in mice. Accordingly, Md was attached to the N-terminal of the peptide via linker 2 and the C-terminal of the AMHa peptide was amidated with the aim of protecting the peptide conjugate from protease degradation (Di, 2015; Diao & Meibohm, 2013; Werle & Bemkop-Schmirch, 2006). This potentially prolonged the in vivo half-life and extended the time that the peptide conjugate was exposed to the target receptor.
  • the thiol provided by linker 2 on the N-terminal of the AMH2 may attach to the reactive C3 of Md via Michael addition. This mode of attachment may negate the alkylating properties of menadione (Klotz et al., 2014) and prevent the adduction of Md to essential cellular biomolecules. Therefore, any damage observed may be attributed to the presence of ROS produced by the redox cycling properties of the Md payload.
  • AMH is secreted by Sertoli cells and acts in a paracrine fashion on AMHr, which is highly expressed within the neighboring population of Leydig cells.
  • AMH binding acts to decrease P450cl7 and LHr expression, both of which suppress testosterone secretion.
  • increased levels of testosterone acts on Sertoli cells via a negative feedback loop to decrease AMH expression (Sansone et al., 2019).
  • the AMH2Md may bind to Leydig cells and, through redox cycling of Md, create sufficient ROS to damage or destroy the Leydig cells.
  • the ROS so generated may also permeate into the seminiferous tubules and create further oxidative damage to Sertoli cells and the developing germ cells they support.
  • mature spermatozoa isolated from the AMH2Md-treated male mice had similar vitality and motility to that of the control mice, and did not exhibit any evidence of oxidative DNA damage (Figs. 30C-E).
  • Figs. 30A-B the spermatozoa from this group of treated males presented with a higher percentage of MSR labelling indicative of mitochondrial superoxide anion generation, and a higher average percentage of susceptibility to DNA fragmentation (Figs. 30A-B) compared to the control group, this response was variable and not statistically significant.
  • AMH2Md can result in expression of two hallmarks of the apoptotic process, the dysregulation of mitochondrial electron transport and nuclease-mediated DNA cleavage in spermatozoa.
  • a decrease in the size and weight of the testes should be observed.
  • histological changes should be observed, including a decrease in the cross-sectional area of the seminiferous tubules and sloughing of the germ cells, neither of which were observed (Figs. 31A-B).
  • a normal spermatogenic cycle takes type A spermatogonia through differentiation to spermatozoa and, in mice, occurs over approximately 34.5 days (Oakberg, 1956). Therefore, if there was no persistent damage induced by the acute administration of AMH2Md, the treated mice should have normal fertility restored by 6 weeks post-injection. To test this possibility, at 6 weeks post-injection, treated males were housed with untreated females for 3 consecutive weeks. Within this period, none of the females became pregnant despite the observation of a mating plug in one of the females. [00409] AMH also has a hormonal role in the circulation where it acts on those neurons in the brain responsible for the control of reproduction.
  • GnRH gonadotropin-releasing hormone
  • a subsequent decrease in testosterone produced by Leydig cells may have led to decreased differentiation in spermatogenesis as well as a decrease in libido, and a slightly lower sperm count, thus impacting the mating behavior and/or fertility of these males.
  • the data from this example recapitulates the phenotype observed in a mutant AMHr mouse model. Only 13% of the homozygous mutant males were able to sire litters. At the same time, spermatogenesis appeared to be normal and the mutant males were able to produce vaginal plugs (Mullen et al., 2019). .
  • an AMHr-targeting peptide was designed that exhibits binding capability and selectivity to Leydig cells in vitro as well as localizing to the testes in vivo. Furthermore, in a proof-of-principle study, using this peptide with a linker designed to enhance redox cycling, Md was successfully delivered mouse testes in vivo. Although this reagent did not produce sufficient ROS to consistently create persistent or significant oxidative damage in the developing male gametes, the treated males exhibited decreased fertility.
  • This peptide might be used either alone or in tandem with other targeting peptides to selectively deliver reagents, such as cytotoxic anticancer agents, or nanoparticles containing cell ablation agents, to the testes to affect permanent sterilization.
  • FSH, LH, and AMH peptides were previously identified, as discussed above.
  • the present example compares different nanoparticle constructs (e.g., varying the materials used in the assembly of the nanoparticles, the type of peptides, peptide density, as well as length of spacer-peptides as well as the testing of cell-specific promoters) to determine the optimal compositions to be tested in vitro and in vivo (mouse) with GFP as a reporter, including optimization of promoters to drive transgene expression.
  • nanoparticles will comprise poly(lactide-co-glycolide) (PLGA) and PEG polymers.
  • PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions in order to effect drug release.
  • DNA can be condensed with protamine or cationic polyethylenimine (PEI, 2kDa) can be incorporated to further enhance transfection efficiency prior to encapsulation.
  • PEG will form the surface coating on the particle matrix to minimize non-specific particle uptake and clearance by immune cells.
  • Targeting peptides will be introduced onto the surface of PEG-PLGA nanoparticles with varied densities.
  • the resulting targeted nanoparticles will be fluorescently labelled and investigated for body distribution and cellular uptake via flow cytometry and fluorescence microscopy, where fluorescently labelled non-targeted nanoparticles will be used as control.
  • the uptake efficiency of nanoparticles will be correlated with the peptide density on particle surface, which will elucidate the optimum peptide density needed to achieve the most efficient targeted delivery. Transfection efficiency can also be used to assess uptake efficiency of the nanoparticles.
  • the nanoparticles with optimum uptake will then be loaded with genetic payloads (EGFP and DTA gene constructs) to confirm that the latter are expressed in a cell specific manner and, in the case of DTA, precipitate a loss of cell viability.
  • Development of the nanoparticles will include examination of materials used in the assembly of the particles, the type of peptide, peptide density, as well as length of spacer-peptides as well as the testing of cell-specific promoters.
  • the nanoparticles are initially designed with an EGFP transgene.
  • the EGFP transgene will be replaced by a DTA transgene.
  • Constructs will utilize well-characterized cell-specific promoters (e.g., ABP, Rhox5, HSD17B3 (male), initially driving EGFP (control vectors), and later, DTA (induction of cell death).
  • promoter regions from cat, dog, human, and mouse may also be evaluated.
  • the promoter/gene sequences will be produced as single molecules of DNA using commercial oligonucleotide synthesis, and embedded into lentiviral constructs using standard cloning techniques.
  • PEGylated polymer/lipid hybrid nanoparticles consisting of PLGA core and DSPE-PEG shell were designed using double emulsion solvent evaporation technique.
  • Cationic polyethylenimine (PEI, 2kDa) were incorporated to improve encapsulation and transfection efficiency of the plasmid DNA (pDNA) encoding RFP.
  • N/P ratio- a ratio of positively charged amine (N) groups of PEI polymer to negatively charged phosphate (P) groups of DNA is the N/P ratio- a ratio of positively charged amine (N) groups of PEI polymer to negatively charged phosphate (P) groups of DNA.
  • N/P ratios a ratio of positively charged amine (N) groups of PEI polymer to negatively charged phosphate (P) groups of DNA.
  • Pdl polydispersity index
  • charge were measured using Zetasizer Pro.
  • NPs exhibited negative charge, low ( ⁇ 0.3) Pdl index, and excellent stability.
  • the N/P ratio of 6: 1 was found to be optimum range for gene transfection.
  • a PicoGreen assay for the quantification of pDNA in the presence of PEI (or other cationic polymers) was developed to determine the encapsulation efficiency of pDNA in the nanoparticles, which was above 99%. Further optimizations of the N/P ratio can be made if required following studies of gene transfection efficiency and cytotoxicity of the NPs.
  • CMV Cytomegalovirus
  • the ubiquitous promoter that was chosen to drive gene expression is the Cytomegalovirus (CMV) promoter.
  • CMV Cytomegalovirus
  • the CMV promoter is a strong mammalian promoter derived from the human cytomegalovirus and is well described to be active in a broad range of cell types and is the most commonly used promoter in mammalian expression plasmids. Training for use of Precision Nanosystems equipment for the reliable and up-scalable production of nanoparticles commenced, and investigations were made into which cell specific promoters would be suitable for induction of cell specific gene expression.
  • Peptides have previously been developed that target the follicle stimulating hormone (FSH) and the luteinizing hormone (LH) receptors of the Sertoli and the Leydig cells, respectively, that are uniquely expressed in the testes.
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • Fig. 32A-D show the in vitro binding of peptide-FITC.
  • Fig. 32A shows LH-FITC on Leydig cell line
  • Fig. 32B shows FSH-FITC on Sertoli cell line.
  • Cultured cells were treated with a range of peptide-FITC concentrations, from 0 to 50 pM, for one hour at 37°C, washed, then stained with Far red Live/Dead before being assessed by FACS (FACSCanto). Data is presented as the % of live cells that were positive for FITC. (Fig. 32C-D). This data confirms the targeting efficacy of our peptides with the TM3 and TM4 cell lines
  • Polymeric nanoparticles were used to encapsulate a fluorescent isothiocyanate (FITC)-dextran cargo. Subsequently, the FSHr and LHr targeting peptides were attached, via their terminal sulfhydryl group, to maleimide on the surface of the nanoparticles. Over 16 hours, the specifically targeted NPs were shown to be taken up by the target cells in vitro at four times the rate of the untargeted NPs and the NPs that had a nonspecific peptide attached, (Figs. 33A-D). The intrinsic phagocytic activity of these cell lines means that over time they will accumulate NPs in a non-specific manner.
  • FITC fluorescent isothiocyanate
  • Figs. 33A-D shows in vitro binding and specificity of peptide-targeted nanoparticles (NPs) encapsulating FITC-dextran.
  • Cultured cells were incubated with 2mg/ml of NP- untargeted NP, nonspecific peptide (nsp)NP, or targeted NP - for 24 hours. Cells were then washed, fixed and counterstained with DAPI before imaging at 40x.
  • Fig. 33A shows LH-NP- FITC.
  • Fig. 33B shows FSH-NP-FITC.
  • Leydig cell line Fig.
  • TM4 is known to exhibit phagocytic activity (Tokuda et al., 1992) .
  • TM4 Sertoli cells Fig. 33D
  • Fig. 33D the non-specific binding that was evident after 24 h with TM4 Sertoli cells
  • Fig. 33D the non-specific binding that was evident after 24 h with TM4 Sertoli cells
  • Fig. 33D the non-specific binding that was evident after 24 h with TM4 Sertoli cells
  • Fig. 33D the non-specific binding that was evident after 24 h with TM4 Sertoli cells (Fig. 33D) could be a reflection of a background level of NP uptake due to the intrinsic phagocytic activity of this cell line.
  • LH-labeled NPs targeting the TM3 Leydig cell line non-specific binding was not as pronounced but even these cells are known to be capable of the non-specific uptake of nanoparticles (diesel exhaust, titanium dioxide and carbon black) ( Komatsu
  • PEGylated polymer/lipid hybrid nanoparticles consisting of PLGA core and DSPE-PEG lipid shell were designed.
  • the formulation was optimized for in vitro transfection of the TM4 Sertoli cell line for which FSH-targeted NPs (N/P ratio of 6: 1) carrying pLenti-RFP plasmid DNA displayed 13% of transfection efficiency. This transfection efficiency is relatively low, and the potency (fluorescent brightness) is also weak compared to TM4s transfected with pLenti-RFP plasmid using Lipofectamine 3000 (resulting in a transfection efficiency of 25%).
  • the hybrid NPs demonstrated slow release of the DNA resulting in delayed transfection times compared to Lipofectamine 3000.
  • lipid nanoparticles composed of cationic lipids and other helper lipids have been assessed. All lipid components being investigated are FDA approved.
  • a lipid nanoparticle formulation containing DC-Cholesterol exhibited transfection with comparable brightness to Lipofectamine 3000 (Fig. 34). The transfection efficiency (percent of overall cells transfected) was approximately 12% at 96 hours post-treatment.
  • lipid nanoparticles composed of ionisable lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) will be further investigated.
  • DLin-MC3-DMA dilinoleylmethyl-4-dimethylaminobutyrate
  • More than 80% of cells may express GFP in a cell specific manner - i.e., >80% of Sertoli cells expressing GFP when targeted by FSH-fiinctionalized NP compared with ⁇ 10% with NP functionalized using an irrelevant peptide sequence.
  • >80% of Leydig cells may express GFP when targeted by LH-fiinctionalized NP compared with ⁇ 10% with NP functionalized with an irrelevant peptide sequence.
  • polymeric NPs enabled gene expression in vitro 7 days after incubation. This is normal as the DNA was encapsulated in the core of polymeric nanoparticles and the polymer slowly degraded to release DNA. To enable more rapid release of DNA, 100 nm lipid NPs have been developed that should degrade much faster. The lipid composition of these particles will be further optimized in order to ensure maximal gene transfection efficiency.
  • lipid and polymeric NP systems can be tested to see which one will work better.
  • the lipid nanoparticles should accelerate the timing of gene expression.
  • the slow and prolonged release characteristic of the polymeric system should have the potential to overcome extracellular barriers and increase the extent and duration of gene expression.
  • DTA gene will be incorporated and again tested in vitro and in vivo to confirm targeting and effectiveness of the nanoparticles construct designed in Example 6.
  • One objective is to test in vitro on cultures expressing the FSH receptor for determination of apoptosis.
  • Another objective is to test lead candidates in vivo with immunohistochemistry and use of an optical dissector to determine the absence of target cells (8 weeks post-injection).
  • peptide density In order to configure the nanoparticles with the appropriate composition, peptide density, peptide coupling/spacing arrangement and genetic payload, in vitro models will be used. These include: primary cultures of Sertoli cells and cell lines expressing the FSH or LH receptor developed in-house.
  • a single cellular model (FSH peptide-functionalized nanoparticles binding to FSH-R-expressing stable cell line) will initially be used to monitor reporter protein (e.g., GFP) expression to determine relative rates of nanoparticle incorporation and expression. This should rapidly lead to identification of exemplary nanoparticle compositions (in terms of the peptide density on the nanoparticle surface and optionally including linkage spacers). These in vitro studies will help identify exemplary SC-targeting and LC-targeting nanoparticles for confirmation of GFP gene expression in vivo.
  • reporter protein e.g., GFP
  • nanoparticles containing a genetic payload encoding DTA under the regulation of cell- and species- specific promoters will be constructed.
  • the ability of these nanoparticles to induce cell death in vitro will be tested by conducting dose- and time- dependent studies using the model systems (LH peptide- functionalized nanoparticles targeting TM3 cells and FSH peptide-functionalized nanoparticles targeting FSH-R-expressing cells). Experiments involving the exposure of control cells (e.g., GC1) not expressing receptors for these gonadotrophins will also be conducted. These in vitro data may confirm the nanoparticles are capable of delivering cell death in a highly selective, targeted, efficient manner.
  • nanoparticle constructs described above to target Sertoli cells (male) and Leydig cells (male) to induce cell ablation can next be assessed in systematic studies.
  • the structure of the nanoparticles will be based on the in vitro studies described above, which will have led to exemplary nanoparticle compositions in terms of lipid-PEG content, the density and configuration of the cell-specific targeting peptides incorporated into the nanoparticle surface and the structure of the genetic pay load inserted into the nanoparticle interior.
  • nanoparticle formulations Using a single dose administration regime, two potential nanoparticle formulations will initially be assessed in vivo'. (1) nanoparticles expressing FSH peptides to target Sertoli cells in males; and (2) LH-functionalized nanoparticles to target Leydig cells in males. Each of these formulations will be assessed at 3 different doses.
  • the in vivo assessment of the biological efficacy of the nanoparticles will be conducted in juvenile adult Swiss mice (6-8 weeks old). The in vivo assessment will be conducted with functionalized nanoparticles initially administered by intraperitoneal injection.
  • target and off-target tissues will be collected, fixed for 24 hr in 4% paraformaldehyde in PBS, sectioned and examined by fluorescence microscopy employing an excitation wavelength of 488 nm.
  • the in vivo studies will involve detailed monitoring of the animals to ensure general health and wellbeing (body weight, posture, vocalization, physical activity) and include, at the time of autopsy, a visual inspection of the major organ systems for signs of systemic damage.
  • any nanoparticle constructs that are found to possess biological activity in the in vivo assessment above will be submitted for fertility trials in male mice to confirm that a state of sterility has been induced. Fertility assessments will be conducted to ensure that target cell ablation is accompanied by permanent infertility. Fertility trials will be conducted 8 weeks after inoculation and repeated at 12 months to ensure that the infertility induced is permanent.
  • mice are naturally resistant to Diphtheria toxin (the murine HBEGF receptor binds the toxin only poorly).
  • a model will be used, in which expression of a transgene for Diphtheria toxin fragment A (DTA) is driven inside cells to induce cell death.
  • DTA diphtheria toxin fragment A
  • This model was previously used to specifically ablate the Sertoli cell population from mouse embryos in vivo, with no off-target effects (Rebourcet et al., 2014).
  • mice Male CD 1 nude mice will be anesthetized by inhalation of isofluorane and castrated/ovarectomised. Removal of gonads ensures that the host mouse gonadotrophins are high in order to drive the maturation of the gonadal tissue; an important and well-characterized interaction between the hypothalamus/pituitary and the xenografted gonad (Honaramooz et al., 2002; Mitchell et al., 2010) . Subsequently, steroid hormone production by the gonads feeds back to the brain to maintain steady state gonadal function (Honaramooz et al., 2002).
  • mice will receive analgesia (Rimadyl LA, Pfizer, New York, USA) in the drinking water for 1 day pre- and 5 days post- surgery, as well as antibiotics (Baytril, Bayer, Germany) for 5 days post-surgery, and will be monitored twice daily.
  • a genetic pay load containing a DNA sequence encoding only fragment A of the toxin is delivered into cells, through binding of nanoparticles to endogenous cell-specific receptors (e.g., FHSR, AmhR etc). Once inside, the cell expression of the transgene for fragment A of the toxin is induced, which then functions to drive cell death (e.g., within 24 hours). Cell targeting and expression of GFP (controls) or induction of cell ablation (DTA nanoparticles) will then be assessed.
  • endogenous cell-specific receptors e.g., FHSR, AmhR etc.
  • mice will be anesthetized until unconscious and then culled by cervical dislocation and grafts will be retrieved by dissection. Xenografts will be weighed and the percentage survival of xenografts will be recorded. Fragments of pre-graft, xenograft and equivalent age-matched control tissues will be snap frozen for molecular analysis, and the remainder fixed for 2 hours in Bonin’s fixative, transferred to 70% ethanol and then processed into paraffin blocks using standard procedures.
  • spermatogenesis recording the most advanced stage of germ cell development observed for each graft, will be determined histologically, and compared to non-grafted age-matched controls, with reference to control data (e.g., previous published studies (Abrishami et al., 2010; Snedaker et al., 2004)).
  • Immunohistochemical markers for each cell-type e.g., SOX9 - Sertoli cells; aSMA -Peritubular Myoid cells/vascular smooth muscle; 3BHSD - Leydig cells, Stra8 - Spermatogonia; SOG1- Spermatocytes; Protamine 1 or PGK1/2 - Spermatids; Mac2/CD68 - Macrophages; CD31 - Endothelial cells
  • Markers reflective of targeting such as GFP, or cleaved caspase 3 (apoptosis marker) will also be examined.
  • the pilot study will include 6 male dogs with one construct. Following a successful pilot study, the wider trial in both genders in both cats and dogs will be completed.
  • GnRH Gonadotropin-releasing hormone
  • Circulating LH and testosterone concentrations will be determined before and after the administration of a single intravenous bolus of a GnRH analogue (Buserelin). Furthermore, semen will be collected and evaluated for volume, progressive motility, morphology and sperm count.
  • GnRH analogue Buserelin
  • the nanoparticles will be administered by a single intravenous injection into the cephalic vein.
  • a general physical examination and andrological examination are performed on a weekly basis. Semen evaluation and a GnRH stimulation test will be performed on a monthly basis after the treatment. A unilateral orchiectomy will be performed under general anesthesia, 6 months after treatment. The removed testis will be examined for the presence of spermatogenesis and overall histological changes. Three and 6 months after hemicastration, the semen will be evaluated and a GnRH stimulation test will be performed to show persistence of the treatment effect.
  • nanoparticles will be administered by a single intravenous injection into the cephalic vein.
  • a GnRH stimulation test will be performed on a monthly basis until 6 months after treatment and at 9 and 12 months after treatment, and the plasma progesterone will be determined every 2 weeks to evaluate reproductive (endocrine) function. If the plasma progesterone concentrations remain ⁇ 1 ng/ml and when no signs of pro-estrus have been detected for a period longer than the mean interestrus interval + 2 months, it will be attempted to induce estrus by using a slow release GnRH analogue implant (Suprelorin®, Virbac). In healthy, untreated female dogs, (pro)estrus can be expected within 10 days after Suprelorin® administration (Fontaine et al., 2011; Maenhoudt et al., 2012) .
  • the female cats will be housed in a group and a vasectomized tomcat will be introduced to enhance estrus detection and induction of ovulation. Before and after the treatment, the female cats will be examined twice weekly for the presence of estrous signs: rolling, head rubbing, treading with hind legs, lordosis and tail deviation. Once a month the plasma progesterone concentration will be determined until the end of the study. A plasma progesterone concentration >1 ng/ml indicates the presence of a corpus luteum. The fecal oestradiol concentration will be determined before- and on a weekly basis after the treatment.
  • the nanoparticles will be administered by a single intravenous injection into the cephalic vein.
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Abstract

Peptides that each bind to a receptor of a reproductive cell (including a follicle stimulating hormone receptor, a luteinizing hormone receptor, or an Anti-Mullerian hormone receptor) are described herein. In addition, nanoparticle constructs and compositions containing one or more of such peptides and an apoptotic or cytotoxic protein or a polynucleotide encoding the apoptotic or cytotoxic protein, such as Diphtheria toxin A ("DTA") are described herein. The nanoparticles comprise surface peptides that can bind to reproductive cell receptors and cause cell death. The methods comprise administering the nanoparticle constructs or compositions thereof for the sterilization of subjects, including male and female cats and dogs.

Description

COMPOSITIONS AND METHODS OF USING TARGETING PEPTIDES AND
NANOPHARMACEUTICALS FOR THE STERILIZATION OF CATS AND DOGS
CROSS-REFERENCE TO RELATED APPLIACTTONS
[001] This application claims priority to US Provisional Patent Application No. 63/136,684, filed January 13, 2021, and to US Provisional Patent Application No. 63/136,688, filed January 13, 2021, each of which are incorporated by reference herein in their entireties.
FIELD
[002] This application relates to compositions and methods of administering nanoparticle constructs containing an apoptotic or cytotoxic protein or a polynucleotide encoding for the apoptotic or cytotoxic protein, such as Diphtheria toxin A (“DTA”), where the nanoparticle constructs comprise one or more peptides that can bind to reproductive cell receptors and cause cell death, and where the compositions and methods are useful for the sterilization of subjects, including male and female cats and dogs.
BACKGROUND OF THE INVENTION
[003] Dogs and cats are the most common domesticated carnivores across the world (Hughes & Macdonald, 2013). Estimates of the world dog population varies widely but is generally believed to be upwards of between 500 - 600 million (Statistics on Dogs - CAROdog, n.d.; Stray Animal Control, n.d.); approximately 75% (~480 million animals) of which are considered to be free-ranging or strays (Chawla et al., 2020). Additionally, the feral cat population is estimated to be at least 100 million, including 60 million in the USA and up to 12 million in Australia alone (Legge et al., 2017). These numbers highlight the magnitude of the worldwide problem posed by free-roaming domestic dogs and cats, which impacts not only human health but also imposes significant economic and environmental costs.
[004] Traditional means of lethal control used to mitigate the impact of free-roaming populations of feral cats and dogs are increasingly becoming unacceptable to communities, nongovernmental agencies and welfare organizations because of their perceived lack of humaneness (Massei & Miller, 2013). Additionally, there remain questions about the efficacy of such strategies and, particularly in the case of poisonous baiting, the impact of the toxicants on nontarget species and the environment at large (Massei & Miller, 2013). Surgical sterilization is one alternative to pest species management that is viewed as both effective (Rowan & Kartal, 2018) and more socially acceptable than culling. However, surgical sterilization is relatively labor intensive and expensive and necessitates the use of anesthesia, experienced personnel, and specialized facilities. An ideal alternative to lethal control or surgical sterilization would be to develop a safe effective means of non-surgically sterilizing pest animal species.
[005] Currently, it is recommended that pet owners and shelters spay or neuter animals, including cats and dogs. Surgical sterilization, e.g., by way of spaying or neutering young animals is considered a responsible way to care for animals. Surgical sterilization is also a key part of the Trap-Neuter-Retum strategy (Robertson, 2008). In males, neutering typically involves removal of the testes. In females, it typically involves abdominal surgery to remove one or both ovaries and/or uterus. It is encouraged (and in some countries required for adopted animals) to prevent the births of unwanted litters, which contribute to the overpopulation of unwanted animals in the rescue system. The ASPCA (American Society for the Prevention of Cruelty to Animals®) has indicated the pet homelessness problem results in millions of healthy dogs and cats being euthanized in the United States each year. This is a worldwide problem that is not limited to just the United States. In addition, the ASPCA also indicates there are medical and behavioral benefits to spaying and neutering animals including: preventing certain infections or tumors (e.g., uterine infections and mammary tumors in females and testicular cancer and prostate problems in males); avoiding female pets going into heat; making it less likely for male pets to roam; and possibly leading to better behaved males. Sterilization procedures such as spaying and neutering are common surgeries, but there can be health risks. General anesthesia, for example, is relatively expensive, and requires specialized equipment, facilities and personnel making it impracticable in remote areas and in low-income countries. Additionally, it takes time for the animals to heal after the surgery. Accordingly, bathing must be avoided for, e.g., at least ten days after surgery; the animal must refrain from running or jumping post-surgery; and the incision site must be monitored to avoid infection and proper healing. Accordingly, the present disclosure provides methods and compositions for sterilizing subjects that can avoid surgery.
[006] Indeed, nonsurgical fertility control is increasingly advocated as both a more humane (Fagerstone et al., 2010; McUaughlin & Aitken, 2011) and cost-effective (Massei & Miller, 2013) solution for free-roaming dog and cat management and to resolve human-wildlife conflicts. Additionally, nonsurgical sterilization may provide a higher throughput, compared to surgical sterilization. Previous attempts to generate non-surgical approaches to animal sterilization have not been successful. This is particularly true of contraceptive vaccines which have been the subject of considerable investment, for instance by governments interested in developing strategies to control feral animal populations. Vaccines, for example, have been used to sterilize females by attempting to permanently remove the primordial follicle population (Aitken et al., 1996). But sterilization via vaccine was somewhat ineffective as infertility takes several months to materialize because the primordial follicle pool is progressively depleted over time. Furthermore, no active immunization approach has ever been found to induce sterility in males.
[007] Currently used non-surgical sterilization technologies include: (i) hormonal methods such as implants (Driancourt & Briggs, 2020); (ii) the induction of immunocontraception that relies on antibody production against reproductive cells or signaling molecules; and (iii) reproductive senescence induced by a chemosterilant (Rafatmah et al., 2019; Silva et al., 2018). Hormonal and immunocontraception require periodic re-administration which may not always be practicable (e.g., where there is not continued access to the animal). Major disadvantages of untargeted chemosterilants are that they are often not species specific, require anesthesia and highly trained administrants (Madbouly et al., 2021; Silva et al., 2018), may have off-target effects at the doses required for sterilization (Abolaji et al., 2016), and/or may be environmental pollutants. The injectable chemical agents, such as zinc gluconate, currently available for sterilization of male animals are effective, but still require specialized equipment and personnel to conduct intratesticular administration (Massei & Miller, 2013). Ideally, an injectable sterilizing agent will be able to be administered either intraperitoneally or intravenously to deliver an agent that will selectively target the gonads.
[008] Accordingly, an aspect of the present invention provides a non-surgical permanent and, in certain embodiments, single dose fertility treatment that is safe and effective in male and female subjects.
[009] The male gonads (i.e., testes) perform two distinct functions, that is, the production of spermatozoa within the seminiferous tubules and the synthesis of steroids by Leydig cells (Ye et al., 2017), which reside within the interstitial space outside the seminiferous tubules (Evans & Ganjam, 2011; Rebourcet et al., 2014). In adults, testicular function is regulated via the hypothalamo-pituitary-gonadal (HPG) axis and secretion of the glycoprotein hormones (GPH), that is, Follicle-Stimulating Hormone (FSH) and Luteinising hormone (LH), by gonadotrophs in the anterior pituitary (Abel et al., 2008; Cheng & Mruk, 2010). These two hormones target the Sertoli cells, expressing the FSH receptor, and the Leydig cells, which express the LH receptor. [0010] A third hormone, Anti -Mullerian hormone (AMH), also known as Mullerian Inhibiting Substance, has a sexually dimorphic expression in Sertoli cells of the testis (Alves et al., 2013) and granulosa cells of the ovary (Josso et al., 2001) and, in the embryo is critical for normal differentiation of the internal reproductive tract structures. The anti -Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors. Although AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood. Nevertheless, AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015). AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Cimino et al., 2016).
[0011] Sertoli and Leydig cells, which are found in the testes are highly differentiated and are necessary for reproductive success, including for example, the maintenance of sperm development and maturation. Without the support function of these two cell types, mature sperm cell pools would not develop, thus resulting in an infertile male. In one aspect, the present invention is directed to permanently disrupting both cell types by delivering a gene, for example, to Sertoli and/or Leydig cells, that will cause cell death. The genetic payload can be delivered intravenously, for example, by a lipid sphere (nanoparticle), which protects the DNA inside until being internalized by the target cell.
[0012] Developing synthetic peptides based on such hormones that specifically target the Sertoli cells and/or Leydig cells offers advantages over whole recombinant proteins since they possess lower immunogenicity and production costs and tend to have higher specificity and selectivity than other smaller compounds (Diao & Meibohm, 2013; Le Joncour & Laakkonen, 2018). Such peptides should, in principle, serve as attractive vectors for the delivery of different cargo payloads to the Sertoli cell population and/or Leydig cell population while minimizing the risk posed by undesirable off-target effects. Accordingly, targeting peptides derived from FSH and LH amino acid sequences within the receptor binding regions are described herein.
[0013] Introducing genes into cells to treat human and animal disease and other conditions is coming of age and has commonly involved the use of viral vector technologies. These vectors require the modification of the virus’s genome to incorporate genes of interest to be introduced. The virus is then manufactured with the DNA or other polynucleotide inside. Viral vectors can be limited, however, in their ability to be modified to introduce targeting peptides on their surface.
