Gene therapy methods for shank3-associated disease

Abstract

Provided are methods of treating or ameliorating the symptoms of SHANK3 deficiencies, such as Phelan-McDermid Syndrome (PMS) by administration of therapeutically effective doses of recombinant adeno-associated viruses (rAAV) containing a transgene encoding a miniSHANK3 protein.

Description

Atty. Docket No. 38061.0013P1GENE THERAPY METHODS FOR SHANK3-ASSOCIATED DISEASE REFERENCE TO SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (38061_0013Pl_SL.xml; Size: 45,709 bytes; and Date of Creation: February 24, 2026) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Provided is a treatment of SHANK3 haploinsufficiency by administration of doses of gene therapy vectors, such as AAV gene therapy vectors, in which the transgene encodes a miniSHANK3 protein.BACKGROUND

[0003] SHANK3 (Src homology 3 [SH3] and multiple ankyrin repeat [ANK] domains 3) encodes a key postsynaptic scaffolding protein that is expressed in neuronal cells. As a “master” scaffolding protein, it forms a key structural component of the postsynaptic density (PSD) of glutamatergic synapses, which are the main excitatory’ synapses in the brain.

[0004] The SHANK3 protein sits beneath the cell membrane, where it functions to connect ion channels and glutamatergic receptors in the postsynaptic membrane to the cytoskeleton and to proteins that facilitate intraneuronal signaling pathways, thereby contributing to the integrity' and molecular composition of the synapse (Phelan, 2008).Adequate synapse function is an essential prerequisite of all neuronal processing, and higher cognitive functions, such as learning and memory.

[0005] Mutations within SHANKS or deletions in the distal long arm (or q arm) of chromosome 22 in the 22ql3.3 region encompassing SHANKS may result in SHANK3 haploinsufficiency.

[0006] Haploinsufficiency occurs when one copy of a gene is deleted or contains a loss of function mutation and the remaining functional copy of the gene cannot produce enough protein to preserve normal function. The loss of a functional copy of SHANK3 can be caused by various errors in genetic coding, including deletions, splice site mutations, and point mutations, such as nonsense, missense, or frameshift mutations. SHANK3 deletions andAtty. Docket No. 38061.0013P1mutations are highly penetrant, meaning that most patients with SHANK3 haploinsufficiency will develop clinical symptoms from the lack of sufficient SHANK3 protein expression.

[0007] When SHANK3 haploinsufficiency occurs, there is less SHANK.3 protein present in the PSD to bind and stabilize key receptors and associated signaling proteins in the neuron’s synaptic membrane. This reduction of key signaling proteins in excitatory' synapses leads to abnormal neuronal morphology, impaired neurotransmission, synaptic plasticity, and potentially toxic imbalances in excitatory transmission. These impacts at the neuronal level result in impaired neuronal communication, which translates functionally into an impaired ability' to leam, process information, and make memories. Clinically, this translates into a heterogeneous but always devastating neurodevelopmental disease that may be characterized by global developmental delay, lack of speech and communication, abnormal sleep, and severely impaired cognitive, social, and motor function disability that ultimately requires 24-hour care and supervision. Further, as these individuals age, they are at increased risk for significant developmental, cognitive, and neurobehavioral regression, which may result in permanent loss of acquired skills.

[0008] Clinically, SHANK3 haploinsufficiency is characterized by lifelong and severe neurobehavioral. developmental, and cognitive impairments. Due to the heterogeneous profile of presenting clinical symptoms and differences in patient diagnostic journeys, individuals may receive initial diagnoses of autism spectrum disorder (ASD), intellectual disability (ID), development delay, or a range of psychiatric disorders. However, once genetic testing confirms SHANKS haploinsufficiency via a loss of function mutation or deletion in the SHANK3 gene, patients can be formally diagnosed with Phelan-McDermid Syndrome (PMS). PMS is exclusively a genetic diagnosis and cannot be diagnosed via clinical diagnostic criteria.

[0009] As of December 2024, the Phelan-McDermid Sy ndrome Foundation (PMSF) had identified approximately 3,600 individuals globally with a diagnosis of PMS. Given that PMS diagnosis requires a genetic confirmation, the prevalence of PMS and SHANKS haploinsufficiency is likely to be substantially larger due to low rates of genetic testing. Beyond the already diagnosed population, SHANK3 genetic screening studies indicate that SHANK3 haploinsufficiency7may be a monogenic cause of ASD in approximately 0.5%-Atty. Docket No. 38061.0013P1may be indicative of more severe ASD, as they are primarily observed in patients with ASD combined with moderate to severe ID. SHANK3 haploinsufficiency remains largely undiagnosed in ASD as genetic screening is not routinely pursued, despite 2010 guidelines establishing this as the standard of care diagnostic test in ASD.

[0010] The diagnostic journey resulting in a genetic confirmation of SHANK3 haploinsufficiency is challenging. Families often observe a constellation of symptoms beginning with abnormal neurodevelopment and delayed cognitive, communicative, social, and motor milestone attainment. When families reach a diagnosis, they learn that there is no effective intervention or treatment that can provide a prospect of improvement or, at minimum, can prevent the loss of acquired skills.

[0011] There are no Food and Drug Administration (FDA)-approved treatments for PMS, despite its severely debilitating manifestations. Parents and caregivers report trying a high number of treatments to address the multitude of individual signs and symptoms, such as sleep difficulties, psychiatric symptoms, and seizures, caused by SHANK3 haploinsufficiency, with very little success and adverse effect profiles that make continuing on treatment difficult. In managing sleep difficulties, some families report utilizing a range of sleep medicines that may help individuals fall asleep, but not stay asleep, while others indicate that treatment benefits are temporary, and medicines eventually stop working. The management of psychiatric symptoms similarly requires combinations of medications including antipsychotics, neuroleptics, and sedatives to manage a range of neurobehavioral and psychiatric challenges. However, side effects associated with these treatments, ranging from development of addiction, weight gain, aggression, and seizures, produce additional challenges for families and make management difficult. For patients experiencing seizures, medication side effects include drowsiness, constipation, and rescue medications for severe or persistent seizures can stop respiration and require resuscitation. For these individuals, contraindications of seizure medications with other symptomatic treatments, together with challenging administration schedules associated with tailored treatment regimens, illustrate the challenges of treating a multitude of symptoms, each of which may require concurrent treatment with multiple drugs that might provide limited or temporary' benefit.

[0012] Provided herein are methods of administering AAV gene therapy vectors to treat PMS and SHANKS haploinsufficiency in human patients at therapeutic doses.Atty. Docket No. 38061.0013P1SUMMARY OF THE INVENTION

[0013] Provided are methods of treating or ameliorating the symptoms of SHANK.3 deficiencies, such as Phelan-McDermid Syndrome (PMS), by administration of therapeutically effective doses of recombinant adeno-associated viruses (rAAV) containing a transgene encoding a miniSHANK3 protein. Disclosed are therapeutic doses of the rAAV miniSHANK3 encoding vectors for unilateral ICV administration for adult or pediatric patients.

[0014] As described below in Example 4, three pediatric patients (Cohort 1) have been administered a single dose by unilateral ICV administration of a pharmaceutical composition comprising 1.91 *1014vector genome copies (vg) of rAAV miniSHANK3 (“AAV9 AAV-hSynl-HumanMmiSHANK3-Vl” (5’ ITR-hSynl- Human mmiSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8)) (also referred to herein as JAG201). All three Cohort 1 patients exhibited moderate to severe developmental impairments in communication, socialization, and motor function at baseline. No treatment-related serious adverse events (SAEs) or dose-limiting toxicities (DLTs) were observed for any of the three Cohort 1 patients as of 5 months to 1 year following administration of JAG201. Vineland-3 scores, PMS-Specific CG1-1 / PMSA-C scores, PMS-Specific CG1-S / PMSA-S scores, VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 1 at baseline (prior to administration of JAG201), and at specified time points following administration of JAG201, and the results of those evaluations are described herein and in Example 4.

[0015] As described below' in Example 5, tw o pediatric patients (Cohort 2) have been administered a single dose by unilateral ICV administration of a pharmaceutical composition comprising 5.73xl014vg of rAAV miniSHANK.3 (“AAV9 AAV-hSynl-HumanMiniSHANK3-Vl” (5’ ITR-hSynl- Human mmiSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8)) (JAG201). All Cohort 2 patients exhibited moderate to severe developmental impairments in communication, socialization, and motor function at baseline. No treatment-related serious adverse events (SAEs) or dose-limiting toxicities (DLTs) were observed for either of the Cohort 2 patients as of two weeks to 3 months following administration of JAG201. Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Specific CGI-S / PMSA-S scores, VEP waveforms, and AEP w aveforms w ere obtained forAtty. Docket No. 38061.0013P1each patient in Cohort 2 at baseline (prior to administration of JAG201), and have been or will be obtained from each patient at specified time points following administration of JAG201, and the results of those evaluations are described herein and in Example 5.

[0016] Provided are methods of treating or ameliorating the symptoms of a SHANK3 deficiency in a human subject in need thereof, comprising administering by intracerebroventricular (ICV) administration, a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle, including an AAV9 particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes aminiSHANK3 protein (including miniSHANK3 proteins having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4), operably linked to a regulatory’ element that promotes expression in central nervous system (CNS), and flanked by AAV ITR sequences (including an artificial genome comprising the nucleotide sequence of SEQ ID NO: 8 (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR)); wherein the therapeutically effective amount of the rAAV particle is administered via unilateral ICV administration at a dose of (i) 2.16x 1014+ / - 10% or 20% rAAV vector genomes (vg)(or between 2.1*1014to 2.2*1014vg), or 5.73xl014+ / - 10% or 20% vg (or between 5.7xl014to 5.8xl014vg), or 6.30xl014+ / - 10% or 20% vg (or between 6.2x1014to 6.4x1014vg) for an adult subject or a pediatric subject age 10 to 17, or (ii) at a dose of 1.91xl014+ / - 10% or 20% vg (or between 1.85xl014to 1.95xl014vg) or 5.73xl014+ / - 10% or 20% vg (or between 5.7xl014to 5.8xl014vg) for a pediatric subject age 2 to 9.

[0017] In embodiments, the therapeutic dosages result in changes in VEP and / or AEP waveforms compared to baseline, as well as improvement in (i) sensory' symptoms associated with SHANK3 mutations; (ii) adaptive behavior; (iii) global cognitive ability; (iv) ability to use language; (v) motor functioning; (vi) autism symptoms; (vii) sleep; and / or(viii) symptoms associated with Phelan-McDermid Syndrome (PMS) in patients having a Class I deletion or a Class II deletion associated with the loss of SHANK.3 activity.

[0018] Also provided is a unit dosage form comprising 2.15mL of a pharmaceutical composition comprising 6.5xl013+ / - 10% or 20% (or between 6.0xl013to 7.0x 1013) vg / mL of a recombinant adeno-associated vector (rAAV) particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a miniSHANK3 protein (including miniSHANK3 proteins having the amino acid sequence of SEQ ID NO: 3 or SEQAtty. Docket No. 38061.0013P1ID NO: 4). operably linked to a regulatory element that promotes expression in central nervous system (CNS) (including an hSynl promoter), and flanked by AAV ITR sequences; wherein the rAAV particle is formulated for intracerebroventricular (ICV) administration.EMBODIMENTS

[0019] Embodiment 1. A pharmaceutical composition for use in treating a SHANKS deficiency or a method of treating a SHANKS deficiency in a human subject in need thereof, comprising administering by intracerebroventricular (ICV) administration a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a miniSHANK3 protein, operably linked to a regulatory element that promotes expression in central nervous system (CNS), and flanked by AAV ITR sequences; wherein the therapeutically effective amount of the pharmaceutical composition is administered via ICV administration(i) to an adult subject or a pediatric subject age 10 to 17 at a dose of 2.16* 1014+ / - 10% or 20% rAAV vector genomes (vg) (or between 2.1 xlO14to 2.2*1014vg), or 5.73 xlO14+ / - 10% or 20% vg (or between 5.7 xlO14to 5.8 xlO14vg), or 6.30x1014+ / - 10% or 20% vg (or between 6.2xl014to 6.4xl014vg); or alternatively(ii) to a pediatric subject age 2 to 9 at a dose of 1.91 xlO14+ / - 10% or 20% vg (or between 1.85xl014to 1.95xl014vg) or 5.73xl014+ / - 10% or 20% vg (or between 5.7x1014to 5.8x1014vg); or alternatively(iii) to an adult subject or a pediatric subject age 10 to 17 at a dose of 2.16xl014vg or 5.73xl014vg or 6.30xl014vg; or alternatively(iv) to apediatric subject age 2 to 9 at a dose of 1.91X1014vg or 5.73xl014vg.

[0020] Embodiment 2. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to an adult subject via ICV administration (i) at a dose of 2.16x 1014+ / - 10% or 20% rAAV vector genomes (vg) (or between 2.1xl014to 2.2 xlO14vg); or alternatively (ii) at a dose of 5.73 xlO14+ / - 10% or 20% vg (or between 5.7xl014to 5.8xl014vg); or alternatively (iii) at a dose of 2.16x 1014vg; or alternatively (iv) at a dose of 5.73x 1014vg-Atty. Docket No. 38061.0013P1

[0021] Embodiment 3. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to an adult subject via ICV administration (i) at a dose of 6.30x 1014+ / - 10% or 20% vg (or between 6.2xl014to 6.4xl014vg); or alternatively (ii) at a dose of 6.30xl014vg.

[0022] Embodiment 4. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to a pediatric subject age 2 to 9 via ICV administration (i) at a dose of 1.91xl014+ / - 10% or 20% vg (or between 1.85xl014to 1.95xl014vg); or alternatively (ii) at a dose of 1.91 xlO14vg.

[0023] Embodiment 5. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to a pediatric subject age 2 to 9 via ICV administration at a dose of (i) 5.73xl014+ / - 10% or 20% vg (or between 5.7x1014to 5.8xl014vg); or alternatively (ii) at a dose of 5.73xl014vg.

[0024] Embodiment 6. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to a pediatric subject age 10 to 17 via ICV administration at a dose of (i) 2.16x 1014+ / - 10% or 20% rAAV vector genomes (vg) (or between 2.1 x 1014to 2.2x 1014vg); or alternatively (ii) at a dose of 5.73xl014+ / - 10% or 20% vg (or between 5.7xl014to 5.8xl014vg); or alternatively (iii) at a dose of 2.16xl014vg; or alternatively (iv) at a dose of 5.73 xlO14vg.

[0025] Embodiment 7. The pharmaceutical composition for use or the method according to embodiment 1, wherein the therapeutically effective amount of the rAAV particle is administered to a pediatric subject age 10 to 17 via ICV administration (i) at a dose of 6.30xl014+ / - 10% or 20% vg (or between 6.2xl014to 6.4xl014vg); or alternatively (ii) at a dose of 6.30xl014vg.

[0026] Embodiment 8. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 7, wherein the miniSHANK3 protein comprises an amino acid sequence at least 70%. 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and retains SHANK3 activity.Atty. Docket No. 38061.0013P1

[0027] Embodiment 9. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 8, wherein the miniSHANK3 protein is encoded by a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] Embodiment 10. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 9, wherein the regulatory element is a promoter that comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

[0029] Embodiment 11. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 10, wherein the transgene comprises a polyadenylation (poly A) signal 3’ of the nucleotide sequence encoding the miniSHANK3 protein.

[0030] Embodiment 12. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 11, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 7.

[0031] Embodiment 13. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 12. wherein the artificial genome comprises a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 8.

[0032] Embodiment 14. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 13, wherein the rAAV particle comprises at least one AAV9 serotype capsid protein.

[0033] Embodiment 15. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 14, wherein the SHANK3 deficiency is (i) a loss of function mutation in SHANKS or a 22ql3.3 deletion classified as a Class I deletion or (ii) a Class II deletion.

[0034] Embodiment 16. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 15, wherein the pharmaceutical composition comprises: (i) about 5 mM to about 25 mM of a buffering agent; (ii) about 0.5 mM MgC12 to about 1.5mM MgC12; (iii) about 50 mM to about 150 mM of atonicity agent; and (iv) from about 0.02% to about 0.2% of a non-ionic surfactant, wherein the pH of the pharmaceutical composition is between about 5.0 and about 9.0.Atty. Docket No. 38061.0013P1

[0035] Embodiment 17. The pharmaceutical composition for use or the method according to embodiment 16, wherein the buffering agent is lOmM Tris.

[0036] Embodiment 18. The pharmaceutical composition for use or the method according to embodiment 16 or 17, wherein the tonicity agent is 150 mM NaCl.

[0037] Embodiment 19. The pharmaceutical composition for use or the method according to any one of embodiments 16 to 18, wherein the pharmaceutical composition has an osmolality is 150 to 450 mOsm / kg.

[0038] Embodiment 20. The pharmaceutical composition for use or the method according to any one of embodiments 16 to 19, wherein the pharmaceutical composition does not comprise a preservative.

[0039] Embodiment 21. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 20, wherein the pharmaceutical composition comprises (i) lOmM Tris; (ii) ImM MgC12; (iii) 150mM NaCl; and (iv) 0.02% (w / v) Poloxamer 188 at pH 8.0.

[0040] Embodiment 22. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 21, wherein the pharmaceutical composition comprises 6.0*1013to 7.0xl013vg / mL, or alternatively 6.5 x 1013vg / mL.

[0041] Embodiment 23. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 22. wherein a change in visual evoked potential (VEP) waveforms is observed within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0042] Embodiment 24. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 23, wherein a change in auditory' evoked potential (AEP) waveforms is observed within 6 months or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0043] Embodiment 25. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 24, wherein sensory symptoms associated with SHANK3 mutations are stabilized or improved within 1 month, 3 months, 6 months, or 12Atty. Docket No. 38061.0013P1months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0044] Embodiment 26. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 25, wherein adaptive behavior is stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0045] Embodiment 27. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 26, wherein there is (i) stabilization of or an increase of at least 2, at least 5, or at least 10 points in the Vineland-3 Adaptive Behavior Scales composite score; or alternatively (ii) stabilization of or an increase of at least 2, at least 5, or at least 10 points in one or more of the socialization, daily living skills, communication, or motor domains of the Vineland-3 Adaptive Behavior Scales, or a combination thereof at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0046] Embodiment 28. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 27, wherein (i) the score for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global Impression Scale of Improvement (CGI-I / PMSA-C), or the overall global CGI-I / PMSA-C score, or combinations thereof, is 4 or lower at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition; or alternatively (ii) the score for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global Impression of Severity (CGI-S / PMSA-S), or the overall global CGI-S / PMSA-S score, or combinations thereof, is stabilized or reduced by at least 1 point, or at least 2 points, or at least 3 points, or at least 4 points, or at least 5 points, or at least 6 points at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition.

[0047] Embodiment 29. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 28. wherein global cognitive ability is stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.Atty. Docket No. 38061.0013P1

[0048] Embodiment 30. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 29, wherein language skills are stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0049] Embodiment 31. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 30, wherein motor functioning is stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0050] Embodiment 32. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 31, wherein autism symptoms are stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0051] Embodiment 33. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 32. wherein duration and / or quality of sleep is stabilized or improved within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0052] Embodiment 34. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 33, wherein symptoms associated with Phelan-McDermid Syndrome (PMS) are stabilized or improved within 1 month, 3 months. 6 months, or 12 months or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

[0053] Embodiment 35. The pharmaceutical composition for use or the method according to any one of embodiments 8 to 34, wherein the SHANK3 protein comprises the amino acid sequence of SEQ ID NO: 3.

[0054] Embodiment 36. The pharmaceutical composition for use or the method according to any one of embodiments 1 to 35, wherein the therapeutically effective amount of the rAAV particle is administered via unilateral ICV administration.

[0055] Embodiment 37. A unit dosage form comprising 2.15mL of a pharmaceutical composition comprising from about 6.0x1013to about 7.0x1013vg / mL, or alternativelyAtty. Docket No. 38061.0013P16.5 x 1013vg / mL. of a recombinant adeno-associated vector (rAAV) particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a miniSHANK3 protein, operably linked to a regulatory element that promotes expression in central nervous system (CNS), and flanked by AAV ITR sequences; wherein the rAAV particle is formulated for intracerebroventricular (ICV) administration.

[0056] Embodiment 38. The unit dosage form of Embodiment 37, wherein the miniSHANK3 protein comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and retains SHANK3 activity.

[0057] Embodiment 39. The unit dosage form of Embodiment 37, wherein the miniSHANK3 protein is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and encodes a miniSHANK3 protein that retains SHANK.3 activity.

[0058] Embodiment 40. The unit dosage form of any one of embodiments 37 to 39, wherein the transgene that encodes a miniSHANK3 protein is operably linked to a regulatory element that is a promoter that comprises or consists of a nucleotide sequence that is at least 85%, 90%, 95%, or 100% identical to a nucleotide sequence of SEQ ID NO: 9, and promotes expression of the miniSEIANK3 protein in cells of the CNS.

[0059] Embodiment 41. The unit dosage form of any one of embodiments 37 to 40, wherein the transgene that encodes a miniSHANK3 protein is operably linked to a regulatory- element that is a promoter that comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

[0060] Embodiment 41. The unit dosage form of any one of embodiments 37 to 41, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of SEQ ID NO: 7

[0061] Embodiment 42. The unit dosage form of any one of embodiments 37 to 41, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of SEQ ID NO: 8.

[0062] Embodiment 43. The unit dosage form of any one of embodiments 37 to 42, wherein the rAAV particle comprises at least one AAV9 serotype capsid protein.Atty. Docket No. 38061.0013P1

[0063] Embodiment 44. The unit dosage form of any one of embodiments 37 to 43, wherein the pharmaceutical composition is a preservative-free, sterile, solution comprising lOmM Tris, ImM Magnesium Chloride Hexahydrate, 150mM Sodium Chloride, and 0.02% (w / v) Poloxamer 188.BRIEF DESCRIPTION OF THE FIGURES

[0064] FIG. 1 is a schematic diagram illustrating a clinical trial study timeline and cohort treatment schematic. The diagram depicts a longitudinal study design extending approximately 5.5 years, from a pre-screening phase through a long-term follow-up period spanning. For a first participant at a given dose level, a monitoring period of eight (8) weeks is implemented to detect potential immune-mediated adverse events (AEs). Upon a determination that the first patient tolerates the JAG201 treatment, subsequent participants are monitored for a period of four (4) weeks. Within this schematic, a person symbol indicates the dosing of a participant, while an arrow represents the monitoring period between participants. The schematic further utilizes a star symbol to denote a data cut for safety review, a diamond symbol to represent a Data and Safety Monitoring Committee (DSMC) review and recommendation, and a square symbol to indicate an interim biomarker analysis.

[0065] FIG. 2 is a schematic diagram illustrating the clinical trial dosage design for pediatric and adult patient populations. The study design comprises a dose escalation from Cohort 1 to Cohort 2 for both populations. Specifically, the pediatric patient dosage is increased from a starting dose of 1.91 xio14vector genomes (vg) in Cohort 1 to a dose of 5.73xl014vg in Cohort 2. Similarly, the adult patient dosage is increased from 2.16xl014vg in Cohort 1 to 6.3 xlO14vg in Cohort 2.

[0066] FIG. 3 is a schematic representation of a JAG201 vector construct map. In the illustrated embodiment, the vector is an adeno-associated virus (AAV) vector comprising, from 5’ to 3’, a first (5‘) AAV2 inverted terminal repeat (ITR), a human synapsin (hSynl) promoter operably linked to polynucleotide sequence encoding a miniature version of the human SHANK3 gene (z.e., a miniSHANK3 protein), a woodchuck hepatitis virus post-transcriptional regulator^' element (WPRE), and a human grow th hormone poly adenylation signal (hGH poly A), and a second (3’) AAV2 ITR.Atty. Docket No. 38061.0013P1DETAILED DESCRIPTION

[0067] Provided are methods of administering rAAV gene therapy particles, particularly AAV9 particles, comprising recombinant genomes (and the cis plasmid for producing rAAV particles with the recombinant genome), said recombinant genomes including transgenes encoding aminiSHANK3 protein, e.g., having an amino acid sequence of SEQ ID NO: 3, operably linked to regulatory elements for expression in CNS cells, for treatment of SHANK3 deficiencies, including PMS. The inventors have identified therapeutic doses of the rAAV miniSHANK.3 encoding particles for unilateral ICV administration for adult or pediatric patients.

[0068] As described below in Example 4, three pediatric patients (Cohort 1) have been administered a single dose by unilateral ICV administration of a pharmaceutical composition comprising 1.91 x 1014vector genome copies (vg) of rAAV miniSHANK3 (“AAV9 AAV-hSynl-HumanMiniSHANK3-Vl” (5’ ITR-hSynl- Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8)) (also referred to herein as JAG201). All three Cohort 1 patients exhibited moderate to severe developmental impairments in communication, socialization, and motor function at baseline. No treatment-related serious adverse events (SAEs) or dose-limiting toxicities (DLTs) were observed for any of the three Cohort 1 patients as of 5 months to 1 year following administration of J AG201. Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Specific CGI-S / PMSA-S scores, VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 1 at baseline (prior to administration of JAG201), and at specified time points following administration of JAG201, and the results of those evaluations are described below.Cohort 1-Results

[0069] Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Specific CGI-S / PMSA-S scores, VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 1 at baseline (prior to administration of JAG201), and at specified time points following administration of JAG201. The preliminary results of these evaluations are summarized below.Cohort l-Vineland-3 Scores

[0070] The Vineland-3 assessment measures adaptive behavior, which are skills needed to function independently at home, school / work, and in the community. TheAtty. Docket No. 38061.0013P1assessment provides scores for four domains (Socialization. Daily Living Skills, Communication, and Motor), as well as a Composite Score. Participants are compared to a normal population with a standard score of 100 + / - one standard deviation (SD) (85-115). PMS patients generally score less than the intellectual disability threshold of 70. Preliminary assessments of Cohort 1 patients indicate stabilization or improvement over baseline for certain Vineland-3 domains following dosing with JAG201 over 3 months, 6 months and / or 9 months from dosing.Cohort 1-PMS-Specific CGI-I / PMSA-C Evaluation

[0071] The PMS-Specific CGI -I (now PMSA-C, and accordingly also referred to herein as CGI-I / PMSA-C) is a clinician-rated tool that measures improvement since baseline in seven (7) PMS-relevant domains. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-I) or Attention and Awareness (for PMSA-C), and (vii) Self-Care. Each patient from Cohort 1 underwent CGI-I / PMSA-C evaluation at baseline (prior to dosing), and at day 28 and month 3, and have or will be evaluated at month 6 following dosing with JAG201. The CGI-I / PMSA-C scale provides a qualitative assessment of clinical change, compared to a baseline (pre-dosing) score for each of the domains, across the seven (7) distinct levels of improvement or worsening, wherein a score of 1 indicates “very much improved,” 2 indicates “much improved,” 3 indicates “minimally improved,” 4 indicates “no change,” 5 indicates “minimally worse,” 6 indicates “much worse,” and 7 indicates “very much worse.”

[0072] All three Cohort 1 patients experienced rapid motor improvements within weeks of JAG201 administration, leading to attainment of new motor skills including running jumping, and ability to navigate stairs. All three Cohort 1 patients also demonstrated increased aw areness of environment and increased interest in social interaction with peers / adults following administration of JAG201. All three Cohort 1 patients were rated as either “minimally improved” (score of 3) or “much improved” (score of 2) for various PMS-CGI-I / PMSA-C domains, as well as for the overall CGI-I / PMSA-C global improvement score, as early as 28 days to 6 months, for those evaluated at 6 months, following administration. One patient exhibited rapid improvement and attainment of skills, show ing “much improved” scores for two CGI-I / PMSA-C domains (Expressive Communication andAtty. Docket No. 38061.0013P1Gross Motor), and “minimally improved” scores for three CGI-I / PMSA-C domains (Receptive Communication, Fine Motor, and Social Interaction) by 28 days following administration of JAG201, and attained an overall CGI-I / PMSA-C global improvement score of 2 (“Much Improved”) at 28 days following administration.Cohort 1-PMS-Specific CGI-S Evaluation

[0073] The PMS-Specific CGI-S (now PMSA-S, and accordingly also referred to herein as CGI-S / PMSA-S) is a clinician-rated tool that measures severity of symptoms in seven (7) PMS-relevant domains, relative to the clinician's total experience with the PMS population, based on domain-specific anchors. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-S) or Attention and Awareness (for PMSA-S), and (vii) Self-Care. The CGI-S / PMSA-S scale provides a qualitative assessment of the severity of symptoms across the relevant domains relative to the total experience with the PMS population, wherein a score of 1 indicates “typical for age / not impaired,” 2 indicates “slightly impaired,” 3 indicates “mildly impaired,” 4 indicates “moderately impaired,” 5 indicates “markedly impaired,” 6 indicates “severely impaired,” and 7 indicates “among most severely impaired.”

[0074] Each patient from Cohort 1 underwent CGI-S / PMSA-S evaluation at baseline (prior to dosing), and at day 28, and month 3, and has or will be evaluated at month 6 following dosing with JAG201. All three Cohort 1 patients generally maintained baseline CGI-S / PMSA-S scores (including a slight (1 point) worsening in certain domain scores for one patient), through three or six months following administration of JAG201. One patient exhibited (i) a consistent improvement in the CGI-S / PMSA-S Gross Motor Skills domain, exhibiting a score of 4 (“moderately impaired) at baseline and a score of 2 (“slightly impaired”) at day 28 and month 3 following administration of JAG201, and (ii) a consistent improvement in the CGI-S / PMSA-S Expressive Communication domain, exhibiting a score of 4 (“moderately impaired) at baseline and a score of 3 (“mildly impaired”) at day 28 and month 3 following administration of JAG201.Cohort 1-Visual Evoked Potential Evaluation

[0075] Visual Evoked Potential (VEP) is a non-invasive method for measuring functionality of the human visual system by detecting neuronal responses to stimuliAtty. Docket No. 38061.0013P1independently of the consciousness and attention state of the patient. VEP waveforms were recorded for all patients in Cohort 1 at baseline (prior to administration of JAG201), and at 3 months and 6 months following administration of JAG201. ERP waveform parameters of interest, including: N75 Amplitude (excitatory response to stimulus), Pl 00 Amplitude (inhibitory response to stimulus), N75 to Pl 00 peak-to-peak amplitude (total initial response to visual stimulus), and N2-P2 peak-to-peak amplitude (secondary response potentially indicative of interpretation of visual stimulus), were analyzed for each patient in Cohort 1.

