SHANK3 gene therapy approach

Abstract

Aspects of the present disclosure relate to non-naturally occurring polynucleotides encoding Shank3 proteins, AAV vectors comprising the polynucleotides, and gene therapy methods.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 066,570, filed August 17, 2020, entitled "SHANK3 Gene Therapy Approach", under 35 U.S.C. § 119(e); the entire disclosure of which is incorporated herein by reference in its entirety. Reference to a Sequence Listing Submitted as a Text File via EFS - Web This application includes a sequence listing submitted in ASCII format via EFS - Web, the entire disclosure of which is incorporated herein by reference. This ASCII copy, created on August 16, 2021, is named B119570108WO00 - SEQ - SXT and is 151,434 bytes in size. Field of the Invention The present disclosure relates to a gene therapy approach for delivering a polynucleotide encoding the Shank3 protein to a subject having, suspected of having, or at risk of having a neurodevelopmental disorder.

Background Art

[0002] Background Deletions and / or mutations involving Shank3 are the primary cause of approximately 0.5 - 1% of all patients with autism spectrum disorder (ASD) and approximately 2% of ASD patients with intellectual disability (ID). However, there is no effective treatment for ASD and / or ID. Several challenges have arisen in developing pharmacological treatments that can modify many of the pathological conditions associated with ASD and ID.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Aspects of the present disclosure relate to the development of an effective gene therapy approach for subjects having a Shank3 mutation.

[0004] Aspects of the present disclosure relate to non-natural polynucleotides encoding Shank3 protein, where the Shank3 protein includes an SH3 domain, a PDZ domain, a Homer binding domain, a cortactin binding domain, and a SAM domain. In some embodiments, the SH3 domain includes at least 90% identity to residues 474 - 525 of SEQ ID NO: 6 or at least 90% identity to residues 473 - 524 of SEQ ID NO: 5. In some embodiments, the PDZ domain includes at least 90% identity to residues 573 - 662 of SEQ ID NO: 6 or at least 90% identity to residues 572 - 661 of SEQ ID NO: 5. In some embodiments, the Homer binding domain includes at least 90% identity to residues 1294 - 1323 of SEQ ID NO: 5 or 6. In some embodiments, the cortactin binding domain includes at least 90% identity to residues 1400 - 1426 of SEQ ID NO: 5 or 6. In some embodiments, the SAM domain includes at least 90% identity to residues 1664 - 1729 of SEQ ID NO: 6 or at least 90% identity to residues 1663 - 1728 of SEQ ID NO: 5. In some embodiments, the polynucleotide is less than 4.7 kb.

[0005] In some embodiments, the polynucleotide further includes a proline-rich region. In some embodiments, the SH3 domain includes residues 474 - 525 of SEQ ID NO: 6 or residues 473 - 524 of SEQ ID NO: 5; the PDZ domain includes residues 573 - 662 of SEQ ID NO: 6 or residues 572 - 661 of SEQ ID NO: 5; the Homer binding domain includes residues 1294 - 1323 of SEQ ID NO: 5 or 6; the cortactin binding domain includes residues 1400 - 1426 of SEQ ID NO: 5 or 6; and / or the SAM domain includes residues 1664 - 1729 of SEQ ID NO: 6 or residues 1663 - 1728 of SEQ ID NO: 5.

[0006] In some embodiments, the polynucleotide contains at least 90% identity with SEQ ID NO: 1 or 2. In some embodiments, the polynucleotide contains SEQ ID NO: 1 or 2.

[0007] In some embodiments, the Shank3 protein encoded by the polynucleotide further comprises an ankyrin repeat domain. In some embodiments, the ankyrin repeat domain comprises at least 90% identity to residues 148-345 of SEQ ID NO: 6, or at least 90% identity to residues 147-313 of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises residues 148-345 of SEQ ID NO: 6 or residues 147-313 of SEQ ID NO: 5.

[0008] In some embodiments, the polynucleotide contains at least 90% identity with SEQ ID NO: 3 or 4. In some embodiments, the polynucleotide contains SEQ ID NO: 3 or 4.

[0009] In some embodiments, the polynucleotides are less than approximately 4.6kb, less than 4.5kb, less than 4.4kb, less than 4.3kb, less than 4.2kb, less than 4.1kb, less than 4.0kb, less than 3.9kb, less than 3.8kb, less than 3.7kb, less than 3.6kb, less than 3.5kb, less than 3.4kb, less than 3.3kb, less than 3.2kb, less than 3.1kb, less than 3.0kb, less than 2.9kb, less than 2.8kb, less than 2.7kb, less than 2.6kb, less than 2.5kb, less than 2.4kb, less than 2.3kb, less than 2.2kb, or less than 2.1kb.

[0010] In some embodiments, the Shank3 protein is less than 65% identical to SEQ ID NO: 5 or 6 throughout its entire length.

[0011] In some embodiments, the Shank3 protein contains an amino acid sequence that is at least 90% identical to one of sequence numbers 17-20. In some embodiments, the Shank3 protein contains an amino acid sequence that includes one of sequence numbers 17-20.

[0012] Further aspects of this disclosure relate to the Shank3 protein encoded by the polypeptide described herein.

[0013] Further aspects of this disclosure relate to vectors comprising polynucleotides as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector comprises a promoter operably ligated to the polynucleotide described herein. In some embodiments, the polynucleotide is adjacent to an AAV reverse terminal repeat (ITR). In some embodiments, the AAV vector comprises a sequence at least 90% identical to SEQ ID NO: 7 or 21 and encodes a protein having Shank3 activity. In some embodiments, the AAV vector comprises the sequence of SEQ ID NO: 7 or 21 and encodes a protein having Shank3 activity. In some embodiments, the AAV vector comprises a sequence at least 90% identical to SEQ ID NO: 2 or 4 and encodes a protein having Shank3 activity. In some embodiments, the AAV vector comprises the sequence of SEQ ID NO: 2 or 4, which encodes a protein having Shank3 activity. In some embodiments, the AAV vector comprises a sequence at least 90% identical to SEQ ID NO: 1 or 3 and encodes a protein having Shank3 activity. In some embodiments, the AAV vector comprises the sequence of SEQ ID NO: 1 or 3, which encodes a protein having Shank3 activity.

[0014] Further aspects of this disclosure relate to AAV particles comprising AAV vectors and capsid proteins, wherein the capsid is of a serotype selected from AAV1, 2, 5, 6, 8, 9, rh10, and PHP.eB. In some embodiments, the serotype is AAV9. In some embodiments, the serotype is AAV10. In some embodiments, the serotype is PHP.eB.

[0015] In some embodiments, the AAV vector further comprises a promoter. In some embodiments, the promoter is a human promoter. In some embodiments, the promoter is hSyn1.

[0016] Further aspects of this disclosure relate to methods of administering the AAV vectors or particles described herein to subjects requiring them. In some embodiments, the subjects are human subjects. In some embodiments, the human subjects are adults. In some embodiments, the human subjects are not adults. In some embodiments, the human subjects are 25 years of age or younger. In some embodiments, the human subjects are 10 years of age or younger. In some embodiments, the subjects have, are suspected of having, or are at risk of having, a neurodevelopmental disorder. In some embodiments, the subjects have, are suspected of having, or are at risk of having autism spectrum disorder (ASD). In some embodiments, the subjects exhibit one or more symptoms of ASD. In some embodiments, the subjects have, are suspected of having, or are at risk of having Phelan McDermid syndrome. In some embodiments, the subjects exhibit one or more of the following: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, speech deprivation, or language delay.

[0017] Further aspects of this disclosure relate to methods for treating subjects with neurodevelopmental disorders. In some embodiments, this disclosure relates to methods for treating subjects with autism spectrum disorder (ASD). In some embodiments, this disclosure relates to methods for treating subjects with Phelan McDermid syndrome. In some embodiments, the treatment method involves administering to a subject an effective amount of a composition comprising an AAV vector containing a polynucleotide encoding the Shank3 protein. In some embodiments, the composition is contained in a pharmaceutically acceptable carrier.

[0018] In some embodiments, the AAV vector is delivered to the brain of the target. In some embodiments, the AAV vector is delivered to the cortex, striatum, and / or thalamus of the target.

[0019] In some embodiments, subjects have, are suspected of having, or are at risk of having reduced Shank3 gene expression compared to control subjects. In some embodiments, control subjects do not have, are not suspected of having, or are not at risk of having neurodevelopmental disorders, autism spectrum disorder (ASD), and / or Phelan McDermid syndrome. In some embodiments, reduced Shank3 gene expression is caused by the disruption of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene includes a deletion in at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene includes one or more mutations within at least one copy of the Shank3 gene.

[0020] Further aspects of this disclosure relate to MiniShank3 proteins comprising sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 17-20. In some embodiments, the MiniShank3 protein comprises any one of SEQ ID NOs: 17-20. [Brief explanation of the drawing]

[0021] [Figure 1] Figures 1A-1B show increased skin lesions and grooming in Shank3B- / - mice.

[0022] [Figure 2AB] Figures 2A-2C show that Shank3B mutant mice exhibited impaired social interaction. "Stranger 1" in Figure 2A represents a partner tested for social contact behavior with the test animals. "Stranger 2" in Figure 2A represents a newly introduced social partner previously placed in an empty wired cage. S1: Stranger 1; S2: Stranger 2; E: Empty cage. [Figure 2C] Figures 2A-2C show that Shank3B mutant mice exhibited impaired social interaction.

[0023] [Figure 3A] Figures 3A-3C show that Shank3B- / - mice exhibited changes in molecular composition in striatal PSD. [Figure 3BC] Figures 3A-3C show that Shank3B- / - mice exhibited changes in molecular composition in striatal PSD.

[0024] [Figure 4AB] Figures 4A–4E show corticostriate synaptic defects in Shank3B mutant mice. PPR: Paired pulse ratio. [Figure 4CD] Figures 4A–4E show corticostriate synaptic defects in Shank3B mutant mice. PPR: Paired pulse ratio. [Figure 4E] Figures 4A–4E show corticostriate synaptic defects in Shank3B mutant mice. PPR: Paired pulse ratio.

[0025] [Figure 5]Figures 5A–5C show the design of miniShank3-v1. Figure 5A shows the protein domain diagram of full-length Shank3. Figure 5B shows a schematic diagram of miniShank3-v1 provided in this disclosure. Figure 5C shows a schematic diagram of GFP-tagged miniShank3-v1 having the human synapsin 1 promoter (hSyn-1). ANK, ankyrin repeat; SH3, src homology 3 domain; PDZ, PDZ domain; Pro, proline-rich region; HBD, Homer-binding domain; CBD, cortactin-binding domain; SAM, sterile alpha motif.

[0026] [Figure 6] Figures 6A–6C show the design of miniShank3-v2. Figure 6A shows the protein domain diagram of full-length Shank3. Figure 6B shows a schematic diagram of miniShank3-v2 provided in this disclosure. Figure 6C shows a schematic diagram of GFP-tagged miniShank3-v2 having the human synapsin 1 promoter (hSyn-1). ANK, ankyrin repeat; SH3, src homology 3 domain; PDZ, PDZ domain; Pro, proline-rich region; HBD, Homer-binding domain; CBD, cortactin-binding domain; SAM, sterile alpha motif.

[0027] [Figure 7AB] Figures 7A–7H show that GFP-miniShank3-v1 was localized to synapses. hSyn1-GFP-miniShank3-v1 was transfected into striatal medium spiny neurons (MSNs) in a corticostriate coculture. Figure 7A shows that GFP-miniShank3-v1 was expressed in MSNs. Figures 7B–7C show the same culture stained with PSD95 to mark synapses (Figure 7B) and stained with MAP2 to show dendrites (Figure 7C). [Figure 7CD]Figures 7A–7H show that GFP-miniShank3-v1 was localized to synapses. hSyn1-GFP-miniShank3-v1 was transfected into striatal medium spiny neurons (MSNs) in a corticostriate coculture. Figures 7B–7C show the same culture stained with PSD95 to mark synapses (Figure 7B), and stained with MAP2 to show dendrites (Figure 7C). Figure 7D shows a merged image of Figures 7A–7C. [Figure 7EFG] Figures 7A–7H show that GFP-miniShank3-v1 was localized to synapses. hSyn1-GFP-miniShank3-v1 was transfected into striatal medium spiny neurons (MSNs) in a corticostriate coculture. Figures 7E–7H are high-magnification images from Figures 7A–7D, showing the precise localization of GFP-miniShank3-v1 (Figure 7E) with PSD95 (Figure 7F) to dendrites (Figure 7G) and dendritic spines (Figure 7H) in the merged image (Figure 7H). [Figure 7H] Figures 7A–7H show that GFP-miniShank3-v1 was localized to synapses. hSyn1-GFP-miniShank3-v1 was transfected into striatal medium spiny neurons (MSNs) in a corticostriate coculture. Figures 7E–7H are high-magnification images from Figures 7A–7D, showing the precise localization of GFP-miniShank3-v1 (Figure 7E) with PSD95 (Figure 7F) to dendrites (Figure 7G) and dendritic spines (Figure 7H) in the merged image (Figure 7H).

[0028] [Figure 8] Figure 8 shows efficient expression of GFP-miniShank3-v1 after facial vein injection on day 0 postnatal. P0 mice were injected with 6.42E+11 units of viral genome (vg) per mouse. Mouse brains were collected two months after AAV injection, and GFP expression was observed in brain sections.

