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WO2024233422A1 - Approches de thérapie génique à shank3 - Google Patents

Approches de thérapie génique à shank3 Download PDF

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WO2024233422A1
WO2024233422A1 PCT/US2024/027909 US2024027909W WO2024233422A1 WO 2024233422 A1 WO2024233422 A1 WO 2024233422A1 US 2024027909 W US2024027909 W US 2024027909W WO 2024233422 A1 WO2024233422 A1 WO 2024233422A1
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Prior art keywords
aav
subject
shank3
seq
primate
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PCT/US2024/027909
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WO2024233422A9 (fr
Inventor
Guoping Feng
Xian Gao
Yuan MEI
Qiangge ZHANG
William MENEGAS
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Massachusetts Institute Of Technology
The Broad Institute, Inc.
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Publication of WO2024233422A1 publication Critical patent/WO2024233422A1/fr
Publication of WO2024233422A9 publication Critical patent/WO2024233422A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to gene therapy approaches for delivering polynucleotides encoding a Shank3 protein to a subject who has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
  • Shank3 accounts for about 0.5-1% of all autism spectrum disorder (ASD) patients and about 2% ASD patients with intellectual disability (ID).
  • ASD autism spectrum disorder
  • ID intellectual disability
  • aspects of the disclosure relate to the development of an effective gene therapy approach for subjects with Shank3 mutations.
  • aspects of the disclosure relate to methods of treating or ameliorating symptoms of an autism spectrum disorder (ASD) or Phelan- McDermid syndrome or a syndrome associated with a SHANK3 mutation in a primate subject in need thereof, the method comprising administering to the primate subject a therapeutically effective amount of a composition comprising an AAV vector, wherein the AAV vector comprises a) an artificial genome comprising a polynucleotide encoding a transgene comprising a miniShank3 protein comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 17-20 and has SHANK3 activity, wherein the transgene is operably linked to an hSYN promoter comprising SEQ ID NO: 22 and b) a capsid protein, wherein the capsid protein is AAV9 or a variant thereof.
  • the transgene comprises an amino acid sequence of any one of SEQ ID NOs
  • the capsid protein or variant thereof crosses the blood-brain barrier.
  • the primate subject has at about 3 months, about 6 months or about one year after said administering a decreased response to negative stimuli, increased motivation, flexibility, and memory, increased response to reward, decreased cognitive deficit and/or intellectual disability compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the changes are a statistically significant and/or clinically significant improvement relative to the pre-treatment subject, or relative to a subject that has a condition and is untreated, or based upon natural history of the disease.
  • the primate subject at about 3 months, about 6 months or about one year or about 18 months or about two years or more than two years after said administering has increased resting fMRI connectivity lower in anterior/medial areas or decreased resting fMRI connectivity lower in posterior/lateral (sensory) areas compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the changes are a statistically significant and/or clinically significant improvement relative to the pre-treatment subject or based upon natural history of the disease.
  • the primate subject is human.
  • the human is an adult human.
  • is human is about 3 years old or about 10 years old.
  • the administration is IV administration. In embodiments, the administration is directly to the brain. In embodiments, the administration is intracerebroventricular (ICV) administration.
  • ICV intracerebroventricular
  • Shank3 protein comprises an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin binding domain, and a SAM domain.
  • the SH3 domain comprises 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.
  • the PDZ domain comprises 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.
  • the Homer binding domain comprises at least 90% identity to residues 1294- at least 90% identity to residues 1400-1426 of SEQ ID NO: 5 or 6 and/or.
  • the SAM domain comprises 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.
  • the polynucleotide is less than 4.7 kb.
  • the polynucleotide further comprises a proline-rich region.
  • the SH3 domain comprises 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
  • 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
  • the SAM domain comprises residues 1664-1729 of SEQ ID NO: 6 or residues 1663-1728 of SEQ ID NO: 5.
  • the polynucleotide comprises at least 90% identity to SEQ ID NO: 1 or 2. In some embodiments, the polynucleotide comprises SEQ ID NO: 1 or 2.
  • the Shank3 protein encoded by the polynucleotide further comprises an ankyrin repeat domain.
  • 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.
  • the polynucleotide comprises residues 148-345 of SEQ ID NO: 6 or residues 147-313 of SEQ ID NO: 5.
  • the polynucleotide comprises at least 90% identity to SEQ ID NO: 3 or 4. In some embodiments, the polynucleotide comprises SEQ ID NO: 3 or 4.
  • the polynucleotide is less than about 4.6 kb, 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4.0 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3.0 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2.4 kb, 2.3 kb, 2.2 kb, or 2.1 kb.
  • the Shank3 protein is less than 65% identical to SEQ ID NO: 5 or 6 over the full length of SEQ ID NO: 5 or 6.
  • the Shank3 protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 17-20. In some embodiments, the Shank3 protein comprises an amino acid sequence comprising any one of SEQ ID NOs: 17-20.
  • Shank3 proteins encoded by polypeptides described herein relate to Shank3 proteins encoded by polypeptides described herein.
  • the vector is a viral vector.
  • the vector is an AAV vector.
  • the vector comprises a promoter operably linked to the polynucleotide described herein.
  • the polynucleotide is flanked by AAV inverted terminal repeat (ITRs).
  • ITRs AAV inverted terminal repeat
  • the AAV vector comprises a sequence that is at least 90% identical to SEQ ID NO: 7 or 21 and encodes a protein with Shank3 activity.
  • the AAV vector comprises the sequence of SEQ ID NO: 7 or 21 and encodes a protein with Shank3 activity.
  • the AAV vector comprises a sequence that is at least 90% identical to SEQ ID NO: 2 or 4 and encodes a protein with Shank3 activity. In some embodiments, the AAV vector comprises the sequence of SEQ ID NO: 2 or 4, which encodes a protein with Shank3 activity. In some embodiments, the AAV vector comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 3 and encodes a protein with Shank3 activity. In some embodiments, the AAV vector comprises the sequence of SEQ ID NO: 1 or 3, which encodes a protein with Shank3 activity.
  • FIG. 1 Further aspects of the disclosure relate to methods of treatment comprising administering a pharmaceutical composition comprising AAV particles comprising an AAV vector and a capsid protein, wherein the capsid is of a serotype selected from AAV1, 2, 5, 6, 8, 9, rhlO, and PHP.eB.
  • the serotype is AAV9 or a variant thereof.
  • the serotype is AAV9.
  • the serotype is AAV10.
  • the serotype is PHP.eB.
  • the capsid is a capsid that crosses the blood-brain barrier, for example, those found in Goertsen, David et al. “AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.” Nature neuroscience vol. 25,1 (2022), incorporated by reference in its entirety.
  • the AAV vector further comprises a promoter.
  • the promoter is a human promoter. In some embodiments, the promoter is hSynl.
  • the subject is a human subject.
  • the human subject is an adult.
  • the human subject is not an adult.
  • the human subject is not older than 25 years old.
  • the human subject is about 10 years old or younger.
  • the human subject is 3 years old or younger.
  • the subject has, is suspected of having, or is at risk of having, a neurodevel opmental disorder.
  • the subject has, is suspected of having, or is at risk of having, an autism spectrum disorder (ASD).