[0014] In contrast to the general practice of using viral vectors to deliver genes, nanoparticle constructs are formulated herein to deliver a gene. Nanoparticles can be formulated with different components and different ratios of components that form the sphere that protects the DNA or other polynucleotide until it enters the cell. Nanoparticles provide versatility of manufacturing. They can be tailored and functionalized to target specific organs by biasing the biodistribution to the site of action of the therapeutic that they deliver. This can also be achieved by grafting or linking ligands to the surface of the nanoparticles, which target a specific receptor expressed by the cells or tissue of interest. Thus, many interactions of the nanoparticle components and targeting ligands bound to the surface of the nanoparticle can be designed for use in the present invention. Nanoparticles themselves are a relatively new drug delivery technology. They are currently used in humans, for example, in the oncology sector for delivering small molecule organic pharmaceuticals (largely chemotherapeutic agents). One exemplary PEGylated liposome is Doxil, indicated for the treatment of AIDS-associated Kaposi’s sarcoma.
[0015] For example, to target Sertoli and/or Leydig cells specifically, nanoparticles can be designed to incorporate small peptide sequences on their surface that bind to receptors present on the target cells (e.g., FSH and LH receptors on Sertoli and/or Leydig cells), thereby adding a level of safety preventing the gene drug from being internalized by non-Sertoli and/or non-Leydig cells. Herein, the inventors have developed a unique array of peptides that are capable of targeting these cells in vitro and in vivo. These cell -targeting peptides were generated through the use of random peptide phage display technology (Eidne et al., 2000) or by the synthesis of peptides capable of binding to receptors that are restricted to the target cell population (e.g., FSH, LH). The nanoparticles can also be utilized, for example, to target Sertoli and/or Leydig cells, gonocyte cells (precursors of spermatogonia), or to target female cells such as primordial follicle cells, or primordial germ cells in utero.
[0016] It is well known in the art that both male and female germ cells are highly vulnerable to ionizing radiation. When tissues are irradiated, highly reactive hydroxyl radicals are generated that induce the rapid onset of lipid peroxidation chain reactions as well as concomitant oxidative damage to proteins and nucleic acids that, together, propel affected cells down an apoptotic pathway leading to cell death (Sakashita et al., 2010). In order to recapitulate the oxidative stress created by ionizing radiation, previous studies included using redox cycling xenobiotics to be carried to target cell types by an appropriate peptide and to induce a local burst of free radical generation (Aitken & Baker, 2013). This approach was previously successful in disrupting spermatogenesis and generated high levels of oxidative stress in the female germ line, but the levels of oxidative stress achievable with the redox cycling quinones was not sufficient to induce cell ablation with a high level of efficiency, particularly in females.
[0017] In certain aspects of the invention, therefore, targeting peptides are incorporated on the surface of nanoparticles that encompass an apoptotic or cytotoxic protein or recombinant viral-based (e.g., recombinant lentiviral-based) DNA construct encoding the apoptotic or cytotoxic protein. Targeting peptides based on, e.g., FSH and LH expressed on the lentiviral coats can be grafted or linked to a nanoparticle to allow specific delivery to, for example, Sertoli and/or Leydig cells in males and primordial follicle cells in females. Species- and cell-specific promoters may also be incorporated to further reduce off-target effects. In certain aspects of the invention, targeting peptides are incorporated on the surface of nanoparticles that encompass a linear DNA construct that encodes an apoptotic or cytotoxic protein.
[0018] As a result of the present invention, sterility may be achieved following a single administration of nanoparticles. Accordingly, sterilization of stray/homeless animals will no longer require a surgical process which can be expensive, time consuming and stressful for the animals. Alongside this, the numbers of animals euthanized as a result of overcrowding in shelters will also be reduced (as a result of fewer litters being produced due to sterilization). Furthermore, this technology could also be used for routine neutering of domestic pets (cats and dogs) as opposed to the surgical processes currently in practice. This technology could both reduce the costs of this procedure and result in a reduced impact on the animals due to lack of anesthetic requirements and surgery.
SUMMARY
[0019] In accordance with the present invention, peptides, conjugated peptides, nanoparticle constructs, pharmaceutical compositions comprising the peptides and nanoparticle constructs, and methods of sterilizing a subject comprising administering to the subject an effective amount of the peptides, nanoparticle constructs or pharmaceutical compositions are provided.
[0020] The disclosure provides an isolated Follicle-Stimulating Hormone (FSH) peptide comprising a fragment of an FSH protein or variant thereof. [0021] In certain aspects, the FSH peptide comprises an amino acid sequence of TVX9GLGPX10Y (SEQ ID No. 95), wherein X9 is R or Q and X10 is S or G.
[0022] In certain aspects, the FSH peptide comprises an amino acid sequence of CTVX9GLGPX10Y (SEQ ID No. 96), wherein X9 is R or Q and X10 is S or G.
[0023] In certain aspects, the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4Q (SEQ ID No. 33), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[0024] In certain aspects, the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 34), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[0025] In certain aspects, the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4Q (SEQ ID No. 35), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[0026] In certain aspects, the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 36), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; and X4 is T or N.
[0027] In certain aspects, the FSH peptide comprises an amino acid sequence of YTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 49), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
[0028] In certain aspects, the FSH peptide comprises an amino acid sequence of CYTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 50), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
[0029] In certain aspects, the FSH peptide comprises an amino acid sequence of CHX7GX8X7DSDSTDX7T (SEQ ID No. 63), wherein X7 is C, S, or A and X8 is K, R, or G.
[0030] In certain aspects, the FSH peptide comprises an amino acid sequence of X8CDSDSTDX7TVX9GL (SEQ ID No. 78), wherein X8 is K, R, or G; X7 is C, S, or A; and X9 is R or Q.
[0031] In certain aspects, the FSH peptide comprises an amino acid sequence of CDSDSTDX7TVX9GL (SEQ ID No. 79), wherein X8 is K, R, or G; X7 is C, S, or A; and X9 is R or Q.
[0032] In certain aspects, the FSH peptide comprises an amino acid sequence of SDSTSX7TVX9GLGPX10Y (SEQ ID No. 89), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G. [0033] In certain aspects, the FSH peptide comprises an amino acid sequence of CSDSTSX7TVX9GLGPX10Y (SEQ ID No. 90), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G.
[0034] In certain aspects, the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
[0035] In certain aspects, the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
[0036] In certain aspects, the FSH peptide comprises the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2. In certain aspects, the FSH peptide comprises an amino acid sequence consisting of SEQ ID No. 1 and/or SEQ ID No. 2.
[0037] In certain aspects, the FSH peptide comprises the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
[0038] In certain aspects, the FSH peptide comprises an amino acid sequence consisting of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
[0039] In certain aspects, the FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and/or SEQ ID No. 18.
[0040] In certain aspects, the isolated FSH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
[0041] The disclosure provides an isolated Luteinizing Hormone (LH) peptide comprising a fragment of an LH protein or variant thereof.
[0042] In certain aspects, the LH peptide comprises an amino acid sequence of SX18SDX7GGPX8X19X20X21 (SEQ ID No. 144), wherein Xis is T, S, orN; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
[0043] In certain aspects, the LH peptide comprises an amino acid sequence of CSX18SDX7GGPX8X19X20X21, (SEQ ID No. 145), wherein Xis is T, S, or N; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S. [0044] In certain aspects, the LH peptide comprises an amino acid sequence of X11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 112), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
[0045] In certain aspects, the LH peptide comprises an amino acid sequence of CX11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 113), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
[0046] In certain aspects, the LH peptide comprises an amino acid sequence of SFPVALX7RX7GPX7RX17 (SEQ ID No. 125), wherein X7 is C, S, or A and X17 is L or R.
[0047] In certain aspects, the LH peptide comprises an amino acid sequence of CSFPVALX7RX7GPX7RX17 (SEQ ID No. 126), wherein X7 is C, S, or A and X17 is L or R.
[0048] In certain aspects, the LH peptide comprises an amino acid sequence of CRX17SX18SDX7G (SEQ ID No. 133), wherein X17 is L or R; Xis is T, S, or N; and X7 is C, S, or A.
[0049] In certain aspects, the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
[0050] In certain aspects, the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
[0051] In certain aspects, the LH peptide comprises the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4. In certain aspects, the LH peptide comprises an amino acid sequence consisting of SEQ ID No. 3 and/or SEQ ID No. 4.
[0052] In certain aspects, the LH peptide comprises the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
[0053] In certain aspects, the LH peptide comprises an amino acid sequence consisting of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
[0054] In certain aspects, the LH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, and/or SEQ ID No. 104.
[0055] In certain aspects, the LH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
[0056] The disclosure provides an isolated Anti-Mullerian Hormone (AMH) peptide comprising a fragment of an AMH protein or variant thereof.
[0057] In certain aspects, the AMH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
[0058] In certain aspects, the AMH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
[0059] In certain aspects, the AMH peptide comprises the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165. In certain aspects, the AMH peptide comprises an amino acid sequence consisting of SEQ ID No. 5 and/or SEQ ID No. 165. [0060] In certain aspects, the AMH peptide comprises the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
[0061] In certain aspects, the AMH peptide comprises an amino acid sequence consisting of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
[0062] In certain aspects, the AMH peptide has one substitution modification, two substitution modifications, three substitution modifications, or four substitution modifications relative to SEQ ID No. 149, SEQ ID No. 153, SEQ ID No. 157, and/or SEQ ID No. 161.
[0063] In certain aspects, the AMH peptide is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
[0064] In certain aspects, the isolated FSH, LH, and/or AMH is amidated. In certain aspects, the peptide comprises a cysteine residue at the N-terminus. In certain aspects, the peptide comprises a linker. In certain aspects, the linker is a maleimide group, 1,6- aminohexanoic acid (Ahx), y-aminobutanoate-mercaptopropionic acid, alanine with 3- mercaptopropionic acid (MPA), y-aminobutanoic acid with MPA, mini-polyethyleneglycol with MPA, or a cathepsin cleavable valine-citrulline(vc)-PABC linker.
[0065] In certain aspects, the isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 18, wherein the peptide is conjugated to a protein, a peptide, a nanoparticle, a label, a small molecule, and/or other moiety. In certain aspects, the protein is an apoptotic protein or a cytotoxic protein. In certain aspects, the small molecule is an auristatin (Aur) molecule, such as such monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), or a menadione (Md) molecule, such as 2-methyl-l,4-naphthoquinone. In certain aspects, the label is fluorescein isothiocyanate (FITC).
[0066] The disclosure provides a composition comprising a peptide conjugate comprising the isolated FSH, LH, and/or AMH peptide of any of the preceding aspects.
[0067] The disclosure provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein.
[0068] The disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein.
[0069] The disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein; and c) a reporter protein or a polynucleotide encoding a reporter protein.
[0070] In certain aspects, the one or more peptides comprises a fragment of a protein or a variant thereof that binds the receptor of the reproductive cell.
[0071] In certain aspects, the one or more peptides is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
[0072] In certain aspects, the nanoparticle construct comprises a polynucleotide encoding the apoptotic or cytotoxic protein.
[0073] In certain aspects, the apoptotic protein is Diphtheria toxin fragment A (DTA).
[0074] In certain aspects, the DTA comprises the amino acid sequence of SEQ ID No: 10 or a variant thereof comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID No: 10.
[0075] In certain aspects, the reporter protein is selected from red fluorescent protein (RFP), green fluorescent protein (GFP), and enhanced green fluorescent protein (EGFP).
[0076] In certain aspects, the reproductive cell is a Sertoli cell and/or a Leydig cell.
[0077] In certain aspects, the one or more peptides bind a receptor of a Sertoli cell and/or a Leydig cell.
[0078] In certain aspects, the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell and/or the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.
[0079] In certain aspects, the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 1 or SEQ ID No: 2.
[0080] In certain aspects, the one or more peptides bind a luteinizing hormone receptor of a Leydig cell. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 3 or SEQ ID No: 4. [0081] In certain aspects, the reproductive cell is a gonocyte, a primordial follicle cell, and/or a primordial germ cell.
[0082] In certain aspects, the one or more peptides bind an anti-Mullerian hormone on a primordial follicle cell. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 5.
[0083] In certain aspects, the one or more peptides bind a primordial germ cell. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 6. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 7. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 8. In certain aspects, the one or more peptides comprise an amino acid sequence of SEQ ID No: 9.
[0084] In certain aspects, the one or more peptides contain a terminal cysteine residue.
[0085] In certain aspects, the polynucleotide encoding the apoptotic or cytotoxic protein is operatively linked to a promoter. In certain aspects, the promoter is a cell-specific promoter. In certain aspects, the promoter is selected from the group consisting of ABP, Rhox5, and HSD17B3. In certain aspects, the promoter is a species-specific promoter.
[0086] In certain aspects, the nanoparticle construct comprises poly(lactide-co-glycolide) (PLGA).
[0087] In certain aspects, the nanoparticle construct comprises polyethylene glycol (PEG). In certain aspects, the nanoparticle construct comprises maleimide-terminated PEG. In certain aspects, the nanoparticle construct comprises DSPE-PEG.
[0088] In certain aspects, the nanoparticle construct comprises cationic lipids, ionizable lipids, and/or helper lipids.
[0089] In certain aspects, the one or more peptides are conjugated to lipids.
[0090] In certain aspects, the mean peptide density is from 5,000 to 50,000 peptides per nanoparticle.
[0091] In certain aspects, the polynucleotide is condensed with protamine, lysine, or poly lysine.
[0092] In certain aspects, the apoptotic or cytotoxic protein or the polynucleotide is encapsulated within the nanoparticle construct.
[0093] In certain aspects, the nanoparticle construct comprises a nanoparticle having a PLGA matrix with maleimide terminated PEG lipids; wherein the one or more peptides are conjugated to the maleimide terminated PEG lipids; and wherein the apoptotic or cytotoxic protein or the polynucleotide is encapsulated in the nanoparticle. [0094] In certain aspects, the nanoparticle construct comprises a viral vector comprising the polynucleotide encoding the apoptotic or cytotoxic protein. In certain aspects, the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector. In certain aspects, the viral vector is a lentiviral vector.
[0095] In certain aspects, the nanoparticle construct further comprises a fluorescent label.
[0096] The disclosure also provides a pharmaceutical composition comprising the isolated FSH, LH, or AMH peptide, the composition, or the nanoparticle construct of any of the preceding aspects.
[0097] In certain aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
[0098] In certain aspects, the pharmaceutical composition comprises an aqueous solution.
[0099] In certain aspects, the isolated FSH, LH, or AMH peptide, composition, or the nanoparticle construct is freeze-dried.
[00100] The disclosure also provides a method of sterilizing a subject comprising administering to the subject an effective amount of the nanoparticle construct or pharmaceutical composition of any one of the preceding aspects.
[00101] In certain aspects, the isolated FSH, LH, or AMH peptide, the nanoparticle construct, or the pharmaceutical composition is freeze-dried as a powder and dispersed in an aqueous medium prior to administration.
[00102] In certain aspects, the composition is administered via injection. In certain aspects, the administering comprises a one-time injection.
[00103] In certain aspects, the administering is intravenous, intraperitoneal, or intratesticular administration. In certain aspects, the administering is intravenous injection. In certain aspects, the administering is a one-time intravenous injection into the cephalic vein.
[00104] In certain aspects, the effective amount comprises a dosage capable of inducing sterilization of the subject.
[00105] In certain aspects, sterilization or ablation occurs within 24 hours, 48 hours, or 72 hours after injection.
[00106] In certain aspects, sterilization is permanent.
[00107] In certain aspects, the effective amount comprises a dosage capable of inducing ablation of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of one or more reproductive cell types of the subject. In certain aspects, the effective amount comprises a dosage capable of inducing ablation of at least about 60% of one or more reproductive cell types of the subject. In certain aspects, the effective amount comprises a dosage capable of inducing ablation of at least about 80% of one or more reproductive cell types of the subject.
[00108] In certain aspects, the one or more reproductive cell types of the subject is Sertoli cells and/or Leydig cells.
[00109] In certain aspects, the one or more reproductive cell types of the subject is gonocytes, primordial follicle cells, and/or primordial germ cells.
[00110] In certain aspects, the subject is a cat or dog. In certain aspects, the subject is a cat. In certain aspects, the subject is a dog. In certain aspects, the subject is a male cat or dog. In certain aspects, the subject is a female cat or dog.
[00111] In certain aspects, the subject is pre-pubescent. In certain aspects, the subject has reached pubertal maturation.
[00112] The disclosure also provides a method of determining uptake efficiency of a nanoparticle construct, comprising: a. administering to a subject the nanoparticle construct of a previous embodiment (comprising a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein) or a composition comprising the nanoparticle construct; and b. assessing ligand density on the nanoparticle surface, wherein increased ligand density or transfection efficiency for the nanoparticle as compared to a control or reference value is indicative of optimal uptake efficiency.
[00113] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
[00114] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
[00115] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiment(s) and together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS
[00116] Figs. 1A-F show exemplary follicle-stimulating hormone (FSH) peptides mapped to the [3-subunit of the FSH protein. The synthetic FSH peptides are indicated by brackets. Fig. 1A: peptide F49 (residues 35-49); Fig. IB: peptide F53 (residues 33-53); Fig. 1C: peptide F95 (residues 81-95); Fig. ID: peptide F99 (residues 86-99); Fig. IE: peptide F103 (residues 89-103); Fig. IF: peptide FSHa (residues 95-103).
[00117] Figs. 2A-B show immunocytochemistry using an anti-FSH receptor antibody with an Alexa Fluor 488 secondary antibody (third column from the left) indicates that the FSH receptor is expressed at high levels by the TM4 Sertoli cell line, but not by the mEcapl8 cell line. Fig. 2A: TM4 Sertoli, 40x objective. Fig. 2B: mEcapl8, 40x objective. Scale bar = 20 pm.
[00118] Figs. 3A-H show in vitro peptide targeting and binding affinity for FSH peptides F49, F53, F95, F99, F103, and FSHa. In Figs. 3A-F, fluorescence -activated cell sorting (FACS) analysis was used to analyze the binding of fluorescein isothiocyanate (FITC)- conjugated FSH peptides to TM4 Sertoli cells (n=3). Fig. 3A: F49; Fig. 3B: F53; Fig. 3C: F95; Fig. 3D: F99; Fig. 3E: F103; Fig. 3F: FSHa. As shown in Figs. 3G-H, FSH peptides exhibited specificity of binding to Sertoli cells when compared to binding to mouse epididymal epithelial (mEcapl8) cells (n=3) at the same concentrations. Fig. 3G shows the shift in FITC median fluorescence intensity of the TM4 Sertoli cells in response to increasing concentrations of FSH- peptides after 1 hour. Fig. 3H shows the shift in FITC median fluorescence intensity of the mEcap 18 cells in response to increasing concentrations of FSH-peptides after 1 hour. A greater shift in median fluorescence indicates greater binding capability.
[00119] Fig. 4 shows representative images of epididymis and excretory organs (kidney, liver, and spleen) 8 hours post-injection. Male adult mice were treated with IP injections of 300 pL of either 1 mM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each treatment group. The peptide-FITCs applied are designated in the first image of each row. Each field includes tissue from a vehicle-treated animal.
[00120] Figs. 5A-C show representative images of peptide-FITC localized to testes 8 hours post-injection compared to the vehicle control. Fig. 5A: FSHa-FITC. Fig. 5B: F49-FITC and F103-FITC. Fig. 5C: F53-FITC, F95-FITC, and F99-FITC.
[00121] Fig. 5D shows corresponding Imagel analysis of mean fluorescence intensity/pixel in testes of peptide-FITC injected males. Mean fluorescence intensity/pixel was normalized to that of the vehicle control within the same image. [00122] Figs. 6A-C show the structure and chemistry of naphthoquinones. Fig. 6A shows the structure of menadione (Md). Fig. 6B show redox chemistry of the naphthoquinones. SOD = superoxide dismutase. Fig. 6C shows linker designs for peptide conjugation. Structures of all linkers attached to the N-terminal of the peptide with menadione attached through a thioether bond to the linker. Structures are drawn using software: Advanced Chemistry Development Inc. ACD/3D Viewer (Freeware) Product Version 12.01 (Build 32890, 18 May 2009).
[00123] Figs. 7A-B show reactive oxygen species (ROS) production by TM4 Sertoli cells in response to FSH-Md and comparison of linkers. TM4 Sertoli cells were treated with 50 pM of the peptide, peptide-Md conjugate or Md alone (as positive control). Regular readings of chemiluminescence were taken over 90 minutes at room temperature. Fig. 7A show total luminescence was calculated by integration of the area under the curve over the first 90 minutes. Three different linkers were used to attach menadione to the FSH peptide (n=l; error bars represent three replicates of observation for the same sample). Fig. 7B shows the rate of production of chemiluminescence (AU/min) at 90 minutes, with all experiments normalized to FSHlMd=l for comparison (n=3). FSHa with linker 2 was shown to consistently outperform FSHa with linker 1 or linker 3.
[00124] Figs. 8A-D show production of ROS and oxidative damage in TM4 Sertoli cells. TM4 Sertoli cells were treated with 50 pM of the peptide, peptide-Md conjugate or Md alone (as positive control) or with vehicle and then incubated at 37°C for 90 minutes. Fig. 8A shows FACS for the detection of Mitosox Red, which is a marker of mitochondrial ROS production (n=3, p<0.05). Fig. 8B shows FACS for anti-4HNE, which is a marker for lipid peroxidation products (n=3, p<5xl0'4). Fig. 8C shows immunocytochemistry using anti-8- hydroxyl-deoxyguanosine (8-OH-dG) to detect DNA oxidation (n=3, p<0.05). Fig. 8D shows immunocytochemistry using anti-caspase 3 to detect apoptotic cells (n=3). No significant difference was observed.
[00125] Figs. 9A-E show analysis of male mice treated with FSH2Md at 10 weeks post-injection. Ten males were treated with IP injections of 300 pL/30g 14.5 mM FSH2Md in 30% Kolliphor/phosphate buffer saline (PBS). Controls were age-matched, untreated males. Fig. 9A: average testes weights (mg). Fig. 9B: sperm motility. Fig. 9C: sperm vitality. In Fig. 9D, the sperm chromatin dispersion test (HALO assay) was used to count the percentage of sperm with DNA damage in the form of strand breaks. p<0.05. Fig. 9E: immunocytochemistry using anti-8- hydroxyl-deoxyguanosine (8-OhdG) to detect sperm with oxidative DNA damage. p<0.05. [00126] Figs. 10A-B show representative images of H and E staining of testes sections showing histology of FSH2Md-treated or control mice at two different magnifications. Seminiferous tubules from FSH2Md-treated mice exhibited uninterrupted spermatogenesis. Fig. 10A: 200x magnification; scale bar = 50 pM. Fig. 10B: 400x magnification; scale bar = 100 pM.
[00127] Fig. 11 show testes section of FSH2Md-treated or control mice. Seminiferous tubules from treated mice exhibited uninterrupted spermatogenesis. The ApopTag assay showed that there was some apoptosis of germ cells.
[00128] Figs. 12A-D show exemplary luteinizing hormone (LH) peptides mapped to the [3-subunit of the LH protein, which targets receptors on the cells in the ovary and Leydig cells of the testes. The synthetic LH peptides are indicated by brackets. Fig. 12A: peptide L57 (residues 38-57); Fig. 12B: peptide L95 (residues 81-95); Fig. 12C: peptide L101 (residues 93- 101); Fig. 12D: peptide LHa (residues 96-107).
[00129] Figs. 13A-B show a Leydig cell model. Fig. 13A shows relative expression of LHr mRNA in the mouse Leydig cell lines, MLTC-1 and TM3 cells, whole testis, and mEcapl8 cells, as assessed by qPCR. Fig. 13B shows immunocytochemistry using an anti- LHr antibody with an Alexa Fluor 488 secondary antibody, which indicates that the LH receptor is expressed at high levels in MLTC-1 cells but not in mEcapl8 cells.
[00130] Figs. 14A-F show in vitro peptide targeting and binding affinity for LH peptides. In Figs. 14A-D, FACS analysis was used to analyze the binding of fluorescein isothiocyanate (FITC)-conjugated LH peptides to mouse Leydig tumor (MLTC-1 Leydig) cells (n=9). Fig. 14A: L57; Fig. L95; Fig. 14C: L101; Fig. 14D LHa. As shown in Figs. 14E-F, LH peptides exhibited specificity of binding to MLTC-1 Leydig cells when compared to binding to the mEcapl8 cells (n=9) at the same concentrations. Fig. 14E shows the shift in FITC median fluorescence intensity of the MLTC-1 Leydig cells in response to increasing concentrations of LH-peptides. Fig. 14F shows the shift in FITC median fluorescence intensity of the mEcapl8 cells in response to increasing concentrations of LH-peptides. A greater shift in median fluorescence indicates greater binding capacity.
[00131] Figs. 15A-B show representative images and mean intensity of fluorescence of peptide-FITC localized to testes 8 hours post-injection compared to the vehicle control. Fig. 15A shows images of testes from LHa-FITC, L57-FITC, L101-FITC, and L95- FITC-treated animals alongside the vehicle control. Fig. 15B shows ImageJ analysis of mean fluorescence intensity/pixel in testes of peptide-FITC injected males. Mean fluorescence intensity/pixel was normalized to that of the vehicle control within the same image (n=3). [00132] Fig. 16 shows representative bright field and fluorescence images of excretory organs (kidney, liver, and spleen) 8 hours post-injection. Male adult mice were treated with IP injections of 300 pL of either 1 mM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each treatment group. Peptide-FITCs applied are as shown in the first image of each row. The fourth (unlabeled) tissue in the bottom three images were from animals treated with an unrelated peptide-FITC.
[00133] Fig. 17 shows lysosomal processing of vcMonomethylAuristatin E (vcMMAE), an anticancer agent that interacts with a-tubulin in a similar way to Vinca alkaloids to block a-tubulin polymerization and prevents the formation of the mitotic apparatus. The linked maleimide allows conjugation of the peptide via the N-terminal thiol group. Between the maleimide and the Aur, there is a cleavable linker made up of valine, citrulline (circled) and para-aminobenzyl carbamate (PABC). In the lysosome, the dipeptide linker is cleaved and then Aur is released from the PABC by 1,6-elimination. Structures drawn using software: Advanced Chemistry Development Inc. ACD/3D Viewer (Freeware) Product Version 12.01 (Build 32890, 18 May 2009).
[00134] Figs. 18A-C show the effect of FSH2Md and LH2Auristatin (LH2Aur) in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. Males were euthanized immediately after the matin period (~I0 weeks post-injection). Fig. 18A shows average testis weights (n=10). Fig. 18B shows representative histology images of H and E staining of tubules from each of the treatment groups. Scale bar = 50 pm. At least 50 tubules from each of the five males from each treatment group was circled and each area in pixels/tubule was calculated, using ImageJ (n=5; p<0.05). Fig. 18C shows the average area occupied by seminiferous tubule in the testes of each treatment group.
[00135] Figs. 19A-C show the effect of FSH2Md and LH2Auristatin (LH2Aur) in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. Males were euthanized immediately after the matin period (~I0 weeks postinjection). Fig. 19A shows representative images of ApopTag staining five males from each treatment group. Scale bar = 50 pm. Fig. 19B shows the average number of apoptotic cells per affected tubule (n=5 sections/treatment group). Fig. 19C shows the percentage of tubules affected (>4 stained cells) per testes section, which were counted with the number of stained cells per affected tubule. NB. Subjects were randomly selected from the treatment groups but those testes weighing less than 60 mg were excluded.
[00136] Figs. 20A-C show the effect of FSH2Md and LH2Aur in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Fig. 20A shows images of the testes of the treated males. Anti-Sox9 was used to stain for Sertoli cells in testis sections from 5 of the 10 males in each treatment group. Inset is secondary only control. Scale bar = 50 pm. Sertoli cells were counted in all tubules of each section for 5 of the 10 males from each treatment (n=5). Fig. 5B shows the average number of Sertoli cells per tubule. NB. Subjects were randomly selected from the treatment groups; subjects whose testes weighed less than 60 mg were excluded. Fig. 20C shows serum FSH levels measured using an anti-FSH ELISA (n=9).
[00137] Figs. 21A-C show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Males were euthanized 10 weeks post-injection and epididymal sperm was collected from all ten males in each treatment group and all samples were assessed for abnormal morphology, vitality, motility, mitochondrial ROS, cytoplasmic ROS, and DNA fractionation with at least one hundred sperm counted in each assay for each sample. Fig. 21 A: Percent sperm with DNA strand breaks was accessed by probing sperm for DNA strand breaks using the HALO assay. Fig. 2 IB: Percent sperm with oxidative DNA damage was assessed using an anti-8-OH-dG antibody to detect oxidized DNA guanosine bases. Fig. 21C: morphology assessment of sperm. Sperm smears were fixed in methanol and then stained using Rapid Diff Kit (Pathtech). Sperm were categorized as either having normal or abnormal morphology.
[00138] Figs. 22A-D show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Fig. LK percent motile sperm. Figs. 22B-D mean motility score. [00139] Figs. 23A-D show the effect of FSH2Md and LH2Aur on epididymal sperm in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Flow cytometry was used with probes to assess mitochondrial ROS (MSR; Fig. 23 A) and cytoplasmic superoxide (DHE; FIG. 23B). Fig. 22C shows caudal sperm vitality as assessed by the eosin exclusion method. Fig. 23D shows percent DNA fragmentation index (DFI) as a measure of susceptibility to acid-mediated DNA fragmentation assessed by the sperm chromatin structure assay (SCSA).
[00140] Figs. 24A-B show the effect of FSH2Md and LH2Aur on fertility in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Five males in each treatment group were mated, with two control females each, six weeks post-injection for three weeks. At 13 days post-coitus, females were euthanized then embryos and resorption moles were counted. Fig. 24A shows the number of embryos per pregnant female. Fig. 24B shows the average number of resorption moles per pregnant female and the proportion of pregnant females that had at least 1 resorption mole.
[00141] Fig. 25A-B show the effect of FSH2Md and LH2Aur on fertility in vivo. Male adult mice were treated with IP injections of (1) 300 pL/30 g of 14.5 mM FSH2Md; (2) 100 pL/30 g of 420 pM LH2Aur; (3) a combination of both, 16 hours apart; or (4) 300 pL/30 g of vehicle (30% Kolliphor/PBS) with 10 mice in each treatment group. Haematoxylin- and eosin-stained sections illustrate normal development of the seminiferous tubules in a vehicle control testis section (Fig. 25A) and abnormal development of the seminiferous epithelia in a testis section from a FSH2Md and LH2Aur-treated male (Fig. 25B). Scale bar = 100 pm.
[00142] Fig. 26 shows an exemplary anti-Mullerian hormone peptide (AMHa) mapped to the surface of the murine AMH protein (residues 93-102). The synthetic AMH peptide is indicated by a circle and an arrow.