[0029] [Figure 9AB]Figures 9A–9F show that a single intravenous injection of AAV-miniShank3-v1 in phase P0 rescued PSD protein deficiency in Shank3-deficient mice. Figure 9A shows the timeline and experimental groups of the AAV-mediated miniShank3-v1 gene therapy experiment. Figure 9B provides Western blots showing miniShank3 expression in striatal synaptosome plasma membrane (SPM) fractions prepared from: wild-type mice (WT) injected with AAV-GFP, InsG3680+ / + mice (mutants) injected with AAV-GFP, InsG3680+ / + mice (miniShank3) injected with AAV-GFP-miniShank3-v1, and lysates from HEK293 cells expressing the cDNA plasmid encoding GFP-miniShank3-v1 (HEK 293-a) and HEK293 cells expressing the cDNA plasmid encoding GFP-p2A miniShank3-v1 (HEK 293-b), which were detected using an anti-Shank3 antibody. [Figure 9C] Figures 9A–9F show that a single intravenous injection of AAV-miniShank3-v1 in P0 rescued PSD protein deficiency in Shank3-deficient mice. Figures 9C and 9E show representative blots of proteins detected by specific antibodies in the striatal (Figure 9C) and cortical (Figure 9E) SPM fractions from the following AAV-injected mice: WT mice injected with AAV-GFP (WT), InsG3680+ / + mice injected with AAV-GFP (mutant), and InsG3680+ / + mice injected with AAV-GFP-miniShank3-v1 (miniShank3). [Figure 9D]Figures 9A–9F show that a single intravenous injection of AAV-miniShank3-v1 at P0 rescued PSD protein deficiency in Shank3-deficient mice. Figures 9D and 9F show quantification of relative protein levels normalized to tubulin protein expression from striatal (Figure 9D) and cortical (Figure 9F) SPMs. (n=4 samples per protein per genotype, each n being pooled tissue from 2 mice). Note that in the Minishank3 group, mutant PSD protein levels were restored to WT levels. *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with Bonferroni post-hoc test (Figures 9D and 9F). [Figure 9E] Figures 9A–9F show that a single intravenous injection of AAV-miniShank3-v1 in P0 rescued PSD protein deficiency in Shank3-deficient mice. Figures 9C and 9E show representative blots of proteins detected by specific antibodies in the striatal (Figure 9C) and cortical (Figure 9E) SPM fractions from the following AAV-injected mice: WT mice injected with AAV-GFP (WT), InsG3680+ / + mice injected with AAV-GFP (mutant), and InsG3680+ / + mice injected with AAV-GFP-miniShank3-v1 (miniShank3). [Figure 9F] Figures 9A–9F show that a single intravenous injection of AAV-miniShank3-v1 at P0 rescued PSD protein deficiency in Shank3-deficient mice. Figures 9D and 9F show quantification of relative protein levels normalized to tubulin protein expression from striatal (Figure 9D) and cortical (Figure 9F) SPMs. (n=4 samples per protein per genotype, each n being pooled tissue from 2 mice). Note that in the Minishank3 group, mutant PSD protein levels were restored to WT levels. *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with Bonferroni post-hoc test (Figures 9D and 9F).

[0030] [Figure 10] Figures 10A–10B show that a single intravenous injection of AAV-miniShank3-v1 at P0 rescued striatal synaptic defects in Shank3-deficient mice. Figure 10A shows that the reduced amplitude of striatal pop spikes in mutant mice was rescued in animals injected with miniShank3-v1. Figure 10B shows representative corticostriate pop spike traces from mice treated as instructed.

[0031] [Figure 11AB] Figures 11A–11E show that systemic delivery of miniShank3-v1 at P0 rescued behavioral disorders in Shank3-deficient mice. Figure 11A shows that in a social interaction test, mutant mice showed no preference for stranger mice (S) over novel objects (O) compared to controls. This behavior was rescued by miniShank3 treatment. Figure 11B shows that miniShank3 treatment rescued the reduced motor activity (reduced distance traveled in an open field test) of mutant mice to wild-type levels. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 11A–11D), two-way ANOVA with Bonferroni post-hoc test (Figure 11E). Data are presented as mean ± SEM (a: n=16 WT+GFP, n=12 mutant+GFP and n=14 mutant+miniShank3; b~e: n=26 WT+GFP, n=29 mutant+GFP and n=19 mutant+miniShank3). [Figure 11CD]Figures 11A–11E show that systemic delivery of miniShank3-v1 at P0 rescued behavioral disorders in Shank3-deficient mice. Figure 11C shows that reduced exploratory behavior (rearing time) in Shank3 mutant mice recovered to WT levels in the miniShank3-treated group. Figure 11D shows that anxiety-like behavior (reduced open-arm time in the elevated zero maze test) in Shank3 mutant mice was also rescued in the miniShank3-treated group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 11A–11D), two-way ANOVA with Bonferroni post-hoc test (Figure 11E). Data are presented as mean ± SEM (a: n=16 WT+GFP, n=12 mutant+GFP and n=14 mutant+miniShank3; b~e: n=26 WT+GFP, n=29 mutant+GFP and n=19 mutant+miniShank3). [Figure 11E] Figures 11A–11E show that systemic delivery of miniShank3-v1 at P0 rescued behavioral disorders in Shank3-deficient mice. Figure 11E shows that the miniShank3-treated group showed a trend toward improvement in motor skills (rotarod test) compared to the Shank3 mutant group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 11A–11D), two-way ANOVA with Bonferroni post-hoc test (Figure 11E). Data are presented as mean ± SEM (a: n=16 WT+GFP, n=12 mutant+GFP and n=14 mutant+miniShank3; b–e: n=26 WT+GFP, n=29 mutant+GFP and n=19 mutant+miniShank3).

[0032] [Figure 12A]Figures 12A–12F show that miniShank3 treatment at postnatal day 28 (P28) selectively rescued social and motor impairments in Shank3 mutant mice. Figure 12A shows the time spent in close social interaction between object (O) and foreign mouse (S) in the phase II social preference assay. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 12A, 12B, 12E, and 12F), two-way ANOVA with Bonferroni post-hoc test (Figures 12C and 12D). Data are presented as mean ± SEM (Figure 12A: WT for n=21, Mut for n=23, and MiniShank3 for n=18; Figure 12B: WT for n=14, Mut for n=20, and MiniShank3 for n=13; Figure 12C: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12D: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12E: WT for n=14, Mut for n=20, and MiniShank3 for n=14; Figure 12F: WT for n=15, Mut for n=18, and MiniShank3 for n=17). WT represents wild type; mut represents Shank3 mutant; MiniShank3 represents Shank3 mutant + miniShank3 treatment. [Figure 12BC]Figures 12A–12F show that miniShank3 treatment at postnatal day 28 (P28) selectively rescued social and motor impairments in Shank3 mutant mice. Figure 12B shows the total distance traveled in the open field test. Figure 12C shows the assessment of motor learning, evaluated via time to fall in the rotarod test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 12A, 12B, 12E, and 12F), two-way ANOVA with Bonferroni post-hoc test (Figures 12C and 12D). Data are presented as mean ± SEM (Figure 12A: WT for n=21, Mut for n=23, and MiniShank3 for n=18; Figure 12B: WT for n=14, Mut for n=20, and MiniShank3 for n=13; Figure 12C: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12D: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12E: WT for n=14, Mut for n=20, and MiniShank3 for n=14; Figure 12F: WT for n=15, Mut for n=18, and MiniShank3 for n=17). WT represents wild type; mut represents Shank3 mutant; MiniShank3 represents Shank3 mutant + miniShank3 treatment. [Figure 12DE]Figures 12A–12F show that miniShank3 treatment at postnatal day 28 (P28) selectively rescued social and motor impairments in Shank3 mutant mice. Figure 12D shows the assessment of motor coordination, evaluated through time to fall in the rotarod test. Figure 12E shows the quantification of time spent on the open arm of an elevated zero maze to assess anxiety-like behavior. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 12A, 12B, 12E, and 12F), two-way ANOVA with Bonferroni post-hoc test (Figures 12C and 12D). Data are presented as mean ± SEM (Figure 12A: WT for n=21, Mut for n=23, and MiniShank3 for n=18; Figure 12B: WT for n=14, Mut for n=20, and MiniShank3 for n=13; Figure 12C: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12D: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12E: WT for n=14, Mut for n=20, and MiniShank3 for n=14; Figure 12F: WT for n=15, Mut for n=18, and MiniShank3 for n=17). WT represents wild type; mut represents Shank3 mutant; MiniShank3 represents Shank3 mutant + miniShank3 treatment. [Figure 12F]Figures 12A–12F show that miniShank3 treatment at postnatal day 28 (P28) selectively rescued Shank3 mutant mice from social and motor impairments. Figure 12F shows the evaluation of grooming time in a 2-hour video recording. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 12A, 12B, 12E, and 12F), two-way ANOVA with Bonferroni post-hoc test (Figures 12C and 12D). Data are presented as mean ± SEM (Figure 12A: WT for n=21, Mut for n=23, and MiniShank3 for n=18; Figure 12B: WT for n=14, Mut for n=20, and MiniShank3 for n=13; Figure 12C: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12D: WT for n=17, Mut for n=14, and MiniShank3 for n=10; Figure 12E: WT for n=14, Mut for n=20, and MiniShank3 for n=14; Figure 12F: WT for n=15, Mut for n=18, and MiniShank3 for n=17). WT represents wild type; mut represents Shank3 mutant; MiniShank3 represents Shank3 mutant + miniShank3 treatment.

[0033] [Figure 13A]Figures 13A–13I show that postnatal (P7) miniShank3 treatment completely rescued all behavioral and sleep disorders in Shank3 mutant mice. Figure 13A shows the time spent in close social interaction between object (O) and stranger mice (S) in a Phase II social preference assay. Figure 13B shows the evaluation of grooming time in a 2-hour video recording. Figure 13C shows the quantification of time spent in the open arm of an elevated zero maze to evaluate anxiety-like behavior. Figures 13D and 13E show the total distance traveled in the open field test. Figure 13F shows the evaluation of motor learning and coordination, assessed through time to fall in the rotarod test. Figure 13G shows the quantification of NREM sleep duration to evaluate sleep behavior in Shank3 mutant mice. Figure 13H shows the quantification of NREM sleep bout length to evaluate sleep behavior in Shank3 mutant mice. Figure 13I shows the quantification of delta power for evaluating sleep behavior in Shank3 mutant mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 13A, 13B, 13C, 13D, 13G, 13H, and 13I), two-way ANOVA with Bonferroni post-hoc test (Figures 13E and 13F). Data are presented as mean ± SEM (Figure 13A: WT of n=16, Mut of n=9, and MiniShank3 of n=14; Figure 13B: WT of n=18, Mut of n=12, and MiniShank3 of n=14; Figure 13C: WT of n=10, Mut of n=10, and MiniShank3 of n=10; Figure 13D: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13E: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13F: WT of n=17, Mut of n=15, and MiniShank3 of n=17; Figures 13G-13I: WT of n=9, Mut of n=8, and MiniShank3 of n=8). WT indicates wild type. Mut indicates Shank3 mutant.MiniShank3 represents the Shank3 mutant + miniShank3 treatment. [Figure 13BC] Figures 13A–13I show that postnatal (P7) miniShank3 treatment completely rescued all behavioral and sleep disorders in Shank3 mutant mice. Figure 13B shows an evaluation of grooming time in a 2-hour video recording. Figure 13C shows a quantification of time spent in the open arm of an elevated zero maze to evaluate anxiety-like behavior. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 13A, 13B, 13C, 13D, 13G, 13H, and 13I), two-way ANOVA with Bonferroni post-hoc test (Figures 13E and 13F). Data are presented as mean ± SEM (Figure 13A: WT of n=16, Mut of n=9, and MiniShank3 of n=14; Figure 13B: WT of n=18, Mut of n=12, and MiniShank3 of n=14; Figure 13C: WT of n=10, Mut of n=10, and MiniShank3 of n=10; Figure 13D: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13E: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13F: WT of n=17, Mut of n=15, and MiniShank3 of n=17; Figures 13G-13I: WT of n=9, Mut of n=8, and MiniShank3 of n=8). WT indicates wild type. Mut indicates Shank3 mutant. MiniShank3 represents the Shank3 mutant + miniShank3 treatment. [Figure 13DE]Figures 13A–13I show that postnatal (P7) miniShank3 treatment completely rescued all behavioral and sleep disorders in Shank3 mutant mice. Figures 13D and 13E show the total distance traveled in the open field study. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 13A, 13B, 13C, 13D, 13G, 13H, and 13I), two-way ANOVA with Bonferroni post-hoc test (Figures 13E and 13F). Data are presented as mean ± SEM (Figure 13A: WT of n=16, Mut of n=9, and MiniShank3 of n=14; Figure 13B: WT of n=18, Mut of n=12, and MiniShank3 of n=14; Figure 13C: WT of n=10, Mut of n=10, and MiniShank3 of n=10; Figure 13D: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13E: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13F: WT of n=17, Mut of n=15, and MiniShank3 of n=17; Figures 13G-13I: WT of n=9, Mut of n=8, and MiniShank3 of n=8). WT indicates wild type. Mut indicates Shank3 mutant. MiniShank3 represents the Shank3 mutant + miniShank3 treatment. [Figure 13FG]Figures 13A–13I show that postnatal (P7) miniShank3 treatment completely rescued all behavioral and sleep disorders in Shank3 mutant mice. Figure 13F shows the assessment of motor learning and coordination, evaluated via time to fall in the rotarod test. Figure 13G shows the quantification of NREM sleep duration to assess sleep behavior in Shank3 mutant mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 13A, 13B, 13C, 13D, 13G, 13H, and 13I), two-way ANOVA with Bonferroni post-hoc test (Figures 13E and 13F). Data are presented as mean ± SEM (Figure 13A: WT of n=16, Mut of n=9, and MiniShank3 of n=14; Figure 13B: WT of n=18, Mut of n=12, and MiniShank3 of n=14; Figure 13C: WT of n=10, Mut of n=10, and MiniShank3 of n=10; Figure 13D: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13E: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13F: WT of n=17, Mut of n=15, and MiniShank3 of n=17; Figures 13G-13I: WT of n=9, Mut of n=8, and MiniShank3 of n=8). WT indicates wild type. Mut indicates Shank3 mutant. MiniShank3 represents the Shank3 mutant + miniShank3 treatment. [Figure 13HI]Figures 13A–13I show that postnatal (P7) miniShank3 treatment completely rescued all behavioral and sleep disorders in Shank3 mutant mice. Figure 13H shows the quantification of NREM sleep bout length for evaluating sleep behavior in Shank3 mutant mice. Figure 13I shows the quantification of delta power for evaluating sleep behavior in Shank3 mutant mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test (Figures 13A, 13B, 13C, 13D, 13G, 13H, and 13I), two-way ANOVA with Bonferroni post-hoc test (Figures 13E and 13F). Data are presented as mean ± SEM (Figure 13A: WT of n=16, Mut of n=9, and MiniShank3 of n=14; Figure 13B: WT of n=18, Mut of n=12, and MiniShank3 of n=14; Figure 13C: WT of n=10, Mut of n=10, and MiniShank3 of n=10; Figure 13D: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13E: WT of n=18, Mut of n=14, and MiniShank3 of n=18; Figure 13F: WT of n=17, Mut of n=15, and MiniShank3 of n=17; Figures 13G-13I: WT of n=9, Mut of n=8, and MiniShank3 of n=8). WT indicates wild type. Mut indicates Shank3 mutant. MiniShank3 represents the Shank3 mutant + miniShank3 treatment.

