Gene therapy for treatment of syngap1-related epileptic encephalopathy

Abstract

The present disclosure describes recombinant adeno-associated virus (rAAV) vectors which comprise a payload sequence encoding a synaptic GTPase-activating protein (SynGAP). The present disclosure also describes methods of treating SynGAP-related diseases, including neurodevelopmental disorders (NDDs) such as epileptic encephalopathies (DEEs).

Description

[0001] DESCRIPTION

[0002] GENE THERAPY FOR TREATMENT OF SYNGAP1-RELATED EPILEPTIC ENCEPHALOPATHY

[0003] [1] The present disclosure describes recombinant adeno-associated virus (rAAV) vectors which comprise a payload sequence encoding a synaptic GTPase-activating protein (SynGAP). The present disclosure also describes methods of treating SynGAP-related diseases, including neurodevel opmental disorders (NDDs) such as epileptic encephalopathies (DEEs).

[0004] [2] In certain embodiments, the present disclosure describes a polynucleotide comprising a promoter sequence operatively linked to a payload sequence, wherein the payload sequence encodes a synaptic GTPase-activating protein (SynGAP). In certain embodiments, the polynucleotide comprises a poly(A) signal sequence.

[0005] [3] In certain embodiments, the payload sequence has at least 85% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence comprises any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence the comprises SEQ ID NO: 7. In certain embodiments, the payload sequence encodes a SynGAP which has at least 85% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP comprising any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP comprising SEQ ID NO: 8.

[0006] [4] In certain embodiments, the promoter sequence has at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the promoter sequence comprises SEQ ID NO: 3. In certain embodiments, the promoter sequence comprises SEQ ID NO: 4.

[0007] [5] In certain embodiments, the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the poly(A) signal sequence comprises SEQ ID NO: 15. In certain embodiments, the poly(A) signal sequence comprises SEQ ID NO: 16.

[0008] [6] In certain embodiments, the payload sequence has at least 90% sequence identity to SEQ ID NO: 7, the promoter sequence has at least 90% sequence identity to SEQ ID NO: 4, and the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16. In certain embodiments, the payload sequence comprises SEQ ID NO: 7, the promoter sequence comprises SEQ ID NO: 4, and the poly(A) signal sequence comprises SEQ ID NO: 16. [7] In certain embodiments, the polynucleotide (e.g., expression cassette) has at least 85% sequence identity to any one of SEQ ID NOs: 17 to 25. In certain embodiments, the polynucleotide comprises any one of SEQ ID NOs: 17 to 25. In certain embodiments, the polynucleotide comprises SEQ ID NO: 21.

[0009] [8] In certain embodiments, the present disclosure describes an adeno-associated viral (AAV) vector genome comprising: a 5' inverted terminal repeat (ITR), an expression cassette, and a 3' ITR; wherein the expression cassette comprises a polynucleotide of the present disclosure. In certain embodiments, the 5' ITR is an AAV2 ITR. In certain embodiments, the 3' ITR is an AAV2 ITR.

[0010] [9] In certain embodiments, the AAV vector genome comprises a sequence which has at least 85% sequence identity to any one of SEQ ID NOs: 26 to 34. In certain embodiments, the AAV vector genome comprises any one of SEQ ID NOs: 26 to 34. In certain embodiments, the AAV vector genome comprises SEQ ID NO: 30.

[0011]

[0010] In certain embodiments, the present disclosure describes an AAV particle comprising an AAV vector genome of the present disclosure and a capsid. In certain embodiments, the capsid has tropism for tissues or cells of the central nervous system (CNS).

[0012]

[0011] In certain embodiments, the present disclosure describes a pharmaceutical composition comprising an AAV particle of the present disclosure, and a pharmaceutically acceptable excipient.

[0013]

[0012] In certain embodiments, the present disclosure describes a method of increasing the expression of synaptic GTPase-activating protein (SynGAP) in a subject, comprising delivering an effective amount of an AAV particle of the present disclosure.

[0014]

[0013] In certain embodiments, the present disclosure describes a method of treating a disease related to a SYNGAP 1 gene mutation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an AAV particle of the present disclosure. In certain embodiments, the disease related to a SYNGAP1 gene mutation is a neurodevelopmental disorder (NDD). In certain embodiments, the disease related to a SYNGAP1 gene mutation is a developmental and epileptic encephalopathy (DEE). In certain embodiments, administering the composition to the subject comprises intracisterna magna (ICM), intra-cerebrospinal fluid (ICSF), intracerebroventricular (ICV), or intraparenchymal administration. In certain embodiments, administering the composition to the subject reduces or eliminates one or more symptoms of the disease. BRIEF DESCRIPTION OF FIGURES

[0015]

[0014] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the present disclosure, as illustrated in the accompanying figures. The figures are not necessarily to scale or comprehensive, with emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure.

[0016]

[0015] FIG. 1 A and FIG. IB show a schematic representation of one example of a signaling pathway regulated by the SynGAPl gene.

[0017]

[0016] FIG. 2A shows one embodiment of a rAAV vector genome which includes a 5' ITR, a promoter, a transgene, a poly A (pA) sequence, and a 3' ITR. FIG. 2B shows embodiments of a rAAV vector genome which includes a 5' ITR, a promoter (hSyn or JeT), a mouse SynGAPl transgene (mSynGAPl), a polyA sequence (SynthPA or SV40PA), and a 3' ITR. FIG. 2C shows embodiments of a rAAV vector genome which includes a 5' ITR, a promoter (hSyn or JeT), a human SynGAPl transgene (hSynGAPl), a polyA sequence (SynthPAor SV40PA), and a 3' ITR.

[0018]

[0017] FIG. 3 shows GFP+ cell measurements (via by flow cytometry) related to a study to evaluate promoter strength using three GFP-expressing plasmids.

[0019]

[0018] FIG. 4 shows ddPCR measurements for expression levels of SynGAPl mRNA (copies / pL) using mSynGAP- and hSynGAP-expressing plasmids.

[0020]

[0019] FIG. 5A shows chemiluminescence intensity measurements (electropherogram) related to the evaluation of SynGAP protein expression for mSynGAP- and hSynGAP- expressing plasmids transfected into cells. FIG. 5B shows a corresponding virtual blot generated from the chemiluminescence peaks shown in FIG. 5 A.

[0021]

[0020] FIG. 6 shows viable cell concentration measurements for mSynGAP or hSynGAP- expressing cassettes at 48 hours post-transfection (hpt).

[0022]

[0021] FIG. 7 shows AAV vector genome titer (VG / mL) in HEK293T crude cell lysate, postpackaging with the mSynGAP or hSynGAP-expressing cassettes.

[0023]

[0022] FIG. 8 shows total AAV particle measurement in HEK293T crude cell lysate, postpackaging with the mSynGAP or hSynGAP-expressing cassettes.

[0024]

[0023] FIG. 9. shows SynGAP protein analysis performed from hippocampal brain lysates of 6-8 weeks old male mice.

[0025]

[0024] FIG. 10 shows MRS analysis in the hippocampus of 7-8 weeks old mice.

[0025] FIG. 11 shows home-cage spontaneous locomotor activity (crosses / hour) of 8-12 weeks old mice during seven consecutive days in the light / dark phase of the day.

[0026]

[0026] FIG. 12 shows epileptiform discharges in the light / dark phase over a 24-hour recording period.

[0027]

[0027] FIG. 13 shows myoclonic-like seizures in the light / dark phase over a 24-hour recording period.

[0028]

[0028] FIG. 14 shows the duration of myoclonic-like seizures in the light / dark phase over a 24-hour recording period.

[0029] DEFINITIONS

[0030]

[0029] Adeno-associated virus'. As used herein, the terms "adeno-associated virus" or "AAV" refer to members of the Dependoparvovirus genus in the Parvoviridae family, as well as particles, sequences, genes, capsids, proteins, or components derived therefrom.

[0031]

[0030] Adeno-associated virus (AAV) particle'. As used herein, the terms "AAV particle" or "AAV vector" refer to a construct which comprises an AAV capsid and a polynucleotide within the AAV capsid (i.e., full capsid). The polynucleotide is referred to as the AAV genome, AAV viral genome, or AAV vector genome (VG). AAV particles of the present disclosure may be produced recombinantly (i.e., rAAV), and may include sequences or proteins based on wild-type adeno-associated virus (AAV) and / or synthetic sequences or proteins.

[0032]

[0031] Approximately / About: As used herein, the terms "approximately" and "about" are used interchangeably herein and refer to a value that is within + / - 10% of the recited value as applied to one or more values of interest. In certain embodiments, the term refers to a range of values that fall within + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, or less of the stated reference value, unless otherwise expressly stated or otherwise clearly evident from the context.

[0033]

[0032] Capsid : As used herein, the term "capsid" refers to the protein shell of a virus which encloses or encapsulates the genetic material (i.e., vector genome) of the virus. Capsids generally comprise a combination of two are more capsid proteins. For AAVs, the AAV capsid generally comprises a combination of VP1, VP2, and VP3 capsid proteins. A capsid may be a wild-type capsid, a recombinant capsid, or an engineered capsid.

[0034]

[0033] Expression'. As used herein, the terms "expression" and "gene expression" refer to the process by which a nucleic acid sequence undergoes successful transcription into a corresponding RNA molecule (e.g., mRNA strand) and, generally, undergoes successful translation of the RNA molecule to produce a protein or polypeptide. Gene expression can thus be measure by measuring the transcription product of the gene expression (e.g., mRNA) and / or measuring the translation product of the gene expression (e.g., polypeptide or protein). Methods of measuring the amount or levels of RNA, mRNA, polypeptide, and proteins are known in the art.

[0035]

[0034] Expression cassette'. As used herein, the terms "expression cassette" and "polynucleotide cassette" refer to a portion of the viral genome between the flanking ITR sequences which comprises at least one payload sequence encoding a payload product (e.g., a therapeutic product), and at least one element to promote the expression of the payload sequence (e.g., a promoter).

[0036]

[0035] Identity: As used herein, the terms "identity," "sequence identity," "percent sequence identity," or "percent identity" refer to the number of identical matched positions shared between two polynucleotide or polypeptide sequences over a comparison window, including gaps, additions, or deletions required for optimal alignment of the two sequences. Percentage of sequence identity can be calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and then converting the value to a percentage of sequence identity. Suitable software programs are available from various sources, including sequence comparison tools from the National Center for Biotechnology Information BLAST web site.

[0037]

[0036] Operatively linked: As used herein, the term "operatively linked" refers to a functional relationship between two or more molecules, constructs, transcripts, moieties, or segments (e.g., nucleic acid segments). Being "operatively linked" can include the functional relationship between a regulatory sequence (e.g., promoter) and a corresponding payload sequence (e.g., transgene payload sequence). For example, a promoter sequence is operatively linked to a payload sequence if it promotes, enhances, or modulates the transcription / expression of the payload sequence in an appropriate host cell or other expression system.

[0038]

[0037] Pharmaceutically acceptable'. As used herein, the phrase "pharmaceutically acceptable" refers to compounds, compositions, carriers, excipients, and / or formulations which are suitable for contact (e.g., injection, ingestion, topical) with the tissues of a subject (e.g., human subject) without creating excessive toxicity, irritation, immuno-response, or other complications, according to reasonable medical judgment.

[0039]

[0038] Serotype'. As used herein, the term "serotype" refers to a distinct variant of a viral capsid (e.g., AAV capsid) related to the combination capsid proteins and surface antigens in the capsid variant which allows for specific epidemiologic classification of the virus (e.g., AAV) at a sub-species level.

[0040]

[0039] Subject: As used herein, the terms "subject" or "patient" refer to any organism to which a composition of the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Examples of subjects include, but are not limited to, mammals such as humans, non-human primates, monkeys, mice, rats, and rabbits.