[00143] Figs. 27A-B show expression of AMH receptor type II (AMHr) mRNA. Fig. 28A shows relative expression of MLTC-1 mRNA by qPCR. Fig. 28B shows immunocytochemistry using an anti-AMHr antibody with an Alexa Fluor 488 secondary antibody, indicating that the AMH receptor is expressed at high levels in MLTC-1 cells (top panels) but not in mEcapl8 cells (bottom panels). Inset: secondary antibody only. Scale bar = 10 pm.
[00144] Fig. 28 shows in vitro binding of AMHa-FITC to MLTC-1 cells. A shift in FITC median fluorescence intensity of the MLTC-1 cells is observed in response to increasing concentrations of AMHa-FITC after 1 hour. AMHa-FITC exhibited specificity of binding to MLTC-1 cells (n=9) when compared to binding to mEcapl9 cells (n=9) at the same concentrations but displayed more favorable specificity at concentrations lower than 50 pM.
[00145] Figs. 29A-I show representative images of AMHa-FITC localization following IP injection of male adult mice with 300 pl of either ImM peptide-FITC in 30% Kolliphor/PBS or 30% Kolliphor/PBS with 3 mice in each group. Representative images from 8 hours post-injection are shown. Fig. 29A: Testis. Fig. 29B: Adrenal. Fig. 29C: Brain. Fig. 29D: Pituitary. Fig. 29E: Heart. Fig. 29F: Lung. Fig. 29G: Kidney. Fig. 29H: Liver. Fig. 291: Spleen.
[00146] Fig. 29J shows ImageJ analysis of mean fluorescence intensity /pixel in testes of AMHa-FITC injected males. Mean fluorescence intensity/pixel was normalized to that of the vehicle control within the same image.
[00147] Figs. 30A-E show analysis of caudal sperm of male mice 10 weeks postinjection with AMH2Md. Fig. 30A shows percent live positive for mitochondrial ROS. Fig. 30B shows percent DNA fragmentation index (DFI) as a measure of susceptibility to acid-mediated DNA fragmentation assessed by SCSA. Fig. 30C shows percent sperm vitality. Fig. 30D shows percent sperm motility. Fig. 30E shows percent oxidative DNA damage as determined by immunocytochemistry using anti-8-OHdG to detect DNA oxidation.
[00148] Figs. 31A-B show histology of the testes of male mice 10 weeks postinjection with AMH2Md or vehicle. Fig. 31A shows testes section of AMH2Md-treated males. Fig. 3 IB shows testes sections of control males. Top rows: 20x magnification; scale bar = 100 pm. Bottom rows: 40x magnification; scale bar = 50 pm.
[00149] Figs. 32A-D show in vitro binding of peptide-FITC. Fig. 32A shows LH- FITC on a Leydig cell line; Fig. 32 shows FSH-FITC on a Sertoli cell line. Cells were cultured in growth specific medium and then, when confluent, incubated for 1 hour at 37°C with 50 pM peptide-FITC, washed, fixed and counterstained with DAPI nuclear stain before imaging at 40x magnification. Figs. 32C-D shows binding of LH-FITC (Fig. 32C) and FSH-FITC (Fig. 32D) to Leydig and Sertoli cell lines. Cultured cells were treated with a range of peptide-FITC concentrations, from 0 to 50 pM, for one hour at 37°C, washed, then stained with Far red Live/Dead before being assessed by FACS (FACSCanto). Data is presented as the % of live cells that were positive for FITC. Error bars =standard error.
[00150] Fig. 33A-D shows in vitro binding and specificity of peptide-targeted nanoparticles (NPs) encapsulating FITC-dextran. Cultured cells were incubated with 2 mg/ml of NP- untargeted NP, nonspecific peptide (nsp)NP, or targeted NP - for 24 hours. Cells were then washed, fixed and counterstained with DAPI before imaging at 40x. Fig. 33A shows LH-NP- FITC; Fig. 33B shows FSH-NP-FITC; Fig. 33C shows a Leydig cell line and Fig. 33D shows a Sertoli cell line incubated with NPs, as above, for each time point at least 100 cells in each of 3 wells, per treatment per time point, were counted. This procedure was repeated 3 times (n=9) for 4 and 16 hours (n=9), once for 24 hours (n=3). Data represents foldchange in binding relative to binding of untargeted NPs at 4 hours. Error bars =standard error.
[00151] Fig. 34 shows transfection efficiency of TM4 Sertoli cells over time. The cells were treated with Lipofectamine + pDNA (first row), empty FSH-targeted lipid NPs (second row), FSH peptide -lacking lipid NPs + pDNA (third row), and FSH peptide -conjugated lipid NPs + pDNA (fourth row).
DESCRIPTION OF THE SEQUENCES
[00152] Table 1 provides a listing of exemplary sequences referenced herein.
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
DESCRIPTION OF CERTAIN EMBODIMENTS
[00153] The present disclosure further relates to nanoparticle constructs (e.g., targeting peptides conjugated to the lipids on the surface of the nanoparticle and a nucleotide sequence encapsulated in the nanoparticle) and compositions thereof. The present disclosure further relates to a method of sterilizing a male or female cat or dog comprising administering to the male or female cat or dog an effective amount of the nanoparticle constructs or compositions thereof, where the nanoparticle constructs comprise one or more peptides that each bind to one or more receptors of one or more reproductive cells; and an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein.
A. Reproductive Organs and Cells
[00154] The male gonads (i.e., testes) perform two distinct functions, that is, the production of spermatozoa within the seminiferous tubules and the synthesis of steroids by Leydig cells (Ye et al., 2017), which reside within the interstitial space outside the seminiferous tubules (Evans & Ganjam, 2011). In adults, testicular function is regulated via the hypothalamic- pituitary-gonadal (HPG) axis and secretion of the glycoprotein hormones (GPH), that is, Follicle-Stimulating Hormone (FSH) and Luteinizing hormone (LH), by gonadotrophs in the anterior pituitary (Abel et al., 2008; Cheng & Mruk, 2010). FSH targets the Sertoli cells expressing the FSH receptor, and LH targets the Leydig cells, which express the LH receptor. [00155] Sertoli and Leydig cells are functionally interdependent and essential for normal testicular function. If Sertoli cells are selectively ablated in the mature animal, then germ cell development is bought to an abrupt halt (Rebourcet et al., 2017). Sertoli cell function is regulated by androgens as well as by FSH (Abel et al., 2008). While androgen receptors are ubiquitously expressed, the FSH receptor is specific to Sertoli cells and the LH receptor is specific to Leydig cells.
[00156] GPHs are generally composed of an a- and a [3-subunit; the former is shared by several related hormones, including FSH, LH, and Thyroid-Stimulating Hormone (TSH), while the latter confers specificity on the binding of a particular GPH to its cognate receptor. [00157] Anti-Mullerian hormone (AMH), also known as Mullerian Inhibiting Substance, has a sexually dimorphic expression in Sertoli cells of the testis (Alves et al., 2013) and granulosa cells of the ovary (Josso et al., 2001) and, in the embryo is critical for normal differentiation of the internal reproductive tract structures. The anti-Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors. Although AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood.
Nevertheless, AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015). AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Cimino et al., 2016).
[00158] AMH belongs to the TGF-J3 superfamily of glycoproteins, which consists of a large number of structurally related polypeptide growth factors involved in the regulation of growth and differentiation (Xu et al., 2019), including the activins and bone morphogenetic proteins (BMPs). The TGF-J3 superfamily bind to paired receptors - a type I receptor that is shared by different members of the family and a type II receptor that endows specificity. AMH binds to BMP receptors la and lb, which it shares with the BMPs and the growth/differentiation factors (GDF), however, AMH also has its own unique receptor type II (Hinck, 2012). There is a paucity of data regarding the AMH amino acid residues that are responsible for binding to the AMH receptor II. However, Greenwald and coworkers (2003) (Greenwald et al., 2003) have published details of receptor binding within the TGF-J3 superfamily, with particular reference to BMP7. Therefore, to design a suitable in AMH targeting peptide, we referred to the published crystal structure of BMP7 with its cognate receptor and specifically the amino acid residues responsible for receptor II binding (Greenwald et al., 2003).
[00159] Anti-Mullerian hormone (AMH), also known as Mullerian Inhibiting Substance, has a sexually dimorphic expression in Sertoli cells of the testis (Alves et al., 2013) and granulosa cells of the ovary (Josso et al., 2001) and, in the embryo is critical for normal differentiation of the internal reproductive tract structures. The anti-Mullerian hormone receptor has a different and more complex structure compared to the GPH receptors. Although AMH has a number of extra-Mullerian functions in the development of the gonads, including control of germ cell maturation, gonadal morphogenesis, and induction of the abdominal phase of testicular descent, the function of AMH in the adult testis is not as well understood. Nevertheless, AMH is expressed by the mature Sertoli cells (Urrutia et al., 2019) and the AMH receptor II is expressed abundantly in the testes (Imhoff et al., 2013), particularly by adult Leydig cells (Racine et al., 1998; Ye et al., 2017), postnatal Sertoli cells (Barbotin et al., 2019), and spermatocytes (Ohyama et al., 2015). AMH receptor II may also be expressed by the GnRH secreting neurons in the brain, albeit at significantly lower levels than that of the testes (Barbotin et al., 2019; Ohyama et al., 2015).
[00160] AMH belongs to the TGF-J3 superfamily of glycoproteins, which consists of many structurally related polypeptide growth factors involved in the regulation of growth and differentiation (Xu et al., 2019), including the activins and bone morphogenetic proteins (BMPs). The TGF-J3 superfamily bind to paired receptors - a type I receptor that is shared by different members of the family and a type II receptor that endows specificity. AMH binds to BMP receptors la and lb, which it shares with the BMPs and the growth/differentiation factors (GDF), however, AMH also has its own unique receptor type II (Hinck, 2012).
B. Exemplary Apoptotic Proteins
[00161] In certain aspects, an apoptotic protein is targeted to reproductive cells or reproductive organs to cause cell death that may lead to sterilization. In certain aspects, the apoptotic protein is Diphtheria toxin fragment A (DTA). DTA has been used in unrelated indications such as glioblastoma multiforme and prostate cancer. Diphtheria toxin (“DTX”) is a single protein composed of two components; fragment A and fragment B. Fragment B is the component responsible for binding to the HBEGF receptor (the heparin-binding EGF-like growth factor receptor), which leads to its entry into the cell. Fragment B binds to the HBEGF receptor present on the surface of many cell-types, leading to internalization and delivery of fragment A into the cells. Once inside the host cell, fragment A is able to block protein synthesis of the host cell. This induces death of the specific host cell. Without fragment B, fragment A cannot enter any other non-host cell, and is thus inactive or inert without it.
[00162] Accordingly, one benefit of using diphtheria toxin for controlled cell ablation of a target cell is that it has not been shown to have a toxic effect on other cell types or systems in vivo (Rebourcet et al., 2017). Diphtheria toxin was previously shown to be a means of selectively ablating Sertoli cells from the testis of adult transgenic mice carrying the diphtheria toxin receptor on the surface of these cells. Use of Diphtheria toxin was shown to be an acute means to specifically induce ablation of Sertoli cells with cell death occurring within 24 hours (Rebourcet et al., 2017). Following induction of Sertoli cell apoptosis by injection of 100 ng DTX (diphtheria toxin), for example, specific and complete ablation of Sertoli cells was observed, which was unchanged 1-day post ablation (Rebourcet et al., 2014). Diphtheria toxin was previously shown to have dose-dependent effects in Sertoli cells in mouse models. As a further example, the Rebourcet 2017 article cited above, which is incorporated by reference herein in its entirety, shows that treatment of neonatal mice with Ing DTX (diphtheria toxin), for example, caused a variable -50% reduction in Sertoli cell numbers and lOng caused complete Sertoli cell ablation. The same article reports treatment of adult mice with lOng and 25ng of DTX caused a clear and variable (mean -50%) reduction in cell numbers and 5 Ong DTX caused complete Sertoli cell ablation. The article further reports that there was no recovery in Sertoli cell number for up to 90 days after partial ablation of Sertoli cells in adult animals, suggesting the effects of DTX may be permanent (Rebourcet et al., 2017).
[00163] As used herein, DTA comprises the amino acid sequence of SEQ ID No. 10 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID No: 10. In general, the variations can comprise modifications, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a “polypeptide” or “peptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental, such as through mutations of hosts which produce the proteins or errors.
[00164] In certain aspects, the nanoparticle construct further comprises a viral vector (i.e., a recombinant viral-based DNA construct). In certain aspects, the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector. In certain aspects, the nanoparticles comprise a lentiviral vector. In certain aspects, the lentiviral constructs are self-inactivating and/or cannot be re-activated by serendipitous viral infections to cause insertional mutagenesis and cannot activate surrounding genes due to the mutations in the 3’ viral long terminal repeat. In certain aspects, the tumorigenic WPRE common to many lentiviral vectors have been replaced with a non-tumorigenic OPRE. This can further negate the already minimal risk of tumor development in treated animals. In certain aspects other viral vectors known in the art (defined elsewhere in this application) can be used. In certain aspects, the nanoparticles comprise a lentiviral vector containing the coding sequence for DTA. [00165] In certain aspects, the nanoparticle construct further comprises a transgene coding a reporter protein (e.g., a fluorescent reporter protein) or a fluorescent label. This labeling is useful, for example, to determine body distribution and cellular uptake (in vitro or in vivo). For example, a fluorescent reporter transgene delivered by these nanoparticles allows for analysis of tissues collected post-mortem using fluorescent imaging equipment and detection of the fluorescent protein within fixed tissues. In certain aspects, the fluorescent protein is red fluorescent protein (RFP), green fluorescent protein (GFP), or enhanced green fluorescent protein (EGFP). In certain aspects, the fluorescent label is fluorescent isothiocyanate (FITC)- dextran. FITC-dextran loaded NPs can be used to optimize targeting ligand density and confirm the targeting specificity and uptake efficacy of NP preparations.
[00166] In certain aspects, the polynucleotide is operatively linked to a promoter (i.e., a specific component controlling delivered gene expression carried within the nanoparticle). In certain aspects, the promoter can be cell-specific or species-specific (e.g., specific for cats or dogs, or specific for a particular breed of cat or dog). Use of the promoter can provide an additional aspect of cell specificity, to enable the delivered gene to be ‘switched on’ when delivered to the cell types of interest. In certain aspects, the promoter is cell-specific. In certain aspects, the cell-specific promoters are selected from ABP, Rhox5, or HSD17B3.
C. Exemplary Peptides
[00167] In general, a peptide used to target reproductive cells or reproductive organs can be from any vertebrate source (e.g., any species or breed of dogs or cats). In certain aspects of the invention, the peptide targets receptors on reproductive cells selected from primordial germ cells or gonocytes, and primordial follicle cells.
[00168] In certain aspects, the peptides target FSH and/or LH receptors on Sertoli and/or Leydig cells. In certain aspects, the peptide is directed to both Sertoli and Leydig cells. It is known in the art that the number of Leydig cell numbers is correlated with Sertoli cell number in adults (mature subjects). It was previously shown in cell ablation studies that ablation (i.e., selective reduction or removal) of Sertoli cells also results in reduction of Leydig cell numbers by 75% (Rebourcet et al., 2014, 2017). A reduction of Leydig cells have been seen when Sertoli cell population is decreased by about 50% or more (Rebourcet et al., 2017). Ablating Leydig cells along with Sertoli cell ablation, may ablate testosterone production to a degree to confer infertility. Ablating Sertoli cells alone will also impact Leydig cell function. In other aspects, the peptide is directed to Sertoli cells alone. [00169] In certain aspects of the invention, the peptide targets the AMH receptor on the primordial follicle.
[00170] Exemplary peptides and modified peptides are presented in Table 1.
[00171] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of TVX9GLGPX10Y (SEQ ID No. 95), wherein X9 is R or Q and X10 is S or G.
[00172] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CTVX9GLGPX10Y (SEQ ID No. 96), wherein X9 is R or Q and X10 is S or G.
[00173] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4Q (SEQ ID No. 33), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[00174] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of RDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 34), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[00175] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4Q (SEQ ID No. 35), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T orN.
[00176] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CRDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 36), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; and X4 is T or N.
[00177] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of YTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 49), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V.
[00178] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CYTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 50), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V. [00179] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CHX7GX8X7DSDSTDX7T (SEQ ID No. 63), wherein X7 is C, S, or A and X8 is K, R, or G. [00180] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of X8CDSDSTDX7TVX9GL (SEQ ID No. 78), wherein Xs is K, R, or G; X7 is C, S, or A; and X9 is R or Q.
[00181] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CDSDSTDX7TVX9GL (SEQ ID No. 79), wherein X8 is K, R, or G; X7 is C, S, or A; and X9 is R or Q.
[00182] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of SDSTSX7TVX9GLGPX10Y (SEQ ID No. 89), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G.
[00183] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence of CSDSTSX7TVX9GLGPX10Y (SEQ ID No. 90), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G.
[00184] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
[00185] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
[00186] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises the amino acid sequence of SEQ ID No. 1 and/or SEQ ID No. 2.
[00187] In certain aspects, an FSH peptide comprises a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises the amino acid sequence of SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
[00188] In certain aspects, the isolated FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and/or SEQ ID No. 18.
[00189] In certain aspects, the isolated FSH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
[00190] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of SX18SDX7GGPX8X19X20X21 (SEQ ID No. 144), wherein Xis is T, S, orN; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
[00191] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CSX18SDX7GGPX8X19X20X21, (SEQ ID No. 145), wherein Xis is T, S, or N; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S.
[00192] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of X11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 112), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
[00193] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CX11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 113), wherein Xu is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V.
[00194] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of SPPVALX7RX7GPX7RX17 (SEQ ID No. 125), wherein X7 is C, S, or A and X17 is L or R. [00195] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CSPPVALX7RX7GPX7RX17 (SEQ ID No. 126), wherein X7 is C, S, or A and X17 is L or R.
[00196] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence of CRX17SX18SDX7G (SEQ ID No. 133), wherein X17 is L or R; Xis is T, S, orN; and X7 is C, S, or A.
[00197] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
[00198] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
[00199] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises the amino acid sequence of SEQ ID No. 3 and/or SEQ ID No. 4.
[00200] In certain aspects, an LH peptide comprises a fragment of an LH protein or variant thereof, wherein the LH peptide comprises the amino acid sequence of SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
[00201] In certain aspects, the isolated FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, and/or SEQ ID No. 104.
[00202] In certain aspects, the isolated LH peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
[00203] In certain aspects, an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
[00204] In certain aspects, an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
[00205] In certain aspects, the isolated AMH peptide has one substitution modification, two substitution modifications, three substitution modifications, or four substitution modifications relative to SEQ ID No. 149, SEQ ID No. 153, SEQ ID No. 157, and/or SEQ ID No. 161.
[00206] In certain aspects, an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the AMH peptide comprises the amino acid sequence of SEQ ID No. 5 and/or SEQ ID No. 165.
[00207] In certain aspects, an AMH peptide comprises a fragment of an AMH protein or variant thereof, wherein the AMH peptide comprises the amino acid sequence of SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
[00208] In certain aspects, the isolated AMH peptide is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
1. Exemplary Modified Peptides
[00209] In certain aspects, the peptide sequences are modified. In certain aspects, the peptide sequences may be modified to include a terminal cysteine or terminal methionine, which may be used to attach the peptide to the nanoparticle, a protein, a peptide, a small molecule, or other moiety. In certain aspects, the terminal cysteine or terminal methionine is at the N- terminus. In certain aspects, the terminal cysteine or terminal methionine is at the C-terminus. [00210] In certain aspects, the peptide is amidated. In certain aspects, the N-terminus is amidated.
[00211] In certain aspects, the peptide comprises a linker. In certain aspects, the linker is a maleimide group, 1,6-aminohexanoic acid (Ahx), a y-aminobutanoate -mercaptopropionic acid (y-aminobutanoate-MPA or y-aminobutanoic acid with MPA) linker, alanine with 3- mercaptopropionic acid (MPA), mini-polyethyleneglycol with MPA, or a cathepsin cleavable valine-citrulline(vc)-linker, such as a vc-PABC linker.
[00212] In certain aspects, the peptide is conjugated to a protein, a peptide, a nanoparticle, a label, a small molecule, and/or other moiety. [00213] In certain aspects, the conjugated protein is an apoptotic protein or a cytotoxic protein.
[00214] In certain aspects, the conjugated small molecule is a cytotoxic agent, an antimimotic agent, or an antineoplastic agent. In certain aspects, the conjugated small molecule is an auristatin molecule, such monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
[00215] In certain aspects, the conjugated small molecule is a redox cycling compound. In certain aspects, the conjugated small molecule is 2-methyl-l,4-naphthoquinone (i.e., menadione (Md)).
[00216] In certain aspects, the conjugated small molecule is a fluorescent label, such as fluorescein isothiocyanate (FITC).
[00217] In certain aspects, the peptide sequences may be modified, for example to purify protein from cell material for use in in vitro experiments.
[00218] In certain aspects, a modified peptide comprises a spacer, such as 1,6- aminohexanoic acid (Ahx). In certain aspects, the spacer is at the N-terminus of the peptide. In certain aspects, the spacer is at the C-terminus of the peptide.
D. Exemplary Nanoparticles
[00219] In general, the compositions comprise a nanoparticle. In certain aspects, the nanoparticle comprises peptides on its surface that target a receptor on a reproductive cell. Nanoparticles can be lipid-based, polymer-based, or be a polymer/lipid hybrid. Nanoparticles provide many unique advantages over conventional delivery systems. For example, nanoparticles can encapsulate various therapeutics, such as nucleic acids, proteins and small compounds, and enhance both drug stability and bioavailability thus leading to substantial reduction in dosage as well as dose frequency. In certain aspects, the nanoparticles encapsulate an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein. Targeting ligands, particularly peptides, can also be incorporated onto the nanoparticle surface in order to enable drug delivery to specific cell populations, achieving improvements in efficacy with a corresponding reduction in unwanted effects. Targeting ligands can be introduced on the surface of the nanoparticles with varied densities, which can affect uptake efficiency of the nanoparticles. As non-limiting examples, the nanoparticles can be polymer-based, lipid-based, a polymer/lipid hybrid, or can comprise biocompatible inorganic nanovectors based on layered- double hydroxide strategy. Nanoparticle constructs (e.g., nanoparticle itself together with the targeting peptides conjugated to the lipids of the nanoparticle and nucleotide sequence encapsulated in the nanoparticle) created from biocompatible materials approved for human use have been widely reported in the literature for targeting human diseases. The activity of a nanoparticle construct can depend on, for example, the materials used in the nanoparticle, the type of peptides, peptide density, as well as length of spacer-peptide ligands as well as the testing of cell-specific promoters.
[00220] Nanoparticles may be prepared using a variety of methods. In certain aspects, a nanoparticle is prepared with methods comprising single or double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation. Methods for making nanoparticles are available to one skilled in the art, such as those described in “Microcapsules and Nanoparticles in Medicine and Pharmacy,” which is incorporated in its entirety herein by reference {Microcapsules and Nanoparticles in Medicine and Pharmacy, n.d.). Related methods are also described in and are incorporated in their entireties by reference (Mathiowitz et al., 1987; Mathiowitz & Langer, 1987; Polyanhydride Microspheres as Drug Carriers. II. Microencapsulation by Solvent Removal - Mathiowitz - 1988 - Journal of Applied Polymer Science - Wiley Online Library, n.d.).
[00221] As used herein, “peptide density” or “ligand density” of targeted nanoparticles refers to the density of targeting peptide/ligand on the nanoparticle surface. Ligand density relates to the ability of the nanoparticles to navigate across biological barriers and promote cell-specific uptake. Generally, there is an optimal ligand density for a nanoparticle construct and targeted receptor that can be determined by a person of ordinary skill in the art. The ligand density of targeted nanoparticles may be indicative of the uptake efficiency of the nanoparticles and may reflect targeted delivery.
[00222] In certain aspects, the mean peptide density is from 5,000 to 50,000 peptides per nanoparticle.
[00223] As used herein, “N/P ratio” refers to the ratio of positively charged amine (N) groups of the polymer to negatively charged phosphate (P) groups of DNA, which is one characteristic of nanoparticles in affecting gene transfection efficiency and cytotoxicity.
[00224] Various other properties characterized, such as surface chemistry, drug loading, stability in blood serum, and release profile of payloads can be indicative of cell expression or viability. Modem spectroscopic (NMR, MS, UV-Vis, HPLC, IR) and other known techniques in the art (dynamic light scattering, size exclusion chromatography, gel electrophoresis, electron microscopy, X-ray photoelectron spectroscopy) can also be used to characterize a full range of nanopharmaceutical properties including surface chemistry, drug loading, stability in blood serum, and release profde of payloads in biological media etc.
1. Exemplary Polymer-based Nanoparticles
[00225] In certain aspects, the nanoparticle is polymer-based. In certain aspects, the nanoparticle comprises components selected from poly(hydroxy acids), polyglycolic acid (PGA), polylactic acid (PLA), poly(D,L-lactic acid), poly(lactide-co-glycolide) (“PLGA”), polyamides, polycarbonates, polyalkylenes, polyethylene, polypropylene, polyethylene glycol), polyethylene oxide), poly(Ethylene terephthalate), polyvinyl compounds, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses, polysaccharides, peptides, proteins, copolymers, PEG+linear polymers, dendritic polymers, or copolymers or blends thereof. In certain aspects, the nanoparticle comprises PLGA. PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions known by a person of ordinary skill in the art to effect drug release. The degradation time of PLGA nanoparticles can be altered from days to years to achieve a safe and sustained action by varying the polymer molecular weight, the composition of the block copolymer and the structure of the nanoparticles. In certain aspects, the nanoparticle comprises PLGA and 1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-poly(ethylene glycol) (“DSPE-PEG”). In certain aspects, the nanoparticles further comprise cationic lipids, ionizable lipids, and other helper lipids. Nonlimiting examples of helper lipids include DC-Cholesterol, dilinoleylmethyl-4- dimethylaminobutyrate, DLin-KC2-DMA, DLin-MC3-DMA, and Lipofectamine 3000.
[00226] Polymeric nanoparticles may be prepared using a variety of methods. In certain aspects, a nanoparticle is prepared with methods comprising single or double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation. Methods for making nanoparticles are available to one skilled in the art, such as those described in “Microcapsules and Nanoparticles in Medicine and Pharmacy,” which is incorporated in its entirety herein by reference (Microcapsules and Nanoparticles in Medicine and Pharmacy, n.d.). Related methods are also described in and are incorporated in their entireties by reference (Mathiowitz et al., 1987; Mathiowitz & Langer, 1987; Polyanhydride Microspheres as Drug Carriers. II. Microencapsulation by Solvent Removal - Mathiowitz - 1988 - Journal of Applied Polymer Science - Wiley Online Library, n.d.).
2. Exemplary Lipid-based Nanoparticles
[00227] In certain aspects, the nanoparticles comprise liposomes. Liposomes using different lipid components may be produced according to methods available to one skilled in the art (Kim et al., 1983; Lee et al., 1992; D. Liu et al., 1992; Wang & Huang, 1989). In certain aspects, the lipids of choice are dissolved in an organic solvent and are mixed and dried onto the bottom of a glass tube under vacuum. The lipid fdm is rehydrated using an aqueous buffered solution containing the material to be encapsulated by gentle swirling. The hydrated lipid vesicles or liposomes are washed by centrifugation and can be filtered and stored at 4°C (Thierry & Dritschilo, 1992).
[00228] In certain aspects, liposomes can be made using the method described in “Production of Unilamellar Vesicles Using an Inverted Emulsion” (Pautot et al., 2003). In some of these embodiments, the liposomes comprise streptavidin-coated lipids, e.g., DPPC, DSPC, and similar lipids.
[00229] In certain aspects, the polynucleotide to be delivered by the nanoparticle construct is condensed with protamine, lysine, or polylysine to further enhance transfection efficiency prior to encapsulation by the nanoparticles. As used herein, “transfection efficiency” refers to the percentage of cells successfully transfected by nanoparticle delivery as evidenced, for example, by the polynucleotide payload delivery to the nucleus resulting in transgene expression as related to an entire population. In certain aspects, the polynucleotide is delivered inside a PLGA nanoparticle, wherein the polynucleotide is condensed with protamine, lysine, or poly lysine.
[00230] In certain aspects, the nanoparticle comprises PEG (“polyethylene glycol)”). PEGylation can minimize non-specific particle uptake and clearance by immune cells. In certain aspects, the nanoparticle comprises a maleimide-terminated PEG, e.g., as a surface coating. Such a coating allows for conjugation of the targeting peptide (synthesized with a terminal cysteine) to the PEG via a covalent thiol -maleimide linkage. Each peptide may be engineered to contain a terminal cysteine for coupling purposes and attachment to the nanoparticle surface may be achieved using maleimide-terminated PEG. The peptide may comprise a terminal cysteine to couple with maleimide-terminated PEG via covalent thiol- maleimide linkage. For example, SEQ ID No. 2 comprises the sequence of SEQ ID No. 1 but modified to include a C-terminal cysteine (FSH receptor of Sertoli cells). SEQ ID No. 4 also comprises the sequence of SEQ ID No. 3, but modified to include the C-terminal cysteine (LH receptor of Leydig cells). In certain aspects, the one or more peptides are conjugated to lipids on the surface of the nanoparticle construct. E. Exemplary Methods of Conjugation
[00231] In many embodiments, a first component, i.e., a protein, an apoptotic protein, a peptide (e.g., an FSH, an LH, and/or an AMH peptide), a nanoparticle, a small molecule, or other moiety; may be modified for conjugation to a second component, i.e., a protein, an apoptotic protein, a peptide, a nanoparticle, a small molecule, or other moiety; using a variety of methods. A first or second component may be modified by reacting the peptide with a modifying agent. A “modifying agent,” as used herein, refers to a suitable organic group e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An “activating group,” as used herein, is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols, include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl dilsulfides, 5- thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
1. Exemplary Covalent Linkers
[00232] In certain aspects, a first component is attached to a second component via a covalent linker. In certain aspects, the covalent linker comprises a 1,2, 3 -triazole linker, an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, a urea or thiourea linker, an amidine linker, an amine linker, or a sulfonamide linker.
[00233] In certain aspects, the covalent linker comprises a 1,2, 3 -triazole linker formed by the 1,3-dipolar cycloaddition reaction of azido groups on the surface of a nanoparticle with a peptide containing an alkyne group. In certain aspects, the 1,2, 3 -triazole linker is formed by the 1,3- dipolar cycloaddition reaction of alkynes on the surface of a first component with a second component containing an azido group. Such cycloaddition reactions may be performed with or without a catalyst. In certain aspects, the reaction includes a Cu(I) catalyst, a suitable Cu(I) -ligand, and a reducing agent to reduce Cu(II) compound to catalytic active Cu(I) compound. This Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC) can also be referred to as the click reaction. [00234] In certain aspects, the covalent linker comprises an amide linker formed via an amide bond between an amine on a first component with the carboxylic acid group of a second component. In certain aspects, the amide bond in the linker can be made using any of the conventional amide bond forming reactions with suitably protected amino acids or peptides and activated carboxylic acid such N-hydroxy succinimide-activated ester.
