WO2019152700A1 - Aptamer-rhdl composites for treating diseases - Google Patents

Abstract

The invention includes in vivo delivery of aptamers by coupling them with reconstituted HDL (rHDL) nanoparticles. Intracellular transport of the aptamer-rHDL nanoparticles may be mediated by the scavenger receptor class B type 1 (SRB 1) also known as SR-BI. The aptamers can be used in downregulation or silencing of genes and receptors associated with cancers, tumors or other diseases, including Alzheimer's.

Description

Aptamer-rHDL Composites for Treating Diseases

FIELD OF INVENTION:

The invention relates to reconstituted high density lipoprotein (HDL) particles for the delivery of aptamers into cells and tissues for the treatment of various cancers and neurodegenerative diseases, including Alzheimer’s.

BACKGROUND:

U.S. Patent No. 8,734,853 describes pharmaceutical compositions that include a) an apolipoprotein; (b) a nucleic acid component comprising a therapeutic nucleic acid segment; and (c) a polypeptide comprising a positively charged region. The therapeutic nucleic acid segment that may be nucleic acid that encodes a therapeutic agent, such as a protein; or a therapeutic nucleic acid that may inhibit the expression of a gene. The nucleic acid component may be a DNA or a RNA. In specific embodiments, the nucleic acid component include an interference RNA. For example, the interference RNA may be a siRNA, or a nucleic acid encoding a siRNA.

Aptamers are a class of small ligands derived from short nucleic acid sequences which are artificially generated using DNA or RNA nucleotides and isolated through laboratory selection techniques. However, unlike the DNA and RNA sequences described in U.S. Pat.No. 8,734,853, aptamers are not capable of encoding for proteins, or of binding to or interfering with the action of rnRNAs. Rather, they act as binding agents (like small molecule drugs) - binding to specific molecular targets with high specificity to bring about therapeutic activity.

Aptamers are relatively short (usually from 20 to 60 nucleotides) single-stranded RNA or DNA oligonucleotides, able to bind target molecules with high affinity and specificity. Currently, a large number of generated aptamers can bind various targets, ranging from simple inorganic molecules to large protein complexes, and entire cells. These targets include lysozyme [Potty, A et al., 2009] thrombin [Long, S et al., 2008] human immunodeficiency virus trans-acting responsive element (HIV TAR), [Darfeuille, F et al., 2006] hemin [Liu, M.; T. Kagahara et al., 2009] interferon g [Min, K. et al., 2008] vascular endothelial growth factor (VEGF) [Ng, E.W.M et al., 2006] prostate specific antigen (PSA) [Savory, N et al. 2010; Jeong, S et al., 2010] dopamine [Walsh, R.; M. DeRosa (2009)] and the non-classical oncogene, heat shock factor 1 (HSF1) [Salamanca, HH (2014)]. Aptamers may be considered nucleotide analogues of antibodies, which are more expensive and difficult to generate with binding specificity, than analogous aptamers. Moreover, aptamers are neither immunogenic nor toxic, making aptamers near-ideal candidates for diagnostic and therapeutic applications, purification of target molecules from complex mixtures, biosensor design, etc.

However, the rapid degradation of aptamers (especially RNA aptamers) by nucleases in biological media, and in blood in particular, limits their practical application. The average time of oligonucleotide decay in blood ranges from several minutes to several tens of minutes depending on the oligonucleotide concentration and conformational structure. Such short circulation times are unacceptable for most therapeutic applications.

The removal of aptamers from the bloodstream via renal filtration also complicates their therapeutic application. Most aptamers have a molecular weight ranging from 5 to 15 kDa (15-50 nucleotides), and they can readily be excreted by the kidneys - which are generally capable of removing substances with a molecular weight below 30-50 kDa. Conjugation of aptamers with polyethylene glycol (PEG) with a molecular weight of 20 or 40 kDa, is one attempted solution to this problem.

