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WO2024178401A1 - Shank3 gene therapy approaches - Google Patents

Shank3 gene therapy approaches Download PDF

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Publication number
WO2024178401A1
WO2024178401A1 PCT/US2024/017176 US2024017176W WO2024178401A1 WO 2024178401 A1 WO2024178401 A1 WO 2024178401A1 US 2024017176 W US2024017176 W US 2024017176W WO 2024178401 A1 WO2024178401 A1 WO 2024178401A1
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WO
WIPO (PCT)
Prior art keywords
subject
seq
human
aav
minishank3
Prior art date
Application number
PCT/US2024/017176
Other languages
French (fr)
Inventor
Guoping Feng
Xian Gao
Yuan MEI
Kevin Foust
Daniel Mccoy
Original Assignee
Massachusetts Institute Of Technology
Jaguar Gene Therapy, Llc
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Publication date
Application filed by Massachusetts Institute Of Technology, Jaguar Gene Therapy, Llc filed Critical Massachusetts Institute Of Technology
Publication of WO2024178401A1 publication Critical patent/WO2024178401A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present disclosure relates to gene therapy approaches for delivering polynucleotides encoding a Shank3 protein to a subject who has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
  • Shank3 accounts for about 0.5-1% of all autism spectrum disorder (ASD) patients and about 2% of ASD patients with intellectual disability (ID).
  • ASD autism spectrum disorder
  • ID intellectual disability
  • aspects of the disclosure relate to the development of an effective gene therapy approach for subjects with Shank3 mutations.
  • aspects of the disclosure provide expression cassettes comprising a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20.
  • the polynucleotide encoding a human miniShank3 protein is operably linked to a human Syn promoter and a polyA signal sequence.
  • the polynucleotide encoding the human miniShank3 protein is operably linked to a WPRE element.
  • the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20.
  • the polynucleotide encoding the miniShank3 protein is at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 2.
  • the polynucleotide encoding the miniShank3 protein comprises the nucleic acid sequence of SEQ ID NO: 2.
  • the present disclosure provides recombinant AAV vectors comprising expression cassettes described herein flanked by AAV inverted terminal repeats (ITRs).
  • ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the nucleic acid sequences of SEQ ID NO:27 and SEQ ID NO: 28, respectively.
  • the recombinant AAV vector comprises the sequence of SEQ ID NO: 21.
  • the recombinant AAV vector comprises the sequence of SEQ ID NO: 30.
  • the present disclosure provides recombinant AAV virions comprising (1) recombinant AAV vectors described herein; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • the present disclosure provides pharmaceutical compositions comprising the recombinant AAV virions described herein and a pharmaceutically acceptable carrier.
  • Further aspects of the disclosure relate to methods of delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof.
  • the methods comprise administering to the CNS of the subject a pharmaceutical composition comprising recombinant AAV virions described herein.
  • the recombinant AAV virion is delivered to the brain of the subject. In some embodiments, the recombinant AAV virion is delivered to the cortex, striatum and/or thalamus of the subject. In some embodiments, the recombinant AAV virion is administered by intracerebroventricular administration (ICV). In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration.
  • ICV intracerebroventricular administration
  • the subject is a human subject. In some embodiments, the human subject is an adult. In some embodiments, the human subject is not an adult. In some embodiments, the human subject is not older than 25 years old. In some embodiments, the human subject is 10 years old or younger. [0013] In some embodiments, the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder. In some embodiments, the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD). In some embodiments, the subject exhibits one or more symptoms of an ASD. In some embodiments the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
  • the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • the recombinant AAV virion is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • the disclosure relates to methods of treating a subject having a neurodevelopmental disorder.
  • the disclosure relates to methods of treating a subject having an autism spectrum disorder (ASD).
  • the disclosure relates to methods of treating a subject having Phelan-McDermid syndrome.
  • the methods of treatment comprise administering to the subject a therapeutically effective amount of a composition comprising an expression cassette or a recombinant AAV virion described herein.
  • the composition is administered by ICV administration.
  • the ICV administration is unilateral administration.
  • the ICV administration is bilateral administration.
  • the autism spectrum disorder comprises autism disorder.
  • the subject has improved sleep efficiency after said administered.
  • the composition is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • Further aspects of the disclosure provide recombinant AAV vectors described herein.
  • the recombinant AAV vector is a plasmid.
  • the methods comprise culturing a host cell that comprise: the recombinant AAV vector described herein; AAV cap; AAV9 rep; and one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell.
  • the AAV cap encodes VP1, VP2, and/or VP3.
  • the AAV9 rep encodes rep78, rep68, rep 52, and/or rep 40.
  • aspects of the disclosure relate to methods of delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof, said method comprising administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • the recombinant AAV virion is delivered to the brain of the subject. In some embodiments, the recombinant AAV virion is delivered to the cortex, striatum and/or thalamus of the subject. In some embodiments, the recombinant AAV virion is administered by intracerebroventricular (ICV) administration. In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration.
  • ICV intracerebroventricular
  • the subject is a human subject.
  • the human subject is an adult.
  • the human subject is not an adult.
  • the human subject is not older than 25 years old.
  • the human subject is 10 years old or younger.
  • the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder. In some embodiments, the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD). In some embodiments, the subject exhibits one or more symptoms of an ASD. In some embodiments, the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome. In some embodiments, the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
  • ID intellectual disability
  • the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • the recombinant AAV virion is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • aspects of the disclosure relate to methods of treating a subject having a neurodevelopmental disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • aspects of the disclosure relate to methods of treating a subject having an autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • aspects of the disclosure relate to methods of treating a subject having Phelan- McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • the subject is a human subject.
  • the human subject is an adult.
  • the human subject is not an adult.
  • the human subject is not older than 25 years old.
  • the human subject is 10 years old or younger.
  • the composition is delivered to the brain of the subject. In some embodiments, the composition is delivered to the striatum and/or thalamus of the subject. In some embodiments, the composition is administered by intracerebroventricular (ICV) administration. In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration. [0033] In some embodiments, the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay. In some embodiments, the autism spectrum disorder (ASD) comprises autism disorder. [0034] In some embodiments, the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • the subject has improved sleep efficiency after said administered. [0035]
  • the composition is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • Embodiment 1 A method of delivering a human miniShank3 protein to the central nervous system of a subject in need thereof, said method comprising administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • Embodiment 2 The method of embodiment 1, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
  • Embodiment 3 The method of embodiment 1 or embodiment 2, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
  • Embodiment 4 The method of any one of embodiments 1 to 3, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
  • Embodiment 5 The method of any one of embodiments 1 to 4, wherein the AAV virion is delivered to the brain of the subject.
  • Embodiment 6 The method of any one of embodiments 1 to 5, wherein the AAV virion is delivered to the cortex, striatum and/or thalamus of the subject.
  • Embodiment 7 The method of any of any one of embodiments 1 to 6, wherein the AAV virion is administered by intracerebroventricular (ICV) administration.
  • ICV intracerebroventricular
  • Embodiment 8 The method of embodiment 7, wherein the ICV administration is unilateral administration.
  • Embodiment 9 The method of embodiment 7, wherein the ICV administration is bilateral administration.
  • Embodiment 10 The method of any one of embodiments 1 to 9, wherein the subject is a human subject.
  • Embodiment 11 The method of embodiment 10, wherein the human subject is an adult.
  • Embodiment 12 The method of embodiment 10, wherein the human subject is not an adult.
  • Embodiment 13 The method of embodiment 10, wherein the human subject is not older than 25 years old.
  • Embodiment 14 The method of embodiment 10, wherein the human subject is 10 years old or younger.
  • Embodiment 15 The method of any one of embodiments 1 to 14, wherein the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
  • Embodiment 16 The method of any one of embodiments 1 to 15, wherein the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD).
  • ASD autism spectrum disorder
  • Embodiment 17 The method of any one of embodiments 1 to 16, wherein the subject exhibits one or more symptoms of an ASD.
  • Embodiment 18 The method of any one of embodiments 1 to 17, wherein the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome.
  • Embodiment 19 The method of any one of embodiments 1 to 18, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
  • ID intellectual disability
  • Embodiment 20 The method of any one of embodiments 1 to 19, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • Embodiment 21 The method of embodiment 20, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • Embodiment 22 The method of embodiment 20 or 21, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • Embodiment 23 The method of embodiment 22, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • Embodiment 24 The method of embodiment 22, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • Embodiment 25 The method of any one of embodiments 1 to 24, wherein the recombinant AAV virion is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • Embodiment 26 A method of treating a subject having a neurodevel opmental disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • Embodiment 27 A method of treating a subject having an autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • Embodiment 28 A method of treating a subject having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • ITRs inverted terminal repeats
  • Embodiment 29 The method of any one of embodiments 26 to 28, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
  • Embodiment 30 The method of any one of embodiments 26 to 28, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
  • Embodiment 31 The method of any one of any one of embodiments 26 to 28, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
  • Embodiment 32 The method of any one of embodiments 26 to 31, wherein the subject is a human subject.
  • Embodiment 33 The method of embodiment 32, wherein the human subject is an adult.
  • Embodiment 34 The method of embodiment 32, wherein the human subject is not an adult.
  • Embodiment 35 The method of embodiment 32, wherein the human subject is not older than 25 years old.
  • Embodiment 36 The method of embodiment 32, wherein the human subject is 10 years old or younger.
  • Embodiment 37 The method of any one of embodiments 26 to 36, wherein the composition is delivered to the brain of the subject.
  • Embodiment 38 The method of embodiment 37, wherein the composition is delivered to the striatum and/or thalamus of the subject.
  • Embodiment 39 The method of any one of embodiments 26 to 38, wherein the composition is administered by ICV administration.
  • Embodiment 40 The method of embodiment 39, wherein the ICV administration is unilateral administration.
  • Embodiment 41 The method of embodiment 39, wherein the ICV administration is bilateral administration.
  • Embodiment 42 The method of any one of embodiments 26 to 41, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
  • ID intellectual disability
  • Embodiment 43 The method of embodiment 42, wherein the autism spectrum disorder (ASD) comprises autism disorder.
  • ASD autism spectrum disorder
  • Embodiment 44 The method of any one of embodiments 26 to 43, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • Embodiment 45 The method of embodiment 44, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • Embodiment 46 The method of embodiment 44 or embodiment 45, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • Embodiment 47 The method of embodiment 46, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • Embodiment 48 The method of embodiment 46, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • Embodiment 49 The method of any one of embodiments 26 to 48, wherein the subject has improved sleep efficiency after said administered.
  • Embodiment 50 The method of any one of embodiments 26 to 49, wherein the composition is administered at a dose of about 1.0 x 10 13 vg to about 1.0 x 10 14 vg.
  • a pharmaceutical composition comprising: (a) a recombinant AAV virion comprising: (i) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (ii) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29; b) 10 mM Tris; c) 1 mM magnesium chloride (MgCh); d) 150 mM sodium chloride (NaCl); and e) 0.02% poloxamer 188; wherein said pharmaceutical composition is at pH 8.0.
  • ITRs inverted terminal repeats
  • Embodiment 52 The pharmaceutical composition of embodiment 51, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
  • Embodiment 53 The pharmaceutical composition of embodiment 51 or embodiment
  • Embodiment 54 The pharmaceutical composition of any one of embodiments 51 to 54.
  • the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
  • Embodiment 55 A method of treating a subject having a neurodevelopmental disorder, having an autism spectrum disorder (ASD), and/or having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising (a) a recombinant AAV virion comprising: (i) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (ii) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29; b) 10 mM Tris; c) 1 mM magnesium chloride (Mg) Mg
  • Embodiment 56 The method of embodiment 55, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
  • Embodiment 57 The method of embodiment 55 or embodiment 56, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
  • Embodiment 58 The method of any one of embodiments 55 to 56, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
  • Embodiment 59 An expression cassette comprising a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence.
  • Embodiment 60 The expression cassette of embodiment 59 wherein the polynucleotide encoding the human miniShank3 protein is operably linked to a WPRE element.
  • Embodiment 61 The expression cassette of embodiment 59 or embodiment 60 wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20.
  • Embodiment 62 The expression cassette of any one of embodiments 59 to 61, wherein the polynucleotide encoding the miniShank3 protein is at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 2.
  • Embodiment 63 The expression cassette of any one of embodiments 59 to 62 comprising the nucleic acid sequence of SEQ ID NO: 2.
  • Embodiment 64 A recombinant AAV vector comprising the expression cassette of any one of embodiments 59 to 63 flanked by AAV inverted terminal repeats (ITRs).
  • Embodiment 65 The recombinant AAV vector of embodiment 64, wherein the ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the nucleic acid sequences of SEQ ID NO:27 and SEQ ID NO: 28, respectively.
  • Embodiment 66 The recombinant AAV vector of embodiment 64 or embodiment 65, wherein the recombinant AAV vector comprises the sequence of SEQ ID NO: 30.
  • Embodiment 67 A recombinant AAV virion comprising (1) the recombinant AAV vector of any one of embodiments 64 to 66; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
  • Embodiment 68 A pharmaceutical composition comprising the recombinant AAV virion of embodiment 67 and a pharmaceutically acceptable carrier.
  • Embodiment 69 The recombinant AAV vector of any one of embodiments 64 to 66 which is a plasmid.
  • Embodiment 70 A host cell comprising the recombinant AAV vector of any one of embodiments 64 to 66 and comprising nucleic acid sequences encoding AAV rep and AAV9 cap.
  • Embodiment 71 A method of producing an AAV virion, the method comprising culturing a host cell comprising: the recombinant AAV vector of any one of embodiments 64 to 66; AAV cap, wherein the AAV cap encodes VP1, VP2, and/or VP3; AAV9 rep, wherein the AAV9 rep encodes rep78, rep68, rep 52, and/or rep 40; and one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell.
  • FIG. 1 shows a plasmid map of pAAV2ITR-SYN-miniShank3Vl-KanR.
  • FIGs. 2A-2C show graphs of the results from an open field assay performed on mice to measure gross motor function and activity 6 weeks post dosing with AAV9- hSynl -Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 knockout mice (KO).
  • FIG. 2A shows the total distance travelled.
  • FIG. 2B shows count of rearings.
  • FIG. 2C shows time binned analysis of distance travelled.
  • AAV9-hSynl-Human miniShank3-Vl was administered at a dose of 2.4 x 10 9 vg/mouse, 1.2 x 10 10 vg/mouse, 6.0 x 10 10 vg/mouse or 2.75 x 10 11 vg/mouse. All dosages tested are shown in FIG. 2A and FIG. 2B and the two highest dosage levels in KO mice are compared to WT and KO vehicle controls in FIG. 2C.
  • FIG. 3 shows a graph of the results from a rotarod assay performed on mice to measure motor function 6 weeks post dosing with AAV9-hSynl-Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 mice.
  • AAV9-hSynl-Human miniShank3- VI was administered at a dose of 2.4 x 10 9 vg/mouse, 1.2 x IO 10 vg/mouse, 6.0 x IO 10 vg/mouse or 2.75 x 10 11 vg/mouse.
  • the latency to fall (seconds) is a measure of the cumulative time the animal maintained its balance prior to falling off the rotarod.
  • FIGs. 4A-4B shows a graph of an electroencephalography (EEG) sleep analysis (EEG Power Delta band) in mice 6 weeks (FIG. 4A) or 10 months (FIG. 4B) after administration of AAV9-hSynl-Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 knockout mice (KO).
  • AAV9-hSynl-Human miniShank3-Vl was administered at a dose of 6.0 x 10 10 vg/mouse or 2.75 x 10 11 vg/mouse. Seizure monitoring only performed in two highest dose levels.
  • FIGs. 5A-5C show the results of the three-chamber social approach test in Phase 2 (7-13 weeks post-dose).
  • Time spent in the compartment with the object (FIG. 5A) or the compartment with the stranger mouse measured over a 10 min observation period (FIG. 5B) for WT and Shank3 KO males treated with vehicle or increasing doses of JAG201 (2.40* 10 9 vg to 2.75* 10 11 vg) 7-13 weeks after injection, and in the stranger compartment, time spent nose-pointing toward the stranger (FIG. 5C).
  • FIGs. 6A-6C show EEG analyses in Phase 2 over 24 hours for WT and Shank3 KO mice treated with vehicle or the mid-high and high doses of AAV9-hSynl- Human miniShank3-Vl (6.00* 10 10 vg and 2.75* 10 11 vg) 14 weeks after injection.
  • Three different EEG patterns were quantified: epileptic-like (FIG. 6A), spike-wave discharges-like (FIG. 6B), and spike-like (FIG. 6C).
  • FIGs. 7A-7C show EEG seizure analyses in Phase 3 over 24 hours for WT and Shank3 KO mice treated with vehicle or the mid-high and high doses of AAV9-hSynl- Human miniShank3-Vl (6.00* 10 10 and 2.75* 10 11 vg) 42-45 weeks after injection.
  • Three different EEG patterns were quantified: epileptic-like (FIG. 7A), spike-wave discharges-like (FIG. 7B), and spike-like (FIG. 7C).
  • FIG. 8 shows miniSHANK3 protein levels in Phase 2 at 12-16 weeks postdosing for WT and Shank3 KO mice treated with vehicle or AAV9-hSynl-Human miniShank3-Vl (at a dose of 2.4 x 10 9 vg/mouse, 1.2 x IO 10 vg/mouse, 6.0 x IO 10 vg/mouse or 2.75 x 10 11 vg/mouse).
  • FIGs. 9A-9C show Homer 1 protein levels (FIG. 9A), GluR2 protein levels (FIG. 9B) and PSD95 protein levels (FIG. 9C) in Phase 2 at 12-16 weeks post-dosing for WT and Shank3 KO mice treated with vehicle or AAV9-hSynl-Human miniShank3-Vl (at a dose of 2.4 x 10 9 vg/mouse, 1.2 x 10 10 vg/mouse, 6.0 x 10 10 vg/mouse or 2.75 x 10 11 vg/mouse).
  • aspects of the disclosure relate to gene therapy approaches for treating neurodevelopmental disorders.
  • the Examples describe administering recombinant AAV virions that comprise expression cassettes that include polynucleotides encoding Shank3 proteins.
  • Gene therapy strategies disclosed herein use an AAV system to deliver a functional copy of the Shank3 gene into brain cells to restore cellular function.
  • SHANK3 encodes a synaptic scaffolding protein, which coordinates the recruitment of signaling molecules and orchestrates assembly of the macromolecular postsynaptic protein complex, which is crucial for proper synaptic development and function. Deletion of SHANK3 is a major cause of the core neurodevelopmental and neurob ehavi oral deficits in Phelan-McDermid syndrome. Human genetic studies also identified SHANK3 mutations as accounting for about 1% of autism spectrum disorder (ASD). Patients with Phelan-McDermid syndrome and other individuals with SHANK3 mutations often exhibit a variety of comorbid traits, which include developmental delay, sleep disturbances, hypotonia, lack of speech or severe language delay, and characteristic features of ASD. Currently, there is no effective treatment for ASD.
  • Shank3 The association of ASD with Shank3 provided an immediate link between synaptic dysfunction and the pathophysiology of ASD.
  • Animal models bridge the human genetics of ASD to brain pathology underlying clinical presentation, and ultimately help to discover and evaluate effective therapeutics.
  • Previous studies in flies, fish, and rodents have revealed synaptic dysfunction and behavioral abnormalities due to loss of SHANK3.
  • disruption of Shank3 in mouse models have resulted in synaptic defects, impaired social interactions, motor difficulties, repetitive grooming and increased anxiety level.
  • Shank3 deficiency causes severe sleep disturbances in rodents, monkeys and human patients, sleep efficiency provides a unique biomarker for ASD.
  • the /a/ J-deficient mouse model presents predictive validity as the synaptic defects and behavioral abnormalities are reversible when Shank3 is restored. Therefore, gene replacement is well suited as a therapeutic strategy for this monogenic disease.
  • rAAVs Novel recombinant adeno-associated viruses
  • rAAVs adeno-associated viruses
  • Shank3 is a large protein with a coding sequence of about 5.7kb, exceeding the packaging capacity of AAV vectors.
  • Miniaturized Shank3 (“MiniShank3”) proteins described herein can be delivered by vector such as AAVs, including AAV9.
  • ICV intracerebroventricular
  • the present disclosure relates to methods and compositions for treating neurodevelopmental disorders by restoring the activity of Shank3 using a miniaturized Shank3 protein (“MiniShank3”).
  • Shank family of proteins are master scaffolding proteins that tether and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five main domain regions: N-terminal ankyrin repeats, SH3 domain, PDZ domain, proline-rich region, and a C-terminal SAM domain. Through these functional domains, Shank proteins interact with many postsynaptic density (PSD) proteins. Without wishing to be bound by any theory, Shank proteins can bind to SAPAP which in turn binds to PSD95 to form the PSD95/SAPAP/Shank postsynaptic complex.
  • PSD postsynaptic density
  • Shank is connected to the mGluR pathway through its binding to Homer.
  • Shank Due to its link to actin-binding proteins, Shank also plays a major role in spine development. It has been found that transfection of Shank3 was sufficient to induce functional dendritic spine synapses in cultured aspiny cerebellar granule cells, indicating a role in spine induction.
  • Shank3 has three primary isoforms including Shank3 a , the longest Shank3 isoform, Shank3p and Shank3 Y . It has been reported that siRNA knockdown of Shank3 reduced the number and increased the length of dendritic spines in DIV18 cultured hippocampal neurons, implicating a role in spine maturation. This proposed function was supported by the finding that overexpression of Shankl enlarged already present dendritic spines in cultured hippocampal neurons. Furthermore, Shankl mutant mice have been reported to have smaller dendritic spines and weaker synaptic transmission.
  • the present disclosure relates to Shank proteins that are capable of restoring synaptic activity in subjects with disrupted Shank protein activity.
  • the disrupted Shank protein activity is present in subjects who have neurodevelopmental disorders, an autism spectrum disorder (ASD), and/or Phelan- McDermid syndrome.
  • the Shank proteins associated with the present disclosure are Shankl proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shankl or a variant of Shankl.
  • the Shank proteins in the present disclosure are Shank2 proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank2 or a variant of Shank2.
  • the Shank proteins in the present disclosure are Shank3 proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank3 or a variant of Shank3. It should be appreciated that Shank proteins associated with the present disclosure can include any Shank protein, including variants or fragments thereof, that function as scaffolding proteins at the synapses of excitatory neurons.
  • Shank proteins (Shankl, Shank 2, and Shank3) for use in gene therapy.
  • Shank3 full length mouse protein sequence corresponding to GenBank Accession No. BAE16756.1 is provided by SEQ ID NO: 5.
  • the Shank3 full length mouse protein sequence corresponding to SEQ ID NO: 5 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_021423, provided by SEQ ID NO: 15.
  • the Shank3 full length human protein sequence corresponding to GenBank Accession No. Q9BYB0.3 is provided by SEQ ID NO: 6.
  • the Shank3 full length human protein sequence corresponding to SEQ ID NO: 6 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_001372044, provided by SEQ ID NO: 16.
  • the full-length Shank3 protein comprises multiple domains and is encoded by a gene that is about 5.2 Kb in size. Due to its size, it is difficult to deliver full-length Shank3 to a tissue or cell of interest via an AAV vector. As reported in PCT Publication No. W02022/040239, entitled “Shank3 Gene Therapy Approaches,” which is incorporated by reference herein in its entirety, specific domains can be removed or truncated from the full- length Shank3 protein to produce MiniShank3 that is efficacious in restoring Shank3 activity in excitatory neurons.
