SHANK3 gene therapy approach

Abstract

Aspects of the present disclosure relate to expression cassettes encoding miniShank3 proteins, AAV vectors containing the expression cassettes, and gene therapy methods, particularly where the expression cassettes are formulated to further contain an AAV9 capsid, and the gene therapy is useful for treating neurodevelopmental disorders such as autism spectrum disorder (ASD) and Phelan-McDermid syndrome.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of Provisional Application No. 63 / 448,199, filed February 24, 2023, entitled "SHANK3 Gene Therapy Approach," the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference The contents of the electronic sequence listing (B119570183WO00-SEQ-VLJ.xml, size: 66,840 bytes, created on: February 23, 2024) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present invention relates to a gene therapy approach for delivering a polynucleotide encoding a Shank3 protein to a subject having, suspected of having, or at risk of having a neurodevelopmental disorder. [Background technology]

[0004] background Shank3-related deletions and / or mutations account for approximately 0.5-1% of all patients with autism spectrum disorder (ASD) and approximately 2% of patients with ASD who also have intellectual disability (ID). However, there are no effective treatments for ASD and / or ID. The development of pharmacological treatments that can correct the numerous pathologies associated with ASD and ID presents several challenges. DISCLOSURE OF THE INVENTION

[0005] overview Aspects of the present disclosure relate to the development of effective gene therapy methods for subjects with Shank3 mutations.

[0006] Aspects of the present disclosure provide an expression cassette comprising a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the 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 a recombinant AAV vector comprising an expression cassette described herein flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the ITRs are the 5' AAV2 ITR and the 3' AAV2 ITR, each 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 a recombinant AAV virion comprising: (1) a recombinant AAV vector described herein; and (2) an AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO: 29. The present disclosure provides a pharmaceutical composition comprising a recombinant AAV virion described herein and a pharmaceutically acceptable carrier.

[0010] A further aspect of the present disclosure relates to a method for delivering a human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof. In some embodiments, the method comprises administering to the CNS of the subject a pharmaceutical composition comprising a recombinant AAV virion as described herein.

[0011] In some embodiments, recombinant AAV virions are delivered to the brain of a subject. In some embodiments, recombinant AAV virions are delivered to the cortex, striatum, and / or thalamus of a subject. In some embodiments, recombinant AAV virions are administered by intracerebroventricular administration (ICV). In some embodiments, ICV administration is unilateral administration. In some embodiments, 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 no 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, or is at risk of having a neurodevelopmental disorder. In some embodiments, the subject has, is suspected of, 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, 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 disorder, hypotonia, speech disorder, or language delay. In some embodiments, the subject has, is suspected of, or is at risk of having decreased expression of the Shank3 gene compared to a control subject.

[0014] In some embodiments, the control subject is a subject who 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.

[0015] In some embodiments, the reduced expression of the Shank3 gene 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 of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

[0016] In some embodiments, the recombinant AAV virions are about 1.0 x 10 13 vg~approx. 1.0 x 10 14 It is administered at a dose of vg.

[0017] Further aspects of the present disclosure provide methods of treating a subject with a neurodevelopmental disorder. In some embodiments, the present disclosure relates to methods of treating a subject with autism spectrum disorder (ASD). In some embodiments, the present disclosure relates to methods of treating a subject with Phelan-McDermid syndrome. In some embodiments, the method of treatment comprises administering to the subject a therapeutically effective amount of a composition comprising an expression cassette or recombinant AAV virion described herein. In some embodiments, the composition is administered by ICV administration. In some embodiments, the ICV administration is unilateral. In some embodiments, the ICV administration is bilateral.

[0018] In some embodiments, the autism spectrum disorder (ASD) comprises autism disorder. In some embodiments, the subject's sleep efficiency is improved after administration. In some embodiments, the composition comprises a dose of about 1.0 x 10 13 vg~approx. 1.0 x 10 14 It is administered at a dose of vg.

[0019] A further aspect of the present disclosure provides a recombinant AAV vector as described herein. In some embodiments, the recombinant AAV vector is a plasmid.

[0020] A further aspect of the present disclosure provides a host cell comprising a recombinant AAV vector described herein, and comprising nucleic acid sequences encoding AAVrep and AAV9cap.

[0021] Another aspect of the present disclosure provides a method for producing AAV virions.In some embodiments, this method comprises culturing a host cell comprising a recombinant AAV vector described herein; AAVcap; AAV9rep; and one or more additional adenovirus helper functions under conditions sufficient to produce AAV virions; and isolating the AAV virions produced by the host cell.In some embodiments, AAVcap encodes VP1, VP2, and / or VP3.In some embodiments, AAV9rep encodes rep78, rep68, rep52, and / or rep40.

[0022] An aspect of the present disclosure relates to a method for delivering human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof, the method comprising administering to the CNS of the subject a pharmaceutical composition comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding 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) a recombinant AAV virion comprising 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, recombinant AAV virions are delivered to the brain of a subject. In some embodiments, recombinant AAV virions are delivered to the cortex, striatum, and / or thalamus of a subject. In some embodiments, recombinant AAV virions are administered by intracerebroventricular (ICV) administration. In some embodiments, ICV administration is unilateral administration. In some embodiments, 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 no 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, or is at risk of having a neurodevelopmental disorder. In some embodiments, the subject has, is suspected of, 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, 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 disorder, hypotonia, speech disorder, or language delay.

[0026] In some embodiments, the subject has, is suspected of, or is at risk of having reduced expression of the Shank3 gene compared to a control subject. In some embodiments, the control subject is a subject who does not have, is not suspected of, or is not at risk of having a neurodevelopmental disorder, an autism spectrum disorder (ASD), and / or Phelan-McDermid syndrome. In some embodiments, the reduced expression of the Shank3 gene 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 of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

[0027] In some embodiments, the recombinant AAV virions are about 1.0 x 10 13 vg~approx. 1.0 x 10 14 It is administered at a dose of vg.

[0028] An aspect of the present disclosure relates to a method of treating a subject having a neurodevelopmental disorder, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 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) a recombinant AAV virion comprising an AAV9 capsid or a capsid having an amino acid sequence 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 present disclosure relate to methods of treating a subject with autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 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) a recombinant AAV virion comprising an AAV9 capsid or a capsid having an amino acid sequence 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] An aspect of the present disclosure relates to a method of treating a subject with Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), where the expression cassette comprises a polynucleotide encoding a human miniShank3 protein comprising an amino acid sequence 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) a recombinant AAV virion comprising an AAV9 capsid or a capsid having an amino acid sequence 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 no 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 disorder, hypotonia, speech disorder, or language delay. In some embodiments, autism spectrum disorder (ASD) comprises autistic disorder.

[0034] In some embodiments, the subject has, is suspected of, or is at risk of having reduced expression of the Shank3 gene compared to a control subject. In some embodiments, the control subject is a subject who does not have, is not suspected of, or is not at risk of having a neurodevelopmental disorder, an autism spectrum disorder (ASD), and / or Phelan-McDermid syndrome. In some embodiments, the reduced expression of the Shank3 gene 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 of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene. In some embodiments, the subject's sleep efficiency is improved after administration.

[0035] In some embodiments, the composition comprises about 1.0 x 10 13 vg~approx. 1.0 x 10 14 It is administered at a dose of vg.

[0036] Aspects Embodiment 1. A method for delivering human miniShank3 protein to the central nervous system of a subject in need thereof, the method comprising administering to the CNS of the subject a pharmaceutical composition comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding human miniShank3 protein comprising an amino acid sequence at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and operably linked to a human Syn promoter and a polyA signal sequence; and (2) a recombinant AAV virion comprising an AAV9 capsid or a capsid having an amino acid sequence 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 the amino acid sequence of SEQ ID NO: 18.

[0038] Embodiment 3. The method of embodiment 1 or 2, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO:26.

[0039] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the polynucleotide of SEQ ID NO: 28.

[0040] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the recombinant 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 virions are delivered to the cortex, striatum, and / or thalamus of the subject.

[0042] Embodiment 7 The method of any one of embodiments 1 to 6, wherein the AAV virions are 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 over 25 years of age.

[0049] Embodiment 14 The method of embodiment 10, wherein the human subject is aged 10 years or less.

[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 neurodevelopmental 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 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 1-18, wherein the subject exhibits one or more of developmental delay, intellectual disability (ID), sleep disorder, hypotonia, speech disorder, 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, decreased expression of the Shank3 gene compared to a control subject.

[0056] Aspect 21. The method of aspect 20, wherein the control subject is a subject who 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 according to embodiment 20 or 21, wherein the 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 the disruption of the Shank3 gene comprises a deletion of at least one copy of the Shank3 gene.

[0059] Embodiment 24 The method of embodiment 22, wherein the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

[0060] 25. The recombinant AAV virions are about 1.0 x 10 13 vg~approx. 1.0 x 10 14 25. The method of any one of aspects 1-24, wherein the antibody is administered at a dose of vg.

[0061] Embodiment 26. A method of treating a subject having a neurodevelopmental disorder, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and 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 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 pharmaceutical composition 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and 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 at least 90% identical or at least 95% identical to SEQ ID NO:29.

[0063] Aspect 28. A method of treating a subject having Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20 and 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 at least 90% identical or at least 95% identical to SEQ ID NO:29.

[0064] Embodiment 29. The method according to any one of embodiments 26 to 28, wherein the human miniShank3 protein comprises the 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 the polynucleotide sequence of SEQ ID NO: 26.

[0066] Embodiment 31 The method of any one of embodiments 26 to 28, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the 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 of age.

[0071] Embodiment 36 The method of embodiment 32, wherein the human subject is aged 10 years or less.

[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 26-41, wherein the subject exhibits one or more of developmental delay, intellectual disability (ID), sleep disorder, hypotonia, speech disorder, 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, decreased expression of the Shank3 gene compared to a control subject.

[0080] Aspect 45. The method of aspect 44, wherein the control subject is a subject who 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 according to embodiment 44 or 45, wherein the 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 the disruption of the Shank3 gene comprises a deletion of at least one copy of the Shank3 gene.

[0083] Embodiment 48 The method of embodiment 46, wherein the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

[0084] Embodiment 49. The method of any one of embodiments 26 to 48, wherein the subject's sleep efficiency is improved after administration.

[0085] Embodiment 50. The composition comprises about 1.0 x 10 13 vg~approx. 1.0 x 10 14 50. The method of any one of aspects 26 to 49, wherein the antibody is administered at a dose of 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and 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 at least 90% identical or at least 95% identical to SEQ ID NO:29; b) 10 mM Tris; c) 1 mM magnesium chloride (MgCl); d) 150 mM sodium chloride (NaCl); and e) 0.02% poloxamer 188; wherein said pharmaceutical composition has a pH of 8.0.

[0087] Embodiment 52. The pharmaceutical composition according to embodiment 51, wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18.

[0088] Embodiment 53. The pharmaceutical composition according to embodiment 51 or 52, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO: 26.

[0089] Embodiment 54. The pharmaceutical composition according to any one of embodiments 51 to 53, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the 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) to (e): The method of claim 1, wherein the pharmaceutical composition has a pH of 8.0.

[0091] Embodiment 56 The method of embodiment 55, wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO: 18.

[0092] Embodiment 57. The method of embodiment 55 or 56, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO: 26.

[0093] Embodiment 58 The method according to any one of embodiments 55 to 56, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the 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, and operably linked to a human Syn promoter and polyA signal sequence.

[0095] Embodiment 60. The expression cassette according to embodiment 59, wherein the polynucleotide encoding the human miniShank3 protein is operably linked to a WPRE element.

[0096] Embodiment 61. The expression cassette according to embodiment 59 or 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).

[0100] Embodiment 65. The recombinant AAV vector according to embodiment 64, wherein the ITRs are the 5' AAV2 ITR and the 3' AAV2 ITR having the nucleic acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0101] Embodiment 66. The recombinant AAV vector according to embodiment 64 or 65, wherein the recombinant AAV vector comprises the sequence of SEQ ID NO: 30.

[0102] Embodiment 67. A recombinant AAV virion comprising: (1) a recombinant AAV vector according to any one of embodiments 64 to 66; and (2) an AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO: 29.

[0103] Embodiment 68. A pharmaceutical composition comprising the recombinant AAV virion of embodiment 67, and a pharmaceutically acceptable carrier.

[0104] Embodiment 69. The recombinant AAV vector according to any one of embodiments 64 to 66, which is a plasmid.

[0105] Embodiment 70. A host cell comprising the recombinant AAV vector of any one of embodiments 64 to 66, and comprising nucleic acid sequences encoding AAVrep and AAV9cap.

[0106] Embodiment 71. A method for producing AAV virions, the method comprising culturing a host cell comprising the recombinant AAV vector of any one of embodiments 64 to 66; an AAVcap encoding VP1, VP2, and / or VP3; an AAV9 rep encoding rep78, rep68, rep52, and / or rep40; and one or more additional adenovirus helper functions, under conditions sufficient to produce AAV virions; and isolating the AAV virions produced by the host cell. [Brief explanation of the drawings]

[0107] [Figure 1] Figure 1 shows the plasmid map of pAAV2ITR-SYN-miniShank3V1-KanR.

[0108] [Figure 2A]Figures 2A-2C show graphs of the results of open field assays performed to measure gross motor function and activity in wild-type (WT) mice and Shank3Δ4-22 knockout (KO) mice after 6 weeks of administration of AAV9-hSyn1-human miniShank3-V1 or vehicle alone. Figure 2A shows the total distance traveled. [Figure 2B] FIG. 2B shows the count of rearings. [Figure 2C] Figure 2C shows a time-bin analysis of distance traveled. AAV9-hSyn1-human miniShank3-V1 was administered at doses 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 doses tested are shown in Figure 2A and Figure 2B, and Figure 2C compares the two highest dose levels in KO mice with WT and KO vehicle controls.

[0109] [Figure 3] Figure 3 shows graphs of the results of rotarod assays performed to measure motor function in wild-type (WT) and Shank3Δ4-22 mice administered AAV9-hSyn1-human miniShank3-V1 or vehicle alone 6 weeks later. AAV9-hSyn1-human miniShank3-V1 was administered at doses of 2.4 x 10 vg / mouse, 1.2 x 10 vg / mouse, 6.0 x 10 vg / mouse, or 2.75 x 10 vg / mouse. The latency to fall (seconds) is a measure of the cumulative time the animal maintained balance before falling from the rotarod.