  • ASSD autism spectrum disorder
  • the subject exhibits one or more symptoms of an ASD.
  • the subject has, is suspected of having, or is at risk of having, Phelan-McDermid syndrome.
  • the subject has a mutation in SHANK3.
  • the subject is SHANK3+/-.
  • the subject exhibits one or more of: developmental delay, intellectual disability (ID), sleep disturbance, hypotonia, lack of speech, or language delay.
  • the disclosure relates to methods of treating a subject having a neurodevelopmental disorder.
  • the disclosure relates to methods of treating a subject having an autism spectrum disorder (ASD).
  • the disclosure relates to methods of treating a subject having Phelan-McDermid syndrome.
  • the methods of treatment comprise administering to the subject an effective amount of a composition comprising an AAV vector that comprises a polynucleotide encoding a Shank3 protein.
  • the composition is in a pharmaceutically acceptable carrier.
  • the AAV vector is delivered to the brain of the subject. In some embodiments, the AAV vector is delivered to the cortex, striatum and/or thalamus of the subject.
  • the subject has, is suspected of having, or is at risk of having, reduced expression of the Shank3 gene relative to a control subject.
  • the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder, an autism spectrum disorder (ASD), and/or Phelan-McDermid syndrome.
  • reduced expression of the Shank3 gene is caused by disruption of at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • MiniShank3 proteins comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 17-20.
  • the MiniShank3 protein comprises the sequence of any one of SEQ ID NOs: 17-20.
  • aspects of the disclosure relate to methods of treating an autism spectrum disorder (ASD) in a primate subject, the method comprising administering to the primate subject an effective amount of a composition comprising an AAV vector, wherein the AAV vector comprises a polynucleotide encoding a Shank3 protein.
  • the composition is administered intravenously.
  • the composition is delivered to the brain of the primate subject.
  • the primate subject is 10 years old or younger.
  • the primate subject is 3 years old or younger.
  • the Shank3 protein is a miniShank3 protein.
  • the miniShank3 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 18 or 20.
  • FIGs. 1A-1E are images and graphs depicting results of behavioral studies involving marmosets administered gene therapy to test the atypical behavior reversal of the gene therapy.
  • Behavioral data was extracted from video in many behavioral settings, demonstrated by the exemplary video collection, trace, and unsupervised behavioral states (e.g., active, social, alone, and climbing) as shown in FIG. 1 A.
  • the frequency of time in different behavioral states was correlated in wild type (WT) and Shank3+/- (SH) marmosets relative to the cage average frequency showing that Shank3+/- marmoset behavior is less correlated to cagemates, as shown in the correlation graphs in FIGs. IB and 1C.
  • Exemplary recordings of four animals and high and low correlations are shown in FIG.
  • FIGs. 2A-2D are images and graphs depicting results of touchscreen-based behavioral stimuli studies involving marmosets administered gene therapy to test the atypical behavior reversal of the gene therapy. Exemplary images and results are shown in FIG. 2A.
  • the correlation with cagemates in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy -treated (GT) marmosets shows that Shank3+/- marmoset touchscreen shape and face behavior is less correlated to cagemates and gene therapy restores it to WT level, as shown in FIGs. 2B and 2C.
  • Similar results in response to touchscreen with reward in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy-treated (GT) marmosets are shown in FIG. 2D.
  • FIGs. 3A-3D are images and graphs depicting results of negative valence behavioral stimuli studies involving marmosets administered gene therapy to test the atypical behavior reversal of the gene therapy. Exemplary images and results are shown in FIG. 3 A.
  • the correlation with cagemates in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy -treated (GT) marmosets shows that Shank3+/- marmoset noise and scary mask response behavior is less correlated to cagemates and gene therapy reverses this behavior, as shown in FIGs. 3B and 3C.
  • Similar results in response to mask (negative stimulus), measured by time near mask in all ages in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy-treated (GT) marmosets are shown in FIG. 3D.
  • FIGs. 4A-4C are images and graphs depicting results of social behavioral stimuli studies involving marmosets administered gene therapy to test the atypical behavior reversal of the gene therapy. Exemplary images and results are shown in FIG. 4A.
  • the correlation with cagemates in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy-treated (GT) marmosets shows that Shank3+/- marmoset novel toys and intruder marmoset response behavior is less correlated to cagemates and gene therapy reverses this behavior, as shown in FIGs. 4B and 4C.
  • FIGs. 5A-5C are images and graphs depicting results of positive valence behavioral stimuli studies involving marmosets administered gene therapy to test the atypical behavior reversal of the gene therapy. Exemplary images and results are shown in FIG. 5A.
  • the correlation with cagemates in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy -treated (GT) marmosets shows that Shank3 novel object and food treat response behavior is less correlated to cagemates and gene therapy reverses this behavior, as shown in FIGs. 5B and 5C.
  • FIGs. 6A-6B are images and graphs depicting results of behavioral studies involving marmosets administered gene therapy to test the atypical social behavior reversal of the gene therapy. Exemplary images and results of control and Shank3 are shown in FIG. 6A.
  • the relative interest before pairing (y-axis) and relative velocity when paired (x-axis) was quantified in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy-treated (GT) marmosets showing that Shank3+/- (SH) animals are less interested in novel animals relative to wild type (WT) animals and gene therapy reverses this behavior, as shown in FIG. 6B.
  • FIGs. 7A-7F are images and graphs depicting results of cognitive task studies involving marmosets administered gene therapy to test the cognitive deficit reversal of the gene therapy.
  • the cognitive tasks studied test motivation, flexibility, and memory.
  • motivational component of cognitive tasks includes free reward, sucrose preference, and progressive ration.
  • Examples of intellectual component cognitive tasks studied were choice task, association updating, location updating, working memory, and foraging.
  • the percent of correct touches was quantified in control (wild type/WT) and Shank3+/- (SH) mice on Day 1 and Day 2 demonstrating that Shank3+/- (SH) animals have lower accuracy and lower trial number than wild type (WT), as shown in FIGs. 7A and 7B.
  • FIG. 7C Exemplary results of fixed position touches in wild type (WT), Shank3+/- (SH), and mini-SHANK3 gene therapy- treated (GT) marmosets are shown in FIG. 7C.
  • the percentage (%) of touches near center in different touch location tasks i.e., free reward, choice, updating, moving, and working memory
  • mini-SHANK3 gene therapy -treated (GT) marmoset has more task- related touches across tasks, as shown in FIG. 7D.
  • the number of trials in different touch location tasks i.e., free reward, choice, updating, moving, and working memory
  • mini-SHANK3 gene therapy -treated (GT) marmoset have increased number of trials across tasks, as shown in FIG. 7E.
  • the percentage (%) total accuracy in different touch location tasks shows that mini- SHANK3 gene therapy -treated (GT) marmoset have improved accuracy in some tasks, as shown in FIG. 7F.
  • FIGs. 8A-8C are fMRI images and graphs depicting results of studies involving marmosets administered gene therapy to test the abnormal connectivity reversal of the gene therapy.
  • aspects of the disclosure relate to gene therapy approaches for treating neurodevelopmental disorders.
