Gene therapy for stxbp1 encephalopathy

Abstract

Aspects of the invention provide an AAV product that delivers an STXBP1 gene supplementation or replacement strategy throughout the human CNS at levels of DNA biodistribution, RNA expression, and hSTXBPlb protein levels expected to restore function in STXBP1 encephalopathy patients.

Description

[0001] GENE THERAPY FOR STXBP1 ENCEPHALOPATHY

[0002] Field of the Invention

[0003] The invention generally relates to virus compositions for treating STXBP1 encephalopathy.

[0004] Background

[0005] Syntaxin-binding protein 1 (STXBPP) encephalopathy is a rare, devastating neurodevelopmental disorder associated with developmental delay and treatment-resistant seizures. STXBP1 encephalopathy is caused by de novo heterozygous mutations in the syntaxinbinding protein 1 gene (STXBPP), which encodes STXBP1 protein. The STXBP1 protein is a SNARE organizer that is expressed in every neuronal synapse throughout the CNS and in neuroendocrine cells and is necessary for presynaptic vesicle and neurotransmitter and neuropeptide release. Homozygous loss of function (LoF) mutations in the STXBP1 gene result in death at birth due to a complete loss of neuronal communication and an inability to breathe. Heterozygous disruptive mutations in the STXBP1 gene impair neuronal communication and occur in ~1 in 30,000 live births and are amongst the top 5 causes of epilepsy and the top 10 causes of neurodevelopmental disorders (NDDs). The major clinical features of STXBP1 encephalopathy are epilepsy, severe intellectual disability, speech and language impairments, movement disturbances, behavioral problems, including autistic traits and absent speech, and in some cases early mortality due to Sudden Unexpected Death in Epilepsy (SUDEP).

[0006] There are currently no treatments for the underlying disorder and few disease-modifying therapies in development; existing treatments are focused on diminishing symptoms of the disease.

[0007] Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and nondividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for therapeutic applications, such as gene therapy. However, systemic delivery of existing AAV serotypes (e.g., intravenous, intrathecal, intraarterial, intracranial, intracisterna magna, intraventricular, intracerebroventricular, or subcutaneous) shows limited transduction of certain cell types and organs, and non-specific, overlapping tropisms in others. This leads to several complications in gene therapy applications, including but not limited to off-target effects due to transduction of unimpactcd organs and cell types (for example, the liver), and the necessity for a larger viral dosage to achieve sufficient therapeutic levels in the tissue or organ of interest.

[0008] Summary

[0009] Compositions and methods of the invention use recombinant adeno-associated viruses (rAAV) to deliver a viral vector comprising a human syntaxin-binding protein 1 (STXBP1 ) gene encoding a functional STXBP1 protein. By allowing cells to produce functional STXBP1, compositions and methods of the invention can be used to treat STXBP1 encephalopathy. In various embodiments, modified rAAVs are used to improve gene delivery and expression and target the central nervous system (CNS) for gene delivery. In certain embodiments, modified rAAVs of the invention may exhibit increased specificity and transduction efficiency in the CNS, allowing for systemic delivery thereof with reduced risk of off-target effects. Such modified rAAVs may exhibit specificity engineered into the capsid structure through iterative rounds of selection in non-human primates (NHPs), yielding variants with tropisms having an increased specificity and transduction efficiency in the CNS, and in some cases, a decreased specificity and transduction efficiency in an off-target environment. The rAAVs described herein achieve widespread transduction in the CNS (e.g., CNS cell types or tissues) in a subject upon systemic delivery (e.g., intravenous, intrathecal, intraarterial, intracranial, intraventricular, intracerebroventricular, or subcutaneous).

[0010] Aspects of the invention provide an AAV product that delivers an STXBP1 gene supplementation or replacement strategy throughout the human CNS at levels of DNA biodistribution, RNA expression, and hSTXBPlb protein levels expected to restore function in STXBP1 encephalopathy patients.

[0011] Specifically, the present invention provides an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1). The vector further comprises (e.g. encapsidates) a nucleic acid encoding a promoter and an STXBP1 transgene. In preferred aspects of the invention, the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1). Aspects of the invention provide an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1).

[0012] The capsid protein may be engineered from an AAV9 capsid protein. For example, the capsid protein may be engineered, relative to an AAV9 capsid protein, by substitution of amino acid position 588 with the amino acid T, insertion between amino acid positions 588 and 589 of the amino acids RNGEVFI, and inclusion of parental AAV9 amino acids AQ in positions 589 and 590.

[0013] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVFIAQ.

[0014] A person of skill in the art will understand the equivalent positions of AAV vectors other than AAV9 in which the sequence ATRNGEVFIAQ (SEQ ID NO: 1) may be adapted.

[0015] Accordingly, in certain embodiments, the AAV vector may comprise an AAV9 backbone. For example, the AAV vector may comprise an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of AAV9 (SEQ ID NO: 2).

[0016] Advantageously, the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS), without being bound to a mechanism of action, thereby increasing the efficacy of AAV approaches to STXBP1 gene supplementation or replacement.

[0017] The AAV capsid protein may be characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

[0018] Further advantageously, an AAV vector targeted to the CNS and / or brain may further comprise a nucleic acid expressing a therapeutic STXBP1 transgene. The STXBP1 transgene may encode a peptide having at least 95% identity to the sequence of STXBP1 isoform b.

[0019] The nucleic acid expressing the transgene may further comprise a CAG promoter. The CAG promoter may comprise human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken -actin promoter, and a chimeric rabbit P-globin (rBG) intron.

[0020] The AAV vector may further comprise at least one of the regulatory elements known to increase expression of the therapeutic gene, poly(A) signal for stability. For example, the regulatory element may be a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE). The poly(A) signal is hGH polyA.

[0021] Accordingly, as detailed above, together aspects of the invention may provide an AAV vector having a sequence of SEQ ID NO: 7 or encoded by a sequence of SEQ ID NO: 8. aspects of the invention may provide an hSTXBPlb peptide having a sequence of SEQ ID NO: 4 or encoded by a sequence of SEQ ID NO: 9. For example, aspects of the invention may provide an AAV vector comprising, in order, a 5’ ITR, CAG promoter, STXBP1 transgene, Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE), hGH polyA signal from the human growth hormone (hGH), and 3’ ITR.

[0022] In some instances, the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and the 3’ ITR each originate from different serotypes, e.g. 5’ ITR from serotype AAV2 and 3’ ITR from AAV5. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency.

[0023] For example, the 5’ ITR (referred to as L-ITR) may comprise a nucleic acid having at least 90% sequence identity with the sequence of SEQ ID NO: 10. The 3’ ITR (referred to as R- ITR) may comprise a nucleic acid having at least 90% sequence identity with the sequence of SEQ ID NO: 11.

[0024] Aspects of the invention also provide methods of treating a disease comprising administration of an AAV vector of the present invention. Accordingly, aspects of the invention provide a method of treatment of a disease that comprises administering a composition comprising an adeno-associated virus (AAV) vector comprising an engineered capsid protein that comprises an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1), further comprising (e.g. encapsidates) a nucleic acid encoding a promoter and an STXBP1 transgene. Methods of the invention are understood to use any of the AAV vectors described in the instant application. Aspects of the invention provide an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1).

[0025] The disease treated may be a disease caused by an STXBP1 mutation, for example STXBP1 encephalopathy. The composition may be administered as an injection. The injection may be an intravenous injection. The intravenous injection may be administered once daily.

[0026] Therapeutically effective amounts of the rAAV may be administered systemically (e.g., intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracistemae magna administration, or subcutaneously). In preferred embodiments, the rAAV is administered intrathecally or intracistemally.

[0027] Brief Description of the Drawings

[0028] FIG. 1 is a table detailing a summary of results from AAV administration to NHPs.

[0029] FIG. 2A-D are graphs showing DNA level changes following AAV administration to NHPs.

[0030] FIG. 3A-D are graphs showing RNA level changes following AAV administration to NHPs.

[0031] FIG. 4A-G are graphs showing protein expression level changes following AAV administration to NHPs.

[0032] FIG. 5A is a graph of histological quantification in CNS tissue following AAV administration to NHPs.

[0033] FIG. 5B is an immunofluorescence assay of the cortex following AAV administration to NHPs.

[0034] FIG. 5C is an immunofluorescence assay of the thalamus following AAV administration to NHPs.

[0035] FIG. 6A-B are graphs showing DNA biodistribution in NHPs following AAV administration.

[0036] FIG. 7A-B are graphs showing RNA expression in NHP CNS tissue following AAV administration.

[0037] FIG. 8A-C are graphs showing DNA biodistribution in liver and RNA expression in NHPs peripheral tissue following AAV administration. FIG. 9 is an immunofluorescence assay of neuronal cells following AAV administration.

[0038] FIG. 10A-C arc graphs of calcium transients that reflect neuronal firing over time following AAV administration.

[0039] FIG. 11A-B show graphs of rates of neuronal transduction and STXBP1 levels in human IPSC-derived wild type or STXBP1 knockout neurons following administration of a capsids of the invention.

[0040] FIG. 12A-B show graphs of synchronized firing and spike intensity following administration of a capsids of the invention.

[0041] FIG. 13A-C show graphs of transduction efficiency in human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0042] FIG. 14A-C show graphs of STXBP1 mean intensity in human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0043] FIG. 15A-C show graphs of STXBP1 mean intensity in neuronal compartments across human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0044] FIG. 16A-C show graphs of hSTXBPl DNA biodistribution across human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0045] FIG. 17A-C show graphs of hSTXBPl RNA biodistribution across human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0046] FIG. 18A-B show graphs of STX1 mean intensity and STXBP1 colocalization measured by immunocytochemistry against Syntaxin- 1 and / or STXBP1 in human IPSC-derived STXBP1 KO neurons following administration of a capsids of the invention.