[0110] [Figure 4A] Figures 4A-4B show graphs of electroencephalogram (EEG) sleep analysis (EEG delta band power) in wild-type (WT) mice and Shank3Δ4-22 knockout (KO) mice administered AAV9-hSyn1-human miniShank3-V1 or vehicle alone at 6 weeks (Figure 4A) or 10 months (Figure 4B). [Figure 4B] Figures 4A-4B show graphs of electroencephalography (EEG) sleep analysis (EEG delta band power) in wild-type (WT) and Shank3Δ4-22 knockout (KO) mice administered AAV9-hSyn1-human miniShank3-V1 or vehicle alone at 6 weeks (Figure 4A) or 10 months (Figure 4B). AAV9-hSyn1-human miniShank3-V1 was administered at doses of 6.0 x 10 vg / mouse or 2.75 x 10 vg / mouse. Seizure monitoring was performed only at the two highest dose levels. p-value 0.05 = *, p-value 0.01 = **, p-value 0.001 = ***, p-value 0.0001 = ****.

[0111] [Figure 5A] Figures 5A-5C show the results of the three-chamber social approach test in Phase 2 (7-13 weeks post-injection). WT and Shank3 KO male mice treated with vehicle or increasing doses of JAG201 (2.40 × 109 vg-2.75 × 1011 vg) were analyzed at 7-13 weeks post-injection. The time spent in the object compartment (Figure 5A) or the stranger compartment (Figure 5B) was measured during a 10-minute observation period, and the time spent pointing their nose toward the stranger compartment (Figure 5C). [Figure 5B] Figures 5A-5C show the results of the three-chamber social approach test in Phase 2 (7-13 weeks post-injection). WT and Shank3 KO male mice treated with vehicle or increasing doses of JAG201 (2.40 × 109 vg-2.75 × 1011 vg) were analyzed at 7-13 weeks post-injection. The time spent in the object compartment (Figure 5A) or the stranger compartment (Figure 5B) was measured during a 10-minute observation period, and the time spent pointing their nose toward the stranger compartment (Figure 5C). [Figure 5C]Figures 5A–5C show the results of the three-chamber social approach test in Phase 2 (7–13 weeks post-injection). WT and Shank3 KO male mice treated with vehicle or increasing doses of JAG201 (2.40 × 109 vg–2.75 × 1011 vg) were analyzed 7–13 weeks post-injection. The time spent in the object compartment (Figure 5A) or the stranger compartment (Figure 5B) was measured during a 10-minute observation period, and the time spent pointing their noses toward the stranger compartment (Figure 5C). ANOVA: A: P = 0.33, B: P = 0.13, C: P < 0.0001. Figures 5A–C: n = 21–23 per group. Results of uncorrected Fisher's LSD multiple comparison tests are indicated in parentheses on the graphs.

[0112] [Figure 6A] Figures 6A-6C show 24-hour EEG analysis of WT and Shank3KO mice treated with vehicle or mid- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 x 10 vg and 2.75 x 10 vg), 14 weeks after injection, in Phase 2. Three distinct EEG patterns were quantified: epileptiform (Figure 6A), spike-wave discharge-like (Figure 6B), and spike-like (Figure 6C). [Figure 6B] Figures 6A-6C show 24-hour EEG analysis of WT and Shank3KO mice treated with vehicle or mid- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 x 10 vg and 2.75 x 10 vg), 14 weeks after injection, in Phase 2. Three distinct EEG patterns were quantified: epileptiform (Figure 6A), spike-wave discharge-like (Figure 6B), and spike-like (Figure 6C). [Figure 6C]Figures 6A-6C show 24-h EEG analysis of WT and Shank3KO mice treated with vehicle or medium- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 × 10 vg and 2.75 × 10 vg), 14 weeks after injection. Three distinct EEG patterns were quantified: epileptiform (Figure 6A), spike-wave discharge-like (Figure 6B), and spike-like (Figure 6C). ANOVA: A: P = 0.47, B: P = 0.14, C: P = 0.35. Figures 6A-6C: n = 8-10 per group. Results of uncorrected Fisher's LSD multiple comparison tests are indicated in parentheses on the graphs.

[0113] [Figure 7A] Figures 7A-7C show 24-hour EEG seizure analysis of WT and Shank3 KO mice treated with vehicle or mid- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 x 1010 and 2.75 x 1011 vg) at 42-45 weeks post-injection in Phase 3. Three distinct EEG patterns were quantified: epileptiform (Figure 7A), spike-wave discharge-like (Figure 7B), and spike-like (Figure 7C). [Figure 7B] Figures 7A-7C show 24-hour EEG seizure analysis of WT and Shank3 KO mice treated with vehicle or mid- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 x 1010 and 2.75 x 1011 vg) at 42-45 weeks post-injection in Phase 3. Three distinct EEG patterns were quantified: epileptiform (Figure 7A), spike-wave discharge-like (Figure 7B), and spike-like (Figure 7C). [Figure 7C]Figures 7A-7C show 24-hour EEG seizure analysis of WT and Shank3 KO mice treated with vehicle or medium- and high-dose AAV9-hSyn1-human miniShank3-V1 (6.00 × 10 and 2.75 × 10 vg) at 42-45 weeks post-injection during Phase 3. Three distinct EEG patterns were quantified: epileptiform (Figure 7A), spike-wave discharge-like (Figure 7B), and spike-like (Figure 7C). Due to the small sample size and the absence of events for most seizure types, statistical analysis was not performed. n = 2-10 per group.

[0114] [Figure 8] Figure 8 shows miniSHANK3 protein levels in WT and Shank3 KO mice treated with vehicle or AAV9-hSyn1-human miniShank3-V1 (doses 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) at 12–16 weeks post-dosing in Phase 2.

[0115] [Figure 9A] Figures 9A-9C show Homer1 (Figure 9A), GluR2 (Figure 9B), and PSD95 (Figure 9C) protein levels in WT and Shank3 KO mice treated with vehicle or AAV9-hSyn1-human miniSHANK3-V1 (doses 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) at 12 to 16 weeks after Phase 2 administration. [Figure 9B]Figures 9A-9C show Homer1 (Figure 9A), GluR2 (Figure 9B), and PSD95 (Figure 9C) protein levels in WT and Shank3 KO mice treated with vehicle or AAV9-hSyn1-human miniSHANK3-V1 (doses 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) at 12 to 16 weeks after Phase 2 administration. [Figure 9C] Figures 9A-9C show Homer1 (Figure 9A), GluR2 (Figure 9B), and PSD95 (Figure 9C) protein levels in WT and Shank3 KO mice treated with vehicle or AAV9-hSyn1-human miniSHANK3-V1 (doses 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) at 12 to 16 weeks after Phase 2 administration.

[0116] Detailed Description Aspects of the present disclosure relate to gene therapy approaches for treating neurodevelopmental disorders.In the examples, the recombinant AAV virion containing the expression cassette that contains the polynucleotide encoding Shank3 protein is administered.The gene therapy strategy disclosed herein uses the AAV system to deliver the functional copy of Shank3 gene to brain cells, and restores cell function.

[0117] SHANK3 encodes a synaptic scaffolding protein, orchestrating the recruitment of signaling molecules and the assembly of macromolecular postsynaptic protein complexes essential for proper synaptic development and function. SHANK3 deletion is the primary cause of core neurodevelopmental and neurobehavioral deficits in Phelan-McDermid syndrome (PMS). Human genetic studies have also found that SHANK3 mutations account for approximately 1% of autism spectrum disorder (ASD). Patients with Phelan-McDermid syndrome and other individuals with SHANK3 mutations often exhibit a variety of comorbidities, including developmental delay, sleep disorders, hypotonia, speech impairment or severe language delay, and features of ASD. Currently, no effective treatments for ASD exist.

[0118] The association of Shank3 with ASD provides a direct link between synaptic dysfunction and the pathophysiology of ASD. Animal models bridge the gap between human genetic features of ASD and the brain pathology underlying clinical symptoms, ultimately aiding in the discovery and evaluation of effective treatments. 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 results in synaptic defects, impaired social interaction, motor deficits, repetitive grooming, and elevated anxiety levels. Because Shank3 deficiency causes severe sleep disturbances in rodents, monkeys, and human patients, sleep efficiency provides a unique biomarker for ASD. Furthermore, Shank3-deficient mouse models offer predictive validity, as restoration of Shank3 reverses synaptic defects and behavioral abnormalities. Therefore, gene replacement is a highly suitable therapeutic strategy for this monogenic disorder.

[0119] Novel recombinant adeno-associated viruses (rAAVs) have become promising gene delivery platforms due to their broad tissue tropism, low immunogenicity, highly efficient and sustained gene transduction, and clinically proven safety profile. However, it is known in the art that Shank3 is a large protein with a coding sequence of approximately 5.7 kb, exceeding the packaging capacity of AAV vectors. The miniaturized Shank3 ("MiniShank3") protein described herein can be delivered by vectors such as AAV, including AAV9. Disclosed are methods for delivering recombinant AAV (including AAV9) carrying a miniShank3 transgene directly to the CNS, including via bilateral or unilateral intracerebroventricular (ICV) administration. Accordingly, the present disclosure relates to methods and compositions for treating neurodevelopmental disorders by restoring Shank3 activity using the miniaturized Shank3 protein ("MiniShank3").

[0120] Shank protein Shank family proteins (e.g., Shank1, Shank2, and Shank3) are master scaffolding proteins that anchor and organize scaffold proteins at synapses of excitatory neurons. Members of this family share at least five major domain regions: N-terminal ankyrin repeats, an SH3 domain, a PDZ domain, a proline-rich region, and a C-terminal SAM domain. Through these functional domains, Shank proteins interact with many postsynaptic density (PSD) proteins. Without being bound by any theory, Shank proteins bind to SAPAP, which in turn binds to PSD95 to form the PSD95 / SAPAP / Shank postsynaptic complex. These multidomain proteins are thought to together form a critical scaffold and coordinate the assembly of macromolecular postsynaptic signaling complexes at glutamatergic synapses. This complex has been shown to play a critical role in targeting, anchoring, and dynamically regulating the synaptic localization of neurotransmitter receptors and signaling molecules. In another example, the Shank family of proteins is connected to the mGluR pathway through binding to Homer.

[0121] Through its association with actin-binding proteins, Shank also plays an important role in spine development. Transfection of Shank3 was found to be sufficient to induce functional dendritic spine synapses in cultured aspiny cerebellar granule cells, suggesting a role in spine induction. Shank3 has three major isoforms: Shank3α, Shank3β, and Shank3γ, which is the longest Shank3 isoform. SiRNA knockdown of Shank3 has been reported to reduce the number and increase the length of dendritic spines in DIV18 cultured hippocampal neurons, suggesting a role in spine maturation. This proposed function was supported by the finding that overexpression of Shank1 enlarges pre-existing dendritic spines in cultured hippocampal neurons. Furthermore, Shank1 mutant mice have been reported to have smaller dendritic spines and weaker synaptic transmission.

[0122] In some embodiments, the present disclosure relates to a Shank protein that can restore synaptic activity in a subject in which Shank protein activity is disrupted. In some embodiments, a subject with a neurodevelopmental disorder, autism spectrum disorder (ASD), and / or Phelan-McDermid syndrome has disrupted Shank protein activity. In some embodiments, the Shank protein related to the present disclosure is a Shank1 protein. In some embodiments, the present disclosure relates to the expression of a polynucleotide encoding Shank1 or a Shank1 variant in a subject in need thereof. In some embodiments, the Shank protein related to the present disclosure is a Shank2 protein. In some embodiments, the present disclosure relates to the expression of a polynucleotide encoding Shank2 or a Shank2 variant in a subject in need thereof. In some embodiments, the Shank protein related to the present disclosure is a Shank3 protein. In some embodiments, the present disclosure relates to the expression of a polynucleotide encoding Shank3 or a Shank3 variant in a subject in need thereof. It should be understood that a Shank protein related to the present disclosure can include any Shank protein, including variants or fragments thereof, that functions as a scaffolding protein at excitatory neuron synapses.

[0123] Also disclosed herein are polynucleotides encoding Shank proteins (Shank1, Shank2, and Shank3) for use in gene therapy.

[0124] The Shank3 full-length mouse protein sequence corresponding to GenBank accession number BAE16756.1 is provided in SEQ ID NO:5.

[0125] In some embodiments, the Shank3 full-length mouse protein sequence corresponding to SEQ ID NO:5 is encoded by the nucleic acid sequence corresponding to GenBank Accession No. NM_021423 and provided by SEQ ID NO:15.

[0126] The Shank3 full-length human protein sequence corresponding to GenBank accession number Q9BYB0.3 is provided in SEQ ID NO:6.

[0127] In some embodiments, the Shank3 full-length human protein sequence corresponding to SEQ ID NO:6 is encoded by the nucleic acid sequence provided by SEQ ID NO:16, which corresponds to GenBank Accession No. NM_001372044.

[0128] The full-length Shank3 protein contains multiple domains and is encoded by a gene approximately 5.2 kb in size. Due to its size, full-length Shank3 is difficult to deliver to target tissues or cells via AAV vectors. As reported in PCT Publication No. WO 2022 / 040239, entitled "Shank3 Gene Therapy Approach," which is incorporated herein by reference in its entirety, specific domains can be deleted or truncated from the full-length Shank3 protein to generate MiniShank3, which is effective in restoring Shank3 activity in excitatory neurons. Shank proteins (e.g., Shank3 proteins) encoded by the polynucleotides described herein can be miniaturized to form truncated variants of the native full-length Shank3 protein. As disclosed herein, miniaturized Shank3 proteins or DNA constructs encoding miniaturized Shank3 proteins are interchangeably referred to as "miniShank3" or "MiniShank3." MiniShank3 proteins include truncated or mutated versions of Shank3 that retain at least some Shank3 activity, eg, when MiniShank3 is introduced into neurons, the effects of Shank3 mutations are mitigated.

[0129] "Shank3 activity" includes activity that ameliorates the effects of Shank3 deficiency or reduction when introduced, for example, by gene therapy into an organism (including neurons of an organism), such as a mouse, non-human primate (NHP), or human, that lacks Shank3 or has reduced Shank3 activity. This activity can be assessed in a Shank3-deficient or -deficient animal model, such as those described in Examples 1 and 2 herein.