[0034] [Figure 14]Figures 14A–14B show that postnatal (P7) miniShank3 treatment did not induce seizure activity in Shank3 mutants. Figure 14A shows representative EEG traces of wild-type animals, mutants injected with the control virus, mutants injected with miniShank3, or Scn2a mutants. Figure 14B shows the quantification of spike-and-slow-wave firing (SWD) observed in the four experimental groups shown in panel a (data are shown as mean ± SEM). ***One-way ANOVA with p<0.001 and Bonferroni post-hoc test.

[0035] [Figure 15] Figure 15 shows a schematic diagram illustrating the plasmid construction of the human miniShank3 gene containing the hSyn1 promoter. [Modes for carrying out the invention]

[0036] Detailed explanation Aspects of this disclosure relate to gene therapy approaches for treating neurodevelopmental disorders. Examples demonstrate a non-natural polynucleotide encoding the Shank3 protein, which can be expressed in a gene delivery vector and administered to a target. The gene therapy strategies disclosed herein use an AAV system to deliver a functional copy of the Shank3 gene to brain cells to restore cellular function.

[0037] SHANK3 encodes a synaptic scaffold protein at excitatory glutamatergic synapses, regulating the recruitment of signaling molecules and the assembly of high-molecular-weight postsynaptic protein complexes; this is crucial for proper synaptic development and function. SHANK3 deletion is a major cause of central neurodevelopmental and neurobehavioral disorders in Phelan McDermid syndrome. Human genetic studies have also identified SHANK3 mutations as a major cause in approximately 1% of autism spectrum disorder (ASD) cases. Individuals with Phelan McDermid syndrome and other individuals with SHANK3 mutations often exhibit a variety of co-occurring traits, including developmental delay, sleep disturbances, hypotonia, speech deficiency or severe language delay, and characteristic features of ASD. Currently, there are no effective treatments for ASD.

[0038] The association between ASD and Shank3 has provided a direct link between synaptic dysfunction and pathophysiology in ASD. Animal models bridge the gap between human genetics of ASD and the neuropathology 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 SHANK3 loss. For example, disruption of Shank3 in mouse models resulted in synaptic defects, impaired social interaction, motor impairment, repetitive grooming, and increased anxiety levels. Since 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 exhibit predictive validity because synaptic defects and behavioral abnormalities are reversible when Shank3 is restored. Therefore, gene replacement is a suitable therapeutic strategy for this monogenetic disorder.

[0039] Novel recombinant adeno-associated viruses (rAAVs) represent a promising gene delivery platform due to their broad tissue affinity, low immunogenicity, highly efficient and sustained gene delivery, and clinically proven safety record. However, Shank3 is known in the art to be a large protein with a coding sequence of approximately 5.7 kb, which exceeds the packaging capacity of AAV vectors. The inventors of this application have found that certain regions of the Shank3 protein are not critical to the protein's function and have therefore designed heterologous Shank3 expression constructs having significantly smaller coding sequences (approximately 2.1 kb to approximately 3.1 kb); these encode versions of the Shank3 protein from which certain non-essential regions have been removed. The resulting miniaturized Shank3 proteins described herein can be delivered by vectors such as AAVs. Surprisingly, the inventors of this application have discovered that miniaturized Shank3 protein can restore defective function caused by deletion or mutation in the gene encoding the Shank3 protein in a mouse model, and thus have the potential to rescue abnormalities caused by diseases associated with Shank3 mutations or deletions. This is in contrast to methods known in the art that focus on repairing or improving partial fragments of the Shank3 protein, but fail to restore the function of the Shank protein. Accordingly, this disclosure relates to methods and compositions for treating neurodevelopmental disorders by restoring Shank3 activity using miniaturized Shank3 protein ("MiniShank3"). Shank Protein

[0040] The Shank family of proteins (e.g., Shank1, Shank2, and Shank3) are major scaffolding proteins that ligate and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five major domain regions: the N-terminal ankyrin repeat, the SH3 domain, the PDZ domain, the proline-rich region, and the C-terminal SAM domain. Through these functional domains, Shank proteins interact with many postsynaptic thickening (PSD) proteins. While not wishing to be bound by any theory, Shank proteins can bind to SAPAP, which in turn binds to PSD95 to form the PSD95 / SAPAP / Shank postsynaptic complex. Collectively, these multi-domain proteins have been proposed to form a crucial scaffold and regulate the assembly of macromolecular postsynaptic signaling complexes at glutamatergic synapses. These complexes have been shown to play a vital role in the targeting, fixation, and dynamic regulation of the synaptic localization of neurotransmitter receptors and signaling molecules. In another example, the Shank family of proteins are linked to the mGluR pathway through their binding to Homer.

[0041] Due to its association with actin-binding proteins, Shank also plays a major role in spine development. Transfection of Shank3 has been found to be sufficient to induce functional dendritic spine synapses in cultured aspiny cerebellar granule cells, demonstrating its role in spine induction. α (Longest Shank3 isoform), Shank3 β , and Shank3 γIt has three major isoforms, including Shank3. 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 is supported by the finding that overexpression of Shank1 enlarges dendritic spines already present in cultured hippocampal neurons. Furthermore, Shank1 mutant mice have been reported to have smaller dendritic spines and weaker synaptic transmission.

[0042] In some embodiments, this disclosure relates to a Shank protein capable of restoring synaptic activity in subjects whose Shank protein activity has been disrupted. In some embodiments, the disruption of Shank protein activity is present in subjects having neurodevelopmental disorders, autism spectrum disorder (ASD), and / or Phelan McDiarmid syndrome. In some embodiments, the Shank protein relevant to this disclosure is the Shank1 protein. In some embodiments, this disclosure relates to the expression of a polynucleotide encoding Shank1 or a variant of Shank1 in subjects that require it. In some embodiments, the Shank protein of this disclosure is the Shank2 protein. In some embodiments, this disclosure relates to the expression of a polynucleotide encoding Shank2 or a variant of Shank2 in subjects that require it. In some embodiments, the Shank protein of this disclosure is the Shank3 protein. In some embodiments, this disclosure relates to the expression of a polynucleotide encoding Shank3 or a variant of Shank3 in subjects that require it. It should be understood that the Shank proteins relevant to this disclosure may include any Shank protein, including its variants or fragments, that function as scaffolding proteins at the synapses of excitatory neurons.

[0043] Further disclosed herein are polynucleotides encoding Shank proteins (Shank1, Shank2, and Shank3) for use in gene therapy.

[0044] The full-length mouse protein sequence of Shank3, corresponding to GenBank accession number BAE16756.1, is provided by SEQ ID NO: 5:

[0045] In some embodiments, the full-length mouse protein sequence of Shank3 corresponding to SEQ ID NO: 5 is encoded by the nucleic acid sequence corresponding to GenBank accession number NM_021423, provided by SEQ ID NO: 15:

[0046]

[0047] The full-length human Shank3 protein sequence corresponding to GenBank accession number Q9BYB0.3 is provided by SEQ ID NO: 6:

[0048] In some embodiments, the full-length human Shank3 protein sequence corresponding to SEQ ID NO: 6 is encoded by the nucleic acid sequence corresponding to GenBank accession number NM_001372044, provided by SEQ ID NO: 16:

[0049]

[0050] The full-length Shank3 protein is encoded by a gene containing multiple domains and measuring approximately 5.2 kb. Due to its size, delivering full-length Shank3 to target tissues or cells via AAV vectors is difficult. The inventors of this application have discovered that specific domains can be removed or cleaved from the full-length Shank3 protein to generate MiniShank3, which is effective for restoring Shank3 activity in excitatory neurons (Figures 5B and 6B). Shank proteins encoded by the polynucleotides described herein (e.g., Shank3 protein) can be miniaturized to form truncated variants of the native full-length Shank3 protein. As disclosed herein, the miniaturized Shank3 protein, or the DNA construct encoding the miniaturized Shank3 protein, is interchangeably referred to as "miniShank3" or "MiniShank3".

[0051] In some embodiments, the Shank3 protein disclosed herein is expressed as a miniaturized Shank3 DNA construct. 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. In some embodiments, the Shank3 protein disclosed herein is encoded by non-native polynucleotides.

[0052] The Shank3 proteins encoded by the polynucleotides described herein may comprise one or more protein domains. For example, a Shank3 protein may comprise one or more of the following: an SH3 domain, a PDZ domain, a Homer-binding domain, a cortactin domain, a SAM domain, and / or an ankyrin repeat domain.

[0053] In some embodiments, the SH3 domain contains 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 with respect to residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5, or 100% identity including all values ​​in between. In some embodiments, the SH3 domain contains at least 90% identity with residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain contains at least 90% identity with residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain includes residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain includes residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain can contain any percentage identity to residues 474-525 of SEQ ID NO: 6 suitable for constructing MiniShank3. In some embodiments, the SH3 domain can contain any percentage identity to residues 473-524 of SEQ ID NO: 5 suitable for constructing MiniShank3.

[0054] In some embodiments, the PDZ domain contains 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 with respect to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5, or 100% identity with respect to all values ​​in between. In some embodiments, the PDZ domain contains at least 90% identity with residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain contains at least 90% identity with residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain includes residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain includes residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain can contain any percentage identity to residues 573-662 of SEQ ID NO: 6 suitable for constructing MiniShank3. In some embodiments, the PDZ domain can contain any percentage identity to residues 572-661 of SEQ ID NO: 5 suitable for constructing MiniShank3.

[0055] In some embodiments, the Homer-binding domain contains 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 with respect to residues 1294–1323 of SEQ ID NO: 5 or 6, or 100% identity including all values ​​in between. In some embodiments, the Homer domain contains at least 90% identity with residues 1294–1323 of SEQ ID NO: 5. In some embodiments, the Homer domain contains at least 90% identity with residues 1294–1323 of SEQ ID NO: 6. In some embodiments, the Homer domain includes residues 1294–1323 of SEQ ID NO: 5 or 6. In some embodiments, the Homer domain may contain any percentage identity to residues 1294–1323 of SEQ ID NO: 5 or 6 suitable for constructing MiniShank3.

[0056] In some embodiments, the cortactin-binding domain contains 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 with respect to residues 1400–1426 of SEQ ID NO: 5 or 6, or 100% identity including all values ​​in between. In some embodiments, the cortactin-binding domain contains at least 90% identity with residues 1400–1426 of SEQ ID NO: 5. In some embodiments, the cortactin-binding domain contains at least 90% identity with residues 1400–1426 of SEQ ID NO: 6. In some embodiments, the cortactin-binding domain includes residues 1400–1426 of SEQ ID NO: 5 or 6. In some embodiments, the cortactin-binding domain may contain any percentage identity to residues 1400–1426 of SEQ ID NO: 5 or 6 suitable for the construction of MiniShank3.

[0057] In some embodiments, the SAM domain contains 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 with respect to residues 1664-1729 of SEQ ID NO: 6 or residues 1663-1728 of SEQ ID NO: 5, or 100% identity including all values ​​in between. In some embodiments, the SAM-binding domain contains at least 90% identity with residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM-binding domain contains at least 90% identity with residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM domain includes residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM domain includes residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM-binding domain can contain any percentage identity to residues 1664-1729 of SEQ ID NO: 6 suitable for the construction of MiniShank3. In some embodiments, the SAM-binding domain can contain any percentage identity to residues 1663-1728 of SEQ ID NO: 5 suitable for the construction of MiniShank3.

[0058] In some embodiments, the ankyrin repeat domain contains 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 with respect to residues 148-345 of SEQ ID NO: 6 or residues 147-313 of SEQ ID NO: 5, or 100% identity with respect to all values ​​in between. In some embodiments, the ankyrin repeat domain contains at least 90% identity with residues 148-345 of SEQ ID NO: 6. In some embodiments, the ankyrin repeat domain contains at least 90% identity with residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain can contain any percentage identity to residues 148-345 of SEQ ID NO: 6, which is suitable for constructing MiniShank3. In some embodiments, the ankyrin repeat domain can contain any percentage identity to residues 147-313 of SEQ ID NO: 5, which is suitable for constructing MiniShank3.

[0059] In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. As used herein, "less than 65%" includes any percentage identity of less than 65% suitable for the construction of MiniShank3. In some embodiments, the MiniShank3 protein is less than 64%, less than 63%, less than 62%, less than 61%, less than 60%, less than 59%, less than 58%, less than 57%, less than 56%, less than 55%, less than 54%, less than 53%, less than 52%, less than 51%, less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 44%, less than 43%, less than 42%, less than 41%, Less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%, or the same.

[0060] In some embodiments, the MiniShank3 protein contains 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%, or 100% identical, including all values ​​in between.

[0061] In some embodiments, the MiniShank3 protein contains one of sequence numbers 17-20. In some embodiments, sequence number 17 is encoded by sequence number 1. In some embodiments, sequence number 18 is encoded by sequence number 2. In some embodiments, sequence number 19 is encoded by sequence number 3. In some embodiments, sequence number 20 is encoded by sequence number 4.

[0062] In some embodiments, the MiniShank3 protein contains an ankyrin repeat domain. In one embodiment in which the MiniShank3 protein contains an ankyrin repeat domain, the MiniShank3 protein contains SEQ ID NOs. 19 and / or 20.