[0076] Changes in VEP waveform observed for Cohort 1 patients included changes in: (i) N75 Amplitude (indicative of changes in excitatory response to stimulus), (ii) P100 Amplitude (indicative of changes in inhibitory response to stimulus), (iii) N75 to P100 peak-to-peak amplitude (indicative of changes in total initial response to visual stimulus), and (iv) N2-P2 peak-to-peak amplitude (indicative of changes in secondary response potentially indicative of interpretation of visual stimulus). In one Cohort 1 patient, the latency of the ERP waveform was observed to decrease from baseline to month 6 post dosing, suggesting a more rapid neuronal response to visual stimulus. In another Cohort 1 patient, the ERP waveform exhibited (i) a greater N75 and Pl 00 amplitude as compared to baseline, (ii) a greater slope from N75-P100 (suggesting a more rapid neuronal response), and (iii) an emerging P2 signal suggesting an improved ability to process visual stimulus.Cohort 1-Auditory Evoked Potential Evaluation

[0077] Auditory Evoked Potential (AEP) is an electrical signal elicited from the brain while an auditory stimulus is presented in a time-locked manner. The AEP signal consists of reproducible positive or negative peaks, latency, amplitude and behavioral correlation. For one patient in Cohort 1, at 6 months following administration of JAG201 changes in AEP waveform as compared to baseline were observed across a number of ERP waveform parameters of interest, including: (i) N1 Amplitude (excitatory response to stimulus), (ii) Pl Amplitude (inhibitory' response to stimulus), (iii) N1 to Pl peak-to-peak amplitude (total initial response to auditory stimulus), and (iv) N2-P2 peak-to-peak amplitude (secondary response potentially indicative of interpretation of auditory stimulus). These changes suggest stronger and more consistent electrical responses in the cortex of the brain, indicating that the auditory information is being communicated from the ears to the parts of the brain in the cortex where they are interpreted.Atty. Docket No. 38061.0013P1Cohort 1-Efficacy Observed Following Administration of JAG201

[0078] Key social improvements observed for Cohort 1 patients at 28 days. 3 months and, in patients evaluated at 6 months, 6 months following administration of JAG201 included: improved eye contact; noticing of and interaction with pet dog and sibling; increased awareness of individuals and environment; initiation of play with peers; smiling in response to interaction; and increased initiation of social interaction (e.g, says “hi,” shows interest in other children, shares enjoyment). Key communication improvements observed for Cohort 1 patients included: expanding vocabulary and better articulation; identification of objects; achievement of phrased speech (<?.g., “come sit down,” or “I want . . .”); pointing to identify needs; and better understanding of directions. Key motor function improvements observed for Cohort 1 patients included: acquiring the ability to run; improvement in stair climbing, including progression from (1) crawling up stairs, to (2) climbing up stairs with support, to (3) alternating feet per step, to (4) walking down stairs with support, to(5) walking downstairs without support; initiation of tricycle riding; improvements in motor coordination; learning to climb a rope ladder at a jungle gym; improved gait / symmetry in movements; and improvements across fine motor skills (e.g., eating, drawing, and using utensils).

[0079] Based on the Cohort 1 results described above, the decision was made to proceed to dosing Cohort 2 patients with JAG201 (AAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR)(SEQ ID NO: 8) via unilateral ICV administration into the lateral ventricle at a single dose of 5.73xl014vg according to the protocol in Example 1.

[0080] As described below in Example 5, two pediatric patients (Cohort 2) have been administered a single dose by unilateral ICV administration of a pharmaceutical composition comprising 5.73xl014vg of rAAV miniSHANK3 (“AAV9 AAV-hSynl-HumanMiniSHANK3-Vl” (5‘ ITR-hSynl- Human miniSHANK3vl-WPRE-hGH poly A- 3' ITR) (SEQ ID NO: 8)) (JAG201). Both Cohort 2 patients exhibited moderate to severe developmental impairments in communication, socialization, and motor function at baseline. No treatment-related serious adverse events (SAEs) or dose-limiting toxicities (DLTs) were observed for either of the Cohort 2 patients as of two w eeks to 3 months follow ing administration of JAG201. Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Atty. Docket No. 38061.0013P1Specific CGI-S / PMSA-S scores. VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 2 at baseline (prior to administration of JAG201), and have been or will be obtained from each patient at specified time points following administration of JAG201, and the results of those evaluations are described below.Cohort 2-Results

[0081] Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Specific CGI-S / PMSA-S scores, VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 2 at baseline (prior to administration of JAG201), and have been or will be obtained from each patient at specified time points following administration of JAG201. The preliminary results of these evaluations are summarized below.Cohort 2-Vineland-3 Scores

[0082] The Vineland-3 assessment measures adaptive behavior, which are skills needed to function independently at home, school / work, and in the community. The assessment provides scores for four domains (Socialization, Daily Living Skills, Communication, and Motor), as well as a Composite Score. Participants are compared to a normal population with a standard score of 100 + / - one standard deviation (SD) (85-115). PMS patients generally score less than the intellectual disability threshold of 70. Preliminary assessments of one Cohort 2 patient at three months following administration of JAG201 indicates stabilization of one Vineland-3 domain (Motor Skills) as compared to baseline, an increase as compared to baseline for two Vineland-3 domains (Daily Living Skills and Socialization), and a decrease as compared to baseline for one domain (Communication).Cohort 2-PMS-Specific CGI-I / PMSA-C Evaluation

[0083] The PMS-Specific CGI-I (now PMSA-C, and accordingly also referred to herein as CGI-I / PMSA-C) is a clinician-rated tool that measures improvement since baseline in seven (7) PMS-relevant domains (as described above in Example 4). Each patient from Cohort 2 has undergone or will undergo CGI-I / PMSA-C evaluation at baseline (prior to dosing), and at various timepoints following administration of JAG201. The CGI-I / PMSA-C scale provides a qualitative assessment of clinical change, compared to a baseline (predosing) score for each of the domains, across the seven (7) distinct levels of improvement or worsening, wherein a score of 1 indicates “very much improved,” 2 indicates “muchAtty. Docket No. 38061.0013P1improved,” 3 indicates '“minimally improved,” 4 indicates “‘no change,” 5 indicates “minimally worse,” 6 indicates “much worse,” and 7 indicates “very much worse.”

[0084] Preliminary assessments of one Cohort 2 patient at day 28 following administration of JAG201 indicates improvement in four domains compared to baseline (Expressive Communication, Receptive Communication, Social Interaction, and Attend on / A areness), as well as an Overall Global CGI-I / PMSA-C score of 2 (“Much Improved”), while the other three domains (Gross Motor. Fine Motor, and Self-Care) remained at baseline. In additional preliminary assessments at day 56 and month 3 following administration of JAG201, this Cohort 2 patient exhibited improvement over baseline in all seven CGI-PMSA-C domains compared to baseline, and maintained an overall global score of 2 (“Much Improved”).Cohort 2-PMS-Specific CGI-S Evaluation

[0085] The PMS-Specific CGI-S (now PMSA-S, and accordingly also referred to herein as CGI-S / PMSA-S) is a clinician-rated tool that measures severity of symptoms in seven (7) PMS-relevant domains relative to the clinician’s total experience with the PMS population, based on domain-specific anchors. The CGI-S / PMSA-S scale provides a qualitative assessment of the severity of symptoms across the relevant domains relative to the total experience with the PMS population, wherein a score of 1 indicates “typical for age / not impaired,” 2 indicates '“slightly impaired,” 3 indicates “mildly impaired,” 4 indicates “moderately impaired,” 5 indicates “markedly impaired,” 6 indicates “‘severely impaired,” and 7 indicates “among most severely impaired.”

[0086] Preliminary assessments of one Cohort 2 patient following administration of JAG201 indicates improvements in three CGI-S / PMSA-S domains (Receptive Communication, Attention / Awareness, and Self-Care) at 3 months following administration of JAG201.Cohort 2-Efficacy Observed Following Administration of JAG201

[0087] Through 3 months following administration of JAG201, one Cohort 2 patient’s caregiver reported that the patient exhibited improvements across many domains, including communication, motor function, and socialization, with examples of the patient being more aware of surroundings and more focused in play. The patient’s caregiver hasAtty. Docket No. 38061.0013P1further reported that the patient is “much improved’7since the start of the study, and is now more focused on daily tasks and routines (for example, sitting on the stairs and putting shoes on), and is more attentive to things that are happening around them.

[0088] Provided are therapeutically effective single doses for unilateral intracerebroventricular (ICV) administration of the rAAV particles (including AAV9 particles) containing the transgenes described herein (including transgenes that encode miniSHANK3-Vl having an amino acid sequence of SEQ ID NO: 3 or miniSHANK3-V2 having an amino acid sequence of SEQ ID NO: 4 operably linked to a Synl promoter, including AAV9- AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8)), which are administered at (i) a dose of 2.16x 1014rAAV vector genomes (vg), or about 2.16* 1014vg, or 2.16* 1014+ / - 10% or 20%. or between 2.1xl014to 2.2xl014vg for an adult subject or a pediatric subject age 10 to 17, or (ii) a dose of 5.73*1014vg, or about 5.73*1014vg, or 5.73*1014+ / - 10% or 20% vg, or between 5.7x1014to 5.8* 1014vg for an adult subject or a pediatric subject age 10 to 17, or (iii) a dose of 6.30 1014vg, or about 6.30xl014vg, or 6.30xl014+ / - 10% or 20% vg, or between 6.2x1014to 6.4xio14vg for an adult subject or a pediatric subject age 10 to 17, or (iv) a dose of 1.91 xlO14vg, or about 1.91 xlO14vg, or 1.91 xlO14+ / - 10% or 20% vg, or between 1.85xl014to 1.95xl014vg for a pediatric subject age 2 to 9; or (v) a dose of 5.73xl014vg, or about 5.73xl014vg, or 5.73xl014+ / - 10% or 20% vg, or between 5.7xl014to 5.8xl014vg for a pediatric subject age 2 to 9. Administration of such therapeutically effective dosages of the rAAVs comprising transgenes described herein (including miniSHANK3-Vl having a nucleotide sequence of SEQ ID NO: 5 or miniSHANK3-V2 having a nucleotide sequence of SEQ ID NO: 6) results in amelioration of one or more indicators of SHANK3 haploinsufficiency, such as, improved sensory symptoms, improved sensory perception, adaptive behavior, global cognition, language acquisition, motor functioning, sleep, and decreased autism symptoms within 12 w eeks. 26 weeks, 36 w eeks, 52 weeks or longer from the administration relative to baseline and also does not result in any serious treatment-related adverse events.

[0089] Accordingly, provided and described herein are methods of administering an rAAV particle, including an rAAV9 particle, comprising a recombinant genome comprising a transgene encoding a miniSHANK3 protein, including the AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8)Atty. Docket No. 38061.0013P1construct, to a subject, including a human subject, in need thereof, wherein the administration is unilateral ICV at a dosage of (i) 2.16* 1014rAAV vector genomes (vg), or about 2.1 x 1014vg, or 2.16xl014+ / - 10% or 20%, or between 2.1 xlO14to 2.2xl014vg for an adult subject or apediatric subject age lO to 17, or (ii) 5.73xl014vg, or about 5.73xl014vg, or 5.73xl014+ / -10% or 20% vg, or between 5.7xl014to 5.8xl014vg for an adult subject or a pediatric subject age 10 to 17, or (iii) 6.30xl014vg, or about 6.30xl014vg, or 6.30xl014+ / - 10% or 20% vg, or between 6.2xl014to 6.4xl014vg for an adult subject or a pediatric subject age 10 to 17, or (iv) 1.91 xlO14vg, or about 1.91 xlO14vg, or 1.91 xlO14+ / - 10% or 20% vg, or between 1.85xl014to 1.95xl014vg for a pediatric subject age 2 to 9; or (v) 5.73xl014vg, or about 5.73xl014vg, or 5.73xl014+ / - 10% or 20% vg, or between 5.7 xlO14to 5.8x1014vg for a pediatric subject age 2 to 9.Definitions

[0090] The articles “a” and "an" are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, '‘an element” means one element or more than one element. For example, “comprising an A, a B, or a C” contemplates and supports embodiments comprising two or more A, two or more B, and two or more C.

[0091] Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments pertain. The preferred materials and methods are described, but it is understood that any methods and materials similar or equivalent to those described can be used in the practice of embodiments. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In describing and claiming the present invention, the following terminology will be used.

[0092] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the embodiments.

[0093] The term “AAV capsid” refers to the AAV protein shell, i.e., a capsid. It may or may not encapsidate a nucleic acid.Atty. Docket No. 38061.0013P1

[0094] The terms "AAV particle7’ or '“AAV virion” can be used interchangeably and refer to an infectious non-replicative virus having an AAV protein shell, i.e., a capsid, encapsidating a nucleic acid. In some embodiments, the AAV particle is active for gene therapy, i.e., upon transduction of an appropriate target cell, a transgene (also “gene of interest” or “GOI”) in the recombinant genome of the AAV particle is expressed in the target cell. Expression of the transgene can be measured by either protein expression or a functional assay, for example, as described herein.

[0095] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.

[0096] AAV ”rep” and “cap” genes are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date, and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are “coupled” together as adjoining or overlapping transcriptional units), and they are generally conserved among AAV serotypes. AAV rep and cap genes are also individually and collectively referred to as “AAV packaging genes.” The AAV cap genes in accordance with the present invention encode Cap proteins which are capable of packaging AAV vectors as recombinant AAV particles in the presence of rep and adeno helper function and are capable of binding target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[0097] A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus El a gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The phrase “adenovirus helper functions” refers to a number of viral helper genes expressed in a cell (as RNA or protein) such that the AAV grows efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of a method disclosed herein,Atty. Docket No. 38061.0013P1AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with one or more plasmid vectors. In some embodiments, the plasmid vector encodes the AAV rep and cap genes. In some embodiments, the plasmid vector encodes the helper genes. In some embodiments, the plasmid vector encodes the rAAV genome. In some embodiments, the plasmid vector encodes the helper genes and the rAAV genome. In some embodiments, the plasmid vector encodes the helper genes and the AAV rep and cap genes.

[0098] In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes.

[0099] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules, or two nucleic acid molecules, such as polynucleotides. When two amino acid sequences have the same residues at the same positions; e.g, if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., ifhalf (e.g., five positions in apolymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. In the case of an insertion or deletion, identity is understood to realign thoseAtty. Docket No. 38061.0013P1thereafter which would be identical and is considered to be not identical at the insertion or deletion.

[0100] By ‘'substantially identical’’ is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, 70%, 75%, 80%. 85%. 90%. 95% or 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0101] Amino acid residues as disclosed herein can be modified by conservative substitutions to maintain, or substantially maintain, overall polypeptide structure and / or function. As used herein, “conservative amino acid substitution” indicates that: hydrophobic amino acids (z.e.. Ala, Cys, Gly, Pro, Met, Vai, lie, and Leu) can be substituted with other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (z.e., Phe, Tyr, and Trp) can be substituted with other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (z. e. , Arg, His, and Lys) can be substituted with other amino acids with positively charged side chains; amino acids with negatively charged side chains (z.e.. Asp and Glu) can be substituted with other amino acids with negatively charged side chains; and amino acids with polar uncharged side chains (z.e., Ser, Thr, Asn, and Gin) can be substituted with other amino acids with polar uncharged side chains.Table 1 Amino Acid SubstitutionsAtty. Docket No. 38061.0013P1Table 2: Amino Acid AbbreviationsAtty. Docket No. 38061.0013P1

[0102] A “nucleic acid,” as used herein, is interchangeable with “polynucleotide” or “a specific sequence of nucleotide or “nucleotide sequences.” These terms refer to a discrete sequence that performs a specific function directly or indirectly in a cell. That function includes encoding a sequence of a gene that is transcribed into mRNA and translated into protein and regulating said transcription (i.e., as a promoter would) and / or translation (i.e., as microRNA would). A nucleic acid inherently has a sequence. Thereby, “a nucleic acid comprising SEQ ID NO.: X” can be used to contemplate and support “a nucleic acid comprising the sequence of SEQ ID NO.: X.” In recombinant molecular biology7, discrete nucleic acids can be combined. In some embodiments, a nucleic acid that encodes a protein can be ligated to a promoter (which is a nucleic acid), and a cis-acting element of a viral vector (i.e., an inverted-terminal repeat (ITR), which is also anucleic acid). For convenience, a “nucleic acid” might be used to refer to the discrete elements within the larger nucleic acid, which could be referred to as “a polynucleotide,” “an expression cassette” i.e., a polynucleotide comprising a promoter and a nucleic acid that encodes a protein), or “a vector” (see definition below).

[0103] “Encoding” refers to the inherent property of a nucleic acid to serve as a template, whether directly (i.e., a sense strand) or indirectly (i.e., an antisense strand) for synthesis of peptide, polypeptides, proteins, or other nucleic acids (i.e., rRNA, tRNA, microRNA). A nucleic acid can “encode” whether it is the sense strand, antisense strand, or a double-stranded segment thereof. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA. The mRNA then serves as the template for translation of a peptide, polypeptide, or protein. The anti-sense strand is generally considered to be the reverse complementary7sequence and is sometimes called a “non-coding” strand in the art (although for present purposes “non-coding” is a misnomer because the non-coding strand still “encodes” the genetic information by perpetuating it during semi-conservative replication by¬Atty. Docket No. 38061.0013P1acting as a template for the polymerization of a new, sense strand). Within semi-conservative replication two single strands in double-stranded nucleic acids are separated, and anew strand is polymerized from the information from each of the single-stranded nucleic acids (i.e., single-stranded template), regardless of whether one single-stranded template is the sense strand (e.g., that which is used to transcribe mRNA and thereby, or directly, encode the translate or protein) or the antisense strand. By perpetuating the genetic information, the antisense strand is still encoding the genetic information for, for example, a protein.Accordingly, “a nucleic acid encoding X”, includes sense and antisense sequences or strands whether X is a peptide, a polypeptide, or a protein or X is a sequence that encodes a rRNA, tRNA, microRNA, antisense RNA, etc. Further, when a recombinant genome is packaged as rAAV particles, the result is a mixture of particles comprising both (i) rAAV particles that contain a “sense” DNA genome (which encodes proteins such as a transgenic protein), and (ii) rAAV particles that contain an “antisense” DNA genome (which is the reverse complement of the “sense” DNA genome). When rAAV particles comprising an “antisense” genome transduce a target cell, the “antisense” DNA genome is converted into a “sense” DNA genome that is capable of expressing proteins such as a transgenic protein.

[0104] Further to which, “nucleic acid encoding X,” includes RNA, DNA, and combinations thereof, since nucleic acids are synthesized from transcription, reversetranscription, and replication, as naturally occurring processes and man-made processes (recombinant biology, molecular biology, etc.).

[0105] With regard to an AAV particle or an AAV virion, the above-noted incorporation of reverse complementary sequences and double-stranded segments into the definition of “a nucleic acid” and the above-noted use of “encoding” as including sense and antisense strands, is intended to incorporate the means by which the AAV vector can introduce an exogenous nucleic acid sequence that encodes nucleic acid or a protein into the cell. It is further intended to incorporate, in some embodiments, processes whereby said introduction results in the expression of said nucleic acid (i.e. miRNA or antisense RNA) or protein.

[0106] Take for example, a nucleic acid encoding a protein, and an AAV vector comprising a nucleic acid encoding said protein. When atypical (i.e. naturally occurring) AAV vector encoding one sense or one antisense strand of the nucleic acid that encodes saidAtty. Docket No. 38061.0013P1protein enters the cell, the inverted-terminal repeats (ITRs) prime the synthesis of a sequence reverse complementary to the sense strand or antisense strand of the nucleic acid that encodes said protein. The polymerization thereby forms a segment of double-stranded DNA comprising the sense and antisense strands, regardless of whether the sense version or antisense version was first introduced to the cell. In this regard, the entire nucleic acid including ITRs and sense and antisense nucleic acids encoding a protein can be one singlestranded DNA, which loops upon itself to form a double-stranded segment, wherein the basepairs the sense and antisense nucleic acids encoding the protein align.

[0107] From this segment of double-stranded DNA, transcription of mRNA and translation of said protein is achieved from said sense strand of DNA, regardless of whether the AAV vector comprised only the sense strand or only the antisense strand when first entering the cell. In this regard, “an AAV vector comprising a nucleic acid encoding protein X” includes, contemplates, and supports embodiments in which the nucleic acid is the sense strand encoding protein X, the antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single stranded nucleic acid comprising sense and antisense strands wherein the sense and antisense strands form a segment of double-stranded nucleic acid.

[0108] The term '‘operably linked’’ refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0109] The terms “final formulation,” and “drug product” as used herein can be used interchangeably and include compositions suitable for administration to mammals, e.g., humans. When the AAV particles of the present disclosure are administered as pharmaceuticals to mammals, e.g.. humans. The term “drug substance” as used herein refers to a purified rAAV composition that needs only to be filtered and / or have added to it pharmaceutically acceptable excipients, diluents or carriers, to make the final formulation. “Pharmaceutical composition” refers to a composition comprising a drug product that is appropriate for administration to subjects, including human subjects in that excipients orAtty. Docket No. 38061.0013P1contaminants are physiologically tolerable, on balance, do not detract from the therapeutic activity of the drug product or cause unduly adverse effects.

[0110] The terms “subject”, “host”, and “patient” are used interchangeably to refer to a human subject. In some embodiments, the term “pediatric subject” refers to a human subject aged 2-9 years old. In some embodiments, the term subject refers to a human subject aged 10-17 years old. In some embodiments, the term “pediatric subject” refers to a human subject under age 10. In some embodiments, the term “adult subject” refers to a human subject age 18 or more.

[0111] As used herein, the term “unit dose” or “unit dosage” refers to a physically discrete unit that contains a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. The unit dose or unit dosage may be in the form of vial comprising a therapeutically effective amount of active ingredient for a single dose for a subject. A dosage may require multiple unit dosage forms.

[0112] The term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, that would cause a severe adverse reaction or side effect, when administered to a human, depending upon the active ingredient and indication to be treated. In embodiments, as used herein, the term “pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0113] The term “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a reduction or loss of a function to be provided by a transgene.

[0114] The terms “promoter” and “regulatory sequence” as used herein, can be used interchangeably and are defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. In some instances, this sequence may be the core promoter and in other instances, this sequence may also include, or be an enhancer alone and / or other regulatory elements which are required for expression of the gene product.Atty. Docket No. 38061.0013P1

[0115] In certain instances the promoter may comprise enhancer elements, exons, and introns from one or a variety of viruses and animals, and thereby the term “promoter’ shall be understood to not be limited to being a non-expressed sequence, nor exclude a non-expressed sequence that is between expressed sequences (i.e. introns), nor be limited to exclude an enhancer alone so long as the combination of sequences used to construct the promoter are capable of initiating the specific transcription of a polynucleotide sequence. Other regulator}’ elements include polyadenylation (poly A) signals and post-transcriptional regulator}’ elements, such as, for example, a woodchuck hepatitis virus post-transcriptional regulatory- element (WPRE).

[0116] A “vector” is a nucleic acid capable of delivering a target gene to the interior of a cell, and includes not only the expression-region (i.e. a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also some cis-acting genetic component. The cis-acting genetic component provides for packaging within a virion, expression in a cell, replication in a cell, or a combination thereof.

[0117] By way of example, inverted-terminal repeats (ITRs) from adeno-associated viruses (AAVs) constitute a vector when adjoined to the nucleic acid encoding a target protein because the ITRs will provide for the nucleic acid encoding the target protein to be packaged within an AAV virion. ITRs also provide other cis-acting functions for expression of the nucleic acid encoding the target protein in the host cell upon entry of the vector into the host cell. Such cis-acting functions of ITRs include aiding in concatemer formation for genomic insertion; initiation of second strand formation in the case of a single-stranded (ss) AAV (ssAAV) vector; or initiation of replication and transcription in the case of ssAAV and self-complementary (sc) AAV (scAAV) vectors. In this regard, the AAV ITRs can be characterized based on the nucleic acid sequences providing such cis-acting functions from the serotypes of AAVs. That is, an ITR isolated from an AAV2 seroty pe can be know n as an AAV2 ITR, even though the ITR generally does not contribute to the serotype of an AAV.

[0118] “Expression cassette” refers to a nucleic acid comprising a transgene, such as a gene encoding a miniSHANK3 protein as described herein, operably linked to regulatory sequences sufficient for expression of the transgene in a target cell. An expression cassette includes a recombinant polynucleotide comprising a nucleic acid that controls expression (i. e. a promoter) and a nucleic acid that encodes the transgene. The transgene includes a nucleicAtty. Docket No. 38061.0013P1acid that encodes a protein. Generally, the promoter is operatively linked to the nucleic acid that encodes the transgene (e.g, miniSHANK3) in a manner that is capable of promoting expression of the protein encoded by the transgene in a target cell, including cells of the CNS. The expression cassette may also include other regulatory elements such as polyadenylation (poly A) signal sequences and other expression elements such as enhancers, introns and post-transcriptional regulatory elements that promote or otherwise increase or decrease expression of the transgene in target cells.

[0119] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7. 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.I. SHANK3 TransgenesA. miniSHANK3 Encoded by the Transgenes

[0120] SHANK3 encodes a synaptic scaffolding protein, which coordinates the recruitment of signaling molecules and orchestrates assembly of the macromolecular postsynaptic protein complex, which is crucial for proper synaptic development and function. Disruption, mutation, or deletion of SHANKS is a major cause of the core neurodevelopmental and neurobehavioral deficits in Phelan-McDermid syndrome.

[0121] Recombinant adeno-associated particles (rAAVs) represent a promising gene delivery platform. However, it is known in the art that SHANK.3 is a large protein with a coding sequence of about 5.4 kb, exceeding the packaging capacity of recombinant AAV particles. Miniaturized SHANK3 (“miniSHANK3”) proteins described herein can be delivered by gene therapy vectors such as rAAV particles, including rAAV9 particles.Disclosed are methods of delivering the recombinant AAV particles, including rAAV9 particles, having a miniSHANK3 transgene directly to the CNS, including via bilateral orAtty. Docket No. 38061.0013P1unilateral intracerebroventricular (ICV) administration. Thus, the present disclosure relates to methods and compositions for treating neurodevel opmental disorders by restoring the activity of SHANK3 using a miniaturized SHANK3 protein (“miniSHANK3”).

[0122] The Shank family of proteins (e.g., SHANK1, SHANK 2, and SHANK3) are master scaffolding proteins that tether and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five main domain regions: N-terminal ankyrin repeats. SH3 domain, PDZ domain, proline-rich region, and aC-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 postsynaptic scaffold proteins of the SAPAP family (SAP90 / PSD-95-associated protein, also called discs-large-associated proteins (DLGAPs)) which in turn bind to PSD95 (postsynaptic density protein 95, a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory) to form the PSD95 / SAPAP / SHANK postsynaptic complex. Together, these multidomain proteins are proposed to form a key scaffold, orchestrating the assembly of the macromolecular postsynaptic signaling complex at glutamatergic synapses. This complex has been shown to play important roles in targeting, anchoring, and dynamically regulating synaptic localization of neurotransmitter receptors and signaling molecules. In another example, the Shank family of proteins is connected to the mGluR pathway through its binding to Homer.

[0123] The SHANK3 full length human protein sequence corresponding to GenBank Accession No. Q9BYB0.3 is provided as SEQ ID NO: 1. The SHANK3 full length human protein sequence corresponding to SEQ ID NO: 1 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_001372044, provided herein as SEQ ID NO: 2.

[0124] The full-length SHANK3 protein comprises multiple domains and is encoded by a gene that is about 5.4 kb in size. Due to its size, it is difficult to deliver a full-length SHANK? protein to a tissue or cell of interest via a recombinant AAV (rAAV) vector, as such vectors have a maximum transgene capacity of ~5 kb to allow for packaging as rAAV particles. As reported in PCT Publication No. W02022 / 040239 and WO2024 / 233422, each entitled “Shank3 Gene Therapy Approaches,’' which are incorporated by reference herein inAtty. Docket No. 38061.0013P1their entirety, specific amino acid domains can be removed or truncated from the full-length SHANK3 protein to produce a miniSHANK3 protein that is efficacious in restoring SHANK3 activity7in excitatory7neurons. SHANK proteins (e.g., SHANK3 proteins) encoded by polynucleotides described herein can be miniaturized to form a shortened variant of the native, full length SHANK3 protein. As disclosed herein, a miniaturized SHANK3 protein, or a DNA construct encoding the miniaturized SHANK3 protein, are referred to interchangeably as "miniSH ANK3” or ‘MiniSHANK3.” MiniSHANK3 proteins include shortened or mutated versions of SHANK3 that have at least some SHANK3 activity, for example, when miniSHANK3 is introduced into neurons, it reduces the effects of SHANK3 mutations,

[0125] “SHANK3 activity” includes, for example, the activity' when introduced into, including by gene therapy, an organism, such as a mouse, non-human primate (NHP) or human, including the neurons of an organism which is deficient for SHANK3 or has reduced SHANK3 activity, that ameliorates the effects of that SHANK3 deficiency or reduction. The activity can be assessed in animal models that are engineered to contain SHANK3 deletions, deficiencies, or mutations that abolish SHANK3 activity, such as those described in WO 2024 / 178401, which is hereby incorporated by reference in its entirety.

[0126] SHANK3 proteins encoded by polynucleotides described herein can include one or more protein domains. For example, SHANK3 proteins can include one or more of an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin domain, a SAM domain, and / or an ankyrin repeat domain.

[0127] In some embodiments, the miniSHANK3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%.98%, or 99% identical, or is 100% identical, including all values in between, to the amino acid sequence of SEQ ID NO: 3 and has SHANK3 activity. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative substitutions are made to SEQ ID NO: 3 and results in a miniSHANK3 protein that substantially maintains SHANK3 activity.

[0128] In some embodiments, the miniSHANK3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to the aminoAtty. Docket No. 38061.0013P1acid sequence of SEQ ID NO: 4 and has SHANK3 activity. In some embodiments. 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, or more conservative substitutions are made to SEQ ID NO: 4 and results in a miniSHANK3 protein that substantially maintains SHANK3 activity.