  • the Examples show that treatment of SHANK3+/- primates with mini-SHANK3 gene therapy restores atypical behavior, atypical social behavior, cognitive deficits, communication deficits, and/or abnormal connectivity to levels at or near that of WT primates.
  • Gene therapy strategies disclosed herein use an AAV system to deliver a functional copy of the Shank3 gene into brain cells to restore cellular function.
  • SHANK3 encodes synaptic scaffolding proteins at the excitatory glutamatergic synapses, coordinates the recruitment of signaling molecules and orchestrates assembly of the macromolecular postsynaptic protein complex, which is crucial for proper synaptic development and function. Deletion of SHANK3 is a major cause of the core neurodevelopmental and neurob ehavi oral deficits in Phelan-McDermid syndrome. Human genetic studies also identified SHANK3 mutations as accounting for about 1% of autism spectrum disorder (ASD).
  • ASSD autism spectrum disorder
  • Shank3 The association of ASD with Shank3 provided an immediate link between synaptic dysfunction and the pathophysiology of ASD.
  • Animal models bridge the human genetics of ASD to brain pathology underlying clinical presentation, and ultimately help to discover and evaluate effective therapeutics.
  • Previous studies in flies, fish, and rodents have revealed synaptic dysfunction and behavioral abnormalities due to loss of SHANK3.
  • disruption of Shank3 in mouse models have resulted in synaptic defects, impaired social interactions, motor difficulties, repetitive grooming and increased anxiety level.
  • Shank3 deficiency causes severe sleep disturbances in rodents, monkeys and human patients, sleep efficiency provides a unique biomarker for ASD.
  • the Nra/AJ-deficient mouse model presents predictive validity as the synaptic defects and behavioral abnormalities are reversible when Shank3 is restored. Therefore, gene replacement is well suited as a therapeutic strategy for this monogenic disease.
  • Novel recombinant adeno-associated viruses represent a promising gene delivery platform because of their wide range of tissue tropism, low immunogenicity, highly efficient and sustained gene transduction, and clinically proven track record in safety.
  • Shank3 is a large protein with a coding sequence of about 5.7kb, exceeding the packaging capacity of AAV vectors.
  • Shank3 protein certain regions of the Shank3 protein were found not to be critical for the function of the protein, and therefore, heterologous Shank3 expression constructs were designed that have a significantly smaller coding sequence (about 2.1 kb to about 3.1 kb) because they encode for a version of the Shank3 protein that has certain non-essential regions removed.
  • the resulting miniaturized Shank3 proteins can be delivered by vector such as AAVs.
  • a miniaturized Shank3 protein can restore defective functions caused by deletion or mutation of the gene encoding the Shank3 protein in a mouse model, and accordingly, can potentially rescue abnormalities caused by diseases that are associated with Shank3 mutations or deletions.
  • the present disclosure relates to methods and compositions for treating neurodevelopmental disorders by restoring the activity of Shank3 using a miniaturized Shank3 protein (“MiniShank3”).
  • Shank family of proteins are master scaffolding proteins that tether and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five main domain regions: N-terminal ankyrin repeats, SH3 domain, PDZ domain, proline-rich region, and a C-terminal SAM domain. Through these functional domains, Shank proteins interact with many postsynaptic density (PSD) proteins. Without wishing to be bound by any theory, Shank proteins can bind to SAPAP which in turn binds to PSD95 to form the PSD95/SAPAP/Shank postsynaptic complex.
  • PSD postsynaptic density
  • these multidomain proteins are proposed to form a key scaffold, orchestrating the assembly of the macromolecular postsynaptic signaling complex at glutamatergic synapses.
  • This complex has been shown to play important roles in targeting, anchoring, and dynamically regulating synaptic localization of neurotransmitter receptors and signaling molecules.
  • the Shank family of proteins is connected to the mGluR pathway through its binding to Homer.
  • Shank Due to its link to actin-binding proteins, Shank also plays a major role in spine development. It has been found that transfection of Shank3 was sufficient to induce functional dendritic spine synapses in cultured aspiny cerebellar granule cells, indicating a role in spine induction. Shank3 has three primary isoforms including Shank3 a , the longest Shank3 isoform, Shank3p and Shank3 y . It has been reported that siRNA knockdown of Shank3 reduced the number and increased the length of dendritic spines in DIV18 cultured hippocampal neurons, implicating a role in spine maturation. This proposed function was supported by the finding that overexpression of Shankl enlarged already present dendritic spines in cultured hippocampal neurons. Furthermore, Shankl mutant mice have been reported to have smaller dendritic spines and weaker synaptic transmission.
  • the present disclosure relates to Shank proteins that are capable of restoring synaptic activity in subjects with disrupted Shank protein activity.
  • the disrupted Shank protein activity is present in subjects who have neurodevelopmental disorders, an autism spectrum disorder (ASD), and/or Phelan- McDermid syndrome.
  • the Shank proteins associated with the present disclosure are Shankl proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shankl or a variant of Shankl.
  • the Shank proteins in the present disclosure are Shank2 proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank2 or a variant of Shank2.
  • the Shank proteins in the present disclosure are Shank3 proteins.
  • the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding Shank3 or a variant of Shank3. It should be appreciated that Shank proteins associated with the present disclosure can include any Shank protein, including variants or fragments thereof, that function as scaffolding proteins at the synapses of excitatory neurons.
  • Shank proteins (Shankl, Shank 2, and Shank3) for use in gene therapy.
  • the Shank3 full length human protein sequence corresponding to SEQ ID NO: 6 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_001372044, provided by SEQ ID NO: 16:
  • the full-length Shank3 protein comprises multiple domains and is encoded by a gene that is about 5.2 Kb in size. Due to its size, it is difficult to deliver full-length Shank3 to a tissue or cell of interest via an AAV vector. As disclosed in and incorporated by reference from PCT Publication No. W02022/040239 and US Patent Publication No.
  • Shank proteins e.g., Shank3 proteins
  • polynucleotides described herein can be miniaturized to form a shortened variant of the native, full length Shank3 protein.
  • a miniaturized Shank3 protein, or a DNA construct encoding the miniaturized Shank3 protein are referred to interchangeably as “miniShank3” or “MiniShank3.”
  • the Shank3 protein disclosed herein is expressed as a miniaturized Shank3 DNA construct.
  • the variant Shank3 DNA constructs and the Shank3 proteins disclosed herein (MiniShank3) comprise fewer domains than the full-length Shank3 gene and protein.
  • the Shank3 protein disclosed herein is encoded by a non-naturally occurring polynucleotide.
  • Shank3 proteins encoded by polynucleotides described herein can include one or more protein domains.
  • Shank3 proteins can include one or more of: an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin domain, a SAM domain, and/or an ankyrin repeat domain.
  • the SH3 domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5.
  • the SH3 domain comprises at least 90% identity to residues 474-525 of SEQ ID NO: 6.
  • the SH3 domain comprises at least 90% identity to residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain can comprise any percent identity to residues 474-525 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SH3 domain can comprise any percent identity to residues 473-524 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the PDZ domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5.
  • the PDZ domain comprises at least 90% identity to residues 573-662 of SEQ ID NO: 6.