[0063] In another embodiment, the MiniShank3 protein does not contain an ankyrin repeat domain. In one embodiment in which the MiniShank3 protein does not contain an ankyrin repeat domain, the MiniShank3 protein includes SEQ ID NOs. 17 and / or 18.

[0064] In some embodiments, the polynucleotide sequences encoding the MiniShank3 proteins related to this disclosure contain 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 to any one of SEQ ID NOs: 1-4, or 100% identity including all values ​​in between, and encode one or more proteins having Shank3 activity. In some embodiments, the polynucleotide sequences encoding the MiniShank3 proteins related to this disclosure contain at least 90% identity to any one of SEQ ID NOs: 1-4, and encode one or more proteins having Shank3 activity. In some embodiments, the sequence of polynucleotides encoding the MiniShank3 protein related to this disclosure comprises one of SEQ ID NOs: 1 to 4. In some embodiments, one of SEQ ID NOs: 1 to 4 encodes one or more proteins having partial Shank3 activity. In some embodiments, one of SEQ ID NOs: 1 to 4 encodes one or more proteins having full Shank3 activity.

[0065] In some embodiments, MiniShank3 is encoded by one of the sequence numbers 1-4 provided in Table 1. Sequence numbers 1 and 3 correspond to mouse MiniShank3 nucleic acid sequences, while sequence numbers 2 and 4 correspond to human MiniShank3 nucleic acid sequences. Sequence numbers 1 and 2 encode MiniShank3 proteins that do not contain an ankyrin repeat domain or an N-terminal domain. Sequence numbers 3 and 4 encode MiniShank3 proteins that contain both an ankyrin repeat domain and an N-terminal domain.

[0066] The polynucleotides described herein that encode the MiniShank3 protein encode a protein having at least partial Shank3 activity.

[0067] As disclosed herein, “identity” of sequences means the measurement or calculation of the percentage of identical match between two or more sequences having gap alignments addressed by mathematical models, algorithms, or computer programs known to those skilled in the art. The percentage identity of two sequences (e.g., nucleic acid or amino acid sequences) can be determined using a Basic Local Alignment Search Tool (BLAST®), such as the NBLAST® and XBLAST® programs (version 2.0). Alignment techniques such as Clustal Omega can be used for multiple sequence alignments. 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).

[0068] In some aspects, the polynucleotides encoding the Shank proteins (Shank1, Shank2, Shank3) disclosed herein are of a size of less than approximately 4.6kb, less than approximately 4.5kb, less than approximately 4.4kb, less than approximately 4.3kb, less than approximately 4.2kb, less than approximately 4.1kb, less than approximately 4.0kb, less than approximately 3.9kb, less than approximately 3.8kb, less than approximately 3.7kb, less than approximately 3.6kb, less than approximately 3.5kb, less than approximately 3.4kb, less than approximately 3.3kb, less than approximately 3.2kb, less than approximately 3.1kb, less than approximately 3.0kb, less than approximately 2.9kb, less than approximately 2.8kb, less than approximately 2.7kb, less than approximately 2.6kb, less than approximately 2.5kb, less than approximately 2.4kb, less than approximately 2.3kb, less than approximately 2.2kb, or less than approximately 2.1kb. In some embodiments, the polynucleotides encoding the Shank proteins (Shank1, Shank2, Shank3) disclosed herein may be of any size suitable for the methods and vectors disclosed herein. Diseases and Disabilities

[0069] This disclosure provides compositions and methods suitable for treating neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan McDiarmid syndrome.

[0070] As used herein, “neurodevelopmental disorder” refers to any disorder that impairs the growth and / or development of the brain and / or central nervous system. In some aspects, neurodevelopmental disorders affect one or more brain functions, such as affect, learning ability, self-control, and memory. It should be understood that aspects of this disclosure may be applicable to the treatment of any neurodevelopmental disorder.

[0071] In some aspects, neurodevelopmental disorders are autism spectrum disorder (ASD). Diagnosis of ASD is primarily based on criteria such as impaired communication, impaired social interaction, and repetitive or restricted interests and behaviors. ASD is a highly heritable disorder, with a concordance rate of up to 90% in identical twins. However, ASD is clinically heterogeneous, covering a variety of distinct disorders with varying degrees of symptom severity. ASD is thought to be etiologically heterogeneous, possibly involving polygenic, monogenic, and environmental factors.

[0072] Alterations in synaptic connectivity and function have been proposed as a key underlying mechanism in ASD. Recent genetic studies have identified numerous candidate genes for ASD, 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 in subsets of ASD. 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 lead to loss of function (LoF) of one of the two normal copies of the Shank3 gene (i.e., haploinsufficiency). Recent genetic screenings have 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). Current estimates suggest that deletions and / or mutations involving Shank3 are the primary cause in approximately 2% of all ASD patients with ID. Therefore, understanding the function of Shank3 may provide insights into the pathological mechanisms of ASD.

[0073] As used herein, “intellectual disability” refers to a disability that results in a deficit in intellectual and / or adaptive functions. Intellectual functions include, for example, reasoning, problem-solving, planning, abstract thinking, judgment, academic learning, and / or experiential learning. Intellectual functions can be measured using any method known in the art, such as IQ tests. Adaptive functions include, for example, the skills necessary to live in an independent and responsible manner, such as communication and social skills. In some cases, intellectual disability may become apparent in childhood or adolescence.

[0074] In some aspects, the neurodevelopmental disorder is Phelan-McDermid syndrome (PMS, 22q13.3 deletion syndrome), which is an autism spectrum disorder characterized by autistic-like behaviors, hypotonia, severe intellectual disability, and speech and language developmental disorders. Shank3 is one of the genes reported to be deleted in Phelan-McDermid syndrome. Disruption of Shank3 is thought to be the cause of the central neurodevelopmental and neurobehavioral disorders in Phelan-McDermid syndrome, because individuals with an intact ring chromosome 22 containing the Shank3 gene exhibit a normal phenotype.

[0075] Other neurodevelopmental disorders may include, but are not limited to, attention-deficit / hyperactivity disorder (ADHD), learning disabilities such as dyslexia or dyscalculia, intellectual disability (mental retardation), conduct disorder or motor disorder, cerebral palsy, visual and hearing impairments, developmental language disorders, neurogenetic disorders such as fragile X syndrome, Down syndrome, Rett syndrome, hypogonadotropic hypogonadism, and traumatic brain injury. subject

[0076] The subjects treated by the methods described herein may be human or non-human subjects. Non-human subjects include, for example, non-human primates; domesticated animals such as cattle, horses, goats, sheep, and pigs; pets such as dogs and cats; and rodents.

[0077] Subjects treated by the methods described herein may be subjects who have, are suspected of having, or are at risk of developing a neurodevelopmental disorder. In some embodiments, subjects have been diagnosed with a neurodevelopmental disorder, while in other embodiments, subjects have not been diagnosed with a neurodevelopmental disorder. In some embodiments, subjects are human subjects who have, are suspected of having, or are at risk of developing autism spectrum disorder (ASD). In some embodiments, subjects are human subjects who have, are suspected of having, or are at risk of developing Phelan McDermid syndrome. In some embodiments, subjects have reduced expression of the Shank3 gene compared to a control subject. In some embodiments, the expression of the Shank3 gene in subjects is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times compared to a control subject. In some embodiments, the control subjects are subjects who do not have, are not suspected of having, or are not at risk of having, a neurodevelopmental disorder. In some embodiments, the reduction in Shank3 gene expression in the subjects is caused by the disruption of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene includes a deletion in at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene includes one or more mutations within at least one copy of the Shank3 gene.

[0078] In some embodiments, subjects are human subjects exhibiting one or more symptoms of ASD. In some embodiments, subjects are human subjects exhibiting developmental delay. In some embodiments, subjects are human subjects exhibiting intellectual disability (ID). In some embodiments, subjects are human subjects exhibiting sleep disorders. In some embodiments, subjects are human subjects exhibiting hypotonia. In some embodiments, subjects are human subjects exhibiting speech deficits. In some embodiments, subjects are human subjects exhibiting language delays. In some embodiments, subjects are human subjects exhibiting any symptoms or signs of ASD.

[0079] In some embodiments, the subject is an adult human subject. In some embodiments, the adult is older than 25 years of age. In some embodiments, the adult is 25 years of age or younger. In some embodiments, the adult is 21 years of age or younger. In some embodiments, the adult is 18 years of age or younger. In some embodiments, the adult is 16 years of age. In some embodiments, the subject is an elderly person (e.g., 65 years of age or older). In some embodiments, the adult may be any age of adulthood suitable for the treatment disclosed herein.

[0080] In some embodiments, the subjects are human subjects who are not adults. In some embodiments, the human subjects are 16 years of age or younger. In some embodiments, the human subjects are not over 10 years of age. In some embodiments, the human subjects are 10 years of age or younger. In some embodiments, the human subjects are children or infants. In some embodiments, the human subjects are toddlers. In some embodiments, the human subjects are in the fetal stage of development. In some embodiments, the human subjects are in the prenatal stage of development. Viral vector

[0081] As disclosed herein, polynucleotides encoding the MiniShank3 protein can be delivered to a target tissue or cell by a viral vector. The vectors described herein can be used to deliver nucleic acids encoding the target protein to a target, including, for example, delivery to a specific organ or the central nervous system (CNS) of the target. In some embodiments, the target protein is the Shank protein. In some embodiments, the target protein is the Shank3 protein. In some embodiments, the target protein is the MiniShank3 protein.

[0082] In some embodiments, the Disclosure provides a vector comprising a polynucleotide encoding the Shank protein disclosed herein. In some embodiments, the 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.

[0083] AAV refers to a replication-deficient dependent parvovirus within the Parvoviridae family of viruses. AAV may be derived from a naturally occurring virus or may be recombinant. AAV can be packaged into a capsid, which may be derived from a naturally occurring capsid protein or a recombinant capsid protein. The single-stranded DNA genome of AAV contains reverse end repeats (ITRs). ITRs are involved in the replication and capsidation of the AAV genome, and its integration into and excision from the host genome. While not wishing to be bound by any theory, an AAV vector may contain one or more ITRs, including 5'ITR and / or 3'ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and / or additional post-transcriptional regulators. The AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to those skilled in the art, such as those described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (2012)); this is incorporated herein by reference in its entirety.

[0084] In some embodiments, AAVs are integrated into the host cell genome. In some embodiments, AAVs are not integrated into the host genome. In some embodiments, the AAV vectors disclosed herein may contain sequences from any known organism. In some embodiments, the AAV vectors disclosed herein may contain 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 transcriptional and translational terminators such as polyadenylation signals. In some embodiments, AAV vectors may contain sequences related to replication and integration.

[0085] In some embodiments, MiniShank3 disclosed herein is delivered to a target tissue or cell via an AAV vector. In some embodiments, the AAV vector delivering MiniShank3 disclosed herein is delivered to the target central nervous system (CNS). As used herein, delivery of an AAV vector to the CNS may include delivery of the AAV vector to any target tissue or cell within the CNS. In some embodiments, delivery of an AAV vector to the CNS includes delivery of the AAV vector to nerve tissue or cells. In some embodiments, delivery of an AAV vector to the CNS includes delivery of the AAV vector to the brain. In some embodiments, delivery of an AAV vector to the CNS includes delivery of the AAV vector to the spinal cord. In some embodiments, delivery of an AAV vector to the CNS includes delivery of the AAV vector to white matter and gray matter. In some embodiments, the AAV vector delivering MiniShank3 disclosed herein is delivered to any target tissue or cell suitable for the treatment disclosed herein.

[0086] As used in this disclosure, “delivering” or “administering” an AAV vector may include any method known in the art for delivering or administering an AAV vector or a composition containing an AAV vector to a subject. Administration includes, but is not limited to, direct administration of the AAV vector or a composition containing an AAV vector, or peripheral administration by passive diffusion or convection-enhanced delivery (CED) known in the art to bypass the blood-brain barrier. The AAV vectors described herein may be administered in any composition that conforms to the aspects of this disclosure.

[0087] The AAV vector can contain any known AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV serotype is AAV9. The clades of AAV viruses are described in Gao et al. (2004) J. Virol. 78(12):6381-6388, which are incorporated herein by reference in their entirety. In some embodiments, any AAV serotype suitable for delivery to the CNS may be selected.

[0088] The AAV vectors of this disclosure may include or be derived from any natural or recombinant AAV serotype. In some embodiments, the AAV vectors may utilize or be based on the AAV serotypes described in WO 2017 / 201258A1, which is incorporated herein by reference in whole; the AAV serotypes described herein include, for example, but are not limited to: AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV 9.16, 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, AAV4 2-1b, 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, AAV223.4, 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, AAV3.1 / 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-4 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、AAVh u.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu .67、AAVhu.14 / 9、AAVhu.t19、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.3 7、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、 AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58 、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R 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, AAV SM10-2, AAV Shuffle100-1, AAV Shuffle100-3, AAV Shuffle100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAV Shuffle100-2, AAV SM10-1, AAV SM10-8, AAV SM100-3, AAV SM100-10, 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 AAV10, 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, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), G2B-26, G2B-13, TH1.1-32 and / or TH1.1-35, and their variants. The AAV vectors are further described in US 9,585,971, US 2017 / 0166926, and WO2020 / 160337, which are incorporated herein by reference in their entirety.

[0089] In some embodiments, MiniShank3 as disclosed herein is delivered by an AAV vector. In some embodiments, the AAV vector comprises a transgene and its regulatory sequence, and optionally 5' and 3' ITRs. In some embodiments, the transgene and its regulatory sequence are adjacent to the 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 regulatory sequences (e.g., poly-A tails). In some embodiments, the AAV vector comprises at least an AAV ITR and the MiniShank3 transgene.

[0090] In some embodiments, AAV may be packaged in AAV particles, 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, which include: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.rh8, AAV.rh10, AAV.rh39, AAV.43, AAV2 / 2-66, AAV2 / 2-84, and AAV2 / 2-125, or any of the aforementioned variants.