[0130] 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 construct and Shank3 protein (MiniShank3) disclosed herein contain fewer domains than the full-length Shank3 gene and protein, but still have Shank3 activity. In some embodiments, the Shank3 protein disclosed herein is encoded by a non-naturally occurring polynucleotide.

[0131] The Shank3 protein encoded by the polynucleotides described herein can include one or more protein domains, for example, the Shank3 protein 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.

[0132] 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 100% identity, to residues 474-525 of SEQ ID NO:6 or residues 473-524 of SEQ ID NO:5, including all values ​​in between. 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 that is suitable for construction of MiniShank3. In some embodiments, the SH3 domain can comprise any percent identity to residues 473-524 of SEQ ID NO: 5 that is suitable for construction of MiniShank3.

[0133] 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 100% identity, to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5, including all values ​​in between. 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 that is suitable for construction of MiniShank3. In some embodiments, the PDZ domain can comprise any percent identity to residues 572-661 of SEQ ID NO: 5 that is suitable for construction of MiniShank3.

[0134] 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, to residues 1294-1323 of SEQ ID NO:5 or SEQ ID NO:6, including all values ​​in between. 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 that is suitable for construction of MiniShank3.

[0135] 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 100% identity, to residues 1400-1426 of SEQ ID NO: 5 or 6, including all values ​​in between. 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 that is suitable for construction of MiniShank3.

[0136] 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 100% identity, to residues 1664-1729 of SEQ ID NO:6 or residues 1663-1728 of SEQ ID NO:5, including all values ​​in between. In some embodiments, the SAM domain comprises at least 90% identity to residues 1664-1729 of SEQ ID NO:6. In some embodiments, the SAM 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 that is suitable for construction of MiniShank3. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1663-1728 of SEQ ID NO: 5 that is suitable for construction of MiniShank3.

[0137] 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 100% identity, to residues 148-345 of SEQ ID NO: 6 or residues 147-313 of SEQ ID NO: 5, including all values ​​in between. 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 that is suitable for constructing MiniShank3. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 147-313 of SEQ ID NO: 5 that is suitable for constructing MiniShank3.

[0138] In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 6 over the entire length of SEQ ID NO: 6. As used herein, "less than 65%" includes percent identities of less than 65% that are appropriate for the construction of 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 entire 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 entire 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 entire length of SEQ ID NO:5 or SEQ ID NO:6.

[0139] 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 100% identical, to any one of SEQ ID NOs: 17-20 set forth in Table 9, including all values ​​in between.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] In some embodiments, the sequence of a polynucleotide encoding a MiniShank3 protein associated with the present disclosure 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 100% identity to any one of SEQ ID NOs: 1-4, including all values ​​in between, and encodes one or more proteins having Shank3 activity. In some embodiments, the sequence of a polynucleotide encoding a MiniShank3 protein associated with the present disclosure comprises at least 90% identity to any one of SEQ ID NOs: 1-4 and encodes one or more proteins having Shank3 activity. In some embodiments, the sequence of a polynucleotide encoding a MiniShank3 protein associated with the present disclosure comprises any one of SEQ ID NOs: 1-4. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins having Shank3 activity.

[0144] In some embodiments, MiniShank3 is encoded by any one of SEQ ID NOs: 1-4 shown in Table 9. SEQ ID NOs: 1 and 3 correspond to the mouse MiniShank3 nucleic acid sequence, and SEQ ID NOs: 2 and 4 correspond to the human MiniShank3 nucleic acid sequence. SEQ ID NOs: 1 and 2 encode MiniShank3 proteins that do not contain ankyrin repeat domains or an N-terminal domain. SEQ ID NOs: 3 and 4 encode MiniShank3 proteins that contain ankyrin repeat domains and an N-terminal domain.

[0145] The polynucleotides encoding the MiniShank3 proteins described herein encode proteins that have Shank3 activity.

[0146] As disclosed herein, sequence "identity" refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by mathematical models, algorithms, or computer programs known to those skilled in the art. The percent identity of two sequences (e.g., nucleic acid sequences or amino acid sequences) can be determined using, for example, Basic Local Alignment Search Tools (BLAST®), such as the NBLAST® and XBLAST® programs (version 2.0). Alignment techniques such as Clustal Omega can be used to align multiple sequences. Other algorithms or alignment methods include, but are not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, or the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0147] In some embodiments, the size of a polynucleotide encoding a Shank protein disclosed herein (Shank1, Shank2, Shank3) is less than about 4.6kb, about 4.5kb, about 4.4kb, about 4.3kb, about 4.2kb, about 4.1kb, about 4.0kb, about 3.9kb, about 3.8kb, about 3.7kb, about 3.6kb, about 3.5kb, about 3.4kb, about 3.3kb, about 3.2kb, about 3.1kb, about 3.0kb, about 2.9kb, about 2.8kb, about 2.7kb, about 2.6kb, about 2.5kb, about 2.4kb, about 2.3kb, about 2.2kb, or about 2.1kb. In some embodiments, polynucleotides encoding the Shank proteins disclosed herein (Shank1, Shank2, Shank3) can be of any size suitable for the methods and vectors disclosed in this disclosure.

[0148] Neurodevelopmental disorders The present disclosure provides compositions and methods suitable for treating neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan-McDermid syndrome.

[0149] As used herein, " neurodevelopmental disorder " refers to any disorder that inhibits the growth and / or development of brain and / or central nervous system.In some embodiments, neurodevelopmental disorder affects one or more brain functions, such as emotion, learning ability, self-control and memory.It should be understood that the aspects of the present disclosure can be applied to the treatment of any neurodevelopmental disorder.

[0150] In some embodiments, the neurodevelopmental disorder is autism spectrum disorder (ASD). The diagnosis of ASD is mainly based on criteria such as communication deficits, impaired social interaction, and repetitive or restricted interests and behaviors. ASD is a highly heritable disorder, with a concordance rate of 90% in identical twins. However, ASD is clinically heterogeneous and encompasses a variety of individual disorders with different symptom severity. ASD is etiologically diverse and is thought to be potentially involved in polygenic, monogenic, and environmental factors.

[0151] Alterations in synaptic connectivity and function have been proposed as a key mechanism underlying ASD. Recent genetic studies have identified numerous candidate ASD genes, many of which encode synaptic proteins, such as Shank3, Neuroligin-3, Neuroligin-4, and Neurexin-1. These findings suggest that synaptic dysfunction may underlie a common mechanism across a subset of ASDs. Various Shank3 mutations have been identified as monogenic causes of ASD with intellectual disability (ID). In ASD patients, all identified Shank3 deletions and / or mutations result in loss of function (LoF) in one of the two normal copies of the Shank3 gene (i.e., haploinsufficiency). As used herein, "haploinsufficiency" refers to a model of dominant gene action in diploid organisms, where a single copy of the wild-type allele at a gene locus heterozygously combined with a mutant allele is insufficient to produce the wild-type phenotype. Haploinsufficiency can occur through de novo mutations or inherited LoF mutations in the mutant allele, resulting in little or no gene product being produced. Recent genetic testing has also identified numerous mutations in the Shank3 gene, including microdeletions, nonsense mutations, and recurrent breakpoints, in ASD patients not diagnosed with Phelan-McDermid syndrome (PMS). These findings suggest that disruption and / or mutations in the Shank3 gene are a monogenic cause of autism spectrum disorder (ASD). It is currently estimated that Shank3-related deletions and / or mutations account for approximately 2% of all ASD patients with ID. Therefore, understanding the function of Shank3 may provide insight into the pathological mechanisms of ASD.

[0152] " Intellectual disability " as used herein refers to the disability that causes the subject to have intellectual and / or adaptive function defects.Intellectual function includes, for example, reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and / or experiential learning.Intellectual function can be measured by any method known in the art, such as IQ test.Adaptive function includes, for example, the skills required to live life independently and responsibly, such as communication and social skills.In some cases, intellectual disability can be apparent in childhood or adolescence.

[0153] In some embodiments, the neurodevelopmental disorder is Phelan-McDermid syndrome (PMS, 22q13.3 deletion syndrome), an autism spectrum disorder characterized by autistic-like behavior, hypotonia, severe intellectual disability, and impaired speech and language development. Shank3 is one of the genes reportedly deleted in Phelan-McDermid syndrome. Disruption of Shank3 is believed to be responsible for the core neurodevelopmental and neurobehavioral defects in Phelan-McDermid syndrome, as individuals with ring chromosome 22 in which the Shank3 gene is intact are phenotypically normal. Thus, provided are methods for treating neurodevelopmental disorders associated with mutations, deletions, or disruptions in the Shank3 gene and reduced Shank3 activity.

[0154] Other neurodevelopmental disorders include, but are not limited to, attention deficit hyperactivity disorder (ADHD), learning disabilities such as dyslexia or dyscalculia, intellectual disabilities, behavioral or motor disorders, cerebral palsy, visual and hearing impairments, developmental language disorders, neurogenetic disorders such as Fragile X syndrome, Down syndrome, Rett syndrome, hypogonadotropic hypogonadism syndrome, and traumatic brain injury.

[0155] subject The subject treated by the methods described herein may be a human or non-human subject. Non-human subjects include, for example, non-human primates; livestock such as cows, horses, goats, sheep and pigs; pets such as dogs and cats; and rodents.

[0156] The subject treated by the methods described herein may be a subject with, suspected of, or at risk of developing a neurodevelopmental disorder. In some embodiments, the subject has been diagnosed with a neurodevelopmental disorder, while in other embodiments, the subject has not been diagnosed with a neurodevelopmental disorder. In some embodiments, the subject is a human subject with, suspected of, or at risk of developing an autism spectrum disorder (ASD). In some embodiments, the subject is a human subject with, suspected of, or at risk of developing Phelan-McDermid syndrome. In some embodiments, the subject has reduced expression of the Shank3 gene compared to a control subject. In some embodiments, the expression of the Shank3 gene in the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 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 who does not have, is not suspected of having, or is not at risk of having neurodevelopmental disorders.In some embodiments, the reduced expression of Shank3 gene in the subject is caused by the disruption of at least one copy of Shank3 gene.In some embodiments, the disruption of Shank3 gene comprises the deletion of at least one copy of Shank3 gene.In some embodiments, the disruption of Shank3 gene comprises one or more mutations in at least one copy of Shank3 gene.

[0157] In some embodiments, the subject is a human subject exhibiting one or more symptoms of ASD. In some embodiments, the subject is a human subject exhibiting developmental delay. In some embodiments, the subject is a human subject exhibiting intellectual disability (ID). In some embodiments, the subject is a human subject exhibiting sleep disorders. In some embodiments, the subject is a human subject exhibiting hypotension. In some embodiments, the subject is a human subject exhibiting language disorders. In some embodiments, the subject is a human subject exhibiting language delay. In some embodiments, the subject is a human subject exhibiting any symptoms or signs of ASD.

[0158] In some embodiments, the subject is an adult human subject. In some embodiments, the adult is 25 years of age or older. In some embodiments, the adult is no more than 25 years of age. In some embodiments, the adult is no more than 21 years of age. In some embodiments, the adult is no more than 18 years of age. In some embodiments, the adult is 16 years of age. In some embodiments, the subject is elderly (e.g., 65 years of age or older). In some embodiments, the adult can be of any age suitable for the treatments disclosed herein.

[0159] In some embodiments, the subject is a non-adult human subject. In some embodiments, the human subject is no more than 16 years of age. In some embodiments, the human subject is no more 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 infant. In some embodiments, the human subject is a toddler. In some embodiments, the human subject is in a fetal stage of development. In some embodiments, the human subject is in a prenatal stage of development.

[0160] viral vectors As disclosed herein, a polynucleotide encoding a MiniShank3 protein can be delivered to a tissue or cell of interest using a viral vector. The vectors described herein can be used, for example, to deliver a nucleic acid encoding a protein of interest to a subject, including delivery to a specific organ or central nervous system (CNS) of the 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.

[0161] In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding the miniShank protein disclosed herein. In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding the 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 some embodiments, the present disclosure provides a recombinant AAV virion comprising an expression cassette encoding the miniShank3 protein and an AAV9 capsid.

[0162] AAV refers to a replication-deficient (e.g., non-replicating) dependoparvovirus of the Parvoviridae virus genus. AAV may be derived from a naturally occurring virus or may be recombinant. AAV can be packaged into capsids that can be derived from naturally occurring or recombinant capsid proteins. The single-stranded DNA genome of AAV contains inverted terminal repeats (ITRs). The ITRs are involved in the replication and encapsidation of the AAV genome and their integration and excision into the host genome. Without being bound by any theory, AAV vectors can include one or more ITRs, including 5' ITRs and / or 3' ITRs, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and / or additional post-transcriptional regulatory elements. The AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to those skilled in the art, for example, as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (2012)), which is incorporated herein by reference in its entirety.

[0163] In some embodiments, AAV is integrated into the genome of host cells. In some embodiments, AAV is not integrated into the host genome. In some embodiments, the AAV vectors disclosed herein can comprise sequences from any known organism. In some embodiments, the AAV vectors disclosed herein can comprise synthetic sequences. AAV vector sequences can be modified in any way known to those skilled in the art, for example, by incorporating insertions, deletions or substitutions, and / or by using post-transcriptional regulatory elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation signals. In some embodiments, AAV vectors can comprise sequences related to replication and integration.

[0164] In some embodiments, the MiniShank3 disclosed herein is delivered to a tissue or cell of interest via a recombinant AAV vector. In some embodiments, the recombinant AAV vector delivering the MiniShank3 disclosed herein is delivered to the central nervous system (CNS) of a subject. As used herein, delivering a recombinant AAV vector to the CNS can include delivering the recombinant AAV vector to any tissue or cell of interest within the CNS. In some embodiments, delivering a recombinant AAV vector to the CNS includes delivering the recombinant AAV vector to a neural tissue or cell. In some embodiments, delivering a recombinant AAV vector to the CNS includes delivering the AAV vector to the brain. In some embodiments, delivering a recombinant AAV vector to the CNS includes delivering the recombinant AAV vector to the spinal cord. In some embodiments, delivering a recombinant AAV vector to the CNS includes delivering the recombinant AAV vector to white matter and gray matter. In some embodiments, the recombinant AAV vector delivering the MiniShank3 disclosed herein is delivered to any tissue or cell of interest in a subject that is suitable for the treatments disclosed herein.