[0041]

[0040] Therapeutic product'. As used herein, the terms "therapeutic product" and "therapeutic agent" refer to any compound, agent, or molecule which, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect on the subject and / or elicits a desired biological and / or pharmacological effect on the subject.

[0042]

[0041] Transfection'. As used herein, the term "transfection" refers to the introduction exogenous molecules (e.g., heterologous nucleotide sequence) into a target cell. Examples of transfection include, but are not limited to, biochemical transfection, physical treatments, lipid-based transfection, vector-based transfection, or combinations thereof.

[0043]

[0042] Vector: As used herein, the term "vector" refers to a molecule, particle, construct, or cell which can transport, transduce, or otherwise carry and transfer a heterologous molecule (e.g., a heterologous nucleotide sequence or a heterologous peptide).

[0044]

[0043] Viral genome : As used herein, the terms "viral genome" or "vector genome" refer to a polynucleotide sequence encapsulated or packaged in a capsid as part of a viral particle (e.g., AAV particle). The terms "AAV viral genome" or "AAV vector genome" refer to the polynucleotide sequence encapsulated in an AAV capsid as part of an AAV particle. AAV vector genomes comprise at least one Inverted Terminal Repeat (ITR) and an expression cassette comprising at least one payload sequence encoding a payload product (e.g., a transgene payload in rAAV vector genome encoding a therapeutic product).

[0045]

[0044] Wild-type: As used herein, the terms "wild-type" or "naturally occurring" refer to forms of construct, compound, sequence, or organism which exists in nature without artificial modification or synthetic formation. ADENO-ASSOCIATED VIRUS (AAV)

[0046]

[0045] Adeno-associated viruses (AAV) are small, non-enveloped viruses of the Parvoviridae family, generally characterized as having a protein capsid surrounding a single stranded DNA viral genome. AAVs are naturally replication defective and can only replicate efficiently in the presence of a helper virus (e.g., adenovirus, herpes virus, baculoviruses, poxviruses). AAV vector genomes can be engineered to produce functional recombinant viral particles which are likewise engineered to target a particular tissue and express a therapeutic payload.

[0047]

[0046] The wild-type AAV (wtAAV) viral genome is a linear, single-stranded DNA (ssDNA) molecule approximately 4,700 nucleotides (nt) in length, capped at both the 5' and the 3' ends with Inverted Terminal Repeats (ITRs). Both the 5' and 3' ITRs in wtAAV include a T-shaped hairpin structure defined by a 145-nt self-complementary sequence which forms the energetically stable double stranded structure of the T-shaped hairpin. The T-shaped ITRs act as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0048]

[0047] The wild-type AAV viral genome also includes two notable open reading frames: a Rep gene encoding four non- structural Rep proteins (Rep78, Rep68, Rep52, Rep40), and a Cap gene encoding three structural capsid proteins (VP1, VP2, VP3). The Rep proteins are important in the replication of the AAV viral genome, and the subsequent packaging of the AAV viral genome into a capsid shell. The capsid proteins assemble to create the AAV capsid protein shell of the AAV particle, typically at a molar ratio of about 1 : 1 : 10 of VP1:VP2:VP3. The capsid is generally composed of 60 subunits arranged into an icosahedral structure. The Cap gene sequence also includes out-of-frame nucleotide sequences which encode other functional proteins, including the assembly activating protein (AAP) and the membrane-associated accessory protein (MAAP).

[0049]

[0048] Recombinant adeno-associated virus (rAAV) is a commonly-used vehicle for therapeutic gene delivery, as the vectors are engineered to be non-integrating and replication deficient. rAAV particles generally include a form of the AAV viral genome which is modified by replacing the Rep gene and Cap gene sequences within the AAV ITR-to-ITR sequence with one or more heterologous payload sequences. During rAAV production, the necessary Rep proteins and Cap proteins for producing the rAAV particles are provided by external replication constructs (e.g., plasmids), such that the Rep and Cap genes are not carried within the rAAV product.

[0049] Upon integration into a target cell, a ITR-flanked expression cassette within rAAV form circular concatemers that persist as episomes in the transduced cell, possibly for several years, making rAAV-based gene therapy a long-lasting therapy that can lead to potential cures for many devastating diseases.

[0050]

[0050] In certain embodiments, the AAV viral genome is a single stranded AAV viral genome (i.e., ssAAV). In certain embodiments, the single-stranded AAV viral genome is converted into double-stranded DNA with a second strand synthesis during transgene expression, which can be considered a limiting step in the onset of transgene expression.

[0051]

[0051] In certain embodiments, the AAV viral genome is a self-complementary AAV viral genome (i.e., scAAV). The scAAV viral genome generally contains DNA strands which anneal together to form double stranded DNA. The double-stranded DNA in scAAVs allows for rapid AAV expression in a target cell by avoiding the need for second strand synthesis before AAV replication (as required in ssAAV). However, the packaging capacity of a self- complementary AAV vector is often reduced.

[0052]

[0052] In certain embodiments, the viral genome is a small viral genome from 2.1 to 3.5 kb in size. In certain embodiments, the viral genome is a medium viral genome from 3.6 to 4.3 kb in size. In certain embodiments, the viral genome is a large viral genome from 4.4 to 5.0 kb in size. In certain embodiments, the viral genome is about 4.7 kb in size (e.g., from 4.6-4.8 kb in size).

[0053]

[0053] Though rAAV viral genomes can range in size, most rAAV treatments have been found to have limited effectiveness with a viral genome outside a range of about 4.5 kb to about 5.0 kb in size, and are generally most effective with a viral genome of about 4.7 kb in size (i.e., around the size of natural AAV). As such, the design process of an rAAV vector genome generally requires the selection of promoters, transgene sequences, and other regulatory elements that fit within the limitations of the packaging size of the expression cassette of 4.7 kb (including the viral ITRs). Exceeding these limits can result in a considerable reduction in viral production yields or cassette truncation and recombination.

[0054]

[0054] In certain embodiments, a viral genome comprises one or more of the following features: Inverted Terminal Repeat (ITR), promoter, enhancer, intron, untranslated region (UTR), a polyadenylation sequence (poly A), linker, spacer, filler, and a payload sequence. The portion of the viral genome between the flanking ITR sequences can be referred to as the AAV expression cassette, which comprises at least one payload sequence encoding a therapeutic product (e.g., protein or polypeptide), at least one element to promote the expression of the payload sequence (e.g., a promoter), and any other elements of a viral genome between the flanking ITR sequences (e.g., polyA sequence). FIG. 2A presents a depiction of an AAV viral genome according to certain embodiments of the disclosure.

[0055]

[0055] In certain embodiments, the present disclosure describes a polynucleotide (e.g., expression cassette) comprising a promoter sequence operatively linked to a payload sequence, wherein the payload sequence encodes a synaptic GTPase-activating protein (SynGAP). In certain embodiments, the polynucleotide (e.g., expression cassette) comprises a poly(A) signal sequence. In certain embodiments, the polynucleotide (e.g., expression cassette) comprises a payload sequence encoding a synaptic GTPase-activating protein (SynGAP), a promoter sequence operatively linked to the payload sequence, and a poly(A) signal sequence. In certain embodiments, the polynucleotide (e.g., expression cassette) comprises: (i) a payload sequence encoding SynGAP, (ii) a promoter sequence operatively linked to the payload sequence, comprising a hSyn or JeT promoter, and (iii) a poly(A) signal sequence comprising SynthPA or SV40PA. In certain embodiments, the polynucleotide (e.g., expression cassette) comprises: (i) a payload sequence encoding SynGAP, (ii) a promoter sequence operatively linked to the payload sequence, comprising a JeT promoter, and (iii) a poly(A) signal sequence comprising SV40PA.

[0056]

[0056] In certain embodiments, the rAAV viral genome comprises a 5' ITR, a polynucleotide comprising an AAV expression cassette, and a 3' ITR. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises a payload sequence encoding a therapeutic payload (e.g., protein or polypeptide), a promoter sequence operatively linked to the payload sequence, and a polyA sequence. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises a payload sequence encoding SynGAP, a promoter sequence comprising hSynapsin (hsyn) or JeT, and a polyA sequence comprising Synth polyA (SynthPA) or SV40 polyA (SV40PA). In certain embodiments, the rAAV viral genome comprises a 5' ITR, an AAV expression cassette, and a 3' ITR, wherein the AAV expression cassette comprises: a payload sequence encoding SynGAP, a promoter sequence comprising hSynapsin (hsyn) or JeT, and a polyA sequence comprising SynthPA or SV40PA.

[0057]

[0057] In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises: (i) payload sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13; (ii) a promoter sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 3 and 4; and (iii) a poly(A) signal sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 15 and 16. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises: (i) payload sequence comprising any one of SEQ ID NOs: 5, 7, 9, 11, and 13; (ii) a promoter sequence comprising any one of SEQ ID NOs: 3 and 4; and (iii) a poly(A) signal sequence comprising any one of SEQ ID NOs: 15 and 16.

[0058]

[0058] In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises a payload sequence which has at least 90% sequence identity to SEQ ID NO: 7, a promoter sequence which has at least 90% sequence identity to SEQ ID NO: 4, and a poly(A) signal sequence which has at least 90% sequence identity to SEQ ID NO: 16. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which comprises a payload sequence comprising SEQ ID NO: 7, a promoter sequence comprising SEQ ID NO: 4, and a poly(A) signal sequence comprising SEQ ID NO: 16.

[0059]

[0059] In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which has at least 85% sequence identity to any one of SEQ ID NOs: 17 to 25. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which has at least 90% sequence identity to any one of SEQ ID NOs: 17 to 25. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which has at least 95% sequence identity to any one of SEQ ID NOs: 17 to 25. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette which has at least 99% sequence identity to any one of SEQ ID NOs: 17 to 25. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising any one of SEQ ID NOs: 17 to 25.

[0060]

[0060] In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 17. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 18. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 19. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 20. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 21. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 22. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 23. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 24. In certain embodiments, the rAAV viral genome comprises an AAV expression cassette comprising SEQ ID NO: 25.

[0061] In certain embodiments, the rAAV viral genome comprises a 5' inverted terminal repeat (ITR), an expression cassette of the present disclosure, and a 3' ITR. In certain embodiments, the rAAV viral genome comprises a sequence which has at least 85% sequence identity to any one of SEQ ID NOs: 26 to 34. In certain embodiments, the rAAV viral genome comprises a sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 26 to 34. In certain embodiments, the rAAV viral genome comprises a sequence which has at least 95% sequence identity to any one of SEQ ID NOs: 26 to 34. In certain embodiments, the rAAV viral genome comprises a sequence which has at least 99% sequence identity to any one of SEQ ID NOs: 26 to 34. In certain embodiments, the rAAV viral genome comprises any one of SEQ ID NOs: 26 to 34.

[0061]

[0062] In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 26. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 27. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 28. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 29. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 30. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 31. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 32. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 33. In certain embodiments, the rAAV viral genome comprises SEQ ID NO: 34.

[0062]

[0063] In certain embodiments, methods for producing, modifying, or engineering rAAV particles include those presented in: US 2003 / 0053990; US 2006 / 0051333; US 6,399,385; and US 2005 / 0053922.

[0063] Inverted Terminal Repeats (ITRs)

[0064]

[0064] In certain embodiments, viral genomes of the present disclosure (e.g., rAAV viral genomes) comprise a nucleic acid sequence with at least one payload region encoding a therapeutic product, and at least one ITR. In certain embodiments, the viral genome comprises two ITR sequences. In certain embodiments, the viral genome comprises a 5' ITR sequence at the 5' end of the viral genome. In certain embodiments, the viral genome comprises a 3' ITR sequence at the 3' end of the viral genome. In certain embodiments, the viral genome comprises a 5' ITR sequence at the 5' end of the viral genome, and a 3' ITR sequence at the 3' end of the viral genome.