[0091] In some embodiments, the miniShank3 transgene coding sequence in the AAV vector is operably ligated 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 tissue-specific transcription. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the tissue-specific regulatory sequence may be a Syn promoter (e.g., hSyn1). In some embodiments, the tissue-specific regulatory sequence may be any promoter or enhancer that is neuron-specific and suitable for the treatment described herein.

[0092] In some embodiments, the miniShank3 transgene coding sequence containing SEQ ID NO: 2 or 4 in the AAV vector is operably ligated to the promoter and adjacent to the AAV ITR. In some embodiments, the miniShank3 transgene coding sequence containing SEQ ID NO: 1 or 3 in the AAV vector is operably ligated to the promoter and adjacent to the AAV ITR.

[0093] Aspects of this disclosure relate to AAV vectors expressing the miniShank3 transgene. In some embodiments, the miniShank3 transgene is adjacent to an AAV ITR. In some embodiments, the AAV ITR includes an AAV2 ITR. In some embodiments, the AAV ITR includes an AAV1 ITR. In some embodiments, the AAV ITR includes an AAV5 ITR. In some embodiments, the AAV ITR includes an AAV6 ITR. In some embodiments, the AAV ITR includes an AAV8 ITR. In some embodiments, the AAV ITR includes an AAV9 ITR. In some embodiments, the AAV ITR includes an rh10 ITR. In some embodiments, the AAV ITR may include a self-complementary ITR.

[0094] It should be understood that the AAV vectors described herein may include DNA constructs containing transgenes such as MiniShank3, 5' and / or 3' ITRs, promoters, introns, and / or other relevant regulatory elements known in the art.

[0095] In some embodiments, the AAV vector contains a woodchuck hepatitis virus posttranscriptional regulator (WPRE) that can enhance miniShank3 transgene expression. In some embodiments, the AAV vector contains an untranslated region, 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.

[0096] In some embodiments, the AAV vectors used herein may be self-complementary vectors.

[0097] Figure 15 shows an example of an AAV vector (referred to as "AAV-hSyn1-human MiniShank3-V1" in Figure 15) containing a human miniShank3 transgene expressed under the control of the human synapsin 1 (hSyn1) promoter. The AAV vector shown in Figure 15 contains the sequence provided as Sequence ID No. 21 in Table 1.

[0098] As shown in Figure 15, Sequence ID No. 21 includes the human Mini-Shank3 gene, 5'-ITR, 3'-ITR, WPRE, hGH poly(A), F1 origin, NeoR / KanR marker, hSyn1 promoter, and PUC origin. In some embodiments, the reverse terminal repeat (ITR) sequence contains approximately 145 nucleotides each. These elements may be useful for effective replication and capsidation in cis. Those skilled in the art will understand that any element of AAV vectors known in the art may be adapted to aspects of this 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 for AAV delivery in DNA constructs similar to those shown in Figure 15. These DNA constructs may contain one or more of the elements shown in Figure 15. For example, in some embodiments, coding sequences containing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of sequence numbers 1-4 are expressed in a DNA construct as shown in Figure 15. In some embodiments, coding sequences containing any one of sequence numbers 1-4 are expressed in a DNA construct as shown in Figure 15. In some embodiments, the DNA construct contains one or more of the elements shown in Figure 15, such as a promoter, 5'-ITR, 3'-ITR, WPRE, hGH poly(A), F1 origin, NeoR / KanR marker, and / or PUC origin.

[0099] In some embodiments, the AAV vectors relating to this disclosure include a nucleic acid sequence encoding the MiniShank3 protein that contains 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 with respect to the sequence of SEQ ID NO: 21, or 100% identity with respect to all values ​​in between. In some embodiments, the AAV vector includes a sequence corresponding to SEQ ID NO: 21, which encodes the MiniShank3 protein containing the sequence of SEQ ID NO: 18.

[0100] In some embodiments, an AAV vector encoding the MiniShank3 protein, which includes a sequence containing 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 to the sequence of SEQ ID NO: 21, or 100% identity including all values ​​in between, and which includes the sequence of SEQ ID NO: 18, may be delivered to human subjects requiring it and may be suitable for treating human subjects with neurodevelopmental disorders.

[0101] As those skilled in the art will understand, any method known in the art for designing AAV vectors for clinical use and for delivering AAV vectors may be adapted to aspects of this disclosure. For example, non-limiting examples of disclosures relating to AAV vectors and delivery are provided in U.S. Patent No. 7,906,111, entitled “Adeno-Associated Virus (AAV) Branches, Sequences, Vectors Containing Them, and Uses Thereof,” and U.S. Patent No. 9,834,788, entitled “AAV Vectors for Use in Gene Therapy for Choroidalemia,” each of which is incorporated herein by reference in whole.

[0102] In some embodiments, the AAV vectors relating to this disclosure include a sequence encoding the MiniShank3 protein for AAV delivery that contains 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 with respect to the sequence of SEQ ID NO: 7 or 21 provided in Table 1, or 100% identity with respect to all values ​​in between. SEQ ID NO: 7 encodes the protein sequence of SEQ ID NO: 11. SEQ ID NO: 8 encodes the protein sequence of SEQ ID NO: 12. SEQ ID NO: 9 encodes the protein sequence of SEQ ID NO: 13. SEQ ID NO: 10 encodes the protein sequence of SEQ ID NO: 14. SEQ ID NO: 21 encodes the protein sequence of SEQ ID NO: 18.

[0103] In some embodiments, an AAV vector encoding the MiniShank3 protein for AAV delivery encodes a protein having a sequence that contains 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 to any one of SEQ ID NOs. 11 or 17-20 provided in Table 1, or 100% identity including all values ​​in between.

[0104] In some embodiments, the vector used to deliver miniShank3 disclosed herein may be a lentiviral vector. In some embodiments, the vector used to deliver miniShank3 disclosed herein may be an adenoviral vector.

[0105] In some embodiments, the vector constructs disclosed herein may include Sequence ID No. 21 shown in Table 1.

[0106] In some embodiments, a vector containing a polynucleotide of the Shank3 protein (i.e., a miniShank3 DNA construct) can be expressed in a specific tissue or cell of interest. In some embodiments, the vector disclosed herein includes a promoter. In some embodiments, the vector includes 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 may be any promoter known in the art and suitable for the construction of miniShank3. In some embodiments, the human promoter may be any promoter having high specificity for nerve tissue and cells. In some embodiments, the promoter may be a constitutive promoter. For example, the constitutive promoter may be a CAG promoter. As those skilled in the art will understand, any promoter may be used as long as the selected promoter is suitable for aspects of this disclosure. Composition and administration

[0107] This disclosure provides compositions comprising a pharmaceutical composition containing a polynucleotide (e.g., miniShank3) delivered by an AAV vector disclosed herein and a pharmaceutically acceptable carrier.

[0108] The compositions of this disclosure may comprise AAV 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 AAVs, each having one or more different Shank proteins.

[0109] A suitable carrier can be readily selected by those skilled in the art, taking into account the indications to which the AAV is directed. For example, one suitable carrier includes saline, which can be compounded with various buffering solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of carriers is not limited to this 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.

[0110] As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antimicrobial and antifungal agent, isotonic and absorption retardant, buffer, carrier solution, suspension, colloid, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Co-active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” means molecular entities and compositions that do not cause an allergic reaction or similar adverse reaction when administered to a host.

[0111] Delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used to introduce the compositions of this disclosure into suitable host cells. In particular, the transgene delivered by the AAV vector may be formulated for delivery, encapsulated in any of the following: lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0112] Such formulations may be preferred for introducing pharmaceutically acceptable formulations of nucleic acids or 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 circulating half-lives have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patents No. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0113] Liposomes are formed from phospholipids that disperse in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution in the core.

[0114] Alternatively, nanocapsule formulations of AAV vectors can be used. Nanocapsules can generally encapsulate 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 is considered.

[0115] In some embodiments, a pharmaceutical composition containing nucleic acids delivered by an AAV vector may contain other pharmaceutical components, 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. Often, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the pharmaceutical composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0116] Suitable pharmaceutical forms for delivering AAV vectors include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Often, the form is sterile and fluid enough to be easily injected. They must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0117] The methods described herein involve transfecting cells of a desired tissue (e.g., the brain) and administering an AAV vector in an amount sufficient to provide a sufficient level of gene transfer and expression without excessive 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 injection and retroorbital injection), intraventricular (ICV), intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Routes of administration may be combined as needed. In some embodiments, the vectors disclosed herein are administered intravenously.

[0118] In some embodiments, this disclosure provides methods for treating subjects having neurodevelopmental disorders. In some embodiments, this disclosure provides methods for treating subjects having autism spectrum disorder (ASD). In some embodiments, this disclosure provides methods for treating subjects having Phelan McDermid syndrome. In some embodiments, the methods provided herein involve administering and delivering an effective amount of a composition comprising a vector containing a polynucleotide encoding the Shank3 protein (e.g., miniShank3) to a target environment or tissue of the subject. 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 an effective amount of a composition comprising a vector containing a polynucleotide encoding the Shank3 protein (e.g., miniShank3) to a target environment or tissue involves delivering the composition to neurons or other brain cell types. In some embodiments, the vector is an AAV vector. In some embodiments, a method for delivering nucleic acids to a target environment or tissue of a subject requiring it comprises providing a composition comprising an AAV vector containing at least a nucleic acid (e.g., miniShank3) to be delivered to the target environment or tissue of the subject, and administering the composition to the subject. Methods of using AAV vectors are 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 may comprise a capsid protein.

[0119] In some embodiments, a composition comprising a vector containing a polynucleotide encoding the Shank3 protein is delivered to a subject via intravenous administration, systemic administration, intraventricular administration, intrauterine administration, intrathecal administration, retroorbital injection, or facial venous injection. In some embodiments, intrauterine administration is used for subjects in the prenatal stage of development. In some embodiments, the composition is delivered to the subject via nanoparticles. In some embodiments, the composition is delivered to the subject via a viral vector. In some embodiments, the composition is delivered to the subject via any carrier suitable for delivering nucleic acid material.

[0120] Any composition comprising a vector containing a polynucleotide encoding a protein that provides some utility or benefit to the subject may be delivered to the target environment or tissue of the subject in accordance with the methods disclosed herein.

[0121] In addition to the delivery methods described above, the following techniques are also envisioned as alternative methods for delivering AAV compositions to the host: sonophoresis (i.e., ultrasound) is used and is described in U.S. Patent No. 5,656,016 as a device for increasing the rate and effectiveness of drug delivery to and through the circulatory system. Other envisioned drug delivery alternatives include intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patents No. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0122] The dose of AAV containing the polynucleotide encoding the Shank3 protein (e.g., miniShank3), required to achieve a specific "therapeutic effect," for example, the dose unit in absolute vector genome (vg) or vector genome per milliliter of pharmaceutical solution (vg / mL), will vary, without limitation, based on several factors including the route of AAV administration, the gene expression level required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene product. A dose that maximizes infection rates without affecting neurodevelopment is also suitable. Those skilled in the art can easily determine the AAV dose range for treating patients with a specific disease or disorder based on the aforementioned and other factors.

[0123] The effective amount of the AAV vector is an amount sufficient to infect an animal or human subject or to target a desired tissue. The effective amount will vary depending on the subject and tissue, as it depends primarily on factors such as the species, age, sex, weight, health status, and tissue to be targeted of the subject. The terms "effective amount" or "effective quantity" in the context of a composition or dosage for administration to a subject refer to the amount of the composition or dosage that produces one or more desired responses in the subject. In some embodiments, the effective amount of the compositions disclosed herein can partially or completely rescue the effects of the mutant Shank3 gene and / or partially or completely restore the loss of function of the Shank3 protein. The effective amount can include reducing the level of an undesired response, but in some embodiments, can include completely preventing an undesired response. The effective amount also includes delaying the occurrence of an undesired response. The effective amount can also be an amount that provides a desired therapeutic endpoint or 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. Any of the foregoing achievements can be monitored by conventional methods and the methods disclosed in this application. Of course, the effective amount will depend on the particular subject being treated; the severity of the condition; individual patient parameters such as age, general condition, size, weight; the duration of treatment; the nature of concurrent treatments (if any); the particular route of administration and like factors.

[0124] For example, in some embodiments, the number of vector genomes administered to a subject is any value between about 6.0×10 11 vg to about 9.0×10 13 vg. In some embodiments, the number of vector genomes administered to a subject is any value between about 6.0×10 13 vg / mL to about 9.0×10 13 vg. In some embodiments, the number of vector genomes administered to a subject is any value between about 1×10 10 to about 1×10 12 vg. In one embodiment, the effective amount of AAV is such that the genome copy number per kg is 10 10 1011 , 10 12 , 10 13 , or 10 14 In one embodiment, the effective dose of AAV is when the genome copy number per subject is 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some cases, approximately 10 11 ~10 13 The dosage of the AAV genome copy is appropriate. In some embodiments, the number of vector genomes administered to the subject can be any dose suitable for the treatments and methods disclosed herein.

[0125] In some embodiments, the dose of AAV is administered to the subject at least once per calendar day (e.g., 24 hours). In some embodiments, the dose of AAV is administered to the subject at least once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of AAV is administered to the subject at least once per calendar week (e.g., 7 calendar days). In some embodiments, the dose of AAV is administered to the subject at least once every 2 weeks (e.g., once every 2 calendar weeks). In some embodiments, the dose of AAV is administered to the subject at least once per calendar month (e.g., once every 30 calendar days). In some embodiments, the dose of AAV is administered to the subject at least once every 6 calendar months. In some embodiments, the dose of AAV is administered to the subject at least once per calendar year (e.g., 365 days or 366 days in a leap year). In some embodiments, the dose of rAAV is administered to the subject at least once every two calendar years (e.g., 730 days or 731 days in a leap year). In some embodiments, the dose of AAV is administered to the subject at least once every three calendar years (e.g., 1095 days or 1096 days in a leap year).