  • Shank proteins e.g., Shank3 proteins
  • a miniaturized Shank3 protein, or a DNA construct encoding the miniaturized Shank3 protein are referred to interchangeably as “miniShank3” or “MiniShank3.”
  • MiniShank3 proteins include shortened or mutated versions of Shank3 that have at least some Shank3 activity, for example, when MiniShank3 is introduced into neurons, it reduces the effects of Shank3 mutations,
  • Shank3 activity includes, for example, the activity when introduced into, including by gene therapy, an organism, such as a mouse, non-human primate (NHP) or human, including the neurons of an organism, which is deficient for Shank3 or had reduced Shank3 activity, that ameliorates the effects of that Shank3 deficiency or reduction.
  • the activity can be assessed in Shank3 deletion or deficient animal models such as described in Examples 1 and 2 herein.
  • the Shank3 protein disclosed herein is expressed from a miniaturized Shank3 DNA construct or expression cassette.
  • the variant Shank3 DNA constructs and the Shank3 proteins disclosed herein (MiniShank3) comprise fewer domains than the full-length Shank3 gene and protein but have Shank3 activity.
  • the Shank3 protein disclosed herein is encoded by a non- naturally occurring polynucleotide.
  • Shank3 proteins encoded by polynucleotides described herein can include one or more protein domains.
  • Shank3 proteins can include one or more of: an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin domain, a SAM domain, and/or an ankyrin repeat domain.
  • the SH3 domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5.
  • the SH3 domain comprises at least 90% identity to residues 474-525 of SEQ ID NO: 6.
  • the SH3 domain comprises at least 90% identity to residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain can comprise any percent identity to residues 474-525 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SH3 domain can comprise any percent identity to residues 473-524 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the PDZ domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5.
  • the PDZ domain comprises at least 90% identity to residues 573-662 of SEQ ID NO: 6.
  • the PDZ domain comprises at least 90% identity to residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain can comprise any percent identity to residues 573-662 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the PDZ domain can comprise any percent identity to residues 572-661 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the Homer binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 5.
  • the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 6. In some embodiments, the Homer domain comprises residues 1294-1323 of SEQ ID NO: 5 or 6. In some embodiments, the Homer domain can comprise any percent identity to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniShank3.
  • the Cortactin binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1400-1426 of SEQ ID NO: 5 or 6.
  • the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 5.
  • the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 6. In some embodiments, the Cortactin binding domain comprises residues 1400-1426 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the Cortactin binding domain can comprise any percent identity to residues 1400-1426 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniShank3.
  • the SAM domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1664-1729 of SEQ ID NO: 6 or to residues 1663-1728 of SEQ ID NO: 5.
  • the SAM binding domain comprises at least 90% identity to residues 1664-1729 of SEQ ID NO: 6.
  • the SAM binding domain comprises at least 90% identity to residues 1663-
  • the SAM domain comprises residues 1664-
  • the SAM domain comprises residues 1663- 1728 of SEQ ID NO: 5. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1664-1729 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1663-1728 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the ankyrin repeat domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 148-345 of SEQ ID NO: 6 or to residues 147-313 of SEQ ID NO: 5.
  • the ankyrin repeat domain comprises at least 90% identity to residues 148-345 of SEQ ID NO: 6.
  • the ankyrin repeat domain comprises at least 90% identity to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 148-345 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 147- 313 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the MiniShank3 protein is less than 65% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. As used herein, “less than 65%” includes any percent identity less than 65% that is suitable for construction of the MiniShank3. In some embodiments, the MiniShank3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%,
  • the MiniShank3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%,
  • the MiniShank3 protein is at least about 35%, at least about 40%, at least about 45%, or at least about 50% identical to SEQ ID NO: 5 or SEQ ID NO: 6 over the full length of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the MiniShank3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 17-20, provided in Table 9.
  • the MiniShank3 protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 17-20.
  • SEQ ID NO: 17 is encoded by SEQ ID NO: 1.
  • SEQ ID NO: 18 is encoded by SEQ ID NO: 2.
  • SEQ ID NO: 19 is encoded by SEQ ID NO: 3.
  • SEQ ID NO: 20 is encoded by SEQ ID NO: 4.
  • the MiniShank3 protein comprises an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein comprises an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NO: 19 and/or SEQ ID NO: 20. [00142] In other embodiments, the MiniShank3 protein does not comprise an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein does not comprise an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NO: 17 and/or SEQ ID NO: 18.
  • sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or are 100% identical, including all values in between, to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity.
  • sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 90% identity to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity. In some embodiments, the sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise any one of SEQ ID NOs: 1-4. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins with Shank3 activity.
  • the MiniShank3 is encoded by any one of SEQ ID NOs: 1-4, provided in Table 9.
  • SEQ ID NO: 1 and SEQ ID NO: 3 correspond to mouse MiniShank3 nucleic acid sequences
  • SEQ ID NO: 2 and SEQ ID NO: 4 correspond to human MiniShank3 nucleic acid sequences.
  • SEQ ID NO: 1 and SEQ ID NO: 2 encode MiniShank3 proteins that do not comprise an ankyrin repeat domain or the N-terminal domain.
  • SEQ ID NO: 3 and SEQ ID NO: 4 encode MiniShank3 proteins that comprise an ankyrin repeat domain and the N-terminal domain.
  • identity refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by a mathematical model, algorithm, or computer program that is known to one of ordinary skill in the art.
  • the percent identity of two sequences may, for example, be determined using Basic Local Alignment Search Tool (BLAST®) such as NBLAST® and XBLAST® programs (version 2.0).
  • BLAST® Basic Local Alignment Search Tool
  • Alignment technique such as Clustal Omega may be used for multiple sequence alignments.
  • Other algorithms or alignment methods may include but are not limited to the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, or Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).
  • a polynucleotide encoding the Shank protein as disclosed herein is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about 4.3 kb, about 4.2 kb, about 4.1 kb, about 4.0 kb, about 3.9 kb, about 3.8 kb, about 3.7 kb, about 3.6 kb, about 3.5 kb, about 3.4 kb, about 3.3 kb, about 3.2 kb, about 3.1 kb, about 3.0 kb, about 2.9 kb, about 2.8 kb, about 2.7 kb, about 2.6 kb, about 2.5 kb, about 2.4 kb, about 2.3 kb, about 2.2 kb, or about 2.1 kb in size.
  • the polynucleotide encoding the Shank protein as disclosed herein is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about
  • compositions and methods suitable for treating a neurodevelopmental disorder such as an autism spectrum disorder (ASD), or Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • Phelan-McDermid syndrome a neurodevelopmental disorder
  • neurodevelopmental disorder refers to any disorder that impairs the growth and/or development of the brain and/or central nervous system.
  • neurodevelopmental disorders impact one or more brain functions, such as emotion, learning ability, self-control, and memory. It should be appreciated that aspects of the disclosure may be applicable for treatment of any neurodevelopmental disorder.
  • the neurodevelopmental disorder is an autism spectrum disorder (ASD).
  • ASSD autism spectrum disorder
  • Diagnosis of ASDs is mainly based on criteria such as deficits in communication, impaired social interaction, and repetitive or restricted interests and behaviors.
  • ASDs are highly heritable disorders with concordance rates as high as 90% for monozygotic twins.
  • ASDs are clinically heterogeneous, covering a wide range of discrete disorders of differential symptomatic severity. ASDs are believed to be etiologically heterogeneous, possibly encompassing polygenic, monogenic and environmental factors.
  • Haploinsufficiency refers to a model of dominant gene action in diploid organisms, in which a single copy of the wildtype allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wildtype phenotype. Haploinsufficiency may arise from a de novo or inherited LoF mutation in the variant allele, such that it yields little or no gene product. Recent genetic screens also identified a large number of mutations in the Shank3 gene including microdeletions, nonsense mutations and recurrent breakpoints in ASD patients not diagnosed with Phelan-McDermid syndrome (PMS).
  • PMS Phelan-McDermid syndrome
  • Shank3 gene disruption and/or mutation as a monogenic cause of autism spectrum disorder (ASD).
  • ASD autism spectrum disorder
  • Intellectual functioning refers to a disability that causes a subject to have deficits in intellectual functioning and/or adaptive functioning.
  • Intellectual functioning can include, for example, reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and/or experiential learning.
  • Intellectual functioning can be measured using any method known in the art, such as by IQ tests.
  • Adaptive functioning can include, for example, skills needed to live in an independent and responsible manner such as communication and social skills. In some instances, intellectual disability can be evident during childhood or adolescence.
  • the neurodevelopmental disorder is Phelan-McDermid syndrome (PMS, 22ql3.3 deletion syndrome), which is an autism spectrum disorder that shows autistic-like behaviors, hypotonia, severe intellectual disability and impaired development of speech and language.
  • Shank3 is one of the genes that has been reported to be deleted in Phelan-McDermid syndrome. Disruption of Shank3 is thought to be the cause of the core neurodevelopmental and neurob ehavi oral deficits in Phelan-McDermid syndrome because individuals carrying a ring chromosome 22 with an intact Shank3 gene are phenotypically normal. Accordingly, provided are methods of treating a neurodevelopmental disorder associated with a mutation, deletion or disruption in the Shank3 gene and a reduction in Shank3 activity.
  • ADHD attention-deficit/hyperactivity disorder
  • learning disabilities such as dyslexia or dyscalculia
  • intellectual disability such as dyslexia or dyscalculia
  • cerebral palsy impairments in vision and hearing
  • developmental language disorder neurogenetic disorders such as Fragile X syndrome, Down syndrome, Rett syndrome, hypogonadotropic hypogonadal syndromes, and traumatic brain injury.
  • a subject to be treated by methods described herein may be a human subject or a non-human subject.
  • Non-human subjects include, for example: non-human primates; farm animals, such as cows, horses, goats, sheep, and pigs; pets, such as dogs and cats; and rodents.
  • a subject to be treated by methods described herein may be a subject having, suspected of having, or at risk for developing a neurodevel opmental disorder.
  • a subject has been diagnosed as having a neurodevel opmental disorder, while in other embodiments, a subject has not been diagnosed as having a neurodevel opmental disorder.
  • the subject is a human subject having, suspected of having, or at risk for developing an autism spectrum disorder (ASD).
  • the subject is a human subject having, suspected of having, or at risk for developing Phelan- McDermid syndrome.
  • the subject is a subject having reduced expression of the Shank3 gene relative to a control subject.
  • the expression of the Shank3 gene is reduced in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder.
  • the reduced expression of the Shank3 gene in a subject is caused by disruption of at least one copy of the Shank3 gene.
  • the disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • the disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • the subject is a human subject who exhibits one or more symptoms of an ASD.
  • the subject is a human subject who exhibits developmental delay.
  • the subject is a human subject who exhibits intellectual disability (ID).
  • the subject is a human subject who exhibits sleep disturbance.
  • the subject is a human subject who exhibits hypotonia.
  • the subject is a human subject who exhibits lack of speech.
  • the subject is a human subject who exhibits language delay.
  • the subject is a human subject who exhibits any symptoms or signs of an ASD.
  • a subject is a human subject who is an adult.
  • the adult is older than 25 years of age. In some embodiments, the adult is not older than 25 years of age. In some embodiments, the adult is not older than 21 years of age. In some embodiments, the adult is not older than 18 years of age. In some embodiments, the adult is 16 years of age. In some embodiments, a subject is elderly (e.g., 65 years old or older). In some embodiments, the adult can be any age of adulthood that is suitable for the treatment disclosed herein.
  • the subject is a human subject who is not an adult. In some embodiments, the human subject is not older than 16 years of age. In some embodiments, the human subject is not older than 10 years of age. In some embodiments, the human subject is 10 years of age or younger. In some embodiments, the human subject is a child or an infant. In some embodiments, the human subject is a toddler. In some embodiments, the human subject is at the fetal stage of development. In some embodiments, the human subject is at the prenatal stage of development.
  • polynucleotides encoding MiniShank3 proteins can be delivered to a tissue or cell of interest in a viral vector.
  • Vectors described herein can be used to deliver a nucleic acid encoding a protein of interest to a subject, including, e.g., delivery to specific organs or to the central nervous system (CNS) of a subject.
  • the protein of interest is a Shank protein.
  • the protein of interest is a Shank3 protein.
  • the protein of interest is a MiniShank3 protein.
  • the present disclosure provides a vector comprising a polynucleotide encoding a miniShank protein disclosed herein. In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a Shank3 protein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the present disclosure provides a recombinant AAV virion comprising a recombinant AAV vector and an AAV capsid. In embodiments, the present disclosure provides a recombinant AAV virion comprising an expression cassette encoding a miniShank3 protein and an AAV9 capsid.
  • AAV refers to a replication-deficient (e.g., nonreplicating) Dependoparvovirus within the Parvoviridae genus of viruses.
  • AAV can be derived from a naturally occurring virus or can be recombinant.
  • AAV can be packaged into capsids, which can be derived from naturally occurring capsid proteins or recombinant capsid proteins.
  • the single-stranded DNA genome of AAV includes inverted terminal repeat (ITRs). ITRs are involved in the replication and encapsidation of the AAV genome, along with its integration in the host genome and its excision.
  • ITRs inverted terminal repeat
  • AAV vectors can comprise one or more ITRs, including a 5’ ITR and/or a 3’ ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and/or additional posttranscriptional regulator elements.
  • AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary skill in the art, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (2012)), which is incorporated herein by reference in its entirety.
  • AAV integrates into a host cell genome. In some embodiments, AAV does not integrate into a host genome.
  • AAV vectors disclosed herein can include sequences from any known organism. In some embodiments, AAV vectors disclosed herein can include synthetic sequences. AAV vector sequences can be modified in any way known to one of ordinary skill in the art, such as by incorporating insertions, deletions or substitutions, and/or through the use of posttranscriptional regulatory elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation signals. In some embodiments, AAV vectors can include sequences related to replication and integration.
  • a MiniShank3 as disclosed herein is delivered to a tissue or a cell of interest via a recombinant AAV vector.
  • the recombinant AAV vector delivering the MiniShank3 as disclosed herein is delivered to the central nervous system (CNS) of a subject.
  • delivering the recombinant AAV vector to the CNS may include delivering the recombinant AAV vector to any tissue or cell of interest in the CNS.
  • delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to neuronal tissues or cells.
  • delivering the recombinant AAV vector to the CNS involves delivering the AAV vector to the brain. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to the spinal cord. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to the white and gray matter. In some embodiments, the recombinant AAV vector delivering the MiniShank3 as disclosed herein is delivered to any tissue or cell of interest of a subject that is suitable for the treatments as disclosed herein.
  • delivering” or “administering” a recombinant AAV vector can include any method known in the art for delivering or administering an AAV vector or a composition comprising an AAV vector to a subject.
  • Administering can include but is not limited to direct administration of a recombinant AAV vector or a composition comprising the recombinant AAV vector, or peripheral administration via passive diffusion or convection-enhanced delivery (CED) to bypass the blood brain barrier as known in the art.
  • CED convection-enhanced delivery
  • Recombinant AAV vectors described herein can be administered in any composition that would be compatible with aspects of the disclosure.
  • AAV vectors can include any known AAV serotype, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.
  • the AAV serotype is AAV9. Clades of AAV viruses are described in, and incorporated by reference, from Gao et al. (2004) J. Virol. 78( 12): 6381 -6388. In some embodiments, any AAV serotype that is suitable for delivery to the CNS may be selected.
  • AAV vectors of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype.
  • the AAV vector may utilize or be based on an AAV serotype described in WO 2017/201258A1, the contents of which are incorporated herein by reference in its entirety, such as, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6
  • a MiniShank3 disclosed herein is delivered by a recombinant AAV vector.
  • the recombinant AAV vector comprises a transgene and its regulatory sequences, and optionally 5' and 3' ITRs.
  • the transgene and its regulatory sequences are flanked by the 5’ and 3’ ITR sequences.
  • the transgene may comprise, as disclosed herein, one or more regions that encode a MiniShank3.
  • the transgene may also comprise a region encoding for another protein.
  • the transgene may also comprise one or more expression control sequences (e.g., a poly-A tail).
  • the transgene may be single stranded.
  • a recombinant AAV vector comprises at least AAV ITRs and a MiniShank3 transgene.
  • the AAV may be packaged into an AAV particle and administered to a subject and/or delivered to a selected target cell.
  • the AAV particle comprises an AAV capsid protein.
  • the AAV particle comprises at least one capsid protein that is selected from the AAV serotypes as disclosed herein including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.eB, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, and AAV2/2-125, or a variant of any of the foregoing.
  • the AAV particle comprises an AAV9 capsid.
  • the miniShank3 transgene coding sequence in the recombinant AAV vector is operably linked to regulatory sequences for tissue-specific gene expression.
  • the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • the tissue-specific regulatory sequence can be a Syn promoter (e.g., hSynl).
  • the hSynl promoter comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 22.
  • the hSynl promoter comprises the nucleic acid sequence of SEQ ID NO: 22.
  • the tissue-specific regulatory sequence can be any promoter or enhancer that is neuron-specific and is suitable for the treatments described herein.
  • a miniShank3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs.
  • a miniShank3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 3 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
  • a miniShank3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 18 or SEQ ID NO: 20 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs.
  • a miniShank3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 17 or SEQ ID NO: 19 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
  • aspects of the disclosure relate to recombinant AAV vectors expressing miniShank3 transgenes.
  • a miniShank3 transgene is flanked by AAV ITRs.
  • the AAV ITRs comprise AAV2 ITRs.
  • the AAV ITRs comprise AAV1 ITRs.
  • the AAV ITRs comprise AAV5 ITRs.
  • the AAV ITRs comprise AAV6 ITRs.
  • the AAV ITRs comprise AAV8 ITRs.
  • the AAV ITRs comprise AAV9 ITRs.
  • the AAV ITRs comprise rhlO ITRs.
  • the AAV ITRs may include one or more modified ITRs which generate self-complementary AAV genomes.
  • the recombinant AAV vector comprises a 5’ AAV2 ITR and a 3’ AAV2 ITR.
  • the 5’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 27.
  • the 3’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 28.
  • the 5’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 27.
  • the 3’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 28.
  • AAV vectors described herein can include DNA constructs or expression cassettes that comprise a transgene such as MiniShank3, 5’ and/or 3’ ITRs, promoters, introns, and/or other associated regulatory elements that are known in the art.
  • the AAV vector comprises a Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element (WPRE), which may enhance miniShank3 transgene expression.
  • WPRE Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element
  • the WPRE comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 23.
  • the WPRE comprises the nucleic acid sequence of SEQ ID NO: 23.
  • the AAV vector comprises an untranslated portion such as an intron or a 5’ or 3’ untranslated region.
  • the intron may be located between the promoter/enhancer sequence and the miniShank3 transgene.
  • the AAV vector used herein may be a self- complementary vector.
  • SEQ ID NO: 21 comprises a human MiniShank3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter.
  • SEQ ID NO: 30 comprises a human MiniShank3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter.
  • the inverted terminal repeat (ITR) sequences comprise about 145 nucleotides each. These elements may be useful in cis for effective replication and encapsidation.
  • a skilled person in the art would appreciate that any elements of AAV vectors known in the art may be compatible with aspects of the disclosure.
  • any of the polynucleotide sequences described herein that encode a functional MiniShank3 protein can be expressed in a DNA construct or expression cassette for AAV delivery.
  • These DNA constructs or expression cassettes may include one or more of the elements described herein.
  • a coding sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette.
  • a coding sequence comprising the sequence of any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette.
  • the DNA construct or expression cassette includes one or more elements such as a promoter, a 5 ’-ITR, a 3 ’-ITR, a Synl promoter, a WPRE, an hGH poly A.
  • Cis plasmids for production of the recombinant AAV virions may have elements such as origin of replications and antibiotic resistance markers, for example, an Fl origin, a NeR/KanR marker and/or a PUC origin.
  • FIG. 1 shows an example of a plasmid for production of recombinant AAV virions described herein.
  • Expression cassettes described herein may comprise a polyA signal.
  • the polyA signal is the hGH polyA signal.
  • the polyA signal comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 24.
  • the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 24.
  • a recombinant AAV vector associated with the disclosure includes a nucleic acid sequence encoding a MiniShank3 protein operably linked to regulatory elements that promote CNS expression and flanking ITRs that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 21 or SEQ ID NO: 30, provided in Table 9.
  • a recombinant AAV vector comprises a sequence corresponding to SEQ ID NO: 21 or SEQ ID NO: 30, which encode a MiniShank3 protein comprising the sequence of SEQ ID NO: 18 and comprises regulatory elements, including a hSynl promoter, an WPRE element and a polyA signal sequence and flanking ITR sequences.
  • a recombinant AAV vector comprising the sequence of SEQ ID NO: 21 or SEQ ID NO: 30 may be delivered to a human subject in need thereof and may be suitable for treating a human subject who has a neurodevelopmental disorder.
  • any method known in the art for designing AAV vectors for clinical use, and for delivery of AAV vectors may be compatible with aspects of the disclosure.
  • disclosure related to AAV vectors and delivery are provided in and incorporated by reference from U.S. Patent No. 7,906,111, entitled “Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor” and U.S. Patent No. 9,834,788, entitled “AAV -vectors for use in gene therapy of choroideremia,” each of which is incorporated by reference herein in its entirety.
  • the recombinant AAV vector encoding a MiniShank3 protein for AAV delivery encodes a protein with a sequence that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, to any one of SEQ ID NOs: 17-20, provided in Table 9.
  • the present disclosure provides recombinant AAV virions comprising: (1) recombinant AAV vectors described herein and (2) an AAV9 capsid.
  • the AAV9 capsid comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, to SEQ ID NO: 29.
  • a “virion” refers to a viral particle that includes genetic material (e.g., RNA or DNA) and a capsid.
  • an expression cassette disclosed herein can comprise SEQ ID NO: 26 or SEQ ID NO: 21 (with flanking ITR sequences) or SEQ ID NO: 30 (with flanking ITR sequences) as shown in Table 9.
  • a recombinant vector comprising an expression cassette that comprises a polynucleotide encoding the Shank3 protein (i.e., the miniShank3 DNA construct) can be expressed in a specific tissue or cell of interest.
  • the expression cassette or vector disclosed herein comprises a promoter.
  • the expression cassette or vector comprises a cell-type specific promoter.
  • the promoter is a human promotor.
  • the human promoter is human Synapsin 1 (hSynl).
  • the hSynlpromotor has a polynucleotide sequence corresponding to SEQ ID NO: 22.
  • the human promoter can be any promotor that is known in the art and is suitable for expression of miniShank3. In some embodiments, the human promoter can be any promoter that has high specificity for neuronal tissues and cells. In some embodiments, the promoter can be a constitutive promoter. For example, the constitutive promoter can be a CAG promoter. As one of ordinary skill in the art would appreciate, any promoter may be used so long as the selected promoter is compatible with aspects of the disclosure.
  • the present disclosure provides methods of producing an AAV virion.
  • the method comprises culturing a host cell comprising an AAV vector described herein, an AAV cap (capsid protein) and an AAV9 rep (replication protein), and optionally one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell.
  • the AAV cap encodes VP1, VP2, and/or VP3.
  • the rep encodes rep78, rep68, rep52, and/or rep40.
  • compositions and Administration [00189]
  • the present disclosure provides compositions, including pharmaceutical compositions, comprising a polynucleotide (e.g., encoding for miniShank3) delivered in a recombinant AAV vector and/or an AAV virion as disclosed herein and a pharmaceutically acceptable carrier.
  • a polynucleotide e.g., encoding for miniShank3
  • compositions of the disclosure may comprise a recombinant AAV vector and/or an AAV virion alone, or in combination with one or more other viruses.