[00235] In certain aspects, the covalent linker comprises a disulfide linker made via the formation of a disulfide (S-S) bond between two sulfur atoms of the form, for instance, of Ri-S-S-R2. A disulfide bond can be formed by thiol exchange of a first component containing thiol/mercaptan group(-SH) with another activated thiol group on a second component containing thiol/mercaptan groups.
[00236] In certain aspects, the covalent linker comprises a thioether linker made by the formation of a sulfur-carbon (thioether) bond in the form, for instance, of R S-R2. Thioether can be made by either alkylation of a thiol/mercaptan (-SH) group on a first component with an alkylating group such as halide or epoxide on a second component. Thioether linkers can also be formed by Michael addition of a thiol/mercaptan group on a first component to an electron-deficient alkene group on a second component containing a maleimide group or vinyl sulfone group as the Michael acceptor. In another way, thioether linkers can be prepared by the radical thiol-ene reaction of a thiol/mercaptan group on a first component with an alkene group a second component.
[00237] In certain aspects, the covalent linker comprises a hydrazone linker made by the reaction of a hydrazide group on a first component with an aldehyde/ketone group on a second component.
[00238] In certain aspects, the covalent linker comprises a hydrazide linker formed by the reaction of a hydrazine group on a first component with a carboxylic acid group on a second component. Such reaction is generally performed using chemistry that is similar to the formation of amide bond where the carboxylic acid is activated with an activating reagent.
[00239] In certain aspects, the covalent linker comprises an imine or oxime linker formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on a first component with an aldehyde or ketone group on a second component.
[00240] In certain aspects, the covalent linker comprises a urea or thiourea linker prepared by the reaction of an amine group on a first component with an isocyanate or thioisocyanate group on a second component. [00241] In certain aspects, the covalent linker comprises an amidine linker prepared by the reaction of an amine group on a first component with an imidoester group on a second component.
[00242] In certain aspects, the covalent linker comprises an amine linker made by the alkylation reaction of an amine group on a peptide with an alkylating group such as halide, epoxide, or sulfonate ester group on a nanoparticle. In certain aspects, the covalent linker comprises an amine linker made by reductive amination of an amine group on a first component with an aldehyde or ketone group on a second component with a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.
[00243] In certain aspects, the covalent linker comprises a sulfonamide linker made by the reaction of an amine group on a first component with a sulfonyl halide (such as sulfonyl chloride) group on a second component.
[00244] In certain aspects, the covalent linker comprises a sulfone linker made by Michael addition of a nucleophile to a vinyl sulfone. In certain aspects, the vinyl sulfone or the nucleophile may be on the surface of or attached to the first or second component.
2. Exemplary Non-covalent Conjugation
[00245] In certain aspects, a first component may be attached to a second component via non-covalent conjugation methods. For example, a negatively charged peptide can be conjugated to a positively charged nanoparticle through electrostatic adsorption. In another example, a peptide containing a metal ligand can also be conjugated to a nanoparticle containing a metal complex via a metal-ligand complex. In certain aspects, a first component is attached to a second component via a (streptjavidin-biotin interaction.
[00246] In certain aspects, a first or second component can be attached to a polymer. In certain aspects, prior to the assembly of a nanoparticle, a peptide can be attached to polylactic acid-block-polyethylene glycol. In certain aspects, a nanoparticle can be formed with reactive or activatable groups on its surface. In the latter case, the peptide is prepared with a group which is compatible with the attachment chemistry that is presented on the surface of the nanoparticle surface.
[00247] In certain aspects, a peptide may be attached to virus-like particles (VLP) or liposomes using a suitable linker. In these embodiments, the linker is a compound or reagent that capable of coupling two molecules together. In certain aspects, the linker can be a homobifimctional or heterobifimctional reagent. For example, an VLP or liposome synthetic nanoparticle containing a carboxylic group on the surface can be treated with a homobifunctional linker, adipic dihydrazide (ADH), in the presence of l-ethyl-3-(3- dimethylamino) propyl carbodiimide, hydrochloride (EDC) to form the corresponding synthetic nanoparticle with the ADH linker. The resulting ADH linked synthetic nanoparticle is then conjugated with an agent containing an acid group via the other end of the ADH linker on the NC to produce the corresponding VLP or liposome peptide conjugate.
[00248] Suitable conjugation methods are known in the art, for example, as described in “Bioconjugate Techniques” (Hermanson, 2013), which is incorporated herein in its entirety by reference.
F. Exemplary Pharmaceutical Compositions
[00249] In certain aspects, the nanoparticle constructs are formulated into compositions comprising a pharmaceutically acceptable carrier. In some aspects, the compositions comprise compendial and widely used excipients. In certain aspects, the composition comprises at least one excipient.
[00250] In certain aspects the compositions comprise a freeze-dried or lyophilized powder, or aqueous solution. In certain aspects, the compositions comprise a freeze-dried powder that can be re-dispersed in aqueous medium prior to administration. This can benefit storage and shelf life.
G. Exemplary Administration of Nanoparticles
[00251] In general, a subject is administered the nanoparticle construct or compositions thereof at an effective dosage. In certain aspects, the subject is administered a composition containing the nanoparticles described above at an effective dosage. In certain aspects, the effective dosage comprises an effective amount of the nanoparticles or compositions thereof sufficient to induce ablation of cells.
[00252] In certain aspects, administration of the effective amount of nanoparticle constructs or compositions thereof described above results in partial or complete cell ablation of the reproductive cells. Even partial ablation of the cells results in significant disruption of the blood testes barrier, which has been shown to lead to infertility in males. In certain aspects, the percentage of cells ablated comprise at least about 50%, at least 60%, at least about 70%, at least about 80%, or at least about 90% of cells. In certain aspects, complete cell ablation occurs. In certain aspects, at least about 60% of cells are ablated.
[00253] In certain aspects, the invention relates to administering to a male cat or dog an apoptotic or cytotoxic protein or transgene encoding an apoptotic or cytotoxic protein. [00254] In certain aspects, administration refers to intravenous, intraperitoneal, or intratesticular administration, although other methods of administration known by persons of ordinary skill in the art in delivering nanoparticle constructs can also be used. In certain aspects, the nanoparticle constructs or compositions thereof are administered by intravenous administration or injection.
[00255] In certain aspects, the nanoparticle constructs or compositions thereof are administered in a single dose or multiple doses. In certain aspects, administration comprises a single dose. In certain aspects, administration comprises a single intravenous injection. In certain aspects, administration comprises a single intravenous injection into the cephalic vein.
[00256] In certain aspects, the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent. Pubertal maturation in males is defined as the presence of motile sperm in the ejaculate (male dogs), or presence of penile spines (male cats). Pubertal maturation in females is defined as the females undergoing at least one normal estrous cycle (female dogs: presence of serosanginuous vulvar discharge, vulvar swelling and increase of the plasma progesterone concentration > 9nmol/L indicating that ovulation has occurred). In certain aspects, the subjects are mature or have reached pubertal maturation.
[00257] In certain aspects, the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent cats or dogs. In certain aspects, the subject is selected from a mature male dog, mature male cat, mature female dog, or mature female cat.
[00258] In certain aspects, induction of cell death occurs within 24 hours, 48 hours, or 72 hours of injection or ablation. In certain aspects, induction of cell death leads to sterilization of the subject. In certain aspects, sterilization of the subject is permanent.
H. Definitions
[00259] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [00260] As used herein, Anti-Mullerian hormone (“AMH”), also known as Mullerian Inhibiting Substance, is a hormone that plays a role in growth differentiation and folliculogenesis. AMH expression inhibits the development of the female reproductive tract in the male embryo and thus is critical to sex differentiation during fetal development.
[00261] As used herein, an “apoptotic protein” is a protein that can cause cell death. In the context of this invention, apoptosis of certain reproductive cells can lead to sterilization.
[00262] As used herein, “gonocytes” relate to precursors of spermatogonia that differentiate from primordial germ cells. As used herein, “nanoparticle” and “nanoparticle construct” refer to materials with overall dimensions in the nanoscale, i.e., under about 100 nm. Nanoparticles can be prepared for a variety of materials, for example, lipids and polymers.
As used herein, “nanoparticle” may be interpreted to also mean “nanoparticle construct” with the proper context set forth above.
[00263] As used herein, the “primordial follicles” are the first class of follicles formed in mammalian ovaries.
[00264] As used herein, “reproductive cell(s)” refer to any cell(s), ablation (i.e., selective reduction or removal) of which could affect or result in sterilization of a subject. Accordingly, “reproductive cells” as used herein include germ cells, as well as cells whose function effects the development or maturation of reproductive systems (“reproductive supportive cells”) during embryogenesis, including but not limited to, for example, Sertoli cells, Leydig cells, cells of the primordial follicle, or granulosa cells. Other such “reproductive cells” or “reproductive supportive cells” would be known to a person of ordinary skill in the art.
[00265] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not.
[00266] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.
[00267] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages, for example, may mean ±5% of the value being referred to. For example, about 100 means from 95 to 105. [00268] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
[00269] The terms “nucleic acid,” “nucleotide,” and “polynucleotide’ are well known in the art. A “nucleic acid” as used herein will generally refer to a molecule (i.e., strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. The term “polynucleotide” refers to at least one molecule of greater than about 100 nucleobases in length. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g. an adenine "A," a guanine "G." a thymine "T" or a cytosine "C") or RNA (e.g. an A, a G. an uracil "U" or a C). The term “oligonucleotide” refers to a molecule of between about 3 and about 100 nucleobases in length. The term “nucleic acid” also refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double -stranded polynucleotides.
[00270] As used herein, the term “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. A “gene” refers to coding sequence of a gene product, as well as non-coding regions of the gene product, including 5’UTR and 3’UTR regions, introns and the promoter of the gene product. These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a doublestranded molecule or a double-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a molecule. The term “gene” refers to the segment of DNA involved in producing a polypeptide chain, it includes regions preceding and following the coding region as well as intervening sequences (introns) between individual coding segments (exons).
[00271] As used herein, the term “binds” (e.g., to a receptor) is a term that is well understood in the art, and methods to determine such binding are also well known in the art. A molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc. [00272] As used herein, the term “vector” includes any genetic element, including, but not limited to, a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, minichromosome, expression vector, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer nucleic acid molecules to cells. Vectors are well known in the art and include, but are not limited to, cloning and expression vectors, as well as viral vectors. As used herein, “viral vector” includes DNA vectors, RNA vectors, and circular or linear vectors and refers to the recombinant viral-based (e.g., recombinant lentiviral -based DNA) construct. The vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper-dependent adenoviral vectors (HD Ad), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, pox virus vectors, and retrovirus vectors. In certain aspects, the nanoparticle constructs and compositions thereof comprise a lentivirus. For a lentivirus, the lentivirus may be integrating or non-integrating.
[00273] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Peptides, oligopeptides, dimers, multimers, and the like, are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include co-translational and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases and prohormone convertases (PCs)), and the like. Furthermore, for purposes of the present invention, a “polypeptide” encompasses a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids. For the purposes of the present invention, the term “peptide” as used herein typically refers to a sequence of amino acids of made up of a single chain of D- or L- amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length.
[00274] As used herein, the term “wild-type” refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof. A wild-type polypeptide may be produced recombinantly.
[00275] As used herein, the term “variant” means a biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide.
[00276] A variant can have, for example, at least 1, 2, 3, 4, or 5 amino acids substituted by a different amino acid. In certain aspects, a variant has at least about 50% sequence identity with the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C- terminus of the polypeptide.
[00277] As used herein, the term “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.
[00278] As used herein, an “amino acid substitution” refers to the replacement of one amino acid in a polypeptide with another amino acid. An amino acid substitution can be a non-conservative substitutions, which will entail exchanging a member of one of these classes with another class. An amino acid substitution can be a conservative substitution.
[00279] Nonlimiting exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.
Figure imgf000065_0001
[00280] Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[00281] To “reduce” or “inhibit” means to decrease, reduce, or arrest an activity, function, parameter, or amount as compared to a reference or control. In certain aspects, “reduce” or “inhibit” refers to the ability to cause an overall decrease of about 20%, about 50%, about 75%, about 85%, about 90%, or about 95%, or greater. In certain aspects, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time. A “reference” or “control” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
[00282] The terms “subject,” “individual,” and “patient” are used interchangeably herein, and refer to the subject to whom treatment with the nanoparticles or compositions thereof according to the present invention, is provided. As used herein, a “subject" means a human or animal. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a cat or a dog. A subject can be male or female. Additionally, a subject can be an adult or can be pre-pubescent.
[00283] As used herein, the terms “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanoparticles or compositions thereof into a subject by a method or route which results in at least partial localization of the nanoparticles at a desired site. The compositions and nanoparticle constructs of the present invention can be administered by the methods described herein and any appropriate route known to one of ordinary skill in the art that results in partial or complete apoptosis of reproductive cells in a subject.
[00284] The term “effective amount” as used herein refers to a sufficient amount of pharmacological composition to provide the desired effect. Thus, it is not possible to specify the exact “effective amount.” However, for any given case as set forth in detail herein, an appropriate “effective amount” can be determined by one of ordinary skill in the art. The efficacy of treatment can be judged by an ordinarily skilled practitioner.
[00285] A “composition” or “pharmaceutical composition” are used interchangeably herein refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells. Exemplary compositions and pharmaceutical compositions are described in detail herein. The cells may be part of a subject, for example for therapeutic, diagnostic, or prophylactic purposes. The cells may also be cultured, for example cells as part of an assay for screening potential pharmaceutical compositions, and the cells may be part of a transgenic animal for research purposes. The composition can also be a cell culture, in which a polypeptide or polynucleotide encoding a metabolic regulator of the present invention is present in the cells and/or in the culture medium.
[00286] “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. The acceptability of treatment can be judged by an ordinarily skilled practitioner.
[00287] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid fdler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. As set forth in detail herein, a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In addition to being “pharmaceutically acceptable” as that term is defined herein, each carrier must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
[00288] Definitions of other common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9). Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:_0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
[00289] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, as defined by the claims. EXAMPLES
Example 1. Materials and methods for Examples 2-4
A. Materials
[00290] Unless otherwise stated, chemicals and reagents were purchased from either Merck Darmstadt, Germany) or Thermo Fisher Scientific (Waltham, Massachusetts, USA). Hydrochloric acid (HC1; 32% w/v AnalaR) was supplied by VWR International (Radnor, PA, USA). Protein Lo-Bind Eppendorf tubes (1.5 mL) were supplied by Eppendorf (AG Hamburg, Germany). Sodium chloride (NaCl), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Astral Scientific, (Sydney, NSW, Australia). Fetal bovine serum was purchased from Interpath (Heidelberg West, Vic, Australia). All peptides were purchased from GenScript (Piscataway, New Jersey, USA). MLTC-1 Leydig cells (CRL-2065), TM3 Leydig cells (CRL-1714), and TM4 Sertoli cells (CRL-1715) were purchased from ATCC (Manassas, Virginia, USA). Mouse epididymal epithelial (mECapI8) cells were obtained as a gift from Dr. Petra Sipila (Institute of Biomedicine, Department of Physiology, University of Turku, Turku, Finland). LSBio rabbit polyclonal anti-FSH receptor antibody (LS-C400751), Bioss rabbit polyclonal anti-LH receptor antibody (bs-6431R), LSBio rabbit polyclonal anti-AMH receptor II antibody (LS-C377389), and donkey serum were purchased from Sapphire Bioscience (Redfern, NSW, Australia). Goat polyclonal anti-8-hydroxydeoxyguanosine (8-OH-dG) was purchased from Millipore (Temecula, CA, USA), Rabbit anti-cleaved Caspase 3 (Asp 175) from Cell Signalling Technologies (Danvers, Massachusetts, USA), and anti-4HNE antiserum from Jomar Diagnostics (Mulgrave, Vic, Australia). Agarose (low melting point) was obtained from Applichem GMBH (Ottoweg 4, 64291 Darmstadt, Germany) and ethanol purchased from POCD Scientific (North Rocks, NSW, Australia). DNA/RNA Damage Antibody (15A3) was obtained from Novus Biologicals (Littleton, Colarado, USA). Maleimide-vcMMAE (Auristatin) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).
B. Synthetic peptides
[00291] In general, cysteine and methionine residues were removed or replaced, unless the cysteine or methionine occurs at the N-terminus or C-terminus of the peptide. In certain aspects, cysteine is replaced by serine, which does not have a thiol group and has a similar structure to cysteine. The 3D structure of the peptide should not be altered by the substitution and the chances of peptide cyclization and peptide oligomerization (which leads to a mixed population of products) should be reduced or eliminated. [00292] The reactive thiol group of a cysteine residue at the N-terminus of a peptide, for example, is useful for attaching the peptide to other moieties or to a nanoparticle, through a linker, such as a maleimide group. See Martinez-Jothar et al., 2018; Varanko et al., 2020. Moreover, removal of internal reactive thiol groups enables a single site is available for linkage, such as a maleimide-thiol mediated coupling of the peptide to the desired pay load, and avoids the potential for mixed end products to be formed. Additionally, in the case of nanoparticles, the peptide may not attach to the surface of the nanoparticle in the correct orientation conducive to peptide-target binding. Furthermore, the C-terminal may be amidated to inhibit protease degradation of the peptide in vivo. See Di, 2015; Werle & Bemkop-Schntirch, 2006.
[00293] In some of the following examples, synthetic peptides linked to maleimide are used. In other of the following examples, synthetic peptides linked to fluorescein isothiocyanate (FITC) are used where the peptide is attached to FITC via an N-terminal spacer, 1,6-aminohexanoic acid (Ahx).
C. Physicochemical properties and structure of synthetic FSH, LH, and AMH peptides
[00294] A comparison was made of the physicochemical properties of FSH, LH, and AMH synthetic peptides using data obtained from PepCalc (PepCalc.com® 2015 Innovagen AB).
[00295] Furthermore, Swiss-Prot Deep Viewer software was used to map each of the peptides onto their reference structures. Synthetic FSH peptides were mapped onto the core structure ofFSHp Swiss-Model Q60687 (FSHB MOUSE) Mus musculus (Mouse) Follitropin subunit beta using Template: 4ay9.1.B "FOLLITROPIN SUBUNIT BETA ” P01225, SMTL Version: 2020-04-01 Seq Identity: 90.09% Seq Similarity: 0.61 (See Figs. 1A-F). Synthetic LH peptides were mapped onto the core structure of LHP Swiss-Model 009108 (LSHB MOUSE) Mus musculus (Mouse) Lutropin subunit beta Template: lhcn.2.B "HUMAN CHORIONIC GONADOTROPIN” P0DN86, SMTL Version: 2019-12-06, Seq Identity: 63.96%, Seq Similarity: 0.52. The synthetic AMHa peptide was mapped onto the structure of BMP7 Swiss- Model P27106 (MIS MOUSE) Mus musculus (Mouse) Muellerian-inhibiting factor, Homology Model: Template:
Figure imgf000069_0001
A A"BONE MORPHOGENETIC PROTEIN 7” P18075, SMTL Version:
2020-04-01, Seq Identity: 26.47%, Seq Similarity: 0.35 (See Figs. 1A-F, 12A-D, and 26). D. Cell culture
[00296] All cell lines were maintained in a humidified atmosphere at 37°C and 5% CO2.
[00297] TM4 Sertoli cells were cultured in growth medium composed of DMEM/F12 supplemented with 5% (v/v) horse serum, 2.5% (v/v) fetal bovine serum, 10 mM HEPES, and 100 U/mL penicillin-streptomycin and subcultured at a ratio of 1:5 into culture flasks.
[00298] MLTC- 1 Leydig cells were cultured in growth medium composed of RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum, and 100 U/mL penicillin-streptomycin. MLTC-1 Leydig cells were subcultured at a ratio of 1:5 into cell culture flasks.
[00299] TM3 Leydig cells were cultured in growth medium composed of F12/Dulbecco’s modified Eagle’s medium, 2.5 mM L-glutamine, 0.5 mM sodium pyruvate, 15 mM HEPES, 2.5% of a mixture of 92.5% horse serum and 5% fetal bovine serum, and 100 U/mL penicillin-streptomycin. TM3 Leydig cells were subcultured at a ratio of 1:5 into cell culture flasks.
[00300] Immortalized mouse proximal caput epididymal epithelial (mE-Capl8) cultures were used as a nonspecific cell control for investigating the specificity of FSH, LH, and AMH peptide interactions (Sipila et al., 2004) and were maintained in growth medium composed of DMEM supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin-streptomycin, 1 mM sodium pyruvate and 50 nM 5-a-androstan-17p-ol-3- one and sub-cultured at a ratio of 1: 10 into culture flasks.
[00301] Cells were grown to 80% confluency before being harvested using 0.5% trypsin-EDTA, quenched, centrifuged at 300x g for 5 min, then washed in warmed growth medium. After centrifugation, the cells were resuspended in warm medium and subjected to incubation with peptide-FITC or peptide-menadione conjugates.
E. Animals and animal care; injections and monitoring
[00302] In these studies, adult (>8 week old) male Swiss (CD1) mice were obtained from a breeding colony at the University of Newcastle Central Animal House and housed under a conventional controlled light and temperature regimen (12-h light: 12-h dark cycle, 21-22°C). All procedures involving mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Additionally, mice were handled, monitored and euthanized with the approval of the University of Newcastle’s Animal Care and Ethics Committee (approval number A-2014-417) in accordance with NSW Animal Research Act 1998, NSW Animal Research Regulation 2010 and the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Ed. Immediately prior to tissue isolation, animals were euthanized by CO2 asphyxiation.
F. RNA extraction, reverse transcription, and qPCR
[00303] Total RNA isolation was performed using two rounds of a modified acid guanidinium thiocyanate-phenol-chloroform protocol (Aitken et al., 1998) followed by isopropanol precipitation. Total RNA was DNase treated prior to reverse transcription to remove genomic DNA. Reverse transcription to cDNA was conducted using M-MLV Reverse Transcriptase (Promega, Ml 701) following the manufacturer’s instructions with subsequent confirmation of transcription using cyclophilin qPCR.
[00304] Taq quantitative PCR (qPCR) was performed using SyBr Green GoTaq qPCR master mix (Promega) according to manufacturer's instructions on a LightCycler 96 SW 1.0 (Roche Diagnostics GmbH, Sandhofer Strasse 116, Mannheim, Germany) thermocycler. Certain qPCR primer sequences (SEQ ID Nos. 173-180) are provided in Table 1. Reactions were performed on cDNA equivalent to 100 ng of total RNA and conducted for 45 amplification cycles. SYBR Green fluorescence was measured after the extension step at the end of each amplification cycle and quantified using LightCycler Analysis Software (Roche). For each sample, replicates omitting the reverse transcription step were undertaken as negative control. Each PCR was performed on at least 3 separate cell or tissue isolations, of which a representative average is shown.
G. Detection of the peptide receptors on cells
[00305] Immunocytochemistry was conducted to confirm expression of the FSH receptor, LH receptor, or AMH receptor in TM4 Sertoli cells, and MLTC-1 Leydig cells. mEcapl8 cells were used as a negative control. Cells were grown to 80% confluence on sterile poly-L-ly sine -coated round coverslips within the wells of 24-well culture plates. Cells were fixed in situ with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). After rinsing with buffer 1 (phosphate buffered saline (PBS) with 3% BSA and 0.015% Triton-X-100), cells were blocked for 1 h at RT with buffer 2 (PBS with 10% goat serum, 3% BSA and 0.015% Triton-X-100) and then incubated overnight at 4°C with anti-FSHr, anti-LHr, or anti-AMHr antibodies (diluted 1/250 in buffer 1). Cells were then re-blocked in buffer 2 for 15 min at RT before incubating for 1 h at RT in 1/200 of goat anti-rabbit AF488 in buffer 2. Finally, cells were washed with buffer 3 (PBS plus 0.015% Triton-X-100) then counterstained using a Far Red nuclear stain (diluted 1/2000 in PBS) for 30 min at RT. After washing in PBS, coverslips were removed from the wells and inverted onto a drop of Mowiol on glass microscope slides then sealed using clear nail varnish before cells were imaged using fluorescence microscopy.
[00306] Quantitative PCR (qPCR), as described above, was also used to assess expression of peptide receptors relative to the house-keeping gene, GAPDH. Subsequently, based on the results obtained in the qPCR, LHr and AMHr protein expression in cultured cells were examined using anti-LHr and anti-AMHr II antibodies, respectively.
H. Assessment of in vitro binding capability and specificity of the synthetic peptides
[00307] TM4 Sertoli cells or MLTC-1 Leydig cells were used to assess the relative binding capabilities and the specificity of targeting of the synthetic FSH, LH, or AMHa peptides. mEcapl8 cells were used as a negative control. All peptides were purchased with an N-terminal spacer, 1,6-aminohexanoic acid (Ahx), attached to fluorescein isothiocyanate (FITC) and dissolved in DMSO to form a 2.5 mM stock solution. Cells were treated for analysis by fluorescence assisted cell sorting (FACS). The median fluorescence intensity was recorded for each treatment group and the shift in median fluorescence intensity was then calculated by subtracting the median fluorescence intensity of the vehicle (0 pM) from the median fluorescence intensity of each dose.
[00308] Cells were grown to 80% confluency in 175 cm2 cell culture flasks before they were harvested using 0.25% trypsin-EDTA, washed, counted and resuspended in growth medium. Cells were then split equally between treatments (in triplicate) of 300,000 cells/200 pL/trcatmcnt in 1.5 mb Protein Lo-bind Eppendorf tubes. Treatments included separate tubes for untreated (non-labeled) and untreated Live/dead stained for each cell type. Cells were treated with FSH-FITC, LH-FITC, or AMHa-FITC in concentrations ranging from 0 to 50 pM, with the 0 pM representing the vehicle (DMSO) control. Treated cells were gently mixed by inversion, and then incubated at 37°C for 1 h. Cells were then centrifuged at 300x g for 5 min, washed with warmed HBSS and resuspended in 180 pL of HBSS. Except for the unstained controls, 20 pL of 1/1000 Live/dead Far Red stain was added to all tubes. Unstained Live/dead tubes were boiled for 3 min and cooled to RT before addition of the Live/Dead stain. Samples were gently mixed and then incubated at 37°C for 20 min before washing and resuspension in HBSS. Cells were then centrifuged, as above, and washed twice with HBSS before being analyzed using FACS. Flow cytometry was performed using a FACSCanto II flow cytometer (BD Biosciences) with a 488-nm argon ion laser. Emission measurements were made using 530/30 band pass (green/FL-1), 585/42 band pass (red/FL-2), and >670 long pass (far red/FL-3) fdters. Forward scatter and side scatter measurements were recorded to generate a scatter plot, which was used to gate for cells only, excluding any contaminating cell debris. All data were acquired and analyzed using BD FACSDiva software (BD Biosciences), with a total of 10,000 events collected per sample.
I. Assessment of in vivo targeting and off-target binding of the synthetic peptides
[00309] All synthetic FSH, LH, and AMHa peptides were assessed in vivo to determine the efficacy of targeting the testes and the extent of off-target binding. A total of 300 pL of 1 mM peptide-FITC or the vehicle (30% Kolliphor/PBS) was injected intraperitoneally (IP) into adult male Swiss CD1 mice, with three males being randomly assigned to each treatment group. Mice were euthanized 8 hours post-injection and tissues (adrenals, brain, epididymis, kidneys, liver, lungs, pituitary, seminal vesicles, spleen, and testes) were collected, weighed and immediately imaged. Tissues were imaged in cold PBS with a Leica MZFLIII microscope with an epifluore scent green filter. All images included the vehicle control to enable comparison of median fluorescence intensity of the treatments relative to the control using ImageJ software [ImageJ version 1.52a; Wayne Rasband, National Institutes of Health, USA. Error! Hyperlink reference not valid.imagei.nih.gov.ii, Java 1.8.0 112 (64-bit)].
J. Conjugation of menadione (Md) to synthetic FSH and AMHa peptides [00310] FSH and AMHa peptides were attached, via the N-terminal thiol, to menadione (Md) as follows. Peptides were weighed and then dissolved 6 mg/mL MilliQ water. The solution was deoxygenated by bubbling through nitrogen gas and then 0.8 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) were added. The mixture was left at RT for 20 min. Meanwhile, 10 molar equivalents of menadione were dissolved in DMSO in a sealed reaction vessel, deoxygenated, and heated in a water bath to 55°C. Whilst stirring with a magnetic bar, and under positive pressure of nitrogen gas, reduced FSH or AMHa peptide was added dropwise to the reaction vessel. After peptide addition to the menadione suspension, stirring of the reaction was continued at 55°C for 2 h. Mass spectrometry was used to confirm the success of the conjugation reaction, using a Bruker Ultraflextreme MALDI-TOF/TOF (mode: reflectron positive; matrix: a-cyano-hydroxy-cinnamic acid). Peptide -menadione conjugates were frozen at -80°C and then freeze dried under vacuum. Once dry, the conjugates were purified using reverse phase HPLC. Samples were eluted from a Waters 250 x 10.0 mm Jupiter 4u Proteo 90A column using a gradient method with solvent A (0. 1 % v/v formic acid in H2O) and Solvent B (90% acetonitrile with 0.1% v/v formic acid) with a flow rate of 2 mL/min over 26 min followed by 2 min at 100% Solvent B before re-equilibration for 7 min. Samples were fdtered through 0.45 pm syringe fdter before injection onto the column. The eluted species were detected by their absorbance at 220 nm. Peak fractions were collected and peptide-quinone identity and purity was verified using MALDI-TOF/TOF mass spectrometry, as above, before the product was freeze dried under vacuum and stored as a powder at -20°C.