[0065]

[0065] In certain embodiments, the ITRs may be derived from the same serotype as the capsid, or a derivative capsid thereof. In certain embodiments, the ITRs may be from a different serotype than the capsid. In certain embodiments, the 5' ITR is the same serotype as the 3' ITR. In certain embodiments, the 5' ITR is a different serotype as the 3' ITR.

[0066] In certain embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 1.

[0066]

[0067] In certain embodiments, the 3' ITR comprises the sequence of SEQ ID NO: 2. SynGAP Payloads

[0067]

[0068] In certain embodiments, rAAV viral genomes of the present disclosure comprise a polynucleotide payload region which comprises at least one payload sequence encoding a polynucleotide product, polypeptide product, or protein product. In certain embodiments, the payload sequence encodes a synaptic GTPase-activating protein (SynGAP).

[0068]

[0069] In certain embodiments, the payload sequence has at least 85% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence has at least 90% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence has at least 85% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence has at least 95% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence has at least 99% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence comprises any one of SEQ ID NOs: 5, 7, 9, 11, and 13. In certain embodiments, the payload sequence comprises SEQ ID NO: 5. In certain embodiments, the payload sequence comprises SEQ ID NO: 7. In certain embodiments, the payload sequence comprises SEQ ID NO: 9. In certain embodiments, the payload sequence comprises SEQ ID NO: 11. In certain embodiments, the payload sequence comprises SEQ ID NO: 13.

[0069]

[0070] In certain embodiments, the payload sequence encodes a SynGAP which has at least 85% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP which has at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP which has at least 95% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP which has at least 99% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP comprises any one of SEQ ID NOs: 6, 8, 10, 12, and 14. In certain embodiments, the payload sequence encodes a SynGAP of SEQ ID NO: 6. In certain embodiments, the payload sequence encodes a SynGAP of SEQ ID NO: 8. In certain embodiments, the payload sequence encodes a SynGAP of SEQ ID NO: 10. In certain embodiments, the payload sequence encodes a SynGAP of SEQ ID NO: 12. In certain embodiments, the payload sequence encodes a SynGAP of SEQ ID NO: 14.

[0071] In certain embodiments, the payload region comprises at least one additional sequence component, including, but not limited to, a promoter region, an intron region, a linker region, or a coding region. In certain embodiments, the payload region comprises a combination of coding and non-coding nucleic acid sequences.

[0070] Regulatory Elements

[0071]

[0072] In certain embodiments, rAAV viral genomes comprise at least one element to promote and / or enhance the expression and / or target specificity of the payload (i.e., payload promoter). Non-limiting examples of elements that promote and / or enhance the payload specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), and enhancers (e.g., CMV enhancers).

[0072]

[0073] In certain embodiments, rAAV viral genomes comprise at least one promoter that is 5' to a payload sequence of the viral genome. In certain embodiments, rAAV viral genomes comprise at least one promoter that is operatively linked to a payload sequence of the viral genome (i.e., payload promoter), and drives expression of the payload encoded in the payload sequence. In certain embodiments, the rAAV viral genome comprises a target-cell-specific promoter, including but not limited to, a promoter that is species specific, tissue-specific, or cell-specific. In certain embodiments, the promoter is a cell cycle-specific promoter.

[0073]

[0074] In certain embodiments, the payload promoter is a naturally occurring promoter. In certain embodiments, the payload promoter is a non-natural, truncated, and / or engineered promoter.

[0074]

[0075] Examples of payload promoters for use in the present disclosure include, but are not limited to: Human synapsin 1 (hSynapsin or hSyn), JeT, cytomegalovirus (CMV) immediate- early; chicken P-actin (CBA); CAG promoter (combination of CMV enhancer, CBA promoter and rabbit beta-Globin splice acceptor site); P glucuronidase (GUSB); GUSB minimal promoter (hGBp); Glial fibrillary acidic protein (GFAP); human elongation factor la-subunit (EFla); human a- 1 -antitrypsin (hAAT); neurofilament heavy (NFH); neurofilament light (NFL); phosphoglycerate kinase 1 (PGK); RNA pol III promoter (e.g., Hl or U6); Sodium Voltage-Gated Channel Alpha Subunit 8 (SCN8A); thyroxine binding globulin (TBG); ubiquitin C (UBC); interleukin-2 (IL-2) minimal promoter; and minimal TATA-Box promoter.

[0075]

[0076] In certain embodiments, the payload promoter comprises a Human synapsin 1 (hSynapsin or hSyn) promoter. In certain embodiments, the payload promoter sequence has at least 90% sequence identity to SEQ ID NO: 3. In certain embodiments, the payload promoter sequence has at least 95% sequence identity to SEQ ID NO: 3. In certain embodiments, the payload promoter sequence has at least 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the payload promoter sequence comprises SEQ ID NO: 3.

[0076]

[0077] In certain embodiments, the payload promoter comprises a JeT promoter. In certain embodiments, the payload promoter sequence has at least 90% sequence identity to SEQ ID NO: 4. In certain embodiments, the payload promoter sequence has at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the payload promoter sequence has at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the payload promoter sequence comprises SEQ ID NO: 4.

[0077]

[0078] In certain embodiments, the payload promoter is less than 1 kb in length. In certain embodiments, the promoter is from 100-800 nucleotides in length. In certain embodiments, the promoter is from 100-500 nucleotides in length. In certain embodiments, the promoter is from 100-200 nucleotides in length.

[0078]

[0079] In certain embodiments, rAAV viral genomes comprise at least one enhancer that is 5' of the promoter. In certain embodiments, the enhancer comprises a CMV enhancer sequence, a GAPDH enhancer sequence, a P-actin enhancer sequence, or an EFl -a enhancer sequence.

[0079] Introns and Untranslated Regions (UTRs)

[0080]

[0080] In certain embodiments, the rAAV viral genome comprises at least one intron element. In certain embodiments, the rAAV viral genome comprises at least one intron element to enhance the transgene target specificity and expression. Examples of intron elements for use in the present disclosure include, but are not limited to, minute virus of mice (MVM) small-intron region, truncated F.IX intron, P-globin SD / IGHC SA, AV SD / IG SA, and SV40 SD / SA. In certain embodiments, the intron or intron portion may be 100-500 nucleotides in length.

[0081]

[0081] In certain embodiments, the rAAV viral genome comprises one or more untranslated regions (UTRs) of a transgene sequence which are transcribed but not translated. In certain embodiments, the rAAV viral genome comprises a 5' UTR which starts at the transcription start site of the transgene and ends at the start codon. In certain embodiments, the rAAV viral genome comprises a 3' UTR which starts immediately following the stop codon of the transgene and continues until the termination signal for transcription.

[0082]

[0082] In certain embodiments, gene elements from target tissues may be engineered into UTRs to enhance the stability and production of the rAAV viral genome in the target tissue. As a non-limiting example, a 5' UTR from mRNA normally expressed in a target cell may be incorporated into a rAAV viral genome to enhance expression of the payload sequence in that target call. In certain embodiments, a 5' UTR includes a Kozak sequence.

[0083]

[0083] In certain embodiments, a 3' UTR includes one or more stretches of adenosines and uridines embedded therein (known in the art as "AU rich elements" or AREs). Examples of 3' UTR AREs for use in the present disclosure include, but are not limited to: c-Myc, MyoD, GM-CSF, TNF-a, c-Jun, and Myogenin.

[0084] Polyadenylation (Poly A) Sequence

[0085]

[0084] In certain embodiments, rAAV viral genomes comprise at least one polyadenylation sequence (i.e., polyA sequence). In certain embodiments, the gene therapy vector genome comprises at least one polyA sequence between the 3' end of the payload coding sequence and the 5' end of the 3' ITR. In certain embodiments, the polyA sequence is from 1-500 nucleotides in length. In certain embodiments, the polyA sequence is from 1-200 nucleotides in length. In certain embodiments, the polyA sequence is from 1-100 nucleotides in length. In certain embodiments, the polyA sequence is a synthetic polyA sequence.

[0086]

[0085] In certain embodiments, the polyA sequence comprises a Synth polyA (SynthPA) sequence. In certain embodiments, the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 15. In certain embodiments, the poly(A) signal sequence has at least 95% sequence identity to SEQ ID NO: 15. In certain embodiments, the poly(A) signal sequence comprises SEQ ID NO: 15.

[0087]

[0086] In certain embodiments, the polyA sequence comprises a SV40 polyA (SV40PA) sequence. In certain embodiments, the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16. In certain embodiments, the poly(A) signal sequence has at least 95% sequence identity to SEQ ID NO: 16. In certain embodiments, the poly(A) signal sequence comprises SEQ ID NO: 16.

[0088] Linkers, Spacers, and Fillers

[0089]

[0087] In certain embodiments, rAAV viral genomes comprise one or more spacer or linker regions. In certain embodiments, the rAAV viral genome comprises one or more spacer or linker regions to separate two or more coding or non-coding regions.

[0090]

[0088] In certain embodiments, the rAAV viral genome comprises one or more linker sequences. In certain embodiments, the linker sequence encodes a cleavable linker, such as a 2 A peptide linker (e.g., T2A linker or P2A linker), or an internal ribosomal entry site (IRES) linker. In certain embodiments, the linker is non-cleavable (e.g., a glycine-rich sequence or a serine-rich sequence).

[0089] In certain embodiments, the rAAV viral genome comprises one or more filler sequences (i.e., stuffer sequences). The filler sequences can be engineered and incorporated to provide the rAAV viral genome with a favorable length (e.g., a length of about 4.7 kb). Examples of filler sequences include, but are not limited to, albumin filler sequences and alpha-1 antitrypsin filler sequences.

[0091] AAV Serotypes

[0092]

[0090] In certain embodiments, AAV particles of the present disclosure have a specific AAV serotype. In certain embodiments, the AAV serotype has tissue targeting capabilities (i.e., tissue tropisms). In certain embodiments, the AAV serotype exhibits increased transduction and / or tropism in one or more target cell types. Examples of target cells for AAV tropism include: adrenals, appendix, blood vessels, bones, bronchi, cardiovascular, CNS (e.g., brain, spinal cord), diaphragm, digestive, endocrine, esophagus, eyes (ocular), gallbladder, gonad, hair, heart (cardiac), hypothalamus, integumentary, kidney, large intestine, larynx, liver, lungs, lymph nodes, lymph vessels, mouth, muscles, nasal, nerves, nails, oil glands, pancreas, pharynx, pineal gland, pituitary gland, prostate, reproductive, respiratory, sense receptors, skin, skeletal, small intestine, spleen, stomach, sweat glands, thymus, thyroid / parathyroid, tonsils, trachea, urinary, urethra, or combinations thereof. In certain embodiments, the AAV serotype exhibits increased transduction and / or tropism CNS cells (e.g., brain, spinal cord).

[0093]

[0091] In certain embodiments, the AAV serotype may utilize or be based on a serotype or include a peptide selected from: AAVPHP.B, AAVPHP.A, AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2, AAVPHP.B3, PHP.B-DGT, AAVPHP.B- EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B- SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B- STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42- 11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4-8 / r 11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5- 3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu. lO, AAV16.12 / hu.l 1, AAV29.3 / bb. l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu. l7, AAV33.4 / hu.l5, AAV33.8 / hu. l6, AAV52 / hu.l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l l, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.l 8, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh. lO, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LKO3, AAV-LK04, AAV-LKO5, AAV-LK06, AAV-LK07, AAV-LKO8, AAV-LK09, AAV-LK1O, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC 12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.5O, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Civ 1-7, AAV Civ 1-8, AAV Civ 1-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAVrh20, AAVrh32 / 33, AAVrh39, AAVrh46, AAVrh73, AAVrh74, AAVhu.26, AAV-AM, variants thereof, or derivatives thereof.