  • a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different recombinant AAV vectors and/or AAV virions.
  • Suitable carriers may be readily selected by one of ordinary skill in the art in view of the indication for which the recombinant AAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the selection of the carrier is not a limitation of the present disclosure.
  • Pharmaceutical compositions comprising AAV vectors are described further in US 9,585,971 and US 2017/0166926, which are incorporated by reference herein in their entireties.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • dispersion media includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the recombinant AAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the recombinant AAV constructs disclosed herein.
  • the formation and use of liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Nanocapsule formulations of the recombinant AAV vector may be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way.
  • ultrafine particles sized around 0.1 pm
  • Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • the pharmaceutical composition comprising a nucleic acid delivered in a recombinant AAV vector comprises other pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, thimerosal, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • isotonic agents for example, sugars or sodium chloride. Prolonged absorption of the pharmaceutical compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical forms suitable for delivering the recombinant AAV vectors include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Methods described herein comprise administering recombinant AAV vector in sufficient amounts to transfect the cells of a desired tissue (e.g., brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • a desired tissue e.g., brain
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ, oral, inhalation, intraocular, intravenous including facial vein injection and retroorbital injection, intracerebroventricular (ICV), intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parental routes of administration.
  • the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via parenteral administration.
  • the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via intravenous administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via ICV administration. In some embodiments, the ICV administration can be unilateral administration. In some embodiments, the ICV administration can be bilateral administration. Recombinant AAV virions described herein delivered via ICV administration may improve the therapeutic effects of miniShank3.
  • Routes of administration may be combined, if desired.
  • the vector as disclosed herein is administered intravenously.
  • the present disclosure provides methods of treating a subject having a neurodevelopmental disorder. In some embodiments, the present disclosure provides methods of treating a subject having an autism spectrum disorder (ASD). In some embodiments, the present disclosure provides methods of treating a subject having Phelan- McDermid syndrome.
  • ASD autism spectrum disorder
  • Methods provided herein comprise administering and delivering an effective amount of a composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue of a subject.
  • the target tissue is cortex.
  • the target tissue is striatum.
  • the target tissue is thalamus cerebellum.
  • the target tissue is hippocampus.
  • the target tissue is any brain structure.
  • methods for administering and delivering an effective amount of a composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue comprise delivering the composition to neurons or other brain cell types.
  • methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof comprise providing a composition comprising a recombinant AAV virion comprising at least a nucleic acid (e.g., miniShank3) to be delivered to the target environment or tissue of the subject and administering the composition to the subject.
  • methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof include delivering an AAV virion by unilateral or bilateral intracerebroventricular administration.
  • Methods of use of AAV vectors are described further in US 9,585,971, US 2017/0166926, and W02020/160337, which are incorporated by reference herein in their entireties.
  • the composition may comprise a capsid protein.
  • the composition comprising a recombinant AAV virion that comprises a polynucleotide encoding a Shank3 protein is delivered to the subject via intravenous administration, systemic administration, intracerebroventricular (ICV) administration, including bilateral or unilateral ICV administration, in utero administration, intrathecal administration, retro-orbital injection, or facial vein injection.
  • in utero administration is used for a subject who is at the prenatal stage of development.
  • the composition is delivered to a subject via a nanoparticle.
  • the composition is delivered to a subject via a viral vector.
  • the composition is delivered to a subject via any carriers suitable for delivering nucleic acid materials.
  • composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a protein that would be of some use or benefit to the subject may be delivered to a target environment or tissue of the subject according to methods disclosed herein
  • Sonophoresis i.e., ultrasound
  • U.S. Pat. No. 5,656,016 has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system.
  • Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
  • the dose of the recombinant AAV vector or recombinant AAV virion comprising a polynucleotide that encodes a Shank3 protein (e.g., miniShank3) required to achieve a particular "therapeutic effect," e.g., the units of dose in absolute vector genomes (vg) or vector genomes per milliliter of pharmaceutical solution (vg/mL) will vary based on several factors including, but not limited to: the route of AAV administration, the level of gene expression required to achieve a therapeutic effect, the specific disorder being treated, and the stability of the gene product. Doses that give maximal percentage of infection without affecting neurodevelopment are also suitable.
  • One of skill in the art can readily determine a recombinant AAV vector or recombinant AAV virion dose range to treat a patient having a particular disorder based on the aforementioned factors, as well as other factors.
  • an effective amount of a recombinant AAV vector or a recombinant AAV virion may be an amount sufficient to infect an animal or human subject or target a desired tissue.
  • the effective amount will depend primarily on factors such as the species, age, gender, weight, health of the subject, and the tissue to be targeted, and may thus vary among subjects and tissues.
  • the term “effective amount” or “amount effective” in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject.
  • an effective amount of a composition disclosed herein may partially or fully rescue the effects of a mutated Shank3 gene and/or partially or fully restore loss of function of the Shank3 protein.
  • An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether.
  • An effective amount can also involve delaying the occurrence of an undesired response.
  • An effective amount can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result.
  • the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result.
  • Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors. It should be appreciated that an effective amount as used herein does not need to be clinically effective.
  • the number of vector genomes administered to the subject is any value between about 6.0 x 10 11 vg and about 9.0 x 10 13 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 6.0 xlO 13 vg/mL and about 9.0 xlO 13 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 1 x 10 10 to about 1 x 10 12 vg. In certain embodiments, the effective amount of AAV is 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 genome copies per kg.
  • the effective amount of AAV is 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some cases, a dosage between about IO 11 to 10 13 AAV genome copies is appropriate. In some embodiments, a dose of about 1.0 x 10 13 to about 1.0 x 10 14 vector genomes is administered to the subject. In some embodiments, the number of vector genomes administered to the subject can be any dose that is suitable for the treatments and methods disclosed herein. In some embodiments, the dose of vector genomes is administered by unilateral or bilateral ICV administration.
  • the dose administered to the subject via unilateral ICV administration is about 3 pl, 4 pl, 5 pl, 6 pl, 7 pl, 8 pl, 9pl, or 10 pl per subject. In some embodiments, the dose administered to the subject via unilateral ICV administration is about 5 pl per subject.
  • the dose administered to the subject via unilateral ICV administration is about 1.4 * 10 10 vg/ml, about 1.4 * 10 11 vg/ml, about 1.4 * 10 12 vg/ml, about 1.4 x 10 13 vg/ml, about 1.4 * 10 14 vg/ml, about 1.4 * 10 15 vg/ml, or about 1.4 * 10 16 vg/ml.
  • the dose administered to the subject via unilateral ICV administration is about 1.4 x 10 13 vg/ml.
  • the dose administered to the subject via unilateral ICV administration is from about 1.4 x 10 10 vg/ml to about 1.4 x 10 16 vg/ml.
  • a dose of recombinant AAV is administered to a subject as a single dose. In some embodiments, a dose of recombinant AAV is administered to a subject as a single dose with the potential to be re-dosed at a later time.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions disclosed herein is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
  • the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • the formulation comprises a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
  • the formulation consists essentially of a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
  • the formulation consists of a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
  • PSD proteins are useful for treating a neurodevel opmental disorder, such as, for example, an autism spectrum disorder (ASD), or Phelan-McDermid syndrome.
  • expression levels of PSD proteins are used to evaluate the efficacy of the administration of the miniShank3.
  • the PSD protein is Homer.
  • the PSD protein is post-synaptic density protein 95 (PSD95).
  • PSD protein is SynGapl.
  • the PSD protein is SAPAP3.
  • the PSD protein is NR1.
  • the PSD protein is NR2B.
  • the PSD protein is GluR2.
  • the PSD protein is any protein that can be improved or restored upon the miniShank3 treatment.
  • an increase of any of the PSD proteins, as compared to an untreated control subject, may indicate efficacy of the miniShank3.
  • expression of Homer in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of post-synaptic protein (PSD95) in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of SynGapl in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of SAPAP3 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of NR1 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of NR2B in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of GluR2 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving sleep efficiency.
  • a subject has improved sleep efficiency after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the sleep efficiency in the subject after being administered an effective amount of a composition described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Improved sleep efficiency can include less sleep disturbance, which includes but is not limited to having trouble falling and staying asleep. Measurement of sleep efficiency can be conducted using any methods known in the art.
  • administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving social impairment.
  • the social impairment of the subject is improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the social impairment in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of social impairment can be conducted using any methods known in the art.
  • social impairment refers to behavioral abnormalities or defects that prohibit a subject from displaying voluntary social interaction.
  • administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving locomotion and/or motor coordination deficits.
  • the locomotion and/or motor coordination deficits of the subject are improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the locomotion and/or motor coordination deficits in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of locomotion and/or motor coordination deficits can be conducted using any methods known in the art.
  • locomotion and/or motor coordination deficits can include, for example, lack of coordination, loss of balance, and/or a shuffling gait.
  • administering can lead to improvement in cortical-striatal synaptic dysfunction.
  • the corti cal -striatal synaptic dysfunction of the subject is improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the corti cal -striatal synaptic dysfunction in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of corti cal -striatal synaptic dysfunction can be conducted using any methods known in the art.
  • cortical-striatal synaptic dysfunction refers to defective corticostriatal circuits in the brain that can cause repetitive and compulsive behaviors, such as in neuropsychiatric disorders and neurodevelopmental diseases such as autism, obsessive- compulsive disorders, and Tourette syndrome.
  • Example 1 Intravenous Administration of an AAV Vector Containing miniShank3 to
  • ASD Autism spectrum disorder
  • Shank postsynaptic scaffold protein family emerges as one cause of ASD, with Shank mutations accounting for approximately 1% of patients with ASD.
  • Prevalence of Shank3 haploinsufficiency due to deletion, truncation or missense mutation of the gene is estimated to be 1 : 15000 globally.
  • Shank3 haploinsufficiency has been identified in patients with chromosome 22ql3.3 deletion syndrome, also known as Phelan-McDermid syndrome (PMS) It has been found that PMS due to Shank3 haploinsufficiency is associated with behavioral and cognitive manifestations of variable severity. Deficits are evident as early as infancy and persist throughout life. Current medical treatments are limited to antipsychotic drugs and treatment of comorbidities. However, treatments to directly address Shank3 haploinsufficiency and PMS, respectively are currently not available.
  • PMS Phelan-McDermid syndrome
  • Shank3-InsG3680 mouse model A study was conducted to test if physiological deficits associated with the Shank3-InsG3680 mouse model can be improved following intravenous delivery of miniShank3 to post-natal day 14 (P14) animals. Specifically, correction of Shank3 protein and binding partner (PSD95, NR1, Homer, GluR2) levels in brain synaptic membrane preparations was assessed by assays including behavioral correction via open field tests.
  • Shank3 protein and binding partner PSD95, NR1, Homer, GluR2
  • AAV9-hSynl-Human miniShank3-Vl refers to a suspension of an adeno-associated viral vector-based gene therapy for parenteral administration. It is a recombinant nonreplicating AAV9 vector containing a single stranded transgene encoding a condensed version of the human Shank3 protein (miniShank3) and under the control of the neuron-specific synapsin promoter. As full length Shank3 is too large to be packaged by conventional AAV methods, miniShank3 was designed to reduce the overall size of the transgene to allow for proper AAV packaging, while preserving critical domains previously identified to be responsible for its proper function as a synaptic scaffold protein.
  • the “Vehicle” control includes the same excipients as the AAV9-hSynl-Human miniShank3-Vl but does not contain the AAV9 vector containing the miniShank3 transgene. [00232] Table 1 below shows the study design:
  • a total of 22 female heterozygous Shank3InsG3680 mice, 5 male Shank3InsG3680 homozygous mice and 5 male wild type mice were bred in trios or doubles. Litter births were monitored daily to time postnatal age of mice and litters remain with dams until weaning. Genotyping biopsies was taken from tail snips from about P3 pups; toe tattooing was used to identify offspring. Genotyping is performed.
  • Subjects e.g., Shank3InsG3680 and WT mice
  • This assay was performed in a custom-made open field apparatus. Each chamber is a 50 by 50 cm square. The 30-minute trial was recorded, and movement was tracked and analyzed using a custom template on Behavior Cloud. The center of the open field is defined as a 13.5 x 13.5 cm square in the geometric center of the arena. For each mouse, the total path length was measured, the time and the path length in the center of the open field was also determined. Each chamber was cleaned between individual mouse testing.
  • Open Field measurements such as total path length, time in center and % path in center were analyzed by One-Way ANOVA with Tukey's post hoc for multiple comparisons. Path length in 5 min bins was analyzed by Two-Way ANOVA with time as a within groups factor and treatment as a between groups factor. Post-hoc pairwise comparisons used Bonferroni corrected comparisons.
  • Shank3InsG3680 mice were given IV injections of AAV- PHP.eB-hSyn-GFP-miniShank3 or a control virus expressing only green fluorescent protein (GFP) under the same promoter.
  • PhP.eB is an AAV9-derived capsid that was evolved to cross the blood brain barrier more efficiently in mice (but not NHP), allowing for transduction of the brain with IV dosing.
  • WT animals administered the control vector were also included. Groups of animals were dosed at postnatal day (P)0-P2, P7, or P28 and evaluated for neurob ehavi oral endpoints.
  • EEG electroencephalogram
  • Shank3 A4-22 mice fall off the rotating bar earlier than WT mice. They also fail to learn motor coordination and balance in the process. These impairments of delta power and motor skills resemble the deficits in sleep and motor activities frequently observed in SHANK3 haploinsuffi cient patients.
  • Impairment of striatal learning is underlined by defects in the postsynaptic signaling complex in the striatum.
  • Miniature evoked postsynaptic currents (mEPSC) in striatal medium spiny neurons (MSN) and spine density are reduced in Shank3A4-22 mice.
  • the PSD is eroded by the loss of scaffolding proteins required for proper anchoring of glutamate receptors, most prominently the SHANK3 binding partner Homerl.
  • Shank3 A4-22 mice displays a milder phenotype, in contrast to the more severe phenotype in SHANK3 haploinsufficient patients. The cause for this difference is not fully understood (Drapeau, 2018).
  • the described animal model parallels many of the clinical features of SHANK3 haploinsufficient patients.
  • the dysfunction in neural connectivity coincides with the current theory that dysfunction of neural circuits and plasticity underly the pathophysiology of SHANK3 haploinsufficiency.
  • Phase 1 assessed genotype-related phenotypes in naive animals
  • Phase 2 evaluated the effect of AAV9-hSynl-Human miniShank3-Vl administered at a range of doses (0, 2.40* 10 9 , 1.2Ox lO 10 , 6.OOx lO 10 , or 2.75xlO n vg) on P14 animals to correct phenotypes identified in Phase 1 at 6 to 16 weeks post- dose
  • Phase 3 evaluated the top two doses from Phase 2 at approximately 7 to 10 months post-dose.
  • Phase 3 In parts of Phase 2 and all of Phase 3, a general increase in dermatitis in the vivarium appeared to predominantly affect AAV9-hSynl-Human mini Shank3 -VI -treated animals. In Phase 3, this led to high mortality that precluded assessment of most efficacy endpoints at the 7 months post-dose timepoint.
  • Table 2 shows a summary of the test article and vehicle used in this study.
  • the test article (AAV9-hSynl -Human miniShank3-Vl) and vehicle are the same as those described in Example 1.
  • the number of animals per group includes 15 males (M) and 15 females (F) per genotype for the Phenotyping Pilot, 17 males and 6 females for the Injection Study of Cohorts 1, 2 and 4 (Phase 2) and 9 males and 3 females for the Injection Study of Cohort 3 (Phase 3).
  • M males
  • F females
  • Phase 2 17 males and 6 females for the Injection Study of Cohorts 1, 2 and 4
  • Phase 3 9 males and 3 females for the Injection Study of Cohort 3
  • mice Up to 3 adult mice were housed per cage and up to 6 pups with one dam per cage after injection. Any additional pups in litters >6 was fostered. Mice were given standard food and water and no acclimation period is given. In general, mice grouped by sex, genotype, and solution were injected when applicable.
  • mice in the injection study were assigned to dosing groups in a rotating manner so that an approximately equal number of mice born each week was assigned to each of the treatment groups. Mice were injected by groups of 4 mice injected successively with one dose on a given day to optimize the use of aliquots. Body weight ⁇ 20% of peak body weight (maximum weight reached for each individual mouse) and Body Condition Score of or below 2 were used as criteria for euthanasia.
  • Phase 1 represents the baseline collection of phenotypes in non-injected mice at 8-13 weeks of age.
  • Phase 2 represents the collection of phenotypes in injected mice at 8-13 weeks of age.
  • Phase 3 represents the baseline collection of phenotypes in injected mice at 7 months of age.
  • Table 6 Cohorts Composition
  • Phase 2 a range of doses of AAV9-hSynl-Human miniShank3-Vl were administered ICV at P14 to WT or Shank3A4-22 KO littermates which were evaluated for neurob ehavi oral endpoints beginning at 6 weeks post-dosing and continuing through 14 weeks post-dose.
  • n 10 males/group had EEG electrodes implanted and were evaluated for seizure-like activity and power spectrum analysis.
  • Phase 2 In Phase 2, several neurob ehavi oral endpoints that had been significant in Phase 1 did not show statistically significant genotype-related differences, including open field vertical activity, and grooming duration. Data from endpoints showing genotype-related differences is shown in the figures below. In Phase 3, the only neurobehavioral endpoint evaluated was EEG assessments.
  • Pups for the study were born from trio (2 females, 1 male) matings of Shank3 heterozygous parents. Matings were set up 19 days prior to the planned birth date. The birth date was planned 14 days prior to the planned injection date. All pups were treatment naive prior to placement on study. Pups were sampled by toe-clip for genotyping between postnatal day 1 and 7.
  • Body Condition Score was determined weekly for mice having lost > 10% of their peak body weight.
  • mice were injected once at P14 ⁇ 2 days. In brief, mice were anesthetized with isoflurane (5% induction, 3% maintenance, in 02). The top of the skull was shaved. Mice were placed on a Stoelting stereotactic table, securing the head with ear bars. A nose cone provides the isoflurane. The shaved area was cleaned with chlorhexidine. Using sterile surgical techniques, an incision was performed on the skin to expose the skull. Hydrogen peroxide is applied to expose the bregma and lambda.
  • a drill was mounted on the articulated arm of the stereotactic table and used to perform an opening on the skull at the selected coordinates: from bregma, 1 mm caudal, 0.4 mm from sagittal suture targeting the right ventricle.
  • a Hamilton syringe was mounted on the Stoelting injector placed in the articulated arm of the table. Needle was moved to x,y coordinates above and lowered to 2.0 mm depth. Injection (unilateral injection of 5 microliters) was initiated at the flow rate selected (1 pL/min). Once injection was complete, the needle was left in place for a least 3 min, then slowly removed. Bupivacaine was applied topically before glue was applied on the skull and sutures, on the skin. 1 mL of warm, sterile saline was given i.p. and mouse was allowed to recover on a warm pad before being returned to the cage. Instruments were bead sterilized before surgery on the next mouse.
  • mice were tested once at 9-10 weeks of age.
  • the apparatus is a square arena ( 40 x 40 x 40 cm) made from clear
  • IR infrared
  • mice in their home cage were habituated to the testing room for 60 minutes prior. To begin, a mouse was placed in the center of the arena and the computer started recording. A typical session lasted 60 min and data is presented as sum of each measurement over bins of 10 min each. At the conclusion of testing, mice were returned to their homecages (mice that are grouped housed are run in parallel in different arenas). The test arena was cleaned with 70% EtOH between subjects.
  • a Ugo-Basile rotarod which consists of a rotating rod that is 3 cm in diameter and is suitably machined to provide a gripping surface for the mice.
  • Six discs divide the drum into five lanes of 5.7 cm width each. This enables the testing of five mice simultaneously.
  • the plate mechanism trips, thereby recording the animal's endurance time in seconds. Height to fall is 16 cm.
  • mice were acclimated in their home cage to the testing room at least 60 minutes prior to testing.
  • the mouse was placed on a rotating rod at 4 rpm.
  • speed was increased linearly to a maximum rpm of 40 over a period of 300 seconds.
  • Mice were run on four successive trials with approximately one-minute inter-trial interval.
  • the rod was cleaned between trials.
  • the procedure was repeated on 2 consecutive days.
  • the animal typically "learns" how to stay in the rod on the first day.
  • the average score obtained on the second day is considered their real ability to stay in the rod. Latency to fall is recorded for each mouse.
  • a Pinnacle Technology headmount was secured on the skull with adhesive, centered over Bregma.
  • a sterile precision drill outfitted with a 0.5 mm sterile bit, was used to bore 4 holes through the skull but not into the dura or the cortex, at the four comers of the headmount. Headmount screws were inserted through the headmount into the holes of the skull.
  • the skull was sealed with dental cement and the implant was embedded into the cement to secure it into place.
  • the skin was closed with size 6-0 nylon sutures and Gluture tissue adhesive to make contact with the cement, so no skull or tissue is exposed.
  • mice were singly housed following surgery. Seven or more days after surgery, a fine, flexible cable (e.g., tether) was attached to the implant such that the mouse can move freely about the cage while its EEG is recorded onto a computer. Recording sessions lasted 72 hrs for one mouse. During this time, food was made available ad libidum as grain on the bottom of the cage and with addition of a DietGel 76A for hydration.
  • a fine, flexible cable e.g., tether
  • the Sirenia Sleep Pro and Sirenia Seizure Pro software were used to analyze EEG recordings. Seizure related EEG events were quantified in Sirenia Seizure Pro and include pre-ictal biphasic spikes, spike-wave discharges (SWD), epileptiform seizure discharges. While EEG was recorded from both the parietal and motor cortex with a 3- electrode system, recording of the motor cortex is screened for seizures and the parietal recording is only used for verification.
  • SWD dominant frequency between 6 and 10 Hz. If the power spectrum did not show a predominant peak at 6-10 Hz, then the event was not categorized as SWD (see below, 4 th class).
  • Flag events were saved with the analyzed recording by the scorers and reviewed by an independent, trained scorers who confirms, changes the class or removed the event. Descriptive statistics of the events for each mouse were exported to a tabulated data file and include the number and duration of each type of event in the light vs. dark phases.
  • Power spectrum analysis was performed using Simia software on final 24h of 72h EEG recordings (400 Hz sample rate, low pass filtered at 50 Hz). Fast Fourier transform calculated power (square of voltage) on 10s epochs for frequency bands delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz) , and beta (12-30 Hz).
  • FIG. 4A AAV9-hSynl- Human miniShank3-Vl (SEQ ID NO: 21) was administered at a dose of 6.0 x IO 10 vg/mouse or 2.75 x 10 11 vg/mouse.
  • EEG assessments included both analysis of seizure-like wave patterns and power analysis as a measure of restorative sleep.
  • representative EEG traces of different types of seizures were recorded in a pilot experiment.
  • Shank3 KO mice were injected with pentylenetetrazole, a known epileptogenic compound; their EEG was recorded, and clinical signs of seizures, including behavioral arrest and convulsions, were observed.
  • Representative traces of three types of seizures in this pilot guided the detection and classification of EEG abnormalities in the AAV9-hSynl-Human mini Shank3 -VI -treated animals: isolated spikes, spike-wave discharges, and epileptic seizures.
  • EEG analyses of WT and KO mice in Phase 2 at 14 weeks after injection revealed no significant changes in the frequency of three specific EEG wave patterns: epileptic-like (FIG. 6A), spike-wave discharges-like (FIG. 6B), and spike-like (FIG. 6C).