K. Conjugation of auristatin (Aur) to synthetic LH2 peptide
[00311] LH2 peptide (see Table 4 in Example 3) with a y-aminobutanoate- mercaptopropionic acid linker and was attached, via the N-terminal thiol, to maleimide- auristatin (Aur) (in the form of maleimide-valine-citrulline- para-aminobenzyloxycarbonyl - monomethyl-auristatin E) as follows. LH2 peptide (MW 1333) was dissolved in 10 mM PBS, pH 7, at a rate of 8.5 mg/250 pL. The peptide was then reduced, using immobilized TCEP disulphide reducing gel, the solution pH adjusted to 7, and then deoxygenated. Meanwhile, 1.2 molar equivalents of maleimide-Aur (MW 1316) were dissolved in 53% dimethyl formamide/10 mM PBS, pH 7, at 10 mg/3 mb and placed in a reaction vessel. The maleimide- Aur solution was then deoxygenated, and the reduced peptide solution was added dropwise, using a syringe, whilst stirring and under positive pressure with nitrogen gas. The reaction was then stirred overnight at RT. The success of the conjugation reaction was confirmed by mass spectrometry, using a Bruker Ultraflextreme MALDI-TOF/TOF (mode: reflectron positive; matrix: a-cyano-hydroxy-cinnamic acid). The solvents were removed from the reaction by freeze drying under vacuum. The dried reaction mix was then reconstituted and purified using reverse phase HPLC. Samples were eluted from the Waters 250 x 10.0 mm Jupiter 4u Proteo 90A column using a gradient method with Solvent A (0. 1% v/v formic acid in H2O) and Solvent B (90% acetonitrile with 0.1% v/v formic acid) with a flow rate of 2 mL/min over 37 min followed by 2 min at 100% Solvent B before re-equilibration for 5 min. The eluted species were detected by their absorbance at 220 nm. Peak fractions were collected and LH2-Aur identity and purity was verified using Orbitrap liquid chromatography /mass spectrometry before the product was freeze-dried under vacuum and stored as a powder at -20°C.
L. Chemiluminometric analysis of peptide-Md conjugates
[00312] Chemiluminometic analysis was used to ascertain whether the Md portion of a peptide-Md conjugate retained its ability to produce hydrogen peroxide. Chemiluminometry was used to measure the production of hydrogen peroxide by a redox cycling agent in the presence of cells. Specifically, after conjugation of Md to the FSH peptides, the FSH-Md conjugates were assessed for retention of redox cycling ability.
[00313] TM4 Sertoli cells were grown to 80% confluency in 175 cm2 cell culture flasks. Cells were harvested and split 500,000 cells/380 pL/treatment (in duplicate or triplicate). Cell free control tubes containing 380 pL of HBSS were also included in each analysis as negative controls. Treatments comprised 50 pM each of the unconjugated peptides and each of the peptides conjugated to Md, as well as Md alone. Luminol-peroxidase dependent assessment of extracellular hydrogen peroxide was recorded on a Berthold 953 luminometer. As soon as possible after addition of the treatment, 8 pL of 2 mg/mL horseradish peroxidase (HRP) in PBS (final peroxidase activity 51.2 U/mL) and 4 pL of Luminol (25 mM in DMSO) were added, the tubes were tapped gently to mix their contents and all tubes were placed immediately in the luminometer where results were recorded for each sample at regular intervals over the duration of the experiment. The area under the curve was then calculated using the Trapezoidal rule to determine the total chemiluminescence produced.
M. Assessment reactive oxygen species (ROS)
[00314] This example describes methods for assessing the impact of reactive oxygen species (ROS) produced by the FSH-Md conjugates on TM4 Sertoli cells. In some of the examples that follow, such as in Example 2F, a y-aminobutanoic acid linker (referred to as linker 2), was used to covalently link FSH to Md. As detailed in Example 2F, this peptide is referred to herein as FSH2Md.
[00315] TM4 Sertoli cells were grown to 8% confluency in 175 cm2 cell culture flasks. Cells were harvested, washed, and resuspended in growth medium and split equally between four treatments (in triplicate): 50 pM of FSH2Md, FSH2 peptide, Md, or the vehicle alone (30% Kolliphor/PBS) and incubated at 37°C for 90 min. After washing in HBSS, treated cells were then either used for immunocytochemistry to detect apoptosis (anti-Caspase 3) and oxidative DNA damage (anti-8-OHdG), or prepared for FACS to assess mitochondrial ROS (MSR) or lipid peroxidation (anti-4HNE). Each experiment was conducted three times in triplicate.
N. Immunocytochemistry
[00316] Cells were fixed in situ with 4% PFA for 10 min at RT. After rinsing with buffer 1 (PBS with 3% BSA and 0.015% Triton-X-100), cells were permeabilized with PBS/0.025% Triton-X-100 at RT. Cells were blocked for 1 h at RT with buffer 2 (PBS with 10% serum (from species in which secondary antibody was raised), 3% BSA and 0.015% Triton-X- 100) and then incubated overnight at 4°C with anti-Caspase 3 (1/400) or anti-8OH-dG (1/200) in buffer 1. Cells were then blocked again in buffer 2 for 15 min at RT before incubating for 1 h at RT in 1/200 of goat anti-rabbit or donkey anti-goat AF488 in buffer 2. Finally, cells were washed with buffer 3 (PBS plus 0.015% Triton-X-100) then counterstained using DAPI (0.5 pg/mL in PBS) for 5 min at RT. After washing in PBS, cells were dropped onto a microscope slide and viewed using fluorescence microscopy. At least 100 cells from each treatment were counted. This experiment was repeated three times in triplicate.
O. Fluorescence-activated Cell Sorting (FACS) for lipid peroxidation
[00317] Cells were analyzed using FACS for lipid peroxidation (anti-4HNE). The presence of 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, was detected using an anti-4HNE antibody. Unstained control cells were resuspended in 50 pL HBSS. Untreated cells, Live/Dead control untreated cells (boiled at 100°C for 35 min) and treated cells were suspended in 49 pL of HBSS. One microliter of anti-4HNE antiserum was added to untreated and treated samples, and 1 pL of Live/Dead stain (1/200 in HBSS) was added to treated, untreated and Live/dead samples, then all cells were incubated for 30 min at 37°C. Cells were centrifuged at 600x g for 5 min and washed twice with HBSS. They were then resuspended in 99 pL of HBSS before 1 pL of secondary antibody (AlexaFluor 488 goat anti-rabbit IgG) was added and incubated at 37°C for 10 min. Cells were centrifuged again, washed twice with HBSS, and then resuspended in 400 pL of HBSS for analysis by flow cytometry as described in preceding sections. Only results for viable cells are reported for the FACS assays.
P. Fluorescence-activated Cell Sorting (FACS) for mitochondrial reactive oxygen species (ROS) generation
[00318] Cells were analyzed using FACS for mitochondrial ROS generation (MitoSOX Red; MSR). Mitochondrial ROS generation was measured using MSR in the presence of LIVE/DEAD™ Fixable Green Dead Cell Stain Kit or SYTOX™ Green Dead Cell Stain to assess cell viability. Briefly, treated TM4 Sertoli cells were resuspended in 200 pL DMEM with additional tubes of untreated cells to create the following controls: untreated cells, unstained cells, MSR untreated, and Live/Dead Green (L/D G) positive (i.e., cells boiled at 100°C for 3 min). A total of 1 pM MSR and 1/10000 L/D G dye was added to untreated and treated cells, and 1/10000 L/D G Dye alone added to L/D G control. All samples were then incubated at 37° C for 20 min. After treatment, the cells were centrifuged at 600x g at RT for 5 min and resuspended in fresh DMEM. The MSR (red) and L/D G (green) fluorescence were then measured on a FACSCanto II flow cytometer (as in preceding sections above). Nonspecific events were gated out, and 10,000 cells were examined. Only results for viable cells are reported for all FACS assays.
Q. Sperm chromatin structure assay (SCSA) and Fluorescence-activated Cell Sorting (FACS) for spermatozoa DNA fragmentation
[00319] Sperm chromatin structure assay (SCSA) and FACS were used to assess spermatozoa susceptibility to DNA fragmentation. The SCSA was performed as previously described (Campbell et al., 1991; Santa Coloma & Reichert, 1990). Briefly, aliquots of spermatozoa were thawed at 37°C and stored on ice, after which 200 pL of acid detergent solution (0.08 N HC1, 0.15 M NaCl, 0.1% Triton X-100, pH of 1.2) was added to 100 pL of sperm suspension. After 30 s, 600 pL of acridine orange staining solution (0.1 M citric acid, 0.2 M NazPOi. 1 mM EDTA, 0.15 M NaCl, 22.6 pM acridine orange, pH 6.0) was added. Samples were immediately separated on a FACS, as above, for 3 min prior to acquiring data. Debris was gated out using a forward scatter/side scatter dot plot with a region drawn around sperm cells. Green fluorescence was detected in FL-1 and red fluorescence was detected in FL- 3. The percentage of cells outside the main population (detectable DNA fragmentation index), the ratio of red fluorescence to total fluorescence (DNA fragmentation index: ratio of single stranded or denatured DNA to total DNA), and the percentage of cells with high green fluorescence (considered to be poorly protaminated) were calculated from the output of CellQuest Pro software, as previously described (Campbell et al., 1991). SCSA is expressed as DNA fragmentation index (DFI).
R. In vivo pilot study on FSH2Menadione (FSH2Md)
[00320] Seven male adult mice were treated with IP injections of 100 pL 14.5 mM FSH2Md (in 30% Kolliphor/PBS) /10 g body weight (with this concentration equating to the solubility of FSH2Md); ostensibly providing a final FSH2Md concentration of 145 pM. Six weeks post injection, 5 of the treated mice and 4 age-matched untreated males were mated with 2 untreated females each for 3 weeks. Females were monitored for mating plugs each day and 12.5 days post-mating, resorption moles and embryos were counted.
[00321] At the end of the mating period, males were sacrificed; testes were collected, weighed and fixed in Bouin’s solution (75 mL picric acid, 40% aqueous solution; 25 mL formalin, 40% aqueous solution; and 5 mL glacial acetic acid). After fixing overnight at 4°C, whole testes were washed with 70% ethanol until the solution was clear and then processed for paraffin embedding, sectioning (5 pm thickness) and haematoxylin and eosin (H and E) staining. An ApopTag Kit was used to detect apoptotic cells in the sections by following the manufacturer’s instructions (Merck).
[00322] Epididymal spermatozoa were also collected by swim up for analysis of motility, vitality, DNA strand breaks (HALO), and oxidative DNA adducts (anti-8-OH-dG). For these assays, a single incision was made in the cauda epididymis with a surgical blade before placing the tissue in prewarmed Biggers-Whitten-Whittingham (BWW) medium (consisting of 95 mM NaCl, 44 mM sodium lactate, 25 mM NaHCOs, 20 mM HEPES, 5.6 mM D-glucose, 4.6 mM KC1, 1.7 mM CaCh, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.27 mM sodium pyruvate, 0.3% (wt/vol) BSA, 5 U/ml penicillin, and 5 mg/ml streptomycin, pH 7.4) at 37°C. Spermatozoa were allowed to swim-out for 10 min before being collected into clean tubes. Sperm motility was assessed using a light microscope at lOOx magnification and sperm vitality was assessed using the eosin exclusion test (WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction \ Obstetrics and Gynecology, Repro, n.d.). At least 100 spermatozoa per sample were analyzed to determine percentage of total motility and vitality of each sample. Sperm motility was assessed using computer-assisted sperm analysis (CASA). The remaining spermatozoa were aliquoted into tubes containing 106 sperm/mL, snap frozen in liquid nitrogen and stored at -80°C until use.
S. In vivo pilot study on FSH2Menadione (FSH2Md) with LH2Auristatin (LH2Aur)
[00323] This example describes methods for assessing the impact of treating mice with an FSH-Md conjugate and a LH-Aur conjugate. In some of the examples that follow, such as Examples 2.E-I, a y-aminobutanoic acid linker (referred to as linker 2), was used to covalently link FSH to Md and LH2 to Aur. These peptides are referred to herein as FSH2Md and LH2Aur.
[00324] Four treatment groups of ten 55-day-old male Swiss male CD 1 mice were treated with IP injections of (1) 300 pL vehicle (30% Solutol (Kolliphor)ZPBS), (2) 300 pL/30 g 14.5 mM FSH2Md, (3) 100 pL/30 g 425 pM LH2Aur, or (4) 300 pL/30 g 14.5 mM FSH2Md plus 100 pL/30 g 425 pM LH2Aur (consecutive injections, 16 h apart). The dose of 1 mg/kg of conjugated Aur was based on previous work finding the maximum tolerated dose (MTD) of conjugated Aur for mice is 1.1 mg/kg (Doronina et al., 2003). Solubility of the FSH2Md was limited to 14.5 mM (25 mg/kg) in the biocompatible reagents (Kolliphor/PBS) delivering a dose much lower than menadione’s MTD of 830 mg/kg. Six weeks post injection, five randomly selected mice from each treatment group were mated with two untreated females (10-12 weeks old) each for three consecutive weeks. Females were monitored for mating plugs each day and on the thirteenth day post-mating plug, or if weight gain indicated approximately 13 days of pregnancy, females were euthanized weighed, embryos and resorption moles were counted, ovaries were collected, weighed, and corpora lutea counted. After the mating period, at ten weeks post-injection, all males were euthanized, weighed and blood collected by cardiac puncture. Blood samples from each animal were decanted into EDTA-coated tubes and plasma was separated (10 min, 400 g). Plasma was stored at -80°C for later measurement of serum FSH levels. An AVIVA FSH ELISA Kit was purchased from Sapphire Bioscience (Redfern, NSW, Australia) and conducted as per manufacturer’s instructions.
[00325] Tissues (seminal vesicles, epididymis, adrenals, kidneys, liver, brain, heart, spleen and testes) were collected and weighed. A sample of each tissue was fixed in Bouin’s solutionfor around 40 min per mg tissue. One testis from each male was fixed and the other snap frozen in liquid nitrogen and stored at -80°C. Fixed samples were processed for H and E staining as described in the preceding section. H and E stained sections were used to evaluate cross-sectional tubule areas as described below. To evaluate the impact on TM4 Sertoli cells numbers, an anti-SOX9 antibody was used to stain sections, while an ApopTag Kit (Merck) was used to detect apoptotic cells in accordance with the manufacturer’s instructions.
[00326] Immediately after dissection, epididymal spermatozoa were also collected for analysis of motility, vitality, DNA strand breaks (HALO), DNA fragmentation (SCSA), the presence of reactive oxygen species (MSR and DHE) and oxidative DNA adducts (anti-8-OH- dG). For these assays, three incisions were made in the cauda epididymis with a surgical blade before placing the tissue in prewarmed Biggers-Whitten- Whittingham (BWW) medium with 5 U/ml penicillin, and 5 mg/mL streptomycin, pH 7.4, at 37°C. Spermatozoa were allowed to swim-out for 10 min before being collected into clean tubes. Sperm motility was assessed using a HTM-IVOS II Computer Assisted Sperm Analysis and sperm vitality was assessed using the eosin exclusion test (WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction \ Obstetrics and Gynecology, Repro, n.d.) and viewed using a light microscope at 40 x magnification. At least 100 spermatozoa per sample were analyzed to determine percentage vitality of each sample. Spermatozoa were stained for FACS analysis of mitochondrial and cytosolic superoxide using MSR and DHE staining (Koppers et al., 2008, 2011). Additionally, sperm smears were made for staining with Rapid Diff (Pathtech, Preston, Vic, Australia), as per manufacturer’s instructions, to evaluate sperm morphology. Sperm were categorized as either having normal or abnormal morphology. Analyses of DNA strand breaks (HALO), and detection of the oxidative DNA adducts (anti-8-OH-dG) were conducted as described in section U below. At least 100 sperm/sample (10,000 sperm/sample for FACS) were assessed for each assay and, where possible, all counts were conducted blind. The remaining spermatozoa were aliquoted into tubes containing 106 sperm/mL, snap frozen in liquid nitrogen and stored at -80°C until use. Thawed sperm cells were later assessed using FACS for DNA fragmentation using a sperm chromatin structure assay (SCSA) as previously described (Evenson & Jost, 2000; Gibb et al., 2014). All FACS data were acquired and analyzed using BD FACSDiva software (BD Biosciences), with a total of 10,000 events collected per sample. Only results for viable cells are reported for all FACS assays.
T. In vivo pilot study on AMH2Menadione (AMH2Md)
[00327] Three male adult mice were twice injected intraperitoneally with 100 pL/10 g 30 mM AMH2Md (in 30% Kolliphor/PBS), with injections spaced 16 h apart. Six weeks post injection, the treated mice along with three aged-matched untreated males were housed with one untreated female each for three weeks. Females were monitored for mating plugs each day and 12.5 days post-mating, resorption moles and embryos were counted. At the end of the mating period, males were sacrificed and testes were collected, weighed, and fixed in Bouin’s solution.
[00328] Samples for fixed and processed for H and E staining as described in preceding sections. After fixing, whole testes were paraffin embedded, sectioned (5-pm thickness) and haematoxylin and eosin stained.
[00329] Epididymal spermatozoa were also collected for analysis of motility and vitality, DNA strand breaks (HALO), and oxidative DNA adducts (anti-8-OH-dG). Spermatozoa were collected as described in the preceding section. epididymiSperm motility and sperm vitality were assessed as described in the preceding section.
U. Sperm chromatin dispersion test (HALO assay)
[00330] The sperm chromatin dispersion test (HALO assay) was used to determine DNA fragmentation in sperm. If the chromatin is intact, it will spring out and form a ‘halo’ around the sperm head, however, if there is DNA fragmentation then the chromatin is unable to spring out and ‘no halo’ effect is observed. Briefly, microscope slides were pre-coated with 0.65% standard agarose. After thawing, 1% low-melting agarose solution was added to the sperm at an agarose to sperm ratio of 7:3. 50-60 pl was then spread evenly across the pre-coated slides and immediately covered with a coverslip (24 x 50 mm). After complete gelling at 4°C for 4 min, the coverslips were removed and the slides were covered with 0.08 N HC1 for 7 min at RT. The HC1 was tapped off and the slide was immersed in solution 1 (0.4 M Tris, 1% (w/v) SDS, 50 mM EDTA, and 800 mM dithiothreitol, pH 7.5) for 10 min at RT, then in solution 2 (2 M NaCl, 0.4 M Tris, 1% SDS, pH 7.5) for 5 min at RT. The slides were then incubated for 2 min at RT in TBE (0.45 M Tris, 0.45 M boric acid, 10 mM EDTA, pH 7.5), then serially with 70%, 90%, and 100% ethanol for 2 min each at RT. Slides were then air dried and stained with DAPI for 10 min at RT. After rinsing in PBS, Mowiol was dropped onto the slides and a coverslip added before sealing with clear nail varnish and viewing using fluorescence microscopy. At least 100 cells on each slide were viewed and classified as either having a halo or no halo.
V. Anti-8-OH-dG fluorescence microscopy
[00331] Sperm DNA oxidation was determined by immunohistochemistry using a DNA/RNA Damage Antibody that detects 8-OHdG (Novus Biologicals # NB110-96878). Snap frozen sperm samples from treated and control mice were thawed at RT and incubated with DNA/RNA Damage Antibody (1/50) at 4°C overnight. Spermatozoa were then washed once in PBS and incubated with Goat Alexa Fluor 488 anti-mouse secondary antibody (1/400 in PBS-T) for 1 h at RT. Cells were washed twice with PBS and a minimum of 100 cells / replicate were assessed for positive 8-OH-dG staining using fluorescence microscopy to determine the overall percentage of spermatozoa harboring oxidized DNA.
W. Microscopy of seminiferous tubules
[00332] To ascertain average seminiferous tubule area, six H and E-stained testes sections were randomly chosen from each treatment group. Sections were imaged using a 10 x objective under bright field microscopy and then images (4080 x 3072 pixels) were imported into ImageJ software [ImageJ version 1.52a; Wayne Rasband, National Institutes of Health, USA. imagej.nih.gov.ij, Java 1.8.0 112 (64-bit)]. At least 50 tubules from each testis section were outlined and the area in pixels/tubule calculated.
X. SOX9 staining
[00333] Testes sections were dewaxed using xylene and then rehydrated in an ethanol gradient. Heat-assisted epitope retrieval was conducted in Tris-EDTA, pH 9. Slides were then incubated in 0.3% hydrogen peroxide in Tris buffered saline (TBS) for 30 min at RT. After washing in TBS (3 x 5 min), the sections were blocked using 5% normal goat serum (NGS) for 30 min at RT in a humidified chamber. The sections were then incubated with rabbit anti-SOX9 antibodies (Merck: AB5535) in NGS (1:500) overnight at 4°C. Slides were washed in TBS (3 x 5 min) prior to incubation with biotinylated goat anti-rabbit antibodies (Abeam, ab6720) in NGS (1:200) for 30 min in a humidified chamber at RT. Slides were washed in TBS (3 x 5 min) then incubated with streptavidin-HRP (Abacus SA-5004) in TBS (1 : 1000) for 30 min at RT before being washed again in TBS (3 x 5 min). Sections were then treated with 3, 3 '-diaminobenzidine (DAB) for up to 5 min or until sufficient color development. Slides were washed with TBS (3 x 5 min) and counterstained with haematoxylin for 8 min before being dehydrated and mounted with Eukitt. Slides were imaged using bright field microscopy and the number of TM4 Sertoli cells counted in all tubules across one section from each slide.
Y. Statistical analysis
[00334] All experiments were conducted at least three times on individual samples in technical triplicate. All quantitative data was expressed as the mean ± standard error from three or more replicate samples. Statistical analysis of data was conducted using Microsoft Excel (vl6.0; Microsoft Corporation, Redmond, WA, USA) as follows: a single factor ANOVA was conducted on the data from all treatments. If the p-value was < 0.05, then pair-wise Student’s T-tests were conducted using two-samples assuming unequal variance if difference between the variances was > 2-fold, otherwise assuming equal variance.
Example 2. Synthetic FSH peptides
A. FSH peptide sequences include residues from the L2 loop or the “seatbelt” receptor binding region of FSH with varying charge and hydrophobicity
[00335] Six new murine FSH peptides that bind to FSHr were designed and are shown in Table 3.
[00336]
Figure imgf000082_0001
Figure imgf000083_0001
[00337] Each of the six FSH peptides were modeled as described in Example 1 above. The peptides were found to map to the surface of the ‘seatbelt’ region and the tip of the PL2 loop of FSH (Figs. 1A-F). Both of these regions are known to contain residues important for FSH receptor binding.
[00338] So as to avoid using high concentrations of organic solvents, such as DMSO that can be cytotoxic, it is important that the peptides have a good aqueous solubility. Additionally, the overall charge on the peptide can influence incidental binding behavior in that positively charged peptides may have a higher attraction to the negatively charged cell surface.
[00339] The physicochemical properties of each peptide was analyzed as described in Example 1 above and the properties are summarized in Table 3, indicating that all are predicted to have good aqueous solubility, the one exception being the FSHa peptide. Despite being predicted to have poor solubility (likely attributable to a high ratio of hydrophobic to hydrophilic residues), the FSHa peptide was nevertheless found to have sufficient solubility. All of the other peptides had a similar ratio of hydrophobic to hydrophillic residues (i.e., 0.6 to 1.1), yet varied widely in terms of their predicted charge at pH 7 (i.e., +2.9 to -1.1) (Table 3).
B. An in vitro model - TM4 Sertoli cells expressing the FSH receptor (FSHr)
[00340] An in vitro TM4 Sertoli model cell line was identified for use in targeting the synthetic FSH peptides to the FSHr. A control cell line that was devoid of FSHr was also identified. The TM4 Sertoli cell line was used, originally purchased from ATCC (derived from 11-14 day old murine testis) and which has been shown in other studies to express the FSH receptor (X. Liu et al., 2014). According to ATCC (atcc.org), the TM4 cell line is reported to respond to FSH with an increase in cAMP production. However, the FSH responsiveness was reportedly much reduced compared to primary Sertoli cell cultures. Nevertheless, immunocytochemistry confirmed abundant FSH receptor protein expression in TM4 Sertoli cell lines but not in the control cell line, comprising a mixed population of epididymal epithelial cells (i.e., mECapl8 cells) (Figs. 2A-B).
C. FSH peptides possess binding capability and specificity in vitro
[00341] Using the FACS method described in Example 1 above, each of the synthetic FSH peptides (shown in Table 3) was found to be capable of binding to TM4 Sertoli cells, with the intensity of labeling increasing in accordance with increasing doses of the applied peptide. This was evidenced by a proportional shift in median fluorescence intensity of the stained TM4 Sertoli cell population across the examined peptide dose-range of 5 to 50 pM (Figs. 3A-F). Additionally, flow cytometry analyses provided evidence that all of the TM4 Sertoli cells exhibited peptide uptake, such that their labeling intensity shifted uniformly as a single population as the peptide dose was increased. Furthermore, each of the peptides showed good specificity with 10-fold higher median fluorescence labeling intensity being detected across the dose range when peptides were applied to TM4 Sertoli cells as compared to the control mECapl8 cells. The peptides with the greatest shift in median fluorescence and, by inference, the greatest binding capability, were identified as F53, F49, and FSHa (Figs. 3G-H).
D. FSH peptides target the testes in vivo and have minimal off-target localization
[00342] Next, localization of the FSH-FITC conjugates in the mouse testes were verified using methods described in Example 1 above. Either a peptide-FITC conjugate or the vehicle was administered via IP injection to each of three adult male mice per treatment. Eight hours after peptide administration to adult males, no significant difference was observed in the weight of mice exposed to either the vehicle or the peptide-FITC treatments. Similarly, no significant difference was observed in the weights or gross morphologies of the representative tissues collected. All six FSH peptides conjugated to FITC showed fluorescence localization within the testes while the vehicle-injected samples showed no fluorescence signal (Fig. 3A-D). This contrasted with findings on the adrenals, brain, lungs, pituitary, or seminal vesicles for which no differences in fluorescence staining were observed between the vehicle control and the treated animal tissues. However, there was evidence of clearance of the peptides through the detoxifying and excretory organs of the liver and kidney, both of which presented with modest fluorescence labeling (Fig. 4). Additionally, there appeared to be modest FITC fluorescence associated with the epididymis in at least a portion of the treated animals (Fig. 4). [00343] Based on these findings, images of the treated testes were imported into ImageJ to compare the mean fluorescence intensity/pixel. The results are expressed as a ratio of the mean fluorescence intensity/pixel of the treated tissue in any image to that of the vehicle control in the same image (Fig. 5D). The testes of FSHa-FITC-treated males exhibited a significantly higher (p<0.005) ratio of mean fluorescence intensity/pixel compared to the testes of animals treated with the alternative array of FSH synthetic peptides. Based on these findings, FSHa was selected as the focus for further studies.
E. Redox cycling of menadione (Md) was facilitated by using y-aminobutanoic acid with a 3-mercaptopropionic acid (MPA) linker
[00344] 2-methyl-l,4-naphthoquinone is a redox cycling compound (also known as menadione, Md, or Vitamin K3) that undergoes one and two electron reduction to produce superoxide radicals. The redox cycling of the naphthoquinone family is facilitated by enzymes, usually flavoenzymes, that use NAD(P)H as an electron source (Gutierrez, 2000a, 2000b). Additionally, the superoxide radicals are catalytically converted by superoxide dismutase (SOD) to hydrogen peroxide (Melov, 2002). The superoxide radicals also have a propensity to release iron from ferritin, while the production of hydrogen peroxide can facilitate the release of iron from heme proteins (Wiseman & Halliwell, 1996). The free iron can then participate in Fenton reactions whereby hydrogen peroxide is converted to hydroxyl and hydroperoxyl radicals that, in turn, act as oxidizing agents. Together with other reactive oxygen species (ROS), the radicals so formed can cause loss of function through oxidative damage to cellular lipids (Halliwell, 1991), proteins, and DNA (Wiseman & Halliwell, 1996).
[00345] Chemiluminometry, as described in Example 1 above, was used to ascertain whether the conjugated form of Md retained the compound’s intrinsic ability to redox cycle upon attachment to FSHa. The results revealed that FSHa-Md retained a very low level of redox activity compared to that of the free native menadione (data not shown). To address the low level of redox activity and improve redox cycling of the attached Md, three different linkers were designed to introduce spacers of varying lengths between the peptide and the Md conjugate (Fig. 6C). The linkers were subsequently attached to the N-terminal of the peptide and thereafter modified through the addition of 3-mercaptopropionic acid (MPA) to maintain a free thiol for attachment of the Md. Specifically, these linkers were: (1) alanine with MPA, (2) y- aminobutanoic acid with MPA, and (3) mini-polyethyleneglycol with MPA. Hereinafter, these linkers are referred to as linkers 1, 2, and 3, respectively. These substitutions were designed to both change the electronic environment of the Md to enhance its potential for reduction (Song & Buettner, 2010) and to position the Md further away from the receptor-bound residues of FSHa and, hence, any factors that might inhibit redox cycling. A further consideration in the design of the linkers was to maintain the aqueous solubility of the FSH peptide-Md conjugate to ensure solvent biocompatibility with end use in the in vitro and in vivo experiments. With higher aqueous solubility, more Md would be expected to be delivered to TM4 Sertoli cells to affect the desired outcome of ROS production and oxidative stress. Hereinafter, the FSHa peptides modified with linkers 1, 2, or 3, are respectively referred to as FSH1, FSH2, or FSH3.
[00346] Notably, the unconjugated FSH peptides displayed only basal levels of redox activity, equivalent to the chemiluminescence generated in the untreated control samples. By contrast, the FSHa peptide, conjugated to Md using linkers 1, 2, or 3 exhibited favorable chemiluminescence compared to the free Md agent over the 90 min time course of this experiment (Fig. 7A). Additionally, FSH2Md exhibited a higher rate of redox cycling activity than the other two peptides conjugates, FSHIMd, and FSH3Md; this hierarchy was maintained throughout the 90 min time course of n=3 experiments (Fig. 7B). Given that FSH2 consistently exhibited higher redox cycling facility, we focused the in vitro and in vivo studies on this peptide/linker system.
F. The conjugation of menadione (Md) to FSH2 was successfully scaled up [00347] The C3 of Md is the most reactive carbon (Fig. 6A) and, therefore, covalent attachment, via Michael addition, was expected to occur between this carbon and the free thiol on the peptide N-terminal. A FSH2Md conjugation reaction was successfully scaled up to produce sufficient material for both in vitro and in vivo experiments. The FSH2Md product was purified by HPLC and its identity confirmed by MALDI-TOF mass spectrometry; the product was found to resolve as a major peak at m/z of 1291.6. Additionally, gas chromatography analyses confirmed that no excess free menadione remained as a contaminant in the final product (results not shown).
G. FSH2-Menadione (FSH2Md) created oxidative stress and induced mitochondrial ROS, lipid peroxidation, and DNA damage in TM4 Sertoli cells in vitro
[00348] The in vitro redox cycling potential of FSH2Md was assessed using the methods described in Example 1 above. Ten percent of TM4 Sertoli cells were positive for mitochondrial ROS after 90 min of co-incubation with FSH2Md (Fig. 8A). This response was significantly higher (P< 0.05) than the response in TM4 Sertoli cells treated with either vehicle or with unconjugated FSH2 peptide. [00349] FACS assessment of TM4 Sertoli cells stained for 4-hydroxynonenal (4- HNE) expression, one of the by-products of lipid peroxidation, revealed that this marker was detected among 7% of the cells treated with FSH2Md (Fig. 8B). This response was significantly higher (P<5xl0‘4) than those 4-HNE positive cells resulting from treatment with either the vehicle or FSH2 peptide alone.