[0047] Detailed Description

[0048] The present invention provides an AAV product that delivers an STXBP1 gene thereby implementing a supplementation or replacement strategy throughout the human CNS at levels of DNA biodistribution, RNA expression, and hSTXBPlb protein levels expected to restore function in STXBP1 encephalopathy patients. STXBP1 and Encephalopathies

[0049] Embodiments of the disclosure concern compositions, including gene therapies, viral particles, and nucleic acids, that are useful for restoring STXBP1 and STXBP1 in individuals with any deficiency, including missense mutations, nonsense mutations, deletions, inversions, insertions, duplications, frameshift mutations, repeat expansions, haploinsufficiencies, or a combination thereof in the STXBP1 gene.

[0050] Syntaxin-binding protein 1 (STXBPP) encephalopathy (also known as STXBP1 Development and Epileptic Encephalopathy” or “STXBP1 Encephalopathy”, used interchangeable herewith) is a rare, devastating neurodevelopmental disorder associated with developmental delay and treatment-resistant seizures. STXBP1 encephalopathy is caused by de novo heterozygous mutations in the syntaxin-binding protein 1 gene (STXBPP), which encodes STXBP1 protein.

[0051] In some embodiments, the composition comprises a viral particle. The viral particle may comprise one or more nucleic acids. The nucleic acids may encode for an STXBP1 gene product. A “STXBP1 gene product” describes a polypeptide generated from transcription and translation of an STXBP1 gene. An STXBP1 gene product may be of any STXBP1 isoform (e.g., isoform a, isoform b, isoform c, isoform d, isoform e, isoform f, isoform g, or isoform h). The nucleic acid that encodes an STXBP1 gene product may be a STXBP1 gene from any organism, including for example, a worm, a fruit fly, a mouse, a rat, any non-human primate, and a human. The nucleic acids that encode an STXBP1 gene product may be an STXBP1 gene with one or more silent mutations. In some embodiments, the nucleic acids encode for isoform a of STXBP1 (also known as STXBPla). In some embodiments, the nucleic acids encode for isoform b of STXBP1 (also known as STXBPlb). In some embodiments, the nucleic acids encode for isoform c of STXBP1 (also known as STXBPlc). In some embodiments, the nucleic acids encode for isoform d of STXBP1 (also known as STXBPld).

[0052] In some embodiments, the viral particle comprises a nucleic acid that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 3, or any range or value derivable therein. In certain embodiments, the viral particle comprises a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:3 (isoform a). In some embodiments, the viral particle comprises a nucleic acid that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 4, or any range or value derivable therein. In certain embodiments, the viral particle comprises a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO: 4 (isoform b).

[0053] In some embodiments, the viral particle comprises a nucleic acid that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5, or any range or value derivable therein. In certain embodiments, the viral particle comprises a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:5 (isoform c).

[0054] In some embodiments, the viral particle comprises a nucleic acid that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6, or any range or value derivable therein. In certain embodiments, the viral particle comprises a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:6 (isoform d).

[0055] Particular sequences of STXBP1 isoform a, STXBP1 isoform b, STXBP1 isoform c, and STXBP1 isoform d are provided below.

[0056] (SEQ ID NO: 3, STXBP1 isoform a):

[0057] MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDSC PDALFNELVK SRAAKVIKTL TEINIAFLPY ESQVYSLDS A DSFQSFYSPH KAQMKNPILE RLAEQIATLC ATLKEYPAVR YRGEYKDNAL LAQLIQDKLD AYKADDPTMG EGPDKARSQL LILDRGFDPS SPVLHELTFQ AMSYDLLPIE NDVYKYETSG

[0058] IGEARVKEVL LDEDDDLWIA LRHKHIAEVS QEVTRSLKDF SSSKRMNTGE

[0059] KTTMRDLSQM LKKMPQYQKE LSKYSTHLHL AEDCMKHYQG TVDKLCRVEQ

[0060] DLAMGTDAEG EKIKDPMRAI VPILLDANVS TYDKIRIILL YIFLKNGITE ENLNKLIQHA QIPPEDSEII TNMAHLGVPT VTDSTLRRRS KPERKERISE QTYQLSRWTP IIKDIMEDTI EDKLDTKHYP YISTRSSASF STTAVSARYG HWHKNKAPGE YRSGPRLIIF ILGGVSLNEM RCAYEVTQAN GKWEVLIGST HILTPTKFLM DLRHPDFRES SRVSFEDQAP TME

[0061] (SEQ ID NO: 4, STXBP1 isoform b):

[0062] MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDSC PDALFNELVK SRAAKVIKTL TEINIAFLPY ESQVYSLDSA DSFQSFYSPH KAQMKNPILE RLAEQIATLC ATLKEYPAVR YRGEYKDNAL LAQLIQDKLD AYKADDPTMG

[0063] EGPDKARSQL LILDRGFDPS SPVLHELTFQ AMSYDLLPIE NDVYKYETSG

[0064] IGEARVKEVL LDEDDDLWIA LRHKHIAEVS QEVTRSLKDF SSSKRMNTGE

[0065] KTTMRDLSQM LKKMPQYQKE LSKYSTHLHL AEDCMKHYQG TVDKLCRVEQ

[0066] DLAMGTDAEG EKIKDPMRAI VPILLDANVS TYDKIRIILL YIFLKNGITE ENLNKLIQHA QIPPEDSEII TNMAHLGVPI VTDSTLRRRS KPERKERISE QTYQLSRWTP IIKDIMEDTI EDKLDTKHYP YISTRSSASF STTAVSARYG HWHKNKAPGE YRSGPRLIIF ILGGVSLNEM RCAYEVTQAN GKWEVLIGST HILTPQKLLD TLKKLNKTDE EISS

[0067] (SEQ ID NO: 5, STXBP1 isoform c):

[0068] MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDYA LFNELVKSRA AKVIKTLTEI NIAFLPYESQ VYSLDSADSF QSFYSPHKAQ MKNPILERLA EQIATLCATL KEYPAVRYRG EYKDNALLAQ LIQDKLDAYK ADDPTMGEGP

[0069] DKARSQLLIL DRGFDPSSPV LHELTFQAMS YDLLPIENDV YKYETSGIGE

[0070] ARVKEVLLDE DDDLWIALRH KHIAEVSQEV TRSLKDFSSS KRMNTGEKTT

[0071] MRDLSQMLKK MPQYQKELSK YSTHLHLAED CMKHYQGTVD KLCRVEQDLA

[0072] MGTDAEGEKI KDPMRAIVPI LLDANVSTYD KIRIILLYIF LKNGITEENL NKLIQHAQIP PEDSEIITNM AHLGVPIVTD STLRRRSKPE RKERISEQTY QLSRWTPIIK DIMEDTIEDK LDTKHYPYIS TRSSASFSTT AVSARYGHWH KNKAPGEYRS GPRLIIFILG

[0073] GVSLNEMRCA YEVTQANGKW EVLIGSTHIL TPQKLLDTLK KLNKTDEEIS S

[0074] (SEQ ID NO: 6, STXBP1 isoform d):

[0075] MHDVIKKVKK KGEWKVLVVD QLSMRMLSSC CKMTDIMTEG ITIVEDINKR REPLPSLEAV YLITPSEKSV HSLISDFKDP PTAKYRAAHV FFTDSCPDAL FNELVKSRAA KVIKTLTEIN IAFLPYESQV YSLDSADSFQ SFYSPHKAQM KNPILERLAE QIATLCATLK EYPAVRYRGE YKDNALLAQL IQDKLDAYKA DDPTMGEGPD KARSQLLILD

[0076] RGFDPSSPVL HELTFQAMSY DLLPIENDVY KYETSGIGEA RVKEVLLDED

[0077] DDLWIALRHK HIAEVSQEVT RSLKDFSSSK RMNTGEKTTM RDLSQMLKKM PQYQKELSKY STHLHLAEDC MKHYQGTVDK LCRVEQDLAM GTDAEGEKIK

[0078] DPMRAIVPIL LDANVSTYDK IRIILLYIFL KNGITEENLN KLIQHAQIPP EDSEIITNMA HLGVPIVTDS TLRRRSKPER KERISEQTYQ LSRWTPIIKD IMEDTIEDKL DTKHYPYIST RSSASFSTTA VSARYGHWHK NKAPGEYRSG PRLIIFILGG VSLNEMRCAY EVTQANGKWE VLIGSTHILT PTKFLMDLRH PDFRESSRVS FEDQAPTME

[0079] In certain embodiments, the nucleic acids encode for part or all of one of SEQ ID NOs:3- 6. In some embodiments, the viral particle comprises a sequence that encodes for an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of SEQ ID NOs:3-6. In some embodiments, the viral particle comprises a nucleic acid sequence encoding one of SEQ ID NOs:3-6, wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer of the codons within one of SEQ ID NOs:3-6 is substituted with another codon, optionally comprising a conservative amino acid substitution or silent mutation, and / or are deleted and / or an insertion (including 5’ and / or 3’ extensions) of 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer codons or any combination of substitutions, deletions and / or insertions, wherein the substitutions, deletions and / or insertions do not unduly impair the structure and / or function of STXBP1.

[0080] Conservative amino acid substitutions are known in the art. In particular embodiments, a conservative amino acid substitution includes substitutions within one or more of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and / or phenylalanine, tyrosine.

[0081] Certain embodiments of the disclosure concern the treatment of an individual with an encephalopathy. The encephalopathy may be an epileptic encephalopathy, such as an early infantile epileptic encephalopathy. In some embodiments, the individual has Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, or Rett syndrome with mutated STXBP1. In some embodiments, the encephalopathy is STXBP1 encephalopathy or early infantile epileptic encephalopathy type 4. In some embodiments, the encephalopathy is STXBP1 encephalopathy. The encephalopathy may be caused by, or associated with, one or more missense mutations, nonsense mutations, deletions, inversions, insertions, duplications, frameshift mutations, repeat expansions, haploinsufficiencies, or a combination thereof in a gene, including STXBP1. The encephalopathy may be caused by, or associated with, one or more mutations in a gene, including STXBP1.