[0165] As used in this 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 includes, but is not limited to, direct administration of a recombinant AAV vector or a composition comprising a recombinant AAV vector, or peripheral administration by passive diffusion or convection-enhanced delivery (CED) to bypass the blood-brain barrier, as known in the art. The recombinant AAV vectors described herein can be administered in any composition compatible with aspects of the present disclosure.

[0166] AAV vectors can include known AAV serotypes, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV serotype is AAV9. AAV virus clades are described in Gao et al. (2004) J. Virol. 78(12):6381-6388, which is incorporated by reference. In some embodiments, any AAV serotype suitable for delivery to the CNS can be selected.

[0167] The AAV vector of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype.In some embodiments, the AAV vector can utilize or be based on the AAV serotype described in WO 2017 / 201258A1 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AV4-4, 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, AAV42-1b , 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, AA V223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AA V3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh. 58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, 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、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AA VC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AA Vrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-1 / hu.1 9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVcy. hu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu. AVhu.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.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、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.10、AAVrh.12、AAVrh.13、AAVrh.13R、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.37 2、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.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R A586R mutation、AAVrh8R R533A variant、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1. 16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AA VhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、A AV-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, AAVSM 10-2, AA 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, AAVSM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, 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 AAV10 serotype, 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-E1, 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-P1, 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-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、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-F1、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 CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV CLv1-7、AAV CLv1-8、AAV CLv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、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, A AVF16 / 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, TH1.1-32 and / or TH1.1-35, and variants thereof. AAV vectors are further described in US 9,585,971, US 2017 / 0166926, and WO2020 / 160337, which are incorporated herein by reference in their entireties.

[0168] In some embodiments, the 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 5' and 3' ITR sequences. The transgene may comprise one or more regions encoding MiniShank3 as disclosed herein. The transgene may also comprise a region encoding another protein. The transgene may also comprise one or more expression control sequences (e.g., a polyA tail). The transgene may be single-stranded. In some embodiments, the recombinant AAV vector comprises at least AAV ITRs and a MiniShank3 transgene.

[0169] In some embodiments, AAV can be packaged into AAV particles and administered to a subject and / or delivered to selected target cells. In some embodiments, the AAV particles comprise an AAV capsid protein. In some embodiments, the AAV particles comprise at least one capsid protein selected from the AAV serotypes disclosed herein, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.eB, AAV.rh8, AAV.rh10, AAV.rh39, AAV.43, AAV2 / 2-66, AAV2 / 2-84, and AAV2 / 2-125, or any variant thereof. In some embodiments, the AAV particles comprise an AAV9 capsid.

[0170] In some embodiments, the miniShank3 transgene coding sequence in the recombinant AAV vector is operably linked to a regulatory sequence for tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces 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., hSyn1). 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% identical, or 100% identical, to the sequence of SEQ ID NO: 22, including all values ​​in between. 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 suitable for the treatments described herein.

[0171] 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 comprising the hSyn promoter and 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 flanked by AAV ITRs.

[0172] 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 comprising the hSyn promoter and 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 flanked by AAV ITRs.

[0173] Aspects of the present disclosure relate to recombinant AAV vectors expressing a miniShank3 transgene. In some embodiments, the 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 rh10 ITRs. In some embodiments, the AAV ITRs may comprise one or more modified ITRs that generate a self-complementary AAV genome.

[0174] 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% identical, or 100% identical, to the sequence of SEQ ID NO:27, including all values ​​in between. 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% identical, or 100% identical, to the sequence of SEQ ID NO: 28, including all values ​​in between. 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.

[0175] It should be understood that the AAV vectors described herein can include DNA constructs or expression cassettes that include a transgene, such as MiniShank3, 5' and / or 3' ITRs, promoters, introns, and / or other associated regulatory elements known in the art.

[0176] In some embodiments, the AAV vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which may enhance expression of the miniShank3 transgene. 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% identical, or 100% identical, to the sequence of SEQ ID NO:23, including all values ​​in between. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO:23.

[0177] 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.

[0178] In some embodiments, the AAV vectors used herein may be self-complementary vectors.

[0179] SEQ ID NO: 21 contains the human MiniShank3 gene, 5'-ITR, 3'-ITR, WPRE, hGH polyA, and hSyn1 promoter. SEQ ID NO: 30 contains the human MiniShank3 gene, 5'-ITR, 3'-ITR, WPRE, hGH polyA, and hSyn1 promoter.

[0180] In some embodiments, the inverted terminal repeat (ITR) sequences each comprise approximately 145 nucleotides. These elements, in cis, may aid in efficient replication and encapsidation. Those skilled in the art will understand that any element of an AAV vector known in the art may be compatible with aspects of the present disclosure. Those skilled in the art will also understand that any of the polynucleotide sequences described herein encoding a functional MiniShank3 protein may be expressed in a DNA construct or expression cassette for AAV delivery. These DNA constructs or expression cassettes may contain 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 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 comprises one or more elements such as a promoter, 5'-ITR, 3'-ITR, Syn1 promoter, WPRE, hGH polyA, etc. Cis plasmids for producing recombinant AAV virions may have elements such as an origin of replication and an antibiotic resistance marker, e.g., an F1 origin, a NeR / KanR marker, and / or a PUC origin. Figure 1 shows an example of a plasmid for producing the recombinant AAV virions described herein.

[0181] The expression cassette described herein can include a polyA signal. In some embodiments, the polyA signal is an 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% identical, or 100% identical, to the sequence of SEQ ID NO: 24, including all values ​​in between. In some embodiments, the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 24.

[0182] In some embodiments, a recombinant AAV vector associated with the present disclosure includes a nucleic acid sequence operably linked to flanking ITRs that encodes a MiniShank3 protein and regulatory elements that promote CNS expression, wherein the nucleic acid sequence 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 100% identical, to the sequence of SEQ ID NO:21 or SEQ ID NO:30 shown in Table 9, including all values ​​in between. In some embodiments, the recombinant AAV vector comprises a sequence corresponding to SEQ ID NO:21 or SEQ ID NO:30, which encodes a MiniShank3 protein comprising the sequence of SEQ ID NO:18 and includes regulatory elements including an hSyn1 promoter, a WPR element, and a polyA signal sequence and flanking ITR sequences. In some embodiments, the 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 with a neurodevelopmental disorder.

[0183] As those skilled in the art can understand, any method known in the art for designing and delivering AAV vectors for clinical use can be compatible with the aspects of the present disclosure.For example, non-limiting examples of the disclosure of AAV vectors and delivery are provided in U.S. Patent No. 7,906,111 "Adeno-associated virus (AAV) clades, sequences, vectors containing them, and their use" and U.S. Patent No. 9,834,788 "AAV vectors for use in gene therapy for congenital choroideremia", each of which is incorporated herein by reference in its entirety.

[0184] In some embodiments, a recombinant AAV vector encoding a MiniShank3 protein for AAV delivery encodes a protein having 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 shown in Table 9.

[0185] The present disclosure provides a recombinant AAV virion comprising (1) a recombinant AAV vector 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 100% identical, to SEQ ID NO: 29. As used herein, "virion" refers to a viral particle that comprises genetic material (e.g., RNA or DNA) and a capsid.

[0186] In some embodiments, the expression cassette disclosed herein may 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.

[0187] In some embodiments, a recombinant vector containing an expression cassette comprising a polynucleotide encoding a Shank3 protein (i.e., a 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 promoter. In some embodiments, the human promoter is human synapsin 1 (hSyn1). In some embodiments, the hSyn1 promoter has a polynucleotide sequence corresponding to SEQ ID NO: 22. In some embodiments, the human promoter can be any promoter known in the art that is suitable for expressing miniShank3. In some embodiments, the human promoter can be any promoter that has high specificity for neural 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 skilled in the art will appreciate, any promoter can be used as long as the selected promoter is compatible with aspects of the present disclosure.

[0188] The present disclosure provides a method for producing AAV virions. In some embodiments, the method comprises culturing a host cell containing the AAV vector described herein, AAVcap (capsid protein) and AAV9rep (replication protein), and optionally one or more additional adenovirus helper functions, under conditions sufficient to produce AAV virions; and isolating the AAV virions produced by the host cell. In some embodiments, AAVcap encodes VP1, VP2, and / or VP3. In some embodiments, rep encodes rep78, rep68, rep52, and / or rep40.

[0189] Composition and Administration The present invention provides compositions (including pharmaceutical compositions) comprising a polynucleotide (e.g., one encoding miniShank3) delivered by a recombinant AAV vector and / or AAV virion disclosed herein and a pharmaceutically acceptable carrier.

[0190] The compositions of the present disclosure may comprise recombinant AAV vectors and / or AAV virions, either alone or in combination with one or more other viruses. In some embodiments, the compositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different recombinant AAV vectors and / or AAV virions.

[0191] A suitable carrier can be easily selected by those skilled in the art, taking into consideration the indication for which the recombinant AAV is intended. For example, one suitable carrier is saline, which can be formulated with various buffer 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 does not limit the present disclosure. Pharmaceutical compositions containing AAV vectors are further described in US 9,585,971 and US 2017 / 0166926, which are incorporated herein by reference in their entirety.

[0192] As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agent, isotonic and absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. 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 composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to a host.

[0193] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by recombinant AAV vectors can be formulated for delivery by encapsulating them in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0194] Such formulations may be preferred for the delivery of pharmaceutically acceptable formulations of the nucleic acids or recombinant AAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulatory half-lives have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for using liposomes and liposome-like formulations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0195] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have diameters of 25 nm to 4 μm. When MLVs are sonicated, small unilamellar vesicles (SUVs) with diameters of 200 to 500 Å are formed, containing aqueous solution in the center.

[0196] Alternatively, nanocapsule formulations of recombinant AAV vectors can be used. Nanocapsules generally confine substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) must be designed using polymers that can be degraded in vivo. The use of biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements has been investigated.

[0197] In some embodiments, pharmaceutical compositions containing nucleic acids delivered by recombinant AAV vectors contain other pharmaceutical ingredients, such as preservatives or chemical stabilizers. Examples of suitable 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 may be preferable to add an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of pharmaceutical compositions can be achieved by using agents that delay absorption of pharmaceutical compositions, such as aluminum monostearate and gelatin, in the composition.

[0198] Pharmaceutical forms suitable for delivering recombinant AAV vectors include sterile aqueous solutions or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and their mixtures, and in oils.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.In most cases, this form is sterile and fluid enough to be easily injected.It must be stable under the conditions of manufacture and storage, and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0199] The methods described herein involve administering a recombinant AAV vector in an amount sufficient to transfect cells of a desired tissue (e.g., the brain) and provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ, oral, inhalation, intravenous (including intraocular, facial vein, and retro-orbital injection), intracerebroventricular (ICV), intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. In some embodiments, the recombinant AAV vector is delivered to cells of a desired tissue (e.g., the brain) via parenteral administration. In some embodiments, the recombinant AAV vector is delivered to cells of a desired tissue (e.g., the brain) via intravenous administration. In some embodiments, the recombinant AAV vector is delivered to cells of a desired tissue (e.g., the brain) via ICV administration. In some embodiments, the ICV administration can be unilateral. In some embodiments, the ICV administration can be bilateral. The recombinant AAV virions described herein, when administered via ICV administration, may improve the therapeutic efficacy of miniShank3.

[0200] Routes of administration can be combined, if desired. In some embodiments, the vectors disclosed herein are administered intravenously.

[0201] In some embodiments, the present disclosure provides a method of treating a subject with a neurodevelopmental disorder. In some embodiments, the present disclosure provides a method of treating a subject with an autism spectrum disorder (ASD). In some embodiments, the present disclosure provides a method of treating a subject with Phelan-McDermid syndrome.

[0202] In some embodiments, the methods provided herein comprise administering and delivering to a target environment or tissue of a subject an effective amount of a composition comprising a recombinant AAV virion comprising an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3). In some embodiments, the target tissue is the cortex. In some embodiments, the target tissue is the striatum. In some embodiments, the target tissue is the thalamus-cerebellum. In some embodiments, the target tissue is the hippocampus. In some embodiments, the target tissue is any brain structure. In some embodiments, the method of administering and delivering to a target environment or tissue an effective amount of a composition comprising a recombinant AAV virion comprising an expression cassette comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) comprises delivering the composition to a neuron or other brain cell type. In some embodiments, the method of delivering a nucleic acid to a target environment or tissue of a subject in need thereof comprises providing a composition comprising a recombinant AAV virion comprising at least the 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, the method for delivering nucleic acid to the target environment or tissue of a subject that requires it comprises delivering AAV virion by unilateral or bilateral intraventricular administration.The method for using AAV vector is further described in US 9,585,971, US 2017 / 0166926 and WO2020 / 160337, which are incorporated herein by reference in their entirety.In some embodiments, the composition can comprise capsid protein.

[0203] In some embodiments, a composition comprising a recombinant AAV virion comprising a polynucleotide encoding Shank3 protein is delivered to a subject via intravenous administration, systemic administration, intracerebroventricular (ICV) administration (including bilateral or unilateral ICV administration), intrauterine administration, intrathecal administration, retro-orbital injection, or facial vein injection. In some embodiments, intrauterine administration is used for subjects in prenatal development. In some embodiments, the composition is delivered to a subject via nanoparticles. 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 carrier suitable for delivering nucleic acid material.

[0204] Any composition comprising a recombinant AAV virion containing an expression cassette comprising a polynucleotide encoding a protein that provides some utility or benefit to a subject can be delivered to a target environment or tissue of a subject according to the methods disclosed herein.

[0205] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering AAV compositions to a host: Sonophoresis (i.e., ultrasound) has been described and used in U.S. Patent No. 5,656,016 as a device to increase the rate and efficacy of drugs penetrating into and through the circulatory system. Other alternative drug delivery methods under consideration include intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), eye drop formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0206] The dose (e.g., dosage units in absolute vector genomes (vg) or vector genomes per milliliter of pharmaceutical solution (vg / mL)) of a recombinant AAV vector or recombinant AAV virion comprising a polynucleotide encoding a Shank3 protein (e.g., miniShank3) required to achieve a particular "therapeutic effect" varies based on several factors, including, but not limited to, the AAV administration route, the level of gene expression required to achieve a therapeutic effect, the particular disorder being treated, and the stability of the gene product. A dose that maximizes infection rate without affecting neurodevelopment is also appropriate. One of skill in the art can readily determine the dose range of a recombinant AAV vector or recombinant AAV virion for treating a patient with a particular disorder based on the aforementioned factors, as well as other factors.