[0129] In some embodiments, the miniSHANK3 protein comprises or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the miniSHANK3 protein comprises or consists of the amino acid sequence of SEQ ID NO: 4.B. Polynucleotides Encoding miniSHANK3 Proteins

[0130] In some embodiments, the sequences of polynucleotides encoding miniSHANK3 proteins associated with the disclosure comprise at least 70%, 71%, 72%, 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or are 100% identical, including all values in between, to SEQ ID NO: 5 or SEQ ID NO: 6, and encode a protein with SHANK3 activity, including a miniSHANK3 protein with an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sequences of polynucleotides encoding miniSHANK3 proteins associated with the disclosure comprise at least 90% identity to SEQ ID NO: 5 or SEQ ID NO: 6, and encode a miniSHANK3 protein with SHANK3 activity. In some embodiments, the sequences of polynucleotides encoding miniSHANK3 proteins associated with the disclosure comprise SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 encodes a mini SH ANKA protein with SHANK3 activity.

[0131] In some embodiments, a polynucleotide encoding the miniSHANK3 protein as disclosed herein is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about 4.3 kb, about 4.2 kb, about 4.1 kb, about 4.0 kb, about 3.9 kb, about 3.8 kb, about 3.7 kb, about 3.6 kb, about 3.5 kb, about 3.4 kb, about 3.3 kb, about 3.2 kb, about 3.1 kb, about 3.0 kb. about 2.9 kb, about 2.8 kb, about 2.7 kb, about 2.6 kb. about 2.5 kb, about 2.4 kb, about 2.3 kb, about 2.2 kb, or about 2.1 kb in size. In some embodiments, the polynucleotide encoding the miniSHANK3 protein as disclosed herein can be in any size that is suitable for the methods and vectors disclosed in the present disclosure.C. Regulatory ElementsAtty. Docket No. 38061.0013P1

[0132] In some embodiments, a polynucleotide encoding a miniSHANK3 transgene coding sequence is operably linked to one or more regulatory sequences, including regulatory sequences necessary for tissue-specific gene expression. In some cases, the tissue-specific regulatory' sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such regulatory sequences (e.g. promoters, enhancers, polyA signals, etc.), including tissue-specific regulatory sequences, are well known in the art. In embodiments, the tissue-specific regulatory sequence can be any promoter or enhancer that is neuron-specific and is suitable for the treatments described herein.

[0133] In some embodiments, a tissue-specific regulatory' element is a Syn promoter (e.g., hSynl) that is operably linked to and drives expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In embodiments, the hSynl promoter comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity', or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 9, and drives expression of a polynucleotide encoding a miniSHANK.3 protein in target cells, including cells of the CNS. In embodiments, the hSynl promoter comprises the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the hSYnl promoter is operably linked to and drives expression of a polynucleotide that encodes a miniSHANK3 protein with an amino acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4 in target cells, including cells of the CNS.

[0134] In some embodiments, the regulatory’ element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) that is operably linked to and facilitates or enhances expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the WPRE comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%.98%, or 99% identity', or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 10, and facilitates or enhances expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the WPRE is operably linked to and facilitates or enhances expression of a polynucleotide thatAtty. Docket No. 38061.0013P1encodes a miniSHANK3 protein with an amino acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4.

[0135] In some embodiments, the regulatory element is a polyA signal that is operably linked to and facilitates or enhances expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the polyA signal is the hGH polyA signal. In some embodiments, the hGH polyA signal comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 11, and facilitates or enhances expression of a polynucleotide encoding a miniSHANK.3 protein in target cells, including cells of the CNS. In some embodiments, the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 11.II. Recombinant AAV (rAAV) Particles Engineered to Express miniSHANK3 Proteins

[0136] Disclosed herein are recombinant AAV viral particles (rAAV particles) comprising polynucleotides (AAV vectors) engineered to express mimSHANK.3 proteins in an organ, tissue, or cell of interest, including, e.g., in specific organs or cells of the central nervous system (CNS) of a subject. In embodiments, the protein of interest is a miniSHANK3 protein. In some embodiments, the miniSHANK3 protein comprises or consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0137] AAV refers to a replication-deficient (e.g.. nonreplicating) Dependoparvovirus within the Parvoviridae genus of viruses. AAV can be derived from a naturally occurring virus or can be recombinant. AAV vectors engineered to express a transgene of interest, such as a miniSHANK3 transgene disclosed herein, can be packaged into rAAV particles comprising capsid proteins, 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.

[0138] AAV vectors comprise an expression cassette engineered to express a transgene of interest, such as a miniSHANK3 transgene disclosed herein, flanked by one orAtty. Docket No. 38061.0013P1more ITRs. including a 5’ ITR (re., an ITR located upstream of the expression cassette) and / or a 3’ ITR (z.e., an ITR located downstream of the expression cassette). The expression cassette itself may comprise one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest (such as a miniSHANK3 protein disclosed herein), and / or additional posttranscriptional regulatory elements. AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary7skill 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.

[0139] 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 regulatory7elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation (poly A) signals.

[0140] In embodiments, the present disclosure provides an rAAV particle comprising an AAV vector and an AAV capsid. In embodiments, the present disclosure provides an rAAV particle comprising (i) an AAV vector comprising AAV inverted terminal repeats (ITRs) flanking an expression cassette comprising one or more regulatory elements (e.g., a promoter, polyA signal sequence, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), or other regulatory7element) operably linked to a polynucleotide encoding a miniSHANK.3 protein (for example, a miniSHANK3 protein having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4), and (li) an AAV9 capsid.

[0141] In some embodiments, a miniSHANK3 protein as disclosed herein is delivered to and expressed in a tissue or a cell of interest via an rAAV particle. In some embodiments, the rAAV particle delivering the miniSHANK3 protein as disclosed herein is delivered to the central nervous system (CNS) of a subject. As used herein, delivering the rAAV particle to the CNS may include delivering the rAAV particle to any tissue or cell of interest in the CNS. In some embodiments, delivering the rAAV particle to the CNS involves delivering the rAAV particle to neuronal tissues or cells. In some embodiments, delivering the rAAV particle to the CNS involves delivering the rAAV particle to the brain. In some embodiments,Atty. Docket No. 38061.0013P1delivering the rAAV particle to the CNS involves delivering the rAAV particle to the white and gray matter.

[0142] As used in the present disclosure, “delivering” or “administering” a recombinant AAV particle can include any method known in the art for delivering or administering an rAAV particle or a composition comprising an rAAV particle to a subject. Administering can include but is not limited to direct administration of an rAAV particle or a composition comprising an rAAV particle. In some embodiments, the administering can be by unilateral intracerebroventricular (ICV) administration. In other embodiments, the administering can be by bilateral ICV administration.

[0143] In some embodiments, an AAV vector engineered to express a miniSHANK3 protein as described herein is packaged as an rAAV particle that is administered to a subject and / or delivered to a selected target cell. In some embodiments, the rAAV particle comprises an AAV capsid protein. In some embodiments, the rAAV particle comprises at least one capsid protein that is an AAV9 capsid protein, or a variant thereof.

[0144] Aspects of the disclosure relate to recombinant AAV vectors expressing miniSHANK3 transgenes. In some embodiments, the AAV vector comprises 5’ and 3’ AAV ITRs flanking an expression cassette engineered to express a miniSHANK3 protein, as described herein. In some embodiments, the AAV ITRs comprise AAV2 ITRs. In some embodiments, the AAV ITRs comprise AAV1 ITRs. In some embodiments, the AAV ITRs comprise AAV5 ITRs. In some embodiments, the AAV ITRs comprise AAV6 ITRs. In some embodiments, the AAV ITRs comprise AAV8 ITRs. In some embodiments, the AAV ITRs comprise AAV9 ITRs. In some embodiments, the AAV ITRs comprise rhlO ITRs. In some embodiments, the AAV ITRs may include one or more modified ITRs which generate self-complementary AAV genomes.

[0145] In some embodiments, the recombinant AAV vector comprises a 5’ AAV2 ITR and a 3’ AAV2 ITR. In some embodiments, the 5’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%.98%. or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 12. In some embodiments, the 3’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,Atty. Docket No. 38061.0013P182%. 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: 13. In some embodiments, the 5’ AAV2 ITR comprises or consists of the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the 3‘ AAV2 ITR comprises or consists of the nucleic acid sequence of SEQ ID NO: 13.A. AAV Vectors and Expression Cassettes

[0146] It should be appreciated that AAV vectors described herein can include 5’ and / or 3’ AAV ITRs flanking expression cassettes that comprise a polynucleotide encoding a miniSHANK.3 protein, said polynucleotide operably linked to promoters, introns, and / or other associated regulatory elements that are known in the art and sufficient to express the miniSHANK3 protein in desired cells and tissues, including those of the CNS. In embodiments, the AAV vector described herein is a single-stranded AAV vector. In other embodiments, the AAV vector described herein is a self-complementary vector.

[0147] In some embodiments, the expression cassette comprises a Syn promoter (e.g, hSynl) that is operably linked to and drives expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In embodiments, the hSynl promoter comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%.92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 9, and drives expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In embodiments, the hSynl promoter comprises the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the hSynl promoter is operably linked to and drives expression of a polynucleotide that encodes a miniSHANK3 protein with an amino acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4 in target cells, including cells of the CNS.

[0148] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory' Element (WPRE), that is operably linked to and facilitates or enhances expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the WPRE comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,Atty. Docket No. 38061.0013P195%. 96%. 97%. 98%. or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 10, and facilitates or enhances expression of a polynucleotide encoding aminiSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the WPRE is operably linked to and facilitates or enhances expression of a polynucleotide that encodes a miniSHANK3 protein with an amino acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4.

[0149] Expression cassettes described herein may also comprise a poly A signal that is operably linked to and facilitates or enhances expression of a polynucleotide encoding a miniSHANK3 protein in target cells, including cells of the CNS. In some embodiments, the polyA signal is the hGH polyA signal. In some embodiments, the hGH polyA signal comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 11, and facilitates or enhances expression of a polynucleotide encoding a miniSHANK.3 protein in target cells, including cells of the CNS. In some embodiments, the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 11.

[0150] In embodiments, the expression cassette comprising a polynucleotide encoding a miniSHANK3 protein as described herein further comprises an untranslated portion such as an intron or a 5' or 3’ untranslated region. In some embodiments, the intron may be located between the promoter / enhancer sequence and the miniSHANK.3 transgene.

[0151] In some embodiments, the expression cassette comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 7, and encodes a miniSHANK3 protein having SHANK3 activity. In some embodiments, the expression cassette comprises SEQ ID NO: 7, and encodes a mimSHANK.3 protein having the amino acid sequence of SEQ ID NO: 3.

[0152] In some embodiments, the inverted terminal repeat (ITR) sequences of the AAV vector comprise about 145 nucleotides each. These elements may be useful in cis forAtty. Docket No. 38061.0013P1effective replication and encapsidation. A skilled person in the art would appreciate that any elements of AAV vectors known in the art may be compatible with aspects of the disclosure. One of skill in the art would also appreciate that any of the polynucleotide sequences described herein that encode a functional miniSHANK3 protein can be expressed in a DNA construct or expression cassette for AAV delivery. These DNA constructs or expression cassettes may include one or more of the elements described herein. For example, in some embodiments a coding sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, or 100% identity to SEQ ID NO: 5 or SEQ ID NO: 6, and encoding a miniSHANK3 protein having SHANK3 activity, is expressed by a DNA construct or expression cassette. In some embodiments a coding sequence comprising the sequence of SEQ ID NO: 5 or SEQ ID NO: 6 is expressed by a DNA construct or expression cassette. In some embodiments, the DNA construct or expression cassette includes one or more elements such as a promoter, a 5’-ITR, a 3’-ITR, a Synl promoter, a WPRE, an hGH poly A. Cis plasmids comprising AAV vector constructs with expression cassettes engineered to express miniSHANK 3 proteins described herein and used for production of the recombinant AAV particles may have elements such as origin of replications and antibiotic resistance markers, for example, an Fl origin, aNeoR / KanR marker and / or a PUC origin. In some embodiments, the Cis plasmid comprises a nucleotide sequence at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identity, including all values in between, to SEQ ID NO: 15, and encodes miniSHANK3 protein having SHANK3 activity. In some embodiments, the Cis plasmid comprises the nucleotide sequence of SEQ ID NO: 15.

[0153] In some embodiments, an AAV vector associated with the disclosure comprises a nucleic acid sequence encoding a miniSHANK3 protein operably linked to regulatory elements that promote CNS expression and flanking ITRs (collectively, an artificial AAV genome), said nucleic acid sequence having 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: 8, and encoding miniSHANK3 protein having SHANK3 activity. In some embodiments, the AAV vector comprises a polynucleotide having the nucleotide sequence of SEQ ID NO: 8, which comprises 5’ and 3’ AAV2 ITRs flanking an expression cassette comprising ahSynlAtty. Docket No. 38061.0013P1promoter operably linked to a polynucleotide encoding a miniSHANK3 protein comprising the amino acid sequence of SEQ ID NO: 5, which in turn is operably linked to a downstream WPRE element and a downstream hGH polyA signal sequence (5’ AAV2 ITR-hSynl -Human miniSHANK3vl-WPRE-hGH polyA-3' AAV2 ITR), as shown in FIG. 3. In some embodiments, a recombinant AAV particle comprising an AAV vector comprising the polynucleotide sequence of SEQ ID NO: 8 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.

[0154] In some embodiments, the recombinant AAV vector encoding a miniSHANK3 protein comprises an expression cassette comprising a polynucleotide sequence that encodes a protein with 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% identity, or is 100% identical, to SEQ ID NO: 3 or SEQ ID NO: 4, and has SHANK3 activity .

[0155] The present disclosure provides recombinant AAV particles comprising: (1) AAV vectors described herein and (2) an AAV9 capsid. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 70%. 71%. 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, to SEQ ID NO: 14. As used herein, a “virion” refers to a viral particle that includes genetic material (e.g, RNA or DNA) and a capsid.

[0156] In some embodiments, an AAV vector comprising an expression cassette that comprises a polynucleotide encoding a miniSHANK3 protein (i.e., the miniSHANK3 DNA construct) can be expressed in a specific tissue or cell of interest. In some embodiments, the expression cassette or vector disclosed herein comprises a promoter. In some embodiments, the human promoter is human Synapsin 1 (hSynl). In some embodiments, the hSynlpromotor has the polynucleotide sequence of SEQ ID NO: 9.III. Methods of Making rAAV Particles

[0157] The present disclosure provides methods of producing an rAAV particle. In some embodiments, the method comprises culturing a host cell compnsing an AAV vector described herein, an AAV cap (capsid protein) and an AAV9 rep (replication protein), andAtty. Docket No. 38061.0013P1optionally one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell. In some embodiments, the AAV cap encodes VP1, VP2, and / or VP3. In some embodiments, the rep encodes rep78, rep68, rep52, and / or rep40.

[0158] Recombinant AAV particles can be made by any method known in the art. Further, the rAAV particle can be made into a final pharmaceutical formulation according to any method known in the art. Methods for obtaining recombinant AAV particles having a desired capsid protein can be obtained from U.S. Patent Application Publication Number 2003 / 0138772, for example, which is incorporated by reference herein in its entirety.Typically, such methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; sufficient helper genes to permit packaging of the recombinant AAV vector into the desired AAV capsid proteins; and a plasmid comprising the AAV vector. Typically, capsid proteins are structural proteins encoded by the cap gene of an AAV. In some aspects, wherein the capsid protein comprises VP1, VP2, and VP3, said VP1, VP2, and VP3 are transcribed from a single cap gene via alternative splicing. In some aspects, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some aspects, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some aspects, capsid proteins protect a viral genome, deliver a genome and / or interact with a host cell. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

[0159] In some aspects, components to be cultured in the host cell to package an AAV vector as an rAAV particle can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g, AAV vector, rep sequences, cap sequences, and / or helper functions) can be provided by a stable host cell which has been engineered to contain one or more of the required components.

[0160] The AAV vector, rep sequences, cap sequences, and helper functions or genes useful for producing the rAAV particles described herein can be delivered to the packaging host cell using any appropriate genetic element (e.g., a plasmid). The selected genetic element can be delivered by any suitable method, including those described herein. The methods used to construct any of compositions disclosed herein are known to those with skillAtty. Docket No. 38061.0013P1in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. (See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., which is incorporated by reference herein in its entirety) Similarly, methods of generating rAAV particles are well known and the selection of a suitable method is not a limitation on the present disclosure. (See. e.g, K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745, which are incorporated by reference herein in their entirety.)

[0161] In some aspects, rAAV particles can be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650, which is incorporated by reference herein in its entirety). Typically, the recombinant AAVs can be produced by transfecting a host cell with a plasmid comprising an AAV vector construct (comprising a transgene and expression sequences flanked by ITRs) to be packaged into rAAV particles, a packaging plasmid comprising AAV helper function sequences, and a plasmid comprising accessory function sequences. An AAV packaging plasmid encodes the “AAV helper function"’ sequences (i. e.. rep and cap), which function in trans for productive AAV replication and encapsidation with the cap gene encoding the capsid proteins of desired serotype, for example, encoding AAV9 capsid proteins as disclosed herein.

[0162] The accessory function plasmid encodes nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the know n helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

[0163] In embodiments, a tangential flow filtration (TFF) process is used to place rAAV particles as described herein into the disclosed pharmaceutical formulations. In TFF, the bulk of the AAV particle composition flows tangentially across the surface of the filter, minimizing membrane fouling while maintaining a high filtration rate. TFF systems are known in the art and commercially available systems are sold by, for example, Millipore, Repligen, Sartorius and Pall.Atty. Docket No. 38061.0013P1IV. Pharmaceutical Compositions

[0164] The present disclosure provides compositions, including pharmaceutical compositions, comprising a polynucleotide (e.g., encoding for miniSHANK3) delivered in a recombinant AAV vector and / or an rAAV particle as disclosed herein and a pharmaceutically acceptable carrier.

[0165] Suitable carriers may be readily selected by one of ordinary skill in the art in view of the indication for which the recombinant AAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure. Pharmaceutical compositions comprising AAV vectors are described further in PCT / US2024 / 019250, which is incorporated by reference herein in its entirety.

[0166] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0167] Buffering Agent

[0168] The disclosed pharmaceutical formulations comprise at least one buffering agent. In embodiments, the buffering agent is Tris HC1, Tris base, sodium phosphate, phosphate buffered saline (PBS), acetate, acetic acid, alanine, arginine, aspartic acid, boric acid, citric acid, glutamic acid, glycine, histidine, lysine, potassium phosphate, sodium acetate, sodium citrate, sodium succinate, succinic acid, tromethamine, HEPES and / or MOPS.

[0169] In embodiments, the buffering agent is present at a concentration between about 5 mM and about 100 mM, about 5 mM and about 50 mM, about 5 mM and about 25 mM, about 5 mM and about 15 mM, about 5 mM and about 10 mM.

[0170] In embodiments, the buffering agent is present at a concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,Atty. Docket No. 38061.0013P1about 40 mM, about 45 mM. about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0171] In embodiments, the buffering agent is appropriate for a pH of about 5.0 to about 9.0, about 6.0 to about 9.0, 6.0 to about 8.5, 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.0, or about 7.5 to about 8.5.

[0172] Magnesium-Containing Salts

[0173] The disclosed pharmaceutical formulations comprise at least one magnesium-containing salt. In embodiments, the magnesium-containing salt is MgCh. In embodiments, the magnesium-containing salt is MgSO-i. In embodiments, the magnesium-containing salt is Magnesium Chloride Hexahydrate.

[0174] In embodiments, the magnesium-containing salt is present at a concentration of about 0.5 mM MgCh, about 1 mM MgCh, about 1.5 mM MgCh, or about 2 mM MgCh.

[0175] Tonicity Modifier

[0176] The disclosed pharmaceutical formulations comprise at least one tonicity modifier. In embodiments, the tonicity modifier is sodium chloride (NaCl), sorbitol or trehalose. In embodiments, the tonicity modifier is dextrose, guanidine, magnesium chloride, maltose, mannitol, potassium chloride, sodium citrate, sodium phosphate, sodium sulfate, and / or sucrose.

[0177] In embodiments, the tonicity modifier is about 100 mM NaCl. about 125 mM NaCl, about 150 mM NaCl, about 175 mM NaCl or about 200 mM NaCl.

[0178] In embodiments, the tonicity modifier is about 2% sorbitol, about 2.5% sorbitol, about 3% sorbitol, about 3.5% sorbitol, about 4% sorbitol, about 4.5% sorbitol, about 5% sorbitol, about 5.5% sorbitol, about 6% sorbitol, about 6.5% sorbitol or about 7% sorbitol. In embodiments, the formulation does not comprise sorbitol.

[0179] In embodiments, the tonicity modifier is about 2% trehalose, about 2.5% trehalose, about 3% trehalose, about 3.5% trehalose, about 4% trehalose, about 4.5% trehalose, about 5% trehalose, about 5.5% trehalose, about 6% trehalose, about 6.5% trehalose or about 7% trehalose. In embodiments, the formulation does not comprise trehalose.Atty. Docket No. 38061.0013P1

[0180] In embodiments, the pharmaceutical composition does not comprise a sugar (sucrose). In embodiments, the pharmaceutical composition does not comprise a sugar substitute.

[0181] In embodiments, the tonicity modifier achieves an osmolality of about 150 to about 450 mOsm / kg, about 175 to about 425 mOsm / kg, about 200 to about 400 mOsm / kg, about 225 to about 375 mOsm / kg, about 250 to about 350 mOsm / kg, about 275 to about 325 mOsm / kg or about 300 to about 400 mOsm / kg.

[0182] N on-ionic Surfactant

[0183] In embodiments, the pharmaceutical composition comprises anon-ionic surfactant. In embodiments, the non-ionic surfactant is poloxamer 188 (Pluronic® F68). In embodiments, the non-ionic surfactant is polysorbate 20 and / or polysorbate 80.

[0184] In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.20% w / v non-ionic surfactant. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%. about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%. about 0.15%. about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% w / v non-ionic surfactant.

[0185] In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.20% w / v poloxamer 188. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%. about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%. about 0.15%. about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% w / v poloxamer 188. In embodiments the concentration of poloxamer 188 is greater than 0.005%, greater than 0.01%, or greater than or including 0.02%, w / v and up to 0.2% or 0.3% or 0.4% or 0.5% w / v. In embodiments, the pharmaceutical composition comprises up to about 0.0460% or up to about 0.0663% w / v poloxamer 188.

[0186] In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.20% w / v polysorbate 20. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, aboutAtty. Docket No. 38061.0013P10.14%. about 0.15%. about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% w / v polysorbate 20.

[0187] In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.20% w / v polysorbate 80. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%. about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%. about 0.15%. about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% w / v polysorbate 80.

[0188] In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% w / v non-ionic surfactant. In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%. from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% w / v poloxamer 188. In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% w / v polysorbate 20. In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% w / v polysorbate 80.

[0189] pH

[0190] In embodiments, the pharmaceutical composition has a pH of about 5.0, about 5.5, about 6.0, about 6.5. about 7.0, about 7.1. about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In embodiments, the pharmaceutical composition has a pH of about 8.0.

[0191] In embodiments, the pharmaceutical composition has a pH of about 5.0 to about 9.0, about 6.0 to about 9.0, 6.0 to about 8.5, 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.0, or about 7.5 to about 8.5.

[0192] In embodiments, the pharmaceutical composition has a pH of about 7.9 to about 8.1, about 7.8 to about 8.2, about 7.8 to about 8.2, about 7.7 to about 8.3. about 7.6 to about 8.4, about 7.5 to about 8.5, about 7.4 to about 8.6, about 7.3 to about 8.7, about 7.2 toAtty. Docket No. 38061.0013P1about 8.8, about 7.1 to about 8.9 or about 7.0 to about 9.0. In embodiments, the pharmaceutical composition has a pH of about 7.0 to about 8.0.

[0193] Osmolality

[0194] In embodiments, the pharmaceutical composition has an osmolality of about 150 to about 450 mOsm / kg, about 175 to about 425 mOsm / kg, about 200 to about 400 mOsm / kg, about 225 to about 375 mOsm / kg, about 250 to about 350 mOsm / kg, about 275 to about 325 mOsm / kg or about 300 to about 400 mOsm / kg.

[0195] In embodiments, the pharmaceutical composition has an osmolality of about 150 mOsm / kg, about 175 mOsm / kg. about 200 mOsm / kg, about 225 mOsm / kg, about 250 mOsm / kg, about 275 mOsm / kg. about 300 mOsm / kg. about 325 mOsm / kg, about 350 mOsm / kg, about 375 mOsm / kg, about 400 mOsm / kg, about 425 mOsm / kg, or about 450 mOsm / kg.

[0196] In embodiments, the pharmaceutical composition does not comprise a preservative.

[0197] In embodiments, the unit dosage form has a volume of about 1 mL, about 1.25 mL, about 1.5 mL, about 1.75 mL, about 2 mL, about 2.15 mL, about 2.25 mL, about 2.5 mL, about 2.75 mL. or about 3 mL. In embodiments, the unit dosage form has a volume of between about 1 mL and about 3 mL, between about 1.5 mL and about 2.5 mL, between about 2 mL and about 2.5 mL, or between about 2mL and about 3 mL. In an embodiment, the unit dosage form has a fill volume of 2.15 mL.

[0198] In embodiments, the rAAV pharmaceutical formulation comprises, consists essentially of, or consists of an rAAV particle comprising (i) an AAV vector comprising an expression cassette flanked by ITRs. wherein the expression cassette comprises a polynucleotide encoding a human miniSHANK3 protein with an amino acid sequence that is at least 90%, 95%, 99% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4 and has SHANK3 activity, operably linked to a human Syn promoter and a polyA signal sequence and (ii) an AAV9 capsid or a capsid having an amino acid sequence that is at least 90% identical to or at least 95% identical to SEQ ID NO: 14. said rAAV particle formulated at a concentration of l x 1013vg / mL to 1 x 1014vg / mL in a solution comprising 10 mM Tris, 1 mM magnesium chloride (MgCh), 150 mM sodium chloride (NaCl) and 0.02% (w / v)Atty. Docket No. 38061.0013P1poloxamer 188, pH 8.0 buffer with. In some embodiments, the rAAV particle is present in the formulation described above at a concentration of 6.5 x 1013vg / mL.

[0199] In some embodiments, the rAAV pharmaceutical formulation is provided as a single unit dosage form. In some embodiments, the single dosage form comprises 2.15 mL at a concentration of 6.5 x 1013vg / mL. In some embodiments, 2.94 mL, 3.32 mL, 8.82 mL or 9.69 mL is administered to the patient from multiple vials to provide the appropriate total dose (z.e., as measured in total number of viral genomes delivered, as described herein).

[0200] In embodiments, the rAAV pharmaceutical formulation is filled into 5 mL cyclic olefin polymer (Crystal Zenith®) vials with a fill volume of 2.15 mL, which contains a 0.15 mL overfill to ensure the labeled fill volume of 2.0 mL is achieved. The vials are stoppered with a pre-sterilized ready -to-use 20 mm chlorobut l rubber serum stopper with FluroTec® B2 coating and sealed with a pre-sterilized, ready -to-use packaged, aluminum seal with a colored plastic flip-off cap.

[0201] In embodiments, the rAAV pharmaceutical formulation is stored frozen at < -60°C. In embodiments, shipment of the rAAV pharmaceutical formulation is performed under dry ice and transferred to < -60°C storage upon receipt.V. Methods of T reatmentA. ICV Administration

[0202] Methods described herein comprise administering a therapeutic dose to pediatric or adult subjects, said therapeutic dose comprising rAAV particles comprising an AAV vector engineered to express a miniSHANK protein as described herein in sufficient amounts to transduce the cells of a desired tissue (e g., brain) and to provide sufficient levels of gene transfer and expression to treat PMS and ameliorate symptoms without undue adverse effects. In some embodiments, the recombinant AAV particle is delivered to the cells of a desired tissue (e.g, brain) via ICV administration. In some embodiments, the ICV administration is unilateral ICV administration. In some embodiments, the ICV administration is bilateral ICV administration.

[0203] In some embodiments, the present disclosure provides methods of treating a subject having a neurodevelopmental disorder. In some embodiments, the present disclosure provides methods of treating a subject having an autism spectrum disorder (ASD). In some embodiments, the present disclosure provides methods of treating a subject having Phelan-Atty. Docket No. 38061.0013P1McDermid syndrome. In some embodiments, the present disclosure provides methods of treating a subject having SHANK3 haploinsufficiency. In some embodiments, the present disclosure provides methods of treating a subject having only SHANK3 deletions or mutations. In other embodiments, the present disclosure provides methods of treating a subject having SHANKS deletions or mutations in combination with RNd and / or ACR and RABL2B deletions or mutations.

[0204] Methods provided herein, in some embodiments, comprise administering and delivering an effective amount of a composition comprising an rAAV particle that comprises an expression cassette comprising a polynucleotide encoding a SHANK3 protein (e.g., miniSHANK3) to a target environment or tissue of a subject. In some embodiments, methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof comprise providing a composition comprising an rAAV particle comprising at least a nucleic acid (e.g., miniSHANK3) to be delivered to the target environment or tissue of the subject and administering the composition to the subject. In some embodiments, methods for delivering a nucleic acid to a target environment or tissue of a subject in need thereof include delivering an rAAV particle by unilateral or bilateral intracerebroventricular (1CV) administration. In embodiments, rAAV particles for use with the methods of treatment provided herein comprise (i) at least one AAV9 capsid protein, and (ii) an artificial AAV genome comprising a nucleic acid sequence encoding a miniSHANK3 protein operably linked to regulatory elements that promote CNS expression and flanking ITRs (collectively, an artificial AAV genome), said nucleic acid sequence having 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: 8, and encoding miniSHANK3 protein having SHANK3 activity. In some embodiments, rAAV particles for use with the methods of treatment described herein comprise (i) at least one AAV9 capsid protein, and (ii) an artificial AAV genome comprising an AAV vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 8. which comprises 5’ and 3‘ AAV2 ITRs flanking an expression cassette comprising ahSynl promoter operably linked to a polynucleotide encoding a miniSHANK3 protein comprising the amino acid sequence of SEQ ID NO: 5, which in turn is operably linked to a downstream WPRE element and aAtty. Docket No. 38061.0013P1downstream hGH polyA signal sequence (5’ AAV2 ITR-hSynl -Human miniSHANK3vl-WPRE-hGH polyA-3’ AAV2 ITR), as shown in FTG. 3.