[0126] The pharmaceutically acceptable excipients and carrier solutions disclosed herein are well known to those skilled in the art, as is the development of appropriate dosages and treatment regimens for using the specific compositions described herein in various treatment regimens. Typically, these formulations may contain at least about 0.1% or more of the active compound, although the percentage of the active ingredient(s) may, of course, vary and may be between about 1 or 2% and about 70% or 80% or more of the total weight or volume of the formulation. Naturally, the amount of the active compound in each therapeutically useful composition may be prepared so that an appropriate dosage is obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are contemplated by those skilled in the art preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desired. Expression of proteins related to the Shank protein network

[0127] The methods and compositions provided herein are useful in several embodiments for treating neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan McDermid syndrome. The inventors of this disclosure have found that delivery of miniShank3 via a viral vector, such as an AAV vector, in a mouse model is effective in restoring the function of postsynaptic thickening (PSD) proteins. In several embodiments, the efficacy of miniShank3 administration is evaluated using the expression level of the PSD protein. In several embodiments, the PSD protein is Homer. In several embodiments, the PSD protein is postsynaptic thickening protein 95 (PSD95). In several embodiments, the PSD protein is SynGap1. In several embodiments, the PSD protein is SAPAP3. In several embodiments, the PSD protein is NR1. In several embodiments, the PSD protein is NR2B. In several embodiments, the PSD protein is GluR2. In several embodiments, the PSD protein is any protein that can be improved or restored by miniShank3 treatment.

[0128] In some embodiments, an increase in any of the PSD proteins may indicate the efficacy of miniShank3 compared to an untreated control. Methods for detecting gene expression and protein levels are well known in the art.

[0129] In some embodiments, Homer expression in subjects treated with miniShank3 increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to controls. In some embodiments, postsynaptic protein (PSD95) expression in subjects treated with miniShank3 increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to controls. In some embodiments, SynGap1 expression in subjects treated with miniShank3 increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to controls. In some embodiments, SAPAP3 expression in subjects treated with miniShank3 increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to controls. In some embodiments, NR1 expression in subjects treated with miniShank3 increases 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 controls.In some embodiments, NR2B expression in subjects treated with miniShank3 increases 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 controls. In some embodiments, GluR2 expression in subjects treated with miniShank3 increases 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 controls.

[0130] In some embodiments, administration of MiniShank3 or a composition containing MiniShank3 may lead to improved sleep efficiency. In some embodiments, subjects have improved sleep efficiency after being administered an effective amount of a composition comprising an expression construct containing a polynucleotide encoding a Shank protein, such as the MiniShank3 protein. In some embodiments, the sleep efficiency of subjects after being administered an effective amount of the composition increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times compared to a control subject. Improved sleep efficiency includes, but is not limited to, a reduction in sleep disturbances, including difficulty falling asleep and maintaining sleep. Sleep efficiency can be measured using any method known in the art.

[0131] In some embodiments, administration of MiniShank3 or compositions containing MiniShank3 may lead to improvement in social impairment. In some embodiments, the social impairment of a subject is improved after administration of an effective amount of a composition containing an expression construct comprising a polynucleotide encoding a Shank protein, such as the MiniShank3 protein. In some embodiments, the social impairment of a subject after administration of an effective amount of the composition is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to a control subject. Measurement of social impairment can be carried out using any method known in the art.

[0132] As used herein, “social impairment” means a behavioral abnormality or deficit that prevents the subject from exhibiting spontaneous social interaction.

[0133] In some embodiments, administration of MiniShank3 or compositions containing MiniShank3 may lead to improvement in locomotion and / or motor coordination. In some embodiments, locomotion and / or motor coordination in a subject is improved after administration of an effective amount of a composition containing an expression construct comprising a polynucleotide encoding a Shank protein, such as the MiniShank3 protein. In some embodiments, locomotion and / or motor coordination in a subject after administration of an effective amount of the composition is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to a control subject. Measurement of locomotion and / or motor coordination can be performed using any method known in the art.

[0134] As used herein, “mobility and / or motor coordination disorder” may include, for example, a lack of coordination, loss of balance, and / or shuffling gait.

[0135] In some embodiments, administration of MiniShank3 or a composition containing MiniShank3 may lead to improvement in corticostriate synaptic dysfunction. In some embodiments, corticostriate synaptic dysfunction in a subject is improved after administration of an effective amount of a composition containing an expression construct comprising a polynucleotide encoding a Shank protein, such as the MiniShank3 protein. In some embodiments, corticostriate synaptic dysfunction in a subject after administration of an effective amount of the composition is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 2x, at least 5x, at least 10x, at least 20x, at least 50x, at least 100x, or at least 1000x compared to a control subject. Measurement of corticostriate synaptic dysfunction can be performed using any method known in the art.

[0136] As used herein, “corticostriate synaptic dysfunction” refers to a defective corticostriate circuit in the brain that may cause repetitive and compulsive behaviors in neuropsychiatric disorders and neurodevelopmental disorders such as autism, obsessive-compulsive disorder, and Tourette syndrome. [Examples]

[0137] Some aspects of the technology described herein may be further understood based on the non-limiting exemplary embodiments described in the following Examples section. The limitations of the embodiments described in the following Examples section are limitations only to the embodiments described in the following Examples section and not to any other embodiments described herein. To further enhance the understanding of the inventions described herein, the following examples are provided. The examples described herein are provided to illustrate the systems and methods provided herein and should not be construed as limiting their scope. Example 1: Mouse models with the Shank3 mutation exhibited synaptic defects and behavioral abnormalities.

[0138] Multiple mouse and monkey models with Shank3 LoF mutations have been previously developed to study how mutations in the Shank3 gene affect brain development, neural structure, synaptic and circuit function, and behavior. These models serve as a basis for testing potential therapeutic approaches.

[0139] Two distinct alleles, Shank3A and Shank3B, were generated in Shank3 mutant mice. In Shank3A mutant mice, a portion of the gene encoding the ankyrin repeat was targeted, resulting in the longest Shank3 isoform, Shank3 α It was completely eliminated. However, the other two isoforms were unaffected (Shank3 in this case). β and Shank3 γ (It was named as such). In the Shank3B mutant, the PDZ domain is targeted, and Shank3 α and Shank3 β Both isoforms were completely ruled out, and the estimated Shank3 γ The number of isoforms was significantly reduced. Further analysis focused primarily on Shank3B mutant mice.

[0140] Shank3B - / - The mice did not show any overall brain abnormalities by histological analysis. However, by 3-6 months of age, Shank3B - / - The mice developed significant skin lesions. The lesions were present in the animals separated during weaning and were not due to excessive in-species grooming, therefore they were self-inflicted; because, Shank3B - / -This is because no lesions were found in wild-type (WT) mice that were housed from birth. 24-hour video recording showed that Shank3B before injury... - / - It was found that mice spent more time grooming compared to WT controls (Figure 1A-1B), which is related to Shank3B. - / - The mice exhibited excessive grooming and self-injurious behavior. In conclusion, Shank3B mutant mice showed repetitive and compulsive grooming leading to skin lesions.

[0141] To study the effects of the Shank3 mutant on social behavior, a three-chamber social arena was used to investigate the animals' ability to spontaneously initiate social interactions and identify social novelty. Initially, the test animals were left alone to explore and initiate social contact with a partner ("stranger 1") held in a wired cage, or they were kept in the same empty wired cage ("empty cage"). Shank3B - / - The mice showed a clear preference for interacting with an empty cage over interacting with a social partner (Figures 2A-2B). In subsequent trials, a new social partner ("stranger 2") was placed in a previously empty wired cage. While WT mice showed a preference for the new animal, Shank3B mice did not. - / - The mutants spent more time in the central chamber (Figures 2A and 2C). In conclusion, Shank3B mutant mice exhibited impaired social interaction.

[0142] We studied the effects of the Shank3 mutant on striatal synapses using Shank3 mutant mice. The basal ganglia is one of the brain regions involved in ASD. Shank3B - / - Repetitive grooming behavior in mice suggested a defect in corticostriatal function. Furthermore, Shank3 was the only Shank family member highly expressed in the striatum (Figure 3A). Therefore, the analysis focused on striatal neurons and corticostriatal synapses.

[0143] To determine how disruption of Shank3 may affect the PSD protein network, purified PSD from the striatum was examined for scaffold proteins and glutamate receptor subunits (Figures 3B-3C). - / - Decreased levels of SAPAP3, Homer-1b / c, and PSD93, as well as glutamate receptor subunits GluR2, NR2A, and NR2B, were observed in mice. This suggests alterations in the molecular composition of striatal PSD and disruption of glutamatergic signaling, supporting the hypothesis that Shank protein is the primary scaffold.

[0144] Using Golgi tracing, Shank3B - / - On MSN Dendritic arbor Neuronal hypertrophy was observed, as indicated by increased complexity, length, and surface area of ​​the dendrites. Furthermore, dye filing of MSNs revealed Shank3B - / - A decrease in vertebral density was observed in mice. EM analysis showed that Shank3B mice were superior to WT mice. - / - The study showed a reduction in the average thickness and length of PSDs from mice. In summary, these results highlight the important role of Shank3 in the development of MSNs and glutamatergic synapses in the striatum.

[0145] To elucidate the functional consequences of disruption in Shank3 on synapses, corticostriate synaptic circuits were recorded in 6-7 week old Shank3B - / - The study was conducted on acute brain slices of mice, and behavioral tests were performed on mice of the same age. Compared to the control group, Shank3B - / -In mice, a significant reduction in field cluster spikes was observed (Figure 4A). Presynaptic function remained clearly unchanged, as indicated by the relationship between stimulus intensity and the amplitude of the action potential component of the response called negative peak 1 (NP1), and the paired pulse ratio (PPR). These results suggest that the reduction in total field response is most likely due to postsynaptic impairment of synaptic function and / or a decrease in the number of functional synapses.

[0146] Whole-cell voltage clamp recordings of AMPAR-mEPSCs were also performed in dorsolateral striatal MSNs. The frequency of mEPSCs was Shank3B - / - Since it was significantly reduced in MSN (Figures 4B-4C) and no defects were observed in PPR (Figure 4E), Shank3B was selected. - / - This suggests a decrease in the number of functional synapses in the MSN. (Shank3B) - / - The peak of mEPSC amplitude in the MSN was also reduced (Figures 4B and 4D), indicating a decrease in postsynaptic responses from available synapses. These data demonstrate the crucial role of Shank3 in postsynaptic function of the corticostriatal circuit.

[0147] Similar trials will be conducted in monkey models and then in clinical trials in human ASD patients with the Shank3 mutation. Example 2: Design and construction of the miniShank3 gene

[0148] The gene therapy strategy for Shank3 mutations disclosed herein involved using AAV to deliver a functional copy of Shank3 cDNA to brain cells and restore Shank3 expression levels in patients. However, Shank3 is a large protein, with a coding sequence of approximately 5.7 kb, exceeding the packaging capacity of AAV vectors. To address this issue, a miniaturized Shank3 (miniShank3) was designed with the aim of reducing its size without compromising its function.

[0149] In the initial version of miniShank3 (miniShank3-v1, Figure 5B), the N-terminal domain and other regions such as the ankyrin repeat, as well as the link sequence between the PDZ domain and the proline-rich domain, including the major portion of the proline-rich domain deemed unimportant, were deleted. This effectively reduced the Shank3 coding sequence to 2.1kb by retaining only the domains predicted to be important for the function of the Shank3 protein.

[0150] While we do not wish to be bound by any theory, the N-terminal domain (NTD) of the Shank3 protein may play a special role in synaptic plasticity. Synaptic plasticity refers to the neuronal ability to regulate synaptic strength in response to various stimuli, and is considered to be the basis of human learning ability and the ability to adapt to environmental changes. Shank3 has been found to have a specific interaction with CaMKIIα, a major synaptic plasticity regulator. A missense mutation identified in human ASD patients with severe ID impairs this interaction. Knock-in mice carrying the same mutation were generated, and these mice had synaptic plasticity defects. Based on these findings, it was hypothesized that adding the NTD to miniShank3-v1 might further enhance the function of miniShank3.

[0151] We designed a second version of miniShank3 containing an NTD (miniShank3-v2, Figure 6B). miniShank3-v2 (3.1kb) is significantly larger than miniShank3-v1 (2.1kb), but still within the AAV packaging capacity. Based on the miniShank3-v1 data, both versions were expected to be effective in restoring Shank3 function. miniShank3-v2 may offer some advantages in restoring synaptic plasticity function, which could potentially extend the scope of gene therapy treatment to multiple developmental stages.

[0152] Corticostriate coculture was performed. Briefly, the primary corticostriate coculture was prepared as previously described in the art. Striatal tissue was P0 Shank3 InsG3680 / InsG3680 Dissections were taken from mutants. Cortical tissue was dissected from P0 wild-type offspring. The tissue was digested with papain (Worthington Biochemical Corporation) and separated using a small glass Pasteur pipette. Approximately 5 million striatal medium spiny neurons (MSNs) were electroporated using a Mouse Neuron Nucleofector® Kit (Lonza) with either 1 μg of AAV-hSyn-GFP plasmid alone, or a mixture of 1 μg of AAV-hSyn1-GFP plasmid 1 and 2 μg of AAV-hSyn1-minishank3 plasmid. Next, striatal MSNs and cortical neurons were mixed in a 3:1 ratio, and poly-D-lysine / laminin (Neuvitro, GG-12-1,5-laminin) was placed inside a 24-well plate containing Neurobasal A medium (Invitrogen) supplemented with 0.5 mM glutamine (Invitrogen), 1 × B27 (Invitrogen), 50 μg / mL penicillin / streptomycin (Invitrogen), 50 ng / mL BDNF (R&D Systems), and 30 ng / mL GDNF (R&D Systems). 5 cells / cm 2 The culture medium was plated onto 12 mm coverslips pre-coated with the specified density. After the initial plating, half of the medium was replaced every 3-4 days with fresh medium that did not contain BDNF and GDNF.