[0207] In some embodiments, an effective amount of a recombinant AAV vector or recombinant AAV virion can be an amount sufficient to infect an animal or human subject or target a desired tissue. The effective amount depends primarily on factors such as the subject's species, age, sex, weight, health, and target tissue, and therefore may vary by subject and tissue. The term "effective amount" or "effective amount" in the context of a composition or dose administered to a subject refers to the 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 can partially or completely rescue the effects of a mutated Shank3 gene and / or partially or completely restore the loss of function of the Shank3 protein. An effective amount can include reducing the level of an undesired response, but in some embodiments, completely preventing an undesired response. An effective amount can also include delaying the onset 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, an effective amount can include increasing the level of a desired response, such as a therapeutic endpoint or result. Achievement of any of the foregoing can be monitored by conventional methods and by the methods disclosed in this application. The effective amount will, of course, vary depending on factors such as the particular subject being treated; the severity of the condition; individual patient parameters such as age, physical condition, size and weight; the duration of treatment; the nature of concurrent treatment (if any); and the particular route of administration. It should be understood that an effective amount, as used herein, does not necessarily have to be clinically effective.

[0208] For example, in some embodiments, the number of vector genomes administered to a subject is about 6.0 x 10 11 vg~approx. 9.0 x 10 13 In some embodiments, the number of vector genomes administered to a subject is about 6.0 x 10 13 vg / mL ~ approx. 9.0 x10 13 In some embodiments, the number of vector genomes administered to a subject is about 1 x 10 10~Approx. 1 x 10 12 In some embodiments, the effective amount of AAV is 100 mg / kg or any value between 100 mg / kg and 100 mg / kg. 10 , 10 11 , 10 12 , 10 13 , or 10 14 In one embodiment, the effective amount of AAV is 10 genome copies per subject. 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 genome copies. In some cases, about 10 11 ~10 13 A dosage of about 1.0 x 10 AAV genome copies is appropriate. In some embodiments, a dosage of about 1.0 x 10 AAV genome copies is appropriate. 13 ~Approx. 1.0 x 10 14 A dose of vector genome is administered to the subject. In some embodiments, the number of vector genomes administered to the subject can be any dose suitable for the treatment and methods disclosed herein. In some embodiments, the dose of vector genome is administered by unilateral or bilateral ICV administration.

[0209] In some embodiments, the dose administered to a subject via unilateral ICV administration is about 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, or 10 μl per subject. In some embodiments, the dose administered to a subject via unilateral ICV administration is about 5 μl per subject.

[0210] In some embodiments, the dose administered to a subject via unilateral ICV administration is about 1.4 x 10 10 vg / ml, approximately 1.4 × 10 11 vg / ml, approximately 1.4 × 10 12 vg / ml, approximately 1.4 × 10 13 vg / ml, approximately 1.4 × 10 14 vg / ml, approximately 1.4 × 10 15 vg / ml, or approximately 1.4 × 10 16In some embodiments, the dose administered to a subject via unilateral ICV administration is about 1.4 x 10 13 In some embodiments, the dose administered to a subject via unilateral ICV administration is about 1.4 x 10 10 vg / ml ~ approx. 1.4 × 10 16 vg / ml.

[0211] In some embodiments, the recombinant AAV dose is administered to the subject as a single dose. In some embodiments, the recombinant AAV dose is administered to the subject as a single dose that may be readministered at a later time.

[0212] The formulation of pharmaceutically acceptable excipients and carrier solutions disclosed herein is well known to those skilled in the art, as is the development of suitable dosage and treatment regimens for the use of specific compositions described herein in various treatment regimens.Of course, the amount of active compound in each therapeutically useful composition can be prepared in such a way that the suitable dosage of compound can be obtained in any given unit dose.Factors such as solubility, bioavailability, biological half-life, administration route, product shelf life and other pharmacological considerations are considered by those skilled in the art of preparing such pharmaceutical preparations, and therefore different dosages and treatment regimens may be desirable.

[0213] In embodiments, the formulation comprises a recombinant AAV virion, the recombinant AAV virion 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and 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 at least 90% identical or at least 95% identical to SEQ ID NO:29, in a buffer of 10 mM Tris, 1 mM magnesium chloride (MgCl), 150 mM sodium chloride (NaCl), and 0.02% poloxamer 188, pH 8.0, at a concentration of about 1.4 × 10 10 vg / ml ~ approx. 1.4 × 10 16 Contains at a concentration of vg / ml.

[0214] In embodiments, the formulation consists essentially of recombinant AAV virions, the recombinant AAV virions 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and operably linked to a human Syn promoter and polyA signal sequence; and 2) an AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO:29, in a buffer of 10 mM Tris, 1 mM magnesium chloride (MgCl), 150 mM sodium chloride (NaCl), and 0.02% poloxamer 188, pH 8.0, at a concentration of about 1.4 × 10 10 vg / ml ~ approx. 1.4 × 10 16 Contains at a concentration of vg / ml.

[0215] In embodiments, the formulation consists of a recombinant AAV virion, the recombinant AAV virion 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 at least 90% identical to SEQ ID NO:18 or SEQ ID NO:20, and operably linked to a human Syn promoter and polyA signal sequence; and 2) an AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO:29, in a buffer of 10 mM Tris, 1 mM magnesium chloride (MgCl), 150 mM sodium chloride (NaCl), and 0.02% poloxamer 188, pH 8.0, at a concentration of about 1.4 × 10 10 vg / ml ~ approx. 1.4 × 10 16 Contains at a concentration of vg / ml. Expression of proteins related to the Shank protein network

[0216] The methods and compositions provided herein are useful, in some embodiments, for treating neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan-McDermid syndrome. In some embodiments, the expression level of a PSD protein is used to assess the effectiveness of administration of miniShank3. In some embodiments, the PSD protein is Homer. In some embodiments, the PSD protein is postsynaptic density protein 95 (PSD95). In some embodiments, the PSD protein is SynGap1. 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 by miniShank3 treatment.

[0217] In some embodiments, an increase in any PSD protein compared to untreated control subjects may indicate the effectiveness of miniShank3. Methods for detecting gene expression and protein levels are well known in the art.

[0218] In some embodiments, expression of Homer in a subject after treatment 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 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 postsynaptic protein (PSD95) in a subject after treatment 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 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 SynGap1 in a subject after treatment 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 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 a subject after treatment 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 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 a subject after treatment 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 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 a subject after treatment 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 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 a subject after treatment 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 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.

[0219] In some embodiments, administration of MiniShank3 or a composition comprising MiniShank3 can result in improved sleep efficiency. In some embodiments, a subject's sleep efficiency is improved after being administered an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein, such as MiniShank3 protein. In some embodiments, the subject's sleep efficiency 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 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 alleviate sleep disorders, including, but not limited to, difficulty falling asleep and shallow sleep. Sleep efficiency can be measured using any method known in the art.

[0220] In some embodiments, administering MiniShank3 or a composition comprising MiniShank3 can result in the improvement of social impairment. In some embodiments, after administering an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein, such as MiniShank3 protein, the social impairment of the subject is improved. In some embodiments, after administering an effective amount of a composition described herein, the social impairment of the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 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 performed using any method known in the art.

[0221] As used herein, "social disorder" refers to a behavioral abnormality or deficit that prevents a subject from exhibiting spontaneous social interaction.

[0222] In some embodiments, administration of MiniShank3 or a composition comprising MiniShank3 can result in improvement of motor and / or motor coordination deficits. In some embodiments, after administration of an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein, such as MiniShank3 protein, the subject's motor and / or motor coordination deficits are improved. In some embodiments, after administration of an effective amount of a composition described herein, the motor and / or motor coordination deficits in the subject are reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 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 motor and / or motor coordination deficits can be performed using any method known in the art.

[0223] As used herein, "movement and / or motor coordination deficits" can include, for example, lack of coordination, loss of balance, and / or a shuffling gait.

[0224] In some embodiments, administration of MiniShank3 or a composition comprising MiniShank3 can result in improvement of corticostriatal synaptic dysfunction. In some embodiments, after administering an effective amount of a composition comprising an expression cassette comprising a polynucleotide encoding a Shank protein, such as MiniShank3 protein, the subject's corticostriatal synaptic dysfunction is improved. In some embodiments, after administering an effective amount of a composition described herein, the subject's corticostriatal synaptic dysfunction is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 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 corticostriatal synaptic dysfunction can be performed using any method known in the art.

[0225] As used herein, "cortico-striatal synaptic dysfunction" refers to defects in the corticostriatal circuits in the brain that can lead to repetitive and compulsive behaviors in neuropsychiatric and neurodevelopmental disorders such as autism, obsessive-compulsive disorder, and Tourette's syndrome.

[0226] Some aspects of the technology described herein can be further understood based on the non-limiting exemplary embodiments described in the Examples section below. Any limitations of the embodiments described in the Examples section below limit only the embodiments described in the Examples section below, and not any other embodiments described herein. [Example]

[0227] In order that the present disclosure may be more fully understood, the following examples are set forth: The examples set forth in this application are provided to illustrate the systems and methods provided herein and should not be construed as limiting the scope thereof.

[0228] Example 1: Intravenous administration of an AAV vector containing miniShank3 into Shank InsG3680 mice Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders, one of the most severe, resulting in significant morbidity and significant socioeconomic impact. The Shank family of postsynaptic scaffolding proteins has emerged as a contributing factor to ASD, with Shank mutations accounting for approximately 1% of ASD patients. The prevalence of Shank3 haploinsufficiency due to gene deletions, truncations, or missense mutations is estimated to be 1:15,000 worldwide. Shank3 haploinsufficiency has been identified in patients with chromosome 22q13.3 deletion syndrome (also known as Phelan-McDermid syndrome (PMS)). PMS due to Shank3 haploinsufficiency has been found to be associated with behavioral and cognitive manifestations of varying severity. Deficits are evident from early childhood and persist throughout life. Current medical treatment is limited to antipsychotic medications and treatment of comorbid conditions. However, there are currently no treatments that directly address Shank3 haploinsufficiency or PMS.

[0229] The ability of the AAV-PHP.eB-hSyn-GFP Mouse MiniShank3 vector, when administered intravenously, to correct behavioral and physiological deficits characteristic of the Shank3-InsG3680 mouse model has been reported in PCT Publication No. WO2022 / 040239 and U.S. Patent Publication No. US2023 / 0340041, which are incorporated by reference herein in their entireties.

[0230] A study was conducted to test whether intravenous administration of miniShank3 to postnatal day 14 (P14) animals could ameliorate the physiological defects associated with the Shank3-InsG3680 mouse model. Specifically, the correction of Shank3 protein and binding partner (PSD95, NR1, Homer, GluR2) levels in brain synaptic membrane preparations was assessed by assays including behavioral modifications in the open field test.

[0231] Test and control substances As used herein, "AAV9-hSyn1-human miniShank3-V1" refers to a suspension of adeno-associated virus vector-based gene therapy for parenteral administration. It is a recombinant, non-replicating AAV9 vector containing a single-stranded transgene encoding a condensed version of the human Shank3 protein (miniShank3) under the control of a neuron-specific synapsin promoter. Because full-length Shank3 is too large to be packaged with conventional AAV methods, miniShank3 was engineered to reduce the overall size of the transgene to allow proper AAV packaging while retaining key domains previously identified for proper function as a synaptic scaffolding protein. The "vehicle" control contains the same excipients as AAV9-hSyn1-human miniShank3-V1, but does not contain the AAV9 vector containing the miniShank3 transgene.

[0232] The study design is shown in Table 1 below. Table 1: Research design [Table 1]

[0233] breeding A total of 22 female heterozygous Shank3InsG3680 mice, 5 male homozygous Shank3InsG3680 mice, and 5 male wild-type mice were housed in pairs of 3 or 2. Littermates were monitored daily for postnatal age and remained with their mothers until weaning. Biopsies for genotyping were taken from the tail tissue of pups at approximately P3, and pups were identified using toe tattoos. Genotyping was performed.

[0234] Registration Subjects (e.g., Shank3InsG3680 mice and WT mice) were enrolled according to genotype results. Homozygous and WT genotypes were randomly enrolled into corresponding groups (both males and females) and identified according to genotype results by toe tattoo. Mice were dosed in an open-label manner via retro-orbital vein injection at P14. Groups were WT vehicle (n=20), homozygous vehicle (n=21), and homozygous AAV9-hSyn1-human miniShank3-V1 (n=22).

[0235] endpoint Open field data collection The assay was performed in a custom-built open field apparatus. Each chamber was a 50 x 50 cm square. A 30-minute trial was recorded, and movement was tracked and analyzed using a custom Behavior Cloud template. The center of the open field was defined as a 13.5 x 13.5 cm square located in the geometric center of the arena. Total path length was measured for each mouse, and time spent in the open field center and path length were also determined. Each chamber was cleaned between trials of individual mice.

[0236] statistical analysis Open field measures, including total path length, time spent in the center, and % path length in the center, were analyzed using one-way ANOVA with Tukey's post hoc multiple comparisons. Path lengths in 5-min bins were analyzed using a two-way ANOVA with time as the within-group factor and treatment as the between-group factor. Bonferroni-corrected comparisons were used for post hoc pairwise comparisons.

[0237] result No AAV9-hSyn1-human miniShank3-V1-dependent effects on body weight or open-field behavior were observed in the Shank3-InsG3680 mouse model when administered as an intravenous dose of 1.0E14 vg / kg on P14 and assessed at 8 weeks of age. These data confirm that intravenous administration of AAV9-hSyn1-human miniShank3-V1 is not suitable for modification in this model.

[0238] Proof-of-principle studies were performed using a mouse surrogate vector and transgene in the Shank3InsG3680 mouse model. Shank3InsG3680 mice were intravenously injected with 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 evolved to more efficiently cross the blood-brain barrier in mice (but not NHPs), allowing transduction into the brain via IV administration. WT animals administered a control vector were also included. Animals were injected at postnatal days (P) 0–P2, P7, or P28 and neurobehavioral endpoints were assessed. A vector encoding the miniSHANK3 protein reversed genotype-dependent effects on open field, rotarod, three-chamber test, elevated zero maze, grooming behavior, and EEG / electromyography (EMG) endpoints, although the specific rescue effect varied somewhat depending on age of injection. Older animals (administered at P28) showed decreased grooming efficacy and elevated zero-maze scores compared to younger animals, suggesting the potential benefit of early intervention. Ex vivo assessment in this study confirmed that mouse miniSHANK3 protein was properly localized to the PSD. Taken together, this study confirms that the disease phenotype can be rescued using a mouse surrogate miniShank3 transgene delivered to young animals.