[0094] AAV Production

[0095]

[0092] In certain embodiments, the present disclosure describes AAV production processes and methods for producing rAAV particles and viral vectors. In certain embodiments, the rAAV particles are be produced using viral production cells, including mammalian cells (e.g., HEK293 mammalian cells).

[0096]

[0093] In certain embodiments, AAV particles are be produced by (a) generating a pool of viral production cells; (b) contacting the viral production cells with a viral production construct encoding necessary AAV capsid (Cap) proteins (e.g., Rep / Cap plasmid), a viral production construct encoding necessary AAV replication (Rep) proteins (e.g., Rep / Cap plasmid), an optional viral production construct encoding AAV production helper proteins (e.g., helper plasmid), and a payload construct which comprises a payload sequence encoding a payload molecule (e.g., payload / transgene plasmid)); (c) culturing the viral production cells under conditions to produce the AAV particles; and (d) isolating (e.g., through lysis of the production cells), clarifying (e.g., using filtration), purifying (e.g., using chromatography), and / or formulating the resulting AAV particles. In certain embodiments, the viral production cell provides the cellular machinery necessary for expression of the proteins and other biomaterials necessary for producing the AAV particles.

[0097]

[0094] In certain embodiments, production, clarification, purification, and / or formulation of rAAVs of the present disclosure can be carried out using the methods or systems presented in US 20020019050, US 20020127582, US 20020136710, US 20020177215, US 20020182723, US 20030032613, US 20030040101, US 20030073232, US 20030138772, US 20040121444, US 20040152183, US 20050153420, US 20130202559, US 2010012940, US 5756283, US 6001650, or US 6258595.

[0098] SynGAP

[0099] Overview

[0100]

[0095] Synaptic GTPase-activating protein (SynGAP) is a neuronal Ras GTPase-activating protein (RasGAP) encoded by the SYNGAP 1 gene located on the 6p21.4 chromosome. SynGAP functions as a postsynaptic density (PSD) scaffold protein and is a downstream component of N-methyl-D-aspartate (NMD A) receptors-associated signaling complexes. SynGAP is electively expressed in the brain and highly enriched at excitatory synapses, where it negatively regulates Ras and Rap activity and their downstream signaling pathways, including signal transduction processes that affect cellular growth, differentiation, survival, and migration.

[0101]

[0096] SynGAP also negatively regulates the a-amino-3-hydroxy-5-methyl-4- isoxazol epropionic acid receptor (AMP A) receptors trafficking to the postsynaptic membrane, having a role in synaptic plasticity and homeostasis, as well as in dendritic spines morphology due to SynGAP protein-protein interactions.

[0102]

[0097] A schematic representation of one example of a signaling pathway regulated by SynGAP is shown in FIG. 1 A and FIG. IB. In FIG. 1 A, SynGAP protein is shown being expressed at the postsynaptic membrane of excitatory synapses. The SynGAP protein inhibits the activation of various small GTPases (Ras and Rap). In the dendritic spines, SynGAP suppresses the Ras and Rap signaling and AMPA receptors exocytosis and trafficking to the membrane. Mutations in the SynGAP 1 gene can lead to decreased levels of SynGAP protein and this elevates basal RasZErk signaling (FIG. IB). Consequently, there is an increase in AMPA receptor incorporation at the membrane, which leads to increased excitatory transmission.

[0103]

[0098] There are four known SynGAP C-termini isoforms: Alphal (SEQ ID NO: 8), Alpha2 (SEQ ID NO: 10), Beta (SEQ ID NO: 12), and Gamma (SEQ ID NO: 14). The SynGAP isoforms arise from alternative splicing of the final three exons of the Syngapl gene. Baseline synaptic phenotypes related to Syngapl gene expression are generally dominated by Alphal (and possibly Alpha2) negative regulation and suppression of excess / excitatory synapse function. See Kilinc et al., "Endogenous Syngapl alpha splice forms promote cognitive function and seizure protection", (Apr 2022) Elife, 11 :e75707. In certain embodiments, the payload sequence encodes a SynGAP. Alphal isoform.

[0104] Treatment of diseases by restoring SynGAP

[0105]

[0099] The SYNGAP 1 gene is considered a high-risk locus for neurodevelopmental disorders (NDDs). Rare variants of SYNGAP 1 have been found in subjects with intellectual disability (ID), severe epilepsy, developmental and epileptic encephalopathies (DEEs), schizophrenia, and autism spectrum disorders (ASDs). In particular, SYNGAP 1 is considered one of the most common single-gene deficiencies related intellectual disability (ID), with an estimated prevalence of 6 out of 100,000 individuals (1 / 16 000). At least 1400 SynGAPl patients were identified in recent census studies.

[0106]

[0100] Around 85% of known patients with pathogenic SYNGAP1 variants have rare, loss- of-function variants that reduce SYNGAP protein expression and function, leading to a neurodevelopmental disorder characterized by substantial developmental delay, cognitive impairment, social-communication disorders, and early-onset seizures, including eyelid myoclonia absence seizures, myoclonic seizures, atypical and drop attacks. Severe autosomal-dominant de novo mutations in SYNGAP 1 may result in haploinsufficiency of the SynGAP protein, usually due to loss of function mutations and protein truncation. SynGAPl haploinsufficiency leads to Mental Retardation-Type 5 (MRD5), reported in up to 2% of non- syndromic ID cases, being the most recognized cause of ID with epilepsy. SynGAPl mutations cause cognitive impairments, social communication disorder, and early-onset seizures; most SYNGAP 1 haploinsufficiency patients have moderate to severe ID, and several have ASD. ID-causing SYNGAP 1 mutations were identified along the entire length of the SYNGAP1 gene, mainly in exons 3-17.

[0107]

[0101] These clinical findings indicate that SYNGAP 1 regulates critical neurodevelopmental processes necessary for sculpting normal brain development. SYNGAP 1 thus plays a role in at least four main phenotypical domains: 1) cognitive function; 2) regulation of excitatory / inhibitory balance, and 3) brain morphology, supported by impaired cognitive function presented by patients; altered neural circuit excitability and epilepsy displayed by patients; and the mild microcephaly or a reduction in head / brain size reported in some MRD5 patients, respectively.

[0108]

[0102] Currently, there is no cure or specific treatment for the underlying conditions related to SYNGAP 1 mutation. The available treatments are symptomatic treatments, including antiseizure medication and behavioral therapies.

[0109]

[0103] In certain embodiments, the present disclosure describes method of increasing the expression of synaptic GTPase-activating protein (SynGAP) in a subject, comprising delivering an effective amount of a SYNGAP 1 AAV vector genome or AAV particle of the present disclosure to the subject. In certain embodiments, delivery of the SYNGAP 1 AAV vector genome or AAV particle to the cell results in an increase in SynGAP production by the cell.

[0110]

[0104] In certain embodiments, the present disclosure describes methods the treating SynGAPl -related diseases in a subject in need thereof, comprising delivering a therapeutically effective amount of a SYNGAP 1 AAV vector genome or AAV particle of the present disclosure to the subject. In certain embodiments, the present disclosure describes methods the treating a neurodevel opmental disorder (NDD) in a subject in need thereof. In certain embodiments, the present disclosure describes methods the treating a developmental and epileptic encephalopathy (DEE) in a subject in need thereof. In certain embodiments, the DEE is Doose syndrome, Jeavons syndrome, Lennox-Gastaut syndrome, or an early infantile DEE. In certain embodiments, the epileptic encephalopathy is caused by, or associated with, one or more mutations in a SYNGAP1 gene.

[0111]

[0105] In certain embodiments, administration of the SYNGAP1 AAV vector genome or AAV particle to the subject increases the level of SynGAP produced by the subject. In certain embodiments, administration of the SYNGAP 1 AAV vector genome or AAV particle to the subject restores the level of SynGAP in the subject to a level found in a healthy subject (i.e., an individual that does not have an SYNGAP 1 insufficiency or mutation). In certain embodiments, administration of the SYNGAP 1 AAV vector genome or AAV particle to the subject increases the amount of SynGAP in a tissue by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 100%.

[0112]

[0106] In certain embodiments, administration the AAV vector genome or AAV particle to the subject reduces or eliminates one or more symptom of the DEE being treated. In certain embodiments, administration of the AAV vector genome or AAV particle to the subject restores one or more cognitive abilities in the subject. In certain embodiments, administration of the AAV vector genome or AAV particle to the subject reduces the number, frequency, and / or severity of seizures (e.g., myoclonic-like seizures) in the subject. In certain embodiments, administration of the AAV vector genome or AAV particle to the subject improves and / or restores one or more motor functions in the subject. In certain embodiments, administration of the AAV vector genome or AAV particle to the subject improves and / or restores one or more cognitive functions in the subject.

[0113]

[0107] The AAV particles of the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to, transdermal, intracerebral, intracerebroventricular (ICV), intracranial, intravenous (IV), intraparenchymal CNS (i.e., into the substance of the CNS), intraci sternal (ICM, within the cisterna magna), intraspinal (i.e., within the vertebral column), and intrathecal (IT, within the cerebrospinal fluid). In certain embodiments, AAV particles are administered by intracistemal (ICM) administration.

[0108] In certain embodiments, the AAV particles are administered so as to be delivered to a target cell or tissue. In certain embodiments, the AAV particles are administered so as to be delivered to a CNS cell.

[0114]

[0109] In certain embodiments, administration of AAV particles in accordance with the present disclosure to cells of the central nervous system (e.g., CNS parenchyma) may comprise a total concentration per subject between about 1 x 106vector genomes (VG) and about 5 x 1014VG. In some embodiments, administration of AAV particles comprises a total concentration per subject of about 1 x 106VG, about 1.5 x 106VG, about 2 x 106VG, about

[0115] 2.5 x 106VG, about 3 x 106VG, about 3.5 x 106VG, about 4 x 106VG, about 4.5 x 106VG, about 5 x 106VG, about 5.5 x 106VG, about 6 x 106VG, about 6.5 x 106VG, about 7 x 106VG, about 7.5 x 106VG, about 8 x 106VG, about 8.5 x 106VG, about 9 x 106VG, about 9.5 x 106VG, about 1 x 107VG, about 1.5 x 107VG, about 2 x 107VG, about 2.5 x 107VG, about 3 x 107VG, about 3.5 x 107VG, about 4 x 107VG, about 4.5 x 107VG, about 5 x 107VG, about 5.5 x 107VG, about 6 x 107VG, about 6.5 x 107VG, about 7 x 107VG, about 7.5 x 107VG, about 8 x 107VG, about 8.5 x 107VG, about 9 x 107VG, about 9.5 x 107VG, about 1 x 108VG, about 1.5 x 108VG, about 2 x 108VG, about 2.5 x 108VG, about 3 x 108VG, about 3.5 x 108VG, about 4 x 108VG, about 4.5 x 108VG, about 5 x 108VG, about 5.5 x 108VG, about 6 x 108VG, about 6.5 x 108VG, about 7 x 108VG, about 7.5 x 108VG, about 8 x 108VG, about 8.5 x 108VG, about 9 x 108VG, about 9.5 x 108VG, about 1 x 109VG, about 1.5 x 109VG, about 2 x 109VG, about 2.5 x 109VG, about 3 x 109VG, about 3.5 x 109VG, about 4 x 109VG, about 4.5 x 109VG, about 5 x 109VG, about 5.5 x 109VG, about 6 x 109VG, about 6.5 x 109VG, about 7 x 109VG, about 7.5 x 109VG, about 8 x 109VG, about 8.5 x 109VG, about 9 x 109VG, about 9.5 x 109VG, about 1 x IO10VG, about 1.5 x IO10VG, about 2 x IO10VG, about 2.5 x IO10VG, about 3 x IO10VG, about 3.5 x IO10VG, about 4 x IO10VG, about 4.5 x IO10VG, about 5 x IO10VG, about 5.5 x IO10VG, about 6 x