[0205] The inventors have determined doses (e.g., the units of dose in absolute vector genomes (vg)) of a recombinant AAV vector or rAAV particle comprising a polynucleotide encoding aminiSHANK3 protein (e.g., a miniSHANK3 protein having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4) as described herein, including “rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl” particles that, as described herein, comprise AAV vectors comprising the polynucleotide sequence of SEQ ID NO: 8 (5’ AAV2 ITR-hSynl -Human miniSHANK3vl-WPRE-hGH polyA-3’ AAV2 ITR), as shown in FIG. 3. which can achieve a particular “therapeutic effect” that outweighs adverse effects in patients suffering from SHANK3 related disorders such as PMS, including pediatric patients aged 2-9years. pediatric patients aged 10-17years, and adult patients (age 18 years or older).

[0206] In some embodiments, the patients have Class I deletions (z.e., deletions including only SHANK3 or SHANK3 in combination with ARSA and / or ACR and RABL2B). In some embodiments, the patients have Class II deletions (all other deletions). See Levy T, Foss-Feig JH, Betancur C, et al. Strong evidence for genotype-phenotype correlations in Phelan-McDermid syndrome: results from the developmental synaptopathies consortium. Hum Mol Genet. 2022;31(4):625-637.

[0207] In some embodiments, the number of vector genomes of rAAV9 AAV-hSyn l-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human mini SH ANKA vl-WPRE-hGH polyA- 3’ ITR) (SEQ ID NO: 8) by unilateral ICV administration administered to an adult subject is (i) about 2.16* 1014rAAV vector genomes (vg), or 2.16x1014vg, or 2.16xl014+ / - 10% or 20% rAAV vg, or between 2. IxlO14to 2.2xl014vg; or (ii) about 5.73xl014vg, or 5.73xl014vg. or 5.73xl014+ / - 10% or 20% vg, or between 5.7x1014to 5.8xl014vg; or (iii) about 6.30xl014vg, or 6.30xl014vg, or 6.30xl014+ / - 10% or 20% vg, or between 6.2xl014to 6.4x 1014vg. In some embodiments, the number of vector genomes administered to an adult subject is between about 2.16xl014vg and about 6.30xl014vg in an adult subject.

[0208] In some embodiments, the number of vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl Human miniSHANK3vl-WPRE-hGH polyA- 3’ ITR) (SEQ ID NO: 8) by unilateral ICV administration administered to a pediatric subject age 2 to 9 is (i) about 1.91 xlO14vg, or 1.91 xlO14vg, or 1.91 xlO14+ / - 10% or 20% vg, orAtty. Docket No. 38061.0013P1between 1.85xl014to 1.95xl014) vg; or (ii) about 5.73*1014vg. or 5.73*1014vg. or 5.73xl014+ / - 10% or 20% vg or between 5.7x114to 5.8x 1014vg. In some embodiments, the number of vector genomes administered to the subject is between about 1.91 x 1014vg and about 5.73 x 1014vg in a pediatric subject age 2 to 9.

[0209] In some embodiments, the number of vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSyn 1 -Human mini SHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) by unilateral ICV administration administered to a pediatric subject age 10 to 17 is (i) about 2.16x 1014Vg, or 2.16x 1014vg, or 2.16x 1014+ / - 10% or 20% rAAV vg, or between 2.1xl014to 2.2xl014vg; or (ii) about 5.73xl014vg, or 5.73xl014vg, or between 5.7xl014to 5.8 xlO14vg; or (iii) about 6.30xl014vg, or 6.30xl014vg, or between 6.2x 1014to 6.4x 1014vg . In some embodiments, the number of vector genomes administered to the subject is between about 2.16xl014vg and about 6.30xl014vg in apediatric subject age 10 to 17.

[0210] In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) administered by unilateral ICV administration is 1.91 xlO14vg per pediatric subject age 2 to 9. In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMimSHANK3-Vl (5’ ITR-hSynl -Human mmiSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 5.73xl014vg per pediatric subject age 2 to 9.

[0211] In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 2.16xl014vg per adult subject. In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5' ITR-hSynl -Human miniSHANK3vI-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 5.73xl014vg per adult subject. In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 6.30xl014vg per adult subject.

[0212] In certain embodiments, the effective amount of rAAV 9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 2.16xl014vg per subject age 10 to 17. In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-Atty. Docket No. 38061.0013P1WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 5.73x 1014vg per subject age 10 to 17. In certain embodiments, the effective amount of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vI -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is 6.30xl014vg per subject age 10 to 17.

[0213] In some embodiments, a dose of about 2.16xl014, or about 5.73xl014, or about 6.30xl014, or between about 2.16xl014and about 6.30xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5' ITR-hSynl -Human miniSHANK3vl -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to an adult subject. In some embodiments, a dose of 2.16x1014, or 5.73xl014, or 6.30xl014, or between 2.16xl014and 6.30xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5‘ ITR-hSyn 1 -Human mini SHANK3vl -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to the adult subject. In some embodiments, a dose of (i) 2.16xl014+ / - 10% or 20%, or between 2.1xl014to 2.2xl014vector genomes; or (ii) 5.73xl014+ / - 10% or 20%, or between 5.7xl014to 5.8xl014vector genomes; or (iii) 6.30xl014+ / - 10% or 20%, or between 6.2xl014to 6.4xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to the adult subject.

[0214] In some embodiments, a dose of about 1.91 xlO14, or about 5.73xl014, or between about 1.91xl014and about 5.73xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSyn 1 -Human mini SHANK3vl -WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to a pediatric subject age 2 to 9. In some embodiments, a dose of 1.91 xlO14, or 5.73 xJO14, or between 1.91 xlO14and 5.73xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5‘ ITR-hSynl-Human miniSHANK3vl -WPRE-hGH poly A- 3' ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to the pediatric subject age 2 to 9. In some embodiments, a dose of (i) 1.91 x 1014+ / - 10% or 20%, or between 1.85xl014to 1.95 xlO14vector genomes; or (ii) 5.73xl014+ / - 10% or 20%, or between 5.7xl014to 5.8xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3' ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to a pediatnc subject age 2 to 9.Atty. Docket No. 38061.0013P1

[0215] In some embodiments, a dose of about 2.16xl014, or about 5.73xl014. or about 6.30x 1014, or between about 2.1 x 1014and about 6.30x ] 014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-h Sy nl -Human mini SH ANKA vl-WPRE-hGH poly A- 3‘ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to a subject age 10 to 17. In some embodiments, a dose of 2.16xl014, or 5.73xl014, or 6.30xl014. or between 2.16xl014and 6.30xl014vector genomes AAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human mini SH ANKA vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to the subject age 10 to 17. In some embodiments, a dose of (i) 2.16xl014+ / - 10% or 20%, or between 2.1xl014to 2.2x l014vector genomes; or (ii) 5.73xl014+ / - 10% or 20%, or between 5.7x1014to 5.8xl014vector genomes; or (iii) 6.30xl014+ / - 10% or 20%, or between 6.2xl014to 6.4xl014vector genomes of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSynl -Human miniSHANKAvl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) is administered via unilateral ICV administration to the subject age 10 to 17.

[0216] In some embodiments, provided are pharmaceutical compositions and unit dosage forms in which the nominal concentration of drug product in the formulation is 6.5 x IO1-5vg / mL. In some embodiments, to achieve the administration of 1.91 xlO14vg, 2.94 mL is administered to the subject. In some embodiments, to achieve the administration of 2.16xl014vg, 3.32 mL is administered to the subject. In some embodiments, to achieve the administration of 5.73x 1014vg, 8.82 mL is administered to the subject. In some embodiments, to achieve the administration of 6.3x 1014vg, 9.69 mL is administered to the subject.

[0217] In some embodiments, the dose administered to the subject via unilateral ICV administration is about IrnL, 1.5 mL, 2mL, 3 mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL or lOmL per subject. In some embodiments, the dose administered to the subject via unilateral ICV administration is about 2.94 mL, 3.3 mL. 8.82 mL. or 9.69 mL per subject.

[0218] In some embodiments, the dose of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSynl -Human miniSHANKAvl-WPRE-hGH poly A- 3' ITR) (SEQ ID NO: 8) administered to an adult subject via unilateral ICV administration is about 2.16xl014vg. In some embodiments, the dose of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSyn 1 -Human mini SHANKAvl-WPRE-hGH poly A- 3’Atty. Docket No. 38061.0013P1ITR) (SEQ ID NO: 8) administered to the adult subject via unilateral ICV administration is about 5.73*1014vg. In some embodiments, the dose administered to the adult subject of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANKAvl-WPRE-hGH poly A- 3‘ ITR) (SEQ ID NO: 8) via unilateral ICV administration is about 6.30* 1014vg. In some embodiments, the dose administered of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSynl -Human mmiSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) to the adult subject via unilateral ICV administration is from about 2.16xl014vg to about 6.30*1014vg.

[0219] In some embodiments, the dose administered of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) to the pediatric subject age 2 to 9 via unilateral ICV administration is about 1.91xl014vg. In some embodiments, the dose administered of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human mini SH ANKA vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) to the pediatric subject via unilateral ICV administration is about 5.73xl014vg. In some embodiments, the dose administered to the pediatric subject age 2 to 9 of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANKAvl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) via unilateral ICV administration is from about 1.91xl014vg to about 5.73xl014vg.

[0220] In some embodiments, the dose of rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) administered to the pediatric subject age 10 to 17 via unilateral ICV administration is about 2.16xl014vg. In some embodiments, the dose of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSynl -Human mmiSHANKAvl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) administered to the pediatric subject age 10 to 17 via unilateral ICV administration is about 5.73 xlO14vg. In some embodiments, the dose administered to the pediatric subject age 10 to 17 of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSynl -Human mini SH ANKA vl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) via unilateral ICV administration is about 6.30x 1014vg. In some embodiments, the dose administered to the pediatric subject age 10 to 17 of rAAV9 AAV-hSynl-HumanMiniSHANKA-Vl (5’ ITR-hSyn 1 -Human miniSHANKAvl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) via unilateral ICV administration is from about 2.16x 1014vg to about 6.30xl014vg.Atty. Docket No. 38061.0013P1

[0221] In some embodiments, a dose of recombinant AAV is administered to a subject as a single dose.B. Patient primary endpoints

[0222] Methods described herein comprise administering rAAV particles (e.g., rAAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl -Human miniSHANK3vl-WPRE-hGH poly A- 3‘ ITR) (SEQ ID NO: 8)) for gene therapy to a subject in need thereof at therapeutically effective dosages. In embodiments, the effect of administration of rAAV particles described herein for gene therapy is determined in comparison to a "baseline" result from a test or other examination described herein that is administered to the subject prior to administration of recombinant AAV to the subject. In other embodiments, the effect of administration of rAAV particles as described herein for gene therapy is determined in comparison to age-matched natural history control subjects who have not been administered the rAAV gene therapy. In embodiments, the effect of administration of rAAV particles as described herein for gene therapy is indicated by an improvement in test results as compared to a baseline result or to age-matched natural history’ control subjects who have not been administered the rAAV gene therapy. In other embodiments, the effect of administration of rAAV particles as described herein for gene therapy is indicated by a stabilization (z.e., lack of meaningful decrease) in test results as compared to a baseline result or to age-matched natural history control subjects who have not been administered the rAAV gene therapy.

[0223] VEP is a non-invasive exploration method of the functionality of the human visual system through detecting neuronal pool activity responding to stimuli independently of the consciousness and attention state of the patient. Individuals with PMS display distinct transient visual evoked potential (VEP) waveform abnormalities in both time and frequency domains that might reflect underlying glutamatergic deficits. Additionally, profound sensory' under-responsiveness has been previously documented in PMS. In embodiments, the gene therapy methods described herein result in changes in, stabilization of, or improvement of visual evoked potential (VEP) w aveform abnormalities as compared to baseline results at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein result in changes in, stabilization of, or improvement of visual evoked potential (VEP) waveform abnormalities as compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein result in changes in, stabilization of, orAtty. Docket No. 38061.0013P1improvement of visual evoked potential (VEP) waveform abnormalities as compared to baseline or age-matched controls at 2 years, 5 years, 10 years, 15 years or more after administration. In some embodiments, the methods described herein lead to changes in, stabilization of, or improvement in one or more visual evoked potential (VEP) waveform parameters, including N75 amplitude, P100 amplitude, N75 to P100 peak to peak amplitude. N2 to P2 peak to peak amplitude, N75 to P100 peak to peak slope, and N2 to P2 peak to peak slope, when compared to baseline or age-matched controls. Changes in VEP may support a change in brain biology' that appears to correlate with other changes observed post-dosing.

[0224] AEP is an electrical signal elicited from the brain while an auditory stimulus is presented in a time-locked manner. AEP signal consists of reproducible positive or negative peaks, latency, amplitude and behavioral correlation. In embodiments, the gene therapy methods described herein result in changes in, stabilization of, or improvement of auditory evoked potential (AEP) waveform abnormalities as compared to baseline results at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein result in changes in. stabilization of, or improvement of auditory evoked potential (AEP) waveform abnormalities as compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein result in changes in, stabilization of, or improvement of auditory evoked potential (AEP) waveform abnormalities as compared to baseline or age-matched controls at 2 years, 5 years, 10 years, 15 years or more after administration. In some embodiments, the gene therapy methods described herein lead to changes in, stabilization of, or improvement in one or more auditory evoked potential (AEP) waveform parameters, including N75 amplitude, P100 amplitude, N75 to P100 peak to peak amplitude, N2 to P2 peak to peak amplitude, N75 to P100 peak to peak slope, and N2 to P2 peak to peak slope, when compared to baseline or age-matched controls. Changes in AEP may support a change in brain biology that appears to correlate with other changes observed post-dosing.

[0225] In some embodiments, the gene therapy methods described herein improve or stabilize sensory symptoms associated with SHANK3 haploinsufficiency as measured by Sensory Assessment for Neurodevelopmental Disorders (SAND) over baseline at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve sensory symptoms associated with SHANK3 haploinsufficiency asAtty. Docket No. 38061.0013P1measured by Sensors’ Assessment forNeurodevelopmental Disorders (SAND) over age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize sensory symptoms associated with SHANK3 haploinsufficiency as measured by Sensory Assessment for Neurodevelopmental Disorders (SAND) over baseline or over age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0226] In some embodiments, the gene therapy methods described herein improve or stabilize adaptive behavior as measured by Vineland Adaptive Behavior Scales, Third Edition (Vineland-3), compared to baseline at 1 month, 3 months, 6 months and / or 12 months after administration. Vineland-3 measures adaptive behavior skills, which are skills needed to function independently at home, school / work. and in the community. The Vineland-3 assessment provides scores for four domains (Socialization, Daily Living Skills, Communication, and Motor), as well as a Composite Score. Participants are compared to a normal population with a standard score of 100 + / - one standard deviation (SD) (85-115). PMS patients generally score less than the intellectual disability threshold of 70. In some embodiments, the gene therapy methods described herein improve or stabilize adaptive behavior as measured by Vineland-3 compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize adaptive behavior as measured by Vineland-3 compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration. In embodiments, the gene therapy methods described herein result in (i) stabilization of or an increase of at least 2, at least 5, or at least 10 points in the Vineland-3 Composite Score; or alternatively (ii) stabilization of or an increase of at least 2, at least 5, or at least 10 points in one or more of the Vineland-3 Socialization, Daily Living Skills, Communication, or Motor domains, or a combination thereof, at 1 month, 3 months, 6 months and / or 12 months, 1 year, 2 years, 5 years, 10 years, 15 years or more after administration, as compared to baseline or age-matched control subjects.

[0227] In some embodiments, the gene therapy methods described herein improve or stabilized adaptive behavior as measured by the Bayley Scales of Infant and Toddler Development, Fourth Edition (Bayley -4), compared to baseline or age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. Bayley-4 is a clinician-administered test of cognitive development (DQ) wherein the patient is asked toAtty. Docket No. 38061.0013P1demonstrate developmental skills and abilities across multiple domains. Participants are compared to a normal population with a standard score of 100 ± 1 SD (85-115). PMS patients generally score less than the intellectual disability threshold of 70. In some embodiments, the methods described herein improve or stabilize adaptive behavior as measured by Bayley-4 compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize adaptive behavior as measured by Bayley -4 compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration. In embodiments, Bayley-4 scores are increased by at least 2 points at 1 month, 3 months, 6 months and / or 12 months after administration compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0228] In some embodiments, the gene therapy methods described herein improve or stabilize global cognitive ability as measured by intelligence quotient (IQ) or developmental quotient (DQ) compared to baseline at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize global cognitive ability as measured by intelligence quotient (IQ) or developmental quotient (DQ) compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize global cognitive ability as measured by intelligence quotient (IQ) or developmental quotient (DQ) compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0229] In some embodiments, the gene therapy methods described herein improve or stabilize language skills as measured by Peabody Picture Vocabulary Test-fifth edition (PPVT-5), Expressive Vocabulary Test-third edition (EVT-3), Vineland Expressive and Receptive Language Subscales, and / or Observer-reported Communication Ability (ORCA) compared to baseline at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize language skills as measured by Peabody Picture Vocabulary Test-fifth edition (PPVT-5), Expressive Vocabulary Test-third edition (EVT-3), Vineland Expressive and Receptive Language Subscales, and / or Observer-reported Communication Ability (ORCA) compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilizeAtty. Docket No. 38061.0013P1language skills as measured by Peabody Picture Vocabulary Test-fifth edition (PPVT-5). Expressive Vocabulary' Test-third edition (EVT-3), Vineland Expressive and Receptive Language Subscales, and / or Observer-reported Communication Ability' (ORCA) compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0230] In some embodiments, the gene therapy methods described herein improve or stabilize motor functioning as measured by Vineland Motor Subscales and / or Visual-Motor Integration (VMI) compared to baseline at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize motor functioning as measured by Vineland Motor Subscales and / or Visual-Motor Integration (VMI) compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize motor functioning as measured by Vineland Motor Subscales and / or Visual-Motor Integration (VMI) compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0231] In some embodiments, the gene therapy methods described herein improve or stabilize autism symptoms as measured by Repetitive Behavior Scales-Revised (RBS-R), Short Sensory Profile-2 Questionnaire (SSP-2) and / or Aberrant Behavior Checklist (ABC) compared to baseline at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize autism symptoms as measured by Repetitive Behavior Scales-Revised (RBS-R), Short Sensory Profile-2 Questionnaire (SSP-2) and / or Aberrant Behavior Checklist (ABC) compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize autism symptoms as measured by Repetitive Behavior Scales-Revised (RBS-R), Short Sensory Profile-2 Questionnaire (SSP-2) and / or Aberrant Behavior Checklist (ABC) compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0232] In some embodiments, the gene therapy methods described herein improve or stabilize sleep behaviors or the frequency / severity of sleep disturbances or disorders, as measured by the Child Sleep Habits Questionnaire (CSHQ) compared to baseline at 1 month,Atty. Docket No. 38061.0013P13 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize sleep behaviors or the frequency / severity of sleep disturbances or disorders, as measured by the Child Sleep Habits Questionnaire (CSHQ) compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the methods described herein improve or stabilize sleep behaviors or the frequency / severity of sleep disturbances or disorders, as measured by the Child Sleep Habits Questionnaire (CSHQ) compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.

[0233] In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by PMS-Specific Clinical Global Impression Scale of Improvement (CGI-I) or PMSA-C (an updated version of the CGI -I scale that incorporates anchoring criteria; CGI-I and PMSA-C may be used interchangeably herein, and collectively are referred to as CGI-I / PMSA-C), including as compared to age-matched controls. In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by PMS-Specific Clinical Global Impression of Severity (CG1-S) or PMSA-S (an updated version of the CG1-S scale; CGI-S and PMSA-S may be used interchangeably herein, and collectively are referred to as CGI-S / PMSA-S), including as compared to baseline or age-matched controls.

[0234] The PMS-Specific CGI-I is a clinician-rated measure designed to assess the change in symptoms associated with Phelan-McDermid Syndrome (PMS) (see, e.g., Guy W. ECDEU Assessment manual for psychopharmacology. US Department of Health, and Welfare, Washington, DC, Publication ADM, 1976, pp. 534-537; Berry-Kravis E, et al. The Development of Phelan-McDermid-specific anchors for the Clinical Global Impression Scales (P17-6.001), 98 Neurology (Supp 18):P17-6.001 (2022) (“Berry-Kravis, 2022”), which are incorporated herein by reference in their entirety). This instrument captures the clinician’s impression of the participant’s change in symptom presentation compared to the baseline visit. The PMS-Specific CGI-I and the PMS-Specific CGI-S were developed by adapting standard clinical global impression scales to enable assessment of the signs and symptoms of PMS. Symptom areas that are clinically and functionally important to people living with PMS were identified based on published literature, input from patient advocacy organizations, and feedback from clinicians. Evaluated symptom areas include expressive communication, receptive communication, gross motor function, fine motor function, socialAtty. Docket No. 38061.0013P1interaction, cognition / leaming, and self-care. Scoring anchors were developed for each domain and reviewed by PMS clinical experts and revised based on feedback in an iterative process. These anchors focus on developmental milestones which are impaired, absent or delayed to varying degrees in individuals with PMS (Berry-Kravis, 2022). More recently, these were refined via cognitive interview, expert advisory panel review and modified Delphi panel validation, following their use in a clinical trial. The updated instruments, termed PMSA-S (formerly PMS-Specific CGI-S) and PMSA-C (formerly PMS-Specific CGI-I), aim to improve clarity, relevance, and usability with the goals of enhancing data quality in clinical trials of PMS (see. e.g., Berry -Kravis E, et al. The development of Phelan-McDermid syndrome-speci fic anchors for the Clinical Global Impression Scale, (Poster presented at American Academy of Neurology 74th Annual Meeting, Seattle, April 2-7, 2022); Berry -Kravis E, et al., Improving Outcome Measures for Trials in Phelan-McDermid Syndrome (PMS): Development of PMS-Specific Clinician and Caregiver Impression-of-Change Measures (Poster presented at the 53rd Child Neurology Society Annual Meeting, San Diego, California, November 11-14, 2024), which are incorporated herein by reference in their entirety).

[0235] The PMS-Specific CGI-I (now PMSA-C, and accordingly also referred to herein as CGI-I / PMSA-C) is a clinician-rated tool that measures improvement since baseline in seven (7) PMS-relevant domains. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-I) or Attention and Awareness (for PMSA-C), and (vii) Self-Care. The CGI-I / PMSA-C scale provides a qualitative assessment of clinical change, compared to a baseline (pre-dosing) score for each of the domains, across the seven (7) distinct levels of improvement or worsening, wherein a score of 1 indicates “very much improved,” 2 indicates “much improved,” 3 indicates “minimally improved,” 4 indicates “no change,” 5 indicates “minimally worse,” 6 indicates “much worse,” and 7 indicates “very much worse.” In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by CGI-I / PMSA-C scores at 1 month, 3 months, 6 months and / or 12 months after administration, including as compared to baseline or age-matched controls. In some embodiments, the gene therapy methods described herein result in a score of 4 or lower for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global ImpressionAtty. Docket No. 38061.0013P1Scale of Improvement (CGI-I / PMSA-C), or the overall global CGI-I / PMSA-C score, or combinations thereof, at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration.

[0236] The PMS-Specific CGI-S (now PMSA-S, and accordingly also referred to herein as CGI-S / PMSA-S) is a clinician-rated tool that measures severity' of symptoms in seven (7) PMS-relevant domains, relative to the clinician's total experience with the PMS population, based on domain-specific anchors. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-S) or Attention and Awareness (for PMSA-S), and (vii) Self-Care. The CGI-S / PMSA-S scale provides a qualitative assessment of the severity of symptoms across the relevant domains relative to the total experience with the PMS population, wherein a score of 1 indicates '‘typical for age / not impaired,” 2 indicates '‘slightly impaired,” 3 indicates “mildly impaired,” 4 indicates “moderately impaired,” 5 indicates “markedly impaired,” 6 indicates “severely impaired,” and 7 indicates “among most severely impaired.” In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by CGI-S / PMSA-S compared to baseline or age-matched controls at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the gene therapy methods described herein result in a score for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global Impression of Severity (CGI-S / PMSA-S), or the overall global CGI-S / PMSA-S score, or combinations thereof, that is stabilized or reduced by at least 1 point, or at least 2 points, or at least 3 points, or at least 4 points, or at least 5 points, or at least 6 points at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition.

[0237] In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by Caregiver-reported Global Impression of Improvement (CaGI-I), Caregiver-reported Global Impression of Severity (CaGI-S), and / or PMS Clinician Domain-Specific Rating Scale (PMS DSRS) compared to baseline or age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration.Atty. Docket No. 38061.0013P1

[0238] In some embodiments, the gene therapy methods described herein improve or stabilize symptoms associated with PMS as measured by CGI-I / PMSA-C, CGI-S / PMSA-S, CaGI-I, CaGI-S, and / or PMS DSRS compared to age-matched control subjects at 1 month, 3 months, 6 months and / or 12 months after administration. In some embodiments, the gene therapy methods described herein result in stabilized or improved symptoms associated with PMS as measured by CGI-I / PMSAC, CGI-S / PMSA-S, CaGI-I, CaGI-S, and / or PMS DSRS compared to baseline or age-matched control subjects at 2 years, 5 years, 10 years, 15 years or more after administration.EXAMPLES

[0239] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope.Example 1: Clinical trial protocol1.1 Study rationale

[0240] Individuals with SHANK3 haploinsufficiency have a debilitating neurodevelopmental disease characterized by global developmental delay, abnormal sleep, lack of speech and communication, and worsening cognitive, social, and motor function disability that ultimately requires 24-hour care and supervision. Further, as these individuals age, they are at increased risk for significant cognitive and neurobehavioral regression, which may result in permanent loss of acquired skills, particularly motor skills.

[0241] As an ICV-administered AAV-based gene therapy aimed at delivering a functional SHANK3 gene, JAG201 has the potential to transduce haploinsufficient neurons, to provide proper SHANK3 levels, and durably restore the synaptic function required for learning and memory, which underlie development and maintenance of cognitive, communicative, social, and motor skills. Intervention early in development, while the affected individuals are acquiring, reinforcing, and building skills, and prior to onset of irreversible and detrimental changes in brain morphology and structure, is critical to ensure that the delivered gene can support the synaptic activity and neuronal development required of early learning and proper neurodevelopment. Although potential for neuronal rescue in anAtty. Docket No. 38061.0013P1adult SHANKS haploinsufficient individual is unclear, transduction of affected adult neurons should yield an increased level of SHANK3 expression with the potential to stabilize membrane associated receptors, proteins and structures required for inter-neuronal communication.

[0242] Nonclinical data (Example 2) demonstrate that JAG201 therapy may lead to improvement in patients with SHANK3 haploinsufficiency. In a rodent ShankS KO disease model, JAG201 led to improvements in neurobehavioral endpoints relating to motor function / exploratory behavior and disrupted sleep. Measurements of synaptic protein levels and functional binding via analysis of synaptic membrane preparations also demonstrate that JAG201 increases SHANK3 function within neurons and improves their capacity to recruit proteins required for glutamatergic signaling and function. Furthermore, the nonclinical data establish prospect for direct benefit (PDB) for human pediatric patients with SHANK3 haploinsufficiency.

[0243] This first-in-human (FIH) Phase 1 / 2 study will evaluate the safety, tolerability, and clinical activity of a single dose of JAG201 administered via ICV administration in pediatric and adult participants with SHANK3 haploinsufficiency.1.2 Potential benefits and risks

[0244] JAG201 is an AAV9 expressing a miniature version of the wild ty pe human SHANK3 (miniSHANK3) complementary' deoxyribonucleic acid (cDNA), designed to deliver functional miniSHANK3 protein to patients with SHANKS haploinsufficiency. JAG201 is proposed to be administered as an ICV administration in this first in human Phase 1 / 2 pediatric and adult clinical study.

[0245] The potential risks for human patients are based on the results of nonclinical toxicology studies with JAG201, the known safety profile for AAV gene therapies, and the known risks associated with ICV administration. The nonclinical studies (Example 2) support clinical evaluation of the safety and efficacy of JAG201 in patients with SHANK3 haploinsufficiency and establish PDB for human pediatric patients with SHANK3 haploinsufficiency.

[0246] The actual risks with JAG201 are not known. Thus, study participants will be monitored periodically with physical examinations, neurologic examination, hematology7,Atty. Docket No. 38061.0013P1chemistry, coagulation, urinalysis. ECGs, cardiac function tests, EEGs, immunogenicity, complement activation testing, MRI, and liver ultrasound.

[0247] The use of ICV route of administration (ROA) is justified as presented in Section 1.8 of this Example. Appropriate measures will be taken to minimize any infectious and noninfectious complications related to the ICV procedure. Perioperative management for approximately 48 hours in a hospital setting will allow for close monitoring and early identification of any acute safety events related to the ICV administration of JAG201.Post-operative brain magnetic resonance imaging (MRI) will be performed for assessment of any potential inflammatory changes.

[0248] The activation of immune system and / or complement pathw ay, w hich are known to be associated with AAV-based gene therapy, will be monitored periodically via clinical assessments, liver ultrasound, and thorough safety labs, including but not limited to: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, activated partial thromboplastin time (aPTT), International Normalized Ratio (INR), and platelet counts. Immunosuppression via corticosteroid prophylaxis will be implemented prior to JAG201 administration to mitigate and manage hepatotoxicity and other immune reactions as presented in Section 1.12.4. Furthermore, safety events related to activation of the immune system and / or complement pathway (such as TMA, HLH) have also been incorporated as study stopping rules (Section 1.13).Attv. Docket No. 38061.0013P1.3 Study objectives and assessment variablesTable 3. Summary of Objectives and Associated Assessment VariablesAtty. Docket No. 38061.0013P11.4 Safety Variables

[0249] As a Phase 1 / 2, FIH study, this study primarily focuses on the safety and tolerability of JAG201. Safety will be monitored via collection of adverse events (AEs) / SAEs and concomitant medications, safety laboratory assessments that include immunogenicity testing for anti-AAV9, anti -transgene antibodies, T-cell reactivity' to transgene and AAV9, complement activation testing, physical examinations, standard 12-lead ECG, echocardiogram, vital signs, and liver ultrasound. Additionally, samples will be collected for viral shedding analysis. Post-operative brain MRI will be performed for assessment of any potential inflammatory' changes. Participants will be monitored for safety' during a follow-up period of at least 5 years from the date of JAG201 administration.