Current examples of aptamers consisting of a particular type of modified oligonucleotides, i.e., those which are SELEX-modified (abbreviation for: Systemic Evolution of Ligands by Exponential Enrichment), which are in clinical use include: Macugen (the only aptamer currently approved for medical application) which targets VEGF (Ng et al. 2006; Siddiqui and Keating 2005). The following aptamers (generated with modified oligonucleotides or as PEG-coupled oligonucleotides) are or have been in clinical trials for a variety of indications, including: ARC1905 (targeting C5) (Biesecker et al. 1999) and E-10030 (targeting PDGF) (Boyer 2013), REG1 (targeting Coagulation factor IXa) (Povsic et al. 2013), ARC1779 (targeting the Al domain of von Willebrand factor) (Schattauer GmbH et al. 2017), NU172 (targeting thrombin) (Lu et al. 2009), AS1411 (targeting nucleolin) (Bates et al. 2009), NOX-A12 (targeting CXCL12) (Hoellenriegel et al. 2014), NOX-E36 (targeting CCL2) (Kulkarni et al. 2007) and NOX-H94 (targeting Hepcidin peptide hormone) (Schwoebel et al. 2013).

The majority of aptamers for clinical use are designed to bind to extracellular targets, as another problem with current aptamer technology is the difficulty of penetrating cell membranes or crossing the blood-brain barrier to deliver aptamers intracellularly. This limitation severely limits their therapeutic potential, particularly for the treatment of diseases such as cancers or Alzheimer’s which are frequently caused or exacerbated by protein dysregulation within the cell. Some advances in the intracellular delivery of aptamers have recently been achieved. Special expression systems may generate aptamers inside cells and ensure their accumulation either in nucleus or in the cytoplasm. Cell-type-specific aptamer synthesis can be achieved by using directional viral expression systems that deliver vectors to particular cells. The concentration of expressed aptamers (also known as intramers) can be increased not only by using strong promoters that ensure a high level of expression, but also by limiting the rate of aptamer degradation by nucleases through protection of the 3’- and 5’- termini with additional structures (e.g., hairpins). Another way of delivering aptamers to intracellular target molecules is by the transfer of aptamers from the bloodstream to cells through receptor-dependent endocytosis. For example, endocytosis of aptamer binding prostate-specific membrane antigen (PSMA) provides effective and specific delivery of conjugated drugs to cancer cells expressing this antigen on their surface. These methods show promise but have not been adequately tested or shown to deliver aptamters intracellularly or across the BBB to cause a pharmaceutical or physiological effect.

A suitable carrier for aptamers is one which helps protect them from degradation, avoid premature elimination by kidneys, and deliver them to intracellular targets. An optimized aptamer carrier system, to reliably deliver them intracellularly and across the BBB is clearly desirable.

SUMMARY

The invention includes in vivo delivery of aptamers by coupling them with reconstituted HDL (rHDL) nanoparticles ( ee U.S. Pat. No. 8,734,853, incorporated by reference). Through transport mediated by the scavenger receptor class B type 1 (SRB 1) also known as SR-BI, the nanoparticles allow delivery of aptamers into cells and tissues. SRB1 is a protein encoded by the SC ARB 1 gene, in humans and functions as a receptor for high-density lipoprotein. High-density lipoprotein (HDL) is a comparatively dense and small lipoprotein that can carry lipids as a multifunctional aggregate in plasma. HDL participates in reverse cholesterol transport in mammals to deliver the peripherally accumulated excess cholesterol to the liver.

Among lipoproteins, HDL particles possess advantageous physicochemical properties, including that they are naturally biosynthesized components; they include amphipathic apolipoproteins associated with efficient lipid-loading and hydrophobic agent-incorporating; they provide specific protein-protein interactions and are heterogenous; and, they are small in size, as they are nanoparticles.

Vickers et al. (in “MicroRNA-27b Is a Regulatory Hub in Lipid Metabolism and Is Altered in Dyslipidemia” Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32: A24; incorporated by reference) showed that HDL transports endogenous miRNA and delivers it to recipient cells, while retaining functional targeting capability. Furthermore, rHDL injected into mice was observed to retrieve distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the targeting of specific messenger RNA reporters. HDL-miRNA complexes from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Overall, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs to and from cells that express SR-B 1 receptor.

The structure of HDL makes it well suited for the transport of cholesteryl esters and lipophilic compounds, including aptamers, in its core compartment. The use of rHDL over liposomes and other artificial complexes as transport vehicles is advantageous because of their payload protection (anti-degradation and anti- elimination) properties, ability to carry a variety of payloads effectively, their smaller size, and their ability to interact with specific cell surface receptors, which aids in their contents being rapidly internalized by receptors of specific cells, including receptors on the surface of tumor tissue. The inventors have demonstrated the successful delivery of aptamers into a variety of nude mice tissues, following intravenous injection.