  • the PDZ domain comprises at least 90% identity to residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain can comprise any percent identity to residues 573-662 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the PDZ domain can comprise any percent identity to residues 572-661 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the Homer binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1294-1323 of SEQ ID NO: 5 or 6.
  • the Homer domain comprises at least 90% identity to residues 1294- 1323 of SEQ ID NO: 5.
  • the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 6.
  • the Homer domain comprises residues 1294-1323 of SEQ ID NO: 5 or 6. In some embodiments, the Homer domain can comprise any percent identity to residues 1294-1323 of SEQ ID NO: 5 or 6 suitable for construction of the MiniShank3.
  • the Cortactin binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1400-1426 of SEQ ID NO: 5 or 6.
  • the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 5.
  • the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 6. In some embodiments, the Cortactin binding domain comprises residues 1400-1426 of SEQ ID NO: 5 or 6. In some embodiments, the Cortactin binding domain can comprise any percent identity to residues 1400-1426 of SEQ ID NOs: 5 or 6 suitable for construction of the MiniShank3.
  • the SAM domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1664-1729 of SEQ ID NO: 6 or to residues 1663- 1728 of SEQ ID NO:5.
  • the SAM binding domain comprises at least 90% identity to residues 1664-1729 of SEQ ID NO: 6.
  • the SAM binding domain comprises at least 90% identity to residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM domain comprises residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM domain comprises residues 1663-1728 of SEQ ID NO:5. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1664-1729 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1663- 1728 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the ankyrin repeat domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 148-345 of SEQ ID NO: 6 or to residues 147-313 of SEQ ID NO: 5.
  • the ankyrin repeat domain comprises at least 90% identity to residues 148-345 of SEQ ID NO: 6.
  • the ankyrin repeat domain comprises at least 90% identity to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 148-345 of SEQ ID NO: 6 suitable for construction of the MiniShank3. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 147- 313 of SEQ ID NO: 5 suitable for construction of the MiniShank3.
  • the MiniShank3 protein is less than 65% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniShank3 protein is less than 65% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. As used herein, “less than 65%” includes any percent identity less than 65% that is suitable for construction of the MiniShank3.
  • the MiniShank3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5.
  • the MiniShank3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 17-20, provided in Table 1.
  • the MiniShank3 protein comprises any one of SEQ ID NOs: 17-20.
  • SEQ ID NO: 17 is encoded by SEQ ID NO: 1.
  • SEQ ID NO: 18 is encoded by SEQ ID NO: 2.
  • SEQ ID NO: 19 is encoded by SEQ ID NO: 3.
  • SEQ ID NO: 20 is encoded by SEQ ID NO: 4.
  • the MiniShank3 protein comprises an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein comprises an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NOs: 19 and/or 20.
  • the MiniShank3 protein does not comprise an ankyrin repeat domain. In certain embodiments in which the MiniShank3 protein does not comprise an ankyrin repeat domain, the MiniShank3 protein comprises SEQ ID NOs: 17 and/or 18.
  • sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or are 100% identical, including all values in between, to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity.
  • sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise at least 90% identity to any one of SEQ ID NOs: 1-4, and encode one or more proteins with Shank3 activity. In some embodiments, the sequences of polynucleotides encoding MiniShank3 proteins associated with the disclosure comprise any one of SEQ ID NOs: 1-4. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins with partial Shank3 activity. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins with full Shank3 activity.
  • the MiniShank3 is encoded by any one of SEQ ID NOs: 1-4, provided in Table 1.
  • SEQ ID NO: 1 and SEQ ID NO: 3 correspond to mouse MiniShank3 nucleic acid sequences
  • SEQ ID NO: 2 and SEQ ID NO: 4 correspond to human MiniShank3 nucleic acid sequences.
  • SEQ ID NO: 1 and SEQ ID NO: 2 encode MiniShank3 proteins that do not comprise an ankyrin repeat domain or the N-terminal domain.
  • SEQ ID NO: 3 and SEQ ID NO: 4 encode MiniShank3 proteins that comprise an ankyrin repeat domain and the N-terminal domain.
  • MiniShank3 proteins encode proteins that have at least partial Shank3 activity.
  • identity refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by a mathematical model, algorithm, or computer program that is known to one of ordinary skill in the art.
  • the percent identity of two sequences may, for example, be determined using Basic Local Alignment Search Tool (BLAST®) such as NBLAST® and XBLAST® programs (version 2.0).
  • BLAST® Basic Local Alignment Search Tool
  • Alignment technique such as Clustal Omega may be used for multiple sequence alignments.
  • Other algorithms or alignment methods may include but are not limited to the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, or Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).
  • a polynucleotide encoding the Shank protein as disclosed herein is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about
  • the polynucleotide encoding the Shank protein as disclosed herein can be in any size that is suitable for the methods and vectors disclosed in the present disclosure.
  • compositions and methods suitable for treating a neurodevelopmental disorder such as an autism spectrum disorder (ASD), or Phelan- McDermid syndrome.
  • ASD autism spectrum disorder
  • Phelan- McDermid syndrome Provided are methods of treating or ameliorating symptoms of an autism spectrum disorder (ASD) or Phelan-McDermid syndrome or a syndrome associated with a SHANK3 mutation in a primate subject in need thereof, the method comprising administering to the primate subject a therapeutically effective amount of a composition comprising an AAV vector, wherein the AAV vector comprises a) an artificial genome comprising a polynucleotide encoding a transgene comprising a miniShank3 protein comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 17-20 and has SHANK3 activity, wherein the transgene is operably linked to an hSYN promoter comprising SEQ ID NO: 22 and b) a capsid protein, wherein the capsid protein is AAV9 or
  • neurodevelopmental disorder refers to any disorder that impairs the growth and/or development of the brain and/or central nervous system.
  • neurodevelopmental disorders impact one or more brain functions, such as emotion, learning ability, self-control, and memory. It should be appreciated that aspects of the disclosure may be applicable for treatment of any neurodevelopmental disorder.
  • the neurodevelopmental disorder is an autism spectrum disorder (ASD).
  • ASD autism spectrum disorder
  • Diagnosis of ASDs is mainly based on criteria such as deficits in communication, impaired social interaction, and repetitive or restricted interests and behaviors.
  • ASDs are highly heritable disorders with concordance rates as high as 90% for monozygotic twins.
  • ASDs are clinically heterogeneous, covering a wide range of discrete disorders of differential symptomatic severity.
  • ASDs are believed to be etiologically heterogeneous, possibly encompassing polygenic, monogenic and environmental factors.
  • Alterations in synaptic connectivity and function have been proposed as a key mechanism underlying ASDs.
  • Shank3 gene disruption and/or mutation as a monogenic cause of autism spectrum disorder (ASD).
  • ASD autism spectrum disorder
  • Intellectual disability refers to a disability that causes a subject to have deficits in intellectual functioning and/or adaptive functioning.
  • Intellectual functioning can include, for example, reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and/or experiential learning.
  • Intellectual functioning can be measured using any method known in the art, such as by IQ tests.
  • Adaptive functioning can include, for example, skills needed to live in an independent and responsible manner such as communication and social skills. In some instances, intellectual disability can be evident during childhood or adolescence.