  • epileptic-like FIG. 6A
  • spike-wave discharges-like FIG. 6B
  • spike-like FIG. 6C
  • mice were assessed in the three-chamber test.
  • 3-chamber mice were tested once at 10-11 weeks of age.
  • the 3-chamber apparatus for mice was a Plexiglas arena (40.5 x 60.0 x 22.0 cm) divided into three equal compartments that are created by removable interior walls. In each of the two end compartments is a cylindrical cage measuring 11 cm H x 10 cm D with 1 cm spacing between cage bars. A same sex "stranger mouse" is placed in one of the two cages.
  • mice were acclimated in their home cage to the testing room for 60 minutes. The subject was habituated to the chamber of the empty apparatus for 10 minutes with no stranger mouse present. Preference for one side was recorded. The tested mouse was removed while dividers are installed and a novel, unfamiliar adult mouse was then placed in a cylindrical cage on one side, and a novel object was placed in the other. The stranger mouse, from a strain that is known to be very calm, and of the same sex as the tested mouse, had been habituated to the test cage for up to 10 min on days prior to the test. The test mouse was placed again in the cage and both dividers are removed, allowing the mouse to explore for another 10 minutes. Time spent in each chamber and entries into each chamber were scored using automated video tracking via an overhead camera.
  • mice were replaced in a holding cage until all cage mates have been tested, at which point all mice were returned to their home cage.
  • the arena was wiped with 70% EtOH between mice to minimize scent-tracking. Data is presented as time spent interacting with the novel object vs. the unfamiliar mouse.
  • Shank3A4-22 mice demonstrated no significant genotypic difference compared to WT animals, as measured by the standard object and stranger time readouts (FIGs. 5A-5B).
  • a genotype-dependent difference was detected in the stranger nose point time, a novel output not previously characterized in Shank3 mouse models, in the three-chamber assessment as shown in FIG. 5C, but this endpoint was not affected by AAV9-hSynl -Human miniShank3-Vl treatment.
  • mice were in an empty cage (no bedding, food or water), placed on a table and video-recorded for future video-tracking by a trained experimenter. Mice were acclimated to the testing room in their home cage for a minimum of one hour. In the habituation phase, mice were placed in an empty cage for 20 minutes. The first 10 minutes are not scored for grooming (habituation). The last 10 minutes were assessed for grooming behavior. The entire experiment is video-recorded. A trained technician scored the grooming behavior of the mice accordingly (cumulative duration and number of grooming bouts with time).
  • mice were tested once at 11-16 weeks of age.
  • mice were habituated to the testing room for a minimum of 30 minutes. A subject was placed inside the cylinder and observed for a maximum exposure time of 30 seconds. Latency to first instance of paw withdrawal, jumping, hind paw lick, or paw shake/flutter was recorded to the nearest 0.1 seconds with the keypad on the device. If no response was observed after 30 seconds, the subject is removed from the hot plate. Subjects are placed in a holding cage until all mice within the home cage have been tested.
  • miniSHANK3 protein levels translate to 21.65%, 49.44%, and 589.20% of WT endogenous SHANK3 protein levels, for KO animals treated with 1.2Ox lO 10 vg, 6.OOx lO 10 vg, and 2.75x lO n vg, respectively (Table B).
  • AAV9-hSynl-Human mini Shank3 -VI -treated KO animals demonstrated a dose-dependent increase in Homerl protein levels, with an increase compared to vehicle-treated KO animals at the 6.00x 10 10 vg dose level and supraphy si ologi cal levels relative to WT Homerl levels achieved at the 2.75x 10 11 vg dose level.
  • PSD95 a protein that in complex with guanylate kinase-associated protein (GKAP) and supported by SHANK3, helps stabilize and regulate ionotropic glutamate N-methyl-D-aspartate (NMD A) receptors (Kim, 1997).
  • GKAP guanylate kinase-associated protein
  • mice of each treatment group are assigned to four necropsy groups.
  • Cardiocentesis and Transcardial perfusion (for BD and Histo subgroups only: SM, SF per treatment group in Phase 2; 7M and 3F in Phase 3)
  • Table 8 below shows the tissues to be collected and the assays to be conducted.
  • Terminal blood samples for Clinical Pathology and ELISA analysis are collected prior to transcardial perfusion as described.
  • Tissues from PBS perfused animals are used. Tissues harvested for fixation is collected, placed in histology cassettes identifying the mouse ID and placed in 10% neutral buffered formalin sample jars containing at least 15 times excess volume to completely submerge the cassette. Cassettes from the same group are put into the same jar. Organ identification is performed at the designated third party. Tissues are fixed for 48-72 hours at room temperature then transferred to 70% ethanol for immediate shipment for processing.
  • spleen tissues is collected for ELISPOT and ELISA sample analysis on 3 male mice and 2 female mice in the BD groups. Blood is processed for clinical chemistry samples.
  • AAV9-hSynl-Human miniShank3-Vl is as an investigational AAV9-based gene therapy in preclinical development intended to deliver a functional version of SHANK3 to treat autism spectrum disorder (ASD), Phelan-McDermid syndrome (PMS), and other neurodevelopmental disorders that result from a mutation or deletion within the gene or from chromosomal rearrangements at 22ql3.3 spanning SHANK3.
  • ASD autism spectrum disorder
  • PMS Phelan-McDermid syndrome
  • other neurodevelopmental disorders that result from a mutation or deletion within the gene or from chromosomal rearrangements at 22ql3.3 spanning SHANK3.
  • NHPs received either a single ICV administration of vehicle or AAV9-hSynl-Human miniShank3-Vl via unilateral or bilateral injection at a dose of 1.0 x 10 13 or 1.0 x 10 14 vg/animal in a total volume of 2.0 mL given via bolus injection.
  • Injection coordinates were determined via MRI followed by stereotaxic administration targeting the lateral ventricles. Animals were followed for a 90-day in-life period, and biodistribution of AAV9-hSynl- Human miniShank3-Vl vector DNA and RNA in CNS and peripheral tissues was analyzed via droplet digital PCR (ddPCR) and supported via RNA fluorescence in situ hybridization (FISH).
  • ddPCR droplet digital PCR
  • FISH RNA fluorescence in situ hybridization
  • AAV9-hSynl-Human miniShank3-Vl The unilateral and bilateral ICV administration procedures and single doses of AAV9-hSynl-Human miniShank3-Vl were well tolerated. At 90 days, animals that received AAV9-hSynl-Human miniShank3-Vl showed robust and widespread rostrocaudal transduction throughout the CNS in a dose-dependent manner compared to vehicle controls. Analysis of AAV9-hSynl-Human miniShank3-Vl vector genome DNA copies in five key brain regions showed the highest levels of transduction in the frontal cortex and hippocampus, followed by the cerebellum and spinal cord, at both dose levels.
  • Betancur C Sakurai T, Buxbaum JD. (2009) The emerging role of synaptic cell-adhesion pathways in the pathogenesis of autism spectrum disorders. Trends Neurosci. 32:402-12.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features.

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Abstract

Aspects of the disclosure relate to expression cassettes encoding miniShank3 proteins, AAV vectors comprising the expression cassettes, and gene therapy methods. In particular, the expression cassettes is formulated to further comprise AAV9 capsids, and the gene therapy is useful for treating neuroveleopmental disorders such as autism spectrum disorder (ASD) and Phelan-McDermid syndrome.

Description

SHANK3 GENE THERAPY APPROACHES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/448,199, filed February 24, 2023, entitled “SHANK3 GENE THERAPY APPROACHES,” the entire disclosure of which is hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The contents of the electronic sequence listing (Bl 19570183 WOOO-SEQ-VLJ.xml; Size: 66,840 bytes; and Date of Creation: February 23, 2024) is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present disclosure relates to gene therapy approaches for delivering polynucleotides encoding a Shank3 protein to a subject who has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
BACKGROUND
[0004] Deletions and/or mutations involving Shank3 account for about 0.5-1% of all autism spectrum disorder (ASD) patients and about 2% of ASD patients with intellectual disability (ID). However, there is no effective treatment for ASD and/or ID. Several challenges have arisen to developing pharmacological treatments that could correct the multitude of pathologies associated with ASD and ID.
SUMMARY
[0005] Aspects of the disclosure relate to the development of an effective gene therapy approach for subjects with Shank3 mutations.
[0006] Aspects of the disclosure provide expression cassettes comprising a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the polynucleotide encoding a human miniShank3 protein is operably linked to a human Syn promoter and a polyA signal sequence.
[0007] In some embodiments, the polynucleotide encoding the human miniShank3 protein is operably linked to a WPRE element. In some embodiments, the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the miniShank3 protein is at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 2. In some embodiments, the polynucleotide encoding the miniShank3 protein comprises the nucleic acid sequence of SEQ ID NO: 2.
[0008] The present disclosure provides recombinant AAV vectors comprising expression cassettes described herein flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the nucleic acid sequences of SEQ ID NO:27 and SEQ ID NO: 28, respectively. In some embodiments, the recombinant AAV vector comprises the sequence of SEQ ID NO: 21. In some embodiments, the recombinant AAV vector comprises the sequence of SEQ ID NO: 30.
[0009] The present disclosure provides recombinant AAV virions comprising (1) recombinant AAV vectors described herein; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29. The present disclosure provides pharmaceutical compositions comprising the recombinant AAV virions described herein and a pharmaceutically acceptable carrier.
[0010] Further aspects of the disclosure relate to methods of delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof. In some embodiments, the methods comprise administering to the CNS of the subject a pharmaceutical composition comprising recombinant AAV virions described herein.
[0011] In some embodiments, the recombinant AAV virion is delivered to the brain of the subject. In some embodiments, the recombinant AAV virion is delivered to the cortex, striatum and/or thalamus of the subject. In some embodiments, the recombinant AAV virion is administered by intracerebroventricular administration (ICV). In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration.
[0012] In some embodiments, the subject is a human subject. In some embodiments, the human subject is an adult. In some embodiments, the human subject is not an adult. In some embodiments, the human subject is not older than 25 years old. In some embodiments, the human subject is 10 years old or younger. [0013] In some embodiments, the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder. In some embodiments, the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD). In some embodiments, the subject exhibits one or more symptoms of an ASD. In some embodiments the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome. In some embodiments, the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay. In some embodiments, the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
[0014] In some embodiments, the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
[0015] In some embodiments, reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
[0016] In some embodiments, the recombinant AAV virion is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
[0017] Further aspects of the disclosure provide methods of treating a subject having a neurodevelopmental disorder. In some embodiments, the disclosure relates to methods of treating a subject having an autism spectrum disorder (ASD). In some embodiments, the disclosure relates to methods of treating a subject having Phelan-McDermid syndrome. In some embodiments, the methods of treatment comprise administering to the subject a therapeutically effective amount of a composition comprising an expression cassette or a recombinant AAV virion described herein. In some embodiments, the composition is administered by ICV administration. In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration.
[0018] In some embodiments, the autism spectrum disorder (ASD) comprises autism disorder. In some embodiments, the subject has improved sleep efficiency after said administered. In some embodiments, the composition is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg. [0019] Further aspects of the disclosure provide recombinant AAV vectors described herein. In some embodiments, the recombinant AAV vector is a plasmid.
[0020] Further aspects of the disclosure provide host cells that comprise the recombinant AAV vector described herein and comprise nucleic acid sequences encoding AAV rep and AAV9 cap.
[0021] Further aspects of the disclosure provide methods of producing an AAV virion. In some embodiments, the methods comprise culturing a host cell that comprise: the recombinant AAV vector described herein; AAV cap; AAV9 rep; and one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell. In some embodiments, the AAV cap encodes VP1, VP2, and/or VP3. In some embodiments, the AAV9 rep encodes rep78, rep68, rep 52, and/or rep 40.
[0022] Aspects of the disclosure relate to methods of delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof, said method comprising administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
[0023] In some embodiments, the recombinant AAV virion is delivered to the brain of the subject. In some embodiments, the recombinant AAV virion is delivered to the cortex, striatum and/or thalamus of the subject. In some embodiments, the recombinant AAV virion is administered by intracerebroventricular (ICV) administration. In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration.
[0024] In some embodiments, the subject is a human subject. In some embodiments, the human subject is an adult. In some embodiments, the human subject is not an adult. In some embodiments, the human subject is not older than 25 years old. In some embodiments, the human subject is 10 years old or younger.
[0025] In some embodiments, the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder. In some embodiments, the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD). In some embodiments, the subject exhibits one or more symptoms of an ASD. In some embodiments, the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome. In some embodiments, the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
[0026] In some embodiments, the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject. In some embodiments, the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome. In some embodiments, reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
[0027] In some embodiments, the recombinant AAV virion is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
[0028] Aspects of the disclosure relate to methods of treating a subject having a neurodevelopmental disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
[0029] Aspects of the disclosure relate to methods of treating a subject having an autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29.
[0030] Aspects of the disclosure relate to methods of treating a subject having Phelan- McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 29. [0031] In some embodiments, the subject is a human subject. In some embodiments, the human subject is an adult. In some embodiments, the human subject is not an adult. In some embodiments, the human subject is not older than 25 years old. In some embodiments, the human subject is 10 years old or younger.
[0032] In some embodiments, the composition is delivered to the brain of the subject. In some embodiments, the composition is delivered to the striatum and/or thalamus of the subject. In some embodiments, the composition is administered by intracerebroventricular (ICV) administration. In some embodiments, the ICV administration is unilateral administration. In some embodiments, the ICV administration is bilateral administration. [0033] In some embodiments, the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay. In some embodiments, the autism spectrum disorder (ASD) comprises autism disorder. [0034] In some embodiments, the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject. In some embodiments, the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome. In some embodiments, reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene. In some embodiments, disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene. In some embodiments, the subject has improved sleep efficiency after said administered. [0035] In some embodiments, the composition is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
EMBODIMENTS
[0036] Embodiment 1. A method of delivering a human miniShank3 protein to the central nervous system of a subject in need thereof, said method comprising administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29. [0037] Embodiment 2. The method of embodiment 1, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
[0038] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
[0039] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
[0040] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the AAV virion is delivered to the brain of the subject.
[0041] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the AAV virion is delivered to the cortex, striatum and/or thalamus of the subject.
[0042] Embodiment 7. The method of any of any one of embodiments 1 to 6, wherein the AAV virion is administered by intracerebroventricular (ICV) administration.
[0043] Embodiment 8. The method of embodiment 7, wherein the ICV administration is unilateral administration.
[0044] Embodiment 9. The method of embodiment 7, wherein the ICV administration is bilateral administration.
[0045] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the subject is a human subject. [0046] Embodiment 11. The method of embodiment 10, wherein the human subject is an adult.
[0047] Embodiment 12. The method of embodiment 10, wherein the human subject is not an adult.
[0048] Embodiment 13. The method of embodiment 10, wherein the human subject is not older than 25 years old.
[0049] Embodiment 14. The method of embodiment 10, wherein the human subject is 10 years old or younger.
[0050] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
[0051] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD).
[0052] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the subject exhibits one or more symptoms of an ASD.
[0053] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome.
[0054] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
[0055] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
[0056] Embodiment 21. The method of embodiment 20, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
[0057] Embodiment 22. The method of embodiment 20 or 21, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
[0058] Embodiment 23. The method of embodiment 22, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
[0059] Embodiment 24. The method of embodiment 22, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene. [0060] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the recombinant AAV virion is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
[0061] Embodiment 26. A method of treating a subject having a neurodevel opmental disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
[0062] Embodiment 27. A method of treating a subject having an autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
[0063] Embodiment 28. A method of treating a subject having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
[0064] Embodiment 29. The method of any one of embodiments 26 to 28, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
[0065] Embodiment 30. The method of any one of embodiments 26 to 28, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26. [0066] Embodiment 31. The method of any one of any one of embodiments 26 to 28, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
[0067] Embodiment 32. The method of any one of embodiments 26 to 31, wherein the subject is a human subject.
[0068] Embodiment 33. The method of embodiment 32, wherein the human subject is an adult.
[0069] Embodiment 34. The method of embodiment 32, wherein the human subject is not an adult.
[0070] Embodiment 35. The method of embodiment 32, wherein the human subject is not older than 25 years old.
[0071] Embodiment 36. The method of embodiment 32, wherein the human subject is 10 years old or younger.
[0072] Embodiment 37. The method of any one of embodiments 26 to 36, wherein the composition is delivered to the brain of the subject.
[0073] Embodiment 38. The method of embodiment 37, wherein the composition is delivered to the striatum and/or thalamus of the subject.
[0074] Embodiment 39. The method of any one of embodiments 26 to 38, wherein the composition is administered by ICV administration.
[0075] Embodiment 40. The method of embodiment 39, wherein the ICV administration is unilateral administration.
[0076] Embodiment 41. The method of embodiment 39, wherein the ICV administration is bilateral administration.
[0077] Embodiment 42. The method of any one of embodiments 26 to 41, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
[0078] Embodiment 43. The method of embodiment 42, wherein the autism spectrum disorder (ASD) comprises autism disorder.
[0079] Embodiment 44. The method of any one of embodiments 26 to 43, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
[0080] Embodiment 45. The method of embodiment 44, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
[0081] Embodiment 46. The method of embodiment 44 or embodiment 45, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
[0082] Embodiment 47. The method of embodiment 46, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
[0083] Embodiment 48. The method of embodiment 46, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
[0084] Embodiment 49. The method of any one of embodiments 26 to 48, wherein the subject has improved sleep efficiency after said administered.
[0085] Embodiment 50. The method of any one of embodiments 26 to 49, wherein the composition is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
[0086] Embodiment 51. A pharmaceutical composition comprising: (a) a recombinant AAV virion comprising: (i) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (ii) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29; b) 10 mM Tris; c) 1 mM magnesium chloride (MgCh); d) 150 mM sodium chloride (NaCl); and e) 0.02% poloxamer 188; wherein said pharmaceutical composition is at pH 8.0.
[0087] Embodiment 52. The pharmaceutical composition of embodiment 51, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
[0088] Embodiment 53. The pharmaceutical composition of embodiment 51 or embodiment
52, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26. [0089] Embodiment 54. The pharmaceutical composition of any one of embodiments 51 to
53, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
[0090] Embodiment 55. A method of treating a subject having a neurodevelopmental disorder, having an autism spectrum disorder (ASD), and/or having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising (a) a recombinant AAV virion comprising: (i) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (ii) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29; b) 10 mM Tris; c) 1 mM magnesium chloride (MgCh); d) 150 mM sodium chloride (NaCl); and e) 0.02% poloxamer 188; wherein said pharmaceutical composition is at pH 8.0.
[0091] Embodiment 56. The method of embodiment 55, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
[0092] Embodiment 57. The method of embodiment 55 or embodiment 56, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
[0093] Embodiment 58. The method of any one of embodiments 55 to 56, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
[0094] Embodiment 59. An expression cassette comprising a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence.
[0095] Embodiment 60. The expression cassette of embodiment 59 wherein the polynucleotide encoding the human miniShank3 protein is operably linked to a WPRE element.
[0096] Embodiment 61. The expression cassette of embodiment 59 or embodiment 60 wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20.
[0097] Embodiment 62. The expression cassette of any one of embodiments 59 to 61, wherein the polynucleotide encoding the miniShank3 protein is at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 2.
[0098] Embodiment 63. The expression cassette of any one of embodiments 59 to 62 comprising the nucleic acid sequence of SEQ ID NO: 2.
[0099] Embodiment 64. A recombinant AAV vector comprising the expression cassette of any one of embodiments 59 to 63 flanked by AAV inverted terminal repeats (ITRs). [00100] Embodiment 65. The recombinant AAV vector of embodiment 64, wherein the ITRs are 5’ AAV2 ITR and 3’ AAV2 ITR, having the nucleic acid sequences of SEQ ID NO:27 and SEQ ID NO: 28, respectively.
[00101] Embodiment 66. The recombinant AAV vector of embodiment 64 or embodiment 65, wherein the recombinant AAV vector comprises the sequence of SEQ ID NO: 30.
[00102] Embodiment 67. A recombinant AAV virion comprising (1) the recombinant AAV vector of any one of embodiments 64 to 66; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
[00103] Embodiment 68. A pharmaceutical composition comprising the recombinant AAV virion of embodiment 67 and a pharmaceutically acceptable carrier.
[00104] Embodiment 69. The recombinant AAV vector of any one of embodiments 64 to 66 which is a plasmid.
[00105] Embodiment 70. A host cell comprising the recombinant AAV vector of any one of embodiments 64 to 66 and comprising nucleic acid sequences encoding AAV rep and AAV9 cap.
[00106] Embodiment 71. A method of producing an AAV virion, the method comprising culturing a host cell comprising: the recombinant AAV vector of any one of embodiments 64 to 66; AAV cap, wherein the AAV cap encodes VP1, VP2, and/or VP3; AAV9 rep, wherein the AAV9 rep encodes rep78, rep68, rep 52, and/or rep 40; and one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[00107] FIG. 1 shows a plasmid map of pAAV2ITR-SYN-miniShank3Vl-KanR.
[00108] FIGs. 2A-2C show graphs of the results from an open field assay performed on mice to measure gross motor function and activity 6 weeks post dosing with AAV9- hSynl -Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 knockout mice (KO). FIG. 2A shows the total distance travelled. FIG. 2B shows count of rearings. FIG. 2C shows time binned analysis of distance travelled. AAV9-hSynl-Human miniShank3-Vl was administered at a dose of 2.4 x 109 vg/mouse, 1.2 x 1010 vg/mouse, 6.0 x 1010 vg/mouse or 2.75 x 1011 vg/mouse. All dosages tested are shown in FIG. 2A and FIG. 2B and the two highest dosage levels in KO mice are compared to WT and KO vehicle controls in FIG. 2C.
[00109] FIG. 3 shows a graph of the results from a rotarod assay performed on mice to measure motor function 6 weeks post dosing with AAV9-hSynl-Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 mice. AAV9-hSynl-Human miniShank3- VI was administered at a dose of 2.4 x 109 vg/mouse, 1.2 x IO10 vg/mouse, 6.0 x IO10 vg/mouse or 2.75 x 1011 vg/mouse. The latency to fall (seconds) is a measure of the cumulative time the animal maintained its balance prior to falling off the rotarod.
[00110] FIGs. 4A-4B shows a graph of an electroencephalography (EEG) sleep analysis (EEG Power Delta band) in mice 6 weeks (FIG. 4A) or 10 months (FIG. 4B) after administration of AAV9-hSynl-Human miniShank3-Vl or vehicle alone in wildtype (WT) and Shank3A4-22 knockout mice (KO). AAV9-hSynl-Human miniShank3-Vl was administered at a dose of 6.0 x 1010 vg/mouse or 2.75 x 1011 vg/mouse. Seizure monitoring only performed in two highest dose levels. P-value of 0.05 = *, p-value of 0.01 = **, p-value of 0.001 =***, p-value of 0.0001 = ****.
[00111] FIGs. 5A-5C show the results of the three-chamber social approach test in Phase 2 (7-13 weeks post-dose). Time spent in the compartment with the object (FIG. 5A) or the compartment with the stranger mouse measured over a 10 min observation period (FIG. 5B) for WT and Shank3 KO males treated with vehicle or increasing doses of JAG201 (2.40* 109 vg to 2.75* 1011 vg) 7-13 weeks after injection, and in the stranger compartment, time spent nose-pointing toward the stranger (FIG. 5C). ANOVA: A: P = 0.33, B: P = 0.13, C: P < 0.0001. FIGs. 5A-C: n = 21-23/group. Results of the uncorrected Fisher’s LSD multiple comparison tests shown with brackets on the graphs.