[00350] The downstream endpoints of oxidative DNA damage and activation of the apoptotic pathway were also assessed using the methods described in Example 1. Oxidative DNA damage was significantly elevated (p<0.05) in FHS2Md-treated TM4 Sertoli cells beyond that of their control counterparts. Specifically, 17% of the FHS2Md-treated cells had detectable levels of oxidative DNA damage (Fig. 8C). While 19% of FSH2Md-treated cells were positively stained for activated Caspase 3 (Fig. 8D), statistical analysis showed that there was no significant difference between the treatments. Taken together, these results indicate that Md conjugated to FSH2 maintained its ability to redox cycle and produced sufficient ROS to induce DNA damage in these cells without inducing significant apoptosis. While it may be desirable, in certain aspects, to induce DNA damage without inducing cell death, in a preferred embodiment, cell death in the testes relating to sterilization in an animal is a desired outcome of treatment. The following section discusses further testing of FSH2Md treatments while Example 3 below discusses combination treatments with FSH2 and LH conjugates for simultaneous targeting of FSHr and LHr.
H. FSH2Menadione (FSH2Md) induces targeted oxidative damage to the male germ line in vivo
[00351] Because FSH2Md targeting was successful in vitro, experiments were carried out to determine if the in vitro results could be recapitulated in vivo. That is, the ability of a menadione-FSH peptide construct to induce high levels of oxidative stress in the testes was examined. Methods used were as described in Example 1 above. Given that the FSH responsiveness of TM4 Sertoli cells is reportedly much reduced compared to primary Sertoli cell cultures, it was noted that the response of Sertoli cells in vivo to FSH2Md may also differ.
[00352] Adult male mice were administered with a single IP injection of FSH2Md at a concentration (14.5 mM) approaching the limit of solubility for FSH2Md. Six weeks postinjection, 5 treated males were housed with 2 females each for 3 weeks. The FSH2Md-treated mice retained fertility, resulting in pregnancies in 9 out of the 10 females, with an average of 11 live embryos per female and just 2 resorption sites in one of their pregnant mates. [00353] The FSH2Md-treated males were characterized by equivalent testicular weights (Fig. 9A), and epididymal sperm motility (Fig. 9B) and vitality (Fig. 9C) compared to those of the control subgroup. These similarities also extended to the histology of the testes, which did not show disruption of spermatogenesis in treated animals (Figs. 10A-B). The use of an Apop-Tag detection kit indicated that there was no elevation in the level of apoptosis occurring within the seminiferous tubules of FSH2Md-treated males (Fig. 11).
[00354] Despite these outcomes, a more detailed analysis of the integrity of the germline revealed pronounced signatures of oxidative damage. In this context, mature spermatozoa harvested from the cauda epididymis of FSH2Md-treated mice presented with significantly (P<0.05) elevated levels of DNA strand breaks, which were accompanied by significant (P<0.05) oxidative DNA damage (Figs. 9D-E). Indeed, over 60% and 40% of the spermatozoa from FSH2Mdtreated males exhibited DNA strand breaks and oxidative DNA damage compared to only <10% and 26% of the spermatozoa from control males (Figs. 9D-E). Taken together, these results provide important proof-of-concept that localized, sub-lethal levels of oxidative stress can be generated in the vicinity of the male germ line, via the coupling of redox active agents to FSH peptide mimics.
[00355] The observations discussed in this example demonstrate that agents (including agents that disrupt spermatogenesis) can be delivered selectively to the testes in mice. Example 3 below builds on this concept and discusses additional targeting of the testes with Auristatin (Aur) towards improved methods for nonsurgical sterilization. As discussed below, Md was used to induce oxidative damage while Aur was used to inhibit mitosis, demonstrating that decreased fertility in male mice is possible when a dual-targeting approach is used.
I. Discussion
[00356] Ideally, a targeting peptide should accumulate in the target tissue but not in off-target tissues, thereby increasing efficacy and minimizing side-effects (Le Joncour & Laakkonen, 2018). Of the three peptide-FITC conjugates that possessed the highest binding capability in vitro, F53 and F49 were designed to encompass the sequence of the PL2 loop binding region of the P-subunit of FSH whereas FSHa was representative of residues residing within the “seatbelt” region of FSH P-subunit (Table 3, Fig. 1). Since both regions of FSHp had been implicated in ligand binding to the cognate FSH receptor (Q. R. Fan & Hendrickson, 2005; Jiang et al., 2014; Keutmann, 1992), differences in in vitro peptide binding efficacy may be attributed to their unique physicochemical properties. In this regard, each of these three peptides have an overall positive charge at neutral pH, whereas those peptides with lower binding capability had an overall negative charge. It is therefore possible that positively charged peptides may be favored in vitro owing to electrostatic attraction between the peptide construct and the negatively charged cell surface(Seelig, 2004). In turn, this may increase the concentration of the peptide that accumulates within the vicinity of the FSH receptors and thus account for the enhanced binding of FSHa, F49 and F53.
[00357] Notably, however, this advantage did not translate directly in vivo where FSHa significantly outperformed all other peptides in targeting of the testis. The overall positive charge on FSHa peptide combined with its relatively high hydrophobicity may confer an advantage in terms of penetrating the cell membrane (Seelig, 2004). Additionally, it is possible that the relative hydrophobicity of FSHa may in part confer a higher in vivo stability by increasing its adsorption to serum albumin. Serum albumin has a in vivo half-life that is days long while unmodified peptides have a half-life on the scale of minutes to hours (Di, 2015; Werle & Bemkop-Schniirch, 2006). The relatively short in vivo half-life of peptides is due to proteolysis and fast renal clearance (Werle & Bemkop-Schniirch, 2006). However, once associated with the serum albumin, proteolysis and filtration-clearance of the peptide from the circulation is limited (Lien & Lowman, 2003). Previously, this property was intentionally harnessed by covalent attachment of peptides to albumin to enhance the in vivo half-life of peptides (Penchala et al., 2015). Increased in vivo stability enhances the plasma residence time (Sato et al., 2006) and, in turn, may allow the peptide to reach the TM4 Sertoli cell target.
[00358] Md was covalently attached to the N-terminus of the FSH peptides via the reactive C3. This mode of attachment negates the alkylating properties of Md meaning that any oxidative damage observed may be attributed to ROS production rather than the adduction of Md to essential biomolecules (Klotz et al., 2014). Additionally, with the C-terminus of the FSH peptides protected by amidation and the N-terminus modified with Md, the peptides may be less susceptible to degradation by exopeptidases (Di, 2015; Diao & Meibohm, 2013) in vivo thereby prolonging the in vivo half-lives and extending the time that the peptides are exposed to the FSHr.
[00359] In testing the utility of FSH peptides to deliver a redox active reagent to the TM4 Sertoli cell line model, the direct FSHaMd conjugate was found to be suboptimal since it retained very poor redox cycling activity relative to that of free Md. Consequently, the Md was distanced away from the FSHa peptide backbone via the introduction of linker molecules. Of these, the y-aminobutanoic acid with MPA linker resulted in the highest production of hydrogen peroxide, as measured indirectly by chemiluminometry. The selection of the y-aminobutanoic acid with MPA linker was supported by the results of an in vitro co-incubation of FSH2Md with TM4 Sertoli cells where, in terms of driving ROS production, it significantly outperformed the vehicle and the peptide only controls. Similarly, TM4 Sertoli cells treated with FSH2Md also exhibited elevated levels of the lipid peroxidation by-product, 4-hydroxynonenal, as well as oxidative DNA lesions. Together, these results suggest that Md, conjugated to FSH peptide via the y-aminobutanoic acid with MPA, maintained its redox cycling ability sufficient to trigger an oxidative stress cascade that likely overwhelmed the TM4 Sertoli cell’s intrinsic antioxidant defenses.
[00360] In view of the in vitro data, an in vivo proof-of-concept experiment was carried out next. The normal spermatogenic cycle in mice occurs over approximately 34.5 days (Oakberg, 1956). This cycle takes all of type A spermatogonia through differentiation to spermatozoa. Therefore, the most significant outcome of this in vivo study was that at ten weeks, or two complete spermatogenic cycles, after a single administration of FSH2Md, the mature epididymal sperm of treated males exhibited a significantly higher level of DNA strand breaks and oxidative DNA damage than that of their control counterparts. Such lesions in the paternal genome did not precipitate an overall reduction in male fertility, however they did induce a significant increase in the incidence of DNA damage with the sperm population.
[00361] In summary, FSHr-targeting peptides were designed that binds with high capability and specificity to a Sertoli cell line in vitro as well as selectively targeting the testes in vivo. Furthermore, modifying these peptides to incorporate an appropriate linker domain and redox active payload (i.e., Md), can be exploited to propagate localized redox cycling within the Sertoli cell population in vivo.
[00362] Example 3 below builds on the method of this Example of targeted delivery of Md to mouse testes. Example 3 discusses additional targeted delivery of Aur to mouse testes to induce germ cell apoptosis, resulting in decreased fertility in male mice.
Example 3. Synthetic LH peptides
A. LH peptide sequences include residues from the L2 loop and “seatbelt” region of the LH -subunit with varying charge and hydrophobicity
[00363] Four new murine LH peptides that bind to LH were designed and are shown in Table 4.
Figure imgf000091_0001
[00364] Each of the LH peptides were modeled as described in Example 1 above.
The peptides were found to map to the (3 L2 loop and the “seatbelt” region of the LH P-subunit , which are the two binding regions on the surface of the P-subunit (Figs. 12A-D). Additionally, their position on the surface of the protein means that they are accessible for receptor binding.
[00365] A comparison of the physicochemical properties of the peptides (Table 4) predicted good aqueous solubility for all of the peptides except for L57. This may be expected as the ratio of hydrophobic to hydrophilic residues for this peptide was 4, which is considerably higher than the ratios of the other, more soluble, peptides which all had an equivalent ratio of less than one. The predicted charge at neutral pH for LHa and L101 were both around +1. L57 was predicted to have a higher overall charge of +1.9, whereas L95 was predicted to possess a very high +3.9 charge at neutral pH. Because hydrophobicity and charge can influence the affinity of a peptide for the cell surface (with positive charges increasing the electrostatic attraction for negative cell surfaces), the binding specificity of each peptide was assessed, as described below in Example 3.C.
B. An in vitro model - MLTC-1 Leydig cells expressing the LH receptor (LHr)
[00366] A cell line with strong expression of the LH receptor gene (Lhcgr) was identified as an in vitro model cell line for use in targeting the synthetic LH peptides to LHr. qPCR and an anti-LHr antibody were used to probe for the presence of the LH receptor in both MLTC-1 and TM3 Leydig cell lines, as well as in the control mECapl8.
[00367] Using qPCR (also described in Example 1 above), we confirmed that the MLT+C1 Leydig cell line expressed the Lhcgr gene (Fig. 13A). By contrast, no Lhcgr transcripts were detected in the TM3 Leydig cell line or in the negative control mECap 18 cell line. These results were validated by immunocytochemistry (also described in Example 1 above), which confirmed LHr expression in the MLTC-1 Leydig cells, but not in mECapl8 cells (Fig. 13B). Given these results, MLTC-1 cells were used as the in vitro Leydig model cell line in the examples that follow.
C. LH peptides demonstrated variable in vitro binding capability and specificity
[00368] MLTC-1 cells and mECapl8 cells were used (as described in Example 1 above) to assess the relative binding capabilities and the targeting specificity of the synthetic LH peptides. Each peptide bound to the MLTC-1 Leydig cells in a dose-dependent manner. This was evidenced by a proportional shift in the median fluorescence intensity of the stained MLTC- 1 cell population across the dose range of applied peptides (Figs. 14A-D). Additionally, each LH peptide displayed suitable specificity with three-fold higher median fluorescence intensity when applied to MLTC-1 Leydig cells as compared to the mECapl8 control cells (Figs. 14E-F). The peptide with the greatest shift in median fluorescence and, by inference, the greatest binding capability, was L95, which possessed more than double the MLTC-1 cell binding affinity than that of the other peptides tested (Fig. 14E). However, L95-FITC showed a lack of specificity at the highest dose tested, 50 pM, with only 1.5-fold relative binding on MLTC-1 cells above that of the background mECapl8 cell binding.
D. LH peptides localized to the testes in vivo
[00369] Given the promising in vitro results, the targeting potential of the LH- FITC conjugates to their testicular target was assessed using in vivo mouse methods. The LH peptides were injected IP into adult male mice and the experiments were carried out as described in Example 1 above.
[00370] No significant difference was found in the weight of mice exposed to either the vehicle or the peptide-FITC treatments eight hours post-injection. Similarly, no difference was observed in the weight or gross morphology of the representative tissues collected. All four LH peptides exhibited localization within the testes with a clear difference in the fluorescence intensity being detected in the testes of the vehicle-injected compared to that of the peptide-FITC injected males (Fig. 15A). This contrasted the findings of the adrenals, brain, lungs, pituitary, or seminal vesicles for which no difference in fluorescence staining was observed between the vehicle control and the treated animal tissues. However, there was evidence of clearance of the peptides through the detoxifying and excretory organs of the liver and kidney, both of which presented with relatively low fluorescence labelling (Fig. 16). Additionally, there appeared to be modest FITC fluorescence associated with the spleen in at least a portion of the treated animals, but particularly within the spleens of the L57-FITC treated animals (Fig. 16).
[00371] Based on these findings, images of the treated testes were imported into ImageJ to compare the mean fluorescence intensity/pixel. The results were expressed as a ratio of the mean fluorescence intensity/pixel of the treated in any image to the vehicle control in the same image (Fig. 15A). All four peptides displayed a normalized fluorescence intensity/pixel ranging from 1.45-fold to 1.6-fold that of the vehicle control (Fig. 15B), albeit with differing levels of significance. These results suggest that the LH peptides have similar capabilities in localizing to the testes and have similar stabilities in vivo. The presence of the FITC-Ahx tag conjugated to the peptide, however, means that important factors that determine the pharmacokinetics, such as the theoretical isoelectric point, the charge and the logP value (which is a measure of hydrophobicity that increases with increasing hydrophobicity) of a modified peptide differs from that of an unmodified peptide and, most likely, that of a peptide with cargo. Therefore, further analysis of the peptides was warranted.
[00372] Aqueous solubility was also considered, both in the context of conjugating with Aur and for use of biocompatible solvents for in vivo delivery. The Aur employed in this study has a valine-citrulline linker (Fig. 17) with a terminal maleimide for ease of conjugation and has very poor aqueous solubility with a logP value of 6.04 (Error!
Hyperlink reference not valid.chemsrc.com/en/cas/646502-53-6_1197872.html). Therefore, so as not to compound this lack of solubility, the targeting peptide needed to possess good aqueous solubility and, as L57 was predicted to have poor water solubility (see Table 4), it was excluded from this study. Additionally, L95 peptide was considered unsuitable as it exhibited a relatively high positive charge (see Table 4) and non-specific binding in vitro (Figs. 14E-FF); both of which are incompatible with selective delivery of the potent cytotoxic payload to be attached to this peptide. Furthermore, Aur was to be conjugated to a y-aminobutanoate-MPA linker (i.e., linker 2) on the N-terminal of the peptide via a thiol-maleimide reaction. The linkers were expected to allow the peptide to bind to the receptor unhampered by the attached Aur. Therefore, a cysteine extra to the N-terminal cysteine, such as in L101 peptide, indicates that a non-ideal mixed product of mono-conjugated and bi-conjugated peptide-Aur, may result. Receptor binding by a bi-conjugated product may be sterically hindered by the extra Aur moiety. Considering these factors, and based on its predicted charge, solubility (Table 4), in vitro selectivity (Figs. 14E-F) and similar in vivo performance to other assessed peptides (Fig. 15B), the LHa peptide was selected for targeted delivery of Aur to the testes.
E. Conjugation of Menadione (Md) to FSH peptide and Auristatin (Aur) to LHa peptide
[00373] In adapting the FSH and LHa peptide sequences for the purpose of carrying a specific payload, linker 2 (i.e., a linker comprised of y-aminobutanoic acid and mercaptopropionic acid (MPA)) was used. This provided a reactive thiol group for attaching the peptide to other moieties or to nanoparticles via a maleimide -thiol coupling reaction (Martinez- Jothar et al., 2018; Varanko et al., 2020). Additionally, the C-terminus of all peptides was amidated with the aim of inhibiting protease degradation, increasing stability (Di, 2015; Werle & Bemkop-Schmirch, 2006), and thereby prolonging the in vivo half-life and extending the time that the peptide is exposed to the target receptor. Additionally, as discussed above, linker 2 allows Md to produce ROS by redox cycling despite its covalent attachment to the peptide. The LHa and FSH peptides with linker 2 are hereinafter referred to as LH2 and FSH2. FSH2 was attached, via the N-terminal cysteine, to C3 of Md and the product, FSH2Md, was isolated and the powdered product was stored at -20°C. LH2 was reacted with maleimide-Aur overnight and the product was successfully purified by HPLC and freeze-dried, and the powdered product was stored at -20°C. The identity and purity of the product was confirmed by LC-MS and showed a single peak with an m/z of -2648. The reagents were resuspended in 30% Kolliphor/PBS immediately before administration to the treatment groups.
F. LH2Auristatin (LH2Aur) localized to the testes and impacted seminiferous tubule morphology and germ cell viability
[00374] Mice were injected IP with FSH2Md, LH2Aur, or FSH2Md and LH2Aur (FSH2Md/LH2Aur) as described in Example 1 above. Treated mice maintained healthy weight gain over a ten-week post-injection surveillance period, with no difference in mean body weights detected between the treatment groups. Additionally, with the exception of the testes, there was no difference in tissue (seminal vesicles, epididymis, adrenals, kidneys, liver, brain, heart, and spleen) weights or morphology between the treatment groups, indicating that the injected peptides did not elicit pronounced off-target effects. In contrast, it appeared that the testes of at least some of the treated mice had been adversely impacted. Specifically, thirty percent of LH2Aur-treated males and an additional twenty percent of FSH2Md/LH2Aur-treated males presented with at least one testis that was significantly smaller (weights ranging from 41 mgto 85 mg) than that of the controls. By comparison, all males that had been treated with either FSH2Md alone or the vehicle had testes weights over 126 mg. However, the overall mean weights of the testes of LH2Aur- and FSH2Md/LH2Aur-treated males were no different than that of the vehicle control group (Fig. 18A).
[00375] To account for the lower testes weights, the morphology of fixed testis sections was examined. Qualitatively, the seminiferous tubules of the LH2Aur- and the FSH2Md/LH2Aur-treated males appeared much smaller than those of the vehicle control- or the FSH2Md-treated males (Fig. 18B). Similarly, a quantitative evaluation using ImageJ software revealed that the average area occupied by seminiferous tubule in the testes of all of the treated animals were lower than that of the vehicle control group (Fig. 18C). This reduction in tubule area proved highly significant (p<0.005), such that the mice treated with LH2Aur and FSH2Md/LH2Aur exhibited a 40% reduction in mean tubule area compared to the testes of the control group. Such changes in tubule area were accompanied by the presence of apoptotic germ cells, which were clearly discerned in the two treatment groups (Fig. 18B).
[00376] To confirm whether germ cell apoptosis had occurred, ApopTag was used to detect single-stranded and double-stranded breaks associated with this form of cell death. The results of this assay confirmed that the testes sections from both the LH2Aur and the FSH2Md/LH2Aur treatment groups exhibited significantly (p<0.05) more tubules containing germ cells undergoing apoptosis (Figs. 19A) and a significantly (p<0.05) greater number of apoptotic cells per affected tubule (Fig. 19B) than the FSH2Md alone or vehicle treatment groups. Given the low number of apoptotic cells in the testes treated with FSH2Md alone, it appears that the apoptosis was mostly precipitated by damage inflicted by the Auristatin.
G. FSH2Menadione (FSH2Md) with LH2Auristatin (LH2Aur) impact on Sertoli cell number
[00377] The number of Sertoli cells per seminiferous tubule were determined by immunolabeling of testes sections with antibodies directed against SOX9, a protein whose testicular expression is restricted to the Sertoli cell population (Figs. 20A-C). Methods used were as described in Example 1 above. This analysis revealed a trend, albeit not statistically significant, of reduced mean numbers of Sertoli cells within the testes of the FSH2Md/LH2Aur treatment group (i.e., ~14 Sertoli cells/tubule) compared to the number of these cells in the vehicle control group (i.e., ~17 Sertoli cells/tubule) (Fig. 20B). This trend was not replicated among Sertoli cell populations in the testes of males treated with either FSH2Md or LH2Aur, neither of which were numerically different from those treated with the vehicle. The absence of impact on the Sertoli cell population was reflected in the serum FSH levels, with an ELISA showing no difference in FSH levels within the cardiac sampled blood serum (Fig. 20C) between the treatment groups. These results indicate that the targeted delivery of low dose Md to Sertoli cells was not sufficient to create a significant reduction in their number.
H. The mature spermatozoa of male mice treated with FSH2Md or FSH2Md/LH2Aur exhibited DNA strand breaks and oxidative DNA damage
[00378] Despite no overt effect on Sertoli cell populations, FSH2Md and FSH2Md/LH2Aur treatments did elicit oxidative DNA damage among the mature spermatozoa of treated males. Indeed, the application of a HALO assay (as described in Example 1 above) indicated that there was a significant increase (p<0.05) in DNA strand breaks harbored by the spermatozoa from FSH2Md- and FSH2Md/LH2Aur-treated animals (Fig. 21A) compared to that of the control group. Specifically, an average of 22% of the spermatozoa from FSH2Md-treated males and 17% from FSH2Md/LH2Aur-treated males displayed DNA strand breaks compared to only 5% of spermatozoa recovered from the cauda epididymis of LH2Aur- alone or vehicle- treated males. Additionally, there was a greater incidence of oxidation of DNA guanosine bases in the spermatozoa from the LH2Aur- and the FSH2Md/LH2Aur-treated treatment groups (averaged between 25 and 31%) compared to the vehicle-treated animals (Fig. 2 IB). Furthermore, the oxidative DNA damage observed in the spermatozoa was evidently not a product of ongoing ROS production as there was no difference in the levels of either mitochondrial ROS or cytoplasmic superoxide detected within these cells (Figs. 23A-B) between the four treatment groups. However, the DNA damage in the caudal spermatozoa of treated males was not accompanied by an increase in the incidence of abnormal sperm gross morphology compared to the control males (Fig. 21C).
[00379] Computer-assisted sperm analysis (CASA) also showed no difference in sperm motility parameters or sperm total motility among the treated males compared to that of the vehicle controls (Figs. 22A-D). Furthermore, there was no difference in the vitality of the caudal spermatozoa (Fig. 23C) or in the DNA fragmentation index using the sperm chromatin structure assay (SCSA) (Fig. 23D) between the treatment groups. Notwithstanding these results, the DNA damage detected in the spermatozoa of some of the treatment groups appeared to be of sufficient magnitude to impinge on the fertility of the treated males.
I. The combination of LH2Auristatin (LH2Aur) and FSH2Menadione (FSH2Md) decreased the fertility of treated males
[00380] Eighty percent of the females that had been housed with the males treated with LH2Aur or the FSH2Md and LH2Aur combination FSH2Md/LH2Aur) became pregnant with live embryos present at 13 days gestation. A mating plug was observed in the remaining 20% of females but no embryos were present at 13 days post-mating. All ovaries exhibited corpora lutea, indicating that ovulation was likely to have taken place in these females. There were significantly fewer average embryos for every pregnant female mated with males from LH2Aur and FSH2Md/LH2Aur treatment groups compared to those females mated with the vehicle control males (Fig. 24A). Additionally, 87.5 % of females mated with males in the FSH2Md/LH2Aur treatment group displayed evidence of embryo resorption, in the form of resorption moles, at 13 days post-mating (Fig. 24B). Whilst all treatment groups produced a higher-than-average number of resorption moles than that of the vehicle control group, the FSH2Md/LH2Aur treatment in particular produced a very high number of resorption moles - 1.9 resorption moles/pregnant female (Fig. 24B), with one female mated from this group exhibiting five resorption moles.
[00381] FSH2Md administration alone was not sufficient to compromise male fertility. In fact, all 5 males in the FSH2Md treatment group were fertile, with each of the 10 females housed with these males becoming pregnant and carrying live embryos at 13 days postmating (Fig. 24A). Only 2 of the females that had been mated with males from this treatment group exhibited one resorption mole each, with none detected in the remaining eight females.
[00382] All 5 males in the vehicle treatment group proved to be fertile with those females that became pregnant having at least 11 (up to 17) and an average of 13.5 embryos present at 13 days, none had resorption moles (Fig. 24A).
J. Discussion
[00383] LHr-targeting peptides were designed to complement the FSHr-targeting peptides described in Example 2 above. Importantly, ten weeks (i.e., two complete spermatogenic cycles) after a single administration of FSH2Md and LH2Aur, the mature epididymal sperm of treated males exhibited a higher level of DNA strand breaks and oxidative DNA damage than that of their control counterparts. Administration of a single dose of LH2Aur resulted in extensive germ cell apoptosis and a reduction in fertility. Although spermatogenesis and fertility persisted, the treatments facilitated enduring damage to the testes and confirmed that the Sertoli cell and Leydig cell populations had been targeted. At the tested peptide concentrations and with administration of a single dose for each peptide, LH2Aur had worked synergistically with FSH2Md to impact the Sertoli cells and decrease the viability of the embryos. However, the extent of the aforementioned effects varied widely within the treatment groups. [00384] To minimize the possibility of adverse off-target effects, a targeting peptide should accumulate in the target tissue but not in off-target tissues(Le Joncour & Laakkonen, 2018). Of the 4 LH peptide-FITC conjugates that were tested, the peptide with the highest binding capability in vitro, L95, along with L101 and LHa were designed to encompass the sequence of the “seatbelt” region of LH P-subunit. The L57 peptide, which comprised residues residing within the L2 loop binding region of the -subunit of LH (Table 4, Fig. 12), displayed higher binding capability than either L101 or LHa in vitro. Since both the “seatbelt” and the PL2 loop regions of LHP have been implicated in ligand binding to the cognate LH receptor (Q. R. Fan & Hendrickson, 2005; Jiang et al., 2014), it is possible that differences in in vitro peptide binding efficacy are attributed to their unique physicochemical properties. L95 peptide, despite displaying a higher binding capability, also lost specificity at higher concentrations (Fig. 14E). This increase in binding capability and lack of specificity at higher concentrations may be attributable to the relatively high positive charge of +2.9 on this peptide at neutral pH (Table 4). A higher positive charge may enable this peptide to more readily bind non-specifically via electrostatic attraction to the negatively charged cell membrane in this in vitro system. Additionally, L57 with a charge of +1.9, exhibited a higher relative binding capability then the LHa and L101 that respectively have predicted charges of +1 and +0.9 (Table 4, Fig. 14E). LH peptides that possess a higher positive charge may be favored in vitro owing to electrostatic attraction between the peptide construct and the negatively charged cell surface (Seelig, 2004). The increased attraction may act to increase the concentration of the peptide that accumulates within the vicinity of the LH receptors and thus account for the enhanced binding capability of L95 and L57. However, this advantage did not translate directly to an in vivo setting in which all four of the peptides displayed similar localization to the testes.
[00385] The LHa peptide with an N-terminal linker 2 (i.e., LH2) was tested for in vivo delivery of the anticancer cell ablation agent, Auristatin (Aur) ( Fig. 17). A hydrophobic form of Aur was chosen because it readily enters the cell and, once within the cell, acts as an antimitotic agent that binds to a-tubulin and prevents the formation of the mitotic spindle, leading to G2-M cell cycle arrest. Aur is highly potent and may be delivered selectively by conjugation to a targeting agent (most commonly an antibody) so as to reduce adverse off-target effects. Here, Aur was attached to the LH2 peptide via a cathepsin cleavable valine- citrulline(vc)-PABC linker (Fig. 17). This linker has been reported to have high stability in serum or plasma and, once the peptide-Aur conjugate binds to the LH receptor, the premise is that it will be taken into the cell where the complex will be metabolized by lysosomal proteases (Sutherland et al., 2006) followed by 1,6-elimination to liberate the active drug (Fig. 17).
[00386] In order to target both the Leydig and the Sertoli cells concurrently,, LH2Aur was used to target the Leydig cells and a FSH2 peptide conjugated to Menadione was used to target the Sertoli cells. After LH2Aur enters the cell, the Aur can be freed from LH2 and released from the cell. Free Aur may then kill surrounding cells via the bystander effect (Li et al., 2020; Lucas et al., 2019). Bystander killing occurs when the drug from atargeting reagent is released either from the target cell following internalization and degradation of the peptide-drug or release of the drug within the extracellular space.
[00387] Sertoli cells are critical for the formation of the stem cell niche within the testis and the subsequent support of all stages of spermatogenesis (Hermo et al., 2010a, 2010b). Spermatogonial germ cells are sensitive to the effects of ionizing radiation (Kangasniemi et al., 1996) that, additional to damage to the DNA in the form of strand breaks by acting directly on the ribose-phosphate backbone, mainly arise from the radiolysis of water leading to the formation of hydroxyl radicals. The hydroxyl radicals subsequently react with DNA bases and cause persistent DNA damage (Eastman & Barry, 1992). Ionizing radiation also generates other ROS, such as superoxide and hydrogen peroxide, all of which can interact with DNA, cellular lipids and proteins (Tominaga et al., 2004) thus facilitating irreversible damage to essential cellular machinery. The addition of Md may recapitulate these effects as the superoxide radicals produced by Md can be catalytically converted by superoxide dismutase (SOD) to hydrogen peroxide (Melov, 2002). Hydrogen peroxide may be converted through a series of reactions to hydroxyl and hydroperoxyl radicals that, in turn, may act as oxidizing agents. Together with other ROS, the radicals so formed can cause loss of function through oxidative damage to cellular lipids (Halliwell, 1991), proteins, and DNA (Wiseman & Halliwell, 1996).
[00388] As discussed in Example 2, FSH2Md led to oxidative DNA damage and DNA strand breaks in the spermatozoa of treated mice. The presence of ROS can also induce oxidative actin cross-linking and dissociation of the cytoskeleton from the plasma membrane. All these alterations may contribute to the multifactorial process underlying the irreversible cell injury caused by oxidative stress. Additionally, as well as its antimitotic effects, Aur has been shown to potentiate the effects of ionizing radiation by acting as a radiosensitizing agent (Adams et al., 2016; Buckel et al., 2015). Challenging both the Sertoli cell and Leydig populations concurrently by the production of localized oxidative stress in the vicinity of the germ cells and in the presence of an anti-mitotic agent may deplete the Leydig cell population as well as enhance the impact of ROS within the testes.