  • the neurodevelopmental disorder is Phelan-McDermid syndrome (PMS, 22ql3.3 deletion syndrome), which is an autism spectrum disorder that shows autistic-like behaviors, hypotonia, severe intellectual disability and impaired development of speech and language.
  • Shank3 is one of the genes that has been reported to be deleted in Phelan-McDermid syndrome. Disruption of Shank3 is thought to be the cause of the core neurodevelopmental and neurobehavioral deficits in Phelan-McDermid syndrome because individuals carrying a ring chromosome 22 with an intact Shank3 gene are phenotypically normal.
  • ADHD attention- deficit/hyperactivity disorder
  • learning disabilities such as dyslexia or dyscalculia
  • intellectual disability mental retardation
  • conduct or motor disorders cerebral palsy
  • impairments in vision and hearing developmental language disorder
  • neurogenetic disorders such as Fragile X syndrome, Down syndrome, Rett syndrome, hypogonadotropic hypogonadal syndromes, and traumatic brain injury.
  • the subject in need of treatment has poor response to negative stimuli, such as noises and scary masks.
  • the primate subject has at about 3 months, about 6 months or about one year after said administering a decreased response to negative stimuli compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the decreased response to negative stimuli is statistically significant and/or clinically significant improvement relative to the pre-treatment subject or based upon natural history of the disease.
  • the subject in need of treatment has low motivation, flexibility and/or memory.
  • the primate subject has at about 3 months, about 6 months or about one year after said administering increased motivation, flexibility, and memory compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the increased motivation, flexibility, and memory is statistically significant and/or clinically significant improvement relative to the pre-treatment subject or based upon natural history of the disease.
  • the subject in need of treatment has a low response to reward stimuli.
  • the primate subject at about 3 months, about 6 months or about one year after said administering has an increased response to reward compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the increased response to a reward is statistically significant and/or clinically significant improvement relative to the pre-treatment subject or based upon natural history of the disease.
  • the subject in need of treatment has a cognitive deficit and/or intellectual disability.
  • the primate subject at about 3 months, about 6 months or about one year after said administering has decreased cognitive deficit and/or intellectual disability compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the decreased cognitive deficit and/or intellectual disability is statistically significant and/or clinically significant improvement relative to the pretreatment subject or based upon natural history of the disease.
  • the subject in need of treatment has abnormal fMRI connectivity in various parts of the brain.
  • the primate subject at about 3 months, about 6 months or about one year after said administering has increased resting fMRI connectivity lower in anterior/medial areas compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the primate subject at about 3 months, about 6 months or about one year after said administering has decreased resting fMRI connectivity lower in posterior/lateral (sensory) areas compared to the primate subject before treatment or a similarly situated untreated control primate.
  • the primate subject after said administering has MRI improvements more than 12 months or more than 18 months or more than two years after administration of the therapeutically effective amount of the composition.
  • the improved fMRI connectivity is statistically significant and/or clinically significant improvement relative to the pre-treatment subject or based upon natural history of the disease.
  • a subject to be treated by methods described herein may be a human subject or a nonhuman subject.
  • Non-human subjects include, for example: non-human primates; farm animals, such as cows, horses, goats, sheep, and pigs; pets, such as dogs and cats; and rodents.
  • the primate subject is human.
  • a subject to be treated by methods described herein may be a subject having, suspected of having, or at risk for developing a neurodevel opmental disorder. In some embodiments, a subject has been diagnosed as having a neurodevel opmental disorder, while in other embodiments, a subject has not been diagnosed as having a neurodevel opmental disorder.
  • the subject is a human subject having, suspected of having, or at risk for developing an autism spectrum disorder (ASD). In some embodiments, the subject is a human subject having, suspected of having, or at risk for developing Phelan- McDermid syndrome. In some embodiments, the subject is a subject having a Shank3 gene mutation. In some embodiments, the subject is a subject having reduced expression of the Shank3 gene relative to a control subject.
  • ASD autism spectrum disorder
  • the subject is a human subject having, suspected of having, or at risk for developing Phelan- McDermid syndrome.
  • the subject is a subject having a Shank3 gene mutation. In some embodiments, the subject is a subject having reduced expression of the Shank3 gene relative to a control subject.
  • the expression of the Shank3 gene is reduced in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevel opmental disorder.
  • the reduced expression of the Shank3 gene in a subject is caused by disruption of at least one copy of the Shank3 gene.
  • the disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene.
  • the disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.
  • the subject is a human subject who exhibits one or more symptoms of an ASD.
  • the subject is a human subject who exhibits developmental delay.
  • the subject is a human subject who exhibits intellectual disability (ID).
  • the subject is a human subject who exhibits sleep disturbance.
  • the subject is a human subject who exhibits hypotonia.
  • the subject is a human subject who exhibits lack of speech.
  • the subject is a human subject who exhibits language delay.
  • the subject is a human subject who exhibits any symptoms or signs of an ASD.
  • a subject is a human subject who is an adult.
  • the adult is older than 25 years of age. In some embodiments, the adult is not older than 25 years of age. In some embodiments, the adult is not older than 21 years of age. In some embodiments, the adult is not older than 18 years of age. In some embodiments, the adult is 16 years of age. In some embodiments, a subject is elderly (e.g., 65 years old or older). In some embodiments, the adult can be any age of adulthood that is suitable for the treatment disclosed herein. [0087] In some embodiments, the subject is a human subject who is not an adult. In some embodiments, the human subject is not older than 16 years of age. In some embodiments, the human subject is not older than 10 years of age.
  • the human subject is 10 years of age or younger. In some embodiments, the human subject is 3 years of age or younger. In some embodiments, the human subject is a child or an infant. In some embodiments, the human subject is a toddler. In some embodiments, the human subject is at the fetal stage of development. In some embodiments, the human subject is at the prenatal stage of development.
  • polynucleotides encoding MiniShank3 proteins can be delivered to a tissue or cell of interest in a viral vector.
  • Vectors described herein can be used to deliver a nucleic acid encoding a protein of interest to a subject, including, e.g., delivery to specific organs or to the central nervous system (CNS) of a subject.
  • the protein of interest is a Shank protein.
  • the protein of interest is a Shank3 protein.
  • the protein of interest is a MiniShank3 protein.
  • the present disclosure provides a vector comprising a polynucleotide encoding a Shank protein disclosed herein. In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a Shank3 protein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector.
  • AAV refers to a replication-deficient Dependoparvovirus within the Parvoviridae genus of viruses.
  • AAV can be derived from a naturally occurring virus or can be recombinant.
  • AAV can be packaged into capsids, which can be derived from naturally occurring capsid proteins or recombinant capsid proteins.
  • the single-stranded DNA genome of AAV includes inverted terminal repeat (ITRs). ITRs are involved in the replication and encapsidation of the AAV genome, along with its integration in the host genome and its excision.
  • ITRs inverted terminal repeat
  • AAV vectors can comprise one or more ITRs, including a 5’ ITR and/or a 3’ ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and/or additional posttranscriptional regulator elements.
  • AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary skill in the art, as described, for example, in Sambrook et. al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (2012)), which is incorporated herein by reference in its entirety.