[00112] FIGs. 6A-6C show EEG analyses in Phase 2 over 24 hours for WT and Shank3 KO mice treated with vehicle or the mid-high and high doses of AAV9-hSynl- Human miniShank3-Vl (6.00* 1010 vg and 2.75* 1011 vg) 14 weeks after injection. Three different EEG patterns were quantified: epileptic-like (FIG. 6A), spike-wave discharges-like (FIG. 6B), and spike-like (FIG. 6C). ANOVA: A: P = 0.47, B: P = 0.14, C: P = 0.35. FIGs. 6A-6C: n = 8-10/group. Results of the uncorrected Fisher’s LSD multiple comparison tests shown with brackets on the graphs.
[00113] FIGs. 7A-7C show EEG seizure analyses in Phase 3 over 24 hours for WT and Shank3 KO mice treated with vehicle or the mid-high and high doses of AAV9-hSynl- Human miniShank3-Vl (6.00* 1010 and 2.75* 1011 vg) 42-45 weeks after injection. Three different EEG patterns were quantified: epileptic-like (FIG. 7A), spike-wave discharges-like (FIG. 7B), and spike-like (FIG. 7C). Statistical analyses were not performed due to the low sample size and absence of events for most seizure types, n = 2-10/group.
[00114] FIG. 8 shows miniSHANK3 protein levels in Phase 2 at 12-16 weeks postdosing for WT and Shank3 KO mice treated with vehicle or AAV9-hSynl-Human miniShank3-Vl (at a dose of 2.4 x 109 vg/mouse, 1.2 x IO10 vg/mouse, 6.0 x IO10 vg/mouse or 2.75 x 1011 vg/mouse).
[00115] FIGs. 9A-9C show Homer 1 protein levels (FIG. 9A), GluR2 protein levels (FIG. 9B) and PSD95 protein levels (FIG. 9C) in Phase 2 at 12-16 weeks post-dosing for WT and Shank3 KO mice treated with vehicle or AAV9-hSynl-Human miniShank3-Vl (at a dose of 2.4 x 109 vg/mouse, 1.2 x 1010 vg/mouse, 6.0 x 1010 vg/mouse or 2.75 x 1011 vg/mouse).
DETAILED DESCRIPTION
[00116] Aspects of the disclosure relate to gene therapy approaches for treating neurodevelopmental disorders. The Examples describe administering recombinant AAV virions that comprise expression cassettes that include polynucleotides encoding Shank3 proteins. Gene therapy strategies disclosed herein use an AAV system to deliver a functional copy of the Shank3 gene into brain cells to restore cellular function.
[00117] SHANK3 encodes a synaptic scaffolding protein, which coordinates the recruitment of signaling molecules and orchestrates assembly of the macromolecular postsynaptic protein complex, which is crucial for proper synaptic development and function. Deletion of SHANK3 is a major cause of the core neurodevelopmental and neurob ehavi oral deficits in Phelan-McDermid syndrome. Human genetic studies also identified SHANK3 mutations as accounting for about 1% of autism spectrum disorder (ASD). Patients with Phelan-McDermid syndrome and other individuals with SHANK3 mutations often exhibit a variety of comorbid traits, which include developmental delay, sleep disturbances, hypotonia, lack of speech or severe language delay, and characteristic features of ASD. Currently, there is no effective treatment for ASD.
[00118] The association of ASD with Shank3 provided an immediate link between synaptic dysfunction and the pathophysiology of ASD. Animal models bridge the human genetics of ASD to brain pathology underlying clinical presentation, and ultimately help to discover and evaluate effective therapeutics. Previous studies in flies, fish, and rodents have revealed synaptic dysfunction and behavioral abnormalities due to loss of SHANK3. For example, disruption of Shank3 in mouse models have resulted in synaptic defects, impaired social interactions, motor difficulties, repetitive grooming and increased anxiety level. Since Shank3 deficiency causes severe sleep disturbances in rodents, monkeys and human patients, sleep efficiency provides a unique biomarker for ASD. Furthermore, the /a/ J-deficient mouse model presents predictive validity as the synaptic defects and behavioral abnormalities are reversible when Shank3 is restored. Therefore, gene replacement is well suited as a therapeutic strategy for this monogenic disease.
[00119] Novel recombinant adeno-associated viruses (rAAVs) represent a promising gene delivery platform because of their wide range of tissue tropism, low immunogenicity, highly efficient and sustained gene transduction, and clinically proven track record in safety. However, it is known in the art that Shank3 is a large protein with a coding sequence of about 5.7kb, exceeding the packaging capacity of AAV vectors. Miniaturized Shank3 (“MiniShank3”) proteins described herein can be delivered by vector such as AAVs, including AAV9. Disclosed are methods of delivering the recombinant AAVs, including AAV9, having a miniShank3 transgene directly to the CNS, including via bilateral or unilateral intracerebroventricular (ICV) administration. Thus, the present disclosure relates to methods and compositions for treating neurodevelopmental disorders by restoring the activity of Shank3 using a miniaturized Shank3 protein (“MiniShank3”).
Shank proteins
[00120] The Shank family of proteins (e.g., Shankl, Shank 2, and Shank3) are master scaffolding proteins that tether and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five main domain regions: N-terminal ankyrin repeats, SH3 domain, PDZ domain, proline-rich region, and a C-terminal SAM domain. Through these functional domains, Shank proteins interact with many postsynaptic density (PSD) proteins. Without wishing to be bound by any theory, Shank proteins can bind to SAPAP which in turn binds to PSD95 to form the PSD95/SAPAP/Shank postsynaptic complex. Together, these multidomain proteins are proposed to form a key scaffold, orchestrating the assembly of the macromolecular postsynaptic signaling complex at glutamatergic synapses. This complex has been shown to play important roles in targeting, anchoring, and dynamically regulating synaptic localization of neurotransmitter receptors and signaling molecules. In another example, the Shank family of proteins is connected to the mGluR pathway through its binding to Homer. [00121] Due to its link to actin-binding proteins, Shank also plays a major role in spine development. It has been found that transfection of Shank3 was sufficient to induce functional dendritic spine synapses in cultured aspiny cerebellar granule cells, indicating a role in spine induction. Shank3 has three primary isoforms including Shank3a, the longest Shank3 isoform, Shank3p and Shank3Y. It has been reported that siRNA knockdown of Shank3 reduced the number and increased the length of dendritic spines in DIV18 cultured hippocampal neurons, implicating a role in spine maturation. This proposed function was supported by the finding that overexpression of Shankl enlarged already present dendritic spines in cultured hippocampal neurons. Furthermore, Shankl mutant mice have been reported to have smaller dendritic spines and weaker synaptic transmission.
[00122] In some embodiments, the present disclosure relates to Shank proteins that are capable of restoring synaptic activity in subjects with disrupted Shank protein activity. In some embodiments, the disrupted Shank protein activity is present in subjects who have neurodevelopmental disorders, an autism spectrum disorder (ASD), and/or Phelan- McDermid syndrome. In some embodiments, the Shank proteins associated with the present disclosure are Shankl proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shankl or a variant of Shankl. In some embodiments, the Shank proteins in the present disclosure are Shank2 proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank2 or a variant of Shank2. In some embodiments, the Shank proteins in the present disclosure are Shank3 proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank3 or a variant of Shank3. It should be appreciated that Shank proteins associated with the present disclosure can include any Shank protein, including variants or fragments thereof, that function as scaffolding proteins at the synapses of excitatory neurons.
[00123] Also disclosed herein are polynucleotides encoding Shank proteins (Shankl, Shank 2, and Shank3) for use in gene therapy.
[00124] The Shank3 full length mouse protein sequence corresponding to GenBank Accession No. BAE16756.1 is provided by SEQ ID NO: 5.
[00125] In some embodiments, the Shank3 full length mouse protein sequence corresponding to SEQ ID NO: 5 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_021423, provided by SEQ ID NO: 15. [00126] The Shank3 full length human protein sequence corresponding to GenBank Accession No. Q9BYB0.3 is provided by SEQ ID NO: 6.
[00127] In some embodiments, the Shank3 full length human protein sequence corresponding to SEQ ID NO: 6 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_001372044, provided by SEQ ID NO: 16.
[00128] The full-length Shank3 protein comprises multiple domains and is encoded by a gene that is about 5.2 Kb in size. Due to its size, it is difficult to deliver full-length Shank3 to a tissue or cell of interest via an AAV vector. As reported in PCT Publication No. W02022/040239, entitled “Shank3 Gene Therapy Approaches,” which is incorporated by reference herein in its entirety, specific domains can be removed or truncated from the full- length Shank3 protein to produce MiniShank3 that is efficacious in restoring Shank3 activity in excitatory neurons. Shank proteins (e.g., Shank3 proteins) encoded by polynucleotides described herein can be miniaturized to form a shortened variant of the native, full length Shank3 protein. As disclosed herein, a miniaturized Shank3 protein, or a DNA construct encoding the miniaturized Shank3 protein, are referred to interchangeably as “miniShank3” or “MiniShank3.” MiniShank3 proteins include shortened or mutated versions of Shank3 that have at least some Shank3 activity, for example, when MiniShank3 is introduced into neurons, it reduces the effects of Shank3 mutations,
[00129] Shank3 activity” includes, for example, the activity when introduced into, including by gene therapy, an organism, such as a mouse, non-human primate (NHP) or human, including the neurons of an organism, which is deficient for Shank3 or had reduced Shank3 activity, that ameliorates the effects of that Shank3 deficiency or reduction. The activity can be assessed in Shank3 deletion or deficient animal models such as described in Examples 1 and 2 herein.
[00130] In some embodiments, the Shank3 protein disclosed herein is expressed from a miniaturized Shank3 DNA construct or expression cassette. In some embodiments, the variant Shank3 DNA constructs and the Shank3 proteins disclosed herein (MiniShank3) comprise fewer domains than the full-length Shank3 gene and protein but have Shank3 activity. In some embodiments, the Shank3 protein disclosed herein is encoded by a non- naturally occurring polynucleotide.
[00131] Shank3 proteins encoded by polynucleotides described herein can include one or more protein domains. For example, Shank3 proteins can include one or more of: an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin domain, a SAM domain, and/or an ankyrin repeat domain. [00132] In some embodiments, the SH3 domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises at least 90% identity to residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises at least 90% identity to residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain can comprise any percent identity to residues 474-525 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SH3 domain can comprise any percent identity to residues 473-524 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
[00133] In some embodiments, the PDZ domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises at least 90% identity to residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises at least 90% identity to residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain can comprise any percent identity to residues 573-662 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the PDZ domain can comprise any percent identity to residues 572-661 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
[00134] In some embodiments, the Homer binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 5. In some embodiments, the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 6. In some embodiments, the Homer domain comprises residues 1294-1323 of SEQ ID NO: 5 or 6. In some embodiments, the Homer domain can comprise any percent identity to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniShank3.
[00135] In some embodiments, the Cortactin binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1400-1426 of SEQ ID NO: 5 or 6. In some embodiments, the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 5. In some embodiments, the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 6. In some embodiments, the Cortactin binding domain comprises residues 1400-1426 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the Cortactin binding domain can comprise any percent identity to residues 1400-1426 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniShank3.
[00136] In some embodiments, the SAM domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1664-1729 of SEQ ID NO: 6 or to residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM binding domain comprises at least 90% identity to residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM binding domain comprises at least 90% identity to residues 1663-
1728 of SEQ ID NO: 5. In some embodiments, the SAM domain comprises residues 1664-
1729 of SEQ ID NO: 6. In some embodiments, the SAM domain comprises residues 1663- 1728 of SEQ ID NO: 5. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1664-1729 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1663-1728 of SEQ ID NO: 5 suitable for construction of the MiniShank3. [00137] In some embodiments, the ankyrin repeat domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 148-345 of SEQ ID NO: 6 or to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain comprises at least 90% identity to residues 148-345 of SEQ ID NO: 6. In some embodiments, the ankyrin repeat domain comprises at least 90% identity to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 148-345 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 147- 313 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
[00138] In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. As used herein, “less than 65%” includes any percent identity less than 65% that is suitable for construction of the MiniShank3. In some embodiments, the MiniShank3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%,
48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%,
32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,
16%, 15%, 14%, 13%, 12%, 11% or 10% identical to SEQ ID NO: 5 over the full length of
SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%,
45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%,
29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11% or 10% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. In some embodiments, the MiniShank3 protein is at least about 35%, at least about 40%, at least about 45%, or at least about 50% identical to SEQ ID NO: 5 or SEQ ID NO: 6 over the full length of SEQ ID NO: 5 or SEQ ID NO: 6.
[00139] In some embodiments, the MiniShank3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 17-20, provided in Table 9.
[00140] In some embodiments, the MiniShank3 protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 17-20. In some embodiments, SEQ ID NO: 17 is encoded by SEQ ID NO: 1. In some embodiments, SEQ ID NO: 18 is encoded by SEQ ID NO: 2. In some embodiments, SEQ ID NO: 19 is encoded by SEQ ID NO: 3. In some embodiments, SEQ ID NO: 20 is encoded by SEQ ID NO: 4.
[00141] In some embodiments, the MiniShank3 protein comprises an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein comprises an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NO: 19 and/or SEQ ID NO: 20. [00142] In other embodiments, the MiniShank3 protein does not comprise an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein does not comprise an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NO: 17 and/or SEQ ID NO: 18.
[00143] In some embodiments, the sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or are 100% identical, including all values in between, to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity. In some embodiments, the sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 90% identity to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity. In some embodiments, the sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise any one of SEQ ID NOs: 1-4. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins with Shank3 activity.
[00144] In some embodiments, the MiniShank3 is encoded by any one of SEQ ID NOs: 1-4, provided in Table 9. SEQ ID NO: 1 and SEQ ID NO: 3 correspond to mouse MiniShank3 nucleic acid sequences, while SEQ ID NO: 2 and SEQ ID NO: 4 correspond to human MiniShank3 nucleic acid sequences. SEQ ID NO: 1 and SEQ ID NO: 2 encode MiniShank3 proteins that do not comprise an ankyrin repeat domain or the N-terminal domain. SEQ ID NO: 3 and SEQ ID NO: 4 encode MiniShank3 proteins that comprise an ankyrin repeat domain and the N-terminal domain.
[00145] Polynucleotides described herein that encode MiniShank3 proteins encode proteins that have Shank3 activity.
[00146] As disclosed herein, “identity” of sequences refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by a mathematical model, algorithm, or computer program that is known to one of ordinary skill in the art. The percent identity of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using Basic Local Alignment Search Tool (BLAST®) such as NBLAST® and XBLAST® programs (version 2.0). Alignment technique such as Clustal Omega may be used for multiple sequence alignments. Other algorithms or alignment methods may include but are not limited to the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, or Fast Optimal Global Sequence Alignment Algorithm (FOGSAA). [00147] In some embodiments, a polynucleotide encoding the Shank protein as disclosed herein (Shank 1, Shank2, Shank3) is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about 4.3 kb, about 4.2 kb, about 4.1 kb, about 4.0 kb, about 3.9 kb, about 3.8 kb, about 3.7 kb, about 3.6 kb, about 3.5 kb, about 3.4 kb, about 3.3 kb, about 3.2 kb, about 3.1 kb, about 3.0 kb, about 2.9 kb, about 2.8 kb, about 2.7 kb, about 2.6 kb, about 2.5 kb, about 2.4 kb, about 2.3 kb, about 2.2 kb, or about 2.1 kb in size. In some embodiments, the polynucleotide encoding the Shank protein as disclosed herein (Shank 1, Shank2, Shank3) can be in any size that is suitable for the methods and vectors disclosed in the present disclosure.
Neurodevelopmental Disorders
[00148] The present disclosure provides compositions and methods suitable for treating a neurodevelopmental disorder, such as an autism spectrum disorder (ASD), or Phelan-McDermid syndrome.
[00149] As used herein “neurodevelopmental disorder” refers to any disorder that impairs the growth and/or development of the brain and/or central nervous system. In some embodiments, neurodevelopmental disorders impact one or more brain functions, such as emotion, learning ability, self-control, and memory. It should be appreciated that aspects of the disclosure may be applicable for treatment of any neurodevelopmental disorder.
[00150] In some embodiments, the neurodevelopmental disorder is an autism spectrum disorder (ASD). Diagnosis of ASDs is mainly based on criteria such as deficits in communication, impaired social interaction, and repetitive or restricted interests and behaviors. ASDs are highly heritable disorders with concordance rates as high as 90% for monozygotic twins. However, ASDs are clinically heterogeneous, covering a wide range of discrete disorders of differential symptomatic severity. ASDs are believed to be etiologically heterogeneous, possibly encompassing polygenic, monogenic and environmental factors.
[00151] Alterations in synaptic connectivity and function have been proposed as a key mechanism underlying ASDs. Recent genetic studies have identified a large numbers of candidate genes for ASDs, many of which encode synaptic proteins including Shank3, Neuroligin-3, Neuroligin-4 and Neurexin-1. These findings suggest that synaptic dysfunction may underlie a common mechanism for a subset of ASDs. Various Shank3 mutations have been identified as a monogenic cause of ASD with intellectual disability (ID). In ASD patients, all Shank3 deletions and/or mutations that have been identified lead to loss of function (LoF) in one of the two normal copies of the Shank3 gene (i.e., haploinsufficiency). “Haploinsufficiency,” as used herein, refers to a model of dominant gene action in diploid organisms, in which a single copy of the wildtype allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wildtype phenotype. Haploinsufficiency may arise from a de novo or inherited LoF mutation in the variant allele, such that it yields little or no gene product. Recent genetic screens also identified a large number of mutations in the Shank3 gene including microdeletions, nonsense mutations and recurrent breakpoints in ASD patients not diagnosed with Phelan-McDermid syndrome (PMS). These implicate Shank3 gene disruption and/or mutation as a monogenic cause of autism spectrum disorder (ASD). The current estimation is that deletions and/or mutations involving Shank3 account for about 2% of all ASD patients with ID. Thus, understanding the function of Shank3 may provide insight into pathological mechanisms of ASD.
[00152] As used herein, “intellectual disability” refers to a disability that causes a subject to have deficits in intellectual functioning and/or adaptive functioning. Intellectual functioning can include, for example, reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and/or experiential learning. Intellectual functioning can be measured using any method known in the art, such as by IQ tests. Adaptive functioning can include, for example, skills needed to live in an independent and responsible manner such as communication and social skills. In some instances, intellectual disability can be evident during childhood or adolescence.
[00153] In some embodiments, the neurodevelopmental disorder is Phelan-McDermid syndrome (PMS, 22ql3.3 deletion syndrome), which is an autism spectrum disorder that shows autistic-like behaviors, hypotonia, severe intellectual disability and impaired development of speech and language. Shank3 is one of the genes that has been reported to be deleted in Phelan-McDermid syndrome. Disruption of Shank3 is thought to be the cause of the core neurodevelopmental and neurob ehavi oral deficits in Phelan-McDermid syndrome because individuals carrying a ring chromosome 22 with an intact Shank3 gene are phenotypically normal. Accordingly, provided are methods of treating a neurodevelopmental disorder associated with a mutation, deletion or disruption in the Shank3 gene and a reduction in Shank3 activity.
[00154] Other neurodevelopmental disorders can include but are not limited to attention-deficit/hyperactivity disorder (ADHD), learning disabilities such as dyslexia or dyscalculia, intellectual disability, conduct or motor disorders, cerebral palsy, impairments in vision and hearing, developmental language disorder, neurogenetic disorders such as Fragile X syndrome, Down syndrome, Rett syndrome, hypogonadotropic hypogonadal syndromes, and traumatic brain injury.
Subjects
[00155] A subject to be treated by methods described herein may be a human subject or a non-human subject. Non-human subjects include, for example: non-human primates; farm animals, such as cows, horses, goats, sheep, and pigs; pets, such as dogs and cats; and rodents.
[00156] A subject to be treated by methods described herein may be a subject having, suspected of having, or at risk for developing a neurodevel opmental disorder. In some embodiments, a subject has been diagnosed as having a neurodevel opmental disorder, while in other embodiments, a subject has not been diagnosed as having a neurodevel opmental disorder. In some embodiments, the subject is a human subject having, suspected of having, or at risk for developing an autism spectrum disorder (ASD). In some embodiments, the subject is a human subject having, suspected of having, or at risk for developing Phelan- McDermid syndrome. In some embodiments, the subject is a subject having reduced expression of the Shank3 gene relative to a control subject. In some embodiments, the expression of the Shank3 gene is reduced in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. In some embodiments, the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder. In some embodiments, the reduced expression of the Shank3 gene in a subject is caused by disruption of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
[00157] In some embodiments, the subject is a human subject who exhibits one or more symptoms of an ASD. In some embodiments, the subject is a human subject who exhibits developmental delay. In some embodiments, the subject is a human subject who exhibits intellectual disability (ID). In some embodiments, the subject is a human subject who exhibits sleep disturbance. In some embodiments, the subject is a human subject who exhibits hypotonia. In some embodiments, the subject is a human subject who exhibits lack of speech. In some embodiments, the subject is a human subject who exhibits language delay. In some embodiments, the subject is a human subject who exhibits any symptoms or signs of an ASD. [00158] In some embodiments, a subject is a human subject who is an adult. In some embodiments, the adult is older than 25 years of age. In some embodiments, the adult is not older than 25 years of age. In some embodiments, the adult is not older than 21 years of age. In some embodiments, the adult is not older than 18 years of age. In some embodiments, the adult is 16 years of age. In some embodiments, a subject is elderly (e.g., 65 years old or older). In some embodiments, the adult can be any age of adulthood that is suitable for the treatment disclosed herein.
[00159] In some embodiments, the subject is a human subject who is not an adult. In some embodiments, the human subject is not older than 16 years of age. In some embodiments, the human subject is not older than 10 years of age. In some embodiments, the human subject is 10 years of age or younger. In some embodiments, the human subject is a child or an infant. In some embodiments, the human subject is a toddler. In some embodiments, the human subject is at the fetal stage of development. In some embodiments, the human subject is at the prenatal stage of development.
Viral Vectors
[00160] As disclosed herein, polynucleotides encoding MiniShank3 proteins can be delivered to a tissue or cell of interest in a viral vector. Vectors described herein can be used to deliver a nucleic acid encoding a protein of interest to a subject, including, e.g., delivery to specific organs or to the central nervous system (CNS) of a subject. In some embodiments, the protein of interest is a Shank protein. In some embodiments, the protein of interest is a Shank3 protein. In some embodiments, the protein of interest is a MiniShank3 protein.
[00161] In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a miniShank protein disclosed herein. In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a Shank3 protein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the present disclosure provides a recombinant AAV virion comprising a recombinant AAV vector and an AAV capsid. In embodiments, the present disclosure provides a recombinant AAV virion comprising an expression cassette encoding a miniShank3 protein and an AAV9 capsid.
[00162] AAV refers to a replication-deficient (e.g., nonreplicating) Dependoparvovirus within the Parvoviridae genus of viruses. AAV can be derived from a naturally occurring virus or can be recombinant. AAV can be packaged into capsids, which can be derived from naturally occurring capsid proteins or recombinant capsid proteins. The single-stranded DNA genome of AAV includes inverted terminal repeat (ITRs). ITRs are involved in the replication and encapsidation of the AAV genome, along with its integration in the host genome and its excision. Without wishing to be bound by any theory, AAV vectors can comprise one or more ITRs, including a 5’ ITR and/or a 3’ ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and/or additional posttranscriptional regulator elements. AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary skill in the art, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (2012)), which is incorporated herein by reference in its entirety.