[0250] During safety' evaluations, differentiation of AEs related to the route of administration and concomitant use of immunosuppression will be performed.1.5 Clinical Activity Variables

[0251] Delivery' of SHANK3 minigene to affected neurons is intended to improve neuronal structure and function and facilitate development and maintenance of intemeuronal pathways and networks. Evidence of improvement and stabilization of neuronal networks will be evaluated as potential evidence of clinical activity that may be a precursor to functional and behavioral improvements. Individuals with PMS display distinct transient visual evoked potential (VEP) waveform abnormalities in both time and frequency domains that might reflect underlying glutamatergic deficits. Additionally, profound sensory underresponsiveness has been previously documented in PMS. Emerging clinical data from interventional studies with insulin-like growth factor- 1 in patients with SHANK3 haploinsufficiency indicate that significant improvements in sensory' perception and VEP may be responsive to pharmacologic intervention and may represent an early predictive clinical marker of benefit. Thus, clinical activity assessments will prioritize evaluation of change from baseline in VEP as well as changes from baseline in sensory perception as measured by Sensory' Assessment for Neurodevelopmental Disorders (SAND), as evidence of changing or improving intemeuronal networks and excitatory and inhibitory neurotransmission in response to JAG201 treatment.

[0252] In order to support neurologic-dependent skill attainment, participants will engage in structured therapies that may include physical, occupational, speech, and ABAAtty. Docket No. 38061.0013P1therapy, for the duration of the study. Further characterization of preliminary clinical acti \ i ty of JAG201 will be evaluated through instruments that will assess each participant’s global adaptive behaviors, cognitive ability, language, motor functioning, symptoms of autism, and other phenotypic manifestations of PMS. Video recordings for some assessments will be performed for independent review. Improvements in cognitive ability will be measured with age-appropriate instruments, including the Stanford-Binet Intelligence Scales (fifth edition, SB-5) for assessment of IQ and / or the Bayley Scales of Infant and Toddler Development (BSID) for assessment of DQ. Adaptive function will be measured with the Vineland Adaptive Behavior Scales, third edition (Vineland-3). In addition, individual milestone attainment and time to milestone attainment will be assessed at regular intervals after gene therapy administration to corroborate potential benefits observed on measures of cognition and adaptive function. As a result, the study proposes to treat pediatrics prior to any history' of regression and adults prior to any history' of post-pubertal regression in this proposed FIH study. However, treated participants will be evaluated regularly for evidence of regression to assess the ability of the JAG201 gene therapy to prevent onset of regression.

[0253] Given the collectively high prevalence of cognitive impairments, adaptive behavior deficits, impaired motor function, and delayed or absent milestone attainment observed in the ongoing prospective natural history' study of the Developmental Synaptopathies Consortium (DSC) (NCT02461420), the assessment of IQ via the SB-5 or DQ via the BSID (as applicable) and the Vineland-3 may allow for clinically meaningful comparisons to the DSC natural history cohort. Additionally, this interventional study will include measures that are also captured yvithin the DSC natural history cohort that have the potential to corroborate observed benefits on measures of cognitive and adaptive function, including milestone attainment, VEPs, PMS-specific rating scales, measures of regression, and sleep assessments, including the Child Sleep Habits Questionnaire (CSHQ). Initial efficacy assessments will focus on 1) demonstrating intra-parti cipant improvements from baseline on measures of cognitive and adaptive function and milestone attainment, 2) if feasible, demonstrating higher performance on measures of cognitive and adaptive function relative to age and genetically matched natural history’ controls (variants, small and large deletions), and 3) if feasible, demonstrating longitudinal change in disease trajectory as evidenced by stabilizing or improving adaptive and cognitive function relative to a matched natural history control population.Atty. Docket No. 38061.0013P1

[0254] Additionally, a structured therapy log will be used to capture information on therapy activities (e.g., speech, physical and occupational therapy, etc., and may also include home videos provided by participant’s caregiver) and a Seizure Log will be used to capture occurrences of seizures.

[0255] Finally, patient reported outcomes involving changes on family impact and caregiver burden will be evaluated.1.6 Study Design

[0256] This is a Phase 1 / 2, FIH, open-label, dose-escalation study to evaluate the safety, tolerability, and clinical activity of a single dose of JAG201 administered via ICV injection in pediatric and adult participants with SHANKS haploinsufficiency resulting from SHANKS loss of function mutation or chromosomal deletions encompassing the SHANKS gene. Clinical data will be evaluated for safety', tolerability, and preliminary clinical activity of JAG201 in pediatrics and adults with SHANK3 haploinsufficiency.

[0257] The study will include 5 periods: Pre-Screening, Screening, Gene Therapy Administration and Perioperative Management, Initial Follow-Up, and Long-Term Follow-Up. In total, all participants will be followed for safety and tolerability for at least 5 years after the date of treatment with JAG201. To avoid unnecessary burden on the participant and caregivers and to avoid the potential for missed visits, early withdrawal from the study, or potentially confounding results, the participant will be encouraged not to enroll in other interventional studies for the duration of this study (FIG. 1).

[0258] A total of 4 dose cohorts are planned for evaluation, 2 cohorts for pediatric participants and 2 cohorts for adult participants A target of 3 and a maximum of approximately 8 participants in pediatric Cohort 1 (i.e., PCI); a target of 3 and a maximum of approximately 8 participants in adult Cohort 1 i.e. , AC l);a target of 3 and a maximum of approximately 10 participants in pediatric Cohort 2 (i.e., PC2); and a target of 3 and a maximum of approximately 5 participants in adult Cohort 2 (i.e., AC2). (FIG. 2). The pediatric cohorts will start enrolling first and the enrollment for adult cohorts may be initiated at a later time point in the study.Atty. Docket No. 38061.0013P1Pediatric cohorts:

[0259] A target of 6 and a maximum of approximately 18 pediatric participants aged 2 to 9 years will be treated in Cohorts 1 and 2 as follows:

[0260] As appropriate, 1 or 2 additional adult participants may be dosed in Cohort 1, if an issue arises that limits or impairs the ability to assess safety' and / or activity' of JAG201 following dosing of any of the first 3 pediatric participants. Furthermore, if 1 out of the 3 pediatric participants in Cohort 1 experiences a dose-limiting toxicity (DLT), then 3 additional pediatric participants will be treated at the same dose level. As appropriate, a dose that is intermediate to the 2 proposed doses of JAG201 for pediatric participants may be selected based on cumulative safety and clinical activity’ data. As appropriate, the Sponsor may elect to dose up to 4 additional pediatric participants in Cohort 2 to further assess safety and / or activity of JAG201. Furthermore, if any of the first 3 pediatric participants in Cohort 2 experiences a dose limiting toxicity7(DLT), then 3 additional pediatric participants will be treated at the same dose level.Adult cohorts:

[0261] A target of 3 and maximum of approximately 13 adult participants aged 18 to 25 years will be treated in Cohorts 1 and 2 as follows:

[0262] As appropriate, 1 or 2 additional adult participants may be dosed in either of the 2 cohorts, if an issue arises that limits or impairs the ability to assess safety and / or activity of JAG201 following dosing of any of the first 3 adult participants. Furthermore, if 1 out of the 3 adult participants in Cohort 1 experiences a dose-limiting toxicity7(DLT), then 3 additional adult participants will be treated at the same dose level. As appropriate, a dose thatAtty. Docket No. 38061.0013P1is intermediate to the 2 proposed doses of JAG201 for adult participants may be selected based on review of cumulative safety and activity data.

[0263] For both pediatric and adult participants, a traditional 3+3 design strategy will be implemented for dose escalation from the 1stcohort to the 2ndcohort (i.e., from PCI to PC2 and from AC1 to AC2; JAG201 doses for these cohorts are defined in the above table). The following rules will be followed for the dose escalation utilizing the 3+3 design for both the pediatric cohorts (i.e., PCI and PC2) and adult cohorts (i.e.. AC1 and AC2):• If none of the 3 participants in the 1stcohort experiences a DLT, then 3 more participants will be treated at the next higher dose level (i.e., the 2ndcohort). However, if 1 out of the 3 participants experiences a DLT, then 3 more participants will be treated at the same dose level.• Dose escalation to the 2ndcohort will continue only if there are no DLTs in these additional participants (i.e., only 1 DLT among the 6 participants in the 1stcohort).• The DSMC will evaluate safety data from the dose escalation cohorts (both pediatric and adult cohorts) and make recommendations regarding continued enrollment and administration of JAG201 based on observed safety data, including the nature, duration, and reversibility of observed adverse events. The DSMC will also review safety data from the dose escalation cohorts (both pediatric and adult cohorts) to make recommendations on dose escalation or de-escalation.• As noted above, the pediatric cohorts will start enrolling first and the enrollment for adult cohorts may be initiated at a later timepoint in the study. It is possible that the enrollment in the adult cohorts is initiated while the dose escalation portion for pediatric participants is ongoing (i.e., pediatric participants are being enrolled in one of the pediatric dose escalation cohorts [i.e., PCI or PC2]). In this scenario where the dose escalation portions for both pediatric participants and adult participants are running concurrently, any DLT occurring in pediatric or adult cohorts would warrant a pause of enrollment in all of the ongoing cohorts (pediatric or adult, as applicable) until the Sponsor and DMSC have reviewed the safety data for all JAG201 -treated participants from all the cohorts. The DSMC will make recommendations regarding continued enrollment (in pediatric and / or adult cohorts, as applicable) and administration of JAG201 and on dose escalation or de-escalation.Atty. Docket No. 38061.0013P1

[0264] The following staggered approach will be used for JAG201 dosing in each of the pediatric and adult cohorts:• Before dosing the second participant in Cohort 1 (e.g, PCI or AC1) and in Cohort 2 (e.g., PC2 or AC2, as applicable), an independent DSMC will provide recommendations to the Sponsor about treatment of the second participant.■ For Cohort 1, the DSMC will review all available safety data after at least 8 weeks following JAG201 dosing of the first participant.■ For Cohort 2, the DSMC will review all available safety data after at least 8 weeks following JAG201 dosing of the first participant in Cohort 2 in addition to all available safety data from participants in Cohort 1.• For both Cohort 1 and Cohort 2, after the second participant has been dosed with JAG201, the Sponsor will review safety' data before treatment of subsequent participants within the respective cohorts.■ For Cohort 1, the Sponsor will review all available safety data after at least 4 weeks following JAG201 dosing of the most recently dosed participant in addition to all available safety data from other participants in Cohort 1.■ For Cohort 2, the Sponsor will review all available safety' data after at least 4 weeks following JAG201 dosing of the most recently dosed participant in addition to all available safety' data from other participants.• Before dose escalation from Cohort 1 to Cohort 2, the DSMC will provide recommendations to the Sponsor about treatment of the first participant in Cohort 2.■ The DSMC will review all available safety data after at least 4 weeks following JAG201 dosing of the third participant in Cohort 1 in addition to all available safety data from other participants in Cohort 1.■ If more than 3 participants have been dosed with JAG201. then the DSMC will review the 4-week post-dose safety' data of the most recently dosed participant in addition to all available safety data from other participants in Cohort 1.• The DSMC will conduct a final review of the safety of JAG201.■ The DSMC will review all available safety' data after at least 4 weeks following JAG201 dosing of the final study participant in addition to all available safety data from other participants.• The DSMC will conduct additional data reviews (e.g. , annual, ad hoc) as per the DSMC Charter.Atty. Docket No. 38061.0013P11.7 Scientific Rationale

[0265] The study objectives, endpoints, and design are considered adequate for assessment of safety, tolerability, and efficacy in the context of a Phase 1 / 2 clinical study.

[0266] This FIH study will have an open-label design with comparison of the treated population to a natural history derived historical control. Assessment of clinical activity responses to JAG201 will focus on VEP and sensory assessment (SAND) and exploration of potentially efficacious responses to JAG201 in participants with SHANK3 haploinsufficiency will be across cognitive, behavioral, and functional measures.

[0267] Intervening early in life before the onset of irreversible developmental and pathological changes to the CNS and the potential onset of developmental regression offers the greatest opportunity for treatment to demonstrate benefit. Additionally, employing robust therapy opportunities after gene therapy treatment will give the participants the learning experiences necessary to build the skills necessary' for demonstrating benefits in cognition and adaptive functioning.1.8 Justification for Intracerebroventricular Route of Administration for JAG201

[0268] Given the disease pathology of SHANK3 haploinsufficiency, the ICV route has been selected as the ROA for JAG201 over any peripheral routes, in order to maximize neuronal transduction in the brain and minimize systemic exposure to the gene therapy vector. It improves the therapeutic index of JAG201 for SHANK3 haploinsufficiency, as the drug is delivered directly to the site of disease pathology'. This also reduces systemic drug exposure which might decrease the likelihood of adverse effects, primarily those mediated through the systemic immune response (e.g, hepatotoxicity). As a centrally-focused delivery’ method, the ICV ROA delivers therapy directly into the cerebral ventricles via an ICV device. This ROA has been used for several decades to provide treatments for pediatric and adult patients who suffer from a broad range of diseases, including infectious meningitis, intractable pain, and various types of cancer. It is an established and globally used method of drug delivery7and numerous studies have demonstrated that employing strict aseptic techniques when using ICV devices can dramatically reduce infectious complication rates. Additionally, following best practices for ICV device use can prevent many infectious and noninfectious complications.Atty. Docket No. 38061.0013P1

[0269] Furthermore, JAG201 nonclinical data in a mouse model with phenotypes similar to SHANK3 haploinsufficient patients demonstrated that the systemic IV infusion ROA was unlikely to provide sufficient transduction of the brain for JAG201 in order to be efficacious, and that a more direct CNS-targeted administration (like the ICV ROA) would be needed to achieve higher levels of transduction in the brain.

[0270] Based on these considerations, the preferred ROA for JAG201 in this study is ICV injection performed unilaterally rather than bilaterally. Data from a 3-month tolerability and biodistribution study was conducted in nonhuman primates in which bilateral ICV administration of JAG201 did not appear to confer a biodistribution benefit compared to unilateral ICV administration. Given the equitable biodistribution in the brain following both unilateral and bilateral ICV administration, selection of the comparably less invasive unilateral ICV administration method was made for JAG201.1.9 Dose Selection Strategy

[0271] In alignment with the American Society of Gene and Cell Therapy DRG Working Group’s recommendation for gene therapy to leverage the International Conference for Harmonisation S9, Nonclinical Risk Assessment Framework for Anticancer Pharmaceuticals (May 2010) in the context of DRG findings, a nonclinical risk assessment using the FDA Guidance for Industry, Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals (March 2019) is warranted for assessing JAG201 in the context of SHANK3 haploinsufficient patients. This guidance states that it is not always necessary to identify a no observed adverse effect level (NOAEL) in general toxicology studies. Instead, the goal is to identify a treatment dose that is expected to have pharmacologic effects and is reasonably safe, and that demonstration of complete recovery is not considered essential. Therefore, the highest nonseverely toxic dose (HNSTD) methodology7was applied for nonclinical safety assessment of JAG201. This approach is further supported by recent publication on reporting of test-article related ganglion pathology7for nonclinical toxicity studies; it identifies the HNSTD as potentially a more suitable approach for setting the threshold value in communicating adversity for AAV-based gene therapies. Additionally, in accordance with Society of Toxicologic Pathology7best practices, only test article-related effects that cause harm to the animal are considered to be adverse.Atty. Docket No. 38061.0013P1

[0272] A comprehensive assessment of JAG201 proof of concept (POC) data and toxicology data have been utilized to select the dose for this clinical study as presented below. The rationale for ICV as an ROA for JAG201 is presented in Section 1.8.1.10 Justification for Immunosuppressive Corticosteroid Prophylaxis for Immunosuppression

[0273] Hepatotoxicity (which may be immune-mediated), generally manifests as elevated ALT and / or AST levels and has been observed in AAV vector clinical studies across different diseases (e.g., spinal muscular atrophy [SMA], Duchenne muscular dystrophy [DMD], myotubular myopathy). Although the exact mechanism of hepatotoxicity has not been elucidated, nonclinical studies suggest an immune response to the vector capsid as a possible mechanism.

[0274] In order to mitigate potential aminotransferase elevations, administration of systemic corticosteroid prophylaxis has been recommended for approved AAV-based gene therapy such as ZOLGENSMA (for SMA) and ELEVIDYS (for DMD); both of these gene therapies are administered intravenously. For ZOLGENSMA, the corticosteroid prophylaxis has been recommended starting 1 day prior to ZOLGENSMA infusion and for 30-day period followed by gradual tapering over next 28 days (ZOLGENSMA Package Insert). For ELEVIDYS, the corticosteroid prophylaxis has been recommended starting 1 day prior to ELEVIDYS infusion and for a minimum of 60 days (unless earlier tapering is clinically indicated) followed by gradual tapering over 2-4 weeks (ELEVIDYS Package Insert).

[0275] Within the JAG201 GLP NHP study, transient elevations in aminotransferase levels were observed that were fully reversible by Week 4 post-dosing.

[0276] These data support the use of corticosteroid prophylaxis for immunosuppression starting 12 to 24 hours prior to JAG201 administration and for at least 28-day period followed by gradual tapering over the next 28 days (see Section 1.12.4 for details).1.11 Study population

[0277] A maximum of approximately 18 pediatric participants and 13 adult participants with SHANKS haploinsufficiency are planned for treatment.Atty. Docket No. 38061.0013P11.11.1 Inclusion and Exclusion Criteria for Pediatric ParticipantsInclusion Criteria for Pediatric Participants

[0278] A pediatric participant must meet all of the following criteria to be eligible for this study:• Is male or female, and 2 to 9 years of age at the time of JAG201 administration • Has a molecular confirmation of a loss of function mutation in SHANK3 or a22ql3.3 deletion classified as a Class I deletion (z.e., deletions including only SHANK3 or SHANK3 in combination with ARSA and / or ACR and RABL2B)• Has evidence of developmental / cognitive delay of at least 2 standard deviations (SD) below the mean (i.e., < 70) via either Full Scale Intelligence Quotient (IQ) on the SB-5 OR Developmental Quotient (DQ) assessment on the BSID-4 (as applicable) • Has an overall Phelan-McDermid Syndrome (PMS) Assessment of Severity (PMSA- S) Score of 3 or greater at Screening• Has a legally authorized representative (LAR) willing to initiate structured therapies for the participant (if not already engaged in these therapies) and continue for the duration of the study as determined by the specific therapist (structured therapies may include, at a minimum, physical therapy, occupational therapy, and speech therapy, and applied behavior analysis [ABA] therapy)• Does not have any contraindications for the IC V procedure (including hydrocephalus or ventricular shunts), as determined by the Investigator and / or neurosurgeon • Has a LAR willing and able to complete the informed consent process• Is stable on any medication regimens (if being administered to control the signs and symptoms of underlying disease) for at least 3 months prior to the planned JAG201 study treatment• If the participant is taking aspirin, nonsteroidal anti-inflammatory drugs, or medications that affect blood clotting, then is willing not to take these medications for at least 7 days prior to until at least 7 days after the ICV procedure• If undergoing any kind of behavioral or therapeutic intervention, then the level of intervention must have remained stable for at least 3 months prior to the planned JAG201 study treatment (exclusive of school vacations / illness)Atty. Docket No. 38061.0013P1• Has a LAR that agrees that the participant will not participate in any other interventional clinical study for the duration of this studyExclusion Criteria for Pediatric Participants

[0279] A pediatric participant who meets any of the following criteria will be excluded from this study:• Has history of developmental regression defined in this study as a prolonged loss of previously acquired skills (defined as skills maintained for at least 3 months) with loss of skills persisting for at least 3 months including, but not limited to, the following domains: language (expressive or receptive), motor (gross or fine), social interaction, cognitive ability, or daily living skills• Has a history of bipolar disorder, schizophrenia or other psychotic disorder, or obsessive-compulsive disorder within 3 years prior to study enrollment (or any history of these in participants < 3 years old)• Has had cancer within 3 years prior to study enrollment• Has known or suspected prion disease (e.g, Creutzfeldt-Jakob Disease)• Has poorly-controlled epilepsy (defined as an increase in the dose or addition of new anti-epileptic medications within the past 3 months) or any history of status epilepticus or seizure-induced hospitalizations within the last 12 months• Has history of acute cerebrovascular episodes• Has active autoimmune disease or prior treatment with immunomodulatory therapy, immunotherapy, and / or immunosuppressive drugs within 3 months prior to study enrollment (Note: Inhaled or topical steroids are permitted in the absence of active autoimmune disease)• Has infection (viral, bacterial, or fungal) that requires treatment < 6 weeks before JAG201 administration (Note: JAG201 administration may be postponed until the infection has resolved and the participant is clinically stable)• Has medical illness or other concern that would cause the Investigator (in discussion with the Sponsor) to conclude that the participant will not be able to perform the study procedures or assessments or would confound interpretation of data obtained during assessments (e.g, VEP waveform abnormalities not consistent with the underlying disease)Atty. Docket No. 38061.0013P1• Has known allerg}' or hypersensitivity to prednisolone or other glucocorticosteroids, or their excipients• Has documented allergy, hypersensitivity, or intolerance to one of the excipients of JAG201• Has received any vaccine < 6 weeks before JAG201 administration• Has received any gene therapy• Has participated in recent clinical studies (with the exception of observational cohort studies or non-interventional studies) within 3 months or 5 half-lives (whichever is longer) or received an investigational or commercial compound, product, or therapy administered with the intent to treat SHANK3 haploinsufficiency prior to study enrollment• Has an average absolute QT interval corrected for heart rate by Fridericia’s formula (QTcF) value of > 460 msec at Screening (Note: may be repeated to confirm) • Has aminotransferase levels of > 2* upper limit of normal (ULN) or total serum bilirubin > 1.5x ULN (> 3* ULN for participants with Gilbert's syndrome)• Has elevated (clinically significant thrombocytosis) or decreased (< 75,000 cells / pL) platelet count• Has abnormal activated partial thromboplastin time (aPTT) > 1.5 x ULN, or International Normalized Ratio (INR) > 1.5 x ULN. Participants who were in the therapeutic window for long-term use of anticoagulants who do not meet these criteria may be permitted to be enrolled if the values are within therapeutic range of intended use of anticoagulants• Is positive for human immunodeficiency virus (HIV), hepatitis B. or hepatitis C serology• Has vital signs outside of the normal range shown in Table 4 (Note: the normal ranges are provided below. Minor deviations from the normal ranges may be allowed if deemed by the Investigator to have no clinical significance.)Table 4. Normal range of vital signs for pediatric participantsAtty. Docket No. 38061.0013P1• Has clinically significant abnormalities at Screening that are not covered under any other Inclusion Criteria or Exclusion Criteria and that in the opinion of the Investigator (in consultation with neurosurgeon, if applicable) create unnecessary risks for centrally administered gene therapy or ICV procedure-related complications• Has contraindications for magnetic resonance imaging (MRI) scans (e.g, cardiac pacemaker, metal fragments in the eye, or aneurysm clip in the brain, etc.)• Has presence of brain malformations or major structural anomalies visible by MRI that would increase risks of ICV administration, as determined by the Investigator and / or neurosurgeon• Has any genetic mutations [other than that related to PMS] that is deemed to be exclusionary after a discussion between the Investigator and the Sponsor’s medical monitor1.11.2 Inclusion and Exclusion Criteria for Adult ParticipantsInclusion Criteria for Adult Participants

[0280] A participant must meet all of the following criteria to be eligible for this study:• Is male or female, and 18 to 25 years of age at the time of JAG201 administration • Has molecular confirmation of a 22ql3.3 deletion classified as a Class I deletion (z.e.. deletions including only SHANK3 or SHANK3 in combination with ARSA and / or ACR and RABL2B)• Has evidence of developmental / cognitive delay of at least 2 standard deviations (SD) below the mean via Full Scale IQ assessment (< 70) on the SB-5• Has an overall PMS Assessment of Severity (PMSA-S) Score of 3 or greater at ScreeningAtty. Docket No. 38061.0013P1• Has a LAR willing to engage in structured therapies that may include, at a minimum, physical therapy, occupational therapy, speech therapy, and applied behavior analysis [ABA] therapy for the duration of the study, and has had some level of engagement in these therapies in the past• Does not have any contraindications for the IC V procedure (including hydrocephalus or ventricular shunts), as determined by the Investigator and / or neurosurgeon • Has a LAR willing and able to complete the informed consent process• Is stable on any medication regimens (if being administered to control the signs and symptoms of underlying disease) for at least 3 months prior to study enrollment • If the participant is taking aspirin, nonsteroidal anti-inflammatory drugs, or medications that affect blood clotting, then is willing not to take these medications for at least 7 days prior to until at least 7 days after the ICV procedure• If undergoing any kind of behavioral or therapeutic intervention, then the level of intervention must have remained stable for at least 3 months prior to study enrollment• Has a LAR that agrees that the participant will not participate in any other interventional clinical study for the duration of this studyExclusion Criteria for Adult Participants

[0281] A participant who meets any of the following criteria will be excluded from this study:• Has history of developmental regression post-puberty defined in this study as a prolonged loss of previously acquired skills (defined as skills maintained for at least 3 months) wi th loss of skills persisting for at least 3 months including, but not limited to, the following domains: language (expressive or receptive), motor (gross or fine), social interaction, cognitive ability, or daily living skills• Has a history of bipolar disorder, schizophrenia or other psychotic disorder, or obsessive-compulsive disorder within 3 years prior to study enrollment• Has had cancer within 3 years prior to study enrollment with the exception of squamous and basal cell carcinomas of the skin and in situ carcinoma of the breast or cervix• Has know n or suspected prion disease (e.g, Creutzfeldt-Jakob Disease)Atty. Docket No. 38061.0013P1• Has poorly-controlled epilepsy (defined as an increase in the dose or addition of new anti-epileptic medications within the past 3 months) or any history of status epilepticus or seizure-induced hospitalizations within the last 12 months• Has history of acute cerebrovascular episodes• Has active autoimmune disease or prior treatment with immunomodulatory therapy, immunotherapy, and / or immunosuppressive drugs within 3 months prior to study enrollment (Note: Inhaled or topical steroids are permitted in the absence of active autoimmune disease)• Has infection (viral, bacterial, or fungal) that requires treatment < 6 weeks before JAG201 administration (Note: JAG201 administration may be postponed until the infection has resolved and the participant is clinically stable)• Has medical illness or other concern that would cause the Investigator (in discussion with the Sponsor) to conclude that the participant will not be able to perform the study procedures or assessments or would confound interpretation of data obtained during assessments (e.g, VEP waveform abnormalities not consistent with the underlying disease)• Has known allergy or hypersensitivity to prednisolone or other glucocorticosteroids, or their excipients• Has documented allergy, hypersensitivity, or intolerance to one of the excipients of JAG201• Has received any vaccine < 6 weeks before JAG201 administration• Has received any gene therapy• Has participated in recent clinical studies (with the exception of observational cohort studies or non-interventional studies) within 3 months or 5 half-lives (whichever is longer) or received an investigational or commercial compound, product, or therapy administered with the intent to treat SHANK3 haploinsufficiency prior to study enrollment• Has an average absolute QT interval corrected for heart rate by Fridericia’s formula (QTcF) value of > 470 msec at Screening (Note: may be repeated to confirm) • Has aminotransferase levels of > 2 upper limit of normal (ULN) or total serum bilirubin > 1.5x ULN (> 3x ULN for participants with Gilbert’s syndrome)Atty. Docket No. 38061.0013P1• Has elevated (clinically significant thrombocytosis) or decreased (< 75,000 cells / gtL) platelet count• Has abnormally activated partial thromboplastin time (aPTT) > 1.5x ULN, or International Normalized Ratio (INR) > 1.5 ULN. Participants who were in the therapeutic window for long-term use of anticoagulants who do not meet these criteria may be permitted to be enrolled if the values are within therapeutic range of intended use of anticoagulants• Is positive for human immunodeficiency virus (HIV), hepatitis B. or hepatitis C serology• Has vital signs outside of the normal range (Note: the normal ranges are provided below. Minor deviations from the normal ranges may be allowed if deemed by the Investigator to have no clinical significance.)o Temperature: 35.8°C to 37.5°Co Blood pressure: systolic (100 to 140 mmHg) / diastolic (60 to 90 mmHg), measured in a sitting position (may be repeated for confirmation in case of a single abnormality)o Heart rate: 45 to 99 beats per minuteo Respiration rate: 12 to 20 breaths per minute• Has clinically significant abnormalities at Screening that are not covered under any other Inclusion Criteria or Exclusion Criteria and that in the opinion of the Investigator (in consultation with neurosurgeon, if applicable) create unnecessary risks for centrally administered gene therapy or ICV procedure-related complications• Has contraindications for magnetic resonance imaging (MRI) scans (e.g., cardiac pacemaker, metal fragments in the eye, or aneurysm clip in the brain, etc.)• Has presence of brain malformations or major structural anomalies visible by MRI that would increase risks of ICV administration, as determined by the Investigator and / or neurosurgeon• If female and is pregnant, lactating, or. if sexually active and not surgically sterile, and is not willing to use a consent-defined acceptable contraception method for the duration of the study• If male and engages in sexual relations with a partner, will not use an acceptable barrier contraception method for the duration of the studyAtty. Docket No. 38061.0013P1• Has any genetic mutations [other than that related to PMSJ that is deemed to be exclusionary after a discussion between the Investigator and the Sponsor’s medical monitorCriteria for Temporarily Delaying Intracerebro ventricular Procedure and JAG201 Administration

[0282] Once a participant becomes eligible for the study, the planned TCV procedure and JAG201 administration should be delayed under the following circumstances:• Participant develops an infection (viral, bacterial, or fungal) that requires treatment within 6 weeks prior to JAG201 administration. In such an event, the ICV procedure and JAG201 administration will be performed once the infection has resolved, and the participant is clinically stable as per the discretion of Investigator and eligibility has been reconfirmed.• Participant develops any clinically significant abnormalities or has abnormal laboratory results that would make them ineligible as per the eligibility criteria defined in Section 1.11. In such an event, the ICV procedure and JAG201 administration will be performed once the event has resolved to a level that would make them again eligible for the study.• Participant develops any clinically significant abnormalities or has abnormal laboratory results that would warrant a delay of the ICV procedure and JAG201 administration (including the ability to initiate corticosteroid prophylaxis) in the opinion of the Investigator and / or neurosurgeon. In such an event, the ICV procedure and JAG201 administration will be performed once it is determined by the Investigator and / or neurosurgeon that it will not pose any unnecessary risks to the participant.• Participant initiates a medication that is prohibited as per the eligibility criteria defined in Section 1.11 taking into account the interval associated with the medication (e.g., 7 days prior to the planned ICV procedure for aspirin, NSAIDs, or medications that affect blood clotting). In such an event, the ICV procedure and JAG201 administration will be performed once the time period associated with not taking that medication is met as per the eligibility criteria.Atty. Docket No. 38061.0013P11.12 Study Intervention(s) Administered1.12.1 JAG201

[0283] JAG201 is an AAV9 expressing a miniature version of the wild-h pe human SHANK3 (jniniSHANKS) complementary deoxyribonucleic acid, designed to deliver functional miniSHANK3 protein to patients with SHANK3 haploinsufficiency.