  • AAV integrates into a host cell genome. In some embodiments, AAV does not integrate into a host genome.
  • AAV vectors disclosed herein can include sequences from any known organism. In some embodiments, AAV vectors disclosed herein can include synthetic sequences. AAV vector sequences can be modified in any way known to one of ordinary skill in the art, such as by incorporating insertions, deletions or substitutions, and/or through the use of posttranscriptional regulatory elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation signals. In some embodiments, AAV vectors can include sequences related to replication and integration.
  • a MiniShank3 as disclosed herein is delivered to a tissue or a cell of interest via an AAV vector.
  • the AAV vector delivering the MiniShank3 as disclosed herein is delivered to the central nervous system (CNS) of a subject.
  • delivering the AAV vector to the CNS may include delivering the AAV vector to any tissue or cell of interest in the CNS.
  • delivering the AAV vector to the CNS involves delivering the AAV vector to neuronal tissues or cells.
  • delivering the AAV vector to the CNS involves delivering the AAV vector to the brain.
  • delivering the AAV vector to the CNS involves delivering the AAV vector to the spinal cord.
  • delivering the AAV vector to the CNS involves delivering the AAV vector to the white and gray matter.
  • the AAV vector delivering the MiniShank3 as disclosed herein is delivered to any tissue or cell of interest of a subject that is suitable for the treatments as disclosed herein.
  • delivering” or “administering” an AAV vector can include any method known in the art for delivering or administering an AAV vector or a composition comprising an AAV vector to a subject.
  • Administering can include but is not limited to direct administration of an AAV vector or a composition comprising the AAV vector, or peripheral administration via passive diffusion or convection-enhanced delivery (CED) to bypass the blood brain barrier as known in the art.
  • AAV vectors described herein can be administered in any composition that would be compatible with aspects of the disclosure.
  • AAV vectors can include any known AAV serotype, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.
  • the AAV serotype is AAV9. Clades of AAV viruses are described in, and incorporated by reference, from Gao et al. (2004) J. Virol. 78(12):6381-6388. In some embodiments, any AAV serotype that is suitable for delivery to the CNS may be selected.
  • AAV vectors of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype.
  • the AAV vector may utilize or be based on an AAV serotype described in WO 2017/201258A1, the contents of which are incorporated herein by reference in its entirety, such as, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6
  • AAV vectors are described further in US 9,585,971, US 2017/0166926, and W02020/160337, which are incorporated by reference herein in their entireties.
  • a MiniShank3 disclosed herein is delivered by an AAV vector.
  • the AAV vector comprises a transgene and its regulatory sequences, and optionally 5' and 3' ITRs.
  • the transgene and its regulatory sequences are flanked by the 5’ and 3’ ITR sequences.
  • the transgene may comprise, as disclosed herein, one or more regions that encode a MiniShank3.
  • the transgene may also comprise a region encoding for another protein.
  • the transgene may also comprise one or more expression control sequences (e.g., a poly-A tail).
  • an AAV vector comprises at least AAV ITRs and a MiniShank3 transgene.
  • the AAV may be packaged into an AAV particle and administered to a subject and/or delivered to a selected target cell.
  • the AAV particle comprises an AAV capsid protein.
  • the AAV particle comprises at least one capsid protein that is selected from the AAV serotypes as disclosed herein including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.eB, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, and AAV2/2-125, or a variant of any of the foregoing.
  • the miniShank3 transgene coding sequence in the AAV vector is operably linked to regulatory sequences for tissue-specific gene expression.
  • the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • the tissuespecific regulatory sequence can be a Syn promoter (e.g., hSynl).
  • the tissue-specific regulatory sequence can be any promoter or enhancer that is neuron-specific and is suitable for the treatments described herein.
  • a miniShank3 transgene coding sequence comprising SEQ ID NO: 2 or 4 in an AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
  • a miniShank3 transgene coding sequence comprising SEQ ID NO: 1 or 3 in an AAV vector is operably linked to a promoter and is flanked by AAV ITRs.
  • aspects of the disclosure relate to AAV vectors expressing miniShank3 transgenes.
  • a miniShank3 transgene is flanked by AAV ITRs.
  • the AAV ITRs comprise AAV2 ITRs.
  • the AAV ITRs comprise AAV1 ITRs.
  • the AAV ITRs comprise AAV5 ITRs.
  • the AAV ITRs comprise AAV6 ITRs.
  • the AAV ITRs comprise AAV8 ITRs.
  • the AAV ITRs comprise AAV9 ITRs.
  • the AAV ITRs comprise rhlO ITRs.
  • the AAV ITRs may include self-complementary ITRs.
  • AAV vectors described herein can include DNA constructs that comprise a transgene such as MiniShank3, 5’ and/or 3’ ITRs, promoters, introns, and/or other associated regulatory elements that are known in the art.
  • a transgene such as MiniShank3, 5’ and/or 3’ ITRs, promoters, introns, and/or other associated regulatory elements that are known in the art.
  • the AAV vector comprises a Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element (WPRE), which may enhance miniShank3 transgene expression.
  • WPRE Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element
  • the AAV vector comprises an untranslated portion such as an intron or a 5’ or 3’ untranslated region.
  • the intron may be located between the promoter/enhancer sequence and the miniShank3 transgene.
  • the AAV vector used herein may be a self- complementary vector.
  • SEQ ID NO: 21 comprises a human Mini-Shank3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, an Fl origin, a NeoR/KanR marker, a hSynl promoter, and a PUC origin.
  • the Inverted terminal repeat (ITR) sequences comprise about 145 nucleotides each. These elements may be useful in cis for effective replication and encapsidation. A skilled person in the art would appreciate that any elements of AAV vectors known in the art may be compatible with aspects of the disclosure.
  • DNA constructs described herein that encode a functional MiniShank3 protein can be expressed in a DNA construct for AAV delivery.
  • DNA constructs may include one or more elements, such as: a 5’ ITR, a 3’ ITR, a WPRE such as WPRE3, a poly A such as an hGH poly A (including modified versions of an hGH poly A), an Fl origin, a NeoR/KanR marker, a hSynl promoter, miRNA binding sites, and/or a PUC origin.
  • a coding sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 1-4 is expressed in a DNA construct.
  • a coding sequence comprising the sequence of any one of SEQ ID NOs: 1-4 is expressed in a DNA construct.
  • the DNA construct includes one or more of elements such as a promoter, a 5 ’-ITR, a 3 ’-ITR, a WPRE, an hGH poly A, an Fl origin, a NeoR/KanR marker, a hSynl promoter, one or more miRNA binding sites and/or a PUC origin.
  • an AAV vector associated with the disclosure includes a nucleic acid sequence encoding a MiniShank3 protein that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 21.
  • an AAV vector comprises a sequence corresponding to SEQ ID NO: 21, which encodes a MiniShank3 protein comprising the sequence of SEQ ID NO: 18.
  • an AAV vector that includes a sequence that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 21, and which encodes a MiniShank3 protein comprising the sequence of SEQ ID NO: 18, may be delivered to a human subject in need thereof and may be suitable for treating a human subject who has a neurodevel opmental disorder.