Preferred rHDL nanoparticles of the invention include a positively charged polyamino acid, or other positively charged molecule, which neutralizes any negatively charged aptamers, thus allowing for successful incorporation of such aptamers into the HDL nonoparticle. Other more preferred embodiments may further include apolipoproteins, including apolipoprotein A-I (Apo A-I), apoplipoprotein A-II (Apo A-II), apolipoprotein A-IV (apo-A-IV), apolipoprotein A-V (apo-V), apolipoprotein B48 (Apo B48), apoplipoprotein B100 (Apo B100), apolipoprotein C-I (Apo C-I), apolipoprotein C-II (Apo C-II), apolipoprotein C-III (Apo C-III), apolipoprotein C-IV, and apolipoprotein D (apoD). In specific embodiments, the apolipoprotein is Apo A-I.

Preferred rHDL nanoparticles of the invention preferably are spherical macromolecular complexes that contain at least three of the lipid and one protein component of the natural circulating HDL. Non-limiting examples of such lipid components of natural circulating HDL include phosphatidyl choline, cholesterol, and cholesteryl ester.

Use of the rHDL nanoparticles of the invention to target intracellularly and positively affect a disease or condition is also described herein. DETAILED DESCRIPTION

The use of the term“or” in the claims and/or specification is used to mean“’and’ as well as“or”’ unless explicitly indicated to refer to alternatives only, or if the alternative are mutually exclusive.

Throughout this application, the term“about” is used to indicate that an associated value includes at least one standard deviation of error for the device and/or method being employed to determine the value.

As used herein the specification and claims,“a” or“an” may mean one or more, unless clearly indicated otherwise. As used herein“another” with a subject, may mean at least a second subject, or more subjects.

An“aptamer” as used below and in the claims, is a modified or specially selected nucleic acid that binds specifically to an intracellular target to cause a pharmaceutical or physiological effect, including gene silencing or downregulation, but does not itself encode or cause a target cell to generate mRNA or proteins, or otherwise bind to or interfere with the action of mRNA in the target cell.

The invention is in part based on the finding that high density lipoprotein (HDL) particles can be employed for efficient delivery of aptamers to certain cells and tissues as therapeutic agents for treating diseases and conditions including hyperproliferative disease, infectious diseases, inflammatory diseases, degenerative diseases, or immune diseases. In particular embodiments, the hyperproliferative disease is a disease associated with neovascularization. In more particular embodiments, the hyperproliferative disease is cancer, including, for example: breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, pancreatic cancer, colon cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia. In other embodiments, the disease is neurodegenerative, including Alzheimer’ s disease.

As noted, the rHDL nanoparticles preferably include a positively charged molecule; which can be a small molecule or a polypeptide containing one or more positively charged groups. In some embodiments, a “polypeptide” as used herein refers to a consecutive series of two or more amino acid residues. The polypeptide may have a length of 2 to 2000 consecutive amino acids, 2 to 1000 consecutive amino acids, 2 to 500 consecutive amino acids, 2 to 400 consecutive amino acids, 2 to 300 consecutive amino acids, 2 to 200 consecutive amino acids, 2 to 100 consecutive amino acids, 2 to 50 consecutive amino acids, 2 to 40 consecutive amino acids, 2 to 30 consecutive amino acids, 2 to 20 consecutive amino acids, or 2 to 15 consecutive amino acids. A positively charged region of a polypeptide is a region that includes a net positive charge that includes at least one positively charged amino acid. In particular embodiments, the polypeptide includes two or more consecutive positively charged amino acid residues. The positively charged region has a net positive charge, and functions to neutralize the negatively charged nucleic acid molecule, which thus facilitates packaging of the nucleic acid molecule into HDL particles. For example, the positively charged amino acids may be lysine residues, histidine residues, arginine residues, positively charged non natural amino acids, such as those described in U.S. Pat. No. 6,783,946 (incorporated by reference), or a mixture of any of these residues. The amino acid segments can include any number of consecutive positively charged residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more residues, or any range of residues derivable therein. In some embodiments, for example, the amino acid segment includes 2 to 40 consecutive lysine residues. In further embodiments, the amino acid segment comprises 2 to 20 consecutive lysine residues, and in yet other embodiments, the amino acid segment comprises 2 to 15 consecutive lysine residues.