[0239] Example 2: Medication study in the Shank3 ASD mouse model As noted in Example 1, the ability of the AAV-PHP.eB-hSyn-GFP Mouse MiniShank3 vector, when administered intravenously, to correct behavioral and physiological deficits characteristic of the Shank3-InsG3680 mouse model has been reported in PCT Publication No. WO2022 / 040239 and U.S. Patent Publication No. US2023 / 0340041, which are incorporated by reference herein in their entireties.

[0240] In this study, we establish the baseline behavioral phenotype of adult Shank3Δ4-22 knockout (KO) mice. These mice have been engineered to remove exons 4-22 of the Shank3 gene, modeling the majority of patients with SHANK3-associated autism / Phelan-McDermid syndrome (PMS), which harbors a mutation that results in a significant truncation of the SHANK3 gene. Furthermore, these mice mirror the behavioral deficits seen in the Shank3-InsG3680 model.

[0241] Adult Shank3Δ4-22 mice are normal in terms of physical characteristics, including body weight. However, their sleep quality, as measured by electroencephalography (EEG)-based delta wave power during the hypoactivity phase, is reduced compared to their WT littermates. In the open field test, Shank3Δ4-22 mice exhibited reduced locomotor activity after the first 10 minutes compared to WT mice, suggesting reduced exploratory behavior and / or increased fatigue.

[0242] Similarly, in the rotarod test, which assesses motor coordination, endurance, and motor learning, Shank3Δ4-22 mice fall off the rotating bar faster than WT mice, and in the process are unable to learn motor coordination and balance. These impairments in delta power and motor skills are similar to the sleep and motor activity disorders commonly observed in SHANK3 haploinsufficient patients.

[0243] Impairments in striatal learning are accentuated by defects in striatal postsynaptic signaling complexes. Shank3Δ4-22 mice exhibit reduced miniature evoked postsynaptic currents (mEPSCs) and spine density in striatal medium spiny neurons (MSNs). The PSD is compromised by the loss of scaffolding proteins required for proper anchoring of glutamate receptors, particularly Homer1, a SHANK3 binding partner.

[0244] Despite a large deletion in Shank3 similar to that seen in patients, heterozygous Shank3Δ4-22 mice exhibit a milder phenotype in contrast to the more severe phenotype seen in SHANK3 haploinsufficient patients, a difference whose cause is not fully understood (Drapeau, 2018).

[0245] In summary, the described animal model resembles many of the clinical features of patients with SHANK3 haploinsufficiency, and the dysfunction of neural connectivity is consistent with current theories that dysfunction of neural circuitry and plasticity underlie the pathophysiology of SHANK3 haploinsufficiency.

[0246] The most definitive POC study consisted of three phases using Shank3Δ4-22 and WT mice: Phase 1 evaluated genotype-related phenotypes in untreated animals; Phase 2 administered various doses (0, 2.40 × 10) to P14 animals to correct the phenotypes identified in Phase 1 6–16 weeks after treatment. 9 , 1.20×10 10 , 6.00×10 10 , or 2.75 × 10 11Phase 3 evaluated the effects of AAV9-hSyn1-human miniShank3-V1 administered at doses of 100 mg / kg (vg); Phase 3 evaluated the two highest doses from Phase 2 at approximately 7–10 months post-dose. During part of Phase 2 and throughout Phase 3, a general increase in dermatitis within the animal facility appeared to primarily affect animals treated with AAV9-hSyn1-human miniShank3-V1. This resulted in high mortality in Phase 3, precluding assessment of most efficacy endpoints at 7 months post-dose.

[0247] In Phase 2, the incidence of dermatitis was reduced, and efficacy was evaluated. No significant changes in efficacy endpoints were observed at the low and low-middle doses. 10 In KO animals, the high dose significantly reversed the genotype-dependent effects in the open field assay (P = 0.049), tended to improve in the rotarod (P = 0.133) and EEG (P = 0.168), had a significant effect on Homer1 recruitment at synaptic membranes (P = 0.014), and restored functional miniSHANK3 expression by 49% compared with WT SHANK3 levels in brain lysates. In KO animals, the high dose resulted in miniSHANK3 expression at 589% of endogenous WT levels, significantly reversed the overexpression phenotype in the open field, EEG (P = 0.005), and Homer1 recruitment (P < 0.0001), and tended to improve in the rotarod assay (P = 0.125).

[0248] The following autism / PMS / anxiety-related behaviors were assessed: 1) open field, 2) chamber social preference, 3) hot plate assay, 4) rotarod, and 5) grooming. After establishing baseline behavioral phenotypes, we tested whether physiological defects associated with the Shank3Δ4-22 mouse model could be ameliorated after unilateral intracerebroventricular (ICV) delivery of AAV9-hSyn1-human miniShank3-V1 to P14 animals. Specifically, levels of Shank3 protein and binding partners (PSD95, NR1, Homer, and GluR2) in brain synaptic membrane preparations were assessed by Western blot, and behavioral assessments were performed using the five assays described above. The presence of seizure activity and changes in sleep patterns were monitored by electroencephalography (EEG). Finally, animal tissues were collected at necropsy for biodistribution, in situ hybridization, clinical pathology, and histopathological analyses.

[0249] Table 2 provides an overview of the test substances and vehicles used in this study. The test substance (AAV9-hSyn1-human miniShank3-V1) and vehicle were the same as those described in Example 1. Table 2: Test substances and vehicles [Table 2]

[0250] Handling of test substances Aliquots of test substance were prepared containing enough solution to administer to four mice (four mice injected with 5 μL each = 20 μL injection + dead volume and excess) and thawed daily. Two aliquots were used on days when eight mice were injected. Aliquots were used the same day (within 6–8 hours of thawing), and excess was discarded if not administered to multiples of four mice. To avoid wasting test substance, P14 animals requiring thawing of aliquots not used for four mice were either dosed on P15 with their newly born littermates the following day or removed from the study.

[0251] The following paragraphs describe the study and experimental design.

[0252] Basic research design The number of animals per group was 15 males (M) and 15 females (F) per genotype for the phenotyping pilot study, 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). After injection, adult mice were housed up to three per cage, and pups were housed up to six per cage per mother after injection. Pups exceeding six per litter were fostered. Mice were fed standard chow and water and were not allowed an acclimation period. In general, mice were grouped by sex, genotype, and solution and injected as needed.

[0253] Mice in injection studies were assigned to treatment groups sequentially, ensuring that approximately equal numbers of mice born each week were assigned to each treatment group. To optimize aliquot use, mice were divided into groups of four and administered one dose per day consecutively. Criteria for euthanasia were a body weight less than 20% of peak body weight (the highest weight an individual mouse had achieved) and a body condition score of 2 or less. Table 3: Study design of the phenotyping pilot study [Table 3] Table 4: Study design for injection study (6 weeks) [Table 4] Table 5: Study design for injection study (7 months) [Table 5]

[0254] Cohort composition In the cohort composition of this study, Phase 1 represents the baseline collection of phenotypes for non-injected mice at 8-13 weeks of age; Phase 2 represents the baseline collection of phenotypes for injected mice at 8-13 weeks of age; and Phase 3 represents the baseline collection of phenotypes for injected mice at 7 months of age. Table 6: Cohort composition [Table 6] Table 7: Study timeline [Table 7]

[0255] In Phase 2, various doses of AAV9-hSyn1-human miniShank3-V1 were administered intravenously to WT or Shank3Δ4-22KO littermates at P14, and neurobehavioral endpoints were assessed starting 6 weeks post-injection and continuing through 14 weeks post-injection. After behavioral assessments were completed, n = 10 males per group were implanted with EEG electrodes and evaluated for seizure-like activity and power spectral analysis. At termination, samples were collected for biodistribution, histopathology, and synaptic membrane preparations to assess SHANK3 binding partners. Blood samples were collected from a subset of animals (n = 0–7 per group) to assess hematology and clinical chemistry. Due to the large size of this phase, multiple cohorts (5 total) were performed.

[0256] In Phase 2, several neurobehavioral endpoints that were significant in Phase 1, including vertical activity in the open field and grooming duration, did not show statistically significant genotype-related differences. Data for endpoints showing genotype-related differences are shown in the figures below. In Phase 3, only EEG assessments were evaluated as neurobehavioral endpoints.

[0257] Management, sampling of pups, and genotyping The pups used in the study were derived from the mating of three Shank3 heterozygous parents (two females, one male). Mating was scheduled 19 days prior to expected delivery. Delivery was scheduled 14 days prior to the scheduled injection date. All pups were naive prior to the start of the study. Pups were sampled for genotyping by toe clip between postnatal days 1 and 7.

[0258] Body Condition Score Body condition scores were measured weekly for mice that had lost 10% or more of their peak body weight.

[0259] Briefly, the BCS of each mouse was assessed as follows: BC1 - Mouse is emaciated A very prominent skeletal structure with little or no flesh covering b.The vertebrae are clearly segmented BC2 - Mouse is poorly adjusted A. The spinal column is clearly segmented b. The dorsal pelvic bones are easily palpable BC3 - Mouse is well adjusted a. The vertebrae and dorsal pelvis are not prominent and can be palpated with light pressure. BC4 - Mice are over-conditioned A spine is a continuous column b. The vertebrae can only be palpated by applying firm pressure BC5 - mice are obese a. The mouse is smooth and chunky B. The bone structure is hidden under the flesh and subcutaneous fat

[0260] For ICV injections, mice were injected once on day P14±2. Briefly, mice were anesthetized with isoflurane (5% induction and 3% maintenance at 02°C). The top of the skull was shaved. Mice were placed on a Stoelting stereotaxic table, and the head was fixed with ear bars. Isoflurane was delivered through a nose cone. The shaved area was cleaned with chlorhexidine. Using sterile surgical techniques, the skin was incised to expose the skull. Hydrogen peroxide was applied to expose bregma and lambda. A drill was attached to the articulating arm of the stereotaxic table, and the skull was opened at the following selected locations: 1 mm caudal to bregma, 0.4 mm from the sagittal suture, and targeted to the right ventricle.

[0261] The Hamilton syringe was attached to a Stoelting injector mounted on an articulating arm on the operating table. The needle was moved along the x and y coordinates and lowered to a depth of 2.0 mm. The injection (5 microliters unilateral injection) was initiated at the selected flow rate (1 μL / min). Upon completion of the injection, the needle was left in place for at least 3 minutes and then slowly removed. Bupivacaine was applied topically before adhesive was applied to the skull and skin sutures. 1 mL of warm sterile saline was administered intraperitoneally, and the mouse was allowed to recover on a warm pad before being returned to its cage. Instruments were bead-sterilized before the next surgery.

[0262] body weight In Phase 2, KO animals treated with the highest dose of AAV9-hSyn1-human miniShank3-V1 had slightly reduced body weight compared to KO vehicle controls (data not shown), confirming the low body weight associated with the KO phenotype.

[0263] Genotype-related effects on body weight were also observed in Phase 3. In males, 2.75 × 10 11 vg KO animals gained less weight than vehicle KO animals. In females, 6.00 x 10 10 vg or 2.75 x 10 11Animals treated with vg tended to gain weight more slowly than vehicle KO animals at various time points during the study, but this finding was significant at only one time point (14 weeks of age) (data not shown).

[0264] Open field test In the open field test, mice were tested once at 9–10 weeks of age.

[0265] Briefly, the apparatus is a square arena (40 x 40 x 40 cm) made of transparent Plexiglas. Data was recorded via a three-dimensional grid system of highly sensitive infrared (IR) photobeams that are invisible to the mice. When the mouse moves or displaces, its body breaks the continuous beam. An automated system then converts the beam breaks into measurements such as distance traveled, number of rearing attempts, and time spent in the center and periphery of the arena.

[0266] Mice in their home cages were allowed to acclimate to the testing room for 60 minutes beforehand. First, a mouse was placed in the center of the arena, and the computer began recording. A typical session lasted 60 minutes, and data were displayed as the sum of each measurement in 10-minute bins. At the end of the test, mice were returned to their home cages (group-housed mice were housed in parallel in different arenas). The testing arena was cleaned with 70% EtOH between subjects.

[0267] Shank3Δ4-22 knockout mice (KO) were injected with AAV9-hSyn1-human miniShank3-V1 (SEQ ID NO: 21) at a high dose level (6.0 x 10 10 vg / mouse or 2.75 x 10 11 After 6 weeks of administration of Shank3Δ4-22 at 100 mg / mouse (vg / mouse), exploratory behavior was significantly increased compared to Shank3Δ4-22 knockout mice (KO) treated with vehicle alone (Figures 2A-2C).

[0268] The reduction in total distance traveled by KO mice did not reach statistical significance in Phase 2 (Figure 2A). 11KO mice treated with AAV9-hSyn1-human miniShank3-V1 (vg) showed a significant increase in distance traveled compared to vehicle-treated KO mice, but not the lowest dose; horizontal activity in the high-dose KO group was also higher than that in vehicle-treated WT mice (Figure 2A). However, when distance traveled was divided into 10-minute time bins (Figure 2C), both KO and WT controls performed similarly during the first 10 minutes of the novel exploration phase, whereas the distance covered after the first 10 minutes of the habituation phase was significantly reduced in KO animals compared to WT controls, consistent with previously published data in the Shank3Δ4-22 model (Drapeau, 2018). Repeated-measures analysis of time bin data revealed that AAV9-hSyn1-human miniShank3-V1 administered at 6.00 × 10 10 vg (P = 0.0493) and 2.75 × 10 11 The results demonstrated a statistically significant improvement in the performance of KO animals treated with the dose level of 2.75 × 10 vg (P < 0.0001). In contrast to the results observed in untreated mice in Phase 1, there was no difference in vertical activity between vehicle-treated WT and KO mice in Phase 2. However, at the high dose (2.75 × 10 11 vg), vertical activity increased significantly (Fig. 2B and Table A). Table A [Table A]

[0269] Rotarod test Mice were tested twice, at 12 and 13 weeks of age.

[0270] Briefly, the Ugo-Basile rotarod was used; it consists of a rotating rod 3 cm in diameter, appropriately machined to provide a surface for the mouse to grasp. Six discs divide the drum into five lanes, each 5.7 cm wide. This allows five mice to be tested simultaneously. When a mouse falls from the cylinder onto the plate below, a mechanism on the plate is activated, recording the animal's endurance time in seconds. The drop height is 16 cm.