[0153] To investigate whether miniShank3 retains the essential function of Shank3, its expression and localization in cultured neurons from Shank3 mutant mice were first examined. Since Shank3 is a synaptic protein, it was important that miniShank3 localize to synapses, similar to the endogenous Shank3 protein. When GFP-tagged miniShank3v1 was expressed in neurons in corticostriate co-cultures, GFP-miniShank3 was found to localize precisely to synapses, as indicated by co-localization with the postsynaptic marker PSD95 (Figure 7A–7H). Therefore, miniShank3 retained this crucial characteristic of a synaptic protein. Example 3: Functional expression of miniShank3 in a mouse model

[0154] To investigate whether miniShank3 captures the full function of endogenous Shank3, we tested the ability of miniShank3-v1 to restore neuronal function in Shank3 mutant mice. It has been previously demonstrated that Shank3 mutant mice exhibit defects in the molecular composition of postsynaptic thickening (PSD), including abnormalities in synaptic electrophysiological properties and behavior. We tested the ability of miniShank3-v1 to restore each of these defects in Shank3 InsG3680 mutant mice. These mice mimic the InsG3680 mutation seen in human ASD patients. Previous studies have shown that these mice exhibited molecular, electrophysiological, and behavioral defects associated with ASD. Therefore, we used this model to test whether miniShank3-v1 can restore the defects observed in these mutant mice.

[0155] GFP-miniShank3-v1 was cloned into the pHP.eB AAV vector. AAV-GFP-miniShank3-v1 virus was prepared and administered to mice 0-2 days post-birth (P0-P2) by facial vein injection. A dose of 6.42E+11 viral genomes (vg) per mouse resulted in high expression of GFP-miniShank3-v1 in the majority of neurons in the brain (Figure 8). AAV-hSyn1-GFP was injected as a control.

[0156] Using standard biochemical approaches, PSDs were isolated from the brains of wild-type mice injected with AAV-hSyn1-GFP, Shank3 mutant mice injected with AAV-hSyn1-GFP, and Shank3 mutant mice injected with AAV-GFP-miniShank3-v1 (Figure 9A). Western blotting assays were used to detect levels of various synaptic proteins in the PSDs (Figure 9B). As previously reported, levels of several synaptic proteins, including Homer, PSD95, SynGap1, SAPAP3, NR1, NR2B, and GluR2, were reduced in the PSDs of Shank3 mutant mice compared to wild-type mice (Figures 9C-9F). Expression of miniShank3-v1 restored the expression levels of these synaptic proteins to wild-type levels, demonstrating that miniShank3-v1 is fully functional in restoring the molecular composition of the PSDs (Figures 9C-9F). In conclusion, miniShank3 restores the molecular defect of postsynaptic thickening (PSD) in Shank3 mutant mice.

[0157] Shank3 is highly expressed in the striatum. Corticostriate synaptic communication is defective in Shank3 mutant mice. To test whether miniShank3-v1 can repair the synaptic defect, we performed electrophysiological recordings of corticostriate synaptic circuits in acute brain slices of Shank3 mutant mice. As previously reported, field cluster spikes were significantly reduced in Shank3 mutant mice compared to controls (Figure 10A). This defect in Shank3 mutant mice was rescued by miniShank3-v1 expression (Figure 10A). Presynaptic function remained unchanged, as indicated by the relationship between stimulus intensity and the amplitude of the action potential component of the response, called negative peak 1 (NP1; Figure 10B). These results suggest that the reduction in total field response is most likely due to postsynaptic impairment of synaptic function and / or a reduction in the number of functional synapses, and these defects could be effectively corrected by miniShank3-v1 expression via AAV at P0. In conclusion, miniShank3 repairs corticostriate synaptic defects in Shank3 mutant mice.

[0158] Previous studies have shown that Shank3 mutant mice exhibit several behavioral phenotypes associated with symptoms seen in patients with Phelan-McDermid syndrome or Shank3 mutations. Treatment with miniShank3-v1 in P0 was found to completely rescue all behavioral impairments tested in Shank3 mutant mice, with the exception of performance in a single assay of motor learning, which showed only a tendency toward improvement. Treatment with miniShank3-v1 completely rescued social interaction impairment as measured by a three-chamber social interaction assay (Figure 11A), motor activity impairment as measured by distance traveled in an open-field test (Figure 11B), exploratory behavior impairment as measured by time to stand up in an open-field test (Figure 11C), and anxiety-like behavior exhibited in an elevated zero maze (Figure 11D). However, motor learning impairment in mutant Shank3 was only slightly improved in the rotarod test (Figure 11E). This may be related to the fact that cerebellar development in mice begins postnatally, which limits AAV infection in P0, resulting in very low levels of miniShank3 expression in the cerebellum when injected in P0. In conclusion, miniShank3 restores behavioral disorders in Shank3 mutant mice. Example 4: Determining the timing of miniShank3 gene therapy

[0159] Previous studies have demonstrated that there is a critical developmental timeframe for rescuing specific behaviors in Shank3 mutants. To determine the effective therapeutic window for AAV-mediated miniShank3 delivery, intravenous viral injections were administered at various developmental stages, and their effects on adult behavior were analyzed. All behavioral experiments were conducted at least 8 weeks after AAV injection into mice. Briefly, wild-type mice, Shank3 mutant mice, and Shank3 mutant mice treated with Shank3 at different ages (P0, P2, P7, and P28) were assigned to experimental groups. P0 and P2 mice were given 6.0 × 10⁶ doses of AAV-pHP.eB-hSyn-GFP or AAV-pHP.eB-hSyn-GFP-MiniShank3 diluted in sterile saline. 11A 20 μl injection mixture consisting of the total viral genome (vg) was administered intravenously. Facial vein injection was used for mice aged P0-P2. P7 and P28 mice received 7.0 × 10⁶ doses of AAV-pHP.eB-hSyn-GFP or AAV-pHP.eB-hSyn-GFP-MiniShank3. 11 The total viral genome (vg) was administered intravenously. For mice aged P7 and P28, retroorbital or intraventricular (ICV) injection was used.

[0160] Treatment with MiniShank3 postnatally (P28) completely rescued social behavioral disorders in the Shank3 InsG3680 mutant, and treated mice showed a strong preference for other mice, unlike untreated mutants which showed no preference (Figure 12A). P28-treated miniShank3 mice also showed a significant increase in locomotor activity compared to mutant mice (Figure 12B). Treatment with miniShank3 at P28 was found to significantly improve motor learning (Figure 12C) and motor coordination (Figure 12D). In contrast to social and motor behaviors, miniShank3 treatment at P28 showed minimal effects on anxiety-like behavior and repetitive grooming (Figure 12D). In the elevated zero maze, miniShank3-treated mice showed no significant difference compared to mutant mice in open-arm exploration time (Figure 12F), and in the grooming assay, miniShank3-treated mice showed only a slight reduction in grooming behavior (Figure 12F).

[0161] Previous studies describing gene rescue of Shank3 expression in adult mice have shown that the grooming phenotype is reversible in adulthood and that dysfunction of the corticostriatal-thalamic-cortical circuit is strongly associated with repetitive / obsessive-compulsive behaviors. To investigate whether the lack of rescue of these behavioral phenotypes in mutants treated with miniShank3 at P28 was due to reduced miniShank3 expression in grooming-related brain regions, we investigated the in vivo distribution of miniShank3 by evaluating GFP expression in P28-treated animals; we found GFP expression to be very low in the thalamus and limited in the striatum. Thus, while miniShank3 treatment at P28 selectively rescued impairments in social behavior, locomotion, and motor coordination, improved strategies for AAV-based targeting of the thalamus and striatum at this age may enable additional therapeutic benefits for anxiety and repetitive behaviors.

[0162] Mutant mice administered miniShank3 on postnatal day 7 (P7) showed a strong preference for other mice compared to their mutant littermates, similar to that observed with P0-P2 injection (Figure 13A). Electrophysiological studies showed that miniShank3 delivery at P7 was sufficient to restore the corticostriate synaptic defects observed in Shank3 mutant mice. Dysfunction of the corticostriate circuit is strongly associated with repetitive and compulsive behaviors related to autism and obsessive-compulsive disorder, and Shank3 mutants exhibited an excessive grooming phenotype. To test this theory, individual wild-type (WT) mice, Shank3 mutant mice, and Shank3 mutant mice treated with miniShank3 at P7 were monitored for 2 hours, and the percentage of time spent grooming during the session was quantified. Shank3 mutant mice were found to have a significantly increased percentage of time spent grooming compared to WT mice. However, miniShank3-treated mice had a significantly reduced grooming time, similar to WT animals (Figure 13B).

[0163] Treatment with MiniShank3 at P7 completely rescued the spontaneous motor phenotypes observed in Shank3 mutants, as measured by total distance traveled in an open-field study (Figure 13C) and anxiety-like behavior in an elevated zero maze (Figures 13D-13E). In contrast to delivery of miniShank3 at P0-P2 (postnatal days 0-2), Shank3 mutant mice treated with miniShank3 at P7 functioned significantly better than mutant littermates in both motor learning and motor coordination (Figure 13F), suggesting that the motor impairments observed in Shank3 mutants are reversible by AAV-mediated miniShank3 gene therapy when administered at the appropriate developmental stage. In summary, these behavioral outcomes demonstrate that miniShank3 gene therapy at P7 can effectively rescue all reported behavioral phenotypes in Shank3 InsG3680 mutant animals. Example 5: Systemic delivery of miniShank3 in P7 leads to improvement of sleep disorders in Shank3 InsG3680 variants.

[0164] Patients with Phelan McDermid syndrome and other individuals with SHANK3 mutations have often been shown to exhibit severe sleep disturbances, including difficulty falling asleep and maintaining sleep. For example, SHANK3 mutations in macaques result in marked sleep disturbances. To evaluate sleep disturbances in Shank3 InsG3680 mutant mice, EEG / EMG-related surgery was performed, and signals from all three experimental groups were examined to determine whether sleep was affected by the Shank3 mutation. InsG3680 homozygotes showed reduced NREM sleep duration with shorter bout length (Figure 13H) (Figure 13G) and reduced delta power of the delta rhythm (1–4 Hz) in frontal EEG during NREM sleep (Figure 13I) compared to WT controls. These results indicated severe sleep disturbances in Shank3 mutant mice. MiniShank3 injected at P7 significantly mitigated both the reduction in NREM sleep (Figure 13G) and the reduction in sleep bout lengths (Figure 13H) observed in untreated mutants. Furthermore, miniShank3 injected at P7 partially rescued attenuated delta power (Figure 13I). In summary, these results demonstrate that miniShank3 can mitigate sleep disturbances in homozygous Shank3 InsG3680 mice, which is consistent with the broader ability of miniShank3 to rescue ASD-related behavioral phenotypes. Example 6: MiniShank3 treatment in P7 does not induce seizure activity of the Shank3 InsG3680 variant.

[0165] To test the potential effects of miniShank3 treatment on seizure activity, we included WT mice, Shank3 mutants injected with a control virus, Shank3 mutants injected with miniShank3, and Scn2a mutants in our study. EEG analysis included measures such as sleep monitoring, spontaneous seizures, and auditory seizures. No spontaneous epileptic EEG abnormalities were observed in Shank3 InsG3680 mutant mice, as evidenced by a stable EEG baseline during 24-hour recording (representative trace, Figure 14A). Sensitivity of Shank3 mutants to auditory seizures (124 dB acoustic stimulation) was investigated, and it was found that the mutant mice lacked behavioral signs of auditory seizures. To assess the safety of miniShank3, spontaneous epileptic EEG abnormalities were investigated in mutant mice injected with miniShank3 at P7, but no detectable hyperexcitatory activity was observed on the EEG (Figure 14A). Furthermore, Shank3 InsG3680 mutant mice and mutant mice injected with miniShank3 exhibited characteristics of absence seizure EEG. thorn slow wave No seizure-induced discharges (SWDs) were observed. However, analysis of animals with heterozygous mutations in Scn2a resulted in frequent absence seizures, identifying approximately 60 SWD episodes per hour, thus validating the effectiveness of the analytical pipeline (Figure 14B). In summary, these results indicate that the Shank3 InsG3680 mutant did not exhibit epileptic-like activity, and miniShank3 expression did not induce detectable seizures. Example 7: Treatment of adult human subjects with miniShank3

[0166] Adult human subjects who have, are suspected of having, or are at risk of having, neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan McDermid syndrome can be treated with miniShank3 using the methods, vectors, and non-natural polynucleotides described herein. Adult human subjects may include adult males or females aged 16 years or older. Adults under 25 years of age may be preferred for the miniShank3 treatment disclosed herein. Methods of administration and delivery may include facial intravenous injection and intraventricular injection, as disclosed herein. Other methods known to those skilled in the art may also be used. Adult human subjects are treated with miniShank3 delivered by a viral vector such as AAV. For example, adult human subjects can be treated with an AAV vector containing the sequence of SEQ ID NO: 21 expressing the miniShank3 transgene. While we do not wish to be bound by any theory, the dose for treatment is 1 × 10⁻⁶. 10 ~1 × 10 12 This may be between viral genomes (vg). Those skilled in the art will understand that various factors such as sex, weight, age, disease status, and disease type can be taken into consideration when determining the dose for a particular adult. The dose is 1 × 10⁻⁶. 10 ~1 × 10 12 It may be outside the range of vg. The route of administration may be, for example, intravenous, intrafacial vein, intracranial, intraventricular, intraocular, or intrathecal. Example 8: Treatment of non-adult human subjects using miniShank3

[0167] Non-adult human subjects who have, are suspected of having, or are at risk of having, neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan McDermid syndrome can be treated with miniShank3 using the methods, vectors, and non-natural polynucleotides described herein. Non-adult human subjects may include any male or female under 16 years of age. While we do not wish to be bound by any theory, the miniShank3 treatment disclosed herein may be preferable for human subjects under 10 years of age, such as infants or toddlers. Methods of administration and delivery may include facial intravenous injection and intraventricular injection, as disclosed herein. Other methods known to those skilled in the art may also be used. Non-adult human subjects are treated with miniShank3 delivered by a viral vector such as AAV. For example, non-adult human subjects can be treated with an AAV vector containing the sequence of SEQ ID NO: 21 expressing the miniShank3 transgene. While we do not wish to be bound by any theory, the dose for treatment is 1 × 10⁻⁶. 10 ~1 × 10 12 This may be between viral genomes (vg). Those skilled in the art will understand that various factors such as sex, weight, age, disease status, and disease type can be taken into consideration when determining the dose for a particular adult. The dose is 1 × 10⁻⁶. 10 ~1 × 10 12 It may be outside the range of vg. The route of administration may be, for example, intravenous, intrafacial vein, intracranial, intraventricular, intraocular, or intrathecal. If the non-adult human subject is a fetus or in the prenatal developmental stage, the route of administration may be intrauterine. Example 9: Treatment of human subjects using miniShank1 or miniShank2