In some embodiments, the rHDL-aptamcr composite of the invention further includes a lipid component; for example, a neutral phospholipid. Non-limiting examples of neutral phospholipids include phosphatidylcholine, phosphatidylethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (“EPC”), dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine (“DSPC”), l-myristoyl-2-palmitoyl phosphatidylcholine (“MPPC”), 1- palmitoyl-2-myristoyl phosphatidylcholine (“PMPC”), l-palmitoyl-2-stearoyl phosphatidylcholine (“PSPC”), l-stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”), dimyristyl phosphatidylcholine (“DMPC”), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”), 1 ,2-diarachidoyl-sn-glycero-3- phosphocholine (“DBPC”), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (“DEPC”), palmitoyloeoyl phosphatidylcholine (“POPC”), lysophosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (“DSPE”), dimyristoyl phosphatidylethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), palmitoyloeoyl phosphatidylethanolamine (“POPE”), and lysophosphatidylethanolamine.

In particular embodiments, the lipid component includes cholesterol. In more particular embodiments, the lipid component includes a combination of cholesterol and cholesterol oleate. Further variation in compositional properties of the lipids can readily be achieved by introducing phosphoglycerides with a desired composition or employing other lipids (e.g., sphingomyelin, cationic lipids) when preparing the HDL-lipid mix. Alteration of surface properties by chemical modification of lipids or apolipoproteins may also be used to alter the specificity of tissue delivery and to enhance the effectiveness of therapies designed for targeting specific metastatic tumors. Because circulating HDL contains apolipoproteins (A-II, A-IV, C- I, C-II, E and F), other than apo-AI, addition of these alone or in combination may be used to enhance specificity of delivery to certain types of metastatic tumors. Peptide analogs of these apolipoproteins may also be employed in the design of specific HDL preparations as described for apo-Al.

The HDL-aptamer composite can be of any size, but in preferred embodiments the particle has a molecular size of from about 100 Angstroms to about 500 Angstroms, and more preferably, from about 100 Angstroms to about 300 Angstroms.

The HDL-aptamer composite particle can have a broad range in molecular weight. The weight is dependent on the size of the nucleic acid incorporated into the particle. For example, in some embodiments, the particle has a molecular weight of between about 100,000 Daltons to about 1,000,000 Daltons, preferably between about 100,000 Daltons to about 500,000 Daltons, more preferably, between about 100,000 Daltons to about 300,000 Daltons. In one embodiment, an aptamer-mRNA conjugate includes an aptamer component for targeting particular cells.

Aptamers resistant to degradation by nucleases are generated using SELEX to isolate degradation-resistant nucleotides. Special DNA and RNA polymerases that are able to utilize nucleoside triphosphate substrates with a modified, for example, 2’ sugar position are used to generate such oligonucleotides. 2’ -Amino pyrimidine nucleosides [Yan X, Gao X et al. (2004)], 2’ -fluoropyrimidine nucleosides [Li N, Nguyen HH et al., 2011], 2’-0-methyl purine, and 2’-0-methyl pyrimidine nucleosides [Lebars I, Richard et al., 2007] are currently used for this purpose.

Modification of nucleotides already included into aptamers could also be performed after the SELEX procedure; however, one must be careful that the inclusion of additional functional groups does not negatively affect the specificity and affinity of the aptamer. The closed ring structures emerging after ligation of the 3’- and 5’-termini of the same aptamer are also highly resistant to degradation by nucleases. Several different aptamers can also be ligated to a closed structure with multiple specificities [Lebars I, Richard T et al., 2007]. The generation of such ring structures is an optimal approach for the regular injection of high amounts of aptamers, since the degradation products of some modified oligonucleotides have the potential of being toxic [Levin AA. Biochim Biophys Acta. 1999]. Another approach to avoiding aptamer degradation by nucleases was provided by the development of“mirror aptamers” (Spiegelmers), which have an oligonucleotide backbone composed entirely of L-ribose (RN A spiegelmers) or L- deoxyribose (DNA spiegelmers) [Eulberg D, Klussmann S et al., 2003].

Conjugation of aptamers with polyethylene glycol (PEG) with a molecular weight of 20 or 40 kDa to help inhibit the kidney’s removal of aptamers, is similar a method currently being used to increase the bloodstream circulation time not only of oligonucleotides, but also of proteins, peptides and low-molecular- weight substances. As an alternative, aptamers can be conjugated with cholesterol molecules to prolong their circulation in the bloodstream.