[00389] The testes of 25-30% of the LH2Aur-treated males were impacted. When the LH2 peptide was used to incorporate and deliver Aur to the Leydig cell population in vivo, the seminiferous tubules in treated mice were significantly reduced in cross-sectional area. This reduction in tubule size translated to a large reduction of testicular weights in 30% of the LH2Aur-treated males. This reduction in tubule size may have resulted from apoptosis of the germ cells observed in the testes of these animals. Yet, this loss of germ cells, most likely precipitated by cell cycle arrest, was insufficient to disrupt spermatogenesis. Nonetheless, the mature spermatozoa in the LH2Aur-treated males harbored elevated levels of oxidative DNA damage.
[00390] Whilst the administration of FSH2Md alone proved sub-lethal to the Sertoli cells, it was nevertheless sufficient to elicit a pronounced and sustained impact on the developing male germ line with the mature gametes exhibiting persistent oxidative DNA damage. However, it did not affect the viability of the embryos sired by these males. These findings reinforce the results described in Example 2 that showed that FSH2Md incurred oxidative damage in the sperm but showed no effect on the fertility of the treated males.
[00391] When LH2Aur was combined with FSH2Md in vivo, the effects of each alone were recapitulated. However, concurrent administration of these two reagents also resulted in a significant increase in embryo resorptions in the pregnant females housed with these males compared to the groups that received either LH2Aur or FSH2Md alone or the vehicle control.
[00392] In accounting for the results observed in LH2Aur treated males, Aur is a potent tubulin inhibitor. This inhibition may provide a devastating effect on Leydig cell function as it binds to microtubules, introduces structural defects, and suppresses microtubule (MT) dynamics, while reducing kinetics and extent of MT assembly. This may lead to suppression of proliferation, mitosis, and disrupted MT network (Best et al., 2021). A depleted Leydig cell population may mean that their support of Sertoli cell health is disrupted. Additionally, the cytoskeleton is responsible for movement of organelles and molecules involved in steroidogenesis (17-P-estradiol synthesis) (Marchlewicz et al., 2004) in the Leydig cells. If, as a result, testosterone levels are reduced, then Sertoli cell nurturing of spermatogenesis (Smith & Walker, 2014) may be disrupted and may contribute to the apoptosis of the germ cells. If Aur indeed reached the Sertoli cells through the aforementioned bystander effect, then its suppression of MT dynamics may also impact Sertoli cell function and cytoskeleton, disrupting the germ cell niche, which, in turn, may adversely affect the germ cell population (Rebourcet et al., 2017). In the case of the Aur entering the tubules, Aur may exert its effects directly on the germ cells by precipitating mitotic arrest (Bourillon et al., 2019) and lead to their apoptosis. Indeed, all stages of spermatogenesis and male fertility are reliant on the dynamic organization/reorganization of microtubules (O’Donnell & O’Bryan, 2014) and the cytoskeleton (Dunleavy et al., 2019). Despite the absence of Md, sperm from LH2Aur-treated males also displayed a higher level of oxidative damage than the control-treated males. A similar observation was made in FSH2Md-treated males. This, too, may have stemmed from MT disruption as 8-oxoguanine DNA glycosylase (OGGI), which excises the oxidized bases from DNA, associates with and relies on microtubules for transport to the site of action during mitosis (Conlon et al., 2004).
[00393] The higher rate of resorption moles in the females housed with FSH2Md/LH2Aur-treated males may be the consequence of DNA damage to the germ line precipitated by the presence of ROS, produced by the menadione, further potentiated by the radiosensitization effect and disruption of microtubules precipitated by Aur. Furthermore, Aur increases the fraction of cells that are in G2/M which is the most radiosensitive phase of the cell cycle (Bourillon et al., 2019). The germ cells, as pachytene spermatocytes and round spermatids, remain adherent to Sertoli cells throughout their development (Mruk & Cheng, 2004). As part of their nurturing role, Sertoli cells are involved in regulating the levels of oxidative stress to which developing germ cells are exposed. ROS, generated via the redox cycling of Md, may have contributed to a level of oxidative stress in the testes that overwhelmed the regulatory mechanisms present within the Sertoli cell population (Y oganathan et al., 1989) so that they were no longer able to protect the germ cells from oxidative stress. At the same time, Sertoli cell function may have been impaired by the impact of Aur on the Leydig cells, thus potentiating the effects of ROS on the germ cells without the protective support normally provided by the Sertoli cells. The persistence of this damage in the DNA may be directly related to the increase in resorption moles as it has been established that the paternal genome (Barton et al., 1984) and genomic imprinting (Thamban et al., 2020) play a vital part in the normal development of the embryo. The accumulation of oxidative DNA damage in the germ cells may have affected key genes within the genome so as to impact the viability of embryos sired by FSH2Md/LH2Auristatin-treated males .
[00394] In accounting for the variation in response within the LH2Aur treatment groups, there are several factors that may adversely impact the amount of Aur that is delivered to the target Leydig cells. Peptides can have a short in vivo half-life due to rapid clearance by the kidneys and degradation by endogenous proteases (Werle & Bemkop-Schntirch, 2006). This can severely limit the proportion of peptide conjugate that reaches the target cells. One method of increasing in vivo stability is to increase the hydrodynamic radius of the peptide by the addition of polyethylene glycol (Werle & Bemkop-Schntirch, 2006) or fusion with albumin (Di, 2015; Diao & Meibohm, 2013) or other serum proteins (Penchala et al., 2015). However, such peptide modifications, including others such as replacement of labile amino acids or replacement of L- amino acids with D-amino acids (Werle & Bemkop-Schntirch, 2006), may disrupt the binding capabilities of the peptide to its cognate receptor. Additionally, the vc-PABC linker that attaches the Aur to the peptide must be stable in vivo. Extracellular cleavage of the vc-PABC linker can be catalyzed by carboxylesterase 1C in mouse semm, thereby releasing the Aur before it reaches the target cell (Dorywalska et al., 2016). Indeed, plasma studies using antibody conjugated-Aur have shown that up to 25% of the Aur is released over the first 96 hours in comparison to less than 2% in human, monkey and rat plasma . Finally, the toxicity of Aur necessitated the delivery of a very low final concentration of 1.42 pM. This dose may not have been high enough to bring the local concentration in the testes to an efficacious level. While in certain aspects, treatment may comprise the administration of more than one dose, in a preferred embodiment, a single injection that results in permanent sterilization is desired.
[00395] In summary, this Example describes the development of LHr-targeting peptides that demonstrated binding capability and specificity to a Leydig cell line in vitro as well as selectively targeting the testes in vivo. An exemplary LHr-targeting peptide was conjugated to Aur for delivery to Leydig cells in vivo, both with and without an FSHr-targeting peptide conjugated to Md for delivery to Sertoli cells. The results showed precipitating germ cell apoptosis with varying outcomes within treatment groups. This variation may have stemmed from susceptibility of enzymatic degradation of the peptide and/or the vc-PABC linker in vivo resulting in a suboptimal quantity of Aur reaching the target cell. Although the levels of embryo resorptions observed in females housed with LH2Aur/FSH2Md treated males may not be acceptable clinically, the results indicate that both the LHr and FSHr targeting peptides selectively localized to the testes and can be used as delivery agents for chemosterilization. For example, the targeting peptides may be attached to the surface of nanoparticles (NPs) encapsulating cytotoxic agents and used for targeted delivery of the cytotoxic agents to testis resulting in targeted testis cell ablation. Targeted delivery of such nanosystems offers the advantage of simultaneously protecting the peptide from clearance and degradation and of increasing the amount of cargo that can be delivered. This targeted approach has the potential to increase local concentrations of the reagents as well as avoid potential off-target effects. Given the promising results of this study, delivery of Md and Aur within targeted NPs as a single injection may increase the local concentration of these two drugs to a level that will elicit a more pronounced and consistent effect.
Example 4. Synthetic AMH peptide
A. AMH peptide maps to solvent accessible portions of AMH protein
[00396] The anti-Mullerian hormone receptor II targeting protein (AMH) and bone morphogenetic protein 7 (BMP7) belong to the same superfamily of proteins and are thought to share similar receptor binding regions (Greenwald et al., 2003). A region in the AMH protein was selected that included several of the residues important for binding of human BMP7 with its cognate receptor II. The mouse AMH peptide, ERISAHHVPNM (SEQ ID No. 164) was identified. The C-terminal methionine was replaced with a glutamate to eliminate the reactive thiol group and to increase peptide solubility, yielding ERISAHHVPNE (AMHa; SEQ ID No. 165). This AMHa peptide was mapped to the surface of the AMH protein model (as described in Example 1) indicating that the selected residues are likely accessible and involved in receptor binding (Fig. 26). The predicted physicochemical properties of the AMHa peptide showed good aqueous solubility, a charge of +0.2 at pH 7, and a ratio of hydrophobic to hydrophilic amino acid residues of 1.
B. An in vitro model - MLTC-1 Leydig cells expressing AMHr
[00397] An in vitro Leydig model cell line was identified for use in targeting the AMHa peptide to the anti -Mullerian hormone receptor II (AMHr). A control cell line that was devoid of AMHr was also identified. The MLTC-1 Leydig cell line was originally purchased from ATCC (established from the M548OP transplantable Leydig cell tumor carried in C57BL/6 mice). According to ATCC (atcc.org/products/crl-2065), the MLTC-1 cells retain the hormonal responsiveness of the original tumor and are stimulated by treatment with human chorionic gonadotropin (hCG) and luteinizing hormone. Although ATCC does not mention the presence of the AMH receptor, both qPCR (Fig. 27A) and immunocytochemistry (Fig. 27B) confirmed that AMHr was expressed in MLTC- 1 Leydig cells but not in the TM4 Sertoli cell line, epididymal epithelial cell line (mECapl8), or the alternative Leydig cell line (TM3). Therefore, the MLTC- 1 cell line was used as an in vitro Leydig model cell line and mECapl8 was used as the negative control cell line. C. AMHa exhibited selective in vitro binding capability
[00398] Using the FACS method described in Example 1 above, the AMHa peptide was confirmed to bind to MLTC-1 cells in vitro in a dose-dependent manner. This was evidenced by a proportional shift in median fluorescence intensity of the cell population across the dose range (Fig. 28). Furthermore, AMHa peptide exhibited selective binding at concentrations of up to 25 pM with four-fold higher median fluorescence intensity when applied to MLTC-1 Leydig cells as compared to the mECapl8 cells across the tested dose range. However, the magnitude of the shift in median fluorescence intensity dropped to two-fold that of the binding to mECapl8 cells at a concentration of 50 pM (Fig. 28), indicating reduced specificity of binding at this higher dose.
D. AMHa target the testes in vivo
[00399] Next, localization of the AMHa-FITC conjugate in the mouse testes was verified using methods described above in Example 1.Either the AMHa-FITC conjugate or the vehicle alone was injected IP into each of three adult male mice per treatment. Eight hours after peptide administration, no significant difference was observed in the weight of mice exposed to either the vehicle or the AMHa-FITC treatments. Similarly, no differences in the weight or gross morphology of the representative tissues collected were observed. AMHa-FITC localized within the testes with strong fluorescence intensity while no fluorescence signal was detected in vehicle-treated males (Fig. 29A). This contrasted with findings on the adrenals (Fig. 29B), brain (Fig. 29C), lungs (Fig. 29F), pituitary (Fig. 29D), or heart (Fig. 29E) for which no difference in fluorescence staining was observed between the vehicle control and the treated animal tissues. However, there was evidence of clearance of the peptides through the detoxifying and excretory organs of the liver (Fig. 29H) and kidney (Fig. 29G), both of which presented with faint fluorescence labelling. White tissue tends to reflect more of the fluorescent signal, therefore, the background fluorescence observed in the adrenal (Fig. 29B), lung (Fig. 29F), and spleen (Fig. 291) tissues is not an indication of the presence of AMHa-FITC.
[00400] Based on these findings, images of the treated testes were imported into ImageJ to compare the mean fluorescence intensity/pixel. The results are expressed as a ratio of the mean fluorescence intensity/pixel of the treated tissue in any image to that of the vehicle control in the same image (Fig. 29 J). This analysis confirmed that the testes of AMHa-FITC- treated males exhibited a significantly higher (p<0.05) ratio of mean fluorescence intensity/pixel compared to the testes of animals treated with the vehicle. E. Male mice treated with AMHa conjugated with Menadione (AMH2Md) exhibited reduced fertility
[00401] Selective targeting of a modified AMH2 peptide conjugated to menadione to the testes was tested in vivo. The AMHa peptide was covalently linked to Md via linker 2 (i.e., y-aminobutanoic acid with MPA). The AMHa-Md conjugate with linker 2 is hereinafter referred to as AMH2Md. The methods used were as described above in Example 1. Specifically, adult male mice were administered with two IP injections of AMH2Md at a concentration (30 mM) approaching that of the compound’s limit of solubility.
[00402] Nine weeks post-injection, male mice treated with AMH2Md were characterized by equivalent testicular weights. However, assessment of mature cauda epididymal spermatozoa revealed an increase in mitochondrial ROS production (Fig. 30A) and propensity for DNA fragmentation (Fig. 30B). These responses were highly variable between the treated males and accordingly, the differences failed to achieve statistical significance. Additionally, treated males presented with equivalent epididymal sperm motility (Fig. 30C) and vitality (Fig. 30D) compared to those of the control subgroup. These similarities also extended to the histology of the testes, which failed to show any notable disruption of spermatogenesis in treated animals (Figs. 31A-B).
[00403] Despite the aforementioned absence of change in the physiology of mature epididymal spermatozoa (i.e., spermatogenesis appears to be normal), there may still be effects on the DNA that may impact fertility. Additionally, AMHr is expressed in the hypothalamus. If the hypothalamo-pituitary-gonadal axis is disrupted, fertility may also be impacted.
[00404] Six weeks post injection, three treated males were housed with 1 untreated female each for 3 consecutive weeks, revealing that the AMH2Md treated male mice exhibited a complete lack of fertility. Indeed, none of the females became pregnant within the 3 weeks despite a mating plug being observed in one of the females (Table 5).
Figure imgf000106_0001
F. Discussion
[00405] As described in Examples 2 and 3 above, synthetic FSH and LH peptides that target the glycoprotein hormone receptors FSHr and LHr, respectively, were developed. This example discusses development of an AMH peptide against a G-protein coupled receptor, AMHr. The synthetic AMHa peptide used exhibited both high binding capacity as well as selectivity in vivo as evidenced by a significantly higher accumulation of fluorescence in treated testes versus vehicle-treated (control) testes, or in any of the other of the representative tissues examined (Figs. 29A-I). After undergoing proteolytic degradation in the blood, liver, kidneys, and small intestine by peptidases and proteases, exogenous peptides are generally eliminated through the detoxifying and excretory organs of the kidneys and liver (Diao & Meibohm, 2013). Indeed, there was some evidence of FITC fluorescence in these excretory tissues that was minimal compared to the vehicle alone (Figs. 29G-H). These data may suggest that AMHa is stable in vivo and persists in the circulation thereby increasing its chance of delivery and binding to the AMHr target. Alternatively, given that the mice were euthanized and the tissues were examined 8 hours post-injection, much of the AMHa-FITC may have already been degraded and excreted leaving AMHa-FITC localized in the testes only.
[00406] Given these in vivo results, AMHa was used to deliver Md, a redox cycling reagent, to the testes in mice. Accordingly, Md was attached to the N-terminal of the peptide via linker 2 and the C-terminal of the AMHa peptide was amidated with the aim of protecting the peptide conjugate from protease degradation (Di, 2015; Diao & Meibohm, 2013; Werle & Bemkop-Schmirch, 2006). This potentially prolonged the in vivo half-life and extended the time that the peptide conjugate was exposed to the target receptor. Additionally, the thiol provided by linker 2 on the N-terminal of the AMH2 (i.e., AMHa that is covalently attached to linker 2), may attach to the reactive C3 of Md via Michael addition. This mode of attachment may negate the alkylating properties of menadione (Klotz et al., 2014) and prevent the adduction of Md to essential cellular biomolecules. Therefore, any damage observed may be attributed to the presence of ROS produced by the redox cycling properties of the Md payload.
[00407] AMH is secreted by Sertoli cells and acts in a paracrine fashion on AMHr, which is highly expressed within the neighboring population of Leydig cells. In Leydig cells, AMH binding acts to decrease P450cl7 and LHr expression, both of which suppress testosterone secretion. Conversely, increased levels of testosterone acts on Sertoli cells via a negative feedback loop to decrease AMH expression (Sansone et al., 2019). In the context of this example, the AMH2Mdmay bind to Leydig cells and, through redox cycling of Md, create sufficient ROS to damage or destroy the Leydig cells. The ROS so generated may also permeate into the seminiferous tubules and create further oxidative damage to Sertoli cells and the developing germ cells they support. Notwithstanding this, mature spermatozoa isolated from the AMH2Md-treated male mice had similar vitality and motility to that of the control mice, and did not exhibit any evidence of oxidative DNA damage (Figs. 30C-E). Although the spermatozoa from this group of treated males presented with a higher percentage of MSR labelling indicative of mitochondrial superoxide anion generation, and a higher average percentage of susceptibility to DNA fragmentation (Figs. 30A-B) compared to the control group, this response was variable and not statistically significant. This result suggests that AMH2Md can result in expression of two hallmarks of the apoptotic process, the dysregulation of mitochondrial electron transport and nuclease-mediated DNA cleavage in spermatozoa. However, if extensive apoptosis had occurred, a decrease in the size and weight of the testes should be observed. Additionally, histological changes should be observed, including a decrease in the cross-sectional area of the seminiferous tubules and sloughing of the germ cells, neither of which were observed (Figs. 31A-B).
[00408] A normal spermatogenic cycle takes type A spermatogonia through differentiation to spermatozoa and, in mice, occurs over approximately 34.5 days (Oakberg, 1956). Therefore, if there was no persistent damage induced by the acute administration of AMH2Md, the treated mice should have normal fertility restored by 6 weeks post-injection. To test this possibility, at 6 weeks post-injection, treated males were housed with untreated females for 3 consecutive weeks. Within this period, none of the females became pregnant despite the observation of a mating plug in one of the females. [00409] AMH also has a hormonal role in the circulation where it acts on those neurons in the brain responsible for the control of reproduction. In the hypothalamus, gonadotropin-releasing hormone (GnRH) neurons express AMHr and on binding AMH these neurons are stimulated to secrete GnRH (Barbotin et al., 2019; Cimino et al., 2016), which is a key regulator of the reproductive axis that signals the hypothalamus to release FSH and LH (Cimino et al., 2016). Although the presence of AMHr in these neurons has been confirmed, the impact of AMH on the hypothalamo-pituitary-gonadal axis has yet to be fully elucidated. Nevertheless, AMH2Md may have bound to neuronal AMHr and adversely impacted the function of these cells. A subsequent decrease in testosterone produced by Leydig cells may have led to decreased differentiation in spermatogenesis as well as a decrease in libido, and a slightly lower sperm count, thus impacting the mating behavior and/or fertility of these males. Additionally, the data from this example recapitulates the phenotype observed in a mutant AMHr mouse model. Only 13% of the homozygous mutant males were able to sire litters. At the same time, spermatogenesis appeared to be normal and the mutant males were able to produce vaginal plugs (Mullen et al., 2019). .
[00410] In this example, an AMHr-targeting peptide was designed that exhibits binding capability and selectivity to Leydig cells in vitro as well as localizing to the testes in vivo. Furthermore, in a proof-of-principle study, using this peptide with a linker designed to enhance redox cycling, Md was successfully delivered mouse testes in vivo. Although this reagent did not produce sufficient ROS to consistently create persistent or significant oxidative damage in the developing male gametes, the treated males exhibited decreased fertility. This peptide might be used either alone or in tandem with other targeting peptides to selectively deliver reagents, such as cytotoxic anticancer agents, or nanoparticles containing cell ablation agents, to the testes to affect permanent sterilization.
Example 5. Design and testing of nanoparticles
[00411] FSH, LH, and AMH peptides were previously identified, as discussed above. The present example compares different nanoparticle constructs (e.g., varying the materials used in the assembly of the nanoparticles, the type of peptides, peptide density, as well as length of spacer-peptides as well as the testing of cell-specific promoters) to determine the optimal compositions to be tested in vitro and in vivo (mouse) with GFP as a reporter, including optimization of promoters to drive transgene expression.
[00412] In general, nanoparticles will comprise poly(lactide-co-glycolide) (PLGA) and PEG polymers. PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions in order to effect drug release. DNA can be condensed with protamine or cationic polyethylenimine (PEI, 2kDa) can be incorporated to further enhance transfection efficiency prior to encapsulation. PEG will form the surface coating on the particle matrix to minimize non-specific particle uptake and clearance by immune cells. Maleimide- terminated PEG will be incorporated into the surface coating, allowing subsequent conjugation of the targeting peptide, synthesized with a terminal cysteine, to the particle surface via a covalent thiol-maleimide linkage (Table 6). Targeting peptides will be introduced onto the surface of PEG-PLGA nanoparticles with varied densities. The resulting targeted nanoparticles will be fluorescently labelled and investigated for body distribution and cellular uptake via flow cytometry and fluorescence microscopy, where fluorescently labelled non-targeted nanoparticles will be used as control. The uptake efficiency of nanoparticles will be correlated with the peptide density on particle surface, which will elucidate the optimum peptide density needed to achieve the most efficient targeted delivery. Transfection efficiency can also be used to assess uptake efficiency of the nanoparticles. The nanoparticles with optimum uptake will then be loaded with genetic payloads (EGFP and DTA gene constructs) to confirm that the latter are expressed in a cell specific manner and, in the case of DTA, precipitate a loss of cell viability. Development of the nanoparticles will include examination of materials used in the assembly of the particles, the type of peptide, peptide density, as well as length of spacer-peptides as well as the testing of cell-specific promoters.
Figure imgf000109_0001
A. Developing the nanoparticle constructs
[00413] The nanoparticles are initially designed with an EGFP transgene. In the best performing constructs, the EGFP transgene will be replaced by a DTA transgene. Constructs will utilize well-characterized cell-specific promoters (e.g., ABP, Rhox5, HSD17B3 (male), initially driving EGFP (control vectors), and later, DTA (induction of cell death). Promoter regions from cat, dog, human, and mouse may also be evaluated. The promoter/gene sequences will be produced as single molecules of DNA using commercial oligonucleotide synthesis, and embedded into lentiviral constructs using standard cloning techniques. [00414] Completed constructs will undergo DNA sequencing and then functional validation (expression of EGFP, or induction of cell death, respectively) via transient transfection into primary cells (Sertoli cells and Leydig cells). The best performing constructs will be packaged into the nanoparticles.
[00415] For packaging into PLGA nanoparticles, it will be assessed whether the condensation of nucleic acid cargo with either protamine or polylysine will further enhance transfection efficiency. Gene delivery to the target cell type by the nanoparticles will be followed by integration of the genetic payload into the cell genome and expression of the transgene (EGFP or DTA respectively). The delivery of vectors containing the EGFP construct will identify targeted cells and be used both to demonstrate efficacy of the targeting technology and the safety of the system by highlighting any off-target gene delivery.
B. Results 1 (N/P Ratio)
[00416] PEGylated polymer/lipid hybrid nanoparticles consisting of PLGA core and DSPE-PEG shell were designed using double emulsion solvent evaporation technique. Cationic polyethylenimine (PEI, 2kDa) were incorporated to improve encapsulation and transfection efficiency of the plasmid DNA (pDNA) encoding RFP.
[00417] One of the key characteristics of nanoparticles in affecting gene transfection efficiency and cytotoxicity is the N/P ratio- a ratio of positively charged amine (N) groups of PEI polymer to negatively charged phosphate (P) groups of DNA. To determine the optimum N/P ratio in terms of transfection efficiency and cytotoxicity, pDNA- PEI/PLGA/DSPE-PEG nanoparticles were prepared with three different N/P ratios (6, 10 and 20) and physicochemical characteristics such as size, polydispersity index (Pdl), and charge were measured using Zetasizer Pro. NPs exhibited negative charge, low (< 0.3) Pdl index, and excellent stability. The N/P ratio of 6: 1 was found to be optimum range for gene transfection.
[00418] A PicoGreen assay for the quantification of pDNA in the presence of PEI (or other cationic polymers) was developed to determine the encapsulation efficiency of pDNA in the nanoparticles, which was above 99%. Further optimizations of the N/P ratio can be made if required following studies of gene transfection efficiency and cytotoxicity of the NPs.
C. Results 2 (Genetic Payloads)
[00419] Plasmid DNA expressing an RFP reporter gene downstream of a ubiquitous promoter was acquired, cloned and bulked up for incorporation into each of the proposed targeted nanoparticle formulations. The ubiquitous promoter that was chosen to drive gene expression is the Cytomegalovirus (CMV) promoter. The CMV promoter is a strong mammalian promoter derived from the human cytomegalovirus and is well described to be active in a broad range of cell types and is the most commonly used promoter in mammalian expression plasmids. Training for use of Precision Nanosystems equipment for the reliable and up-scalable production of nanoparticles commenced, and investigations were made into which cell specific promoters would be suitable for induction of cell specific gene expression.
D. Results 3 (Targeting Peptides)
[00420] Peptides have previously been developed that target the follicle stimulating hormone (FSH) and the luteinizing hormone (LH) receptors of the Sertoli and the Leydig cells, respectively, that are uniquely expressed in the testes. To demonstrate that these peptides specifically target Sertoli and Leydig cell lines in vitro, cells were cultured in growth specific medium and then, when confluent, incubated for 1 hour at 37°C with 50 pM peptide- FITC, washed, fixed and counterstained with DAPI nuclear stain before imaging at 40x magnification.
[00421] Fig. 32A-D show the in vitro binding of peptide-FITC. Fig. 32A shows LH-FITC on Leydig cell line, while Fig. 32B shows FSH-FITC on Sertoli cell line. Cultured cells were treated with a range of peptide-FITC concentrations, from 0 to 50 pM, for one hour at 37°C, washed, then stained with Far red Live/Dead before being assessed by FACS (FACSCanto). Data is presented as the % of live cells that were positive for FITC. (Fig. 32C-D). This data confirms the targeting efficacy of our peptides with the TM3 and TM4 cell lines
[00422] Polymeric nanoparticles were used to encapsulate a fluorescent isothiocyanate (FITC)-dextran cargo. Subsequently, the FSHr and LHr targeting peptides were attached, via their terminal sulfhydryl group, to maleimide on the surface of the nanoparticles. Over 16 hours, the specifically targeted NPs were shown to be taken up by the target cells in vitro at four times the rate of the untargeted NPs and the NPs that had a nonspecific peptide attached, (Figs. 33A-D). The intrinsic phagocytic activity of these cell lines means that over time they will accumulate NPs in a non-specific manner.
[00423] Figs. 33A-D shows in vitro binding and specificity of peptide-targeted nanoparticles (NPs) encapsulating FITC-dextran. Cultured cells were incubated with 2mg/ml of NP- untargeted NP, nonspecific peptide (nsp)NP, or targeted NP - for 24 hours. Cells were then washed, fixed and counterstained with DAPI before imaging at 40x. Fig. 33A shows LH-NP- FITC. Fig. 33B shows FSH-NP-FITC. Leydig cell line (Fig. 33C) and TM4 Sertoli cell line (Fig 33D) were incubated with NPs, as above, for each time point at least 100 cells in each of 3 wells, per treatment per time point, were counted. This procedure was repeated 3 times (n=9) for 4 and 16 hours (n=9), once for 24 hours (n=3). Data represents fold change in binding relative to binding of untargeted NPs at 4 hours. Error bars =standard error. Sertoli cells are naturally phagocytic since they phagocytose the residual body during spermatogenesis and share many features in common with macrophages. The cell line used in the FSH-binding studies, TM4 is known to exhibit phagocytic activity (Tokuda et al., 1992) . Thus, the non-specific binding that was evident after 24 h with TM4 Sertoli cells (Fig. 33D) could be a reflection of a background level of NP uptake due to the intrinsic phagocytic activity of this cell line. With LH-labeled NPs targeting the TM3 Leydig cell line, non-specific binding was not as pronounced but even these cells are known to be capable of the non-specific uptake of nanoparticles (diesel exhaust, titanium dioxide and carbon black) (Komatsu et al., 2008). This intrinsic activity would account for the low background level of non-specific particle uptake depicted in Fig. 33C.
E. Results 4 (Transfection Efficiency)
[00424] PEGylated polymer/lipid hybrid nanoparticles consisting of PLGA core and DSPE-PEG lipid shell were designed. The formulation was optimized for in vitro transfection of the TM4 Sertoli cell line for which FSH-targeted NPs (N/P ratio of 6: 1) carrying pLenti-RFP plasmid DNA displayed 13% of transfection efficiency. This transfection efficiency is relatively low, and the potency (fluorescent brightness) is also weak compared to TM4s transfected with pLenti-RFP plasmid using Lipofectamine 3000 (resulting in a transfection efficiency of 25%). The hybrid NPs demonstrated slow release of the DNA resulting in delayed transfection times compared to Lipofectamine 3000.
[00425] To increase the speed of DNA release and to further enhance transfection, lipid nanoparticles composed of cationic lipids and other helper lipids have been assessed. All lipid components being investigated are FDA approved. A lipid nanoparticle formulation containing DC-Cholesterol exhibited transfection with comparable brightness to Lipofectamine 3000 (Fig. 34). The transfection efficiency (percent of overall cells transfected) was approximately 12% at 96 hours post-treatment.
[00426] To enhance transfection efficiency, lipid nanoparticles composed of ionisable lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) will be further investigated.
[00427] More than 80% of cells may express GFP in a cell specific manner - i.e., >80% of Sertoli cells expressing GFP when targeted by FSH-fiinctionalized NP compared with <10% with NP functionalized using an irrelevant peptide sequence. In addition, >80% of Leydig cells may express GFP when targeted by LH-fiinctionalized NP compared with <10% with NP functionalized with an irrelevant peptide sequence.
[00428] Additionally, polymeric NPs enabled gene expression in vitro 7 days after incubation. This is normal as the DNA was encapsulated in the core of polymeric nanoparticles and the polymer slowly degraded to release DNA. To enable more rapid release of DNA, 100 nm lipid NPs have been developed that should degrade much faster. The lipid composition of these particles will be further optimized in order to ensure maximal gene transfection efficiency.
[00429] Both the lipid and polymeric NP systems can be tested to see which one will work better. The lipid nanoparticles should accelerate the timing of gene expression. The slow and prolonged release characteristic of the polymeric system should have the potential to overcome extracellular barriers and increase the extent and duration of gene expression.