[0284] As shown in FIG. 3, JAG201 consists of a single-stranded genome containing a miniSHANK3 expression cassette comprising the nucleotide sequence of SEQ ID NO: 7 flanked by adeno-associated virus serotype 2 (AAV2) inverted terminal repeats (ITRs), such that the entire AAV genome comprises the nucleotide sequence of SEQ ID No. 8 (AAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR).Transgene expression is under regulation of the human synapsin I (hSynl) promoter.Efficient polyadenylation of transgene mRNA is enabled via the human growth hormone polyadenylation signal (hGH poly A) sequence and increased transgene expression is supported by the woodchuck hepatitis virus post-transcriptional regulatory element. The ITR sequences enable packaging of the genome into the AAV9 capsid to produce JAG201. The full capsid molecular weight (theoretical) is 4.99 MDa. For simplicity, the AAV2 / 9 vector is described as an AAV9 vector.

[0285] Both JAG201 Drug Substance and Drug Product for clinical studies are manufactured under current Good Manufacturing Practice. The JAG201 Drug Substance recombinant AAV vector is produced by transient expression of vector genome and AAV9 capsid proteins in human embryonic kidney7293 suspension cell culture using an animal-derived-component-free and chemically -defined medium. Cells are expanded in a production bioreactor, triple transfected with plasmid DNA and harvested by cell lysis. The proposed purification process includes 7 downstream steps designed to reduce product- and process-related impurities, enrich for full AAV9 capsids and generate stable drug substance which can be readily formulated into a stable drug product of sufficient concentration for ICV administration.

[0286] JAG201 Drug Product is a single-dose, preservative-free, sterile, solution for ICV administration, of non-replicating AAV9 vector in a composition having lOrnM Tris, ImM Magnesium Chloride Hexahydrate, 150mM Sodium Chloride, and 0.02% (w / v) Poloxamer 188 at the target Phase 1 / 2 clinical vector genome concentration (6.5 x 1013Atty. Docket No. 38061.0013P1vg / mL). JAG201 Drug Product is filled into 5 mL cyclic olefin polymer (Crystal Zenith® ) vials with a fill volume of 2.15 mL, which contains a 0.15 mL overfill to ensure the labeled fill volume of 2.0 mL is achieved. The vials are stoppered with a pre-sterilized ready -to-use 20 mm chlorobutyl rubber serum stopper with Fl uroTec " B2 coating and sealed with a presterilized, ready -to-use packaged, aluminum seal with a colored plastic flip-off cap.

[0287] JAG201 Drug Product is stored frozen at < -60°C prior to shipment to the clinical sites. Shipment is performed under dry ice and upon receipt, the clinical sites will transfer JAG201 Drug Product to < -60°C storage.1.12.2 JAG201 Intracerebroventricular Administration

[0288] Commercially available medical devices will be provided for use by the clinicians for the preparation and ICV administration of JAG201. Study drug will be administered via unilateral ICV injection to the ventricle using best practice technique and following the site procedures.1.12.3 Selection and Timing of Dose for Each Participant

[0289] For both pediatric and adult participants, a traditional 3+3 design strategy will be implemented for dose escalation from the 1stcohort to the 2ndcohort (i.e., from PCI to PC2 and from AC1 to AC2). The following rules will be followed for the dose escalation utilizing the 3+3 design for both the pediatric cohorts (i.e., PCI and PC2) and adult cohorts (i.e., AC1 and AC2) (see Section 1.6 for the details on the 3+3 design strategy for dose escalation). Refer to Section 1.13 for the study stopping rules.1.12.4 Prophylactic MedicationsSystemic Corticosteroids for Immunosuppression

[0290] The risk for hepatotoxicity (which may be immune-mediated) will be managed with prophylactic systemic corticosteroids.

[0291] On Day -1, approximately 12-24 hours before dosing with JAG201, treatment-eligible participants will begin treatment with systemic corticosteroids for the 28-day period dosed as following:• For pediatric participants: equivalent to oral prednisolone at 1 mg / kg of body weight per day (Note: systemic corticosteroids equivalent to this could also be used as per Investigator's discretion).Atty. Docket No. 38061.0013P1• For adult participants: equivalent to oral prednisolone at 40 mg / day (Note: systemic corticosteroids equivalent to this could also be used as per Investigator’s discretion).

[0292] If oral corticosteroid therapy is not tolerated, intravenous corticosteroid (e.g, dexamethasone) is permitted. Dosing is to continue for at least 4 weeks, followed by gradual tapering over the next 4 weeks (or longer, as needed). Supportive measures for corticosteroids treatment (e.g., antacids to prevent and reduce digestive disturbances) should be implemented according to the local standard of care.

[0293] The Investigator should monitor liver function by clinical examination and by laboratory testing (ALT, AST, total bilirubin, aPTT, PT, and INR levels) on a regular basis, and at other times as clinically indicated.

[0294] At the end of the 28-day period of systemic corticosteroid treatment, the gradual tapering of corticosteroids will be initiated. While there are no standard evidencebased guidelines for tapering of corticosteroid treatment, it is recommended that the corticosteroid treatment is gradually tapered as follows:• For pediatric participants using oral prednisolone: tapered to 0.5 mg / kg / day for 2 weeks and then to 0.25 mg / kg / day for next 2 weeks.• For pediatric participants using systemic corticosteroids equivalent to oral prednisolone: tapered 10 to 20% every 3 to 7 days over a 28-day tapering penod.• For adult participants: tapered 10 to 20% every 3 to 7 days over a 28-day tapering period.

[0295] The tapering period may be extended as clinically indicated. Clinical judgement should be used while implementing the tapering and specialists (e.g, endocrinologist, gastroenterologist, etc. [pediatric or adult, as applicable]) should be consulted, as needed.

[0296] The use of corticosteroids (both the treatment and the w ithdraw al) has numerous metabolic, immunologic, and end-organ manifestations indicated by different adverse events and laboratory abnormalities. Therefore, the following important guidelines / recommendations are to be implemented:• Prior to initiating the corticosteroid taper, the Investigator should check liver status clinically and by assessing ALT, AST, total bilirubin, aPTT, PT, and INR. The Investigator is to promptly assess and closely monitor participants with w orseningAtty. Docket No. 38061.0013P1liver function test results and / or signs or symptoms of acute illness (e.g, vomiting, deterioration in health).o For participants with unremarkable findings (normal clinical examination and ALT, AST, total bilirubin, aPTT, and INR levels below 2* ULN): Taper the corticosteroid dose gradually over the next 28 days. Do not stop systemic corticosteroids abruptly.o If liver function abnormalities persist, continue systemic corticosteroids (equivalent to oral prednisolone at 1 mg / kg of body weight per day for pediatric participants or equivalent to oral prednisolone at 40 mg / day for adult participants, as applicable) until AST and ALT values are both below 2x ULN and all other assessments return to normal range, and then taper the corticosteroid dose gradually over the next 28 days or longer if needed. Do not stop systemic corticosteroids abruptly.o If liver function abnormalities continue to persist > 2x ULN after the 28-day period of systemic corticosteroids, promptly consult a gastroenterologist or hepatologist (pediatric or adult, as applicable).• Furthermore, hypothalamic-pituitary-adrenal (HP A) axis suppression (referred to as adrenal suppression) often occurs following abrupt discontinuation of the corticosteroids therapy. The clinical presentation of adrenal suppression is variable; many of the signs and symptoms are non-specific and can be mistaken for symptoms of intercurrent illness or the underlying condition being treated with the corticosteroids therapy (such as fatigue, nausea, diarrhea, abdominal pain, fever, myalgia, psychiatric symptoms, etc.) (Liu, 2013). While the gradual tapering of corticosteroids therapy is being recommended to minimize the risk of adrenal suppression, participants should be monitored closely for adverse effects associated with adrenal suppression. Additional tests (i.e., blood morning cortisol levels) should be considered to assess adrenal function as the corticosteroids therapy is being withdrawn as per the local standard of care and endocrinologist (pediatric or adult, as applicable) should be consulted, as needed. Tapering should be modified as clinically indicated if there is any suspicion of evidence of adrenal suppression. Symptomatic adrenal suppression should be treated with daily physiologic replacement doses of corticosteroids as per the local standard of care.Atty. Docket No. 38061.0013P1• Also, depending on results of analyses of T-cell reactivity to AAV9 and anti-transgene as measured by ELISpot, the duration of corticosteroid prophylaxis could be extended beyond 28 days. In such a case, the duration of the extension and the timing of tapering will be determined after discussion with the Investigator and in consideration of ELISpot and other relevant clinical laboratory findings. The investigator may have already initiated the tapering of corticosteroids while the Day 21 results for ELISpot are pending. Based on the Day 21 ELISpot results, the investigator may re-escalate the corticosteroids dose or continue the tapering as per clinical judgement and after discussion with the Sponsor's medical monitor. Similarly, the Day 56 results for ELISpot will also guide the corticosteroids dosing (e.g. , if to escalate or re-initiate corticosteroids) based on the clinical judgement and after discussion with the Sponsor's medical monitor.• Participants should be monitored closely for adverse effects associated with corticosteroid use including, but not limited to, behavioral and mood disturbances, adrenal suppression, Cushing’s syndrome, hyperglycemia, and infections. Adverse effects should be managed appropriately as per local standard of care, and specialists (e.g., endocrinologist, psychiatrist, etc. [pediatric or adult, as applicable]) should be consulted, as needed.

[0297] Corticosteroids can also be re-introduced later at any time during the study as per the clinical / laboratory findings and per Investigator’s discretion. If liver function test levels are elevated or increased during corticosteroid prophylaxis or at any later time, and / or other evidence of immunogenic reaction(s) to the capsid or transgene emerges, corticosteroid doses may be increased (e.g., oral prednisolone at 1.5 or 2 mg / kg of body weight per day for pediatric participants) followed by close monitoring. IV bolus corticosteroids may also be considered in those participants who continue to have further increases in transaminases despite increasing their daily steroid dosage (e.g., IV methylprednisolone [30-mg / kg infusion] for pediatric participants). In addition, a treatment plan involving administration of other immunomodulator(s) may be started after a discussion between the Investigator and the medical monitor. Should such a situation occur, details of each case and all interventions will be discussed with the DSMC to determine whether dosing for any future eligible participants for the study to be continued at that level, or be reduced, and / or if additional prophylactic orAtty. Docket No. 38061.0013P1reactive immune-modulatory measures should be initiated in all ongoing and future participants.

[0298] Note: while participants may also get immunosuppression treatment other than the corticosteroids, the study protocol provides guidance specifically on the usage of corticosteroids. For other treatments, investigator / treating physician should use clinical j udgement and follow local standard of care.Antibiotic Prophylaxis

[0299] Participants may be given standard antibiotic prophylaxis for a neurosurgical procedure (i.e., ICV administration of JAG201) at the discretion of the neurosurgeon.1.13 Discontinuation of Study Intervention and Participant Discontinuation / Withdrawal Post-DosingDose-Limiting Toxicities

[0300] A DLT is defined as any Grade > 3 AE and excludes toxicities clearly related to PMS, prophylactic systemic corticosteroid use, or any intercurrent illness. Toxicity grades will be defined by the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 (NCI-CTCAE v5.0) (NIH 2017).

[0301] During the study. non-DLT safety concerns will be considered.Study Stopping Rules

[0302] This study may be paused or prematurely terminated at any time.

[0303] Study enrollment will be interrupted should the following occur after JAG201 administration:• Any AE > Grade 3 (severe), independent of attribution• Any SAE, independent of attribution• Any AE involving a participant’s death, independent of attribution• Any severe local complication in the injected area, independent of attribution • Any AST or ALT > 3* ULN with concurrent increase in total bilirubin > 2* ULN, independent of attribution• Any persistence of liver function abnormalities (AST or ALT values > 3x ULN) in participants on prophylactic systemic corticosteroids that requires increasing the dose or adding supplemental immunomodulationAtty. Docket No. 38061.0013P1• Any event of suspected or confirmed TMA

[0304] If any stopping rule is met, dosing will be halted. All available safety information, including all AEs and safety laboratory' results, will be reviewed to determine whether the study should be continued, terminated early following the discontinuation rules, or if protocol revisions are recommended. Any occurrence of AEs related to activation of the immune system and / or complement pathway will be considered along with the interventions used to manage such events. Based on that, it will be determined whether any additional prophylactic or reactive immune-modulatory measures should be initiated in all ongoing and future participants.

[0305] Written notification documenting the reason for study pause, temporary suspension, or termination will be provided to the Investigators and regulatory authorities (as required per local and national regulations). If the study is paused or prematurely terminated, the Investigator will promptly inform the Institutional Review Board (IRB)ZIndependent Ethics Committee (IEC) and will provide the reason(s) as per local requirements.1.14 Study assessment and procedures

[0306] Protocol waivers or exemptions are not allowed. Adherence to the study design requirements, including those specified in the Schedules of Events, is essential and required for study conduct.

[0307] Immediate safety concerns should be discussed immediately upon occurrence or awareness to determine if any additional safety monitoring measures should be implemented in the study for all ongoing and future participants.

[0308] All Pre-Screening / Screening evaluations must be completed and reviewed to confirm that potential participants meet all eligibility criteria. The Investigator will maintain a screening log to record details of all participants screened and to confirm eligibility' or record reasons for screening failure, as applicable.

[0309] The Pre-Screening Period will be up to 90 days before Day 1 (JAG201 Dosing Day) and will begin when the first assessment is performed. The Pre-Screening Period enables the earliest possible assessment of objective, critical eligibility criteria whose analyses and interpretations may require significant time to complete. Determining these critical eligibility criteria as early as possible avoids burdening the participant withAtty. Docket No. 38061.0013P1completing all Screening assessments only to leam of study ineligibility based on objective criteria. During the Pre-Screening Period, the following assessments will be prioritized for determination of eligibility : medical history, genetic testing, and MRI. If unable to collect all scheduled Pre-Screening laboratory samples in 1 day, samples may be collected over multiple days during the Pre-Screening Period taking into consideration the duration required to receive results.

[0310] The Screening Period will be up to 28 days before Day 1 (JAG201 Dosing Day). The Screening Period may be lengthened to perform Pre-Screening Period assessments or, if necessary', due to participant’s transient illness, unavoidable logistical challenges, or other factors determined by the Investigator. Treatment eligibility will be confirmed on Day -1.

[0311] Participants may be rescreened for determination of eligibility after discussion. Rescreening assessments are to be conducted during Screening (Day -28 to -2) if not conducted during the Pre-Screening Period or if > 90 days have elapsed since the initial PreScreening assessment. Obtain informed re-consent, if appropriate. Note: any of these assessments also could be repeated during Screening (Day -28 to -2) at the Investigator’s discretion (e.g., clinically indicated, there is a reason to believe it has substantially changed, etc.) even if they were performed during the Pre-Screening Period within 90 days.1.14.1 Confirmation of Treatment Eligibility

[0312] After completion of the Pre-Screening / Screening procedures, treatment eligibility' of the participant will be re-confirmed on Day 1. Participants will come to the clinic for the pre-treatment baseline procedures. Hospitalization of the participant will be at the discretion of the Investigator. Participants who are eligible for treatment with JAG201 will begin prophylactic systemic corticosteroids at least 12-24 hours before JAG201 administration.1.14.2 Medical / Surgical History

[0313] Assessment of medical history should be prioritized during the Pre-Screening Period for determination of eligibility.

[0314] Medical history (including PMS history ), surgical history7, and prior medications and / or structured therapy interventions (including physical, speech, and occupational therapy) will be obtained during Screening and will include collection ofAtty. Docket No. 38061.0013P1information, as available, on any of the following conditions and / or body systems: allergies, cardiovascular, dermatologic, endocrine and metabolic, gastrointestinal and hepatic, head, eyes, ears, nose, throat (HEENT), hematologic and lymphatic, immune and inflammatory, musculoskeletal, neurologic (other than primary neurologic condition with which participant is diagnosed), including history of seizures and medications used for control of seizures, psychiatric, respiratory7, urinary and reproductive.

[0315] A participant’s medical records may be used for the collection of relevant data (e.g., medical history ), as appropriate.1.14.3 Seizure History

[0316] An assessment of seizure history7and medications used for control of seizures will be obtained during Screening. Also, as discussed in Section 1.14.8, a Seizure Log will capture occurrence of seizures post-JAG201 dosing.1.14.4 Physical Examinations and Assessments

[0317] All physical examinations (PEs) will be performed at Screening and at specified additional time points.

[0318] Full PEs will include the following body systems: Skin, HEENT, Respiratory, Cardiovascular , Gastrointestinal, Blood and lymphatic, Musculoskeletal

[0319] Directed physical examinations / limited physical assessments will include the following body systems (at a minimum): Skin, Cardiovascular, Respiratory7, Gastrointestinal

[0320] Assessments of weight (kg) and height (cm) will be conducted at specified time points. Assessment of height will be conducted at Screening, yearly, and at ET.1.14.4.1 Vital Signs

[0321] Vital sign measurements will include blood pressure (mmHg), pulse (beats per minute), temperature (°C), and respiratory rate (breaths per minute).

[0322] For the purposes of determining study eligibility only, normal ranges are disclosed in Section 1.11.1 (Note: minor deviations from the normal ranges may be allowed if deemed by the Investigator to have no clinical significance.)

[0323] On Day 1 (Dosing), vital signs will be recorded within 15 minutes before ICV administration of JAG201. After the injection has ended, vital signs will be recorded approximately every7hour for the next 12 hours or according to local practice ( / .<?., 1 hour (±Atty. Docket No. 38061.0013P115 minutes), 2 hours (± 15 minutes), and so on. Thereafter, vital sign assessments will be performed in accordance with local clinical practice until discharge and will be captured in the eCRF.1.14.4.2 Neurological Examination

[0324] A neurological examination will be conducted at specified time points and will include the following domains: Mental status, Language, Cranial nerves, Motor function and musculoskeletal. Reflexes. Sensory, and Coordination.

[0325] As part of the neurological examination, neuro-ophthalmological testing will be conducted and will include: Extra-ocular movements, Visual tracking and Pupillary response.

[0326] Fundoscopic examination is not required unless clinically indicated.1.14.4.3 Electrocardiogram

[0327] A standard 12-lead ECG will be conducted at specified time points.1.14.5 Imaging Studies1.14.5.1 Liver Ultrasound

[0328] Ultrasound of the liver will be performed at specified time points as part of the assessment of immunogenicity.1.14.5.2 Magnetic Resonance Imaging

[0329] MRI will be performed at specified time points specified to determine study eligibility, to determine the best trajectory for catheter placement as part of the dosing procedure, and to monitor post-operative safety.

[0330] Pre-Screening / Screening MRI is not required if an MRI obtained within 6 months of Day 1 (JAG201 Dosing Day) could be used to determine the study el igibility and to determine the best trajectory for catheter placement as part of the dosing procedure.Otherwise. MRI should be performed during the Pre-Screening / Screening Period. Every effort should be made to ensure that only 1 MRI is performed (preferably during the PreScreening Period) which could be used for both determination of eligibility’ and catheter placement trajectory. Additionally, at the neurosurgeon’s discretion, optional alternative imaging (e.g, CT) may be performed prior to surgery or on the morning of Day 1 (prior toAtty. Docket No. 38061.0013P1JAG201 dosing) to assist with catheter placement. A neuronavigational system must be used for surgery.

[0331] Post-operative MRI may be performed at any time during the visit window. Post-operative brain MRI will be performed for assessment of any potential inflammatory changes.1.14.5.3 Echocardiogram

[0332] Echocardiogram will be performed at specified time points.1.14.6 Laboratory Assessments

[0333] Collect baseline laboratory samples before initiating corticosteroid prophylaxis.

[0334] Fasting is not required before laboratory sample collection.

[0335] The Investigator must review the laboratory report, document this review, and record any clinically significant changes occurring during the study as an AE. The laboratory reports must be filed with the source documents. Abnormal laboratory findings associated with the underlying disease are not considered clinically significant unless judged by the Investigator to be more severe than expected for the participant’s condition.

[0336] All laboratory' tests with values considered clinically significantly abnormal during participation in the study should be repeated until the values return to normal or baseline or are no longer considered clinically significant by the Investigator or the medical monitor. If clinically significant values do not return to normal / baseline within a period of time judged reasonable by the Investigator, the etiology7should be identified, and additional parties notified.1.14.6.1 Routine Laboratory Assessments

[0337] Routine laboratory assessments (hematology, blood chemistry, urinalysis, and coagulation) are listed below. Requirements for routine laboratory assessments pertaining to study eligibility are specified in Section 1.11 of the protocol.

[0338] During the Pre-Screening and Screening Periods, routine clinical laboratory testing and complement testing will be performed centrally unless needed to be performed locally for eligibility7verification or unless approved by the Sponsor (specialty7labs will always been sent centrally). During the initial 2-3 months post-JAG201 administrationAtty. Docket No. 38061.0013P1(depending on how long corticosteroid prophylaxis use and tapering lasts), routine clinical laboratory testing and complement testing will be performed locally for close safety monitoring while the participant continues corticosteroid prophylaxis but may be transitioned to central testing at the Sponsor's approval after discussion with the Investigator. After the participant is weaned from corticosteroid prophylaxis, routine clinical laboratory testing and complement testing will be performed centrally unless otherwise approved by the Sponsor.1.14.6.1.1 Hematology

[0339] Standard hematology assessments will include the following: white blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count differential (absolute and relative numbers of neutrophils, eosinophils, lymphocytes, monocytes, and basophils).1.14.6.1.2 Blood Chemistry

[0340] Clinical blood chemistry laboratory analytes will include the following: albumin, alkaline phosphatase, ALT, AST, amylase, bicarbonate (or total carbon dioxide), bilirubin (fractionated and total), blood urea nitrogen (BUN), calcium, ferritin, chloride, creatinine, gamma-glutamyl transferase (GGT), glucose, lactate dehydrogenase, lipase, potassium, sodium, and total protein.1.14.6.1.3 Urinalysis

[0341] Standard urinalysis assessments will include, but are not limited to, the following: appearance, pH, specific gravity, protein, glucose, ketones, and microscopic examination of sediment.

[0342] If a participant is not toilet trained, then the participant’s urine may be collected at home prior to a study visit. In such cases, urine will be collected in a provided specimen cup or in a urine bag within a diaper.1.14.6.1.4 Coagulation

[0343] Standard coagulation assessments will include the following: aPTT and INR.1.14.6.1.5 Pregnancy Testing

[0344] Pregnancy testing will be performed only for females of childbearing potential. Serum pregnancy testing will be performed during Screening, and urine pregnancy testing will be performed at other specified time points. A positive urine pregnancy test will be confirmed by a serum pregnancy test.Atty. Docket No. 38061.0013P11.14.6.1.6 Pathogen Screening (Serology)

[0345] Assessments will include screening for current or past presence of HTV-1 , HIV-2, and hepatitis B and hepatitis C.1.14.6.2 Genetic Testing

[0346] Genetic testing to confirm SHANK3 haploinsufficiency will be conducted on all participants at Pre-Screening or Screening visits and will be prioritized for determination of eligibility. Genetic testing will also include a panel of genes associated with complement-mediated TMAs to identify potential participants genetically pre-disposed who may have increased risk of developing the condition. Genetic testing does not need to be repeated for participants who are rescreening and have already had this sample drawn for the trial.1.14.6.3 Immunogenicity Testing

[0347] Immunogenicity assessments will include testing to detect: anti-AAV9 total antibodies by MSD, anti -transgene product total antibodies by MSD, T cell reactivity to AAV9 and transgene product by ELISpot. Development of antibodies to AAV2 will not be assessed because of the design of the vector construct.1.14.6.4 Cerebrospinal Fluid

[0348] Samples of CSF will be collected first as part of the ICV procedure and thereafter by lumbar puncture to support potential evaluation of immunogenicity. The procedure will be performed according to local standard of care (e.g., use of anesthesia, etc.). A separate informed consent may be required.1.14.6.5 Complement Activation Testing

[0349] Samples for assessment of complement factors will be collected.1.14.6.6 Vector Shedding and Blood Biodistribution

[0350] Blood biodistribution and vector shedding will be assessed, including detection of vector genomes in samples of the participant’s blood, saliva, urine, and feces.

[0351] If the participant does not produce a urine or fecal sample on the day of the visit, an attempt should be made to collect the sample(s) within the given visit window. Sample collection and measurement for viral shedding will continue until the values of 3 consecutive samples are below the lower limit of quantification.Atty. Docket No. 38061.0013P1

[0352] If a participant is not toilet trained, then the participant’s urine may be collected at home prior to a study visit. In such cases, urine will be collected in a provided specimen cup or in a urine bag within a diaper.1.14.7 Structured Therapy

[0353] A structured therapy program will be established to define standards and best practices related to at a minimum physical therapy, occupational therapy, speech therapy, and ABA therapy (Note: in certain circumstances, a therapy might not be utilized for a participant [e.g., participant not eligible for the ABA therapy, etc.]; sites should consult with the Sponsor’s medical monitor). Structured therapy interviews will occur in accordance with the Structured Therapy Manual. Structured therapy will initiate within 1 -month post-JAG201 administration in accordance with the Structured Therapy Manual. Structured therapy interviews will be conducted pre-dose and quarterly throughout the study duration; for further details, refer to the Structured Therapy Manual.1.14.8 Seizure Log

[0354] A log will be provided to the caregiver to contemporaneously capture the occurrence of seizure episodes post-JAG201 dosing. Sites are required to review the Seizure Log at specified visits and record worsening of seizures (e.g., increased frequency, duration, etc.) or new onset of seizures as AEs, as applicable.1.14.9 Electroencephalogram and Visual Evoked Potentials

[0355] EEG is an assessment that measures the electrical impulses of the brain, indicating how cells within the brain interact with each other. VEP is a non-invasive exploration method of the functionality of the human visual system through detecting neuronal pool activity responding to stimuli independently of the consciousness and attention state of the patient.

[0356] EEG and VEP will be performed at specified timepoints. Additional VEP / AEP / EEG acquisitions may be requested by the Sponsor due to issues including, but not limited to, participant engagement, data quality, etc. EEG is an assessment that measures the electrical impulses of the brain, indicating how cells within the brain interact with each other. VEP is a non-invasive exploration method of the functionality of the human visual system through detecting neuronal pool activity responding to stimuli independently of theAtty. Docket No. 38061.0013P1consciousness and atention state of the patient. Video recordings and photos will be taken for the cap placement.1.14.10 Auditory Evoked Potential

[0357] AEP is an electrical signal elicited from the brain while an auditory stimulus is presented in a time-locked manner. AEP signal consists of reproducible positive or negative peaks, latency, amplitude and behavioral correlation. AEP will be performed at specified timepoints. Additional VEP / AEP / EEG acquisitions may be requested by the Sponsor due to issues including, but not limited to, participant engagement, data quality, etc.1.14.11 Administered Developmental Tests

[0358] Each participant’s neurodevel opmental status will be assessed through administration of scales of motor, cognitive and language development, and living and functioning with PMS. Age- and ability-appropriate scales will be administered (Note: a scale for a participant for any given assessment may change from one to another as the participant ages and is out of range for that assessment). The caregiver might act as proxy for the participant for all the PROs. All assessments will be conducted specified time points.

[0359] To ensure collection of the highest quality of data for analysis, all scales and instruments will be administered to study participants by qualified raters, who are individuals with particular skills, expertise, and knowledge, and who meet specific educational and experiential criteria to administer the scales and instruments. The method of administration includes training materials and / or applicable operational manuals.

[0360] Video recordings for some Clinician-Rated developmental tests assessments will be performed for independent review. Data will be captured in electronic systems and devices provided to the sites and caregivers, research staff.1.14.11.1 Clinician-Rated Developmental Tests

[0361] Every effort should be made to perform assessments on all participants in accordance with the Rater Reference Manual. If a participant is unable to perform a specific assessment due to inability (e.g., cognitive inability), this will not be considered a protocol deviation. Refer to the Rater Reference Manual regarding administration instructions including details specific to scale transitions.Atty. Docket No. 38061.0013P11.14.11.1.1 Sensory Assessment for Neurodevelopmental Disorders with Video

[0362] The SAND is a clinician-administered observation and corresponding caregiver interview that captures sensory' symptoms based on Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition criteria for ASD. Children are presented with a series of visual, tactile, and auditory stimuli that probe for sensory behaviors, which are rated by a trained examiner on an algorithm measuring sensory hyperreactivity, hyporeactivity, and seeking behaviors. The corresponding caregiver interview consists of 36 corresponding items that assess presence and severity of sensory behaviors. Administration of this assessment will be video recorded for independent review.1.14.11.1.2 Vineland-3 with Video

[0363] The Vineland Adaptive Behavior Scales, Third Edition, Comprehensive Interview Form (Vineland-3) measures adaptive behavior of individuals from birth to age of 90 years on 3 domains, including communication, daily living skills, socialization, and optional domains of motor skills, and maladaptive behavior. Individual domains will be collected at appropriate ages as per developer’s guidance. Administration of this assessment will be video recorded for independent review.1.14.11.1.3 Intelligence Quotient (Stanford-Binet Intelligence Scale, Fifth Edition) with Video

[0364] IQ, or intelligence quotient, is a measure of one’s ability to reason and solve problems. It reflects how well an individual performs on a specific test compared to other people of the same age group. The Stanford-Binet Intelligence Scale (SB-5), Fifth Edition will be used to measure the IQ. Full scale IQ (FSIQ, along with NVIQ, VIQ) will be measured. SB-5 is an adaptive measure that assesses general intellectual ability' and information processing for individuals 2-85 years old (Becker, 2003). Both the Early Childhood version and the Comprehensive versions will be used as outlined per developer's guidance. Administration of this assessment will be video recorded for independent review.1.14.11.1.4 Developmental Quotient (Bayley Scales of Infant and Toddler Development) with Video

[0365] The Developmental Quotient (DQ), most frequently used with infants or preschool children, is a numerical indicator of a child’s growth to maturity across a range ofAtty. Docket No. 38061.0013P1psychosocial competencies and will be measured using the Bayley Scales of Infant and Toddler Development (BSID). BSID is a set of individually administered developmental scales designed to measure current developmental functioning in the areas of cognition, motor skills, and behavior. This instrument measuring cognitive functioning was designed to be used with children from 16 days to 42 months. The 4thedition of BSID (Bayley™-4) will be used. It consists of a Cognitive scale, Language scale. Motor scale, Social-Emotional Scale, and Adaptive Behavior Scale (the adaptive behavior scale utilizes the Vineland behavior assessment system). Administration of this assessment will be video recorded for independent review.