[0116] 1010VG, about 6.5 x IO10VG, about 7 x IO10VG, about 7.5 x IO10VG, about 8 x IO10VG, about 8.5 x IO10VG, about 9 x IO10VG, about 9.5 x IO10VG, about 1 x 1011VG, about 1.5 x

[0117] 1011VG, about 2 x 1011VG, about 2.5 x 1011VG, about 3 x 1011VG, about 3.5 x 1011VG, about 4 x 1011VG, about 4.5 x 1011VG, about 5 x 1011VG, about 5.5 x 1011VG, about 6 x 1011VG, about 6.5 x 1011VG, about 7 x 1011VG, about 7.5 x 1011VG, about 8 x 1011VG, about 8.5 x 1011VG, about 9 x 1011VG, or about 9.5 x 1011VG, about 1 x 1012VG, about

[0118] 1.5 x 1012VG, about 2 x 1012VG, about 2.5 x 1012VG, about 3 x 1012VG, about 3.5 x 1012VG, about 4 x 1012VG, about 4.5 x 1012VG, about 5 x 1012VG, about 5.5 x 1012VG, about 6 x 1012VG, about 6.5 x 1012VG, about 7 x 1012VG, about 7.5 x 1012VG, about 8 x 1012 VG, about 8.5 x 1012VG, about 9 x 1012VG, about 9.5 x 1012VG, about 1 x 10° VG, about 1.5 x 10° VG, about 2 x 10° VG, about 2.5 x 1013VG, about 3 x 1013VG, about 3.5 x 1013VG, about 4 x 1013VG, about 4.5 x 1013VG, about 5 x 1013VG, about 5.5 x 1013VG, about 6 x 1013VG, about 6.5 x 1013VG, about 7 x 1013VG, about 7.5 x 1013VG, about 8 x 1013VG, about 8.5 x 1013VG, about 9 x 1013VG, about 9.5 x 1013VG, about 1 x 1014VG, about 1.5 x 1014VG, about 2 x 1014VG, about 2.5 x 1014VG, about 3 x 1014VG, about 3.5 x 1014VG, about 4 x 1014VG, about 4.5 x 1014VG, or about 5 x 1014VG.

[0119] EMBODIMENTS

[0120] [HO] Certain embodiments of the present disclosure may also be defined according to any one of the following exemplary, non-limiting embodiments:

[0121] [Hl] Embodiment 1. A polynucleotide comprising a promoter sequence operatively linked to a payload sequence, wherein the payload sequence encodes a synaptic GTPase-activating protein (SynGAP).

[0122]

[0112] Embodiment 2. The polynucleotide of Embodiment 1, wherein the payload sequence has at least 85% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

[0123]

[0113] Embodiment 3. The polynucleotide of Embodiment 1, wherein the payload sequence has at least 90% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

[0124]

[0114] Embodiment 4. The polynucleotide of Embodiment 1, wherein the payload sequence has at least 95% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

[0125]

[0115] Embodiment 5. The polynucleotide of Embodiment 1, wherein the payload sequence comprises any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

[0126]

[0116] Embodiment 6. The polynucleotide of Embodiment 1, wherein the payload sequence comprises SEQ ID NO: 7.

[0127]

[0117] Embodiment 7. The polynucleotide of any one of Embodiments 1-6, wherein the payload sequence encodes a SynGAP which has at least 85% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

[0128]

[0118] Embodiment 8. The polynucleotide of Embodiment 7, wherein the payload sequence encodes a SynGAP which has at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

[0129]

[0119] Embodiment 9. The polynucleotide of Embodiment 7, wherein the payload sequence encodes a SynGAP which has at least 95% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

[0120] Embodiment 10. The polynucleotide of Embodiment 7, wherein the payload sequence encodes a SynGAP comprising any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

[0130]

[0121] Embodiment 11. The polynucleotide of Embodiment 7, wherein the payload sequence encodes a SynGAP comprising SEQ ID NO: 8.

[0131]

[0122] Embodiment 12. The polynucleotide of any one of Embodiments 1-11, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 3.

[0132]

[0123] Embodiment 13. The polynucleotide of any one of Embodiments 1-11, wherein the promoter sequence comprises SEQ ID NO: 3.

[0133]

[0124] Embodiment 14. The polynucleotide of any one of Embodiments 1-11, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 4.

[0134]

[0125] Embodiment 15. The polynucleotide of any one of Embodiments 1-11, wherein the promoter sequence comprises SEQ ID NO: 4.

[0135]

[0126] Embodiment 16. The polynucleotide of any one of Embodiments 1-15, wherein the polynucleotide comprises a poly(A) signal sequence.

[0136]

[0127] Embodiment 17. The polynucleotide of Embodiment 16, wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 15.

[0137]

[0128] Embodiment 18. The polynucleotide of Embodiment 16, wherein the poly(A) signal sequence comprises SEQ ID NO: 15.

[0138]

[0129] Embodiment 19. The polynucleotide of Embodiment 16, wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16.

[0139]

[0130] Embodiment 20. The polynucleotide of Embodiment 16, wherein the poly(A) signal sequence comprises SEQ ID NO: 16.

[0140]

[0131] Embodiment 21. The polynucleotide of any one of Embodiments 1-20, wherein the payload sequence has at least 90% sequence identity to SEQ ID NO: 7, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 4, and wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16.

[0141]

[0132] Embodiment 22. The polynucleotide of any one of Embodiments 1-20, wherein the payload sequence comprises SEQ ID NO: 7, wherein the promoter sequence comprises SEQ ID NO: 4, and wherein the poly(A) signal sequence comprises SEQ ID NO: 16.

[0142]

[0133] Embodiment 23. The polynucleotide of any one of Embodiments 1-22, wherein the polynucleotide has at least 85% sequence identity to any one of SEQ ID NOs: 17 to 25.

[0143]

[0134] Embodiment 24. The polynucleotide of any one of Embodiments 1-22, wherein the polynucleotide has at least 90% sequence identity to any one of SEQ ID NOs: 17 to 25.

[0135] Embodiment 25. The polynucleotide of any one of Embodiments 1-22, wherein the polynucleotide has at least 95% sequence identity to any one of SEQ ID NOs: 17 to 25.

[0144]

[0136] Embodiment 26. The polynucleotide of any one of Embodiments 1-22, wherein the polynucleotide comprises any one of SEQ ID NOs: 17 to 25.

[0145]

[0137] Embodiment 27. The polynucleotide of any one of Embodiments 1-22, wherein the polynucleotide comprises SEQ ID NO: 21.

[0146]

[0138] Embodiment 28. An adeno-associated viral (AAV) vector genome comprising: a 5' inverted terminal repeat (ITR), an expression cassette, and a 3' ITR; wherein the expression cassette comprises the polynucleotide of any one of Embodiments 1-27.

[0147]

[0139] Embodiment 29. The AAV vector genome of Embodiment 28, wherein the 5' ITR is an AAV2 ITR.

[0148]

[0140] Embodiment 30. The AAV vector genome of Embodiment 28 or Embodiment 29, wherein the 3' ITR is an AAV2 ITR.

[0149]

[0141] Embodiment 31. The AAV vector genome of any one of Embodiments 1-30, wherein the AAV vector genome comprises a sequence which has at least 85% sequence identity to any one of SEQ ID NOs: 26 to 34.

[0150]

[0142] Embodiment 32. The AAV vector genome of any one of Embodiments 1-30, wherein the AAV vector genome comprises a sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 26 to 34.

[0151]

[0143] Embodiment 33. The AAV vector genome of any one of Embodiments 1-30, wherein the AAV vector genome comprises a sequence which has at least 95% sequence identity to any one of SEQ ID NOs: 26 to 34.

[0152]

[0144] Embodiment 34. The AAV vector genome of any one of Embodiments 1-30, wherein the AAV vector genome comprises any one of SEQ ID NOs: 26 to 34.

[0153]

[0145] Embodiment 35. The AAV vector genome of Embodiment 28, wherein the AAV vector genome comprises SEQ ID NO: 30.

[0154]

[0146] Embodiment 36. An AAV particle comprising the AAV vector genome of any of Embodiments 28-35 and a capsid.

[0155]

[0147] Embodiment 37. An AAV particle of Embodiment 36, wherein the capsid has tropism for tissues or cells of the central nervous system (CNS).

[0156]

[0148] Embodiment 38. A pharmaceutical composition comprising an AAV particle of Embodiment 36 or Embodiment 37, and a pharmaceutically acceptable excipient.

[0149] Embodiment 39. A method of increasing the expression of synaptic GTPase- activating protein (SynGAP) in a subject, comprising delivering an effective amount of an AAV particle of Embodiment 38 to the subject.

[0157]

[0150] Embodiment 40. A method of treating a disease related to a SYNGAPl gene mutation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of Embodiment 38.

[0158]

[0151] Embodiment 41. The method of Embodiment 40, wherein the disease related to a SYNGAPl gene mutation is a neurodevelopmental disorder (NDD).

[0159]

[0152] Embodiment 42. The method of Embodiment 40, wherein the disease related to a SYNGAPl gene mutation is a developmental and epileptic encephalopathy (DEE).

[0160]

[0153] Embodiment 43. The method of any one of Embodiments 40-42, wherein administering the composition to the subject comprises intracisterna magna (ICM), intra- cerebrospinal fluid (ICSF), intracerebroventricular (ICV), or intraparenchymal administration.

[0161]

[0154] Embodiment 44. The method of any one of Embodiments 40-43, wherein administering the composition to the subject reduces or eliminates one or more symptoms of the disease.

[0162]

[0155] Embodiment 45. A polynucleotide of any one of Embodiments 1-27, an AAV vector genome of any one of Embodiments 28-35, an AAV particle of Embodiment 36 or Embodiment 37, or a pharmaceutical composition of Embodiment 38, for use in the prevention and / or treatment of a disease related to a SYNGAPl gene mutation in a subject.

[0163]

[0156] Embodiment 46. The polynucleotide, AAV vector genome, AAV particle, or pharmaceutical composition for use according to Embodiment 45, wherein the disease related to a SYNGAP 1 gene mutation is a neurodevelopmental disorder (NDD).

[0164]

[0157] Embodiment 47. The polynucleotide, AAV vector genome, AAV particle, or pharmaceutical composition for use according to Embodiment 45, wherein the disease related to a SYNGAPl gene mutation is a developmental and epileptic encephalopathy (DEE).

[0165]

[0158] Embodiment 48. Use of a polynucleotide of any one of Embodiments 1-27, an AAV vector genome of any one of Embodiments 28-35, an AAV particle of Embodiment 36 or Embodiment 37, or a pharmaceutical composition of Embodiment 38 for the treatment of a disease related to a SYNGAPl gene mutation in a subject.

[0166]

[0159] Embodiment 49. Use of a polynucleotide of any one of Embodiments 1-27, an AAV vector genome of any one of Embodiments 28-35, an AAV particle of Embodiment 36 or Embodiment 37, or a pharmaceutical composition of Embodiment 38 for the manufacture of a medicament for the treatment of a disease related to a SYNGAP 1 gene mutation in a subject.

[0167]

[0160] Embodiment 50. The use according to Embodiment 48 or Embodiment 49, wherein the disease related to a SYNGAP 1 gene mutation is a neurodevelopmental disorder (NDD).

[0168]

[0161] Embodiment 51. The use according to Embodiment 48 or Embodiment 49, wherein the disease related to a SYNGAP1 gene mutation is a developmental and epileptic encephalopathy (DEE).