F. In vitro and in vivo testing of nanoparticle comprising DTA gene [00430] In this example, the DTA gene will be incorporated and again tested in vitro and in vivo to confirm targeting and effectiveness of the nanoparticles construct designed in Example 6. One objective is to test in vitro on cultures expressing the FSH receptor for determination of apoptosis. Another objective is to test lead candidates in vivo with immunohistochemistry and use of an optical dissector to determine the absence of target cells (8 weeks post-injection).
G. In vitro
[00431] In order to configure the nanoparticles with the appropriate composition, peptide density, peptide coupling/spacing arrangement and genetic payload, in vitro models will be used. These include: primary cultures of Sertoli cells and cell lines expressing the FSH or LH receptor developed in-house.
[00432] A single cellular model (FSH peptide-functionalized nanoparticles binding to FSH-R-expressing stable cell line) will initially be used to monitor reporter protein (e.g., GFP) expression to determine relative rates of nanoparticle incorporation and expression. This should rapidly lead to identification of exemplary nanoparticle compositions (in terms of the peptide density on the nanoparticle surface and optionally including linkage spacers). These in vitro studies will help identify exemplary SC-targeting and LC-targeting nanoparticles for confirmation of GFP gene expression in vivo.
[00433] Once the ability of the nanoparticle construct to achieve reporter protein expression in vitro is confirmed, nanoparticles containing a genetic payload encoding DTA under the regulation of cell- and species- specific promoters will be constructed. [00434] The ability of these nanoparticles to induce cell death in vitro will be tested by conducting dose- and time- dependent studies using the model systems (LH peptide- functionalized nanoparticles targeting TM3 cells and FSH peptide-functionalized nanoparticles targeting FSH-R-expressing cells). Experiments involving the exposure of control cells (e.g., GC1) not expressing receptors for these gonadotrophins will also be conducted. These in vitro data may confirm the nanoparticles are capable of delivering cell death in a highly selective, targeted, efficient manner.
H. In vivo
[00435] The ability of the nanoparticle constructs described above to target Sertoli cells (male) and Leydig cells (male) to induce cell ablation can next be assessed in systematic studies. The structure of the nanoparticles will be based on the in vitro studies described above, which will have led to exemplary nanoparticle compositions in terms of lipid-PEG content, the density and configuration of the cell-specific targeting peptides incorporated into the nanoparticle surface and the structure of the genetic pay load inserted into the nanoparticle interior.
[00436] Using a single dose administration regime, two potential nanoparticle formulations will initially be assessed in vivo'. (1) nanoparticles expressing FSH peptides to target Sertoli cells in males; and (2) LH-functionalized nanoparticles to target Leydig cells in males. Each of these formulations will be assessed at 3 different doses.
[00437] The in vivo assessment of the biological efficacy of the nanoparticles will be conducted in juvenile adult Swiss mice (6-8 weeks old). The in vivo assessment will be conducted with functionalized nanoparticles initially administered by intraperitoneal injection.
[00438] The nanoparticles will only be administered on a single occasion and the effects of the treatment will be monitored at autopsy 8 weeks later. Controls animals will receive injections of identical nanoparticles minus the genetic payload. A total of 10 animals will be included in each experimental group (n=10 SC-targeting; n=10 LC-targeting; n=10 controls, x 2 promoters for each cell-type; total mice = 60) in order to provide sufficient data for statistical analyses by ANOVA.
[00439] To assess EGFP expression, target (testes) and off-target (liver, brain, kidney) tissues will be collected, fixed for 24 hr in 4% paraformaldehyde in PBS, sectioned and examined by fluorescence microscopy employing an excitation wavelength of 488 nm.
[00440] To assess DTA expression, two different promoters and three doses will be tested, the identical dose to the GFP -expressing nanovectors plus two others to be determined empirically with a view to identifying the minimal dose to achieve sterility. (n=10 SC-1285 targeting per dose; n=10 LC -targeting per dose; n=10 controls per dose, per dose = 90 mice in total). Cell counts will be conducted using stereological methods as described previously(Rebourcet et al., 2017). These cell counts will focus on the particular cell type targeted for ablation - Sertoli cells and Leydig cells.
[00441] A histological assessment of the quality of spermatogenesis and gonadal architecture respectively will occur in the same tissues.
[00442] Immunohistochemical determination of induced apoptosis will be supported by stereological analyses employing the optical disector technique, to assess the induction of target cell ablation by quantifying the number of Sertoli and Leydig- cells in each testis. The nanoparticles may be capable of inducing cell death in > 80% of the targeted cell population (Sertoli and Leydig cells) in vivo.
[00443] The in vivo studies will involve detailed monitoring of the animals to ensure general health and wellbeing (body weight, posture, vocalization, physical activity) and include, at the time of autopsy, a visual inspection of the major organ systems for signs of systemic damage.
I. Long-term fertility assessment
[00444] Any nanoparticle constructs that are found to possess biological activity in the in vivo assessment above will be submitted for fertility trials in male mice to confirm that a state of sterility has been induced. Fertility assessments will be conducted to ensure that target cell ablation is accompanied by permanent infertility. Fertility trials will be conducted 8 weeks after inoculation and repeated at 12 months to ensure that the infertility induced is permanent.
[00445] These fertility trials will involve housing treated male mice (n=20 SC- targeting; n=20 LC targeting; n=20 controls; + 60 female partners) with mating partners and monitoring the incidence of pregnancy in addition to a full histological examination of the major organ systems, 10 days after mating and 8 weeks after inoculation with the nanoparticles.
J. Xenograft study in mice
[00446] In vivo mouse testing to confirm nanoparticle activity will be performed with the cat and dog gonadal tissue xenograft model in which immunologically compromised nude mice are xenografted with cat and dog ovarian and testicular tissue. This permits testing of efficacy directly in cat and dog gonadal tissue in vivo.
[00447] Mice are naturally resistant to Diphtheria toxin (the murine HBEGF receptor binds the toxin only poorly). A model will be used, in which expression of a transgene for Diphtheria toxin fragment A (DTA) is driven inside cells to induce cell death. This model was previously used to specifically ablate the Sertoli cell population from mouse embryos in vivo, with no off-target effects (Rebourcet et al., 2014).
K. Xenografting procedure
[00448] Owner-consented cat and dog ovaries and testes will be collected at 4 months of age during routine neutering.
[00449] Male CD 1 nude mice will be anesthetized by inhalation of isofluorane and castrated/ovarectomised. Removal of gonads ensures that the host mouse gonadotrophins are high in order to drive the maturation of the gonadal tissue; an important and well-characterized interaction between the hypothalamus/pituitary and the xenografted gonad (Honaramooz et al., 2002; Mitchell et al., 2010) . Subsequently, steroid hormone production by the gonads feeds back to the brain to maintain steady state gonadal function (Honaramooz et al., 2002).
[00450] In males, small pieces (3 mm3 approx.) of donor testis tissue from a total of six male dogs and six male cats will be inserted subcutaneously under the dorsal skin after aseptic preparation of the skin of the mice using povidone or chlorhexidine scrubbing plus swabbing with ethanol. Between 4-6 testis grafts from a single cat/dog will be inserted on either side of the midline; six mice per cat/dog donor (72 nude mice in total). All surgical procedures will be carried out within a Class II containment cabinet and mice will be housed in individually ventilated cages (IV C) in order to provide a sterile environment and avoid any potential for transmission of pathogens within/between species. Mice will receive analgesia (Rimadyl LA, Pfizer, New York, USA) in the drinking water for 1 day pre- and 5 days post- surgery, as well as antibiotics (Baytril, Bayer, Germany) for 5 days post-surgery, and will be monitored twice daily.
L. Characterization of xenografts
[00451] A genetic pay load containing a DNA sequence encoding only fragment A of the toxin is delivered into cells, through binding of nanoparticles to endogenous cell-specific receptors (e.g., FHSR, AmhR etc). Once inside, the cell expression of the transgene for fragment A of the toxin is induced, which then functions to drive cell death (e.g., within 24 hours). Cell targeting and expression of GFP (controls) or induction of cell ablation (DTA nanoparticles) will then be assessed.
[00452] Host mice will be anesthetized until unconscious and then culled by cervical dislocation and grafts will be retrieved by dissection. Xenografts will be weighed and the percentage survival of xenografts will be recorded. Fragments of pre-graft, xenograft and equivalent age-matched control tissues will be snap frozen for molecular analysis, and the remainder fixed for 2 hours in Bonin’s fixative, transferred to 70% ethanol and then processed into paraffin blocks using standard procedures.
[00453] For testes, the presence of spermatogenesis, recording the most advanced stage of germ cell development observed for each graft, will be determined histologically, and compared to non-grafted age-matched controls, with reference to control data (e.g., previous published studies (Abrishami et al., 2010; Snedaker et al., 2004)). Immunohistochemical markers for each cell-type (e.g., SOX9 - Sertoli cells; aSMA -Peritubular Myoid cells/vascular smooth muscle; 3BHSD - Leydig cells, Stra8 - Spermatogonia; SOG1- Spermatocytes; Protamine 1 or PGK1/2 - Spermatids; Mac2/CD68 - Macrophages; CD31 - Endothelial cells) will be assayed to establish any changes in cellular ratios. Markers reflective of targeting such as GFP, or cleaved caspase 3 (apoptosis marker) will also be examined.
M. Preliminary clinical trials- male and female cats and dogs
[00454] The two most efficacious nanoparticle constructs based on the studies performed above (male and female) will be used in in vivo testing in dogs and cats.
[00455] The pilot study will include 6 male dogs with one construct. Following a successful pilot study, the wider trial in both genders in both cats and dogs will be completed.
[00456] In general, to investigate whether steroid hormone production is only affected in the gonadal cells and not in adrenocortical cells, before and every 2 months after the treatment, or if clinical symptoms suggestive of abnormal adrenal steroidogenesis are present, an ACTH stimulation test (with measurement of cortisol and aldosterone) will be performed. In addition, the circulating ACTH/cortisol ratio and aldosterone/renin ratio will be determined in dogs and cats in which the gene(s) encoding steroidogenic enzymes have been targeted before and every 2 months after the treatment.
N. Male dogs
[00457] A total of 6 adult male beagle dogs with two scrotal testes will be used in this study. Before treatment, a general physical examination will be followed by an andrological examination involving ultrasonographical measurement of prostate dimensions. In addition, a pre-treatment Gonadotropin-releasing hormone (“GnRH”) stimulation test (de Gier et al., 2012), will be performed to study the effects of the treatment on the hypothalamic -pituitary-gonadal (HPG) axis and spermatogenesis.
[00458] Circulating LH and testosterone concentrations will be determined before and after the administration of a single intravenous bolus of a GnRH analogue (Buserelin). Furthermore, semen will be collected and evaluated for volume, progressive motility, morphology and sperm count.
[00459] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.
[00460] After the treatment, a general physical examination and andrological examination are performed on a weekly basis. Semen evaluation and a GnRH stimulation test will be performed on a monthly basis after the treatment. A unilateral orchiectomy will be performed under general anesthesia, 6 months after treatment. The removed testis will be examined for the presence of spermatogenesis and overall histological changes. Three and 6 months after hemicastration, the semen will be evaluated and a GnRH stimulation test will be performed to show persistence of the treatment effect.
O. Male cats
[00461] Six adult Domestic Shorthaired tomcats will be used. Only cats with two scrotal testes will be included in the study. Treatment will occur similarly to that for the dogs. Before- and on a monthly basis after the treatment, a GnRH stimulation test as described above will be performed, the testes size will be determined and the penis will be examined for the presence of penile spines, which is correlated to the plasma testosterone concentration. Semen will be collected by urethral catheterization (Zambelli et al., 2008).
[00462] The removed testis will be examined using identical endpoints to those used for dogs (see above). Three and 6 months after hemicastration the semen will be evaluated and GnRH stimulation test will be performed.
P. Female dogs
[00463] Six adult anestrous female dogs will be used. All dogs will be examined thrice weekly for swelling of the vulva and serosanguineous vaginal discharge, signifying the onset of pro-estrus (Schaefers-Okkens, 2000). Plasma progesterone concentration will be measured thrice weekly from the start of pro-estrus until it exceeded 13-16 nmol/1, at which time ovulation is assumed to occur (Concannon et al., 1977; Okkens et al., 1985; Wildt et al., 1979). Anestrus is defined as the period from 100 days after ovulation to the onset of pro-estrus, as indicated by vulvar swelling and serosanguineous discharge. Only female dogs who showed regular ovulatory estrous cycles will be included in this study.
[00464] A general physical examination will be performed before the start of the study, as well as a GnRH stimulation test (but measuring estradiol instead of testosterone). [00465] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.
[00466] Additionally, a GnRH stimulation test will be performed on a monthly basis until 6 months after treatment and at 9 and 12 months after treatment, and the plasma progesterone will be determined every 2 weeks to evaluate reproductive (endocrine) function. If the plasma progesterone concentrations remain <1 ng/ml and when no signs of pro-estrus have been detected for a period longer than the mean interestrus interval + 2 months, it will be attempted to induce estrus by using a slow release GnRH analogue implant (Suprelorin®, Virbac). In healthy, untreated female dogs, (pro)estrus can be expected within 10 days after Suprelorin® administration (Fontaine et al., 2011; Maenhoudt et al., 2012) . One month after Suprelorin® administration a unilateral ovariectomy will be performed under general anesthesia. The dissected ovary will be examined for targeting success, the presence of steroidogenic enzymes and follicular development. In case of complete disruption of ovarian function, it is expected that (pro) estrus will fail to occur, the plasma progesterone concentration will remain below 1 ng/ml, which indicates infertility.
Q. Female cats
[00467] Six adult Domestic Shorthaired female cats will be used. The female cats will be housed in a group and a vasectomized tomcat will be introduced to enhance estrus detection and induction of ovulation. Before and after the treatment, the female cats will be examined twice weekly for the presence of estrous signs: rolling, head rubbing, treading with hind legs, lordosis and tail deviation. Once a month the plasma progesterone concentration will be determined until the end of the study. A plasma progesterone concentration >1 ng/ml indicates the presence of a corpus luteum. The fecal oestradiol concentration will be determined before- and on a weekly basis after the treatment.
[00468] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.
[00469] Before and every two months until 6 months after treatment, and at 3 and 6 months after unilateral ovariectomy, a GnRH stimulation test will be performed. If estrous signs failed to occur at 6 months after the treatment, it will be attempted to induce estrus by using Suprelorin®. In anestrous female cats, estrus can be expected within 12 days (Zambelli et al., 2008). One month after Suprelorin® administration a unilateral ovariectomy will be performed under general anesthesia and six months later, the other ovary will be removed. The ovaries will be histologically examined for the presence of folliculogenesis. References
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[00470] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
[00471] While various specific embodiments have been illustrated and described, it will be appreciated that changes can be made without departing from the spirit and scope of the invention(s).

Claims

What is Claimed is:
1. An isolated Follicle-Stimulating Hormone (FSH) peptide comprising a fragment of an FSH protein or variant thereof, wherein the FSH peptide comprises: a) an amino acid sequence of TVX9GLGPX10Y (SEQ ID No. 95), wherein X9 is R or Q and X10 is S or G; b) an amino acid sequence of CTVX9GLGPX10Y (SEQ ID No. 96), wherein X9 is R or Q and X10 is S or G; c) an amino acid sequence of RDLVYX1DX2ARPX3X4Q (SEQ ID No. 33), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; and X4 is T or N; d) an amino acid sequence of RDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 34), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T or N; e) an amino acid sequence of CRDLVYX1DX2ARPX3X4Q (SEQ ID No. 35), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T or N; f) an amino acid sequence of CRDLVYX1DX2ARPX3X4QX1 (SEQ ID No. 36), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, orN; and X4 is T or N; g) an amino acid sequence of YTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 49), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V; h) an amino acid sequence of CYTRDLVYX1DX2ARPX3X5QX1X6 (SEQ ID No. 50), wherein Xi is K or G; X2 is A or P; X3 is K, S, G, or N; X5 is I, T, or N; and Xe is T or V; i) an amino acid sequence of CHX7GX8X7DSDSTDX7T (SEQ ID No. 63), wherein X7 is C, S, or A and Xs is K, R, or G; j) an amino acid sequence of X8CDSDSTDX7TVX9GL (SEQ ID No. 78), wherein Xs is K, R, or G; X7 is C, S, or A; and X9 is R or Q; k) an amino acid sequence of CDSDSTDX7TVX9GL (SEQ ID No. 79), wherein Xs is K, R, or G; X7 is C, S, or A; and X9 is R or Q; l) an amino acid sequence of SDSTSX7TVX9GLGPX10Y (SEQ ID No. 89), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G; and/or m) an amino acid sequence of CSDSTSX7TVX9GLGPX10Y (SEQ ID No. 90), wherein X7 is C, S, or A; X9 is R or Q; and X10 is S or G.
2. An isolated Follicle-Stimulating Hormone (FSH) peptide comprising a fragment of an FSH protein or a variant thereof, wherein the FSH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No.
41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No.
67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
3. The isolated FSH peptide of claim 1 or claim 2, wherein the FSH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and/or SEQ ID No. 18.
4. The isolated FSH peptide of any one of claims 1 to 3, wherein the peptide comprises the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No.
42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SE ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No.
68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 91, SEQ ID No. 92, SEQ ID No. 93, and/or SEQ ID No. 94.
5. The isolated FSH peptide of any one of claims 1 to 4, wherein the peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
6. An isolated Luteinizing Hormone (LH) peptide comprising a fragment of an LH protein or variant thereof, wherein the LH peptide comprises: a) an amino acid sequence of SX18SDX7GGPX8X19X20X21 (SEQ ID No. 144), wherein Xis is T, S, or N; X7 is C, S, or A ; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S; b) an amino acid sequence of CSX18SDX7GGPX8X19X20X21, (SEQ ID No. 145), wherein Xis is T, S, or N; X7 is C, S, or A; Xs is K, R, or G; X19 is D, T, or A; X20 is H or Q; and X21 is P or S; c) an amino acid sequence of X11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 112), wherein X11 is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V; d) an amino acid sequence of CX11RVLX12AX13LPPX14PX15X16 (SEQ ID No. 113), wherein X11 is M or V, X12 is Q, P, or G; X13 is V or A; X14 is V or L; X15 is Q or G; and Xi6 is P or V; e) an amino acid sequence of SFPVALX7RX7GPX7RX17 (SEQ ID No. 125), wherein X7 is C, S, or A and X17 is L or R; f) an amino acid sequence of CSFPVALX7RX7GPX7RX17 (SEQ ID No. 126), wherein X7 is C, S, or A and X17 is L or R; and/or g) an amino acid sequence of CRX17SX18SDX7G (SEQ ID No. 133), wherein X17 is L or R; Xis is T, S, orN; and X7 is C, S, or A.
7. An isolated Follicle-Stimulating Hormone (LH) peptide comprising a fragment of an LH protein or a variant thereof, wherein the LH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
8. The isolated LH peptide of claim 6 or claim 7, wherein the LH peptide has one substitution modification, two substitution modifications, three substitution modifications, four substitution modifications, five substitution modifications, or six substitution modifications relative to SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, and/or SEQ ID No. 104.
9. The isolated LH peptide of any one of claims 6 to 8, wherein the peptide comprises the amino acid sequence of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. I l l, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, SEQ ID No. 124, SEQ ID No. 127, SEQ ID No. 128, SEQ ID No. 129, SEQ ID No. 130, SEQ ID No. 131, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 135, SEQ ID No. 136, SEQ ID No. 137, SEQ ID No. 138, SEQ ID No. 139, SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, and/or SEQ ID No. 143.
10. The isolated LH peptide of any one of claims 5 to 9, wherein the peptide is 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids in length.
11. An isolated Anti-Mullerian Hormone (AMH) peptide comprising a fragment of an AMH protein or a variant thereof, wherein the AMH peptide comprises an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to the amino acid sequence of SEQ ID No. 5, SEQ ID No. 165, SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
12. The isolated AMH peptide of claim 11, wherein the AMH peptide has one substitution modification, two substitution modifications, three substitution modifications, or four substitution modifications relative to SEQ ID No. 149, SEQ ID No. 153, SEQ ID No. 157, and/or SEQ ID No. 161.
13. The isolated AMH peptide of claim 11 or claim 12, wherein the peptide comprises the amino acid sequence of SEQ ID No. 5, SEQ ID No. 165, SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 167, SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 170, and/or SEQ ID No. 171.
14. The isolated AMH peptide of any one of claims 11 to 13, wherein the isolated AMH peptide is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length.
15. The isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 14, wherein the peptide is amidated.
16. The isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 15, wherein the peptide comprises a cysteine residue at the N-terminus.
17. The isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 16, wherein the peptide comprises a linker.
18. The isolated FSH, LH, and/or AMH peptide of claim 17, wherein the linker is a maleimide group, 1,6-aminohexanoic acid (Ahx), y-aminobutanoate-mercaptopropionic acid, alanine with 3 -mercaptopropionic acid (MPA), y-aminobutanoic acid with MPA, minipolyethyleneglycol with MPA, or a cathepsin cleavable valine-citrulline(vc)-PABC linker.
19. The isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 18, wherein the peptide is conjugated to a protein, a peptide, a nanoparticle, a label, a small molecule, and/or other moiety.
20. The isolated FSH, LH, and/or AMH peptide of claim 19, wherein the protein is an apoptotic protein or a cytotoxic protein.
21. The isolated FSH, LH, and/or AMH peptide of claim 19, wherein the small molecule is an auristatin (Aur) molecule, such as such monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), or a menadione (Md) molecule, such as 2-methyl-l,4-naphthoquinone.
22. The isolated FSH, LH, and/or AMH peptide of claim 19, wherein the label is fluorescein isothiocyanate (FITC).
23. A composition comprising a peptide conjugate comprising the isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 22.
24. A nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell, wherein the one or more peptides comprises a fragment of a protein or a variant thereof that binds the receptor of the reproductive cell, optionally wherein the one or more peptides is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length; and b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein.
25. A nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell, wherein the one or more peptides comprises a fragment of a protein or a variant thereof that binds the receptor of the reproductive cell, optionally wherein the one or more peptides is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length; and b) a reporter protein or a polynucleotide encoding a reporter protein.
26. A nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell, wherein the one or more peptides comprises a fragment of a protein or a variant thereof that binds the receptor of the reproductive cell, optionally wherein the one or more peptides is 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, or 8 amino acids in length; b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein; and c) a reporter protein or a polynucleotide encoding a reporter protein.
27. The nanoparticle construct of claim 24 or 26, wherein the nanoparticle construct comprises a polynucleotide encoding the apoptotic or cytotoxic protein.
28. The nanoparticle construct of claim 24, 26 or 27, wherein the apoptotic protein is Diphtheria toxin fragment A (DTA).
29. The nanoparticle construct of claim 28, wherein the DTA comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:
142
30. The nanoparticle construct of claims 25 or 26, wherein the reporter protein is selected from red fluorescent protein (RFP), green fluorescent protein (GFP), and enhanced green fluorescent protein (EGFP).
31. The nanoparticle construct of any one of claims 24 to 30, wherein the reproductive cell is a Sertoli cell and/or a Leydig cell.
32. The nanoparticle construct of any one of claims 24 to 31, wherein the one or more peptides bind a receptor of a Sertoli cell and/or a Leydig cell.
33. The nanoparticle construct of any one of claims 24 to 32, wherein the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell and/or the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.
34. The nanoparticle construct of any one of claims 24 to 33, wherein the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell.
35. The nanoparticle construct of any one of claims 24 to 34, wherein the one or more peptides comprises the isolated FSH, LH, and/or AMH peptide of any one of claims 1 to 22.
36. The nanoparticle construct of any one of claims 24 to 35, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 1 or SEQ ID No: 2.
37. The nanoparticle construct of any of claims 24 to 36, wherein the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.
38. The nanoparticle construct of any one of claims 24 to 37, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 3 or SEQ ID No: 4.
39. The nanoparticle construct of any one of claims 24 to 38, wherein the reproductive cell is a gonocyte, a primordial follicle cell, and/or a primordial germ cell.
40. The nanoparticle construct of any one of claims 24 to 39, wherein the one or more peptides bind an anti-Mullerian hormone on a primordial follicle cell.
41. The nanoparticle construct of any one of claims 24 to 40, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 5 or SEQ ID No. 165.
42. The nanoparticle construct of any one of claims 24 to 41, wherein the one or more peptides bind a primordial germ cell.
43. The nanoparticle construct of any one of claims 24 to 42, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 6.
44. The nanoparticle construct of any one of claims 24 to 43, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 7.
143
45. The nanoparticle construct of any one of claims 24 to 44, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 8.
46. The nanoparticle construct of any one of claims 24 to 45, wherein the one or more peptides comprise an amino acid sequence of SEQ ID No: 9.
47. The nanoparticle construct of any one of claims 24 to 46, wherein the one or more peptides contain an N-terminal cysteine residue.
48. The nanoparticle construct of any of claims 24 to 47, wherein the polynucleotide encoding the apoptotic or cytotoxic protein is operatively linked to a promoter.
49. The nanoparticle construct of claim 48, wherein the promoter is a cell-specific promoter.
50. The nanoparticle construct of claim 48 or 49, wherein the promoter is selected from the group consisting of ABP, Rhox5, and HSD17B3.
51. The nanoparticle construct of any one of claims 48 to 50, wherein the promoter is a species-specific promoter.
52. The nanoparticle construct of any of claims 24 to 51, wherein the nanoparticle construct comprises poly(lactide-co-glycolide) (PLGA).
53. The nanoparticle construct of any one of claims 24 to 52, wherein the nanoparticle construct comprises polyethylene glycol (PEG).
54. The nanoparticle construct of any one of claims 24 to 53, wherein the nanoparticle construct comprises maleimide-terminated PEG.
55. The nanoparticle construct of any one of claims 24 to 54, wherein the nanoparticle construct comprises DSPE-PEG.
56. The nanoparticle construct of any one of claims 24 to 55, wherein the nanoparticle construct comprises cationic lipids, ionizable lipids, and/or helper lipids.
57. The nanoparticle construct of any one of claims 24 to 56, wherein the one or more peptides are conjugated to lipids.
58. The nanoparticle construct of any one of claims 24 to 57, wherein the mean peptide density is from 5,000 to 50,000 peptides per nanoparticle.
59. The nanoparticle construct of any one of claims 24 to 58, wherein the polynucleotide is condensed with protamine, lysine, or polylysine.
60. The nanoparticle construct of any one of claims 24 to 59, wherein the apoptotic or cytotoxic protein or the polynucleotide is encapsulated within the nanoparticle construct.
61. The nanoparticle construct of any one of claims 24 to 60, wherein the nanoparticle construct comprises a nanoparticle having a PLGA matrix with maleimide terminated PEG
144 lipids; wherein the one or more peptides are conjugated to the maleimide terminated PEG lipids; and wherein the apoptotic or cytotoxic protein or the polynucleotide is encapsulated in the nanoparticle.
62. The nanoparticle construct of any one of claims 24 to 61, wherein the nanoparticle construct comprises a viral vector comprising the polynucleotide encoding the apoptotic or cytotoxic protein.
63. The nanoparticle construct of claim 62, wherein the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector.
64. The nanoparticle construct of claim 62 or 63, wherein the viral vector is a lentiviral vector.
65. The nanoparticle construct of any one of claims 24 to 64, wherein the nanoparticle construct further comprises a fluorescent label.
66. A pharmaceutical composition comprising the isolated FSH, LH, or AMH peptide of any one of claims 1 to 22, the composition of claim 23, or the nanoparticle construct of any one of claims 24 to 65.
67. The pharmaceutical composition of claim 66, further comprising a pharmaceutically acceptable carrier.
68. The pharmaceutical composition of claim 66 or claim 67, comprising an aqueous solution.
69. The pharmaceutical composition of any one of claims 66 to 68, wherein the pharmaceutical composition, the isolated FSH, LH, or AMH peptide, the composition, or the nanoparticle construct is freeze-dried.
70. A method of sterilizing a subject comprising administering to the subject an effective amount of the isolated FSH, LH, or AMH peptide of claim 21 or claim 22, the nanoparticle construct of any one of claims 24 to 65, or the pharmaceutical composition of any one of claims 66 to 69.
71. The method of claim 70, wherein the isolated peptide, the nanoparticle construct, or the pharmaceutical composition is freeze-dried as a powder and dispersed in an aqueous medium prior to administration.
72. The method of any one of claims 70 or 71, wherein the composition is administered via injection.
73. The method of any one of claims 70 to 72, wherein the administering comprises a onetime injection.
145
74. The method of any one of claims 70 to 73, wherein the administering is intravenous, intraperitoneal, or intratesticular administration.
75. The method of any one of claims 70 to 74, wherein the administering is intravenous injection.
76. The method of any one of claims 70 to 75, wherein the administering is a one-time intravenous injection into the cephalic vein.
77. The method of any one of claims 70 to 76, wherein the effective amount comprises a dosage capable of inducing sterilization of the subject.
78. The method of any one of claims 70 to 77, wherein sterilization or ablation occurs within 24 hours, 48 hours, or 72 hours after injection.
79. The method of any one of claims 70 to 78, wherein sterilization is permanent.
80. The method of any one of claims 70 to 79, wherein the effective amount comprises a dosage capable of inducing ablation of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of one or more reproductive cell types of the subject.
81. The method of any one of claims 70 to 80, wherein the effective amount comprises a dosage capable of inducing ablation of at least about 60% of one or more reproductive cell types of the subject.
82. The method of any one of claims 70 to 81, wherein the effective amount comprises a dosage capable of inducing ablation of at least about 80% of one or more reproductive cell types of the subject.
83. The method of any one of claims 80 to 82, wherein the one or more reproductive cell types of the subject is Sertoli cells and/or Leydig cells.
84. The method of any one of claims 80 to 83, wherein the one or more reproductive cell types of the subject is gonocytes, primordial follicle cells, and/or primordial germ cells.
85. The method of any one of claims 70 to 84, wherein the subject is a cat or dog.
86. The method of any one of claims 70 to 85, wherein the subject is a cat.
87. The method of any one of claims 70 to 85, wherein the subject is a dog.
88. The method of any one of claims 70 to 85, wherein the subject is a male cat or dog.
89. The method of any one of claims 70 to 85, wherein the subject is a female cat or dog.
90. The method of any one of claims 70 to 89, wherein the subject is pre-pubescent.
91. The method of any one of claims 70 to 89, wherein the subject has reached pubertal maturation.
146 A method of determining uptake efficiency of a nanoparticle construct, comprising: a) administering to a subject the nanoparticle construct of claim 25 or a composition comprising the nanoparticle construct of claim 25; b) assessing ligand density on the nanoparticle surface, wherein increased ligand density or transfection efficiency for the nanoparticle as compared to a control or reference value is indicative of optimal uptake efficiency.
147
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