[0366] At each IQ assessment using SB-5, full-scale IQ (along with NVIQ and VIQ) will be collected. The BSID-4 is the preferred developmental assessment tool for pediatric participants up to 3 years and 6 months old and should be used in these participants if the Investigator deems it appropriate based on the individual’s development ability’. The transition from the BSID-4 to the SB-5 would initiate based on a participants’ mental age equivalent (as derived from the Vineland-3 Adaptive Behavior Composite age-equivalence tables) is within 6 months of 42 months and 30 days. For participants aged greater than 3 years and 6 months who do not meet the mental age equivalent threshold, SB-5 will be deferred until the participant achieves the mental age equivalent that is within 6 months of 42 months and 30 days, and the BSID-4 should be used. In certain circumstances, collection of both SB-5 and BSID-4 is allowed after discussion with the Sponsor's medical monitor. Refer to the Rater Reference Manual regarding administration details specific to scale transitions.1.14.11.1.5 Peabody Picture Vocabulary Test with Video

[0367] The Peabody Picture Vocabulary Test-fifth edition (PPVT-5) is a norm-referenced and individually administered measure of receptive vocabulary based on words in Standard American English. The PPVT-5 measures listening and understanding of singleword vocabulary and was developed for individuals 2.5 years old through greater than 90 years old. The PPVT-5 does not require the participant to speak, read, or write, and the test can be administered to individuals with significant motor impairment. Administration of this assessment will be video recorded for independent review;1.14.11.1.6 Expressive Vocabulary Test with VideoAtty. Docket No. 38061.0013P1

[0368] The Expressive Vocabulary Test-third edition (EVT-3) is an individually administered, norm-referenced instrument that assesses expressive vocabulary and word retrieval and was developed for individuals 2.5 years through greater than 90 years old. The EVT-3 can be used in conjunction with the PPVT-5. Administration of this assessment will be video recorded for independent review.1.14.11.1.7 Visual-Motor Integration with Video

[0369] The Beery-Buktenica Developmental Test of Visual-Motor Integration (VMI), Sixth Edition (BEERY™ VMI) is a standardized, norm-referenced assessment involving copying geometric forms that is used to determine the level of integration between visual and motor systems in people of all ages. The Beery-Buktenica Developmental Test of Visual-Motor Integration (VMI) Long Form Test, Sixth Edition (BEERY™ VMI), is a standardized, norm-referenced assessment involving copying a series of geometric forms starting with simple line drawings and progressing to more complex figures that is used to determine the level of integration between visual and motor systems in people of all ages. Administration of this assessment will be video recorded for independent review.1.14.11.1.8 Short Sensory Profile-2 Questionnaire

[0370] The Short Sensory Profile-2 (SSP-2) Questionnaire is a shortened form of Dunn’s Sensory Profile caregiver questionnaire originally developed as a screening tool to identify children with sensory processing difficulties. SSP-2 is only required for participants aged 3years to 14 years and 11 months (however, SSP 2 should be collected at screening for all pediatric participants irrespective of age). The SSP-2 contains 38 items organized into seven subscales (Tactile sensitivity, Taste / Smell sensitivity. Movement sensitivity. Under responsive / Seeks sensation, Auditory filtering. Low Energy / Weak, and Visual / Auditory sensitivity). The SSP-2 total score and the score on each subscale can be used to classify7children’s level of sensory abnormality7(Typical, Probably Difference, or Definite Difference) based on score percentiles from a large normative sample of children without disabilities.1.14.11.1.9 PMS-Specific Clinical Global Impression

[0371] The PMS-specific Assessment of Change (PMSA-C) (formerly known as PMS-specific CGI-I) and PMS-specific Assessment of Severity7(PMSA-S) (formerly know n as PMS-specific CGI-S) are brief, clinician-rated measures that consist of CGI assessments ofAtty. Docket No. 38061.0013P1individual PMS-relevant domains including Expressive Communication, Receptive Communication, Gross Motor Function, Fine Motor Function, Social Interaction, Attention and Awareness, and Self-Care.1.14.11.1.9.1 PMS-Specific Assessment of Change (PMSA-C)

[0372] The PMS-specific Assessment of Change (PMSA-C) (formerly known as PMS-specific CGI-I) asks the clinician to rate the participant's total improvement since baseline, respectively, whether or not the improvement is judged to be due entirely to the experimental treatment. The items are rated on a 7-point scale, with a range of responses from 1 (very much improved) to 7 (very7much worse).1.14.11.1.9.2 PMS-Specific Assessment of Severity (PMSA-S)

[0373] The PMS-specific Assessment of Severity (PMSA-S) (formerly known as PMS-specific CGI-S) asks the clinician to rate the participant’s current severity of illness based on the clinician’s total clinical experience with the PMS population. The scale is structured as a CGI scale and is rated on a 7-point scale, with a range of responses from 1 (normal, not at all impaired) through 7 (amongst the most extremely impaired patients).1.14.1.10 PMS Domain-Specific Rating Scale

[0374] The PMS domain-specific rating scale (PMS-DSRS; Baseline and Follow Up versions) will be conducted with the PMSA-S.

[0375] The PMS domain-specific rating scale is a clinician administered rating scale with scores ranging from 0-4 based on overall severity of symptoms in key PMS domains over the past week, including Gastrointestinal Issues, Seizures, Sleep Issues, Sensory Sensitivity, and Challenging Behavior. Key symptoms or clinical observations contributing to the rating, including impact on functionality, are documented in comments.1.14.11.2 Caregiver-reported Developmental Tests

[0376] For all the Caregiver-reported Developmental Tests, the caregivers will be given ± 1 week allowance for completion. Instructions to caregivers will include a request to complete the Caregiver-Reported outcomes (CaROs) prior to when procedures are performed.1.14.11.2.1 Observer-reported Communication AbilityAtty. Docket No. 38061.0013P1

[0377] The Observer-reported Communication Ability (ORCA) is a questionnaire that assesses the communication ability of individuals with neurodevelopmental disorders that significantly impact verbal speech, designed to be completed by the rimary caregiver who is most familiar with the individual and the ways they communicate.1.14.11.2.2 Aberrant Behavior Checklist

[0378] The Aberrant Behavior Checklist (ABC) is an informant rating instrument for patients aged 5 years and older (but will be used across all participants in the study), designed to be performed by someone who knows the person well, that contains 58 items that resolve onto 5 subscales. The subscales and the respective number of items are as follows: (a) Irritability (15 items), (b) Social Withdraw al (16 items), (c) Stereotypic Behavior (7 items), (d) Hyperactivity / Noncompliance (16 items), and Inappropriate Speech (4 items).1.14.11.2.3 Children’s Sleep Habits Questionnaire

[0379] The Children’s Sleep Habits Questionnaire (CSHQ) is a parent / caregi ver-reported questionnaire that assesses the frequency of behaviors associated with common sleep difficulties. This instrument w as designed for patients 4-10 years of age but will be used across all participants in the study.1.14.11.2.4 Repetitive Behaviors Scale

[0380] The Repetitive Behavior Scales-Revised (RBS-R) is a 43-item parent / caregi ver-reported questionnaire that is used to measure the breadth of repetitive behavior in children, adolescents, and adults with ASD. The RBS-R provides a quantitative, continuous measure of the full spectrum of repetitive behaviors (unpublished data, ClinicalTrials.gov 2023b).1.14.11.2.5 Caregiver-reported Global Impression

[0381] The PMS-Specific CaGI-I and PMS-Specific CaGI-S are caregiver-completed assessment of the individual’s overall function, wellbeing, and change in specific symptom severity. Items are rated on a 7-point scale ranging from very much improved to very much worse. Symptom-specific assessment domains including communication, social interaction, behavior, motor abilities, seizures, cognition, self-care, gastrointestinal issues, and sensory sensitivity.1.14.11.2.6 Patient Reported Outcome: Pediatric Quality of Life Inventory (Family Impact Module)Atty. Docket No. 38061.0013P1

[0382] Pediatric Quality of Life Inventory (Family Impact Module) (PedsQL™-FIM) will measure the impact of the disease on parents and family members.1.14.2.7 Patient Reported Outcome: Caregiver Burden (Family Appraisal of Caregiving Questionnaire)

[0383] The Patient Reported Outcome (PRO) - Family Appraisal of Caregiving Questionnaire will assess caregiver burden through questions about the caregiver experiences of caring for a relative (such as a child).1.14.2.8 Developmental Milestone Historical Caregiver Interview

[0384] The Developmental Milestones Historical Caregiver Interview will be completed to better understand developmental history of critical milestones across key functional domains impacted by PMS for each participant prior to dosing. Historical milestones trajectories will be assessed to establish an individual participant baseline to determine if any new gains or regains in gross and fine motor, social skills, communication, and self-care occur post dosing.

[0385] Developmental milestones gain, loss and regain will be assessed only once at Screening using the Developmental Milestones Historical Caregiver Interview to determine the participants’ developmental milestone attainment (Note: For participants already dosed in the study, the site will perform this interview once approved by an IRB [it will be noted as an unscheduled visit] and the Caregiver will be asked to recall prior to JAG201 dosing).Historical milestone information will be collected by a memory-aided, clinician-facilitated caregiver interview, in which the following will be recorded for each of the milestones assessed: historical ability or lack thereof, age at milestone gain, age at loss and time since loss, and age if ever regained. This interview will be completed once, with ongoing milestone assessment determined using existing video evidence from other study assessments, as applicable.11.14.2.9 Caregiver Exit Interview

[0386] Caregivers of participants will be asked to participate in up to two interviews. The interviews will be conducted at 2 timepoints: occurring within approximately 2 weeks of the months 24 and 60 visits. Interviews will be requested from caregivers of participants who discontinue ahead of months 24 or 60. The objectives of the patient interview s are:Atty. Docket No. 38061.0013P1• To gather qualitative data directly from caregivers to help interpret clinical trial outcomes and the meaningfulness of these outcomes according to caregivers • To better understand participants' experiences with PMS (e.g., symptoms, impacts) both before and during the clinical trial including the meaningfulness of any changes in caregiver observations of signs, symptoms, and impacts of PMS during the trial

[0387] Study sites should remind caregivers of the two interviews at the time of study consent. The same caregiver will be asked to participate in both interviews. Please refer to the Study Reference Manual for additional details.1.14.3 Home Video Collection by Caregiver

[0388] Home videos may be collected by the caregiver as per Sponsor's discretion to evaluate the behavior, function, and / or improvement in naturalistic environment.1.15 Safety

[0389] AEs, SAEs. and concomitant medication use will be monitored continuously. Standard safety assessments, laboratory measures (including albumin, AST, ALT, GGT, total and direction bilirubin, hematology, coagulation, complete blood count, and kidney function), PEs (full and directed), standard 12-lead ECG, echocardiogram, and liver ultrasound will be performed as denoted in the Schedules of Events.

[0390] Also, immunogenicity7testing for anti-AAV9 and anti -transgene antibodies, as well as complement activation testing, will be performed. Vector shedding in saliva, urine, and feces will be monitored.

[0391] Participants and / or their LARs will be encouraged to freely report any changes in the participant’s health from the time of providing informed consent through completion of the study. Study staff will also inquire about any changes in participants’ health.

[0392] Testing or other evaluations not specified in the study but deemed necessary by the Investigator for participant safety7are permitted. Data will be entered as an unscheduled visit in the clinical database. All AEs will be recorded in source documents and the eCRF.1.15.1 Definition of Adverse EventAtty. Docket No. 38061.0013P1

[0393] The definitions in this section are in accordance with 21 CFR 312.32 and Good Clinical Practice (GCP) guidelines of the International Conference on Harmonization (ICH).1.15.1.1 Adverse Event

[0394] An AE is any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related. An AE can, therefore, be any unfavorable and unintended sign (including an abnormal laboratory finding) or disease temporally associated with the use of a drug and does not imply any judgment about causality. An AE can arise with the use of any drug or medicinal product.1.15.1.1.1 Events Meeting the Adverse Event Definition

[0395] Adverse Events include:• Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the Investigator (z.e., not related to progression of underlying disease).• Exacerbation of a chronic or intermittent pre-existing condition, including either an increase in frequency and / or intensity of the condition.• New conditions detected or diagnosed after study intervention administration even though it may have been present before the start of the study.• Signs, symptoms, or the clinical sequelae of a suspected intervention-intervention interaction.• Signs, symptoms, or the clinical sequelae of a suspected overdose of either study intervention or a concomitant medication. Overdose per se will not be reported as an AE / SAE unless it is an intentional overdose taken with possible suicidal / self- harming intent. Such overdoses should be reported regardless of sequelae.1.15.1.1.2 Events Not Meeting the Adverse Event Definition Adverse Events do not include:• Any clinically significant abnormal laboratory findings or other abnormal safety assessments that are associated with the underlying disease, unless judged by the Investigator to be more severe than expected for the participant’s condition.Atty. Docket No. 38061.0013P1• The disease / disorder being studied or expected progression, signs, or symptoms of the disease / disorder being studied, unless more severe than expected for the participant’s condition.• Medical or surgical procedure (e.g., endoscopy, appendectomy): the condition that leads to the procedure is the AE.• Situations in which an untoward medical occurrence did not occur (social and / or convenience admission to a hospital).• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen.1.15.1.2 Treatment Emergent Adverse Event

[0396] A treatment-emergent adverse event (TEAE) is an AE that starts or worsens at any time from the initiation of the administration of JAG201.1.15.1.3 Serious Adverse Event

[0397] An AE is considered serious if. in the view of the Investigator, it satisfies any of the following criteria:• Results in death• Is life-threateningo The term life threatening in the definition of serious refers to an event in which the participant was at risk of death at the time of the event. It does not refer to an event, which hypothetically might have caused death, if it were more severe.• Requires inpatient hospitalization > 24 hours or prolongs existing hospitalization o In general, hospitalization signifies that the participant has been admitted (usually involving at least an overnight stay) at the hospital or emergency ward for observation and / or treatment that would not have been appropriate in the physician’s office or outpatient setting. Complications that occur during hospitalization are AEs. If a complication prolongs hospitalization or fulfills any other serious critena, the event is serious. When in doubt as to whether hospitalization occurred or was necessary, the AE should be considered serious.Atty. Docket No. 38061.0013P1o Hospitalization for the planned ICV procedure and post-operative management (per the study protocol) or for elective treatment of a preexisting condition that did not worsen from baseline is not considered an AE.• Results in persistent or significant disability or incapacity or substantial disruption of the ability to conduct normal life functionso The term disability means a substantial disruption of a person’s ability to conduct normal life functions.o This definition is not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental trauma (e.g., sprained ankle) that may interfere with or prevent everyday life functions but do not constitute a substantial disruption.• Is a congenital anomaly / birth defect• Is an important medical evento Note: important medical events that may not result in death, be lifethreatening, or require hospitalization may be considered an SAE when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent 1 of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home or blood dyscrasias or convulsions that do not result in inpatient hospitalization.

[0398] Adverse events that require emergency room care but that do not result in hospital admission are not SAEs unless assessed by the Investigator to be an important medical event.1.15.1.4 Unexpected Adverse Event and Unexpected Suspected Drug Reaction

[0399] An AE or suspected adverse reaction (an AE where there is a reasonable possibility that JAG201 caused the event; suspected drug related) is considered “unexpected” if it is not listed as “expected” or is not listed at the specificity or severity that has been observed.1.15.1.5 Laboratory and Diagnostic Abnormalities as Adverse EventsAtty. Docket No. 38061.0013P1

[0400] Clinically significant abnormal laboratory findings or other abnormal diagnostic assessments (e.g., ECGs, echocardiography vital signs, etc.) that are detected in participants who receive JAG201 or that significantly worsen relative to baseline in participants who receive JAG201 will be reported as AEs.

[0401] Clinical significance is based on the Investigator’s judgment but will ty pically include findings that result in study withdrawal, require active medical management, or are associated with clinical signs.1.15.2 Recording Adverse Events

[0402] Caregivers will be encouraged to spontaneously report any changes in the participant’s health from the time of signing the ICF through completion of the study. Study staff will also inquire about any changes in the participants' health.

[0403] All AEs will be recorded in source documents and the eCRF. It is the responsibility of the Investigator to review all documentation (e.g., hospital progress notes, laboratory and diagnostic reports) for the presence of AEs for a complete evaluation of known AEs.

[0404] At minimum for each AE, the Investigator will evaluate and report the event name / term / description, onset (date and time), resolution (date and time), event severity / intensity, relationship to study drug, action taken, and whether the event is a serious adverse event (SAE). The event time is only required while the participant is inpatient.1.15.2.1 Adverse Event Term / Name / Description

[0405] The Investigator will attempt to establish a diagnosis of each AE based on signs, symptoms, and / or other clinical information. Whenever possible, the diagnosis (not the individual signs / symptoms or therapeutic measures taken to treat the AE) will be documented as the AE.1.15.2.2 Adverse Event Intensity / Severity Grading• AEs will be classified as follows according to the NCI-CTCAE version 5.0:• Grade 1 (Mild): Experiences that are usually transient, requiring no special treatment, and do not interfere with the participant’s daily activities.Atty. Docket No. 38061.0013P1• Grade 2 (Moderate): Experiences that introduce some level of inconvenience or concern to the participant, and may somewhat interfere with daily activities, but are usually ameliorated by simple therapeutic measures (may include drug therapy).• Grade 3 (Severe): Experiences that are unacceptable or intolerable, significantly interrupt the participant’s usual daily activity, and require systemic drug therapy or other treatment.• Grade 4 (Life-threatening): Experiences that cause the participant to be in imminent danger of death.• Grade 5 (Fatal): Experiences that result in death.1.15.2.3 Relationship to Study Drug

[0406] The Investigator is obligated to assess the relationship between:• The occurrence of each TEAE and JAG201• The occurrence of each AE or TEAE and prophylactic systemic corticosteroids • The occurrence of each AE or TEAE and the ICV procedure

[0407] The Investigator will use clinical judgment to determine the relationship.

[0408] Alternative causes, such as natural history of the underlying disease, concomitant therapy, other risk factors, and the temporal relationship of the event to prophylactic glucocorticoid administration or ICV procedure or injection of JAG201, will be considered and investigated. The Investigator will also consult the IB and / or Product Information for marketed products (e g., prophylactic systemic corticosteroids) in the determination of the assessment.

[0409] The Investigator will assess causality about whether the event is related to JAG201 based on the following definitions:• Not Related: If no valid reason exists for suggesting a relationship to JAG201or the AE was more likely explained by causes other than JAG201.• Unlikely to be Related: Onset of the event has a reasonable temporal relationship to JAG201 and, although a causal relationship is unlikely, it is biologically plausible.• Possibly Related: Onset of the event has a strong temporal relationship to JAG201 and a causal relationship is biologically plausible.• Related: The injection of JAG201 and AE were closely related in time and the AE may be explained by exposure to JAG201.Atty. Docket No. 38061.0013P1

[0410] The Investigator will assess causality about whether the event is related or not related to prophylactic systemic corticosteroids based on the following definitions:• Not Related: If no valid reason exists for suggesting a relationship to prophylactic systemic corticosteroid or the AE was more likely explained by causes other than prophylactic systemic corticosteroids.• Unlikely to be Related: Onset of the event has a reasonable temporal relationship to prophylactic systemic corticosteroids and, although a causal relationship is unlikely, it is biologically plausible.• Possibly Related: Onset of the event has a strong temporal relationship to prophylactic systemic corticosteroids and a causal relationship is biologically plausible.• Related: The administration of prophylactic systemic corticosteroid and AE were closely related in time and the AE may be explained by exposure to prophylactic systemic corticosteroids (e.g, known pharmacological effect or recurrence on rechallenge).

[0411] The Investigator will assess causality about whether the event is related or not related to the ICV procedure itself based on the following definitions:• Not Related: If no valid reason exists for suggesting a relationship to the ICV procedure or the AE was more likely explained by causes other than the ICV procedure.• Unlikely to be Related: Onset of the event has a reasonable temporal relationship to the ICV procedure and, although a causal relationship is unlikely, it is biologically plausible.• Possibly Related: Onset of the event has a strong temporal relationship to the ICV procedure and a causal relationship is biologically plausible.• Related: The ICV procedure and AE were closely related in time and the AE may be explained by the ICV procedure (e.g. AEs due to catheter insertion).

[0412] There may be situations, particularly when an SAE has occurred, where the Investigator has minimal information to make an assessment in an initial SAE report.However, it is very important that the Investigator always makes an assessment of causality7for every SAE before transmission of the SAE report. The Investigator may change opinionAtty. Docket No. 38061.0013P1of causality- in light of follow-up information, amending the SAE report accordingly. Any assessment of causality made by the Investigator must also be documented in the participant’s source medical record.1.15.2.4 Action Taken with JAG201 Injection

[0413] JAG201 is given as a one-time ICV injection. If an AE occurs the action taken with regard to JAG201 administration will be assessed by the Investigator as described in Table 5Table 5. Action Taken with JAG201 Injection1.15.2.5 Assessment of Outcome

[0414] The result or conclusion of the AE is to be assessed and recorded by the Investigator as described in Table 6.Table 6. Assessment of Outcome1.16 Statistical Considerations1.16.1 Sample Size

[0415] No formal sample size or power calculations were performed for this study with primary objectives of safety and tolerability. A maximum of approximately 31Atty. Docket No. 38061.0013P1participants with SHANK3 haploinsufficiency in the dose-escalation cohorts (a maximum of approximately 18 pediatric participants and 13 adult participants) are planned for JAG201 treatment.1.16.2 Analysis Populations

[0416] The following analysis sets will be used:• The All Participants Analysis Set will consist of all participants enrolled in the doseescalation cohorts who have signed the informed consent, including screen failures.• The Safety Analysis Set will consist of all treated participants who receive JAG201.• The Full Analysis Set will consist of all participants in the Safety Analysis Set who have any post-baseline clinical activity measurements available.

[0417] Safety analyses will be based on the Safety Analysis Sets. The main analysis set for analyses of clinical activity will be based on the Full Analysis Set. Additional sensitivity analyses may be performed in the event important protocol deviations occur that impact analysis conclusions. In all analyses, participants will be classified according to the dose received.1.16.3 General Statistical Methods

[0418] Continuous variables will be summarized using descriptive statistics including but not limited to number of observations, mean, standard deviation, median, minimum, and maximum. Categorical variables will be summarized using number and percentage.

[0419] In general, baseline will be defined as the last available non-missing value prior to administration of JAG201. The SAP will outline baseline definitions for each endpoint and analy sis.

[0420] All analyses will be presented for pediatric and adult participants separately. Select analyses will be presented for all participants combined, where applicable, and will be outlined in the SAP.1.16.4 Participant Disposition and Demographic Characteristics

[0421] Participant disposition will include the number of participants screened, screen failed, and the number and percentage of participants included in each analysis population. The frequency and percentage of participants who prematurely discontinue from the study, along with the primary reason for discontinuation, will also be summarized.Atty. Docket No. 38061.0013P1

[0422] The comparability of treatment groups and cohorts with respect to participant demographics and baseline characteristics will be assessed in a descriptive manner with summary' statistics by Cohort (Dose) and Overall. No formal statistical testing will be performed. Standard continuous or categorical variable summaries will be presented by Cohort (Dose) and Overall for demographic and baseline characteristics in each of the analysis populations. Standard descriptive statistics will be presented for the demographic continuous variables (e.g., age, weight, body mass index) as well as total counts and percentages of participants presented for categorical variables including sex, race, ethnicity', etc.1.16.5 Preliminary Disease Response Analyses (Secondary Endpoints)

[0423] Preliminary response analyses will be based on the Full Analysis Set. All data will be included in participant data listings.

[0424] Due to the small sample size and open-label nature of the study, longitudinal trends will be primarily assessed using descriptive statistics. Mixed models and other repeated-measure methods (e.g. generalized estimating equations) may also be used to characterize the trajectory over time, particularly if the data structure and variability warrant more formal statistical modeling, and if the sample size allows for stable model estimation. Correlations between SAND scores and VEP measurements will be analyzed.1.16.5.1 Visual Evoked Potentials

[0425] VEP will produce the measurements using standard duration tVEPs (long condition) and short duration tVEPs (short condition). The following measurements will be produced:• Amplitude: Amplitudes (in pV) will be measured peak-to-trough at P60-N75 and N75 Pl 00 (subscripted numbers indicate typical time-to-peak values)• Eatency: Latencies (in ms) will be measured by time-to-peak at P60, N75, and P 100 (subscripted numbers indicate t pical time-to-peak values)• Frequency domain analyses

[0426] Descriptive statistics for change from baseline in the amplitude, latency and frequency variables listed above will be presented. Summary statistics will be presented longitudinally by visit for observed results and change from baseline for each Cohort (Dose) and Overall.Atty. Docket No. 38061.0013P11.16.5.2 Sensory Assessment for Neurodevelopmental Disorders

[0427] SAND yields the following composite scores:• Total score is based on the sum of all items across the Observation and Interview, including Severity Items. Total scores range from 0 to 90.• Scores can be combined into Domain (Hyperreactivity, Hyporeactivity, and Seeking) and Modality (Visual. Tactile, and Auditory) scales (each ranging from 0 to 30).• The individual and modality' scales produce the nine subscales (Modality x Domain).Subscale scores range from 0 to 10.

[0428] Total score (ranging from 0 to 90). domain and modality scores (each ranging from 0 to 30), and the 9 subscales (each ranging 0 to 10) will be summarized descriptively for both the raw score and corresponding percentile. Summary statistics will be presented longitudinally by visit for observed results and change from baseline for each Cohort (Dose) and Overall.

[0429] In addition to tabular presentations, graphical displays of results over time will be presented per participant in addition to participant data listings.1.16.6 Preliminary Clinical Activity and Patient Reported Outcomes Analyses (Exploratory Endpoints)

[0430] All exploratory endpoints including those assessing clinical activity as well as patient reported outcomes will be presented longitudinally by visit for each Cohort (Dose) and Overall. In addition to tabular presentations, graphical displays of results over time may be presented per participant in addition to participant data listings. Exploratory analyses will be based on the Full Analysis Set.1.16.7 Safety Analyses

[0431] Statistical methods for the safety analyses will be primarily descriptive in nature and based on the Safety Analysis Set. No imputation of missing safety data will be performed, other than of missing or partial dates, to be described in further detail in the SAP.

[0432] All AEs will be classified using the Medical Dictionary for Regulatory7Activities (MedDRA) classification. All TEAEs will be summarized by Cohort (Dose) and Overall. All TEAE summaries will include the number and percentage of participantsAtty. Docket No. 38061.0013P1experiencing an event, and the number of AEs experienced by the participants. Additional summaries will be produced such as TEAEs by severity and relationship to study drug.

[0433] Clinically significant changes in PE findings, laboratory assessments, vital signs, and ECGs will be reported as AEs. Changes from baseline in clinically significant abnormalities in laboratory values will be summarized by Cohort (Dose) and Overall using descriptive statistics and shifts from normal ranges.

[0434] Incidence of changes from baseline in antibody and T-cell response to AAV9 capsid protein and transgene product (miniSHANK3 protein) will also be summarized descriptively by Cohort (Dose) and Overall.

[0435] Concomitant medications data will be listed and summarized by Cohort (Dose) and Overall.

[0436] All safety data will be included in participant data listings.Example 2: Nonclinical Proof of Concept Data (Murine studies 1)

[0437] The nonclinical development program for JAG201 includes a pre-proof of concept (POC) and POC studies in two distinct but similar rodent models of SHANKS haploinsufficiency. Early work in one of these models at The Broad Institute of MIT and Harvard with a construct that encoded the same miniSHANK.3 protein as JAG201 provided initial proof of a mouse miniShankS gene therapy product’s proof of principle. An additional study in this model used JAG201 by intravenous (IV) administration, which did not show any treatment-related effects, suggesting direct CNS administration would be needed.

[0438] A Definitive POC study was conducted in a similar rodent model of Shank3 deficiency that carries more genetic relevance to the human patient population where large deletions are commonly present (Shank3A4~22mice). Shank3A4'22mice carry a deletion of all protein-coding exons and recapitulate several clinical features of SHANKS haploinsufficient patients. The efficacy, durability, safety, and dose-response of JAG201 administered via ICV injection to pre- weaned juvenile animals were evaluated. The minigene retains key functional domains required to address the structural impairments of the PSD and restore the glutamate receptor function required for neuronal transmission and synaptic plasticity. An additional nonclinical Expansion POC study was conducted to further define both the probability (e.g..Atty. Docket No. 38061.0013P1statistical significance) and magnitude (e.g.. 3 neurobehavioral outcomes) of benefit and demonstrate PDB. The Expansion POC study was designed to be compatible with the Definitive POC study. The same Shank3 KO model was used, with the same route of administration (ICV) and the mice were handled by the same technicians and surgeons, and there were no statistically significant differences seen in control animal baseline behavioural assessments across studies. The complete POC neurobehavioral data from the Shank3A4~22mouse model in the POC studies encompasses three metrics that correlate to manifestations of SHANK3 haploinsufficiency: EEG (sleep), rotarod (balance and motor learning), and open field tests (locomotor ability / exploratory behaviour). Together, these similarities and consistent neurobehavioral baseline were noted as supportive of combining the data from both studies.