Examples of aptamers which include oligonucleotides modified using some of these approaches and which are in clinical use include: Macugen, the only aptamer currently approved for medical application targets VEGF (Ng et al. 2006; Siddiqui and Keating 2005). Numerous additional aptamers, generated with similar use of modified oligonucleotides or PEG configurations, with discrete targets are or have been in clinical trials for a variety of indications, including: ARC1905 (targeting C5) (Biesecker et al. 1999) and E-10030 (targeting PDGF) (Boyer 2013), REG1 (targeting Coagulation factor IXa) (Povsic et al. 2013), ARC1779 (targeting the Al domain of von Willebrand factor) (Schattauer GmbH et al. 2017), NU172 (targeting thrombin) (Lu et al. 2009), AS1411 (targeting nucleolin) (Bates et al. 2009), NOX-A12 (targeting CXCL12) (Hoellenriegel et al. 2014), NOX-E36 (targeting CCL2) (Kulkarni et al. 2007), and NOX-H94 (targeting Hepcidin peptide hormone) (Schwoebel et al. 2013).

In some embodiments, the aptamer component is a nucleic acid aptamer, synthesized from DNA or RNA nucleotides. The nucleic acid aptamers will have been selected using the SELEX protocol, or a derivation thereof (Ellington and Szostak 1990; Tuerk and Gold 1990). The aptamer will be selected from a combinatorial library of sufficient diversity, ranging from 10¹³-10¹⁵ sequences containing degenerate regions of at least 25 nucleotides in length. Selection rounds in SELEX will proceed using a recombinant form of the either partially or fully-expressed protein, and will proceed through up to 15 rounds of selection.

Many factors are important for successful aptamer selection by SELEX. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool, and the SELEX process for a particular target may need to be repeated with multiple different starting libraries. The SELEX process may also need to be expanded and repeated using either DNA or RNA combinatorial libraries with increasing levels of diversity by increasing the length of the degenerate region, to select a suitable aptamer.

The nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof. In some embodiments, nucleotides or modified nucleotides of the nucleic acid aptamer can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid aptamer is not substantially reduced by the substitution.

Nucleic acids in accordance with the embodiments described herein may include nucleotides entirely of the types found in naturally occurring nucleic acids, or may instead include one or more nucleotide analogs or have a structure that otherwise differs from that of a naturally occurring nucleic acid. U.S. Pat. Nos. 6,403,779, 6,399,754, 6,225,460, 6,127,533, 6,031,086, 6,005,087, 5,977,089, disclose a wide variety of specific nucleotide analogs and modifications that may be used, and are hereby incorporated by reference as if fully set forth herein. Also see Crooke, S. (ed.) Antisense Drug Technology: Principles, Strategies, and Applications (1st ed.), Marcel Dekker; ISBN: 0824705661 ; 1st edition (2001), which is also hereby incorporated by reference as if fully set forth herein. For example, 2'-modifications include halo, alkoxy and allyloxy groups. In some embodiments, the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Examples of modified linkages include phosphorothioate and 5 '-N -phosphor amidite linkages.

Nucleic acids having a variety of different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages can be utilized in accordance with the embodiments described herein. Nucleic acids may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxy thymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of modified nucleotides include base modified nucleoside (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'- deoxyuridine, 3-nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2- thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7- deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, Ml- methyladenosine, pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine, 5- propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'- azidoribose, 2'-0-methylribose, L-enantiomeric nucleosides arabinose, and hexose), modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids comprising such modifications display improved properties relative to nucleic acids consisting only of naturally occurring nucleotides. In some embodiments, nucleic acid modifications described herein are utilized to reduce and/or prevent digestion by nucleases (e.g. exonucleases, endonucleases, etc.). For example, the structure of a nucleic acid may be stabilized by including nucleotide analogs at the 3' end of one or both strands order to reduce digestion.

Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid. The nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially affected. To give but one example, modifications may be located at any position of an aptamer component such that the ability of the aptamer to specifically bind to the target is not substantially affected. The modified region may be at the 5 '-end and/or the 3 '-end of one or both strands. For example, modified nucleic acid aptamers in which approximately 1-5 residues at the 5' and/or 3' end of either of both strands are nucleotide analogs and/or have a backbone modification have been employed. The modification may be a 5' or 3' terminal modification.