[0168] Human subjects having, suspected of having, or at risk of having, neurodevelopmental disorders, autism spectrum disorder (ASD), or Phelan McDermid syndrome can be treated with miniShank1 or miniShank2 using the methods, vectors, and non-natural polynucleotides described herein. Adult human subjects may include any male or female human subjects. The treatment method is the same as the methods described herein, except that the miniShank3 construct (SEQ ID NOs. 1-4 and 21) is modified to include Shank1 (miniShank1) or Shank2 (miniShank2). Adult or non-adult human subjects are treated with miniShank1 or miniShank2 delivered by a viral vector such as AAV. For example, adult or non-adult human subjects can be treated with an AAV vector containing the sequence of SEQ ID NO. 21 expressing the miniShank3 transgene. While we do not wish to be bound by any theory, the dose for treatment is 1 × 10⁻⁶. 10 ~1 × 10 12 This may be between viral genomes (vg). Those skilled in the art will understand that various factors such as sex, weight, age, disease status, and disease type can be taken into consideration when determining the dose for a particular adult. The dose is 1 × 10⁻⁶. 10 ~1 × 10 12 It may be outside the range of vg. The route of administration may be, for example, intravenous, intrafacial vein, intracranial, intraventricular, intraocular, or intrathecal. If the non-adult human subject is a fetus or in the prenatal developmental stage, the route of administration may be intrauterine. Example 10: Delivery of miniShank3 using a lentiviral viral vector

[0169] Human subjects who have, are suspected of having, or are at risk of having, neurodevelopmental disorders such as autism spectrum disorder (ASD) or Phelan McDermid syndrome can be treated with miniShank1 or miniShank2 using the methods, vectors, and non-natural polynucleotides described herein. Adult or non-adult human subjects are treated with miniShank3 delivered by a viral vector such as a lentivirus. While we do not wish to be bound by any theory, the dose for treatment is 1 × 10⁻⁶. 10 ~1 × 10 12 This may be between viral genomes (vg). Those skilled in the art will understand that various factors such as sex, weight, age, disease status, and disease type can be taken into consideration when determining the dose for a particular adult. The dose is 1 × 10⁻⁶. 10 ~1 × 10 12 It may be outside the range of vg. The route of administration may be, for example, intravenous, intrafacial vein, intracranial, intraventricular, intraocular, or intrathecal. If the non-adult human subject is a fetus or in the prenatal developmental stage, the route of administration may be intrauterine. Table 1. Mouse and human miniShank3 sequences and vector sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16

[0170] References Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6

[0171] In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or made clear from the context. A claim or description containing “or” between one or more members of a group is deemed satisfied if, unless otherwise indicated or made clear from the context, one, two or more, or all of the group members are present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which two or more, or all, of the group members are present in, used in, or otherwise related to a given product or process.

[0172] Furthermore, this 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, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same basic claim. Where elements are presented as a list (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) may be removed from a group. In general, where this disclosure or an aspect of this disclosure is referred to as containing certain elements and / or features, it should be understood that certain aspects of this disclosure or an aspect of this disclosure consist of, or essentially consist of, such elements and / or features. For simplicity, these aspects are not described verbatim in this specification. Note that the terms “contains” and “includes” are intended to be open and may include additional elements or steps. Where a scope is given, unless otherwise specified, endpoints are included within that scope. Furthermore, unless otherwise indicated, or unless evident from the context and the understanding of those skilled in the art, values ​​expressed as ranges may be assumed to be any specific value or subrange within the ranges described in different embodiments of this disclosure, up to one-tenth of the lower limit of the range, unless the context explicitly indicates otherwise.

[0173] This application refers to various published patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between the incorporated references and this specification, this specification shall prevail. Furthermore, any particular aspect of this disclosure contained in the prior art may be expressly excluded from any one or more claims. Such aspects are considered to be known to those skilled in the art and may be excluded even if the exclusion is not expressly stated herein. Any particular aspect of this disclosure may be excluded from any claim for any reason whatsoever, whether or not it relates to the existence of the prior art.

[0174] Those skilled in the art will recognize or be able to identify many equivalents to the particular embodiments described herein by means of routine experiments alone. The scope of the embodiments described herein is not intended to be limited to the above description, but is as set out in the appended claims. Those skilled in the art will understand that various changes and modifications to this description may be made without departing from the spirit or scope of this disclosure, as defined in the following claims.

Claims

1. A polynucleotide that codes for the Shank3 protein, Here, the Shank3 protein comprises the SH3 domain, PDZ domain, Homer-binding domain, cortactin-binding domain, and SAM domain; Here, the SH3 domain contains at least 90% identity with residues 474-525 of SEQ ID NO: 6, or at least 90% identity with residues 473-524 of SEQ ID NO: 5; Here, the PDZ domain contains at least 90% identity with residues 573-662 of SEQ ID NO: 6, or at least 90% identity with residues 572-661 of SEQ ID NO: 5; Here, the Homer-binding domain contains at least 90% identity with residues 1294-1323 of SEQ ID NO: 5 or 6; Here, the cortactin-binding domain contains at least 90% identity with residues 1400-1426 of SEQ ID NO: 5 or 6; and / or Here, the SAM domain contains at least 90% identity with residues 1664-1729 of SEQ ID NO: 6, or at least 90% identity with residues 1663-1728 of SEQ ID NO: 5; Here, the amino acid sequence of the Shank3 protein encoded by the polynucleotide is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs. 17-20, or consists of any one of the sequences of SEQ ID NOs. 17-20: and Here, the polynucleotide is less than 4.7 kb.

2. The polynucleotide codes for the Shank3 protein, and here The SH3 domain includes residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5; The PDZ domain comprises residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5, and the Homer-binding domain comprises residues 1294-1323 of SEQ ID NO: 5 or 6; The cortactin-binding domain comprises residues 1400–1426 of SEQ ID NO: 5 or 6; and / or The polynucleotide according to claim 1, wherein the SAM domain comprises residues 1664-1729 of SEQ ID NO: 6 or residues 1663-1728 of SEQ ID NO:

5.

3. The polynucleotide according to claim 1 or 2, wherein the sequence of the polynucleotide is at least 90% identical to sequence number 1 or 2, or consists of sequence number 1 or 2.

4. The amino acid sequence of the Shank3 protein encoded by the polynucleotide is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 or 20, and the Shank3 protein encoded by the polynucleotide contains an ankyrin repeat domain. The polynucleotide according to claim 1, wherein the ankyrin repeat domain contains at least 90% identity with residues 148-345 of SEQ ID NO: 6, or at least 90% identity with residues 147-313 of SEQ ID NO: 5, or contains residues 148-345 of SEQ ID NO: 6 or residues 147-313 of SEQ ID NO:

5.

5. The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is at least 90% identical to sequence number 3 or 4, or consists of sequence number 3 or 4.

6. The polynucleotide according to claim 1, wherein the amino acid sequence of the Shank3 protein is at least 90% identical to the amino acid sequence of SEQ ID NO:

17.

7. The polynucleotide according to claim 6, wherein the amino acid sequence of the Shank3 protein consists of the amino acid sequence of SEQ ID NO:

17.

8. The polynucleotide according to claim 6 or 7, wherein the sequence of the polynucleotide encoding the Shank3 protein is at least 90% identical to or consists of SEQ ID NO:

1.

9. The polynucleotide according to claim 1, wherein the amino acid sequence of the Shank3 protein is at least 90% identical to the amino acid sequence of SEQ ID NO:

18.

10. The polynucleotide according to claim 9, wherein the amino acid sequence of the Shank3 protein consists of the amino acid sequence of SEQ ID NO:

18.

11. The polynucleotide according to claim 9 or 10, wherein the sequence of the polynucleotide encoding the Shank3 protein is at least 90% identical to or consists of SEQ ID NO:

2.

12. The polynucleotide according to claim 1, wherein the amino acid sequence of the Shank3 protein is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

13. The polynucleotide according to claim 12, wherein the amino acid sequence of the Shank3 protein is the sequence number 19.

14. The polynucleotide according to claim 12 or 13, wherein the sequence of the polynucleotide encoding the Shank3 protein is at least 90% identical to or consists of SEQ ID NO:

3.

15. The polynucleotide according to claim 1, wherein the amino acid sequence of the Shank3 protein is at least 90% identical to the amino acid sequence of SEQ ID NO:

20.

16. The polynucleotide according to claim 15, wherein the amino acid sequence of the Shank3 protein consists of the amino acid sequence of SEQ ID NO:

20.

17. The polynucleotide according to claim 15 or 16, wherein the sequence of the polynucleotide encoding the Shank3 protein is at least 90% identical to or consists of SEQ ID NO:

4.

18. A Shank3 protein encoded by a polynucleotide according to any one of claims 1 to 17.

19. A vector comprising a polynucleotide according to any one of claims 1 to 17, wherein the vector is a viral vector or an AAV vector.

20. The vector according to claim 19, wherein the vector comprises a promoter operably linked to a polynucleotide according to any one of claims 1 to 17.

21. The vector according to claim 20, wherein the polynucleotide is adjacent to the AAV reverse terminal repeat (ITR).

22. The vector according to claim 20, wherein the promoter is hSyn1.

23. A vector according to any one of claims 19 to 22, comprising a woodchuck hepatitis virus post-transcriptional regulator (WPRE) operably linked to a polynucleotide according to any one of claims 1 to 17.

24. A vector according to any one of claims 19 to 23, and AAV particles comprising a capsid protein, wherein the capsid is of a serotype selected from AAV1, 2, 5, 6, 8, 9, rh10, and PHP.eB.

25. The AAV particle according to claim 24, wherein the serotype is AAV9.

26. A pharmaceutical composition comprising a vector according to any one of claims 19 to 23 or an AAV particle according to claim 24 or 25, for use in treating neurodevelopmental disorders, autism spectrum disorder (ASD), or Phelan McDiarmid syndrome in subjects requiring such treatment.

27. The pharmaceutical composition according to claim 26, wherein a vector or AAV particles are delivered to the target brain.

28. The subject exhibits one or more symptoms of ASD; and / or The pharmaceutical composition according to claim 26 or 27, wherein the subject exhibits one or more of the following conditions: developmental delay, intellectual disability (ID), sleep disorder, hypotonia, speech deficiency, or language delay.

29. If the subject has, is suspected of having, or is at risk of having a decrease in Shank3 gene expression compared to the control subject, Here, the decrease in Shank3 gene expression is caused by the disruption of at least one copy of the Shank3 gene, and / or The pharmaceutical composition according to any one of claims 26 to 28, wherein the disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene, or one or more mutations in at least one copy of the Shank3 gene.

30. A pharmaceutical composition comprising an effective amount of AAV particles for use in the treatment of neurodevelopmental disorders, autism spectrum disorder (ASD), or Phelan McDermid syndrome in a subject, wherein the AAV particles comprise a polynucleotide as described in any one of claims 1 to 17.

31. A pharmaceutical composition for use in the treatment of neurodevelopmental disorders, autism spectrum disorder (ASD), or Phelan McDermid syndrome in subjects, comprising an effective amount of AAV particles, wherein the AAV particles comprise a polynucleotide encoding a Shank3 protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 or 20, wherein: The pharmaceutical composition wherein the Shank3 protein comprises an ankyrin repeat domain, wherein the ankyrin repeat domain has at least 90% identity with residues 148-345 of SEQ ID NO: 6, or at least 90% identity with residues 147-313 of SEQ ID NO:

5.

32. A pharmaceutical composition for use in the treatment of neurodevelopmental disorders, autism spectrum disorder (ASD), or Phelan McDermid syndrome in a subject, comprising an effective amount of AAV particles, wherein the AAV particles comprise a polynucleotide encoding a Shank3 protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or 18.

33. The pharmaceutical composition according to claim 32, wherein the AAV particles contain a polynucleotide encoding a Shank3 protein having an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

17.

34. The pharmaceutical composition according to claim 32, wherein the AAV particles contain a polynucleotide encoding a Shank3 protein having an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

18.

35. A pharmaceutical composition according to any one of claims 31 to 34, wherein the polynucleotide comprises a promoter.

36. The pharmaceutical composition according to claim 35, wherein the promoter is hSyn1.

37. The pharmaceutical composition according to claim 35 or 36, wherein the polynucleotide comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

38. A pharmaceutical composition according to any one of claims 26 to 37, wherein the polynucleotide is less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4.0 kb, less than 3.9 kb, less than 3.8 kb, less than 3.7 kb, less than 3.6 kb, less than 3.5 kb, less than 3.4 kb, less than 3.3 kb, less than 3.2 kb, less than 3.1 kb, less than 3.0 kb, less than 2.9 kb, less than 2.8 kb, less than 2.7 kb, less than 2.6 kb, less than 2.5 kb, less than 2.4 kb, less than 2.3 kb, less than 2.2 kb, or less than 2.1 kb.

39. A MiniShank3 protein wherein the amino acid sequence of the MiniShank3 protein is at least 95% identical to or consists of SEQ ID NO: 18 or 20, and wherein the MiniShank3 protein has Shank3 activity.

40. The MiniShank3 protein according to claim 39, wherein the amino acid sequence of the Shank3 protein is the sequence of SEQ ID NO:

18.

41. The MiniShank3 protein according to claim 39, wherein the amino acid sequence of the Shank3 protein is the sequence of SEQ ID NO:

20.

42. AAV particle according to claim 24, (i) AAV9 capsid protein, and (ii) The polynucleotide comprising an AAV2 ITR adjacent to the polynucleotide encoding the Shank3 protein consisting of the amino acid sequence of SEQ ID NO: 18, Herein, the AAV particle is characterized in that the polynucleotide encoding the Shank3 protein is operably linked to the hSyn1 promoter, the WPRE sequence, and the hGH polyA sequence.

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