[0082] Accordingly, provided herein are methods of treating STXBP1 encephalopathy, or a symptom of STXBP1 encephalopathy, in a subject, comprising: (a) diagnosing a subject with STXBP1 encephalopathy affecting a target in vivo environment; and (b) treating STXBP1 encephalopathy by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with increased transduction, transgene expression, or specificity for the target in vivo environment.

[0083] Transgene delivery

[0084] In some embodiments, an individual is treated by a method comprising administering a therapeutically effective amount of one or more compositions encompassed herein, including any viral particle herein, to the individual. The composition may increase the level of a heterologous transgene in the individual, including in cells of the individual. In some embodiments, the composition administered to the individual restores the level of the transgene to a level found in a control individual. In some embodiments, the compositions restore cognitive abilities in the individual. In some embodiments, the compositions reduce the number and / or severity of seizures in the individual. In some embodiments, the compositions restore motor functions in the individual. In some embodiments, the compositions restore psychiatric and behavioral functions in the individual.

[0085] Transgene delivery strategies may result in gene replacements or gene supplementation, used interchangeably herein. Specifically, gene replacement specifically refers to introduction of exogenous nucleic acids to host cells to restore gene expression of a mutated or deleted gene. Gene supplementation refers to the introduction of exogenous nucleic acids to host cells to increase a specific gene's expression in the context of mutations or deletions that result in reduced gene expression from host DNA.

[0086] The transgene may be in cis with two inverted terminal repeats (ITRs) flanking the transgene. Due to the limited packaging capacity of the rAAV (~5kB), in some cases, the transgene may be split between two AAV vectors, the first with 3’ splice donor and the second with a 5 ’ splice acceptor. Upon co-infection of a cell, concatemers form, which are spliced together to express a full-length transgene.

[0087] Effective dosages of the viral particles to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid to be delivered, and can be determined in a routine manner. Examples of effective doses for achieving therapeutic effects include virus titers of at least about 105, 106, 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015transducing units or more.

[0088] In some embodiments, the viral particle is administered directly to the CNS, e.g., the brain or the spinal cord. Direct administration can result in high specificity of transduction of CNS cells, e.g., wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are CNS cells. Any method known in the art to administer vectors directly to the CNS can be used. The vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The vector may also be administered to different regions of the eye such as the retina, cornea, or optic nerve. The vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector.

[0089] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the ail, including but not limited to, intrathecal, intracerebral, intra-cistema magna, intraventricular, intranasal, intra- aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri ocular (e.g., sub-Tenon's region) delivery or any combination thereof.

[0090] Typically, the viral vector will be administered in a liquid formulation by direct injection to the desired region or compartment in the CNS. In some embodiments, the vector can be delivered via a reservoir and / or pump. In other embodiments, the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets. As a further alternative, the vector may be administered as a solid, slow-release formulation.

[0091] In some embodiments, one can inject the AAV particles directly into the brain tissues. In some embodiments, one can deliver the particles into cerebrospinal fluid (CSF), such as by injection into the ventricle or lumbar intrathecal space. In some embodiments, one can deliver the particles systemically, such as by injection into a blood vessel, and then let the AAV particles cross the blood brain barrier (BBB). In particular embodiments, one or more AAV particles of the disclosure have the ability to cross the BBB. In embodiments wherein any AAV particle is considered to have very weak or no ability to cross BBB, one can inject the AAV particles directly into the brain tissues, such as by intrap arenchymal injection.

[0092] In general, methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some instances, methods comprise administering a therapeutic rAAV composition by intraperitoneal injection. In some instances, methods comprise administering a therapeutic rAAV composition by intravenous (“IV”) administration. It is conceivable that one may also administer therapeutic rAAV compositions disclosed herein by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intracerebroventricular administration, intrathecally, or any other suitable parenteral administration. Routes, dosage, time points, and duration of administrating therapeutics may be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition.

[0093] The term “CNS” or “central nervous system” means a tissue selected from brain, including thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, and from brainstem and spinal cord. The brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital, and parietal lobes.

[0094] The term “systemic delivery” is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes both blood or cerebrospinal fluid circulatory systems. Examples of systemic administration for the CNS include intraarterial, intravenous or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location, in the spine (i.e., but not limited to lumbar) or brain (i.e., but not limited to cistema magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0095] In some embodiments, routes for administration include administration into the CSF, for example, via an intracerebroventricular (ICV), intrathecal cisternal, intra cistema magna, or intrathecal lumbar route. Particular embodiments result in delivery to neurons and glial cells of the brain. Other routes of delivery to the CNS / brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, intranasal administration, endovascular administration, and intraparenchymal administration.

[0096] An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition. Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject. An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity observed in normal individuals (e.g. individuals who do not suffer from the disease or condition). The dosage amount and / or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition. In some embodiments, as a patient is started on a regimen of a therapeutic rAAV composition, the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.

[0097] In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases, the concentration of infectious particles is 2xl07, 2xl08, 2xl09, 2xlO10, 2xlOn, 2xl012, 2xl013, 2xl014, 2xl015, 2xl016, or 2xl017. In some cases, the concentration of the infectious particles is 3xl07, 3xl08, 3xl09, 3xlO10, 3xlOn, 3xl012, 3xl013, 3xl014, 3xl015, 3xl016, or 3xl017. In some cases, the concentration of the infectious particles is 4xl07, 4xl08, 4xl09, 4xlO10, 4xlOn, 4xl012, 4xl013, 4xl014, 4xl015, 4xl016, or 4xl017. In some cases, the concentration of the infectious particles is 5xl07, 5xlO8, 5xl09, 5xlO10, 5xlOn, 5xl012, 5xl013, 5xl014, 5xl015, 5xl016, or 5xl017. In some cases, the concentration of the infectious particles is 6xl07, 6xl08, 6xl09, 6xlO10, 6xlOn, 6xl012, 6xl013, 6xl014, 6xl015, 6xl016, or 6xl017. In some cases, the concentration of the infectious particles is 7xl07, 7xl08, 7xl09, 7xlO10, 7xlOn, 7xl012, 7xl013, 7xl014, 7xl015, 7xl016, or 7xl017. In some cases, the concentration of the infectious particles is 8xl07, 8xlO8, 8xl09, 8xlO10, 8xlOn, 8xl012, 8xlO13, 8xl014, 8xl015, 8xl016, or 8xl017. In some cases, the concentration of the infectious particles is 9xl07, 9xl08, 9xl09, 9xlO10, 9xlOn, 9xl012, 9xl013, 9xl014, 9xl015, 9xl016, or 9xl017.

[0098] Disclosed herein, in some embodiments are formulations of pharmaceutically-acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In some instances, the rAAV compositions are suitably formulated pharmaceutical compositions disclosed herein, to be delivered cither intraocularly, intravitrcally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, intraperitoneally, by nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.

[0099] In some embodiments, the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0100] In some cases, for administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular-, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

[0101] Disclosed herein are sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example by intravenous administration.

[0102] Also provided herein are formulations in a neutral or salt form. Pharmaceutically- acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0103] Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0104] The amount of AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing. For example, the number of infectious particles administered to a mammal may be on the order of about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, or even higher, infectious particlcs / ml given either as a single dose or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In fact, in certain embodiments, it may be desirable to administer two or more different AAV vector compositions, either alone, or in combination with one or more other therapeutic drugs, to achieve the desired effects of a particular therapy regimen. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the therapeutic rAAV composition used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0105] The effective dosage ranges may be adjusted based on subject’s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes.

[0106] In certain embodiments, the daily dosage range and / or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized.

[0107] Viral Vectors

[0108] Certain embodiments of the disclosure concern methods of producing viral particles. In some embodiments, the method comprises providing to a cell in vitro, (a) a template comprising (i) a nucleic acid encoding for a gene product, and (ii) packaging signal sequences sufficient for the encapsidation of an AAV template into virus particles (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) AAV sequences sufficient for replication and encapsidation of the template into viral particles (e.g., the AAV rep and AAV cap sequences encoding an AAV capsid). The template and AAV replication and capsid sequences are provided under conditions such that recombinant virus particles comprising the template packaged within the capsid are produced in the cell. The method can further comprise the step of collecting the virus particles from the cell. Virus particles may be collected from the medium and / or by lysing the cells. Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an cpisomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). For example, upon delivery to a host in vivo environment, a rAAV may ( 1 ) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid, (5) convert of the heterologous nucleic acid from single- stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell. rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell), transduction efficiency (the effectiveness of a virus, engineered or naturally occurring, at delivering its DNA component to a host cell), and transgene expression (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0109] An rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., therapeutic nucleic acid, gene editing machinery). The AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in an approximately 1:1:10 ratio to form the viral capsid. VP1 covers the whole of VP2 protein in addition to a -137 amino acid N-terminal region (VPlu), VP2 covers the whole of VP3 in addition to -65 amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0110] While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In certain embodiments, a parent AAV capsid sequence comprises a VP1, VP2 and / or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

[0111] The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded P-barrel motif (PB-pi) and a small a-helix (aA). The loop regions inserted between the P-strands consist of the distinctive HI loop between P-strands H and I, the DE loop between P-strands D and E, and nine variable regions (VRs), which are typically surface exposed on the capsid structure. These VRs, such as VR-VIII, which contains AA588 in AAV9, can be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigenic specificity. Disclosed herein are AAV capsids comprising AAV capsid proteins with a substitution at the AA588 and peptide insertion between AA588 and AA589 that confer a desired tropism characterized by a higher efficiency and specificity for transduction in specific cell-types, including, for example, cells within the CNS or brain cell types (e.g., brain endothelial cells, neurons, astrocytes). In particular, the AAV capsid proteins disclosed herein enable rAAV- mediated transduction of a heterologous nucleic acid (e.g., transgene) in the CNS of a subject. The AAV capsids of the present disclosure, or the AAV capsid proteins, may be formulated as a pharmaceutical composition. In addition, the AAV capsids or the AAV capsid proteins can be isolated and purified to be used for a variety of applications. Disclosed herein are recombinant AAV (rAAV) capsids which comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein. In some embodiments, the AAV capsids are used in the methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some instances, the rAAV capsids have desired AAV tropisms rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.