  • any method known in the art for designing AAV vectors for clinical use, and for delivery of AAV vectors may be compatible with aspects of the disclosure.
  • disclosure related to AAV vectors and delivery are provided in and incorporated by reference from U.S. Patent No. 7,906,111, entitled “Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor” and U.S. Patent No. 9,834,788, entitled “AAV-vectors for use in gene therapy of choroideremia,” each of which is incorporated by reference herein in its entirety.
  • an AAV vector associated with the disclosure includes a sequence encoding a MiniShank3 protein for AAV delivery that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 7 or 21, provided in Table 1.
  • 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.
  • the AAV vector encoding a MiniShank3 protein for AAV delivery encodes a protein with a sequence that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, to any one of SEQ ID NOs: 11 or 17-20, provided in Table 1.
  • the vector used for delivering the miniShank3 as disclosed herein can be a lentivirus vector. In some embodiments, the vector used for delivering the miniShank3 as disclosed herein can be an adenovirus vector.
  • the vector construct disclosed herein can comprise SEQ ID NO: 21 as shown in Table 1.
  • the vector comprising the polynucleotide of the Shank3 protein can be expressed in a specific tissue or cell of interest.
  • the vector disclosed herein comprises a promoter.
  • the vector comprises a cell-type specific promoter.
  • the promoter is a human promotor.
  • the human promoter is human Synapsin 1 (hSynl).
  • the hSynlpromotor has a polynucleotide sequence corresponding to SEQ ID NO: 22.
  • the human promoter can be any promotor that is known in the art and is suitable for construction of the miniShank3.
  • the human promoter can be any promoter that has high specificity for neuronal tissues and cells.
  • the promoter can be a constitutive promoter.
  • the constitutive promoter can be a CAG promoter.
  • any promoter may be used so long as the selected promoter is compatible with aspects of the disclosure.
  • compositions including pharmaceutical compositions, comprising a polynucleotide (e.g., miniShank3) delivered in an AAV vector as disclosed herein and a pharmaceutically acceptable carrier.
  • a polynucleotide e.g., miniShank3
  • compositions of the disclosure may comprise an AAV alone, or in combination with one or more other viruses.
  • a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different AAVs each having one or more different Shank protein.
  • Suitable carriers may be readily selected by one of ordinary skill in the art in view of the indication for which the AAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the selection of the carrier is not a limitation of the present disclosure.
  • Pharmaceutical compositions comprising AAV vectors are described further in US 9,585,971 and US 2017/0166926, which are incorporated by reference herein in their entireties.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • dispersion media includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the AAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the AAV constructs disclosed herein.
  • liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • nanocapsule formulations of the AAV vector may be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • the pharmaceutical composition comprising a nucleic acid delivered in an AAV vector comprises other pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, thimerosal, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • isotonic agents for example, sugars or sodium chloride. Prolonged absorption of the pharmaceutical compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical forms suitable for delivering the AAV vectors include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Methods described herein comprise administering AAV vector in sufficient amounts to transfect the cells of a desired tissue (e.g., brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • a desired tissue e.g., brain
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ, oral, inhalation, intraocular, intravenous including facial vein injection and retroorbital injection, intracerebroventricular (ICV), intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.
  • the vector as disclosed herein is administered intravenously.
  • the present disclosure provides methods of treating a subject having a neurodevel opmental disorder. In some embodiments, the present disclosure provides methods of treating a subject having an autism spectrum disorder (ASD). In some embodiments, the present disclosure provides methods of treating a subject having Phelan- McDermid syndrome. Methods provided herein, in some embodiments, comprise administering and delivering an effective amount of a composition comprising a vector that comprises a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue of a subject. In some embodiments, the target tissue is cortex. In some embodiments, the target tissue is striatum.
  • ASD autism spectrum disorder
  • the target tissue is thalamus cerebellum. In some embodiments, the target tissue is hippocampus. In some embodiments, the target tissue is any brain structure. In some embodiments, methods for administering and delivering an effective amount of a composition comprising a vector that comprises a polynucleotide encoding a Shank3 protein (e.g., miniShank3) to a target environment or tissue comprise delivering the composition to neurons or other brain cell types. In some embodiment, the vector is an AAV vector.
  • methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof comprise providing a composition comprising an AAV vector comprising at least a nucleic acid (e.g., miniShank3) to be delivered to the target environment or tissue of the subject and administering the composition to the subject.
  • AAV vector comprising at least a nucleic acid (e.g., miniShank3) to be delivered to the target environment or tissue of the subject and administering the composition to the subject.
  • Methods of use of AAV vectors are described further in US 9,585,971, US 2017/0166926, and W02020/160337, which are incorporated by reference herein in their entireties.
  • the composition may comprise a capsid protein.
  • the composition comprising a vector that comprises a polynucleotide encoding a Shank3 protein is delivered to the subject via intravenous administration, systemic administration, intracerebroventricular administration, in utero administration, intrathecal administration, retro-orbital injection, or facial vein injection.
  • in utero administration is used for a subject who is at the prenatal stage of development.
  • the composition is delivered to a subject via a nanoparticle.
  • the composition is delivered to a subject via a viral vector.
  • the composition is delivered to a subject via any carriers suitable for delivering nucleic acid materials.
  • composition comprising a vector that comprises a polynucleotide encoding a protein that would be of some use or benefit to the subject may be delivered to a target environment or tissue of the subject according to methods disclosed herein
  • Sonophoresis i.e., ultrasound
  • U.S. Pat. No. 5,656,016 has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system.
  • Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
  • the dose of AAV comprising a polynucleotide that encodes a Shank3 protein (e.g., miniShank3) required to achieve a particular "therapeutic effect," e.g., the units of dose in absolute vector genomes (vg) or vector genomes per milliliter of pharmaceutical solution (vg/mL) will vary based on several factors including, but not limited to: the route of AAV administration, the level of gene expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene product. Doses that give maximal percentage of infection without affecting neurodevelopment are also suitable.
  • An effective amount of AAV vector is an amount sufficient to infect an animal or human subject or target a desired tissue.
  • the effective amount will depend primarily on factors such as the species, age, gender, weight, health of the subject, and the tissue to be targeted, and may thus vary among subjects and tissues.
  • the term “effective amount” or “amount effective” in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject.
  • an effective amount of a composition disclosed herein may partially or fully rescue the effects of a mutated Shank3 gene and/or partially or fully restore loss of function of the Shank3 protein.
  • An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether.
  • An effective amount can also involve delaying the occurrence of an undesired response.
  • An effective amount can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result.
  • the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. The achievement of any of the foregoing can be monitored by routine methods and the methods as disclosed in the present application. Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors.
  • the number of vector genomes administered to the subject is any value between about 6.0xl0 n vg and about 9.0xl0 13 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 6.0 xlO 13 vg/mL and about 9.0 xlO 13 vg. In some embodiments, the number of vector genomes administered to the subject is any value between about lxl0 10 to about lxl0 12 vg. In certain embodiments, the effective amount of AAV is 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 genome copies per kg.
  • the effective amount of AAV is 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some cases, a dosage between about 10 11 to 10 13 AAV genome copies is appropriate. In some embodiments, the number of vector genomes administered to the subject can be any dose that is suitable for the treatments and methods disclosed herein.