In some embodiments, the aptamer targets proteins which play key roles in the growth of tumor cells, e.g., aptamers which dysregulate the activity of the target proteins by interfering with the targets ability to enact its cancer-supporting function. Other suitable aptamers target transcription factors which are either cause or heighten cancer progression. These transcription factors are known to drive transcription of genes which directly result in the proliferation or survival of tumor cells. Non-limiting examples of such genes include Stat3, c-Myc, FOXOl, FOXM1, among others. In other embodiments, the aptamer targets oncogenic proteins responsible for intracellular signaling critical for growth of the cancer cell. Non-limiting examples of such oncogenic proteins include: AKT, mTOR, Janus Kinases, among others.

In some embodiments, the aptamer targets proteins which may be associated with progression of Alzheimer’s, or other neurodegenerative diseases, and would dysregulate the activity of the target proteins by interfering with the disease-progressing activity of the targets. Such targets include asparagine endopeptidase (AEP) and tau proteins, among others. The HDL-aptamer composite of the invention may include a single type of aptamer, or more than one type of aptamer. The particles of the invention may further include one or more additional therapeutic agents incorporated into the particle, which may or may not be aptamer. For example, the additional therapeutic agent may be a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, and so forth.

In some embodiments, the HDL-aptamer composite further includes one or more attached ligands to target the particle to a particular cell type or tissue type in a subject. The targeting ligand can be attached to the particle using any method known to those of ordinary skill in the art. In specific embodiments, the targeting ligand is attached to the protein component of the apolipoprotein by a covalent bond. Non-limiting types of targeting ligands include a small molecule, a peptide, a polypeptide, a protein, an antibody, or an antibody fragment. In some embodiments, the targeting ligand targets the particle to a tumor cell.

The invention includes compositions that include any of rHDL-aptamers of the invention (which may include an apolipoprotein, an aptamer, and a molecule that includes a positively-charged region which interacts with or is associated with the aptamer) and one or more pharmaceutically acceptable carriers. The carrier can be any pharmaceutically acceptable carrier including water or saline solution. The invention further includes treating a subject with a disease or condition by administering to the subject a pharmaceutically effective amount of such pharmaceutical compositions. Suitable subjects include: rats, rabbits, cats, dogs, cattle, horses, sheep, goats, pigs, chickens, and humans and other primates.

The pharmaceutical compositions can be administered using any method known to those of ordinary skill in the art. For example, the composition may be administered to the subject intravenously, topically, locally, systemically, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In specific embodiments, the composition is administered intravenously.

The invention also concerns methods of delivering an aptamer payload into a cell that involves contacting the cell with an effective amount of an rHDL-aptamer of the invention, wherein the aptamer component is delivered into the cell. Suitable cells include tumor cells and neurons. In particular embodiments, the cell expresses a receptor that binds to an apolipoprotein. In a specific embodiment, the cell expresses the SR- B 1 receptor (Connelly et al, 2004). Examples:

Example I: Demonstration that rHDL encapsulated aptamer can be delivered to cell cytoplasm

Method of Making: Lyophilized oligonucleotide with the sequence: TGCCGCCATT CACACGGATT AATCGCCGTA G A A A AGC AT G TCAAAGCCGG AATTAAAUGC CCGCCAUGAC CAG (SEQ ID. NO: l) was spun down and reconstituted with HEPES Buffer according to the manufacturer’s instruction, then heated at boiling for 15 minutes, moved to room temperature, and allowed to cool for 20 minutes. 2.5pg/mL of the reconstituted oligonucleotide and poly-L-lysine were mixed in a 1:5 ratio (w/w) and incubated at 37°C for 30 minutes. In a sterile, RNAse Away-treated glass vial with lid, l5mg EYCP, 0. l5mg CE, 0.35mg FC, and l.5mg PEG-PE were mixed together. The mixture was dried down to a thin film under N2 gas. To the dried mixture, the aptamer/poly-L -lysine mixture, 60pL of 3% DMSO, and enough Cholate Dialysis Buffer to make the volume up to lmL was added. l40mg sodium cholate and 5mg ApoA-l was also added. The rHDL product was incubated at 4°C overnight.