[0112] The rAAV capsid proteins are engineered for optimized transduction and transgene expression in the CNS, for example the brain, of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid proteins are engineered to have tropisms that eliminate the need for intracranial injection, while also achieving widespread and efficient transduction of an encapsidated transgene. In particular, the tropisms comprise at least one of an increased specificity and efficiency (e.g., of viral transduction) in the CNS of a subject, as compared to a reference AAV.

[0113] The engineered AAV capsid proteins described herein have, in some cases, a peptide insertion and amino acid substitution that is heterologous to the parental AAV capsid protein at the amino acid positions 587 and 590 in AAV9 (SEQ ID NO: 2). In some embodiments, the amino acids flanking the peptide insertion do not originate from the parental AAV capsid protein. The amino acids flanking the insertion may share sequence identity with the amino acids at the same position within the parental serotype or equivalent amino acid position as the substitution and peptide insertion in alternative AAV serotypes or engineered variant capsid proteins. Also disclosed herein are rAAVs with engineered capsid proteins that are optimized for targeting specific organ or tissue within a subject. In a non-limiting example, the rAAVs of the present embodiment have increased specificity, transduction, and transgene expression in the CNS.

[0114] Seven amino acids comprise the peptide insertion sequence (7-mer, respectively) that is inserted or substituted within VR-VIII in the parental AAV capsid protein. Aspects provided herein provide amino acid insertions comprising seven amino acid polymer (7-mer) inserted between AA588-589 and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and / or AA589-590) to produce an eleven amino acid polymer (11-mer) at the 588 loop of a parental AAV capsid protein.

[0115] The capsid protein is engineered, relative to an AAV9 capsid protein, by substituting amino acid position 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 the amino acids RNGEVFI, and parental AAV9 amino acids AQ in positions 589 and 590.

[0116] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVFIAQ.

[0117] Peptide insertion sequences of the disclosure include sequences that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) alter binding affinities, and (3) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., equivalent, conservative or nonconservative substitutions, deletions, or additions) may be made in a sequence.

[0118] An AAV vector can comprise a viral genome comprising a nucleic acid sequence encoding the recombinant AAV (rAAV) capsid protein described herein. The viral genome can comprise a Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some instances, the Cap gene is modified encoding a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1- VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rcp68, Rcp52, Rcp40 and AAP proteins.

[0119] In some instances, the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and the 3’ ITR each originate from different serotypes, e.g. 5’ ITR from serotype AAV2 and 3’ ITR from AAV5. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency.

[0120] A conservative amino acid substitution refers to the substitution of an amino acid in an insertion sequence with a functionally similar amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity.

[0121] In some embodiments, methods of increasing transduction of an encoded gene in a target in vivo environment comprise delivering a rAAV particle described herein, the rAAV engineered to have an increased transduction enrichment in a target in vivo environment (e.g., tissue or cell type). In some instances, the increased transduction enrichment comprises a 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50- fold or 100-fold increase, or more, relative to a reference AAV. In some instances, the increased transduction enrichment is at least 2-fold. In some instances, the increased transduction enrichment is at least 10-fold. In some instances, the increased transduction enrichment is at least 20-fold.

[0122] Methods of delivering a heterologous nucleic acid to a target in vivo environment are also provided comprising delivering the rAAV particle described herein that has been engineered to have an increased expression or specificity in an in vivo environment (e.g., tissue or cell type), as compared to a reference AAV. Methods, in some cases, comprise detecting whether a rAAV possesses more specificity for an in vivo environment, including measuring a level of gene expression product expressed from the vector encapsidated by the rAAV in a tissue sample obtained from the in vivo environment in a subject.

[0123] In some instances, the reference AAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof. Provided herein are methods of delivering a heterologous nucleic acid to a target in vivo environment comprising delivering a composition to the target in vivo environment selected from a CNS in a subject, the composition comprising a rAAV particle with a rAAV capsid protein, the rAAV capsid protein encapsidating a viral vector encoding a heterologous nucleic acid (e.g., therapeutic nucleic acid). In some embodiments, the rAAV particle encapsidating the heterologous nucleic acid comprises a rAAV capsid protein engineered with an increased transduction enrichment when measured in the CNS of the subject, even when administered to the subject systemically.

[0124] Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased transduction and transgene expression efficiency when measured in the CNS in the subject. In some embodiments, delivery is systemic. Alternatively, delivery is direct (e.g., into the affected area of the CNS).

[0125] Promoter / Enhancers

[0126] A variety of promoter / enhancer elements may be used depending on the level and tissuespecific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0127] Promoter / enhancer elements can be native to the target cell or subject to be treated and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0128] Promoters are DNA regions that initiate gene transcription by controlling the binding of RNA polymerase to the vector DNA to begin the process toward expression of the encoded protein. Promoters control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein. Thus, the promoter region controls when and where in the organism the gene of interest is expressed. Exemplary promoters include CMV, CBh, human synapsin I, EFla, SV40, PGK1, Ubc, human beta actin, and CAG. In preferred embodiments, the vector comprises a promoter selected from a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter. Sec Miyazaki, J; Takaki, S; Araki, K; Tashiro, F; Tominaga, A; Takatsu, K; Yamamura, K (Jul 15, 1989). "Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5". Gene. 79 (2): 269-77; Grey et al., Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors, Hum Gene Ther. 2011 Sep; 22(9): 1143— 1153; Glover et al., Adenoviral-mediated, High-Level, Cell-Specific Transgene Expression: A SYN1-WPRE Cassette Mediates Increased Transgene Expression With No Loss of Neuron Specificity, Mol Ther. 2002 May; 5(5 Pt l):509-16; the content of each of which is incorporated herein by reference.

[0129] In some instances, the vector may comprise a promoter and / or enhancer, for example, a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter may be CMV promoter, a CMV-P-Actin-intron-P-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, Hl promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter. As a non-limiting example, promoters can be tissue- specific expression elements that include, but are not limited to, human elongation factor la-subunit (EFlot), immediate-early cytomegalovirus (CMV), chicken P-actin (CBA) and its derivative CAG, the P glucuronidase (GUSB), and ubiquitin C (UBC). The vector may include a tissue-specific expression elements for neurons such as, but not limited to, neuronspecific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor Bchain (PDGF-P), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2),Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The vector may comprise a tissue-specific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The vector may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.

[0130] Various regulatory elements may be included in vectors of the invention including posttranscriptional regulatory elements (PREs) such as those derived from hepatitis B virus (HPRE), woodchuck hepatitis virus (WPRE), human heat shock protein 70 mRNA (Hsp70), the vascular endothelial growth factor (SP163), the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM), or the first intron of human cytomegalovirus immediate early gene (Intron A). Posttranscriptional regulatory elements can help enhance gene expression when included in expression vectors such as those described herein. Particular PREs may exhibit cell-specific and / or gene-specific regulatory enhancement and those factors are considered when selecting a PRE.

[0131] Examples

[0132] Vector and STXBP1 sequences

[0133] The peptides and nucleic acids were engineered for expression of an AAV vector of the invention for delivery of the STXBP1 transgene.

[0134] AAV vector capsid sequence comprising ATRNGEVFIAQ

[0135] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSATRNGEVFIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPS PLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENS KRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 7)

[0136] Nucleic acid sequence encoding capsid ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAG

[0137] GAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAA

[0138] CAACATCAAGACAACGCTaGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCC

[0139] GGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCG

[0140] AGCACGACAAaGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAG

[0141] TACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGG

[0142] GGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTG

[0143] GTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAG

[0144] TCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCT

[0145] AAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCA

[0146] ACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTC

[0147] AGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTT

[0148] CCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACC

[0149] AGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCC

[0150] AACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCC

[0151] TGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAG

[0152] CGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTt

[0153] AACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAA

[0154] CCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCT

[0155] CGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCC

[0156] TCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTT

[0157] TTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTT

[0158] CAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCT

[0159] GGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCTAGAACTAT

[0160] TAACGGCAGCGGCCAAAACCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCA

[0161] ACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGT

[0162] GTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCT

[0163] TCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCtctCA

[0164] CAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACA

[0165] AGGTACtGGCAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAG AAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAAC CACCAGAGTGCCACCAGAAACGGCGAAGTCTTCATCGCCCAAGCCCAAACCGGTTG GGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACC TGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTC CGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAAC ACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTC ATCACCCAGTATTCTACTGGtCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAG GAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTC TAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCAT TGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ ID NO: 8) hSTXBPlb gene peptide sequence

[0166] MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMTDIMTEGITIV EDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTDSCPDALFNELV KSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKNPILERLAEQIATLC ATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGPDKARSQLLILDRGFD PSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLLDEDDDLWIALRHKHIAE VSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQYQKELSKYSTHLHLAEDCM KHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPILLDANVSTYDKIRIILLYIFLK NGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDSTLRRRSKPERKERISEQTYQLSR WTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTAVSARYGHWHKNKAPGEYRSGPRLI IFILGGVSLNEMRCAYEVTQANGKWEVLIGSTHILTPQKLLDTLKKLNKTDEEISS (SEQ ID NO: 4)

[0167] DNA sequence encoding hSTXBPlb cargo

[0168] ATGGCCCCCATTGGCCTCAAAGCTGTTGTCGGAGAGAAGATTATGCATGATGTGATA AAGAAGGTCAAGAAGAAGGGGGAATGGAAGGTGCTGGTGGTGGATCAGTTAAGCA TGAGGATGCTGTCCTCCTGCTGCAAGATGACAGACATCATGACCGAGGGCATAACG ATTGTGGAAGATATCAATAAGCGCAGAGAGCCGCTCCCCAGCCTGGAGGCTGTGTA TCTCATCACTCCATCCGAGAAGTCCGTCCACTCTCTCATCAGTGACTTTAAGGACCC GCCGACTGCTAAATACCGGGCTGCACACGTCTTCTTCACTGACTCTTGTCCAGATGC CCTGTTTAATGAACTGGTAAAATCCCGAGCAGCCAAAGTCATCAAAACTCTGACGG