[00163] In some embodiments, AAV integrates into a host cell genome. In some embodiments, AAV does not integrate into a host genome. In some embodiments, AAV vectors disclosed herein can include sequences from any known organism. In some embodiments, AAV vectors disclosed herein can include synthetic sequences. AAV vector sequences can be modified in any way known to one of ordinary skill in the art, such as by incorporating insertions, deletions or substitutions, and/or through the use of posttranscriptional regulatory elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation signals. In some embodiments, AAV vectors can include sequences related to replication and integration.
[00164] In some embodiments, a MiniShank3 as disclosed herein is delivered to a tissue or a cell of interest via a recombinant AAV vector. In some embodiments, the recombinant AAV vector delivering the MiniShank3 as disclosed herein is delivered to the central nervous system (CNS) of a subject. As used herein, delivering the recombinant AAV vector to the CNS may include delivering the recombinant AAV vector to any tissue or cell of interest in the CNS. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to neuronal tissues or cells. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the AAV vector to the brain. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to the spinal cord. In some embodiments, delivering the recombinant AAV vector to the CNS involves delivering the recombinant AAV vector to the white and gray matter. In some embodiments, the recombinant AAV vector delivering the MiniShank3 as disclosed herein is delivered to any tissue or cell of interest of a subject that is suitable for the treatments as disclosed herein. [00165] As used in the present disclosure, “delivering” or “administering” a recombinant AAV vector can include any method known in the art for delivering or administering an AAV vector or a composition comprising an AAV vector to a subject. Administering can include but is not limited to direct administration of a recombinant AAV vector or a composition comprising the recombinant AAV vector, or peripheral administration via passive diffusion or convection-enhanced delivery (CED) to bypass the blood brain barrier as known in the art. Recombinant AAV vectors described herein can be administered in any composition that would be compatible with aspects of the disclosure. [00166] AAV vectors can include any known AAV serotype, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV serotype is AAV9. Clades of AAV viruses are described in, and incorporated by reference, from Gao et al. (2004) J. Virol. 78( 12): 6381 -6388. In some embodiments, any AAV serotype that is suitable for delivery to the CNS may be selected. [00167] AAV vectors of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. In some embodiments, the AAV vector may utilize or be based on an AAV serotype described in WO 2017/201258A1, the contents of which are incorporated herein by reference in its entirety, such as, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42- 11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43- 23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7/rh.48, AAVl-8/rh.49, AAV2-15/rh.62, AAV2- 3/rh.61, AAV2-4/rh.5O, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3- 1 l/rh.53, AAV4-8/rl 1.64, AAV4- 9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.lO, AAV16.12/hu. l l, AAV29.3/bb.l, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.4O, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.1O/hu.6O, AAV161.6/hu.61, AAV33.12/hu. l7, AAV33.4/hu. l5, AAV33.8/hu.l6, AAV52/hu. l9, AAV52.1/hu.2O, AAV58.2/hu.25, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV- DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-l/hu.l, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l l, AAVhu.13, AAVhu.15, AAVhu.l 6, AAVhu.l 7, AAVhu.l 8, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV- LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV- LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV- LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV- PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA- 101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr- 7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr- E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt- 6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt- Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd- N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg- F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl- 1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Civ 1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv- Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv- R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp- 8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP. A), G2B-26, G2B-13, THE 1-32 and/or THE 1-35, and variants thereof. AAV vectors are described further in US 9,585,971, US 2017/0166926, and W02020/160337, which are incorporated by reference herein in their entireties. [00168] In some embodiments, a MiniShank3 disclosed herein is delivered by a recombinant AAV vector. In some embodiments, the recombinant AAV vector comprises a transgene and its regulatory sequences, and optionally 5' and 3' ITRs. In some embodiments, the transgene and its regulatory sequences are flanked by the 5’ and 3’ ITR sequences. The transgene may comprise, as disclosed herein, one or more regions that encode a MiniShank3. The transgene may also comprise a region encoding for another protein. The transgene may also comprise one or more expression control sequences (e.g., a poly-A tail). The transgene may be single stranded. In some embodiments, a recombinant AAV vector comprises at least AAV ITRs and a MiniShank3 transgene.
[00169] In some embodiments, the AAV may be packaged into an AAV particle and administered to a subject and/or delivered to a selected target cell. In some embodiments, the AAV particle comprises an AAV capsid protein. In some embodiments, the AAV particle comprises at least one capsid protein that is selected from the AAV serotypes as disclosed herein including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.eB, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, and AAV2/2-125, or a variant of any of the foregoing. In some embodiments, the AAV particle comprises an AAV9 capsid.
[00170] In some embodiments, the miniShank3 transgene coding sequence in the recombinant AAV vector is operably linked to regulatory sequences for tissue-specific gene expression. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the tissue-specific regulatory sequence can be a Syn promoter (e.g., hSynl). In some embodiments, the hSynl promoter comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 22. In some embodiments, the hSynl promoter comprises the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the tissue-specific regulatory sequence can be any promoter or enhancer that is neuron-specific and is suitable for the treatments described herein.
[00171] In some embodiments, a miniShank3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs. In some embodiments, a miniShank3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 3 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
[00172] In some embodiments, a miniShank3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 18 or SEQ ID NO: 20 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs. In some embodiments, a miniShank3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 17 or SEQ ID NO: 19 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
[00173] Aspects of the disclosure relate to recombinant AAV vectors expressing miniShank3 transgenes. In some embodiments, a miniShank3 transgene is flanked by AAV ITRs. In some embodiments, the AAV ITRs comprise AAV2 ITRs. In some embodiments, the AAV ITRs comprise AAV1 ITRs. In some embodiments, the AAV ITRs comprise AAV5 ITRs. In some embodiments, the AAV ITRs comprise AAV6 ITRs. In some embodiments, the AAV ITRs comprise AAV8 ITRs. In some embodiments, the AAV ITRs comprise AAV9 ITRs. In some embodiments, the AAV ITRs comprise rhlO ITRs. In some embodiments, the AAV ITRs may include one or more modified ITRs which generate self-complementary AAV genomes.
[00174] In some embodiments, the recombinant AAV vector comprises a 5’ AAV2 ITR and a 3’ AAV2 ITR. In some embodiments, the 5’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 27. In some embodiments, the 3’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 28. In some embodiments, the 5’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the 3’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 28.
[00175] It should be appreciated that AAV vectors described herein can include DNA constructs or expression cassettes that comprise a transgene such as MiniShank3, 5’ and/or 3’ ITRs, promoters, introns, and/or other associated regulatory elements that are known in the art. [00176] In some embodiments, the AAV vector comprises a Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element (WPRE), which may enhance miniShank3 transgene expression. In some embodiments, the WPRE comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 23. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO: 23.
[00177] In some embodiments, the AAV vector comprises an untranslated portion such as an intron or a 5’ or 3’ untranslated region. In some embodiments, the intron may be located between the promoter/enhancer sequence and the miniShank3 transgene.
[00178] In some embodiments, the AAV vector used herein may be a self- complementary vector.
[00179] SEQ ID NO: 21 comprises a human MiniShank3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter. SEQ ID NO: 30 comprises a human MiniShank3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter. [00180] In some embodiments, the inverted terminal repeat (ITR) sequences comprise about 145 nucleotides each. These elements may be useful in cis for effective replication and encapsidation. A skilled person in the art would appreciate that any elements of AAV vectors known in the art may be compatible with aspects of the disclosure. One of skill in the art would also appreciate that any of the polynucleotide sequences described herein that encode a functional MiniShank3 protein can be expressed in a DNA construct or expression cassette for AAV delivery. These DNA constructs or expression cassettes may include one or more of the elements described herein. For example, in some embodiments a coding sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette. In some embodiments a coding sequence comprising the sequence of any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette. In some embodiments, the DNA construct or expression cassette includes one or more elements such as a promoter, a 5 ’-ITR, a 3 ’-ITR, a Synl promoter, a WPRE, an hGH poly A. Cis plasmids for production of the recombinant AAV virions may have elements such as origin of replications and antibiotic resistance markers, for example, an Fl origin, a NeR/KanR marker and/or a PUC origin. FIG. 1 shows an example of a plasmid for production of recombinant AAV virions described herein. [00181] Expression cassettes described herein may comprise a polyA signal. In some embodiments, the polyA signal is the hGH polyA signal. In some embodiments, the polyA signal comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 24. In some embodiments, the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 24.
[00182] In some embodiments, a recombinant AAV vector associated with the disclosure includes a nucleic acid sequence encoding a MiniShank3 protein operably linked to regulatory elements that promote CNS expression and flanking ITRs that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 21 or SEQ ID NO: 30, provided in Table 9. In some embodiments, a recombinant AAV vector comprises a sequence corresponding to SEQ ID NO: 21 or SEQ ID NO: 30, which encode a MiniShank3 protein comprising the sequence of SEQ ID NO: 18 and comprises regulatory elements, including a hSynl promoter, an WPRE element and a polyA signal sequence and flanking ITR sequences. In some embodiments, a recombinant AAV vector comprising the sequence of SEQ ID NO: 21 or SEQ ID NO: 30 may be delivered to a human subject in need thereof and may be suitable for treating a human subject who has a neurodevelopmental disorder.
[00183] As one of ordinary skill in the art would appreciate, any method known in the art for designing AAV vectors for clinical use, and for delivery of AAV vectors, may be compatible with aspects of the disclosure. For example, non-limiting examples of disclosure related to AAV vectors and delivery are provided in and incorporated by reference from U.S. Patent No. 7,906,111, entitled “Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor” and U.S. Patent No. 9,834,788, entitled “AAV -vectors for use in gene therapy of choroideremia,” each of which is incorporated by reference herein in its entirety.
[00184] In some embodiments, the recombinant AAV vector encoding a MiniShank3 protein for AAV delivery encodes a protein with a sequence that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, to any one of SEQ ID NOs: 17-20, provided in Table 9. [00185] The present disclosure provides recombinant AAV virions comprising: (1) recombinant AAV vectors described herein and (2) an AAV9 capsid. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, to SEQ ID NO: 29. As used herein, a “virion” refers to a viral particle that includes genetic material (e.g., RNA or DNA) and a capsid.
[00186] In some embodiments, an expression cassette disclosed herein can comprise SEQ ID NO: 26 or SEQ ID NO: 21 (with flanking ITR sequences) or SEQ ID NO: 30 (with flanking ITR sequences) as shown in Table 9.
[00187] In some embodiments, a recombinant vector comprising an expression cassette that comprises a polynucleotide encoding the Shank3 protein (i.e., the miniShank3 DNA construct) can be expressed in a specific tissue or cell of interest. In some embodiments, the expression cassette or vector disclosed herein comprises a promoter. In some embodiments, the expression cassette or vector comprises a cell-type specific promoter. In some embodiments, the promoter is a human promotor. In some embodiments, the human promoter is human Synapsin 1 (hSynl). In some embodiments, the hSynlpromotor has a polynucleotide sequence corresponding to SEQ ID NO: 22. In some embodiments, the human promoter can be any promotor that is known in the art and is suitable for expression of miniShank3. In some embodiments, the human promoter can be any promoter that has high specificity for neuronal tissues and cells. In some embodiments, the promoter can be a constitutive promoter. For example, the constitutive promoter can be a CAG promoter. As one of ordinary skill in the art would appreciate, any promoter may be used so long as the selected promoter is compatible with aspects of the disclosure.
[00188] The present disclosure provides methods of producing an AAV virion. In some embodiments, the method comprises culturing a host cell comprising an AAV vector described herein, an AAV cap (capsid protein) and an AAV9 rep (replication protein), and optionally one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell. In some embodiments, the AAV cap encodes VP1, VP2, and/or VP3. In some embodiments, the rep encodes rep78, rep68, rep52, and/or rep40.
Compositions and Administration [00189] The present disclosure provides compositions, including pharmaceutical compositions, comprising a polynucleotide (e.g., encoding for miniShank3) delivered in a recombinant AAV vector and/or an AAV virion as disclosed herein and a pharmaceutically acceptable carrier.
[00190] The compositions of the disclosure may comprise a recombinant AAV vector and/or an AAV virion alone, or in combination with one or more other viruses. In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different recombinant AAV vectors and/or AAV virions.
[00191] Suitable carriers may be readily selected by one of ordinary skill in the art in view of the indication for which the recombinant AAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure. Pharmaceutical compositions comprising AAV vectors are described further in US 9,585,971 and US 2017/0166926, which are incorporated by reference herein in their entireties.
[00192] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
[00193] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the recombinant AAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
[00194] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the recombinant AAV constructs disclosed herein. The formation and use of liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
[00195] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
[00196] Alternatively, nanocapsule formulations of the recombinant AAV vector may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
[00197] In some embodiments, the pharmaceutical composition comprising a nucleic acid delivered in a recombinant AAV vector comprises other pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, thimerosal, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the pharmaceutical compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[00198] The pharmaceutical forms suitable for delivering the recombinant AAV vectors include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
[00199] Methods described herein comprise administering recombinant AAV vector in sufficient amounts to transfect the cells of a desired tissue (e.g., brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ, oral, inhalation, intraocular, intravenous including facial vein injection and retroorbital injection, intracerebroventricular (ICV), intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parental routes of administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via parenteral administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via intravenous administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via ICV administration. In some embodiments, the ICV administration can be unilateral administration. In some embodiments, the ICV administration can be bilateral administration. Recombinant AAV virions described herein delivered via ICV administration may improve the therapeutic effects of miniShank3.
[00200] Routes of administration may be combined, if desired. In some embodiments, the vector as disclosed herein is administered intravenously.
[00201] In some embodiments, the present disclosure provides methods of treating a subject having a neurodevelopmental disorder. In some embodiments, the present disclosure provides methods of treating a subject having an autism spectrum disorder (ASD). In some embodiments, the present disclosure provides methods of treating a subject having Phelan- McDermid syndrome.
[00202] Methods provided herein, in some embodiments, comprise administering and delivering an effective amount of a composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue of a subject. In some embodiments, the target tissue is cortex. In some embodiments, the target tissue is striatum. In some embodiments, the target tissue is thalamus cerebellum. In some embodiments, the target tissue is hippocampus. In some embodiments, the target tissue is any brain structure. In some embodiments, methods for administering and delivering an effective amount of a composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue comprise delivering the composition to neurons or other brain cell types. In some embodiments, methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof comprise providing a composition comprising a recombinant AAV virion comprising at least a nucleic acid (e.g., miniShank3) to be delivered to the target environment or tissue of the subject and administering the composition to the subject. In some embodiments, methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof include delivering an AAV virion by unilateral or bilateral intracerebroventricular administration. Methods of use of AAV vectors are described further in US 9,585,971, US 2017/0166926, and W02020/160337, which are incorporated by reference herein in their entireties. In some embodiments, the composition may comprise a capsid protein.
[00203] In some embodiments, the composition comprising a recombinant AAV virion that comprises a polynucleotide encoding a Shank3 protein is delivered to the subject via intravenous administration, systemic administration, intracerebroventricular (ICV) administration, including bilateral or unilateral ICV administration, in utero administration, intrathecal administration, retro-orbital injection, or facial vein injection. In some embodiments, in utero administration is used for a subject who is at the prenatal stage of development. In some embodiments, the composition is delivered to a subject via a nanoparticle. In some embodiments, the composition is delivered to a subject via a viral vector. In some embodiments, the composition is delivered to a subject via any carriers suitable for delivering nucleic acid materials.
[00204] Any composition comprising a recombinant AAV virion that comprises an expression cassette comprising a polynucleotide encoding a protein that would be of some use or benefit to the subject may be delivered to a target environment or tissue of the subject according to methods disclosed herein
[00205] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the AAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
[00206] The dose of the recombinant AAV vector or recombinant AAV virion comprising a polynucleotide that encodes a Shank3 protein (e.g., miniShank3) required to achieve a particular "therapeutic effect," e.g., the units of dose in absolute vector genomes (vg) or vector genomes per milliliter of pharmaceutical solution (vg/mL) will vary based on several factors including, but not limited to: the route of AAV administration, the level of gene expression required to achieve a therapeutic effect, the specific disorder being treated, and the stability of the gene product. Doses that give maximal percentage of infection without affecting neurodevelopment are also suitable. One of skill in the art can readily determine a recombinant AAV vector or recombinant AAV virion dose range to treat a patient having a particular disorder based on the aforementioned factors, as well as other factors.
[00207] In some embodiments, an effective amount of a recombinant AAV vector or a recombinant AAV virion may be an amount sufficient to infect an animal or human subject or target a desired tissue. The effective amount will depend primarily on factors such as the species, age, gender, weight, health of the subject, and the tissue to be targeted, and may thus vary among subjects and tissues. The term “effective amount” or “amount effective” in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject. In some embodiments, an effective amount of a composition disclosed herein may partially or fully rescue the effects of a mutated Shank3 gene and/or partially or fully restore loss of function of the Shank3 protein. An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether. An effective amount can also involve delaying the occurrence of an undesired response. An effective amount can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. In other embodiments, the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. The achievement of any of the foregoing can be monitored by routine methods and the methods as disclosed in the present application. Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors. It should be appreciated that an effective amount as used herein does not need to be clinically effective.
[00208] For example, in some embodiments, the number of vector genomes administered to the subject is any value between about 6.0 x 1011 vg and about 9.0 x 1013 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 6.0 xlO13 vg/mL and about 9.0 xlO13 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 1 x 1010 to about 1 x 1012vg. In certain embodiments, the effective amount of AAV is 1010, 1011, 1012, 1013, or 1014 genome copies per kg. In certain embodiments, the effective amount of AAV is 1010, 1011, 1012, 1013, 1014, or 1015 genome copies per subject. In some cases, a dosage between about IO11 to 1013 AAV genome copies is appropriate. In some embodiments, a dose of about 1.0 x 1013 to about 1.0 x 1014 vector genomes is administered to the subject. In some embodiments, the number of vector genomes administered to the subject can be any dose that is suitable for the treatments and methods disclosed herein. In some embodiments, the dose of vector genomes is administered by unilateral or bilateral ICV administration.
[00209] In some embodiments, the dose administered to the subject via unilateral ICV administration is about 3 pl, 4 pl, 5 pl, 6 pl, 7 pl, 8 pl, 9pl, or 10 pl per subject. In some embodiments, the dose administered to the subject via unilateral ICV administration is about 5 pl per subject.
[00210] In some embodiments, the dose administered to the subject via unilateral ICV administration is about 1.4 * 1010 vg/ml, about 1.4 * 1011 vg/ml, about 1.4 * 1012 vg/ml, about 1.4 x 1013 vg/ml, about 1.4 * 1014 vg/ml, about 1.4 * 1015 vg/ml, or about 1.4 * 1016 vg/ml. In some embodiments, the dose administered to the subject via unilateral ICV administration is about 1.4 x 1013 vg/ml. In some embodiments, the dose administered to the subject via unilateral ICV administration is from about 1.4 x 1010 vg/ml to about 1.4 x 1016 vg/ml.
[00211] In some embodiments, a dose of recombinant AAV is administered to a subject as a single dose. In some embodiments, a dose of recombinant AAV is administered to a subject as a single dose with the potential to be re-dosed at a later time.
[00212] Formulation of pharmaceutically-acceptable excipients and carrier solutions disclosed herein is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Naturally, the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[00213] In embodiments, the formulation comprises a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
1.4 x IO10 vg/ml to about 1.4 * 1016 vg/ml in a 10 mM Tris, 1 mM magnesium chloride (MgCh), 150 mM sodium chloride (NaCl) and 0.02% poloxamer 188, pH 8.0 buffer.
[00214] In embodiments, the formulation consists essentially of a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
1.4 x 1O10 vg/ml to about 1.4 x 1016 vg/ml in a 10 mM Tris, 1 mM magnesium chloride (MgCE), 150 mM sodium chloride (NaCl) and 0.02% poloxamer 188, pH 8.0 buffer.
[00215] In embodiments, the formulation consists of a recombinant AAV virion comprising 1) a recombinant AAV vector comprising 1) an expression cassette flanked by ITRs, wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence and 2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 29 at a concentration of from about
1.4 x 1O10 vg/ml to about 1.4 x 1016 vg/ml in a 10 mM Tris, 1 mM magnesium chloride (MgCE), 150 mM sodium chloride (NaCl) and 0.02% poloxamer 188, pH 8.0 buffer.
Expression of Proteins Associated with the Shank Protein Network
[00216] Methods and compositions provided herein, in some embodiments, are useful for treating a neurodevel opmental disorder, such as, for example, an autism spectrum disorder (ASD), or Phelan-McDermid syndrome. In some embodiments, expression levels of PSD proteins are used to evaluate the efficacy of the administration of the miniShank3. In some embodiments, the PSD protein is Homer. In some embodiments, the PSD protein is post-synaptic density protein 95 (PSD95). In some embodiments, the PSD protein is SynGapl. In some embodiments, the PSD protein is SAPAP3. In some embodiments, the PSD protein is NR1. In some embodiments, the PSD protein is NR2B. In some embodiments, the PSD protein is GluR2. In some embodiments, the PSD protein is any protein that can be improved or restored upon the miniShank3 treatment. [00217] In some embodiments, an increase of any of the PSD proteins, as compared to an untreated control subject, may indicate efficacy of the miniShank3. Methods for detecting gene expression and protein levels are well-known in the art.
[00218] In some embodiments, expression of Homer in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of post-synaptic protein (PSD95) in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of SynGapl in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of SAPAP3 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of NR1 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of NR2B in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, expression of GluR2 in the subject after treated with miniShank3 delivered by a recombinant AAV vector or recombinant AAV virion described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. [00219] In some embodiments, administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving sleep efficiency. In some embodiments, a subject has improved sleep efficiency after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein. In some embodiments, the sleep efficiency in the subject after being administered an effective amount of a composition described herein is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. Improved sleep efficiency can include less sleep disturbance, which includes but is not limited to having trouble falling and staying asleep. Measurement of sleep efficiency can be conducted using any methods known in the art.
[00220] In some embodiments, administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving social impairment. In some embodiments, the social impairment of the subject is improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein. In some embodiments, the social impairment in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. Measurement of social impairment can be conducted using any methods known in the art.
[00221] As used herein, “social impairment” refers to behavioral abnormalities or defects that prohibit a subject from displaying voluntary social interaction.
[00222] In some embodiments, administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving locomotion and/or motor coordination deficits. In some embodiments, the locomotion and/or motor coordination deficits of the subject are improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein. In some embodiments, the locomotion and/or motor coordination deficits in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. Measurement of locomotion and/or motor coordination deficits can be conducted using any methods known in the art.
[00223] As used herein, “locomotion and/or motor coordination deficits” can include, for example, lack of coordination, loss of balance, and/or a shuffling gait.
[00224] In some embodiments, administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improvement in cortical-striatal synaptic dysfunction. In some embodiments, the corti cal -striatal synaptic dysfunction of the subject is improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein. In some embodiments, the corti cal -striatal synaptic dysfunction in the subject after being administered an effective amount of a composition described herein is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. Measurement of corti cal -striatal synaptic dysfunction can be conducted using any methods known in the art.
[00225] As used herein, “cortical-striatal synaptic dysfunction” refers to defective corticostriatal circuits in the brain that can cause repetitive and compulsive behaviors, such as in neuropsychiatric disorders and neurodevelopmental diseases such as autism, obsessive- compulsive disorders, and Tourette syndrome.
[00226] Some aspects of the technology described herein may be understood further based on the non-limiting illustrative embodiments described in the below Examples section. Any limitations of the embodiments described in the below Examples section are limitations only of the embodiments described in the below Examples section and are not limitations of any other embodiments described herein.