The resulting solution was filtered through a Bio-Rad Econo-Pac® Gravity Flow Column packed with 5mL bed volume of Bio-Gel P-10 Gel resin. Fifteen lmL fractions were collected, centrifuged at 4K rpm for 5miutes, and filtered through a 0.2m PVDF filter. The resulting filtrates were assessed for protein concentration. The fractions containing the highest concentration of protein were pooled, forming encapsulated rHDL-aptamer, and were used in subsequent cell treatments.

MDA MB 231 cells were maintained in a petri dish, passed and plated on a 24-well plate.

Cell Treatment: Cells were treated with 0.75ug of naked aptamer (SEQ ID. NO: l) or encapsulated aptamer in 200uL of culture media and incubated at 37°C for 45minutes. Cells were rinsed, DNA stained, and visualized on the Leica Microscope using LAS-X software. More of the encapsulated aptamer appeared to be taken up in the cells, as compared with the naked aptamer; indicating that more of the encapsulated aptamer was delivered to the cytoplasm of the cells.

Example II: Deliver a variety of distinct, clinically relevant aptamers intracellulary

Several different clinically relevant aptamers targeting critical cellular functions are chosen; e.g., aptamers which target intracellular transcription factors and known to drive transcription of genes which directly result in the proliferation or survival of tumor cells, such as: Stat3, c-Myc, FOXOl, and FOXM1 ; or, aptamers targeting oncogenic proteins responsible for intracellular signaling critical for growth of the cancer cell, such as: AKT, mTOR, and Janus Kinases. The aptamers are separately encapsulated into rHDL particles using methods as described in Example I. Concentrations of aptamers pre- and post-encapsulation solution are determined using absorbance spectrophotometry. The aptamer loaded rHDL particles are added separately to cells in culture. At least two cell lines expressing SR-B 1 receptors and one cell line not expressing SR-B 1 receptor are chosen for this purpose. Control rHDL particles without aptamers, and naked aptamers, are used as controls, where the different combinations with controls are set forth below.

Lor each aptamer, an appropriate readout is obtained for the cell line, depending on which component is being targeted. Lor aptamers targeting cell division, cell viability is determined using the MTS assay. Lor aptamers blocking a point in a cellular pathway, the concentration of proteins involved downstream of that point is measured through immunohistochemistry (IHC) and subsequent image analysis, as well as through Western Blots.

Example III: rHDL aptamers can be used to inhibit cell division in cancer cells

As a specific instance of the use of rHDL-aptamer combination, we demonstrate its utility in cancer, especially those cancers which have an over-expression of SR-B 1 receptor. One such cancer is certain forms of breast cancer. This can be particularly useful for breast cancers which are refractory to hormone-based chemotherapy, the so-called ER-PR negative cancers or triple -negative cancers. In order to inhibit cell division, we target E2L transcription factors.

These factors were originally characterized as cellular proteins activated by the viral oncoprotein El A. They are now recognized as central players in the control of animal cell cycle differentiation and transformation. All E2Ls share a highly conserved DNA binding domain (DB) encompassing a stretch of basic residues, along with overlapping helix-loop-like and putative leucine -zipper-like domains. The C- terminal region of all E2Ls, except E2L6, is a transactivation region (TA). The C-terminal 18 residues of this TA are necessary and sufficient to bind to the pRB tumor suppressor protein family. This is controlled by the cyclin/cdk (D/k4, E/k2, A/k2) -dependent phosphorylation of the 'pocket proteins' and of E2L/DP. This control exerted by cyclin/cdks directly links the cell-cycle machinery to gene transcription, and as such, defines a critical regulatory pathway that gates cell cycle progression through positive and negative regulation of E2L-regulated genes. Consistent with this picture, mammalian tumors typically show a

IS number of distinct genetic aberrations in this Cyclin/CDK-pocket protein/E2F pathway that might cause a deregulation of E2F activity, thus indicating that E2Fs are key signal transducers whose deregulation is required for tumor development. An anti-E2F aptamer (Apt5) has been previously described ( Oncogene volumel8, pages 4357-4363) that strongly inhibited E2F activity. Here, we show that this E2F inhibitor, when delivered using rHDL, strongly inhibits breast cancer cell proliferation by blocking cells in Gl.