[0169] AAATCAATATTGCATTTCTCCCGTATGAATCCCAGGTCTATTCCTTGGACTCTGCTGA

[0170] CTCTTTCCAAAGCTTCTACAGTCCCCACAAGGCTCAGATGAAGAATCCTATACTGGA

[0171] GCGCCTGGCAGAGCAGATCGCGACCCTTTGTGCCACCCTGAAGGAGTACCCGGCTG

[0172] TGCGGTATCGGGGGGAATACAAGGACAATGCCCTGCTGGCTCAGCTAATCCAGGAC

[0173] AAGCTCGATGCCTATAAAGCTGATGATCCAACAATGGGGGAGGGCCCAGACAAGGC

[0174] ACGCTCCCAGCTCCTGATCCTGGATCGAGGCTTTGACCCCAGCTCCCCTGTGCTCCA

[0175] TGAATTGACTTTTCAGGCTATGAGTTATGATCTGCTGCCTATCGAAAATGATGTATA

[0176] CAAGTATGAGACCAGCGGCATCGGGGAGGCACGGGTGAAGGAGGTGCTCCTGGACG

[0177] AGGACGACGACCTGTGGATAGCACTGCGCCACAAGCACATCGCAGAGGTGTCCCAG

[0178] GAAGTCACCCGGTCTCTGAAAGATTTTTCTTCTAGCAAGAGAATGAATACTGGAGAG

[0179] AAGACCACCATGCGGGACCTGTCCCAGATGCTGAAGAAGATGCCTCAGTACCAGAA

[0180] AGAGCTCAGCAAGTACTCCACCCACCTGCACCTTGCTGAGGACTGTATGAAGCATTA

[0181] CCAAGGCACCGTAGACAAACTCTGCCGAGTGGAGCAGGACCTGGCCATGGGCACAG

[0182] ATGCTGAGGGAGAGAAGATCAAGGACCCTATGCGAGCCATCGTCCCCATTCTGCTG

[0183] GATGCCAATGTCAGCACTTATGACAAAATCCGCATCATCCTTCTCTACATCTTTTTGA

[0184] AGAATGGCATCACGGAGGAAAACCTGAACAAACTGATCCAGCACGCCCAGATACCC

[0185] CCGGAGGATAGTGAGATCATCACCAACATGGCTCACCTCGGCGTGCCCATCGTCACC

[0186] GATTCCACGCTGCGTCGCCGGAGCAAGCCGGAGCGGAAGGAACGCATCAGCGAGCA

[0187] GACCTACCAGCTCTCACGGTGGACTCCGATTATCAAGGACATCATGGAGGACACTAT

[0188] TGAGGACAAACTTGACACCAAACACTACCCTTATATCTCTACCCGTTCCTCTGCCTC

[0189] CTTCAGCACCACCGCCGTCAGCGCCCGCTATGGGCACTGGCATAAGAACAAGGCCC

[0190] CAGGCGAGTACCGCAGTGGCCCCCGCCTCATCATTTTCATCCTTGGGGGTGTGAGCC

[0191] TGAATGAGATGCGCTGCGCCTACGAGGTGACCCAGGCCAACGGAAAGTGGGAGGTG

[0192] CTGATAGGATCCACACACATCCTCACCCCACAGAAACTGCTGGACACACTGAAGAA

[0193] ACTGAATAAAACAGATGAAGAAATAAGCAGTTAG (SEQ ID NO: 9)

[0194] 5’ ITR (L-1TR)

[0195] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGAC

[0196] CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT

[0197] CCATCACTAGGGGTTCCT (SEQ ID NO: 10) 3 ’ ITR (R-ITR) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG AGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 11)

[0198] Example 1: Capsid administration

[0199] The rAAVs of the invention disclosure were designed as a modified adeno-associated virus serotype 9 (AAV9, SEQ ID NO: 2) capsid containing an amino acid substitution in AA588 and 7-mer insertion between AA588 and AA589 (together, ATRNGEVFIAQ, SEQ ID NO: 1) to the rAAV capsid protein and has been engineered to deliver the human syntaxin binding protein 1, isoform b (hSTXBP lb) gene to the CNS following a single intravenous injection. rAAVs of the invention were used to deliver a DNA construct (CAG-hSTXBPlb-WPRE-hGHpA) that encodes the b isoform of the wild-type (WT) human STXBP1 transgene (hSTXBP 1 b. NCBI Reference Sequence NMOO 1032221.6) expressed from a chicken beta-actin promoter with CMV intermediate-early enhancer element and hybrid chicken beta-actin and rabbit P-globin intron (all together named CAG), a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE), and a human growth hormone polyadenylation signal (hGHpA). The rAAVs of the invention have been engineered for preferential blood-brain barrier (BBB) penetrance and CNS transduction in primates for delivery of the hSTXBP lb DNA cargo. The rAAVs of the invention were produced in HEK293 cells and formulated in 0.01% pluronic acid in PBS. For all dosing, the rAAVs of the invention were diluted to the appropriate titer using 0.01% pluronic acid in PBS.

[0200] This work establishes significantly improved BBB penetrance and CNS transduction in NHPs - achieving levels of cargo expression expected to surpass efficacy requirements for STXBP1 gene supplementation or replacement in the human STXBP1 encephalopathy patient population.

[0201] Study 1: NHP administration

[0202] Syntaxin-binding protein 1 (STXBP1) encephalopathy (also known as genetic epilepsy due to STXBP1 mutations) is a rare and severe genetic epilepsy and neurodevelopmental disorder that impacts approximately 1 in 30,000 newborns. De novo heterozygous mutations in the STXBP1 gene result in impaired synaptic vesicle release and neuronal communication. Patients suffer from treatment-resistant epilepsy, profound intellectual disabilities, speech and motor impairments, behavioral issues, and increased risk of early mortality due to sudden unexpected death in epilepsy (SUDEP). Currently, no approved disease-modifying therapies exist for this debilitating disorder.

[0203] A study was conducted to assess the transduction rates and cell type tropism of rAAVs of the invention in male and female cynomolgus macaques and subsequently examine the efficacy of the rAAVs of the invention in crossing the primate BBB for CNS delivery of the hSTXBPl gene supplementation or replacement strategy.

[0204] In this NHP capsid characterization study, NHPs were assigned to Group 1 (1.5E13 vg / kg, n=3) or Group 2 (2.9E13 vg / kg, n=3) (doses based on previous titer method using WPRE primers are 1.25E13 and 2.5E13 vg / kg, respectively), of the rAAVs of the invention delivered in a single intravenous (IV) injection.

[0205] FIG. 1 is a study timeline overview from NHP administration. About one week before administration, the following exams were conducted: physical and blood samples were collected for the following readouts: nAb titer assessment (Nabs), cytokine / complement analysis, hematology (complete blood count; CBC), serum chemistry (clinical chemistry), and PBMC’s for ELISpot assay analysis. Following dosing (Day 1) experimental observations were taken over 44 days. At study end on Day 42, gross observations, organ weights, and tissue collections were conducted.

[0206] Throughout a 6-week in-life period, safety of the drug product was assessed via in-life measurements, including weekly body weights and physical exams, routine body-temperature measurements, daily clinical observations, blood collection for clinical chemistry and CBC analyses, cytokine and complement analyses, nAb titer assessment, and ELISpot assay. At the conclusion of the in-life period, all animals were euthanized and assessed for transgene DNA biodistribution, RNA expression using WPRE probes, and STXBP1 protein expression via ELISA. Histopathology (CNS & peripheral organs, including DRG / liver, immunogenicity (ELISpot, Nabs, Cytokines), in-life measures (physical, neurological, clinical chemistry, and hematology), DNA-level (biodistribution), and protein-level expression (ELISA) were analyzed.

[0207] All safety read-outs corroborate that engineered capsids and DNA cargo were well tolerated at both doses. No notable in-life findings were observed, including clinical observations, physical examination, food intake, body weight, body temperature, neurological assessments, hematology, serum chemistry, gross observations, and organ weights.

[0208] No notable histopathology findings were observed from all sampled organs, including brain, meninges, spinal cord, dorsal root ganglia, heart, kidneys, liver, lungs, pancreas, skeletal muscle, sciatic nerve, vagus nerve, and spleen.

[0209] No notable immunogenicity findings were observed. All animals were AAV9 seronegative at dosing. No evidence was observed of adaptive immune response to capsid or cargo (ELISpot). No evidence was observed of significant cytokine / complement response 24 hours post-dosing.

[0210] FIG. 2A-D are graphs showing DNA level changes following AAV administration to NHPs. DNA Biodistribution (qPCR) was conducted to provide a quantitative measurement to detect transgene DNA from tissue lysates. STXBP1 cargo DNA biodistributed widely throughout key regions of interest (ROIs) of the NHP CNS, including cortical regions, for example the frontal (Fr) cortex (Ctx) (Level 1 and Level 2), Corpus Striatum, Hypothalamus, Primary Motor Ctx, Hippocampus, Temporal Ctx, Parietal Ctx, Thalamus, Midbrain, Occipital Ctx, and Cerebellum, at levels indicating an increased efficacy in comparison to previous AAV approaches to STXBP1 gene supplementation or replacement. DNA biodistribution of rAAVs of the invention surpassed parental AAV9 throughout the NHP cortex following IV delivery and was significantly liver de-targeted across all administered doses compared to AAV9. rAAVs of the invention were identified as a superior capsid to parental AAV9. DNA biodistribution confirms widespread CNS transduction (>4 times higher than wild type AAV9 at 2.9E13 vg / kg).