[0271] Mice were acclimated to the testing room in their home cages for at least 60 minutes before testing. In a typical trial, mice were placed on a rod rotating at 4 rpm. All mice were placed on the rod, and the speed was increased linearly to a maximum of 40 rpm over 300 seconds. Mice performed four consecutive trials with an interval of approximately 1 minute. The rod was cleaned between trials. This procedure was repeated on two consecutive days. Animals typically "learn" how to stay on the rod on the first day. The average score obtained on the second day was considered to be the actual ability to stay on the rod. The latency to fall was recorded for each mouse.

[0272] Shank3Δ4-22 knockout mice (KO) were injected with AAV9-hSyn1-human miniShank3-V1 (SEQ ID NO: 21) at a high dose level (6.0 x 10 10 vg / mouse or 2.75 x 10 11 After 6 weeks of treatment with Shank3Δ4-22 (vg / mouse), motor function was significantly improved compared to Shank3Δ4-22 knockout mice (KO) treated with vehicle alone (Figure 3).

[0273] The latency to fall in the rotarod assessment was reduced in the KO animals as expected, reaching 6.00 × 10 10 vg / animal and 2.75 × 10 11 There was a trend towards correction with vg / animal, but this was not statistically significant.

[0274] EEG electrode implantation and recording Mice were tested once after completing behavioral testing (13–18 weeks of age).

[0275] Briefly, 10 male mice were implanted per experimental group. Mice were anesthetized with isoflurane inhalation (5% induction, 2–3% maintenance) and administered carprofen subcutaneously at 5 mg / kg. The hair on the back of the head was shaved, and the skin was disinfected with 70% ethanol and chlorexidine. Mice were placed in a stereotaxic apparatus, and a temperature-controlled heat pad provided supplemental warming throughout the procedure. To expose the skull, a vertical incision was made in the skin from just anterior to the eye to just caudal to the ear. Topical analgesic, 0.1% bupivacaine, was applied to the periosteum and exposed tissue around the wound. The skull was cleaned with 3% hydrogen peroxide and 70% ethanol.

[0276] A Pinnacle Technology head mount was glued to the skull, centered on the bregma. A sterile precision drill with a 0.5 mm sterile bit was used to drill four holes through the skull at the four corners of the head mount, but not through the dura or cortex. Head mount screws were inserted through the head mount and into the holes in the skull. The skull was sealed with dental cement, and the implants were embedded in the cement to hold them in place. To avoid exposing the skull or tissue, the skin was sutured with size 6-0 nylon sutures and Gluture tissue adhesive to contact the cement.

[0277] Mice were housed singly after surgery. At least 7 days after surgery, a thin, flexible cable (e.g., tether) was attached to the implant, allowing the mouse to move freely around the cage, and its EEG was recorded by a computer. Recording sessions lasted 72 hours per mouse. During this time, grain was sprinkled on the bottom of the cage, DietGel 76A was added for hydration, and food was available ad libitum.

[0278] EEG analysis EEG recordings were analyzed using Sirenia Sleep Pro and Sirenia Seizure Pro software. Seizure-related EEG events were quantified with Sirenia Seizure Pro, including preictal biphasic spikes, spike-wave discharges (SWDs), and epileptiform discharges. EEG was recorded from both the parietal and motor cortices using a three-electrode system; motor cortex recordings were used for seizure screening, and parietal cortex recordings were used for validation only.

[0279] Recordings from the motor cortex were manually screened by blinded observers trained to detect abnormal changes in the signal with amplitudes approximately twice the baseline amplitude observed during the waking phase. Visual screening was performed using a 1-minute sliding time-span window. Screening was aided by immediate visualization of the power spectrum calculated in selected regions of suspected events. If an event was detected in the motor channel, scorers confirmed that an event was also present in the parietal channel. Only events in the motor channel that matched those in the parietal channel were selected. Scorers flagged events and assigned them to one of four classes.

[0280] 1. Spike: A sudden rise, often followed by a gradual decline.

[0281] 2. SWD: dominant frequency between 6 and 10 Hz. If the power spectrum did not show a significant peak between 6 and 10 Hz, the event was not classified as SWD (see fourth class below).

[0282] 3. Seizure: spike frequency increases during the event, followed by a decrease in amplitude and a slow, regular baseline wave.

[0283] 4. Unclassified event: A seizure event that is distinguishable from the surrounding pattern and appears as an interruption in ongoing activity, but does not fit into categories 1-3 at all.

[0284] Flagged events are saved along with the scorer's analysis record and reviewed by an independent trained scorer to confirm, reclassify, or delete the event. Descriptive statistics of events for each mouse are exported to a tabular data file, including the number and duration of each type of event in the light vs. dark phase.

[0285] Power spectral analysis was performed on the final 24 hours of the 72-hour EEG recording using Sirnia software (400 Hz sample rate, 50 Hz low-pass filtering). Fast Fourier transforms were used to calculate power (voltage squared) in 10-second epochs for the frequency bands delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), and beta (12-30 Hz). Automated power analysis was performed for six default bands (i.e., full, delta, theta, alpha, beta, and gamma). Analysis was performed on the final 48 hours of recording (after 24 hours of acclimation) and covered a 12-hour light and 12-hour dark period.

[0286] Six weeks after administration of AAV9-hSyn1-human miniShank3-V1 (SEQ ID NO: 21), Shank3Δ4-22 knockout mice (KO) showed a significant dose-dependent improvement in recovered delta band power during the 12-hour rest period compared to Shank3Δ4-22 knockout mice (KO) treated with vehicle alone (Figure 4A). AAV9-hSyn1-human miniShank3-V1 (SEQ ID NO: 21) was administered at 6.0 x 10 10 vg / mouse or 2.75 x 10 11 The dose was 100 mg / mouse.

[0287] In Phase 3 (10 months after administration), delta power in vehicle-treated KO mice was significantly reduced compared to WT mice. 10 vg and 2.75 × 10 11Delta power in KO mice treated with vg remained lower than that in WT mice. The dose-dependent increase in delta power observed in Phase 2 was not observed in Phase 3. The small number of mice recorded in Phase 3 may have prevented meaningful conclusions from being drawn regarding delta power (Figure 4B).

[0288] EEG evaluation included both analysis of seizure-like waveforms and power analysis as a measure of recovery sleep. Prior to EEG analysis, representative EEG traces of various seizure types were recorded in a pilot experiment. Shank3 KO mice were injected with pentylenetetrazole, a known epileptogenic compound, and EEG was recorded to observe clinical signs of seizures, including behavioral arrest and convulsions. In this pilot study, EEG abnormalities (solitary spikes, spike-wave discharges, and epileptic seizures) were detected and classified based on representative traces of three types of seizures in animals treated with AAV9-hSyn1-human miniShank3-V1.

[0289] Phase 2 EEG analysis of WT and KO mice at 14 weeks post-injection revealed no significant changes in the frequency of three specific EEG waveform patterns: epileptiform (Fig. 6A), spike-like (Fig. 6B), and spike-like (Fig. 6C). Overall, the incidence of abnormal EEG waveforms was very low, and no abnormal waveforms were observed during 24 hours of recording in multiple mice from each treatment group.

[0290] Similarly, EEG analysis showed no effect on AAV9-hSyn1-human miniShank3-V1-associated seizures in phase 3. Quantification of EEG abnormalities revealed no epileptic seizures or single spikes in mice in phase 3. A small number of spike-wave discharges were detected, but statistical analysis was not possible due to the small number of mice and events. 11 In the vg-treated WT group, the incidence of spike-wave discharges was high in one of four recorded mice ( Fig. 7<em>B ). statistical methods

[0291] Statistical analysis was performed using GraphPad Prism version 8.0. Two-way analysis of variance for vehicle-treated mice of different genotypes was used to identify statistically significant differences between genotypes (genotype effect). Two-way analysis of variance (independent analysis of WT and HOM mice) for mice of the same genotype treated with AAV9-hSyn1-human miniShank3-V1 and vehicle was used to identify statistically significant differences between treatment groups (treatment effect). Sidak's multiple comparison test was used to identify statistically significant differences between specific groups.

[0292] Social Approach (3-chamber) Test Because impairments in social interaction have been previously reported in several models of SHANK3 deficiency, mice were assessed in the three-chamber test in phase 2. For the social approach (three-chamber) test, mice were tested once at 10–11 weeks of age.

[0293] Briefly, the three-chamber mouse apparatus consisted of a Plexiglas arena (40.5 x 60.0 x 22.0 cm) divided into three equal compartments by removable inner walls. Each of the two compartments contained a cylindrical cage measuring 11 cm high and 10 cm deep, with 1 cm spacing between the cage bars. One of the two cages contained a "stranger mouse" of the same sex. Mice were allowed to acclimate to the testing chamber in their home cage for 60 minutes. Subjects were allowed 10 minutes to acclimate to an empty chamber of the apparatus without any unfamiliar mice. Side preference was recorded. The test mouse was removed while the divider was installed, and a new, unfamiliar adult mouse was placed on one side of the cylindrical cage, with a novel object on the other side. A stranger mouse of the same sex as the test mouse, from a strain known to be very calm, was allowed to acclimate to the test cage for up to 10 minutes before the test day. The test mouse was then returned to the cage, and both dividers were removed, allowing the mouse to explore for an additional 10 minutes. Time spent in and entries into each chamber were recorded using automated video tracking with an overhead camera. After testing, mice were placed in a holding cage until all mice in the cage had completed testing, at which point all mice were returned to their home cages. The arena was wiped with 70% EtOH to minimize scent tracking between mice. Data are presented as the duration of contact with the novel object vs. the unfamiliar mouse.

[0294] As previously reported (Drapeau, 2018), Shank3Δ4-22 mice showed no significant genotype differences compared to WT animals, as measured by the time to point to the standard object and the stranger mouse (Figures 5A-5B). As shown in Figure 5C, in the three-chamber assessment, genotype-dependent differences were detected in the time to point to the stranger mouse's nose, a novel endpoint not previously characterized in the Shank3 mouse model, and this endpoint was unaffected by AAV9-hSyn1-human miniShank3-V1 treatment.

[0295] Quantification of spontaneous grooming Mice were tested once at 11-12 weeks of age.

[0296] Briefly, for grooming observations, mice were placed in empty cages (without bedding, food, or water), placed on a table, and videotaped for future video tracking by a trained experimenter. Mice were allowed to acclimate to the testing room in their home cages for a minimum of 1 hour. During the habituation phase, mice were placed in empty cages for 20 minutes. Grooming was not scored during the first 10 minutes (habituation). Grooming behavior was assessed during the final 10 minutes. The entire experiment was videotaped. Trained technicians scored the mice's grooming behavior (cumulative duration and number of grooms over time).

[0297] There was no statistically significant difference in spontaneous grooming between wild-type and Shank3Δ4-22 knockout mice (data not shown).

[0298] Hot Plate Test For the hot plate test, mice were tested once at 11–16 weeks of age.

[0299] The clear Plexiglas cylinder was placed on a hot plate (ie, Harvard Apparatus LE7406 or equivalent model) and the surface temperature was maintained at 52±2°C. Mice were allowed to acclimate to the testing room for a minimum of 30 minutes. Subjects were placed in the cylinder and observed for a maximum exposure time of 30 seconds. The latency to the first behavior, such as paw withdrawal, jumping, hind paw licking, or paw shaking / flapping, was recorded to the nearest 0.1 second using the device's keypad. If no response was observed after 30 seconds, the subject was removed from the hot plate. Subjects remained in a waiting cage until testing of all mice in their home cage was completed.

[0300] There was no statistically significant difference in the results of the hot plate test between wild-type mice and Shank3Δ4-22 knockout mice (data not shown).

[0301] Transgene expression and binding partners To assess transgene expression of miniSHANK3 at the protein level and its effect on synaptic physiological deficits via SHANK3 binding partners (Homer1, glutamate ionotropic receptor AMPA subunit 2 [GluR2], and postsynaptic density protein 95 [PSD95]), synaptic membrane preparations from brain lysates were evaluated by automated Western blotting. A dose-dependent increase in miniSHANK3 protein levels in the brain was observed in both WT and KO animals after AAV9-hSyn1-human miniShank3-V1 treatment compared with vehicle-treated animals (Figure 8). These miniSHANK3 protein levels were 1.20 × 10 10 vg, 6.00 x 10 10 vg, and 2.75 × 10 11 in vg-treated KO animals, corresponding to 21.65%, 49.44%, and 589.20% of WT endogenous SHANK3 protein levels, respectively (Table B). Table B - MiniSHANK3 protein levels relative to WT endogenous SHANK3 levels in the final POC study [Table B]

[0302] To assess the impact of AAV9-hSyn1-human miniShank3-V1 and the resulting miniSHANK3 protein expression on synaptic physiological deficits, we also performed protein quantification of SHANK3 binding partners in synaptic membrane preparations from brain lysates (Figures 9A-9C). Homer1 directly binds to the Homer1-binding region of SHANK3, which together function as a scaffold to stabilize metabotropic glutamate receptors (mGluRs) in the PSD (Xiao, 1998). The presence of synaptic deficits resulting from loss of SHANK3 was supported by Homer1 protein quantification, which demonstrated a significant reduction in genotype in vehicle-treated KO animals compared to vehicle-treated WT animals. In KO animals treated with AAV9-hSyn1-human miniShank3-V1, Homer1 protein levels increased in a dose-dependent manner, reaching 6.00 × 10 10 At the vg dose level, an increase was observed compared to vehicle-treated KO animals, with 2.75 × 10 11 At the vg dose level, physiological levels exceeding those of wild-type Homer1 were achieved. Comparable significant genotypic loss and AAV9-hSyn1-human miniShank3-V1-induced improvement were also observed in GluR2 protein levels, a subunit of the ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor that is stabilized by SHANK3 through its intermediate binding partner, glutamate receptor-interacting protein (GRIP) (Sheng, 2000). Finally, no significant genotypic differences were observed in PSD95, a protein that forms a complex with guanylate kinase-associated protein (GKAP) and helps stabilize and regulate the ionotropic glutamate N-methyl-D-aspartate (NMDA) receptor with the assistance of SHANK3 (Kim, 1997).