[0169] EXAMPLES

[0170] Example 1: rAAV Plasmid Production and Testing a) Overview

[0171]

[0162] Mouse and human SynGAPl cDNA were cloned into test plasmids, driven by different mammalian promoters and polyA. Two different promoters — hSynapsin (hsyn) and JeT — and two polyA sequences — SynthPA and SV40PA — were used for the expression of cDNA of mouse and human SynGAPl. A total of six different SynGAPl AAV vector genome expressing plasmids were designed, three to express mouse SynGAP protein (FIG. 2B) and three to express human SynGAP protein (FIG. 2C). The AAV vector genome containing the promoter hSyn driving the expression of human or mouse SynGAPl and SynthPA were synthesized and used as the backbone plasmid to swap the JeT promoter and / or SV40PA. The AAV vector genome containing the hSyn promoter and SynthPA were synthetized containing the cDNA for human and mouse SynGAPl. These plasmids were used as backbone to swap the JeT promotor and / or SV40PA.

[0172]

[0163] The AAVs were designed to fit the large SynGAPl transgene with a shorter, functional promoter and a polyA region to fit in the 4.7kb AAV cassette.

[0173]

[0164] As a control, the SynGAPl cDNA was replaced with GFP. HEK 293T cells, adapted to serum-free (SF) media BalanCD (FUJIFILM), were transfected with the nine plasmids produced. GFP expression was evaluated by flow cytometry (FC) and SynGAPl expression was evaluated by dd(mRNA) and Jess Protein Simple (quantitative western for protein). b) Production and amplification of AAV plasmids

[0174]

[0165] Plasmids containing the cDNA sequences for human and mouse SynGAPl, human synapsin (hSyn) promoter, JeT promoter, SynthPA, and SV40PA were synthesized and cloned into AAV production plasmids using available restriction sites, Nhel and d.scl or Mfel and Hpa , respectively.

[0166] A GFP-Zeo fusion (Invivogen, USA) was amplified by PCR and cloned into constructed backbones using Asci and Mfel sites, in place of human SynGAPl. All cloning procedures were performed using In-Fusion seamless cloning (Takara Bio). The results was nine test plasmids as follows: (1) hsyn-mSynGAPlalphal-SynthPA; (2) JeT- mSynGAPlalphal-SV40PA; (3) JeT-mSynGAPlalphal-SynthPA; (4) hsyn- hSynGAPlalphal-SynthPA; (5) JeT-hSynGAPlalphal-SV40PA; (6) JeT-hSynGAPlalphal- SynthPA; (7) JeT-GFP-SV40PA; (8) JeT-GFP-SV40PA; (9) JeT-GFP-SV40PA.

[0175]

[0167] The nine resulting test plasmids were transformed into chemically competent E. coli Stbl3 (Thermo Fischer Scientific). Each plasmid coding sequence was confirmed by restriction enzyme digestion and sequence analysis for mouse and human SynGAPl -AAV transgenes (Stabvida). c) HEK 293 T cell transfection and SynGAPl expression detection

[0176]

[0168] Expression of GFP, human and mouse SynGAPl was evaluated by transient transfection of HEK 293 T suspension cells, in serum-free conditions. The subculturing of HEK 293T cells was performed twice a week with seeding at 0.35 x 106viable cells / mL, for a 4-day culture and 0.50 x 106viable cells / mL for a 3-day culture, into BalanCD medium (FUJIFILM), supplemented with 6 mM GlutaMAX (Thermo Fisher Scientific), using a

[0177] 1 :6.25 - 1 :4.0 culture / flask volume ratio. Shaker flasks were placed into a Multitron (INFORS, HT), with a 25 mm throw, at 130 rpm, 37 °C, 8% CO2. Cell viability was determined by trypan blue exclusion method using CytoSMART equipment (Corning).

[0178]

[0169] One day prior to transfection, cells were seeded at 1.4 * 106cells / mL. After 24 h, cells were diluted to 2 x 106cells / mL and transfected with 1 pg of plasmid DNA per 106viable cells, at a 1 : 1.5 (w / w) DNA:PEIpro (Polyplus) ratio (1 / 10 vol of the culture volume of media). Cells were incubated at 37° C, 8% CO2, and a 130-rpm shaking. GFP-expressing plasmids cells were harvested at 24-, 48- and 72-hours post-transfection (hpt) to measure percentage of GFP-positive cells by flow cytometry (FC) (Celesta, BD Biosciences).

[0179]

[0170] GFP-positive cells measurements at 24-, 48- and 72-hours post-transfection are shown in FIG. 3. The plotted data represents the average of two independent transfections. Error bars represent standard error. d) mRNA detection

[0180]

[0171] Total RNA was extracted from 2 x 106cells, pelleted at 300 g for 10 minutes and washed with DPBS. Then QIAamp® RNeasy Mini Kit (Qiagen, Hilden, Germany) was used according to manufacturer instructions. To eliminate contaminant DNA, cell extracts samples were treated twice with DNase using on-column digestion (RNase-free DNase Set, Qiagen). Resulting RNA was eluted in RNase-free water. RNA yields were quantified using Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific), and purity was accessed through the absorbance ratios at 260 / 280 nm and 260 / 230 nm. Only 260 / 280 nm absorbance ratios of 2.0 ± 0.2 of the RNA samples were considered to be pure.

[0181]

[0172] RNA was immediately used for complementary DNA (cDNA) synthesis. cDNA synthesis was performed using Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany), following manufacturer’s instructions. 2 pg of total RNA and anchored-oligo (dT) primers for total mRNA reverse transcription were used. The cDNA products were 4x diluted in ultrapure water (Milli-Q®, Merck Millipore, Billerica, MA, USA) and then stored at -20 °C until further use. e) cDNA quantification by ddPCR

[0182]

[0173] The primer sets designed and used are listed in Table 1.

[0183] TABLE 1 - ddPCR Primers (Set B)

[0184]

[0174] To optimize PCR conditions, an initial set of primers and probes designed (Set A) was tested using 20 ng / pL of linearized Human hSyn-SynthPA plasmid (XmnI + Sall). Gradient PCR was performed with an increasing annealing temperature (Ta) from 58 °C to 64 °C. The basic PCR conditions were set according to the protocol provided by the manufacturer (Phusion High-Fidelity DNA Polymerase, NEB). The amplicons were subjected to electrophoresis in a 2% agarose gel with 0.05 pL / mL RedSafe Nucleic Acid Staining Solution (INtRON Biotechnology). The DNA was visualized in GelDoc XR+ system (BioRad). No unspecific products or primer dimers were detected, anda quantitative PCR (qPCR) reaction was performed to validate primer efficiency.

[0185]

[0175] In qPCR, 2.5 pL of each DNA sample and plasmid standard curve (10-fold dilutions of linearized human hSyn-SynthPA plasmid, ranging from 108-102 copies / pL) were added to a 12.5 pL final volume qPCR reaction with FastStart Essential DNA Probes and primerprobes Set A for SynGAPl or for UBC (housekeeping gene Ubiquitin C) at 0.5 pM and 0.25 pM final concentration, respectively. Thermal cycling was performed in a LightCycler 480 Instrument (Roche), at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 10 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 6 seconds (single acquisition) and a cooling step at 40 °C.

[0186]

[0176] As primer-probe Set A delivered an amplification efficiency below 90%, the method was considered not valid resulting in the quantification assessment by other method, the droplet digital PCR (ddPCR). A different set of primer-probe, primer-probe set B (Table 1) was designed, tested, and evaluated in parallel with set A by ddPCR assay. 3 pL of sample were added to a 22 pL final volume and mixed in a multiplex reaction containing target primer-probe set A or B with a primer-probe set specific for housekeeping gene UBC, at 900 nM for each primer and 250 nM for probe. Droplets were generated by an Automated Droplet Generator (BioRad) followed by PCR reactions in a Cl 000 Touch Thermal Cycler (BioRad), at 95 °C for 10 minutes, followed by 40 cycles of 94 °C for 30 seconds, annealing / extension from 54 °C to 68 °C for 60 seconds, and enzyme deactivation at 98 °C for 600 seconds and a cooling step at 4 °C. A QX200 droplet reader (Bio-Rad) using the QuantaSoft software package (Bio-Rad) was used to detect fluorescent signals in each droplet. For validation, cDNA of human JeT-SV40PA cDNA was used.

[0187]

[0177] ddPCR measurement results for expression levels of SynGAPl mRNA (copies / pL) are shown in FIG. 4. f) Protein detection

[0188]

[0178] For total protein extraction, 2 x 106cells were harvested, pelleted at 300 g for 10 minutes and washed with DPBS. Cell lyses was conducted using 200 pL Mammalian Protein Extraction Reagent (M-PER) (Thermo Fisher Scientific) with lx cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche Applied Science). The mixture was vortexed, incubated for 10 minutes at room temperature and agitation (350 g). Extracts clarification was done by centrifugation (14000 g for 10 minutes). Samples were stored at -20 °C until analysis. Total protein quantification was performed with BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Bovine serum albumin (BSA) (Thermo Fisher Scientific) was used to establish the standard calibration curve. Samples were diluted and applied in duplicates.

[0189]

[0179] To detect SynGAPl protein in transfected HEK 293 T cells, manufacturer’s standard method was followed for 12-230-kDa Jess separation module, using the Streptavidin-HRP Conjugate. The Jess™ Simple Western system (Protein Simple) is an automated capillarybased size separation system that can detect chemiluminescence and fluorescence signals. The cell extracts were diluted in PBS to a concentration of 1 pg / pL and 2.5 pg of total protein added per lane. The primary antibodies used were human SynGAP antibody, diluted 1 :50 (Cell Signaling Technology, CS #3200), herein named CS. The secondary antibody used was an anti-rabbit secondary antibody (Protein Simple). Detection was performed by chemiluminescence. A digital image of the chemiluminescence signal of the capillary was captured with Compass Simple Western software 6.1.0 (Protein Simple) that automatically calculated heights (chemiluminescence intensity), area, and signal / noise ratio. Results could be visualized as electropherograms format representing peak of chemiluminescence intensity and as lane view (virtual blots). Virtual blots convert electropherogram data into images of band densities similar to images of standard Western blots.

[0190]

[0180] Chemiluminescence intensity results obtained from electropherogram data are shown in FIG. 5A. A corresponding virtual blot generated from the chemiluminescence peaks is shown in FIG. 5B.

[0191] Example 2: rAAV Packaging and Viral Titering a) rAAV production

[0192]

[0181] pAAV transgene plasmids were produced according to the general procedures of Example 1. Rep / Cap (7330 bp) plasmids were also obtained, which contained the replicase from serotype 2 (Rep2) and capsid (Cap) proteins.

[0193]

[0182] Packaging plasmids, pHelper (11 635 bp), were acquired from AAV Helper Free System (Agilent), which contained Adenoviral helper factors (i.e., E2A, E4, VA RNA genes). Packaging plasmids were transformed into chemically competent E. coli DH5a (Invitrogen - TFS, EU) and Stellar (Takara Bio, EU), respectively. The bacteria were selected in LB, supplemented with 100 pg / mL of Ampicillin (Amp), at 37 °C. Liquid cultures grew at 37 °C with aeration by shaking (150 - 180 rpm). Commercially available kits were used to purify plasmid high-pure grade DNA, to be used in bacteria transformation and mammalian cells transfection. Plasmid DNA purity was assessed by NanoDrop and only a ratio of A260 / A280 ~ 1.8 and of A260 / A230 2.0 - 2.2 was considered acceptable and used. Plasmid DNA integrity was evaluated by restriction enzyme analysis. The plasmid coding sequence was also confirmed by sequencing analysis (Eurofins Genomics).