[0211] FIG. 3A-D are graphs showing RNA level changes following systemic administration of rAAVs of the invention to NHPs. RNA biodistribution (determined via qPCR assay) was conducted to provide a quantitative measurement to detect transgene RNA from tissue lysates. STXBP1 cargo delivered by rAAVs of the invention resulted in robust RNA biodistribution throughout key areas of the NHP CNS, including cortical regions. RNA biodistribution of rAAVs of the invention far surpasses parental AAV9 throughout the NHP cortex following i.v. delivery and STXBPI RNA is expressed significantly less in the liver across all doses of engineered A A Vs administered in comparison to AAV9. rAAVs of the invention were again identified as a superior capsid to parental AAV9 and capable of delivering STXBP1 throughout the CNS. RNA levels in liver following systemic administration with rAAVs of the invention are >2 times lower than wild type AAV9 at a dose of 2.9E13 vg / kg. RNA biodistribution confirms widespread CNS transduction for rAAVs of the invention and is >86 times higher than parental AAV9. In analyzing brain RNA enrichment vs. DNA liver de-targeting, rAAVs of the invention are >500 times improved relative to AAV9 levels of RNA enrichment vs. DNA liver-detargeting, providing evidence that rAAVs of the invention at both doses could achieve efficacy.

[0212] FIG. 4A-G are graphs showing protein expression level changes following systemic administration of rAAVs of the invention to NHPs, including initial (FIG. 4F) and final (FIG. 4G) total STXBP1 ELISA quantitative protein measurements.

[0213] All doses shown are in vg / kg. Average protein levels in the cortex normalized to baseline levels of STXBP1 are shown. To enable quantification of % increase in STXBP1 protein levels after systemic administration of rAAVs of the invention in dosed animals, region- specific comparisons were made to age-matched AAV-dosed (non-STXBPl cargo) control NHPs with the same genotype (wild type; WT) as the dosed NHPs (referred to as “Controls” and “wild-type; WT” in the Figure legend). One-Way ANOVA was conducted with Multiple comparisons of dosed groups vs. Controls. Primary Motor Cortex was not collected in the Control NHPs - instead, the data presented originates from the Control cortical average.

[0214] STXBP1 ELISA quantitative protein measurement was conducted. The rAAVs 2.9E13 dose group showed increases in STXBP1 in various cortical ROIs, with statistically significant increases above Control levels of STXBP1 protein levels in the Primary Motor, Parietal, and Occipital cortices. Statistically significant increases in STXBPI protein were detected in the CNS on average after systemic administration of rAAVs of the invention. rAAVs of the invention dosed at 2.9E13 vg / kg increased STXBPI protein levels in 3 of 5 cortical regions (primary motor, parietal, occipital) compared to Controls.

[0215] ELISA studies confirmed that rAAVs of the invention are capable of significantly increasing STXBPI levels throughout the cortex of wild type NHPs. The rAAVs of the invention systemically administered at a dose of 2.9E13 vg / kg showed a significant increase in average cortical STXBP1 levels, demonstrating potential therapeutic efficacy with the rAAVs of the invention. The significant increases in STXBP1 total protein above Control levels (quantified by ELISA) after systemic administration with rAAVs of the invention are statistically significant at the 2.9E13 vg / kg dose, supporting potential efficacy for patients.

[0216] FIG. 5A is a graph of histological quantification in CNS tissue following AAV administration to NHPs.

[0217] Due to STXBP1 cellular localization, immunohistochemical techniques do not allow for quantification of neuronal transduction at the protein level, when carrying STXBP1 cargo. Use of an HA-tagged surrogate cargo (gene of interest, GOI) delivered by rAAVs capsid proteins of the invention allows for representative neuronal transduction quantification at the protein level. The rAAVs capsid proteins of the invention encapsidating CAG-GOI-HA at an IV administered dose of 1.5E13 vg / kg was assayed in NHPs.

[0218] Histological quantification of HA positive (HA+) neurons with rAAV s capsid proteins of the invention carrying an HA-tagged surrogate cargo at two comparable doses tested with the rAAVs capsid proteins of the invention achieves neuronal transduction levels expected to achieve efficacy in patients with STXBP1 encephalopathy.

[0219] FIG. 5B is an immunofluorescence assay of the cortex following systemic administration of rAAVs capsid proteins of the invention encapsidating CAG-GOI-HA to NHPs. Anti-HA immunofluorescence in the cortex showing representative histological staining of HA-tagged cargo demonstrated robust expression and neuronal bias of rAAVs capsid proteins of the invention throughout the cortex.

[0220] FIG. 5C is an immunofluorescence assay of the thalamus following systemic administration of rAAV capsid proteins of the invention to NHPs. Anti-HA immunofluorescence in the thalamus also showed representative histological staining of HA-tagged cargo demonstrated robust expression and neuronal bias of rAAV capsid proteins of the invention throughout the thalamus. rAAV capsid proteins of the invention achieve similarly high levels of transduction and transgene expression brain-wide at both 1.5E13 vg / kg and 2.9E13 vg / kg doses.

[0221] Significant increases were found of the cargo delivered by rAAV s of the invention in DNA, RNA, and protein levels throughout the CNS, at levels expected to increase efficacy in comparison to previous AAV approaches to STXBP1 gene supplementation or replacement in the human STXBP1 Encephalopathy patient population.

[0222] FIG. 6A-B are graphs showing DNA biodistribution in NHPs following administration of rAAVs capsid proteins of the invention.

[0223] FIG. 7A-B are graphs showing RNA expression in NHP CNS tissue following administration of rAAVs capsid proteins of the invention.

[0224] FIG. 8A-C are graphs showing DNA biodistribution in liver and RNA expression in NHP peripheral tissue following administration of rAAVs capsid proteins of the invention.

[0225] IV administration of capsids of the invention to NHPs at 4 doses produced dosedependent increases in DNA transduction and RNA expression in the brain across all doses. RNA expression achieved by 2.9E13 vg / kg and by 4.7E13 and 5.9E13 is expected to fully correct seizures and provide meaningful correction to motor and cognitive deficits.

[0226] Compared to AAV9 at an equivalent dose to 2.9E13 vg / kg, DNA transduction in the liver was significantly decreased (20-fold) and RNA expression in the DRGs was significantly decreased (143-fold).

[0227] Discussion

[0228] In NHPs, an engineered AAV vector delivering STXBP1 achieved widespread DNA and RNA biodistribution, and increased STXBP1 protein expression, at low to moderate doses. The AAV capsid delivering a gene of interest showed protein expression in up to 70% of neurons brain- wide at low to moderate doses. The AAV vector delivering STXBP1 is well tolerated and has reduced liver targeting compared to traditional AAV9 vectors, enabling lower dosing and a broader therapeutic window.

[0229] These studies demonstrate potential for a first- and best-in-class, disease-modifying gene therapy for STXBP1 encephalopathy mutations. The gene therapy achieved levels of STXBP1 RNA expression brain-wide in NHPs that are expected to produce meaningful clinical benefit.

[0230] Study 2: human iPSC-derived STXBP1 knockout neurons administration

[0231] Capsids of the invention were administered to iPSC-derived STXBP1 knockout (KO) neurons at 3.3E5 vg / neuron, 1E6 vg / neuron, and 3.3E6 vg / neuron. FIG. 9 is an immunofluorescence assay of neuronal cells following administration of capsids of the invention. Confocal images of STXBP1 wild type (WT) and KO iPSC-dcrivcd human neurons were stained for STXBP1 (green), MAP2 (red) to image cell bodies and dendrites, and SV2A (white) to image synaptic terminals. STXBP1 protein is absent in KO neurons. Transduction with capsids of the invention delivering STXBP1 restored STXBP1 protein expression levels and cellular distribution at all doses.

[0232] FIG. 10A-C are graphs of calcium transients reflecting neuronal firing over time in neuronal cells following administration of capsids of the invention. Live calcium imaging demonstrated that capsids of the invention delivering STXBP1 restored synchronous network activity in KO neurons. Traces show the timing of activity-dependent calcium transients (red dots) in ~33 neurons per condition; spontaneous action potentials in vehicle (VEH)-treated KO neurons are desynchronized compared to synchronous firing of WT neurons. All doses are in vg / neuron.

[0233] Overall, gene supplementation with capsids of the invention delivering STXBP1 in human iPSC-derived STXBP1 KO neurons restored STXBP1 protein expression and neuronal network activity

[0234] FIG. 11A-B show graphs of STXBP1 levels measured by immunocytochemistry against STXBP1 in human IPSC-derived wild type or STXBP1 KO neurons following administration of a capsids of the invention.

[0235] Dose-dependent increases were shown in the percent of neurons transduced in human IPSC-derived STXBP1 KO neurons across the three doses of capsids tested. Dose-dependent increases were also shown in total STXBP1 protein in human IPSC-derived STXBP1 KO neurons. The data demonstrates that capsids of the invention can robustly transduce human neurons and generate STXBP1 cargo protein expression in human IPSC-derived STXBP1 KO neurons, in a dose-dependent manner.

[0236] FIG. 12A-B show graphs of synchronized firing and spike intensity at peak measured by live calcium imaging measuring real-time activity dependent changes in neuronal firing following administration of a capsids of the invention.

[0237] Restoration of synchronized neuronal activity (compared to human IPSC-derived KO neurons) was shown following administration of capsids of the invention in STXBP1 human IPSC-derived KO neurons to near-WT levels. Dose-dependent increases were shown inducing recovery of spike intensity, compared to human IPSC-derived KO neurons. The data demonstrates that expression of hSTXBPl cargo protein by capsids of the invention restores neuronal function in human IPSCderived STXBP1 KO neurons.

[0238] FIG. 13A-C show graphs of transduction efficiency measured by immunocytochemistry against STXBP1 in human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of a capsids of the invention.

[0239] Capsids of the invention transduced human IPSC-derived STXBP1 KO, HET, and WT neurons in a dose-dependent manner, with genotype-based effect on capsid transduction. The data demonstrates that capsids of the invention can comparably transduce human IPSC-derived neurons across three STXBP1 genotypes (KO, HET, or WT), in a dose-dependent manner.

[0240] FIG. 14A-C show graphs of STXBP1 mean intensity measured by immunocytochemistry against STXBP1 in human IPSC-derived STXBP1 KO, HET, and WT neurons following administration of capsids of the invention.