[0439] EEG testing was conducted over a 24-hour period at 11-18 weeks post-dosing to evaluate changes in delta-band power as a measure of restorative sleep, as abnormal sleep is frequently observed in SHANK3 haploinsufficient patients. A statistically significant decrease in delta-band power was observed in Shank3 KO animals relative to WT controls, demonstrating recapitulation of abnormal sleep in the ShankS KO model. Statistically significant improvement in Shank3 KO animals over untreated ShankS animals was seen in both the 6.00* IO10(P = 0.027), which is equivalent to the starting dose, and 1.20* 1011vg (P = 0.018) groups. The improvement in the highest dose group (2.75xlOnvg) was just shy of significance (P = 0.065) compared to the KO + vehicle cohort.

[0440] Rotarod testing was performed at 9 to 13 weeks post-dosing to evaluate balance and motor learning performance, as abnormal motor function is frequently observed in SHANK3 haploinsufficient patients. A statistically significant decrease in rotarod performance was observed in Shank3 KO animals relative to WT controls (P < 0.0001), demonstrating recapitulation of motor deficits in the Shank3 KO model. Statistically significant improvement in Shank3 KO animal time on the rotarod was observed in the JAG201 6.00* 1010vg group (P = 0.047), which is equivalent to the starting dose in humans. Significance was also reached in the highest dose 2.75xl0nvg group (P = 0.045).

[0441] Open field testing was performed over a 1-hour period at 6-11 weeks postdosing to evaluate changes in motor activity, as abnormal motor function is frequently observed in SHANK3 haploinsufficient patients. A statistically significant decrease inAtty. Docket No. 38061.0013P1distance covered was observed in vehicle treated Shank3 KO animals relative WT control animals (P = 0.037), demonstrating recapitulation of motor deficits in the Shank3 KO model. In all but the two lowest dose cohorts, treatment with JAG201 at dose levels of > 6.00x IO10vg significantly improved the distance traveled in the open field test, indicating increased motor activity and explorative behavior in treated mice.

[0442] In neurobehavioral assessments in this model, as further summarized in Table 7, JAG201 -treated Shank34~22animals demonstrated dose-dependent improvements across many of these neurological categories at the mid (6.00x 1010vg), mid-high (1.20X1011vg) and / or high (2.75xlOnvg) dose levels when compared to vehicle-treated controls.

[0443] Improvements in neurobehavioral endpoints were supported by JAG201 biodistribution analysis, demonstrating sustained and widespread JAG201 vector genome deoxyribonucleic acid (DNA) distribution and transgene messenger ribonucleic acid (mRNA) expression out to 10-months post-dosing. Following a single ICV administration, expression was persistent throughout the CNS, including target brain regions associated with described motor and cognitive deficits such as striatum, thalamus, cerebellum, and prefrontal cortex. Further, this miniSHANK3 mRNA expression translated to increased miniSHANK3 protein localization to neuronal synaptic densities in a dose-dependent manner. These JAG201-dependent increases in miniSHANK3 protein corresponded with significant increases in the SHANK3 protein binding partner Homerl which, when bound to SHANK3, acts as a scaffold to stabilize metabotropic glutamate receptors at the PSD. This enrichment demonstrates overall improvement of the neuronal structure and synaptic s tabi 1 i ty required for proper glutamatergic signaling and cell function. Together, neurobehavioral and synaptic membrane preparation data provide evidence of clinically translatable functional improvements in Shank3 deficient rodents that are supported by mechanistic data demonstrating miniSHANK3 function at the glutamatergic neuronal synapse.

[0444] Within the definitive POC study, there was evidence that the 2.75x 1011vg dose was not as well-tolerated, despite having signs of increased efficacy as measured by EEG delta pow er assessment. Findings at this dose level in JAG201 -treated KO mice include potential hyperactivity in the open field assessment, supraphysiological levels of miniSHANK3 protein (589% of endogenous SHANK3 WT levels), and mortality' ofAtty. Docket No. 38061.0013P1undetermined cause in nonclinical POC longer-term 9-month study arm. To mitigate these potential overexpression risks, a starting clinical dose level has been selected equivalent to where these findings were not observed (6.00x IO10vg in mouse). At the 6.00x IO10vg / animal dose in the POC studies, safety findings included issues associated with the injection procedure and eosinophilic inclusions in Purkinje cells. These inclusions did not appear to affect cell function, and improvements were noted in endpoints for which impairment in Purkinje function would be expected to be observed (e.g., in the rotarod as a measure of balance). Because the 6.00* IO10vg / dose was also associated with improvements in disease-related phenotypes and resulted in a relative expression level of 44% vs. WT mice, the clinical equivalent of this dose is considered appropriate for investigation in patients.Table 7. JAG201 Treatment of KO Mice Leads to Improvement in Neurobehavioral OutcomesAtty. Docket No. 38061.0013P1Example 3: Nonclinical Toxicology Data

[0445] Five nonclinical studies have been conducted which contribute to the safety assessment for JAG201. These were conducted in WT mice, ShankS '4--- KO mice, and nonhuman primates (NHPs). Consistent with the intended route of administration, these studies used ICV administration. Studies were conducted using a mix of sexes and extended to 10 months (mice) or 6 months (NHP) post-dose.

[0446] In Example 2, there was evidence that the 1.20*10“ vg dose and 2.75*10“ vg dose were not as well tolerated, despite being efficacious, with signs of overexpression risk in the open field assessment at both 1.20*10“ vg dose and 2.75*10“ vg dose levels, and increased mortality of undetermined cause in the preclinical POC longer-term 9-month study arm at 2.75*10“ vg dose level in a WT genetic background. Increased overall mortality was also seen in WT animals dosed at the 1.20x10“ vg dose level (but not in KO animals). Of note, these findings appeared to be species specific as no overexpression or increased mortality observations were observed at any dose level tested in NHPs with the same WT genetic background. To mitigate these potential overexpression risks, a starting clinical dose level was selected equivalent to where these findings were not observed (6.00* 1010vg in mouse). At the 6.00* 1010vg / animal dose in Example 2. safety findings included issues associated with the injection procedure and eosinophilic inclusions in Purkinje cells. These inclusions did not appear to affect cell function, and improvements w ere noted in endpoints for which impairment in Purkinje function would be expected to be observed (e.g., in the rotarod as a measure of balance). As the 6.00* 1010vg / dose was also associated with improvements in disease-related phenotypes and resulted in a relative expression level ofAtty. Docket No. 38061.0013P144% vs. WT mice; the clinical equivalent of this dose is considered appropriate for investigation in patients.

[0447] Safety was further evaluated in two studies in NHP. In the 3-month, non-GLP, NHP tolerability and distribution study with a three-month post-dosing observation period, JAG201 was well tolerated following single-dose ICV administration of l.OOxlO13vg and l.OOxlO14vg in 16 cynomolgus monkeys. No JAG201 -related mortalities, adverse events, adverse clinical observations (body weight, body temperature, heart rate, respiration, dermatitis), or adverse neurological observations (level of consciousness, motor function, proprioception, ataxia, cranial nerve assessments, cervical spinal assessments, and lumbosacral spinal assessments) were observed on study. JAG201 -related transient increases in liver aminotransferase enzymes demonstrated recoverability to baseline pre-study values at four weeks post-dosing. The transient nature of these increases was supported by the absence of JAG201 dose-dependent microscopic findings in the liver at 3 months post-dosing. No JAG201 -related changes in hematology7, coagulation parameters, or cerebrospinal fluid (CSF) chemistry were noted. Administration of JAG201 at a dose of l.OOxlO14vg was associated with development of humoral immune response via antidrug antibody enzyme-linked immunosorbent assay (ELISA) and cellular immune response via enzyme-linked immunosorbent spot (ELISpot) assay against the AAV9 capsid, but with no consistent anti-transgene response. Minimal to mild dorsal root ganglia / trigeminal ganglia / spinal cord (DRG / TG / SC) neuronal degeneration was observed but was not considered adverse in this study due to the lack of correlating clinical observations. Results of the non-GLP NHP study supported further dose characterization of JAG201 in the pivotal GLP NHP toxicology7study at dose levels up to l.OOxlO14vg.

[0448] In the 6-month, pivotal, GLP NHP toxicology study, toxicity and biodistribution was assessed after a single ICV administration of JAG201 at l.OOx 1013, 2.50xl013, 5.00xl013, and 9.30xl013vg dose levels to 44 cynomolgus monkeys at 3- and 6-months post-dosing. No JAG201 -related mortalities, adverse in-life events, adverse clinical observations (body weight, ophthalmology, electroencephalogram, electrocardiogram), or adverse neurological observations in JAG201 -dosed animals were observed on study.JAG201 -related transient and reversible increases in liver aminotransferase enzymes resolved by 4 weeks post-dosing, mirroring non-GLP NHP study findings. The transient nature of these increases was supported by the absence of JAG201 -related microscopic findings in theAtty. Docket No. 38061.0013P1liver at 3- and 6-months post-dosing. Cellular immune response analysis via ELISpot assay also mirrored non-GLP NHP study findings in relation to AAV9 capsid and demonstrated rare (2 animals) and very low magnitude (< 14 spots / well) non-dose-dependent responses to the transgene. Also consistent with the non-GLP study, microscopic evaluation at necropsy demonstrated minimal to mild DRG / TG / SC neuronal degeneration findings at > LOOxlO13vg, but in contrast to the non-GLP study pathologist’s determination, these findings were deemed adverse solely due to the non-regenerative nature of neurons and did not consider the complete absence of observed clinical correlates. Of note, rare and focal mild decreases in brain cellularity were observed in a non-dose-dependent manner in 2 of 12 animals at the 2.50xl013vg dose level and in 1 of 12 animals at the 9.30*1013vg dose level (total of3 of 44 animals dosed with JAG201). Similar to the DRG / TG / SC neuronal degeneration findings, these findings were not associated with any clinical correlates. The microscopic findings highlighted above were deemed by study pathologists to be consistent with effects on neurons and nerve fibers of the CNS and ganglia associated with administration of AAV test articles. It is possible that these non-dose-dependent observations of brain cellularity in 3 of44 animals may have occurred as a result of focal supraphysiological SHANK3 / miniSHANK3 expression generated in the WT background NHPs. Based on these results, a NOAEL was not identified in the study report because the minimal neuronal degeneration / necrosis in the ganglia occurred at the lowest dose evaluated, and this effect was considered adverse to the animals in the context of the study. Given the rare incidence of adverse neuronal observations, the minimal to mild severity' and non-progressive nature of the findings, and the lack of clinical correlates associated with these findings, JAG201 is considered well tolerated in WT NHPs at dose levels up to 9.30x 1013vg.

[0449] Based on these results, the NOAEL could not be established considering the conditions of the study, but the HNSTD was considered to be the high dose given the lack of any clinical correlates to the microscopic findings (9.30xl013vg).3.1 Selection of JAG201 Doses for Adults with SHANK3 haploinsufficiency

[0450] The HNSTD of 9.30x 1013vg for JAG201 in NHPs is approximately equivalent to 2.06x 1015vg in adult humans when normalized to brain mass. This HNSTD represents an approximately 9-fold safety margin above the 6.00x1010vg dose level in theAtty. Docket No. 38061.0013P1definitive POC study as the 6.OOxlO10vg rodent dose equates to approximately l.OOxlO13vg in NHPs and 2.16x]014vg in a 16- to 40-year-old human based on total brain mass (Table 8) Table 8: JAG201 Dose Extrapolation Based on Brain Mass Leads to an Effective Starting Dose of 2.16xf014vg for Adults in the First-in-Human Study

[0451] Overall, the nonclinical safety data support the use of JAG201 at a starting dose of 2.1 x 1014vg / parti cipant for Cohort 1 adult participants in this FIH clinical study. This dose is equivalent to the lowest dose tested in the GLP toxicology7study and is 9-fold lower than the HNSTD in that study.3.2 Selection of JAG201 Doses for Pediatric Subjects with SHANK3 haploinsufficiency

[0452] To extrapolate the dose in pediatric patients, scaling by brain weight was used since brain weight is relatively stable over the age range of the intended patient population, and providing a single dose level is desired. Using the 3-year-old human brain weight as a reference point, the maximum variance in brain weight for the intended patient population of 2-9 years of age is estimated to be 11.8% (Table 9).Table 9: Summary of Average Brain Weight from 2-9 Years of Age

[0453] The HNSTD of 9.30x 1013vg for JAG201 in NHPs is approximately equivalent to 1.82xl015vg in pediatric humans when normalized to brain mass. This HNSTD represents an approximately 9-fold safety margin above the 6.00x1010vg dose level in theAtty. Docket No. 38061.0013P1Definitive POC study as the 6.00* IO10vg rodent dose equates to 9.75* 1012vg in NHPs and 1.91 x1014vg in a 2- to 9-year-old pediatric human based on total brain mass (Table 10). Overall, the nonclinical safety data support the use of JAG201 at a starting dose of1.91 x 1014vg / participant for Cohort 1 pediatric participants in this FIH clinical study.Table 10: JAG201 Dose Extrapolation Based on Brain Mass Leads to an Effective Starting Dose of 1.91X1014vg for Pediatric Participants in the First-in-Human StudySemple, 2013:bLi, 2023;cChuang, 2011:dAmato, 2022;cDekaban, 1978

[0454] For the Cohort 2 pediatric participants, thrice the starting dose (i.e., 5.73xl014vg / participant) is being proposed, which is ~3-fold lower than the HNSTD in the GLP toxicology study. Furthermore, as mentioned under the study design, an intermediate dose of JAG201 may be selected for Cohort 2 based on its review of cumulative safety and activity data.Example 4: Cohort 1 Clinical Trial Results

[0455] Example 4 describes preliminary clinical safety and efficacy results following administration of JAG201 to a cohort (Cohort 1) consisting of 3 pediatric patients aged 2-9 years. The initial reported results continue to be assessed and the patients followed for testing at later time points. Accordingly, the results are subject to refinement and change. All three Cohort 1 patients exhibited moderate to severe developmental impairments in communication, socialization, and motor function at baseline (prior to dosing).Cohort 1-Safety

[0456] Each patient in Cohort 1 was dosed with JAG201 (AAV9 AAV-hSynl-HumanMiniSHANK3-Vl (5’ ITR-hSynl-WPRE-hGH poly A- 3’ ITR) (SEQ ID NO: 8) via unilateral ICV administration to the lateral ventricle at a single dose of 1.91xl014vg, according to the methods described in the protocol of Example 1. No treatment-relatedAtty. Docket No. 38061.0013P1serious adverse events (SAEs) or dose-limiting toxicities (DLTs) were observed for any of the three Cohort 1 patients as of 5 months to 1 year following administration of JAG201.Cohort 1-Results

[0457] Vineland-3 scores, PMS-Specific CGI-I / PMSA-C scores, PMS-Specific CGI-S / PMSA-S scores, VEP waveforms, and AEP waveforms were obtained for each patient in Cohort 1 at baseline (prior to administration of JAG201), and at specified time points following administration of JAG201. The results of these evaluations are summarized below.Cohort l-Vineland-3 Scores

[0458] The Vineland-3 assessment measures adaptive behavior, which are skills needed to function independently at home, school / work, and in the community. The assessment provides scores for four domains (Socialization, Daily Living Skills, Communication, and Motor), as well as a Composite Score. Participants are compared to a normal population with a standard score of 100 + / - one standard deviation (SD) (85-115). PMS patients generally score less than the intellectual disability’ threshold of 70. Preliminary assessments of Cohort 1 patients indicate stabilization or improvement over baseline for certain Vineland-3 domains following dosing with JAG201 over 3 months, 6 months and / or 9 months from dosing.Cohort 1-PMS-Specific CGI-I / PMSA-C Evaluation

[0459] The PMS-Specific CGI-I (now PMSA-C, and accordingly also referred to herein as CGI-I / PMSA-C) is a clinician-rated tool that measures improvement since baseline in seven (7) PMS-relevant domains. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-I) or Attention and Awareness (for PMSA-C), and (vii) Self-Care. Each patient from Cohort 1 underwent CGI-I / PMSA-C evaluation at baseline (prior to dosing), and at day 28 and month 3, and have or will be evaluated at month 6 following dosing with JAG201. The CGI-I / PMSA-C scale provides a qualitative assessment of clinical change, compared to a baseline (pre-dosing) score for each of the domains, across the seven (7) distinct levels of improvement or worsening, wherein a score of 1 indicates “very much improved,’72 indicates “much improved,” 3 indicates “minimally improved,” 4 indicates “no change,” 5 indicates “minimally worse,” 6 indicates “much worse,” and 7 indicates “very much worse.”Atty. Docket No. 38061.0013P1

[0460] All three Cohort 1 patients experienced rapid motor improvements within weeks of JAG201 administration, leading to attainment of new motor skills including running jumping, and ability to navigate stairs. All three Cohort 1 patients also demonstrated increased awareness of environment and increased interest in social interaction with peers / adults following administration of JAG201. All three Cohort 1 patients were rated as either “minimally improved’’ (score of 3) or “much improved” (score of 2) for various PMS-CGI-I / PMSA-C domains, as well as for the overall CGI-I / PMSA-C global improvement score, as early as 28 days to 6 months, for those evaluated at 6 months, following administration. One patient exhibited rapid improvement and attainment of skills, showing “much improved” scores for two CGI-I / PMSA-C domains (Expressive Communication and Gross Motor), and “minimally improved” scores for three CGI-I / PMSA-C domains (Receptive Communication, Fine Motor, and Social Interaction) by 28 days following administration of JAG201, and attained an overall CGI-I / PMSA-C global improvement score of 2 (“Much Improved”) at 28 days following administration.Cohort 1-PMS-Specific CGI-S Evaluation

[0461] The PMS-Specific CGI-S (now PMSA-S, and accordingly also referred to herein as CGI-S / PMSA-S) is a clinician-rated tool that measures severity of symptoms in seven (7) PMS-relevant domains, relative to the clinician's total experience with the PMS population, based on domain-specific anchors. The seven measured domains are (i) Expressive Communication, (ii) Receptive Communication, (iii) Gross Motor Skills, (iv) Fine Motor Skills, (v) Social Interaction, (vi) Cognition and Learning (for CGI-S) or Attention and Awareness (for PMSA-S), and (vii) Self-Care. The CGI-S / PMSA-S scale provides a qualitative assessment of the severity' of symptoms across the relevant domains relative to the total experience with the PMS population, wherein a score of 1 indicates “typical for age / not impaired,” 2 indicates "slightly impaired,” 3 indicates “mildly impaired,” 4 indicates “moderately impaired,” 5 indicates “markedly impaired,” 6 indicates “severely impaired,” and 7 indicates “among most severely impaired.”

[0462] Each patient from Cohort 1 underwent CGI-S / PMSA-S evaluation at baseline (prior to dosing), and at day 28, and month 3, and has or will be evaluated at month 6 following dosing with JAG201. All three Cohort 1 patients generally maintained baseline CGI-S / PMSA-S scores (including a slight (1 point) worsening in certain domain scores for one patient), through three or six months following administration of JAG201. One patientAtty. Docket No. 38061.0013P1exhibited (i) a consistent improvement in the CGI-S / PMSA-S Gross Motor Skills domain, exhibiting a score of 4 (“moderately impaired) at baseline and a score of 2 (“slightly impaired”) at day 28 and month 3 following administration of JAG201, and (ii) a consistent improvement in the CGI-S / PMSA-S Expressive Communication domain, exhibiting a score of 4 (“moderately impaired) at baseline and a score of 3 (“mildly impaired”) at day 28 and month 3 following administration of JAG201.Cohort 1-Visual Evoked Potential Evaluation

[0463] Visual Evoked Potential (VEP) is a non-invasive method for measuring functionality of the human visual system by detecting neuronal responses to stimuli independently of the consciousness and attention state of the patient. VEP waveforms were recorded for all patients in Cohort 1 at baseline (prior to administration of JAG201), and at 3 months and 6 months following administration of JAG201. ERP waveform parameters of interest, including: N75 Amplitude (excitatory response to stimulus), Pl 00 Amplitude (inhibitory response to stimulus), N75 to P100 peak-to-peak amplitude (total initial response to visual stimulus), and N2-P2 peak-to-peak amplitude (secondary response potentially indicative of interpretation of visual stimulus), were analyzed for each patient in Cohort 1.

[0464] Changes in VEP waveform observed for Cohort 1 patients included changes in: (i) N75 Amplitude (indicative of changes in excitatory response to stimulus), (ii) P100 Amplitude (indicative of changes in inhibitory response to stimulus), (iii) N75 to Pl 00 peak-to-peak amplitude (indicative of changes in total initial response to visual stimulus), and (iv) N2-P2 peak-to-peak amplitude (indicative of changes in secondary response potentially indicative of interpretation of visual stimulus). In one Cohort 1 patient, the latency of the ERP waveform was observed to decrease from baseline to month 6 post dosing, suggesting a more rapid neuronal response to visual stimulus. In another Cohort 1 patient, the ERP waveform exhibited (i) a greater N75 and Pl 00 amplitude as compared to baseline, (ii) a greater slope fromN75-P100 (suggesting a more rapid neuronal response), and (iii) an emerging P2 signal suggesting an improved ability to process visual stimulus.Cohort 1-Auditory Evoked Potential Evaluation

[0465] Auditory Evoked Potential (AEP) is an electrical signal elicited from the brain while an auditory stimulus is presented in a time-locked manner. The AEP signal consists of reproducible positive or negative peaks, latency, amplitude and behavioral correlation. ForAtty. Docket No. 38061.0013P1one patient in Cohort 1, at 6 months following administration of JAG201 changes in AEP waveform as compared to baseline were observed across a number of ERP waveform parameters of interest, including: N1 Amplitude (excitatory response to stimulus), Pl Amplitude (inhibitory response to stimulus), N1 to Pl peak-to-peak amplitude (total initial response to auditory stimulus), and N2-P2 peak-to-peak amplitude (secondary response potentially indicative of interpretation of auditory stimulus). These changes suggest stronger and more consistent electrical responses in the cortex of the brain, indicating that the auditory information is being communicated from the ears to the parts of the brain in the cortex where they are interpreted.Cohort 1-Efficacy Observed Following ...

Claims

Atty. Docket No. 38061.0013P1CLAIMSWhat is claimed is:

1. A method of treating a SHANKS deficiency in a human subject in need thereof, comprising administering by intracerebroventricular (ICV) administration a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a miniSHANK3 protein, operably linked to a regulatory element that promotes expression in central nervous system (CNS) and flanking AAV ITR sequences; wherein the therapeutically effective amount of the pharmaceutical composition is administered(i) to an adult subject or a pediatric subject age 10 to 17 at a dose of 2.16* 1014+ / - 10% or 20% rAAV vector genomes (vg) (or between 2.1 xlO14to 2.2*1014vg), or 5.73 xlO14+ / - 10% or 20% vg (or between 5.7 x 1014to 5.8x1014vg), or 6.30x1014+ / - 10% or 20% vg (or between 6.2xl014to 6.4xl014vg); or alternatively(ii) to a pediatric subject age 2 to 9 at a dose of 1.91 xlO14+ / - 10% or 20% vg (or between 1.85xl014to 1.95xl014vg) or 5.73xl014+ / - 10% or 20% vg (or between 5.7x1014to 5.8x1014vg); or alternatively(iii) to an adult subject or a pediatric subject age 10 to 17 at a dose of 2.16xl014vg or 5.73xl014vg or 6.30xl014vg; or alternatively(iv) to apediatric subject age 2 to 9 at a dose of 1.91xl014vg or 5.73xl014vg.

2. The method according to claim 1 , wherein the therapeutically effective amount of the rAAV particle is administered via unilateral ICV administration.

3. The method according to claim 1, wherein the miniSHANK3 protein comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and retains SHANK3 activity.

4. The method according to claim 1, wherein the miniSHANK3 protein comprises an amino acid sequence of SEQ ID NO: 3.

5. The method according to claim 1, wherein the miniSHANK3 protein is encoded by a nucleotide sequence at least 70% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 and has SHANK3 activity.Atty. Docket No. 38061.0013P16. The method according to claim 1, wherein the regulatory element is a promoter that comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

7. The method according to claim 1, wherein the transgene comprises a polyadenylation (poly A) signal 3’ of the nucleotide sequence encoding the miniSHANK3 protein.

8. The method according to claim 1, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 7.

9. The method according to claim 1, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 8.

10. The method according to claim 9, wherein the rAAV particle is an AAV9 serotype.

11. The method according to claim 1, wherein the SHANK3 deficiency is (i) a loss of function mutation in SHANK3 or a 22ql3.3 deletion classified as a Class I deletion or (ii) a Class II deletion.

12. The method according claim 10, wherein the pharmaceutical composition comprises(i) about 5 mM to about 25 mM of a buffering agent;(ii) about 0.5 mM MgC12 to about 1.5mM MgC12:(iii) about 50 mM to about 150 mM of a tonicity agent; and(iv) from about 0.02% to about 0.2% of a non-ionic surfactantwherein the pH of the pharmaceutical composition is between about 5.0 and about 9.0.

13. The method according to claim 12, wherein the buffering agent is lOmM Tris.

14. The method according to claim 12. wherein the tonicity agent is 150 mM NaCL 15. The method according to claim 12, wherein the pharmaceutical composition has an osmolality is 150 to 450 mOsm / kg.

16. The method according to claim 12, wherein the pharmaceutical composition does not comprise a preservative.

17. The method according to claim 12, wherein the pharmaceutical composition comprises (i) lOmM Tris; (ii) ImM MgCb; (iii) 150mM NaCl; (iv) 0.02% (w / v) Poloxamer 188 at pH 8.0.

18. The method according to claim 12, wherein the pharmaceutical composition comprises 6.0*1013to 7.0*1013vg / mL, or alternatively 6.5 x 1013vg / mL.Atty. Docket No. 38061.0013P119. The method according to claim 12. wherein a change in visual evoked potential (VEP) waveforms is observed within 1 month, 3 months, 6 months, or 12 months or more after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history controls.

20. The method according to claim 12, wherein a change in auditory evoked potential (AEP) waveforms is observed within 6 months, or 12 months or more after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history controls.

21. The method according to claim 12. wherein sensory symptoms associated with SHANKS mutations are stabilized or improved within 1 month, 3 months. 6 months, or 12 months or more after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history' controls.

22. The method according to claim 12, wherein adaptive behavior is stabilized or improved within 1 month. 3 months, 6 months, or 12 months or more after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history controls.

23. The method according to claim 12, wherein global cognitive ability is stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history’ controls.

24. The method according to claim 12, wherein language skills are stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history’ controls.

25. The method according to claim 12, wherein motor functioning is stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history' controls.

26. The method according to claim 12, wherein autism symptoms are stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, compared to baseline or age-matched natural history’ controls.

27. The method according to claim 12, wherein sleep behaviors, or the fre uency / se verily of sleep disturbances or disorders, are stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, relative to baseline or age-matched natural history controls.Atty. Docket No. 38061.0013P128. The method according to claim 12. wherein symptoms associated with Phelan-McDermid Syndrome (PMS) are stabilized or improved within 1 month, 3 months, 6 months, or 12 months after administration of the pharmaceutical formulation, relative to baseline or age-matched natural history controls.

29. The method according to claim 12, wherein there is (i) stabilization of or an increase of at least 2, at least 5, or at least 10 points in the Vineland-3 Adaptive Behavior Scales composite score; or alternatively (ii) stabilization of or an increase of at least 2, at least 5, or at least 10 points in one or more of the socialization, daily living skills, communication, or motor domains of the Vineland-3 Adaptive Behavior Scales, or a combination thereof at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition, compared to baseline or age-matched control subjects.

30. The method according to claim 12, wherein the score for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global Impression Scale of Improvement (CGI-I / PMSA-C), or the overall global CGI-I / PMSA-C score, or combinations thereof, is 4 or lower at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition.

31. The method according to claim 12, wherein the score for one or more of the 7 PMS-relevant domains of the PMS-Specific Clinical Global Impression of Severity (CGI-S / PMSA-S), or the overall global CGI-S / PMSA-S score, or combinations thereof, is stabilized or reduced by at least 1 point, or at least 2 points, or at least 3 points, or at least 4 points, or at least 5 points, or at least 6 points at 3 months, 6 months, 12 months, 2 years, 5 years 10 years, or 15 years or more after administration of the pharmaceutical composition.

32. A unit dosage form comprising 2.15mL of a pharmaceutical composition comprising from about 6.0xl013to about 7.0x]013vg / mL, or alternatively 6.5xl013vg / mL, of a recombinant adeno-associated vector (rAAV) particle, wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a miniSHANK3 protein, operably linked to a regulatory’ element that promotes expression in central nervous system (CNS), and flanked by AAV ITR sequences; wherein the rAAV particle is formulated for intracerebroventricular (ICV) administration.

33. The unit dosage form of claim 32, wherein the miniSHANK3 protein comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and retains SHANK3 activity.Atty. Docket No. 38061.0013P134. The unit dosage form of claim 32, wherein the miniSHANK3 protein is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and retains SHANK3 activity.

35. The unit dosage form of claim 32, wherein the transgene that encodes a miniSHANK3 protein is operably linked to a regulatory element that is a promoter that comprises or consists of a nucleotide sequence that is at least 85%, 90%, 95%, or 100% identical to a nucleotide sequence of SEQ ID NO: 9, and promotes expression of the miniSHANK3 protein in cells of the CNS.

36. The unit dosage form of claim 32, wherein the transgene that encodes a miniSHANK3 protein is operably linked to a regulatory element that is a promoter that comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

37. The unit dosage form of any one of claim 32, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of SEQ ID NO: 8.

38. The unit dosage form of any one of claim 32, wherein the artificial genome comprises a nucleotide sequence comprising or consisting of SEQ ID NO: 7.

39. The unit dosage form of any one of claims 32 to 38, wherein the rAAV particle comprises at least one AAV9 serotype capsid protein.

40. The unit dosage form of any one of claims 32 to 39, wherein the pharmaceutical composition is a preservative-free, sterile, solution comprising lOmM Tris, ImM Magnesium Chloride Hexahydrate, 150mM Sodium Chloride, and 0.02% (w / v) Poloxamer 188.

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