[0303] End-of-life care and autopsy Mice from each treatment group are assigned to four necropsy groups. Cardiac puncture and transcardial perfusion (BD and Histo subgroups only: SM, SF per treatment group in Phase 2, 7M and 3F in Phase 3)

[0304] Animals are terminally anesthetized by controlled CO2 anesthesia. After proper anesthesia, mice are fixed in a supine position. The maximum amount of blood that can be obtained is obtained by cardiac puncture, and processed as described herein for CBC and clinical chemistry analysis.

[0305] After cardiac puncture, a 5-6 cm lateral incision is made in the outer skin and abdominal wall just below the rib cage. Using curved, blunt scissors, the diaphragm and rib cage are incised. An 18- to 27-gauge needle is inserted into the left ventricular apex and secured with a hemostat. A small incision is made in the right atrium using standard scissors to drain the perfusate from the circulation. The animal is administered 20 mL of PBS perfusate either manually with a plastic syringe or using a perfusion pump.

[0306] Table 8 below lists the tissues collected and the assays performed. Table 8: Tissue collection table [Table 8-1] [Table 8-2]

[0307] Collection method of vector DNA / RNA / carrier samples (BD samples: 3 males and 2 females in each group in Phase 2, 4 males and 1 female in each group in Phase 3) All frozen tissue samples for vector DNA and RNA ddPCR analysis are shipped on dry ice. Retention samples will be stored at -60°C or below until the end of the project. Tissues will be placed in labeled 2mL screw-cap tubes and frozen on dry ice. Tissues will be divided into three equal portions (one for DNA, one for RNA, and one for retention). The following tissues may not be splittable: ovaries. Tissues will be kept at -60°C or below during storage and transport. Histology / ISH sample collection method (histology samples: 2 males and 3 females per group in Phase 2, 3 males and 2 females per group in Phase 3)

[0308] Tissues from PBS-perfused animals are used. Tissues harvested for fixation are collected and placed in histology cassettes labeled with mouse ID and placed in a 10% neutral buffered formalin sample jar containing at least a 15-fold excess to completely immerse the cassettes. Cassettes from the same group are placed in the same jar. Organ identification is performed by a designated third-party laboratory. Tissues are fixed at room temperature for 48-72 hours, then transferred to 70% ethanol and immediately shipped for processing.

[0309] Collection method for synaptic membrane samples (SMP samples: Phase 2 only, 2 males and 1 female per group) Before craniotomy to remove the brain, perform humane euthanasia by CO2 asphyxiation, divide the brain into two hemispheres, freeze them on dry ice, and maintain them at approximately -60 °C until processing. Thaw each hemisphere and homogenize it in one Dounce homogenizer (approximately 200 mg of tissue). Prepare synaptic membranes.

[0310] Additionally, spleen tissues will be collected from three male and two female mice in the BD group for ELISPOT and ELISA sample analysis, and blood will be processed for clinical chemistry samples.

[0311] In summary, the most reliable POC data for the Shank3Δ4-22 model are 6.00 × 10 10 Our results suggest that doses of ≥ 1000 vg / animal result in improvement of disease-associated phenotypes across multiple neurobehavioral outcomes, measures of functional protein efficacy (indicating improved synaptic stability), and persistent transgene expression.

[0312] Example 3: Biodistribution evaluation in non-human primates (NHPs) of AAV9-hSyn1-human miniShank3-V1, a SHANK3 AAV9 vector delivered by ICV injection, for ASD, Phelan-McDermid syndrome, and other SHANK3 mutation- or deletion-associated conditions. AAV9-hSyn1-human miniShank3-V1 is an investigational AAV9-based gene therapy in preclinical development that delivers a functional version of SHANK3 to treat autism spectrum disorder (ASD), Phelan-McDermid syndrome (PMS), and other neurodevelopmental disorders caused by intragenic mutations or deletions or chromosomal rearrangements at 22q13.3 spanning SHANK3. Here, we used a non-GLP study in nonhuman primates (NHPs) to evaluate the biodistribution of AAV9-hSyn1-human miniShank3-V1 vector genome DNA and RNA transgene expression in the CNS and peripheral organs after unilateral and bilateral intracerebroventricular (ICV) administration.

[0313] The biodistribution of AAV9-hSyn1-human miniShank3-V1 following ICV administration was studied in 24 NHPs (12 males, 12 females) approximately 2–3 years of age. NHPs received a single ICV dose of vehicle or AAV9-hSyn1-human miniShank3-V1 via unilateral or bilateral injections of 1.0 x 10 13 or 1.0 x10 14 A dose of 1000 mg / animal was administered via a bolus injection in a total volume of 2.0 mL. Injection coordinates were determined by MRI, followed by stereotactic administration targeting the lateral ventricle. Animals were followed for a 90-day survival period, and the biodistribution of AAV9-hSyn1-human miniShank3-V1 vector DNA and RNA in CNS and peripheral tissues was analyzed by droplet digital PCR (ddPCR) and corroborated by RNA fluorescence in situ hybridization (FISH).

[0314] Unilateral and bilateral ICV administration procedures and a single dose of AAV9-hSyn1-human miniShank3-V1 were well tolerated. At day 90, animals administered AAV9-hSyn1-human miniShank3-V1 demonstrated potent and widespread rostrocaudal transduction throughout the CNS in a dose-dependent manner compared with vehicle controls. Analysis of AAV9-hSyn1-human miniShank3-V1 vector genomic DNA copies in five major brain regions showed that the highest levels of transduction were observed in the frontal cortex and hippocampus at both dose levels, followed by the cerebellum and spinal cord. Strong transduction of the striatum and thalamus was observed at higher AAV9-hSyn1-human miniShank3-V1 dose levels. These findings were supported and reflected by AAV9-hSyn1-human miniShank3-V1 RNA quantification and RNA FISH analysis, which demonstrated dose-dependent expression in all brain and spinal cord regions evaluated. Furthermore, unilateral and bilateral AAV9-hSyn1-human miniShank3-V1 administration resulted in comparable overall levels of vector DNA and RNA copies throughout the brain, with comparable levels in both the contralateral and ipsilateral sides of the brain. Analysis of vector DNA copies in peripheral tissues confirmed that the vector leaked out of the CNS; however, in contrast to the CNS, significantly lower relative levels of RNA expression were observed, likely a result of the highly neuron-specific activity of the human synapsin promoter, which limits off-target transgene expression.

[0315] These data provided evidence that both unilateral and bilateral ICV administration of AAV9-hSyn1-human miniShank3-V1 resulted in comparable broad biodistribution throughout the CNS at doses predicted to be clinically relevant for patients. Furthermore, AAV9-hSyn1-human miniShank3-V1 RNA expression analysis confirmed robust expression throughout the brain and spinal cord, while limiting off-target expression in peripheral tissues. In summary, these results confirm the use of unilateral ICV administration for further evaluation of AAV9-hSyn1-human miniShank3-V1, a SHANK3 AAV9-based gene therapy targeting SHANK3 mutation- or deletion-associated disorders. Table 9. Mouse and human miniShank3 sequences and vector sequences [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11] Table 9-12 Table 9-13 Table 9-14 Table 9-15 Table 9-16 Table 9-17 Table 9-18 Table 9-19 Table 9-20 Table 9-21 Table 9-22 Table 9-23 References

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[0370] In the claims, articles such as "a," "an," and "the" can mean one or more unless stated otherwise or clear from context. A claim or description containing "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or associated with a particular product or process, unless stated otherwise or clear from context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or associated with a particular product or process. The present disclosure includes embodiments in which more than one or all of the group members are present in, employed in, or associated with a particular product or process.

[0371] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, a claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends from the same base claim. Where elements are presented as a list (e.g., in Markush group format), each subgroup of elements is also disclosed, and any element(s) can be deleted from the group. In general, when a disclosure or an aspect of the disclosure is referred to as comprising certain elements and / or features, it should be understood that a particular embodiment of the disclosure or aspect of the disclosure consists of or consists essentially of such elements and / or features. For the sake of brevity, these embodiments have not been specifically described herein. It should also be noted that the terms "comprise" and "contain" are intended to be open and permit the inclusion of additional elements or steps. Where ranges are presented, the endpoints are included in such ranges unless otherwise specified. Additionally, unless otherwise stated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can take any particular value or subrange within the ranges described in different aspects of this disclosure to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0372] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Furthermore, certain aspects of the present disclosure that constitute prior art may be expressly excluded from any one or more of the claims. Such aspects are deemed to be known to those of skill in the art and may therefore be excluded even if the exclusion is not expressly set forth herein. Certain aspects of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0373] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications can be made to this description without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

1. A method for delivering human miniShank3 protein to the central nervous system (CNS) of a subject in need thereof, the method comprising administering to the CNS of the subject a pharmaceutical composition comprising: (1) a recombinant AAV vector comprising an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette comprises a polynucleotide encoding human miniShank3 protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and 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 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 the amino acid sequence of SEQ ID NO:

18.

3. 3. The method of claim 1 or 2, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO:

26.

4. 4. The method of any one of claims 1 to 3, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the polynucleotide of SEQ ID NO:

28.

5. 5. The method of any one of claims 1 to 4, wherein recombinant AAV virions are delivered to the brain of the subject.

6. 6. The method of any one of claims 1 to 5, wherein the recombinant AAV virions are delivered to the cortex, striatum and / or thalamus of the subject.

7. The method of any one of claims 1 to 6, wherein the recombinant AAV virions are administered by intracerebroventricular (ICV) administration.

8. 8. The method of claim 7, wherein the ICV administration is unilateral administration.

9. 8. 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. 11. The method of claim 10, wherein the human subject is an adult.

12. 11. The method of claim 10, wherein the human subject is not an adult.

13. 11. The method of claim 10, wherein the human subject is no older than 25 years of age.

14. 11. The method of claim 10, wherein the human subject is 10 years of age or younger.

15. 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 neurodevelopmental disorder.

16. 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. 17. The method of any one of claims 1 to 16, wherein the subject exhibits one or more symptoms of ASD.

18. 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. 19. The method of any one of claims 1-18, wherein the subject exhibits one or more of developmental delay, intellectual disability (ID), sleep disorder, hypotonia, speech disorder, or language delay.

20. 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, decreased expression of the Shank3 gene compared to a control subject.

21. 21. The method of claim 20, wherein the control subject is a subject who 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.

22. The method of claim 20 or 21, wherein the 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 the disruption of the Shank3 gene comprises deletion of at least one copy of the Shank3 gene.

24. The method of claim 22, wherein the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

25. Recombinant AAV virions were approximately 1.0 x 10 13 vg ~ approx. 1.0 x 10 14 25. The method of any one of claims 1 to 24, wherein the method is administered at a dose of vg.

26. A method for treating a subject having a neurodevelopmental disorder, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and 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 at least 90% identical or at least 95% identical to SEQ ID NO:

29.

27. A method for treating a subject with autism spectrum disorder (ASD), the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and 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 at least 90% identical or at least 95% identical to SEQ ID NO:

29.

28. A method for treating a subject with Phelan-McDermid syndrome, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and 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 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 the amino acid sequence of SEQ ID NO:

18.

30. 29. The method of any one of claims 26 to 28, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO:

26.

31. 29. The method of any one of claims 26 to 28, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the 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. 33. The method of claim 32, wherein the human subject is an adult.

34. 33. The method of claim 32, wherein the human subject is not an adult.

35. 33. The method of claim 32, wherein the human subject is no older than 25 years of age.

36. 33. The method of claim 32, wherein the human subject is 10 years of age or younger.

37. 37. The method of any one of claims 26 to 36, wherein the composition is delivered to the brain of the subject.

38. 38. The method of claim 37, wherein the composition is delivered to the striatum and / or thalamus of the subject.

39. 39. The method of any one of claims 26 to 38, wherein the composition is administered by intracerebroventricular (ICV) administration.

40. 40. The method of claim 39, wherein the ICV administration is unilateral administration.

41. 40. The method of claim 39, wherein the ICV administration is bilateral.

42. 42. The method of any one of claims 26-41, wherein the subject exhibits one or more of developmental delay, intellectual disability (ID), sleep disorder, hypotonia, speech disorder, or language delay.

43. 43. The method of claim 42, wherein the autism spectrum disorder (ASD) comprises autism disorder.

44. 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, decreased expression of the Shank3 gene compared to a control subject.

45. 45. The method of claim 44, wherein the control subject is a subject who 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.

46. The method of claim 44 or 45, wherein the 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 the disruption of the Shank3 gene comprises deletion of at least one copy of the Shank3 gene.

48. 47. The method of claim 46, wherein the disruption of the Shank3 gene comprises one or more mutations in at least one copy of the Shank3 gene.

49. 49. The method of any one of claims 26 to 48, wherein the subject's sleep efficiency is improved following administration.

50. The composition is about 1.0 x 10 13 vg ~ approx. 1.0 x 10 14 50. The method of any one of claims 26 to 49, wherein the method is administered at a dose of vg.

51. 1. A pharmaceutical composition comprising: Recombinant AAV virions, which include: 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and operably linked to a human Syn promoter and polyA signal sequence; and AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO: 29 10 mM Tris; 1 mM magnesium chloride (MgCl 2 ); 150 mM sodium chloride (NaCl); and 0.02% Poloxamer 188; wherein the pharmaceutical composition has a pH of 8.

0.

52. 52. The pharmaceutical composition of claim 51, wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO:

18.

53. 53. The pharmaceutical composition of claim 51 or 52, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO:

26.

54. 54. The pharmaceutical composition of any one of claims 51 to 53, wherein the ITR comprises a 5' ITR and a 3' ITR, wherein the 5' ITR comprises the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the polynucleotide of SEQ ID NO:

28.

55. 1. 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: Recombinant AAV virions, which include: 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 at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 20 and operably linked to a human Syn promoter and polyA signal sequence; and AAV9 capsid or a capsid having an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO: 29 10 mM Tris; 1 mM magnesium chloride (MgCl 2 ); 150 mM sodium chloride (NaCl); and 0.02% Poloxamer 188; wherein the pharmaceutical composition has a pH of 8.

0.

56. 56. The method of claim 55, wherein the human miniShank3 protein comprises the amino acid sequence of SEQ ID NO:

18.

57. 57. The method of claim 55 or 56, wherein the expression cassette comprises the polynucleotide sequence of SEQ ID NO:

26.

58. 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 the polynucleotide of SEQ ID NO: 27 and the 3' ITR comprises the polynucleotide of SEQ ID NO: 28.

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