[0241] Capsids of the invention express cargo STXBP1 protein comparably across STXBP1 KO, HET, and WT human IPSC-derived neurons, in a dose dependent manner. The data demonstrates that capsids of the invention can comparably express the hSTXBPl cargo in human IPSC- derived neurons across three STXBP1 genotypes (KO, HET, or WT), in a dose-dependent manner.

[0242] FIG. 15A-C show graphs of STXBP1 mean intensity measured by immunocytochemistry against STXBP1 in neuronal compartments across STXBP1 KO, HET, and WT human IPSC- derived neurons following administration of capsids of the invention.

[0243] The cargo STXBP1 protein localized across the cell comparably across KO, HET, and WT human IPSC-derived neurons, in a dose dependent manner. The data demonstrates that the capsid-mediated cargo STXBP1 protein can localize across compartments of human IPSC- derived neurons across three STXBPI genotypes (KO, HET, or WT), in a dose-dependent manner.

[0244] FIG. 16A-C show graphs of hSTXBPl DNA biodistribution quantitatively measured by PCR to detect vector DNA across STXBPI KO, HET, and WT human IPSC-derived neurons following administration of capsids of the invention. FIG. 17A-C show graphs of hSTXBPl RNA biodistribution quantitatively measured by ddPCR to detect cargo RNA from in vitro lysates across STXBP1 KO, HET, and WT human IPSC-derived neurons following administration of a capsids of the invention.

[0245] Capsid vector DNA and cargo RNA levels increased in a dose-dependent manner, comparably across KO, HET, and WT human IPSC-derived neurons. The data demonstrates that capsids of the invention can achieve comparable levels of vector DNA and cargo RNA in human-derived iNeurons across three STXBP1 genotypes (KO, HET, or WT), in a dosedependent manner.

[0246] FIG. 18A-B show graphs of STX1 mean intensity measured by immunocytochemistry against Syntaxin- 1 and / or STXBP1 in STXBP1 KO human IPSC-derived neurons following administration of a capsids of the invention.

[0247] Capsids of the invention restored Syntaxin- 1 protein levels in STXBP1 KO human IPSC- derived neurons, in a dose-dependent manner. Capsids of the invention-mediated STXBP1 cargo protein colocalized with Syntaxin- 1 in human IPSC-derived STXBP1 KO neurons. The data demonstrates that restoration of STXBP1 via capsids of the invention subsequently restores Syntaxin- 1 protein levels, in a dose-dependent manner. Moreover, capsids of the invention- mediated cargo STXBP1 proteins interact with Syntaxin- 1.

[0248] Discussion

[0249] The rAAVs of the invention deliver the STXBP1 gene supplementation or replacement strategy throughout the human CNS at levels of DNA biodistribution, RNA expression, and hSTXBPlb protein levels expected to restore function in STXBP1 encephalopathy patients, demonstrated to provide an increase in the efficacy shown by previous AAV approaches to STXBP1 gene supplementation or replacement based on the preclinical NHP studies. Capsids of the invention were further demonstrated to transduce and express the hSTXBPl therapeutic cargo in human IPSC-derived neurons in a dose-dependent manner, comparably across three genotypes of STXBP1 (WT, HET, and KO). Furthermore, hSTXBPlgene supplementation via capsids of the intervention restored neuronal function in human IPSC-derived STXBP1 KO neurons by restoring neuronal network synchronicity. These data demonstrate that the capsids of the invention are capable of functionally restoring STXBP1 levels in a model STXBP1 human diseased neurons. Example 2: Background to treatment of Pediatric patients with syntaxin-binding protein 1 (STXBP1) encephalopathy

[0250] Construct structure

[0251] The construct of the invention, as detailed in Example 1 , is administered a gene therapy product that consists of a novel recombinant adeno-associated virus (rAAV) capsid enclosing a single- stranded DNA construct.

[0252] The DNA construct (CAG-hSTXBPlb-WPRE-hGHpA) encodes the b-isoform of the wild type (WT) human syntaxin-binding protein 1 (STXBP1) transgene (hSTXBPlb', NCBI Reference Sequence NMOO 1032221 .6) expressed from a chicken beta-actin promoter with cytomegalovirus (CMV) intermediate-early enhancer element and hybrid chicken beta-actin and rabbit P globin intron (all together named CAG), a woodchuck hepatitis virus (WHV) Posttranscriptional Regulatory Element (WPRE) mut6 version to prevent WHV-X protein synthesis, and a human growth hormone polyadenylation signal (hGHpA). This sequence was synthesized between AAV2 inverted terminal repeats (ITRs), with the 5’ ITR containing an 11 base pair deletion. The total length of the packaged sequence is 4362 base pairs. This construct was selected based on preclinical studies demonstrating the safety and pharmacodynamic effects of this promoter and transgene combination in mouse models with Stxbpl gene disruption.

[0253] The DNA construct is packaged into an engineered variant of AAV9 containing a 7 amino acid insertion after amino acid 588. The VP1, VP2, and VP3 capsid proteins are expressed from the AAV2 rep sequence under the control of a tetracycline-controlled transactivator (tTA), poly adenylation signal (pABGH), and a Tet-responsive element (TRE), together termed induced capsid gene (iCAP), for inducible replication capsid gene (Rep / CAP).

[0254] Indication and context of production development

[0255] Syntaxin-binding protein 1 (STXBP1) encephalopathy is a rare autosomal dominant neurodevelopmental disorder caused by de novo disruptive mutations occurring in one STXBP1 allele. Heterozygous disruptive mutations in the STXBP1 gene impair neuronal communication and occur in 3.3 to 3.8 / 100,000 live births globally, are the top 5 causes of epilepsy in patients referred for epilepsy genetic testing, and among the top 10 causes of neurodevelopmental disorders in patients with pathogenic mutations in a single gene. Additionally, STXBP1 encephalopathy is one of the most common causes of developmental epileptic encephalopathy.

[0256] The construct is a novel recombinant adeno-associated virus (rAAV) gene therapy product that is administered for the treatment of pediatric patients with STXBP1 encephalopathy, >18 months to <8 years of age.

[0257] Therapy

[0258] The therapy is administered as a single IV administration to symptomatic STXBP1- deficient pediatric patients >18 months to <8 years of age. The therapy consists of a novel central nervous system (CNS)-enriched AAV9-derived capsid designed to deliver the functional hSTXBPlb (b-isoform of hSTXBPl; referred to as hSTXBPl) gene to cells in the CNS, thereby providing a permanent source of STXBP1 protein in the brain and allowing for long-term correction of synaptic vesicle release throughout the CNS.

[0259] Quality Assurance Assays

[0260] The stability of the construct drug product is evaluated with product-contact components used for preparation and administration of the infusion of the construct to patients in the clinical trial. The study evaluates material loss in the lowest dose intended for the clinical trial due to the higher risk of material absorbance at lower concentrations. Additionally, infusion set compatibility for use in preclinical studies is performed.

[0261] A STXBP1- syntaxin binding assay is conducted as a functional potency assay to assess dose-dependent restoration of syntaxin binding upon restored expression of STXBP1 in an immortalized cell line. STXBP1 (Muncl8-1) is present in every neuron and synapse in the mammalian CNS. The primary function of STXBP1 is to bind syntaxin- 1 (“syntaxin”; 1 of 3 neuronal SNARE proteins), chaperone syntaxin to the synaptic terminal, and then help scaffold the correct formation of the SNARE complex. In STXBP1 encephalopathy, reductions in STXBP1 protein (haploin sufficiency) directly result in insufficient syntaxin-binding and incorrect formation of SNARE complexes which causes dysregulated synaptic vesicle release and impaired neuronal communication, resulting in the major clinical features of STXBP1 encephalopathy. The assay involves transduction of an immortalized cell line that does not express STXBP1 at baseline (either a cell line that lacks endogenous STXBP1 expression or an engineered KO cell line) and is amenable for use in a CMC manufacturing environment. Cells are transduced at multiple multiplicities of infection (MMOI) with the candidate construct or empty vector, and binding of exogenously applied tagged syntaxin to cell lysates are measured.

[0262] Phannacology / Toxicology

[0263] A non-Good Laboratory Practice (GLP) Dose-Range Finding (DRF) study was conducted to (1) characterize the safety (defined by tolerability of the candidate and potential for adverse findings) and biodistribution of the candidate delivered as a single intravenous (IV) administration into WT NHP, (2) test two immunomodulatory regimens used in conjunction with the candidate for subsequent selection and characterization in a GLP Toxicology study, and (3) identify doses of the candidate that are expected to provide prospect of direct benefit (PDB) for further characterization in the GLP-Toxicology study and support dose- selection of the FIH trial. The non-GLP DRF study was conducted with the clinically representative formulation of the candidate in study-naive WT nonhuman primates. The safety and biodistribution of the candidate was characterized over a range of candidate doses (1.25E13 vg / kg to 5.0E13 vg / kg) and immunomodulation regimens. All animals received a prophylactic immunomodulatory regimen, and an additional group treated with 2.5E13 vg / kg of the candidate also received an additional, combinatorial immunomodulatory regimen. Results from the non-GLP DRF study enabled candidate dose selection for further characterization

[0264] A GLP toxicology and biodistribution study in NHPs is conducted to guide dose selection and safety monitoring for the proposed first-in-human (FIH) clinical trial, using a single IV administration of the candidate, at 1 of 3 doses (3.8E13 vg / kg, 5.9E13 vg / kg, or 7.4E13 vg / kg), which is administered in conjunction with a prophylactic immunomodulatory drug regimen, modeling the clinical dosing regimen. Briefly, the GLP Toxicology and biodistribution study in WT NHPs assesses candidate safety, biodistribution, and expression at 3-month and 6-month terminal timepoints. Briefly, the GLP Toxicology Study is conducted in seronegative and study- naive NHPs aged 1 .5 to 3.5 years old and includes 4 dose groups: (1) a vehicle-treated group and (2-4) the candidate administered at 1 of 3 doses (3.8E13 vg / kg, 5.9E13 vg / kg, or 7.4E13 vg / kg). Each dose group has 6 animals, using a mixed-sex design (2:1 or 1:2) with a sex distribution balance designed to achieve 3 males and 3 females in each dose group. Dose selection is based on results from the non-GLP DRF to support testing 3 doses of the candidate that maximally informs therapeutic index and support dose selection for a first-in-human (FIH) clinical trial.