[0194]

[0183] rAAV production using the designed transgene plasmids was evaluated using suspension cultures of 293T cells, adapted to serum free media BalanCD. One day prior transfection, cells were seeded at 1.4 * 106cells / mL. Before transfection, cells were diluted and inoculated at a density of 2.0 x 106cells / mL and transfected with 1 pg of plasmid DNA per 106viable cells. A 1 : 1.5 (w / w) DNA:PEIpro (Polyplus) ratio was used in 1 / 10 volume of the cells. Transfections were conducted in 15 mL suspension cultures for each experimental condition. The plasmid ratio for pHelper, Rep / Cap and pAAV transgene was 1 : 1 : 1 by mass. To allow viral production, cells were incubated for 3 days at 37 °C, 8% CO2, shaking at 130 rpm (25 mm throw; Multitron). Viable cell concentration was assessed by trypan blue exclusion method, in Cytosmart equipment (Corning). Viable cell concentration measurement at 48 hours post-transfection (hpt) are shown in FIG. 6, with the plotted data representing one independent transfection.

[0195]

[0184] 72 hours post-transfection (hpt), cells were harvested to measure the transfection efficiency (JeT-GFP-SV40PA was used for testing), by flow cytometry (Celesta, BD Biosciences) and concentration of viable cells, by trypan blue exclusion method (CytoSMART, Coming).

[0196]

[0185] The resulting virus from each test batch was collected following protocols known to those in the art. Briefly, cells were lysed in the shake-flask using a lysis buffer (50 mM of Tris, 1% (v / v) Tween 20, 2 mM of MgC12, pH 8.0). Nuclease treatment was performed using 50 U / mL (Benzonase) with an incubation period of 1 hour after which NaCl (0.5 M) was added for vims stabilization for additional 30 minutes. Upon harvest, rAAVs were clarified by filtration and analyzed by ELISA and ddPCR or stored at -80 °C. b) rAAV characterization

[0197]

[0186] Each batch of produced rAAV vectors (Total Particles, TP) were quantified using an AAV ELISA assay (PROGEN). The sandwich ELISA recognizes a specific surface epitope on the assembled capsid via a conformational change that is not present on unassembled capsid proteins. The TPs were calculated using a 4-parameter logistic (4PL) regression.

[0198]

[0187] To quantify full particles (i.e., capsids with full viral genomes (VG)), the crude samples were treated with DNase to eliminate free DNA contamination. Viral DNA was then purified using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH). VGs were quantified by ddPCR, using the primer-probe set B (Table 1) specific for the syngapl gene from Example 1, following the thermal cycler conditions described in Examples 1 (e). At 72 hours, a transfection efficiency of 63.2% was observed, and the doubling of transfected cells. The productivity was similar for all tested syngapl transgenes as well as the full / empty ratio, below 30%.

[0199]

[0188] AAV vector genome titers (VG / mL) in the HEK293T crude cell lysate, postpackaging with the six mSynGAP or hSynGAP-expressing cassettes, is shown in FIG. 7. Total AAV particles in the HEK293T cells crude cell lysate, post-packaging with the six mSynGAP or hSynGAP-expressing cassettes, is shown in FIG. 8.

[0200]

[0189] To evaluate the quality of produced virus by different analytics, a small-scale purification with magnetic beads (Dynabeads™ Capture Select™ AAV Magnetic Beads, Thermo) was performed following the manufacturer instructions. The purified viruses were analyzed by mass photometry and the obtained % of full particles was similar in crude and purified samples.

[0201] Example 3: In vivo testing in heterozygous SynGAPl mouse model

[0202]

[0190] Augmenting SynGAPl in the CNS of a subject was studied using an AAV vector (AAV-hSynGAPl) as a therapeutic strategy for SynGAPl related epileptic encephalopathy. a) Heterozygous SynGAPl mouse model

[0203]

[0191] A heterozygous SynGAPl mouse model (SynGAPl+ / ‘), that recapitulates the human SynGAPl haploinsufficiency, was acquired from The Jackson Laboratories (Strain #: 008890). Mice were housed in standard laboratory conditions with a 12-hour light / dark cycle with chow diet and water ad libitum. Mice exhibit nocturnal behaviors and to quantify behaviors accurately, a 24-hour period was divided into two equal 12-hour periods: light phase is when the lights were turned on and is the less active period (7 AM - 7 PM) and dark phase is when the lights were turned off and is the more active period (7 PM - 7 AM). The light phase is characterized by reduced locomotor activity and increased resting behavior, whereas the dark phase is the active phase and is characterized by increased locomotor activity and exploratory behaviors. b) AAV administration

[0204]

[0192] Viral vector (AAV-hSynGAPl; AAV) or vehicle (PBS) was administered by bilateral intracerebroventricular (ICV) injection into the brain of neonatal mice at post-natal day 1-2.

[0205]

[0193] Experiments were conducted in three groups of animals: control littermate wild-type (WT) mice injected with vehicle PBS (SynGAPl+ / + PBS), heterozygous SynGAPl mice injected with vehicle PBS (SynGAPH- / - PBS) and heterozygous SynGAPl mice transduced with AAV-hSynGAPl (SynGAPH- / - AAV).

[0194] c) Protein Quantification (Quantitative Western Blot)

[0206]

[0195] At 6-8 weeks post-injection, brain tissues were collected from the mice. For biochemical analysis, a sample of hippocampus was dissected and homogenized in RIPA buffer (R0278, Sigma) with a protease inhibitor cocktail (P8849, Sigma) using the Precellys Touch Homogenizer (Bertin Technologies). Afterwards, samples were centrifuged, and the supernatant was collected as the final protein total brain extract.

[0207]

[0196] Protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific) and samples (0.4mg / nL of protein) were loaded into a Jess Automated Western Blot (Bio-Techne).

[0208]

[0197] Primary antibodies observed / identified were SynGAP antibody (1 : 10 dilution; #3200, Cell Signaling Technology) and beta-actin antibody (1 :25 dilution, #4970, Cell Signaling Technology). Secondary antibody that was observed / identified was an anti-rabbit secondary antibody (Protein Simple).

[0209]

[0198] The antibody detection was performed by chemiluminescence. A digital image of the chemiluminescence signal of the capillary was captured with Compass Simple Western software 6.1.0 (Protein Simple) that automatically calculated height (chemiluminescence intensity), area, and signal / noise ratio. d) Hippocampal neurometabolite profile (1H-MRS)

[0210]

[0199] In vivo proton magnetic resonance spectroscopy (1H-MRS) was performed on the mice at the age of 7-8 weeks.

[0211]

[0200] Magnetic resonance imaging (MRI) was performed in a horizontal 11 ,7T Bruker BioSpec (Bruker Biospin GmbH) high-field preclinical MRI scanner with bore size 160 mm equipped with a gradient set capable of max. gradient strength 750 mT / m and interfaced to a Bruker Avance III console (Bruker Biospin GmbH). A volume coil (Bruker Biospin GmbH) was used for transmission and a two-element surface array coil for receiving (Rapid Biomedical GmbH). Animals were anesthetized with 5% isoflurane (in 70% N2 and 30% 02; flow 300 mL / min). During the MRI acquisition the concentration of isoflurane was reduced to 1.5 - 2.0%. Total duration of anesthesia was < 45min. Animals’ temperature was monitored and maintained at 36.5±1.5°C with a homeothermic blanket system. Isoflurane anesthetized animals were fixed to a custom-made holder and positioned in the magnet bore in a standard orientation relative to the gradient coils.

[0212]

[0201] For the 1H-MRS measurement, a volume of interest (voxel) was placed in the hippocampus based on T2-weighted localizer images. Automatic FASTMAP shimming algorithm was used to adjust BO homogeneity in the voxel. The water signal was suppressed using variable power RF pulses with optimized relaxation delays (VAPOR) to obtain Bl and T1 insensitivity. A PRESS sequence (TE = 10 ms) combined with outer volume suppression (OVS) was used for the pre-localization. Three OVS blocks were used interleaved with water suppression pulses. Data were collected by averaging 512 excitations (frequency corrected for each FID) with TR of 4s, number of points 2048 and spectral width 5 kHz. Excitation frequency was shifted 2 ppm, to minimize the chemical shift phenomenon within the selected voxel. In addition, a reference spectrum without water suppression (NT=8) was collected from the identical voxel using the same acquisition parameters.

[0213]

[0202] Peak areas for metabolites were analyzed using LCModel (Stephen Provencher Inc.) and results were given relative to water content in tissue. Glutamine and glutamate neurometabolites were included in the analysis. Criteria of < 20% SD in the Cramer-Rao lower bounds were used in the reporting of metabolite values. e) Anesthesia and Implantable Telemetry Surgical Preparations for physiological monitoring

[0214]

[0203] Animals were anesthetized (75 mg / kg ketamine and 1 mg / kg medetomidine, intraperitoneally) and given 7.5 mg / kg subcutaneous carprofen. A midline 2-cm skin incision on the head and neck was made and the subcutaneous tissue was bluntly separated.

[0215] Radiotelemetry ETA-F10 implants (Data Sciences International) were implanted into a subcutaneous dorsal pocket and leads were coiled into a loop and anchored to surrounding tissue. Two burr holes (0.7 mm in diameter) were positioned with an electric high-speed drill. Electrodes were shortly bent at the tip and placed directly on the dura mater (epidural lead placement) in a bipolar deflection following manufacturer instruction coordinates: -1 / +1 mm to bregma (left hemisphere); +1 / -1 mm caudal of bregma (right hemisphere). Electrodes were secured on the skull with glass ionomer cement (VOCO GmbH). After the cement dried, the scalp was closed. Animals were allowed to recover for one-week prior to experiments. f) Locomotor activity

[0216]

[0204] Home-cage locomotor activity of individually housed freely moving mice was obtained with ETA-F10 implants (Data Science International). Data was collected continuously for 24 hours over a period of 7 days (Ponemah software, Data Sciences International). Locomotor activity in the light / dark cycle (number of crosses per hour) was analyzed using NeuroScore software (Data Sciences International). g) Video-EEG analysis

[0217]

[0205] EEG recording was performed with ETA-F10 implants (Data Science International). Data were collected continuously for 24 hours. Raw data acquisition was performed with Ponemah software (Data Sciences International). EEG data was collected at a sampling rate of 500 Hz, using a bandpass filter with a high-pass cut-off at 0.1 Hz and a low-pass cut-off at 100 Hz. Data was analyzed using NeuroScore software (Data Sciences International). Spikes were identified based on an absolute threshold of 200 pV, with a minimum spike duration of

[0218] 1 ms and a maximum spike duration of 200 ms. The spike interval ranged from a minimum of 0.05 s to a maximum of 0.5 s. Myoclonic-like seizures are characterized by brief, sudden involuntary movements, such as muscle jerks or irregular muscle twitching, accompanied by high brain activity. Myoclonic-like seizures were identified and validated manually by considering the animal's locomotor activity, brain activity, and video analysis. h) Statistical analysis

[0219]

[0206] Statistical analysis was performed using Prism v8 (GraphPad). Data were expressed as mean ± standard error of the mean (SEM). Differences between groups were analyzed using 1-way ANOVA followed by Fisher’s LSD post-hoc test. A p-value of <0.05 was considered statistically significant. i) Results - Protein quantification

[0220]

[0207] The potential of an intra-cerebrospinal fluid injection of a recombinant AAV vector encoding SynGAPl as a therapeutic approach for SynGAPl related epileptic encephalopathy was investigated. First, the ability to restore SynGAP protein levels in a SynGAPl heterozygous mouse model was evaluated. Second, the therapeutic effect of the gene delivery method was assessed by measuring the correction of functional phenotypes in a mouse model that recapitulates human disease clinical symptoms.