  • a dose of AAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of AAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of AAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of AAV is administered to a subject no more than bi-weekly (e.g., once in a two calendar week period). In some embodiments, a dose of AAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days).
  • a dose of AAV is administered to a subject no more than once per six calendar months.
  • a dose of AAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).
  • a dose of rAAV is administered to a subject no more than once per two calendar years (e.g., 730 days or 731 days in a leap year).
  • a dose of AAV is administered to a subject no more than once per three calendar years (e.g., 1095 days or 1096 days in a leap year).
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions disclosed herein is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
  • these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Methods and compositions provided herein, in some embodiments, are useful for treating a neurodevelopmental disorder, such as, for example, an autism spectrum disorder (ASD), or Phelan-McDermid syndrome.
  • ASD autism spectrum disorder
  • Phelan-McDermid syndrome a neurodevelopmental disorder
  • delivery of a miniShank3 via a viral vector such as AAV vector in a mouse model is effective in restoring functionality of postsynaptic density (PSD) proteins.
  • expression levels of PSD proteins are used to evaluate the efficacy of the administration of the miniShank3.
  • the PSD protein is Homer.
  • the PSD protein is post-synaptic density protein 95 (PSD95). In some embodiments, the PSD protein is SynGapl. In some embodiments, the PSD protein is SAPAP3. In some embodiments, the PSD protein is NR1. In some embodiments, the PSD protein is NR2B. In some embodiments, the PSD protein is GluR2. In some embodiments, the PSD protein is any protein that can be improved or restored upon the miniShank3 treatment.
  • an increase of any of the PSD proteins, as compared to an untreated control subject, may indicate efficacy of the miniShank3.
  • Methods for detecting gene expression and protein levels are well-known in the art.
  • expression of Homer in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50- fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of post-synaptic protein (PSD95) in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100- fold, or at least 1000-fold compared to a control.
  • expression of SynGapl in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of SAPAP3 in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of NR1 in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2- fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of NR2B in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • expression of GluR2 in the subject after treated with the miniShank3 is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
  • a subject has improved sleep efficiency after being administered an effective amount of a composition comprising an expression construct comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the sleep efficiency in the subject after being administered to an effective amount of the composition is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Improved sleep efficiency includes less sleep disturbance, which includes but is not limited to having trouble falling and staying asleep. Measurement of sleep efficiency can be conducted using any methods known in the art. [00137] In some embodiments, administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving social impairment. In some embodiments, the social impairment of the subject is improved after being administered an effective amount of a composition comprising an expression construct comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the social impairment in the subject after being administered to an effective amount of the composition is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of social impairment can be conducted using any methods known in the art.
  • social impairment refers to behavioral abnormalities or defects that prohibit a subject from displaying voluntary social interaction.
  • administration of a MiniShank3 or a composition comprising a MiniShank3 can lead to improving locomotion and/or motor coordination deficits.
  • the locomotion and/or motor coordination deficits of the subject are improved after being administered an effective amount of a composition comprising an expression construct comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the locomotion and/or motor coordination deficits in the subject after being administered to an effective amount of the composition is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of locomotion and/or motor coordination deficits can be conducted using any methods known in the art.
  • locomotion and/or motor coordination deficits can include, for example, lack of coordination, loss of balance, and/or a shuffling gait.
  • administering can lead to improvement in cortical-striatal synaptic dysfunction.
  • the cortical-striatal synaptic dysfunction of the subject are improved after being administered an effective amount of a composition comprising an expression construct comprising a polynucleotide encoding a Shank protein such as a MiniShank3 protein.
  • the corti cal -striatal synaptic dysfunction in the subject after being administered to an effective amount of the composition is decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject.
  • Measurement of corti cal -striatal synaptic dysfunction can be conducted using any methods known in the art.
  • cortical-striatal synaptic dysfunction refers to defective corticostriatal circuits in the brain that can cause repetitive and compulsive behaviors, such as in neuropsychiatric disorders and neurodevelopmental diseases such as autism, obsessive- compulsive disorders, and Tourette syndrome.
  • the goal of this project is to develop a platform for studying neuropsychiatric disorders in nonhuman primates and testing the effectiveness of treatments. This should serve to focus human trials on the most promising options.
  • This effort was begun by establishing a monogenic Shank3 ASD model of mutant marmosets. Natural breeding was used to produce 16 young Shank3+/- marmosets. A wide range of assays was designed to study the animals’ natural social behavior, vocalizations, cognition, responses to a variety of stimuli, gait, and sleep quality. By comparing over 100 control animals with the 16 Shank3+/- marmosets, it was determined how their behaviors differed across all these assays.
  • the Shank3+/- marmosets had an increased fear of threatening stimuli, decreased interest in social stimuli, performed worse on cognitive tasks requiring flexibility, and atypical social interactions. Based on these results, a gene replacement therapy method was tested using an AAV injected via IV to express miniShank3 in neurons across the brain.
  • Shank3+/- animals which received gene therapy injections were found to have reduced fear of threatening stimuli, had increased interest in social stimuli, performed better on certain cognitive tasks, and had more typical social interactions.
  • a goal of this study was to determine if Shank3+/- marmosets exhibit ASD phenotypes and if these phenotypes were reverted by gene therapy.
  • the behavioral phenotypes tested were atypical behavior (restricted/repetitive behavior, unusual patterns of attention, unlike others), atypical social behavior (either uninterested, not understanding, or disliking social interactions), cognitive deficits (ASD often associated with severe intellectual disability), and abnormal connectivity (difference in resting-state fMRI scan or task-related fMRI scan).
  • Shank3+/- marmoset exhibit atypical behavior measured by unsupervised behavioral states (e.g., active, social, alone, and climbing) and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • unsupervised behavioral states e.g., active, social, alone, and climbing
  • miniShank3 SEQ ID NO: 21
  • FIGs. 2A-2D show that Shank3+/- marmoset exhibit atypical behavior measured by touchscreen-based behavioral stimuli and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • FIGs. 3 A-3D show that Shank3+/- marmoset exhibit atypical behavior measured by negative valence behavioral stimuli and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • FIGs. 4A-4C show that Shank3+/- marmoset exhibit atypical behavior measured by social behavioral stimuli and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • FIGs. 5A-5C show that Shank3+/- marmoset exhibit atypical behavior measured by positive valence behavioral stimuli and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • FIGs. 6A-6B show that Shank3+/- marmoset exhibit atypical social behavior measured by novel animal studies and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this behavior.
  • FIGs. 7A-7F show that Shank3+/- marmoset exhibit cognitive deficit measured by cognitive task studies and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this cognitive deficit.
  • FIGs. 8A-8C show that Shank3+/- marmoset exhibit abnormal functional connectivity measured by fMRI and gene therapy delivery of miniShank3 (SEQ ID NO: 21) reverses this cognitive deficit.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features.

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Abstract

Des aspects de la divulgation portent sur des polynucléotides d'origine non naturelle codant pour une protéine Shank3, des vecteurs de virus adéno-associés (VAA) comprenant les polynucléotides, et des méthodes de thérapie génique.
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