EXAMPLES
[00227] In order that the present disclosure may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope. Example 1: Intravenous Administration of an AAV Vector Containing miniShank3 to
Shank InsG3680 Mice
[00228] Autism spectrum disorder (ASD) is a heterogenous group of neurodevelopmental disorders and is one of the most severe disorders that results in significant morbidity to patients and carries a large socioeconomical impact. The Shank postsynaptic scaffold protein family emerges as one cause of ASD, with Shank mutations accounting for approximately 1% of patients with ASD. Prevalence of Shank3 haploinsufficiency due to deletion, truncation or missense mutation of the gene is estimated to be 1 : 15000 globally. Shank3 haploinsufficiency has been identified in patients with chromosome 22ql3.3 deletion syndrome, also known as Phelan-McDermid syndrome (PMS) It has been found that PMS due to Shank3 haploinsufficiency is associated with behavioral and cognitive manifestations of variable severity. Deficits are evident as early as infancy and persist throughout life. Current medical treatments are limited to antipsychotic drugs and treatment of comorbidities. However, treatments to directly address Shank3 haploinsufficiency and PMS, respectively are currently not available.
[00229] The ability of an AAV-PHP.eB-hSyn-GFPmouseMiniShank3 vector to correct behavioral and physiological deficits characteristic of the Shank3-InsG3680 mouse model when administered intravenously was reported in PCT Publication No. W02022/040239 and US Patent Publication No. US2023/0340041, which are incorporated by reference herein in their entireties.
[00230] A study was conducted to test if physiological deficits associated with the Shank3-InsG3680 mouse model can be improved following intravenous delivery of miniShank3 to post-natal day 14 (P14) animals. Specifically, correction of Shank3 protein and binding partner (PSD95, NR1, Homer, GluR2) levels in brain synaptic membrane preparations was assessed by assays including behavioral correction via open field tests.
Test and Control articles
[00231] As used herein, “AAV9-hSynl-Human miniShank3-Vl” refers to a suspension of an adeno-associated viral vector-based gene therapy for parenteral administration. It is a recombinant nonreplicating AAV9 vector containing a single stranded transgene encoding a condensed version of the human Shank3 protein (miniShank3) and under the control of the neuron-specific synapsin promoter. As full length Shank3 is too large to be packaged by conventional AAV methods, miniShank3 was designed to reduce the overall size of the transgene to allow for proper AAV packaging, while preserving critical domains previously identified to be responsible for its proper function as a synaptic scaffold protein. The “Vehicle” control includes the same excipients as the AAV9-hSynl-Human miniShank3-Vl but does not contain the AAV9 vector containing the miniShank3 transgene. [00232] Table 1 below shows the study design:
Table 1: Study design
Figure imgf000048_0001
Breeding
[00233] A total of 22 female heterozygous Shank3InsG3680 mice, 5 male Shank3InsG3680 homozygous mice and 5 male wild type mice were bred in trios or doubles. Litter births were monitored daily to time postnatal age of mice and litters remain with dams until weaning. Genotyping biopsies was taken from tail snips from about P3 pups; toe tattooing was used to identify offspring. Genotyping is performed.
Enrollment
[00234] Subjects (e.g., Shank3InsG3680 and WT mice) were enrolled depending on genotype results. Homozygous and WT genotypes were enrolled randomly to their corresponding groups (both sexes) and identified following genotype results by toe tattoo. Mice were dosed in a non-blinded fashion at P14 via retroorbital intravenous injection. Groups were WT vehicle (n=20), Homozygous vehicle (n=21) and Homozygous AAV9- hSyn 1 -Human mini Shank3- VI (n=22).
Endpoints
Collection of Open Field Data
[00235] This assay was performed in a custom-made open field apparatus. Each chamber is a 50 by 50 cm square. The 30-minute trial was recorded, and movement was tracked and analyzed using a custom template on Behavior Cloud. The center of the open field is defined as a 13.5 x 13.5 cm square in the geometric center of the arena. For each mouse, the total path length was measured, the time and the path length in the center of the open field was also determined. Each chamber was cleaned between individual mouse testing.
Statistical Analyses
[00236] Open Field measurements such as total path length, time in center and % path in center were analyzed by One-Way ANOVA with Tukey's post hoc for multiple comparisons. Path length in 5 min bins was analyzed by Two-Way ANOVA with time as a within groups factor and treatment as a between groups factor. Post-hoc pairwise comparisons used Bonferroni corrected comparisons.
Results
[00237] No observable AAV9-hSynl-Human mini Shank3 -VI -dependent effects on body weight or open field behavior were seen in the Shank3-InsG3680 mouse model when administered as an intravenous dose of 1.0E14 vg/kg at P14 and assessed at 8 weeks of age. These data confirm that intravenous delivery of AAV9-hSynl-Human miniShank3-Vl is not suitable for correction in this model.
[00238] Proof-of-principle was demonstrated in the Shank3InsG3680 mouse model using a mouse surrogate vector and transgene. Shank3InsG3680 mice were given IV injections of AAV- PHP.eB-hSyn-GFP-miniShank3 or a control virus expressing only green fluorescent protein (GFP) under the same promoter. PhP.eB is an AAV9-derived capsid that was evolved to cross the blood brain barrier more efficiently in mice (but not NHP), allowing for transduction of the brain with IV dosing. WT animals administered the control vector were also included. Groups of animals were dosed at postnatal day (P)0-P2, P7, or P28 and evaluated for neurob ehavi oral endpoints. Vector encoding the miniSHANK3 protein reversed genotype-dependent effects on open field, rotarod, the three-chamber test, elevated zero maze, grooming behavior, and EEG/electromyography (EMG) endpoints, though the specific rescue effects differed somewhat between dosing ages. Older animals (dosed at P28) showed reduced efficacy in the grooming and elevated zero maze assessments compared to younger animals, suggesting a potential benefit of early intervention. Ex vivo assessments in this study confirmed proper localization of the mouse miniSHANK3 protein in the PSD. Taken together, this study confirmed that rescue of disease phenotypes was possible using the mouse surrogate miniShank3 transgene delivered in young animals.
Example 2: Dosing Study in a Shank3 ASP Mouse Model
[00239] As discussed in Example 1, the ability of an AAV-PHP.eB-hSyn-GFP- mouseMiniShank3 vector to correct behavioral and physiological deficits characteristic of the Shank3-InsG3680 mouse model when administered intravenously was reported in PCT Publication No. WO 2022/040239 and US Patent Publication No. US2023/0340041, which are incorporated by reference herein in their entireties.
[00240] This study establishes the baseline behavioral phenotype of adult Shank3A4- 22 knockout mice (KO). These mice have been engineered to have exons 4-22 of the Shank3 gene removed and model a large proportion of SHANK3 -associated Autism/Phelan McDermid syndrome (PMS) patients with mutations resulting in large truncations of the SHANK3 gene. Furthermore, these mice mirror the behavioral deficits seen in the Shank3- InsG3680 model.
[00241] Adult Shank3A4-22 mice score normally for physical characteristics like bodyweight. Reduced sleep quality is observed compared to WT littermates as measured by electroencephalogram (EEG)-based delta wave power during low activity periods. Reduced spontaneous locomotion compared to WT mice in open field testing was observed after the first 10 minutes, suggesting lower explorative behavior and/or higher fatiguability of Shank3A4-22 mice.
[00242] Similarly, in the rotarod test, which assesses motor coordination, endurance, and motor learning, Shank3 A4-22 mice fall off the rotating bar earlier than WT mice. They also fail to learn motor coordination and balance in the process. These impairments of delta power and motor skills resemble the deficits in sleep and motor activities frequently observed in SHANK3 haploinsuffi cient patients.
[00243] Impairment of striatal learning is underlined by defects in the postsynaptic signaling complex in the striatum. Miniature evoked postsynaptic currents (mEPSC) in striatal medium spiny neurons (MSN) and spine density are reduced in Shank3A4-22 mice. The PSD is eroded by the loss of scaffolding proteins required for proper anchoring of glutamate receptors, most prominently the SHANK3 binding partner Homerl.
[00244] Despite having a large deletion in Shank3 that is similar to those in patients, the heterozygotic genotype in Shank3 A4-22 mice displays a milder phenotype, in contrast to the more severe phenotype in SHANK3 haploinsufficient patients. The cause for this difference is not fully understood (Drapeau, 2018).
[00245] In summary, the described animal model parallels many of the clinical features of SHANK3 haploinsufficient patients. The dysfunction in neural connectivity coincides with the current theory that dysfunction of neural circuits and plasticity underly the pathophysiology of SHANK3 haploinsufficiency.
[00246] The definitive POC study used Shank3A4-22 and WT mice and consisted of three phases: Phase 1 assessed genotype-related phenotypes in naive animals; Phase 2 evaluated the effect of AAV9-hSynl-Human miniShank3-Vl administered at a range of doses (0, 2.40* 109, 1.2Ox lO10, 6.OOx lO10, or 2.75xlOn vg) on P14 animals to correct phenotypes identified in Phase 1 at 6 to 16 weeks post- dose; and Phase 3 evaluated the top two doses from Phase 2 at approximately 7 to 10 months post-dose. In parts of Phase 2 and all of Phase 3, a general increase in dermatitis in the vivarium appeared to predominantly affect AAV9-hSynl-Human mini Shank3 -VI -treated animals. In Phase 3, this led to high mortality that precluded assessment of most efficacy endpoints at the 7 months post-dose timepoint.
[00247] In Phase 2, there was a lower incidence of dermatitis, and efficacy was considered evaluable. There were no significant changes in efficacy endpoints at the low and low-mid dose. At the high-mid dose (6.00x 1010 vg) in KO animals, there was a significant rescue of genotype- dependent effects in the open field assay (P = 0.049), trends towards improvements in the rotarod (P = 0.133) and EEG (P = 0.168), a significant effect on recruitment of Homerl in synaptic membranes (P = 0.014), and a 49% restoration of functional miniSHANK3 expression relative to WT SHANK3 levels in brain lysates. At the high dose in KO animals, where miniSHANK3 expression was 589% of endogenous WT levels, there was an overexpression phenotype in open field, a significant rescue of the EEG phenotype (P = 0.005) and Homerl recruitment (P < 0.0001), and a trend towards improvement in the rotarod assay (P = 0.125).
[00248] The following autism/PMS/anxiety-related behaviors were assessed: 1) Open Field, 2) Chamber social preference, 3) Hotplate assay, 4) Rotarod, and 5) Grooming. Once baseline behavioral phenotyping was established, whether physiological deficits associated with the Shank3A4-22 mouse model can be improved upon following unilateral intracerebroventricular (ICV) AAV9-hSynl-Human miniShank3-Vl delivery to P14 animals was tested. Specifically, Shank3 protein and binding partner (PSD95, NR1, Homer, GluR2) levels in brain synaptic membrane preparations were assessed by western blot, and behavioral assessment was carried out using the five assays mentioned above. The presence of seizure activity and changes to sleep patterns was monitored via electroencephalogram (EEG). Finally, at necropsy, animal tissues were harvested for biodistribution, in situ hybridization, clinical pathology, and histopathological analysis.
[00249] Table 2 shows a summary of the test article and vehicle used in this study. The test article (AAV9-hSynl -Human miniShank3-Vl) and vehicle are the same as those described in Example 1.
Table 2: Test Article and Vehicle
Figure imgf000052_0001
Figure imgf000053_0001
Test article handling
[00250] Test article aliquots were prepared to contain enough solution for the dosing of 4 mice (4 mice injected with 5 pL each= 20 pL injected + dead volumes and overage) and were thawed daily. On days where 8 mice were injected, 2 aliquots were used. Aliquots were used the same day (within 6-8hrs of thaw) with any extra discarded if a multiple of 4 mice was not dosed. To avoid discarding test article, P14 animals that would require the thaw of an aliquot that was not used for 4 mice, were lumped together with any new litters the next day and dosed at Pl 5, or be excluded from the study.
[00251] The following paragraphs discuss the study and experimental designs.
Basic Study Design
[00252] The number of animals per group includes 15 males (M) and 15 females (F) per genotype for the Phenotyping Pilot, 17 males and 6 females for the Injection Study of Cohorts 1, 2 and 4 (Phase 2) and 9 males and 3 females for the Injection Study of Cohort 3 (Phase 3). Up to 3 adult mice were housed per cage and up to 6 pups with one dam per cage after injection. Any additional pups in litters >6 was fostered. Mice were given standard food and water and no acclimation period is given. In general, mice grouped by sex, genotype, and solution were injected when applicable.
[00253] Mice in the injection study were assigned to dosing groups in a rotating manner so that an approximately equal number of mice born each week was assigned to each of the treatment groups. Mice were injected by groups of 4 mice injected successively with one dose on a given day to optimize the use of aliquots. Body weight < 20% of peak body weight (maximum weight reached for each individual mouse) and Body Condition Score of or below 2 were used as criteria for euthanasia.
Table 3: Study Design for the Phenotyping Pilot
Figure imgf000054_0001
Table 4: Study Design for the injection study (6wk)
Figure imgf000054_0002
Figure imgf000055_0001
Table 5: Study Design for the injection study (7mo)
Figure imgf000055_0002
Cohorts composition
[00254] For cohort composition in this study, Phase 1 represents the baseline collection of phenotypes in non-injected mice at 8-13 weeks of age. Phase 2 represents the collection of phenotypes in injected mice at 8-13 weeks of age. Phase 3 represents the baseline collection of phenotypes in injected mice at 7 months of age. Table 6: Cohorts Composition
Figure imgf000056_0001
*Hom = homozygous
Table 7: Study Timeline
Figure imgf000056_0002
Figure imgf000057_0001
[00255] In Phase 2, a range of doses of AAV9-hSynl-Human miniShank3-Vl were administered ICV at P14 to WT or Shank3A4-22 KO littermates which were evaluated for neurob ehavi oral endpoints beginning at 6 weeks post-dosing and continuing through 14 weeks post-dose. After completion of the behavioral assessments, n = 10 males/group had EEG electrodes implanted and were evaluated for seizure-like activity and power spectrum analysis. At termination, samples were taken for biodistribution, histopathology, and synaptic plasma membrane preparations to evaluate SHANK3 binding partners. Blood samples were also taken from a subset of animals (n = 0- 7/group) to evaluate hematology and clinical chemistry. Due to the large size of this phase, it was conducted in several cohorts (5 total).
[00256] In Phase 2, several neurob ehavi oral endpoints that had been significant in Phase 1 did not show statistically significant genotype-related differences, including open field vertical activity, and grooming duration. Data from endpoints showing genotype-related differences is shown in the figures below. In Phase 3, the only neurobehavioral endpoint evaluated was EEG assessments.
Husbandry, pup sampling and genotyping
[00257] Pups for the study were born from trio (2 females, 1 male) matings of Shank3 heterozygous parents. Matings were set up 19 days prior to the planned birth date. The birth date was planned 14 days prior to the planned injection date. All pups were treatment naive prior to placement on study. Pups were sampled by toe-clip for genotyping between postnatal day 1 and 7.
Body Condition Score [00258] Body condition score was determined weekly for mice having lost > 10% of their peak body weight.
[00259] In brief, BCSs were assessed for each mouse:
• BC 1 - Mouse is emaciated a. Skeletal structure extremely prominent; little or no flesh cover b. Vertebrae distinctly segmented
• BC2 - Mouse is underconditioned a. Segmentation of vertebral column evident. b. Dorsal pelvic bones are readily palpable
• BC3 - Mouse is well-conditioned a. Vertebrae and dorsal pelvis not prominent; palpable with slight pressure
• BC4 - Mouse is overconditioned a. Spine is a continuous column b. Vertebrae palpable only with firm pressure
• BC5 - Mouse is obese a. Mouse is smooth and bulky b. Bone structure disappears under flesh and subcutaneous fat
[00260] For ICV injections, mice were injected once at P14±2 days. In brief, mice were anesthetized with isoflurane (5% induction, 3% maintenance, in 02). The top of the skull was shaved. Mice were placed on a Stoelting stereotactic table, securing the head with ear bars. A nose cone provides the isoflurane. The shaved area was cleaned with chlorhexidine. Using sterile surgical techniques, an incision was performed on the skin to expose the skull. Hydrogen peroxide is applied to expose the bregma and lambda. A drill was mounted on the articulated arm of the stereotactic table and used to perform an opening on the skull at the selected coordinates: from bregma, 1 mm caudal, 0.4 mm from sagittal suture targeting the right ventricle.
[00261] A Hamilton syringe was mounted on the Stoelting injector placed in the articulated arm of the table. Needle was moved to x,y coordinates above and lowered to 2.0 mm depth. Injection (unilateral injection of 5 microliters) was initiated at the flow rate selected (1 pL/min). Once injection was complete, the needle was left in place for a least 3 min, then slowly removed. Bupivacaine was applied topically before glue was applied on the skull and sutures, on the skin. 1 mL of warm, sterile saline was given i.p. and mouse was allowed to recover on a warm pad before being returned to the cage. Instruments were bead sterilized before surgery on the next mouse.
Body Weights
[00262] In Phase 2, there was a slight decrease in body weights in KO animals treated with the highest dose of AAV9-hSynl-Human miniShank3-Vl compared to KO vehicle controls (data not shown). This study confirmed lower body weights associated with the KO phenotype.
[00263] In Phase 3, the genotype-related effect on body weights was also observed. In males, the 2.75* 1011 vg KO animals gained less weight than vehicle KO animals. In females, animals given 6.00x IO10 vg or 2.75xlOn vg tended to gain weight slower than the vehicle KO animals at various timepoints during the study, although this finding was only significant at a single timepoint (14 weeks of age) (data not shown).
Open field test
[00264] For the Open Field test, mice were tested once at 9-10 weeks of age.
[00265] In brief, the apparatus is a square arena ( 40 x 40 x 40 cm) made from clear
Plexiglas. Data were recorded via a sensitive infrared (IR) photobeam three-dimensional grid system that is invisible to mice. When the mouse moves or travels, its body breaks the otherwise continuous beam. The automated system then translates the beam breaks into measurements of the distance traveled, number of rearings, and time spent in the center versus perimeter of the arena.
[00266] Mice in their home cage were habituated to the testing room for 60 minutes prior. To begin, a mouse was placed in the center of the arena and the computer started recording. A typical session lasted 60 min and data is presented as sum of each measurement over bins of 10 min each. At the conclusion of testing, mice were returned to their homecages (mice that are grouped housed are run in parallel in different arenas). The test arena was cleaned with 70% EtOH between subjects.
[00267] There was a significant increase in explorative behavior in Shank3A4-22 knockout mice (KO) six weeks after administration of AAV9-hSynl-Human miniShank3-Vl (SEQ ID NO: 21) at higher dose levels (6.0 x 1010 vg/mouse or 2.75 x 1011 vg/mouse) compared to Shank3A4-22 knockout mice (KO) treated with vehicle alone (KO). FIGs. 2A- 2C. [00268] The reduction in total distance travelled of KO mice did not reach statistical significance in Phase 2 (FIG. 2A). The distance travelled was significantly increased in the high dose, 2.75* 1011 vg-treated KO mice compared to vehicle-treated KO mice, but not in the lowest dose groups; horizontal activity in the high-dose KO group was also higher than in vehicle-treated WT mice (FIG. 2A). However, when distance traveled was separated into 10-minute time bins (FIG. 2C), both KO and WT controls were observed to perform comparable for the first 10 minutes during the novel exploration phase, whereas the distance covered after the initial 10 minutes during the habituation phase was significantly reduced in KO animals compared to WT controls, mirroring previously published data in the Shank3 Ad- 22 model (Drapeau, 2018). Repeated measures analysis of the time binned data demonstrated statistically significant improvement in the performance of KO animals treated with AAV9- hSynl-Human miniShank3-Vl at dose levels of 6.00* 1010 vg (P = 0.0493) and 2.75* 1011 vg (P < 0.0001). Vertical activity in vehicle-treated WT and KO mice was not different in Phase 2, contrary to the observation made in the naive mice of Phase 1. The vertical activity was however significantly increased in the high dose (2.75* 1011 vg) (FIG. 2B and Table A). Table A
Figure imgf000060_0001
Rotarod test
[00269] Mice were tested twice at 12 and 13 weeks of age.
[00270] In brief, a Ugo-Basile rotarod was used, which consists of a rotating rod that is 3 cm in diameter and is suitably machined to provide a gripping surface for the mice. Six discs divide the drum into five lanes of 5.7 cm width each. This enables the testing of five mice simultaneously. When a mouse falls off its cylinder section onto the plate below, the plate mechanism trips, thereby recording the animal's endurance time in seconds. Height to fall is 16 cm.
[00271] Mice were acclimated in their home cage to the testing room at least 60 minutes prior to testing. For a typical single test, the mouse was placed on a rotating rod at 4 rpm. When all mice have been placed on the rod, speed was increased linearly to a maximum rpm of 40 over a period of 300 seconds. Mice were run on four successive trials with approximately one-minute inter-trial interval. The rod was cleaned between trials. The procedure was repeated on 2 consecutive days. The animal typically "learns" how to stay in the rod on the first day. The average score obtained on the second day is considered their real ability to stay in the rod. Latency to fall is recorded for each mouse.
[00272] There was a significant improvement in motor function in Shank3A4-22 knockout mice (KO) six weeks after administration of AAV9-hSynl-Human miniShank3-Vl (SEQ ID NO: 21) at higher dose levels (6.0 x IO10 vg/mouse or 2.75 x 1011 vg/mouse) compared to Shank3A4-22 knockout mice (KO) treated with vehicle alone (KO). FIG. 3.
[00273] The latency to fall in the rotarod assessment showed the expected decrease in KO animals which showed a trend towards correction at 6.00* 1010 vg/animal and 2.75* 1011 vg/animal, as shown in FIG. 3, which was not statistically significant.
EEG electrodes implantation and recording
[00274] Mice were tested once after completion of the behavioral tests (13-18 week of age).
[00275] In brief, ten (10) males per experimental group were implanted. The mouse was anesthetized by isoflurane inhalation (5% induction, 2-3% maintenance) and carprofen was given at 5mg/kg subcutaneously. The fur from the dorsal head was shaved and the skin was disinfected with 70% EtOH and chlorexhidine. The mouse was placed within a stereotaxic setup and given supplemental heat via a temperature-controlled heat pad during the procedure. A longitudinal incision was made in the skin from just rostral to the eyes to just caudal the ears to expose the skull. The topical analgesic 0.1% bupivacaine was applied to the periosteum and any exposed tissues at the periphery of the wound. The skull was cleaned with 3% hydrogen peroxide and 70% EtOH.
[00276] A Pinnacle Technology headmount was secured on the skull with adhesive, centered over Bregma. A sterile precision drill, outfitted with a 0.5 mm sterile bit, was used to bore 4 holes through the skull but not into the dura or the cortex, at the four comers of the headmount. Headmount screws were inserted through the headmount into the holes of the skull. The skull was sealed with dental cement and the implant was embedded into the cement to secure it into place. The skin was closed with size 6-0 nylon sutures and Gluture tissue adhesive to make contact with the cement, so no skull or tissue is exposed.
[00277] Mice were singly housed following surgery. Seven or more days after surgery, a fine, flexible cable (e.g., tether) was attached to the implant such that the mouse can move freely about the cage while its EEG is recorded onto a computer. Recording sessions lasted 72 hrs for one mouse. During this time, food was made available ad libidum as grain on the bottom of the cage and with addition of a DietGel 76A for hydration.