[0221]

[0208] At 6-8 weeks post-treatment, western blot protein quantification analysis by Jess Automated Western Blot confirmed a statistically significant increase in the expression of SynGAP protein levels in heterozygotic SynGAPl mice transduced with AAV-hSynGAPl (SynGAPl+ / - AAV), as compared to heterozygous SynGAPl mice injected with vehicle PBS (SynGAP 1+ / - PBS).

[0222]

[0209] Results are illustrated in FIG. 9. Data is shown for control wild-type mice treated with vehicle PBS (SynGAP 1+ / + PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPl+ / - PBS) and heterozygous SynGAPl mice transduced with AAV-hSynGAPl (SynGAPl+ / - AAV). Data were expressed as relative expression normalized to beta-actin levels as a loading control. n=2-9 / group. **p < 0.01.

[0210] Results show that heterologous expression of SynGAPl in the CNS of the heterozygous SynGAPl mouse restored the SynGAP protein levels close to WT levels. The protein levels were restored in notable brain areas implicated in the disease, like the hippocampus. i) Results - Neurometabolites

[0223]

[0211] Expression of SynGAP in notable brain areas was associated with functional correction of phenotypes in the mouse model. Mice treated with AAV expressing SynGAP (SynGAPH- / - AAV) showed correction in neurometabolite deficits in Glutamate and Glutamine levels in the hippocampus. In vivo 'H-MR.S was performed at the age of 7-8 weeks. MRS voxel analysis was localized on dorsal hippocampus, bilaterally. There was a statistically significant difference in glutamine and glutamate concentration in the hippocampus between heterozygous SynGAPl mice injected with vehicle PBS (SynGAPH- / - PBS) and heterozygotic SynGAPl mice transduced with AAV-hSynGAPl (SynGAPH- / - AAV).

[0224]

[0212] Results are illustrated in FIG. 10. Data are shown for control wild-type mice treated with vehicle PBS (SynGAP 1+ / + PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPH- / - PBS) and heterozygous SynGAPl mice transduced with AAV- hSynGAPl (SynGAPH- / - AAV). Data presented as group mean ± SEM. n=8 / group. **p < 0.01. k) Results - Hyperactivity

[0225]

[0213] Heterozygous SynGAPl mice display a hyperactivity phenotype in a general exploratory locomotion assay. To confirm the therapeutic potential of augmentation of SynGAP levels, AAV-hSynGAPl treated heterozygous SynGAPl mice were tested in a locomotion assay. Home-cage spontaneous locomotor activity during the dark phase (z.e., active period) showed that heterozygous SynGAPl mice treated with vehicle PBS (SynGAPH- / - PBS) have a significant increase in activity (hyperactivity) compared to control wild-type littermate mice injected with vehicle PBS (SynGAPl+ / + PBS). AAV-hSynGAPl transduction (SynGAPH- / - AAV) significantly decreased the hyperactivity observed in the heterozygous SynGAPl mice (SynGAPH- / - PBS). No difference was observed between the groups during the light phase (i.e., less active period).

[0226]

[0214] Results are shown in FIG. 11. Data is shown for control wild-type mice treated with vehicle PBS ( SynGAP 1+ / + PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPH- / - PBS) and heterozygous SynGAPl mice transduced with AAV-hSynGAPl (SynGAPl+ / - AAV). Data presented as group mean ± SEM. n=7-8 per group. *p < 0.05, **p < 0.01. l) Results - Electroencephalography (EEG)

[0227]

[0215] Altered electroencephalography (EEG) signatures are emerging as potential endpoints in several neurodevelopmental disorders. Epileptiform discharges are abnormal electrical activities in the brain that can be measured by EEG, characterized by sharp waves or spikes. Heterozygous SynGAPl mice (SynGAPl+ / - PBS) show characteristic epileptiform discharges. However, these discharges were absent in littermate WT mice (SynGAPl+ / + PBS). AAV treated heterozygous SynGAPl mice showed around 50% reduction in the total number of epileptiform discharges in the light phase, when the mice are less active.

[0228]

[0216] Results are shown in FIG. 12. Data is shown for control wild-type mice treated with vehicle PBS (SynGAPl + / + PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPl+ / - PBS) and heterozygous SynGAPl mice transduced with AAV-hSynGAPl (SynGAPl + / - AAV). Data presented as group mean ± SEM normalized to SynGAPl + / - PBS group. n=3-9 per group. *p < 0.05, **p < 0.01. m) Results - Myoclonic-like seizures

[0229]

[0217] Additionally, myoclonic-like seizures were detected in all heterozygous SynGAPl mice treated with vehicle PBS. AAV treated heterozygous SynGAPl mice showed a reduced number of myoclonic-like seizures in the light phase (less active period), as well as a decrease in the duration of these events, compared to heterozygous SynGAPl mice treated with vehicle PBS. These seizures were absent in the WT mice.

[0230]

[0218] Results are shown in FIG. 13 for myoclonic-like seizures in the light / dark phase over a 24-hour recording period. Data is shown for control wild-type mice treated with vehicle PBS (SynGAPl+ / +PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPl+ / " PBS) and heterozygous SynGAPl mice transduced with AAV-hSynGAPl (SynGAPl+ / ‘ AAV). Data presented as group mean ± SEM. n=3-9 per group. *p < 0.05.

[0231]

[0219] Results are shown in FIG. 14 for the duration of myoclonic-like seizures in the light / dark phase over a 24-hour recording period. Data is shown for control wild-type mice treated with vehicle PBS (SynGAPl+ / + PBS), heterozygous SynGAPl mice treated with vehicle PBS (SynGAPl+ / - PBS) and heterozygous SynGAPl mice transduced with AAV- hSynGAPl (SynGAPl+ / - AAV). Data presented as group mean ± SEM as a percentage of total detected seizures duration. n=3-9 per group. *p < 0.05. SEQUENCES

Claims

CLAIMS1. A polynucleotide comprising a promoter sequence operatively linked to a payload sequence, wherein the payload sequence encodes a synaptic GTPase-activating protein (SynGAP).

2. The polynucleotide of claim 1, wherein the payload sequence has at least 85% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

3. The polynucleotide of claim 1, wherein the payload sequence has at least 90% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

4. The polynucleotide of claim 1, wherein the payload sequence has at least 95% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

5. The polynucleotide of claim 1, wherein the payload sequence comprises any one of SEQ ID NOs: 5, 7, 9, 11, and 13.

6. The polynucleotide of claim 1, wherein the payload sequence comprises SEQ ID NO:7.

7. The polynucleotide of any one of claims 1-6, wherein the payload sequence encodes a SynGAP which has at least 85% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

8. The polynucleotide of claim 7, wherein the payload sequence encodes a SynGAP which has at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

9. The polynucleotide of claim 7, wherein the payload sequence encodes a SynGAP which has at least 95% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

10. The polynucleotide of claim 7, wherein the payload sequence encodes a SynGAP comprising any one of SEQ ID NOs: 6, 8, 10, 12, and 14.

11. The polynucleotide of claim 7, wherein the payload sequence encodes a SynGAP comprising SEQ ID NO: 8.

12. The polynucleotide of any one of claims 1-11, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 3.

13. The polynucleotide of any one of claims 1-11, wherein the promoter sequence comprises SEQ ID NO: 3.

14. The polynucleotide of any one of claims 1-11, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 4.

15. The polynucleotide of any one of claims 1-11, wherein the promoter sequence comprises SEQ ID NO: 4.

16. The polynucleotide of any one of claims 1-15, wherein the polynucleotide comprises a poly(A) signal sequence.

17. The polynucleotide of claim 16, wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 15.

18. The polynucleotide of claim 16, wherein the poly(A) signal sequence comprises SEQ ID NO: 15.

19. The polynucleotide of claim 16, wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16.

20. The polynucleotide of claim 16, wherein the poly(A) signal sequence comprises SEQ ID NO: 16.

21. The polynucleotide of any one of claims 1-20, wherein the payload sequence has at least 90% sequence identity to SEQ ID NO: 7, wherein the promoter sequence has at least 90% sequence identity to SEQ ID NO: 4, and wherein the poly(A) signal sequence has at least 90% sequence identity to SEQ ID NO: 16.

22. The polynucleotide of any one of claims 1-20, wherein the payload sequence comprises SEQ ID NO: 7, wherein the promoter sequence comprises SEQ ID NO: 4, and wherein the poly(A) signal sequence comprises SEQ ID NO: 16.

23. The polynucleotide of any one of claims 1-22, wherein the polynucleotide has at least 85% sequence identity to any one of SEQ ID NOs: 17 to 25.

24. The polynucleotide of any one of claims 1-22, wherein the polynucleotide has at least 90% sequence identity to any one of SEQ ID NOs: 17 to 25.

25. The polynucleotide of any one of claims 1-22, wherein the polynucleotide has at least 95% sequence identity to any one of SEQ ID NOs: 17 to 25.

26. The polynucleotide of any one of claims 1-22, wherein the polynucleotide comprises any one of SEQ ID NOs: 17 to 25.

27. The polynucleotide of any one of claims 1-22, wherein the polynucleotide comprises SEQ ID NO: 21.

28. An adeno-associated viral (AAV) vector genome comprising: a 5' inverted terminal repeat (ITR), an expression cassette, and a 3' ITR; wherein the expression cassette comprises the polynucleotide of any one of claims 1-27.

29. The AAV vector genome of claim 28, wherein the 5' ITR is an AAV2 ITR.

30. The AAV vector genome of claim 28 or claim 29, wherein the 3' ITR is an AAV2 ITR.

31. The AAV vector genome of any one of claims 1-30, wherein the AAV vector genome comprises a sequence which has at least 85% sequence identity to any one of SEQ ID NOs:26 to 34.

32. The AAV vector genome of any one of claims 1-30, wherein the AAV vector genome comprises a sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 26 to 34.

33. The AAV vector genome of any one of claims 1-30, wherein the AAV vector genome comprises a sequence which has at least 95% sequence identity to any one of SEQ ID NOs: 26 to 34.

34. The AAV vector genome of any one of claims 1-30, wherein the AAV vector genome comprises any one of SEQ ID NOs: 26 to 34.

35. The AAV vector genome of claim 28, wherein the AAV vector genome comprises SEQ ID NO: 30.

36. An AAV particle comprising the AAV vector genome of any of claims 28-35 and a capsid.

37. An AAV particle of claim 36, wherein the capsid has tropism for tissues or cells of the central nervous system (CNS).

38. A pharmaceutical composition comprising an AAV particle of claim 36 or claim 37, and a pharmaceutically acceptable excipient.

39. A method of increasing the expression of synaptic GTPase-activating protein (SynGAP) in a subject, comprising delivering an effective amount of an AAV particle of claim 38 to the subject.

40. A method of treating a disease related to a SYNGAP 1 gene mutation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of claim 38.

41. The method of claim 40, wherein the disease related to a SYNGAP 1 gene mutation is a neurodevelopmental disorder (NDD).

42. The method of claim 40, wherein the disease related to a SYNGAP1 gene mutation is a developmental and epileptic encephalopathy (DEE).

43. The method of any one of claims 40-42, wherein administering the composition to the subject comprises intraci sternal (ICM), intrathecal (IT), intracerebroventricular (ICV), intra- cerebrospinal fluid (ICSF), or intraparenchymal administration.

44. The method of any one of claims 40-43, wherein administering the composition to the subject reduces or eliminates one or more symptoms of the disease.

45. The method of any one of claims 40-43, wherein administering the composition to the subject improves glutamate and glutamine neurometabolite levels in the hippocampus of the subject.

46. The method of any one of claims 40-43, wherein administering the composition to the subject decreases hyperactivity in the subject.

47. The method of any one of claims 40-43, wherein administering the composition to the subject decreases abnormal epileptiform discharges in the brain of the subject, as measured by electroencephalography (EEG).

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