Apt5 aptamers are synthesized using standard methodology. These aptamers are encapsulated in rHDL particles as described in Example I. The Apt5/rHDL are characterized, including by having the size and surface charge of the particles measured using dynamic light scattering. The encapsulation efficiency of the particles is measured by measuring the concentration of Apt5 before and after encapsulation, using spectrophotometry. Two separate breast cancer cell lines which express SR-B 1 receptors (MCF-7 and MDA-MB-231), and one cell line which does not (HaCat cells) are cultured and plated. The cells are incubated in separate wells with PBS, Apt5, rHDL, and rHDL- Apt5 for 1 hour at 37°C. Cells are then incubated for a further 24 hours.

Cell numbers in each well at the finish of the incubation are estimated using an MTS assay.

Example IV : rHDL/Apt5 can inhibit tumor growth in mice with SR-B 1 expressing tumor models Mouse tumor models can be used to assess the utility of anticancer treatments. In a mouse model of triple negative breast cancer, using MDA-MB-231 cells, synthesis and characterization of Apt5/rHDL particles is carried out as described in Example I. Also, the culturing of MDA-MB-231 cells is done as described previously. Subsequently, 10^L6 cells are subcutaneously implanted in the left flank of each of 24 6-week old BALB/c mice.

The 24 mice are divided into four groups with six mice in each group, and the groups are administered on of: PBS only, rHDL (empty), Apt5, and Apt5/rHDL. After two weeks, tumor sizes and the animal’s weight are determined. The measurements are again taken after another 14 days pass. Then animals are sacrificed, and the tumors are harvested, fixed, and sent for H&E processing.

It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may or may not apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. It should be understood, however, that the description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. The invention is defined only in the claims which follow.

The following references and all other articles, papers, patents and patent applications set forth in the specification are hereby incorporated by reference.

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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising an aptamer incorporated in or carried by an rHDL nanoparticle.

2. The pharmaceutical composition of claim 1 further including a positively charged polyamino acid, or other positively charged molecule.

3. The pharmaceutical composition of claim 1 further including an apolipoprotein.

4. The pharmaceutical composition of claim 3 wherein the apolipoprotein is one or more of: A-I (Apo A-I), apoplipoprotein A-II (Apo A-II), apolipoprotein A-IV (apo-A-IV), apolipoprotein A-V (apo-V), apolipoprotein B48 (Apo B48), apoplipoprotein B100 (Apo B100), apolipoprotein C-I (Apo C-I), apolipoprotein C-II (Apo C-II), apolipoprotein C-III (Apo C-III), apolipoprotein C-IV, and apolipoprotein D (apoD).

5. The pharmaceutical composition of claim 1 wherein the rHDL nanoparticles include one or more of: phosphatidyl choline, cholesterol and cholesteryl ester.

6. The pharmaceutical composition of claim 1 wherein the rHDL nanoparticles include at least three lipid and one protein component of natural circulating HDL.

7. The pharmaceutical composition of claim 1 wherein the rHDL nanoparticles include a neutral phospholipid.

8. The pharmaceutical composition of claim 7 wherein the neutral phospholipid is one or more of: phosphatidylcholine, phosphatidylethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (“EPC”), dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine (“DSPC”), l-myristoyl-2-palmitoyl phosphatidylcholine (“MPPC”), 1- palmitoyl-2-myristoyl phosphatidylcholine (“PMPC”), l-palmitoyl-2-stearoyl phosphatidylcholine (“PSPC”), l-stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”), dimyristyl phosphatidylcholine (“DMPC”), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”), 1 ,2-diarachidoyl-sn-glycero-3- phosphocholine (“DBPC”), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (“DEPC”), palmitoyloeoyl phosphatidylcholine (“POPC”), lysophosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (“DSPE”), dimyristoyl phosphatidylethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), palmitoyloeoyl phosphatidylethanolamine (“POPE”), and lysophosphatidylethanolamine.

9. The pharmaceutical composition of claim 1 wherein the nucleic acid of the aptamer is formed into a closed ring structure.

10. The pharmaceutical composition of claim 1 wherein the nucleic acid of the aptamer is composed of L-ribose or L-deoxyribose.

11. The pharmaceutical composition of claim 1 wherein the nucleic acid of the aptamer is conjugated with cholesterol or PEG.

12. A method of treating a disease or condition by administering a pharmaceutically effective amount of a pharmaceutical composition of any of claims 1 to 11 to a patient.

13. The method of claim 12 wherein the disease is cancer or Alzheimer’s.