EEG analysis
[00278] The Sirenia Sleep Pro and Sirenia Seizure Pro software were used to analyze EEG recordings. Seizure related EEG events were quantified in Sirenia Seizure Pro and include pre-ictal biphasic spikes, spike-wave discharges (SWD), epileptiform seizure discharges. While EEG was recorded from both the parietal and motor cortex with a 3- electrode system, recording of the motor cortex is screened for seizures and the parietal recording is only used for verification.
[00279] The recording from the motor cortex was screened manually by blinded observers trained to detect abnormal variations of the signal with an amplitude of approximately twice the baseline amplitude observed during the wake stage. Visual screening was performed on a sliding time span windows of 1 min. Screening was assisted by an instant visualization of the power spectrum calculated on selected areas suspected to be an event. When an event was detected in the motor channel, the scorer verified in the parietal channel that events were also present. Only events in the motor channel matching events in the parietal channel were selected. Scorer flagged events and assigned them to one of 4 classes. [00280] 1. Spike: sharp upswing, often followed by slower dip.
[00281] 2. SWD: dominant frequency between 6 and 10 Hz. If the power spectrum did not show a predominant peak at 6-10 Hz, then the event was not categorized as SWD (see below, 4th class).
[00282] 3. Seizure: increasing frequency of spiking during the event, followed by lower amplitude, slow & regular baseline waves.
[00283] 4. Unsorted events: Seizure events that are distinguishable from surrounding pattern, appear as interruption to ongoing activity, but did not all into categories 1-3.
[00284] Flag events were saved with the analyzed recording by the scorers and reviewed by an independent, trained scorers who confirms, changes the class or removed the event. Descriptive statistics of the events for each mouse were exported to a tabulated data file and include the number and duration of each type of event in the light vs. dark phases. [00285] Power spectrum analysis was performed using Simia software on final 24h of 72h EEG recordings (400 Hz sample rate, low pass filtered at 50 Hz). Fast Fourier transform calculated power (square of voltage) on 10s epochs for frequency bands delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz) , and beta (12-30 Hz). Automated power analysis on 6 default bands (i.e., full, delta, theta, alpha, beta, and gamma) was performed. Analyses were performed for the last 48 hrs of the recording (following 24hrs of habituation) and covered 12 hours of light and 12 hours of dark phase.
[00286] There was significant dose-dependent improvement in restorative Delta band power during the 12 hour restful period in Shank3 A4-22 knockout mice (KO) 6 weeks after administration of AAV9-hSynl-Human miniShank3-Vl (SEQ ID NO: 21) compared to in Shank3A4-22 knockout mice (KO) treated with vehicle alone. FIG. 4A. AAV9-hSynl- Human miniShank3-Vl (SEQ ID NO: 21) was administered at a dose of 6.0 x IO10 vg/mouse or 2.75 x 1011 vg/mouse.
[00287] In Phase 3 (10 months post-dose), delta power was significantly lower in vehicle-treated KO mice compared to WT mice. Delta power for AAV9-hSynl-Human miniShank3-Vl 6.00* 1010 vg and 2.75* 1011 vg-treated KO mice remained lower than in WT mice. The dose-dependent increase of the delta power in KO mice, observed in Phase 2, was not present in Phase 3. The low number of mice recorded in Phase 3 likely prevented the ability to draw meaningful conclusions regarding delta power (FIG. 4B).
[00288] EEG assessments included both analysis of seizure-like wave patterns and power analysis as a measure of restorative sleep. Prior to analyzing EEG recordings, representative EEG traces of different types of seizures were recorded in a pilot experiment. Shank3 KO mice were injected with pentylenetetrazole, a known epileptogenic compound; their EEG was recorded, and clinical signs of seizures, including behavioral arrest and convulsions, were observed. Representative traces of three types of seizures in this pilot guided the detection and classification of EEG abnormalities in the AAV9-hSynl-Human mini Shank3 -VI -treated animals: isolated spikes, spike-wave discharges, and epileptic seizures.
[00289] EEG analyses of WT and KO mice in Phase 2 at 14 weeks after injection revealed no significant changes in the frequency of three specific EEG wave patterns: epileptic-like (FIG. 6A), spike-wave discharges-like (FIG. 6B), and spike-like (FIG. 6C). Overall, the incidence of abnormal EEG waves was very low, with multiple mice in each treatment group showing no abnormal wave at all over the 24 hours of recording.
[00290] In Phase 3, EEG analysis showed a similar lack of AAV9-hSynl-Human mini Shank3 -VI -related effects on seizures. Quantification of EEG abnormalities revealed no epileptic seizures nor single spikes in mice of Phase 3. A few spike-wave discharges were detected but the low number of mice and events prevented statistical analyses. A single mouse out of the four recorded in the 2.75 x 1011 vg-treated WT group showed a higher incidence of spike-wave discharges (FIG. 7B).
Statistical Methods
[00291] Statistical analyses were performed with GraphPad Prism, Version 8.0. Two- way ANOVA across Vehicle-treated mice of different genotypes were used to identify statistically significant differences between genotypes (genotype effect). Two-way ANOVA across AAV9-hSynl -Human miniShank3-Vl- and Vehicle-treated mice of one genotype (independent analyses for WT and HOM mice) were used to identify statistically significant differences between treatment groups (treatment effect). Sidak's multiple comparison tests were used to identify statistically significant differences between two specific groups.
Social approach (3-chamher) test
[00292] As impaired social interaction has previously been reported in some models of SHANK3 deficiency, Phase 2 mice were assessed in the three-chamber test. For social approach (3-chamber) test, mice were tested once at 10-11 weeks of age.
[00293] In brief, the 3-chamber apparatus for mice was a Plexiglas arena (40.5 x 60.0 x 22.0 cm) divided into three equal compartments that are created by removable interior walls. In each of the two end compartments is a cylindrical cage measuring 11 cm H x 10 cm D with 1 cm spacing between cage bars. A same sex "stranger mouse" is placed in one of the two cages.
Mice were acclimated in their home cage to the testing room for 60 minutes. The subject was habituated to the chamber of the empty apparatus for 10 minutes with no stranger mouse present. Preference for one side was recorded. The tested mouse was removed while dividers are installed and a novel, unfamiliar adult mouse was then placed in a cylindrical cage on one side, and a novel object was placed in the other. The stranger mouse, from a strain that is known to be very calm, and of the same sex as the tested mouse, had been habituated to the test cage for up to 10 min on days prior to the test. The test mouse was placed again in the cage and both dividers are removed, allowing the mouse to explore for another 10 minutes. Time spent in each chamber and entries into each chamber were scored using automated video tracking via an overhead camera. Following the test, mice were replaced in a holding cage until all cage mates have been tested, at which point all mice were returned to their home cage. The arena was wiped with 70% EtOH between mice to minimize scent-tracking. Data is presented as time spent interacting with the novel object vs. the unfamiliar mouse. [00294] As previously published (Drapeau, 2018), Shank3A4-22 mice demonstrated no significant genotypic difference compared to WT animals, as measured by the standard object and stranger time readouts (FIGs. 5A-5B). A genotype-dependent difference was detected in the stranger nose point time, a novel output not previously characterized in Shank3 mouse models, in the three-chamber assessment as shown in FIG. 5C, but this endpoint was not affected by AAV9-hSynl -Human miniShank3-Vl treatment.
Spontaneous Grooming quantification
[00295] Mice were tested once at 11-12 weeks of age.
[00296] In brief, for grooming observations, mice were in an empty cage (no bedding, food or water), placed on a table and video-recorded for future video-tracking by a trained experimenter. Mice were acclimated to the testing room in their home cage for a minimum of one hour. In the habituation phase, mice were placed in an empty cage for 20 minutes. The first 10 minutes are not scored for grooming (habituation). The last 10 minutes were assessed for grooming behavior. The entire experiment is video-recorded. A trained technician scored the grooming behavior of the mice accordingly (cumulative duration and number of grooming bouts with time).
[00297] There was no statistically significant difference in spontaneous grooming between wild-type mice and Shank3A4-22 knockout mice (data not shown).
Hotplate test
[00298] For the hotplate test, mice were tested once at 11-16 weeks of age.
[00299] A clear Plexiglass cylinder was placed atop a hot plate (i.e. Harvard Apparatus
LE7406 or comparable model) with the surface temperature maintained at 52° ± 2° C.
Mice were habituated to the testing room for a minimum of 30 minutes. A subject was placed inside the cylinder and observed for a maximum exposure time of 30 seconds. Latency to first instance of paw withdrawal, jumping, hind paw lick, or paw shake/flutter was recorded to the nearest 0.1 seconds with the keypad on the device. If no response was observed after 30 seconds, the subject is removed from the hot plate. Subjects are placed in a holding cage until all mice within the home cage have been tested.
[00300] There was no statistically significant difference in in the results of the hot plate test between wild-type mice and Shank3 A4-22 knockout mice (data not shown).
Transgene Expression and Binding Partners [00301] To assess transgene expression of miniSHANK3 at the protein level and impact on synaptic physiological deficits via SHANK3 binding partners (Hom er 1, glutamate ionotropic receptor AMPA type subunit 2 [GluR2] and postsynaptic density protein 95 [PSD95]), synaptic membrane preparation from brain lysates were assessed by automated western blot. A dose-dependent increase in brain miniSHANK3 protein levels were observed in both WT and KO animals following AAV9-hSynl -Human miniShank3-Vl treatment, compared to vehicle-treated animals (FIG. 8). These miniSHANK3 protein levels translate to 21.65%, 49.44%, and 589.20% of WT endogenous SHANK3 protein levels, for KO animals treated with 1.2Ox lO10 vg, 6.OOx lO10 vg, and 2.75x lOn vg, respectively (Table B). Table B - MiniSHANK3 Protein Levels Relative to WT Endogenous SHANK3 Levels in the Definitive POC Study
Figure imgf000066_0001
[00302] To assess the impact of AAV9-hSynl-Human miniShank3-Vl and resulting miniSHANK3 protein expression on synaptic physiological deficit, protein quantification of SHANK3 binding partners in synaptic membrane preparations from brain lysates was also carried out (FIGs. 9A-9C). Homer 1 directly binds to SHANK3’s Hom er 1 -binding region acting together as a scaffold to stabilize metabotropic glutamate receptors (mGluR) at the PSD (Xiao, 1998). The presence of a synaptic deficit resulting from loss of SHANK3 was supported by Homerl protein quantification, which demonstrated a significant genotypic reduction in vehicle-treated KO animals when compared to vehicle-treated WT animals. AAV9-hSynl-Human mini Shank3 -VI -treated KO animals demonstrated a dose-dependent increase in Homerl protein levels, with an increase compared to vehicle-treated KO animals at the 6.00x 1010 vg dose level and supraphy si ologi cal levels relative to WT Homerl levels achieved at the 2.75x 1011 vg dose level. Comparable significant genotypic deficit and AAV9-hSynl-Human miniShank3-Vl- induced improvements were also observed in GluR2 protein levels, a subunit of ionotropic glutamate a-amino-3-hydroxy-5-methyl-4- isoxazol epropionic acid (AMP A) receptors that is stabilized by SHANK3 via intermediatory binding partner glutamate receptor-interacting protein (GRIP) (Sheng, 2000). Finally, no significant genotypic difference was seen in PSD95, a protein that in complex with guanylate kinase-associated protein (GKAP) and supported by SHANK3, helps stabilize and regulate ionotropic glutamate N-methyl-D-aspartate (NMD A) receptors (Kim, 1997).
Terminal Procedures and Necropsy
[00303] Mice of each treatment group are assigned to four necropsy groups.
Cardiocentesis and Transcardial perfusion (for BD and Histo subgroups only: SM, SF per treatment group in Phase 2; 7M and 3F in Phase 3)
[00304] Animals are terminally anesthetized by regulated CO2 narcosis. After adequate anesthesia, the mouse is secured in a supine position. Maximal obtainable blood volume is collected by cardiocentesis and processed as described herein for CBC and clinical chemistry analyses.
[00305] Following cardiocentesis, a 5-6 cm lateral incision is made through the integument and abdominal wall just beneath the rib cage. Using a curved, blunt scissor, an incision of the diaphragm and rib cage is made. An 18-27-gauge needle is inserted into the apex of the left ventricle and clamped in place with an hemostat. To allow the perfusate to exit the circulation, a small incision on the right atrium is made using standard scissors. The animal is given a PBS perfusate of 20 mL either manually with a plastic syringe or with a perfusion pump.
[00306] Table 8 below shows the tissues to be collected and the assays to be conducted.
Table 8: Tissue Collection Table
Figure imgf000067_0001
Figure imgf000068_0001
* Terminal blood samples for Clinical Pathology and ELISA analysis are collected prior to transcardial perfusion as described.
** For Biodistribution Only, Ipsi and Contralateral to Injection *** Spleen will be collected for both Biodistribution and ELISPOT analysis as described.
Collection method for Vector DNA/RNA/Retain samples (BD Samples: 3 males, 2 females per group in Phase 2; 4 males, 1 female per group in Phase 3)
[00307] All frozen tissue samples for Vector DNA and RNA ddPCR analysis are shipped on dry ice. Retain samples are stored until the end of the project at < -60°C. Tissues are placed in 2 mL labelled screw-cap tubes and frozen on dry ice. Tissues are evenly divided into 3 pieces (1 for DNA, 1 for RNA, and 1 retain). The following tissue may not be divided: ovary. Tissues are held at <-60°C for storage and shipment.
Collection method for Histology/ISH samples (Histo samples: 2 males, 3 females per group in Phase 2; 3 males, 2 females per group in Phase 3)
[00308] Tissues from PBS perfused animals are used. Tissues harvested for fixation is collected, placed in histology cassettes identifying the mouse ID and placed in 10% neutral buffered formalin sample jars containing at least 15 times excess volume to completely submerge the cassette. Cassettes from the same group are put into the same jar. Organ identification is performed at the designated third party. Tissues are fixed for 48-72 hours at room temperature then transferred to 70% ethanol for immediate shipment for processing.
Collection method for Synaptic Membrane Preparation samples (SMP samples: 2 males, 1 female per group in Phase 2 only)
[00309] Humane euthanasia via CO2 asphyxiation is performed prior to removal of the brain via craniotomy and bisection into two hemispheres frozen on dry ice and maintained at about -60°C until processing. Each hemisphere is thawed and used for homogenization with a single Dounce homogenizer (about 200 mg of tissue). Synaptic membrane preparation is conducted.
[00310] Additionally, spleen tissues is collected for ELISPOT and ELISA sample analysis on 3 male mice and 2 female mice in the BD groups. Blood is processed for clinical chemistry samples.
[00311] In summary, the data from the definitive POC study in the Shank3A4-22 model suggests that doses of > 6.00* 1010 vg/animal provide improvement of disease-related phenotypes across multiple neurobehavioral outcomes, measures of functional protein efficacy (which represent improved synaptic stability), and durable transgene expression. Example 3: Biodistribution Assessment in Non-Human Primates (NHPj of AAV9-hSynl- Human nuniShank3-Vl, a SHANK3 AAV9 Vector Delivered via ICV Injection for ASP, Phelan-McDernud Syndrome, and Other SHANK3 Mutation or Deletion Related Conditions
[00312] AAV9-hSynl-Human miniShank3-Vl is as an investigational AAV9-based gene therapy in preclinical development intended to deliver a functional version of SHANK3 to treat autism spectrum disorder (ASD), Phelan-McDermid syndrome (PMS), and other neurodevelopmental disorders that result from a mutation or deletion within the gene or from chromosomal rearrangements at 22ql3.3 spanning SHANK3. Here, using a non-GLP study in non-human primates (NHPs) biodistribution of vector genome DNA and RNA transgene expression of AAV9-hSynl-Human miniShank3-Vl in the CNS and peripheral organs following unilateral and bilateral intracerebroventricular (ICV) administration was evaluated. [00313] The biodistribution of AAV9-hSynl-Human miniShank3-Vl was tested after ICV administration in 24 NHPs (12 males and 12 females) aged approximately 2-3 years. NHPs received either a single ICV administration of vehicle or AAV9-hSynl-Human miniShank3-Vl via unilateral or bilateral injection at a dose of 1.0 x 1013 or 1.0 x 1014 vg/animal in a total volume of 2.0 mL given via bolus injection. Injection coordinates were determined via MRI followed by stereotaxic administration targeting the lateral ventricles. Animals were followed for a 90-day in-life period, and biodistribution of AAV9-hSynl- Human miniShank3-Vl vector DNA and RNA in CNS and peripheral tissues was analyzed via droplet digital PCR (ddPCR) and supported via RNA fluorescence in situ hybridization (FISH).
[00314] The unilateral and bilateral ICV administration procedures and single doses of AAV9-hSynl-Human miniShank3-Vl were well tolerated. At 90 days, animals that received AAV9-hSynl-Human miniShank3-Vl showed robust and widespread rostrocaudal transduction throughout the CNS in a dose-dependent manner compared to vehicle controls. Analysis of AAV9-hSynl-Human miniShank3-Vl vector genome DNA copies in five key brain regions showed the highest levels of transduction in the frontal cortex and hippocampus, followed by the cerebellum and spinal cord, at both dose levels. Robust transduction of the striatum and thalamus was observed at the higher AAV9-hSynl-Human miniShank3-Vl dose level. These findings were supported and mirrored by AAV9-hSynl- Human miniShank3-Vl RNA quantification and RNA FISH analysis, with dose-dependent expression observed in all evaluated brain and spinal cord regions. Additionally, unilateral and bilateral AAV9-hSynl-Human miniShank3-Vl administration resulted in comparable overall levels of vector DNA and RNA copies throughout the brain, including comparable levels in both the contralateral and ipsilateral sides of the brain. Analysis of vector DNA copies in peripheral tissues confirmed leakage of vector from the CNS; however, in contrast to the CNS, significantly lower relative levels of RNA expression were observed as a result of the highly neuron-specific activity of the human synapsin promoter limiting off-target transgene expression.
[00315] These data provided evidence that both unilateral and bilateral ICV administration of AAV9-hSynl-Human miniShank3-Vl result in comparable widespread biodistribution throughout the CNS at doses predicted to be clinically relevant in patients. Further, AAV9-hSynl-Human miniShank3-Vl RNA expression analysis confirms robust expression throughout the brain and spinal cord while limiting off-target expression in peripheral tissues. In summary, these findings confirm the selection of unilateral ICV administration for further evaluation of AAV9-hSynl-Human miniShank3-Vl, a SHANK3 AAV9-based gene therapy targeting SHANK3 mutation or deletion related disorders.
Table 9. Mouse and Human miniShank3 Sequences and Vector Sequences
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
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[00370] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [00371] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included in such ranges unless otherwise specified. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00372] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[00373] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the disclosure, as defined in the following claims.

Claims

CLAIMS What is claimed is:
1. A method of delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof, said method comprising administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
2. The method of claim 1, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
3. The method of claim 1 or claim 2, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
4. The method of any one of claims 1 to 3, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
5. The method of any one of claims 1 to 4, wherein the recombinant AAV virion is delivered to the brain of the subject.
6. The method of any one of claims 1 to 5, wherein the recombinant AAV virion is delivered to the cortex, striatum and/or thalamus of the subject.
7. The method of any of any one of claims 1 to 6, wherein the recombinant AAV virion is administered by intracerebroventricular (ICV) administration.
8. The method of claim 7, wherein the ICV administration is unilateral administration.
9. The method of claim 7, wherein the ICV administration is bilateral administration.
10. The method of any one of claims 1 to 9, wherein the subject is a human subject.
11. The method of claim 10, wherein the human subject is an adult.
12. The method of claim 10, wherein the human subject is not an adult.
13. The method of claim 10, wherein the human subject is not older than 25 years old.
14. The method of claim 10, wherein the human subject is 10 years old or younger.
15. The method of any one of claims 1 to 14, wherein the subject has, is suspected of having, or is at risk of having, a neurodev el opmental disorder.
16. The method of any one of claims 1 to 15, wherein the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD).
17. The method of any one of claims 1 to 16, wherein the subject exhibits one or more symptoms of an ASD.
18. The method of any one of claims 1 to 17, wherein the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome.
19. The method of any one of claims 1 to 18, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
20. The method of any one of claims 1 to 19, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
21. The method of claim 20, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
22. The method of claim 20 or 21, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
23. The method of claim 22, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
24. The method of claim 22, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
25. The method of any one of claims 1 to 24, wherein the recombinant AAV virion is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
26. A method of treating a subject having a neurodevel opmental disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a poly A signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
27. A method of treating a subject having an autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a poly A signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
28. A method of treating a subject having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV virion comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a poly A signal sequence; and (2) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29.
29. The method of any one of claims 26 to 28, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
30. The method of any one of claims 26 to 28, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
31. The method of any one of any one of claims 26 to 28, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
32. The method of any one of claims 26 to 31, wherein the subject is a human subject.
33. The method of claim 32, wherein the human subject is an adult.
34. The method of claim 32, wherein the human subject is not an adult.
35. The method of claim 32, wherein the human subject is not older than 25 years old.
36. The method of claim 32, wherein the human subject is 10 years old or younger.
37. The method of any one of claims 26 to 36, wherein the composition is delivered to the brain of the subject.
38. The method of claim 37, wherein the composition is delivered to the striatum and/or thalamus of the subject.
39. The method of any one of claims 26 to 38, wherein the composition is administered by intracerebroventricular (ICV) administration.
40. The method of claim 39, wherein the ICV administration is unilateral administration.
41. The method of claim 39, wherein the ICV administration is bilateral administration.
42. The method of any one of claims 26 to 41, wherein the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
43. The method of claim 42, wherein the autism spectrum disorder (ASD) comprises autism disorder.
44. The method of any one of claims 26 to 43, wherein the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
45. The method of claim 44, wherein the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
46. The method of claim 44 or 45, wherein reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
47. The method of claim 46, wherein disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
48. The method of claim 46, wherein disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
49. The method of any one of claims 26 to 48, wherein the subject has improved sleep efficiency after said administered.
50. The method of any one of claims 26 to 49, wherein the composition is administered at a dose of about 1.0 x 1013 vg to about 1.0 x 1014 vg.
51. A pharmaceutical composition comprising: a recombinant AAV virion comprising: a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a poly A signal sequence; and an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29
10 mM Tris;
1 mM magnesium chloride (MgCh);
150 mM sodium chloride (NaCl); and
0.02% pol oxamer 188; wherein said pharmaceutical composition is at pH 8.0.
52. The pharmaceutical composition of claim 51, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
53. The pharmaceutical composition of claim 51 or claim 52, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
54. The pharmaceutical composition of any one of claims 51 to 53, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
55. A method of treating a subject having a neurodevelopmental disorder, having an autism spectrum disorder (ASD), and/or having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a composition comprising: a recombinant AAV virion comprising: a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20, operably linked to a human Syn promoter and a polyA signal sequence; and an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 29
10 mM Tris;
1 mM magnesium chloride (MgCh);
150 mM sodium chloride (NaCl); and 0.02% pol oxamer 188; wherein said pharmaceutical composition is at pH 8.0.
56. The method of claim 55, wherein the human miniShank3 protein comprises an amino acid sequence of SEQ ID NO: 18.
57. The method of claim 55 or claim 56, wherein the expression cassette comprises a polynucleotide sequence of SEQ ID NO: 26.
58. The method of any one of claims 55 to 57, wherein the ITRs comprise a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises a polynucleotide of SEQ ID NO: 27 and the 3’ ITR comprises a polynucleotide of SEQ ID NO: 28.
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Citations (2)

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WO2020250081A1 (en) * 2019-06-13 2020-12-17 Alma Mater Studiorum - Università di Bologna New gene therapy constructs
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