[0265] The 3-month post-dose terminal time point provides the most relevant safety readout to inform FIH clinical trials because the anticipated toxicities associated with rAAV systemic administration are often observed within the first few months after dosing. The 3-month analysis provide information on potential toxicities related to administration of the candidate product and potential innate immune response related to the capsid component of candidate. Since the NHPs are screened to be seronegative against the candidate capsid component of candidate, the potential for toxicities related to a pre-existing humoral response against the capsid is reduced. No overt toxicities (serious adverse events [SAEs] / deaths) have been observed in any study using the candidate. In addition, no candidate- specific dorsal root ganglia (DRG) pathology has been observed in dosed NHPs to date. Delayed toxicities are not found related to immune responses to the transgene product given complete homology between murine, NHP, and human STXBP1. In addition, tested mice and NHPs, and potential patients, all have at least one functional copy of the gene.

[0266] The STXBP1 protein sequence is identical in mice, NHPs, and humans, and it is not possible to differentiate endogenous STXBP1 (in mice or NHPs) from candidate cargo-derived STXBP1 protein. Conversely, candidate-derived cargo RNA can be quantitatively and directly measured using unique elements not present endogenously (e.g. WPRE). Therefore, the sponsor plans to use cargo RNA as the primary bridge between mouse and NHP studies to support selecting doses of the candidate expected to provide a PDB for an FIH clinical trial.

[0267] An infusion Set Compatibility Assessment Strategy is conducted as follows. To ensure dose administration accuracy, pre- and post-dose retains are taken from each study group in the non-GLP DRF and GLP toxicology studies and tested for titer using the transgene-specific digital droplet polymerase chain reaction (ddPCR) assay used for clinical material.

[0268] Clinical Relevance of Nonhuman Primales

[0269] NHPs are considered the best animal model to predict candidate biodistribution in humans due to the phylogenetic and physiological similarities of BBB function and CNS organization. Additionally, this species has shown responses to the canddiate or similar class of compound. Consequently, use of this species will maximize the likelihood of identifying responses that are quantitatively and qualitatively similar to those that may be expected in humans. The novel capsid, used in the candidate to deliver the hSTXBPl therapeutic cargo, was evolved to cross the BBB of NHP. Therefore, the only way to fully evaluate the biodistribution of the candidate and the potential for toxicities is in NHPs.

[0270] In the FIH clinical study, weight-adjusted doses are administered to the indicated pediatric patient population. Young NHPs reflect the size and physiology of the pediatric patient population in an FIH clinical study of STXBP1 encephalopathy. The use of traditional PK concepts and allometric scaling for rAAV systemic administration are not widely used because they are generally not applicable to A AV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction, transgene expression under the control of a non-endogenous promoter, and translation of the transgene-derived protein.

[0271] The age of the target patient population for the clinical trial is >18 months to <8 years of age at the time of treatment, as these patients have both the most significant unmet medical need and are the most likely to receive substantial benefit from the candidate. To best approximate clinical responses to the candidate, NHPs in the GLP Toxicology and biodistribution study are selected to be approximately between the ages of 1.5 to 3.5 years of age at the time of dosing with weights between 1.8 and 4.0 kg. According to the World Health Organization (WHO) growth standards, the median weight for a male at 18 months of age is -10.2 kg and for a female, approximately -9.75 kg. Children at 7 years of age have a median weight of 27.5 kgs. The difference in weight between the treated NHP and prospective patient population is likely to range between 3- and 10-fold, and not more than 20-fold (for reference, mouse [0.02 kg] to young NHP [2.0 kg] is a 100-fold difference). Without appropriate models for allometric scaling of rAAV therapies and the low multiple of weight differences between NHP and potential patients in an FIH clinical study, the weight-based doses found to be safe and efficacious in the GLP toxicology study, conducted in young NHPs, are in the indicated pediatric population without additional scaling.

[0272] Lastly, some toxicities reported with rAAV vector administration such as dorsal root ganglion (DRG) and peripheral nerve toxicities, have only been elucidated in humans, NHP, and piglets. Characterizing the DRG safety profile in NHPs is an important aspect of characterizing the benefit-to-risk profile of the candidate. Therefore, NHPs are the most appropriate species to assess the potential for safety and biodistribution.

[0273] Non-GLP Dose-Range Finding Study in NHPs

[0274] The objective of the non-GLP DRF study was to:

[0275] 1. Test the safety (defined by tolerability of the candidate and potential for adverse findings) and biodistribution of 4 doses of the candidate (manufactured using the representative clinical process) delivered IV in cynomolgus macaques.

[0276] 2. Test two immunomodulatory regimens used in conjunction with the candidate for subsequent selection and characterization in a GLP Toxicology study.

[0277] The study was conducted with clinically representative material of the candidate in study- naive WT NHPs that were seronegative for AAV9 and the candidate capsids. The NHPs were cynomolgus monkeys (Macaca fascicularis) of Mauritius origin, aged 18 to 30 months at time of dosing.

[0278] 3.3.3.1 Study Design

[0279] The safety and biodistribution of the candidate was characterized over a range of candidate doses (1.25E13, 2.5E13, 4.0E13, and 5.0E13 vg / kg) administered with one of two prophylactic immunomodulation regimens.

[0280] NHPs were dosed with the candidate in a manner similar to that proposed for the clinical setting. Following dosing, NHPs were monitored for a 6-week period for clinical observations and measurements. Immunogenicity, biodistribution, and histopathology analyses were conducted at the conclusion of the 6-week-in-life period.

[0281] The dose formulations are administered to appropriate animals by IV infusion over 30 minutes (± 3 minutes) once on Day 1. The infusion rate is not to exceed 1 mL / min. Dose formulations are administered using a temporary catheter inserted into a saphenous or cephalic vein connected to an infusion line. The appropriate volume is delivered using an infusion pump. Dose administration is initiated within 180 minutes from the time of removal of bulk test article from frozen storage. This administration route of exposure is consistent with the route of administration in humans.

[0282] To ensure dose administration accuracy, pre- and post-dose retains are taken from each study group and tested for titer using the transgene-specific ddPCR assay used for clinical material.

[0283] In addition to the above candidate dose levels, all groups receive a prophylactic dose of an immunomodulatory agent, and one group receives additional immunomodulation treatments. Immunomodulation may be incorporated in the candidate FIH clinical trial to decrease the risk of immunogenicity-related AEs that have been observed with gene therapies, such as thrombotic microangiopathy (TMA). To understand the potential impact of immunomodulation on the safety and efficacy of the candidate, the impact of 2 immunomodulation regimens is assessed in both the Non-GLP DRF Study in NHPs.

[0284] The results of she immunomodulation and dosing strategy directly inform the immunomodulation and dosing regimen in the GLP Toxicology Study in NHPs.

[0285] Clinical observations and examinations are performed pre- and post-dose on all animals. In the immediate 5-day period post-dose, clinical observations are performed and include twice- daily monitoring for mortality and morbidly, daily monitoring of food consumption and body temperature, and 48 hours post-dose blood sampling for neutralizing antibody, cytokine, and complement monitoring, hematology, and coagulation parameters. Subsequent monitoring includes twice-daily mortality and morbidity checks, daily food consumption checks, and weekly clinical examination, including body weight and temperature measurements. Blood samples are collected at pre- and post-dose intervals for measurement of hematology and coagulation parameters and serum chemistries. Blood is collected at scheduled intervals to evaluate the immune response (neutralizing antibody) to the administered product.

[0286] Animals are euthanized and analyzed after 6-weeks post-dosing. Planned study readouts include cargo DNA and RNA biodistribution in CNS and peripheral organs, total STXBP1 protein expression in CNS, clinical observations, and histopathology. Analyses are performed for the detection of cargo DNA and RNA levels in major perfused organs (liver, heart, kidney, spleen, lung), as well as multiple regions of interest in the brain (brainstem, cerebellar cortex, globus pallidus, basal ganglia, substantial nigra) and the spinal cord at multiple levels is evaluated. Additionally, a comprehensive gross pathology and histopathology assessments is made for all animals in the following tissues:

[0287] Multiple regions of the brain (brainstem, cerebellar cortex, globus pallidus, basal ganglia, substantial nigra);

[0288] Spinal cord (cervical, thoracic, and lumbar areas);

[0289] Dorsal root ganglion (cervical, thoracic, and lumbar areas);

[0290] All gross lesions.

[0291] In the case of unscheduled deaths, the planned comprehensive gross pathology and histopathology is performed on a complete panel of tissues to determine the possible cause of death.

[0292] Planned Good Laboratory Practice Toxicology Study in Nonhuman Primates

[0293] A GLP Toxicology Study in NHPs is performed in compliance with FDA Good Laboratory Practice (GLP) regulations for Nonclinical Laboratory Studies.

[0294] The GLP Toxicology Study in NHPs is designed to guide dose selection and safety monitoring for the FIH clinical trial. The study design includes 2 terminal time points: 3 months and 6 months post-dose. It is conducted in study-naive WT NHPs that are seronegative for AAV9 and candidate capsids; NHPs are cynomolgus monkeys (Macaca fascicularis) of Mauritius origin, aged 18 to 24 months at time of dosing. The GLP toxicology study includes 4 dose groups: vehicle-treated, Low, Medium, and High groups. Each group has 6 animals, using a mixed-sex design (2:1 or 1:2) with 3 animals allotted for each terminal timepoint, and with a sex distribution balance designed to achieve 3 males and 3 females in each dose group (3 month and 6 month; see Table below). Dose levels are selected based results from the non-GLP DRF Study in NHPs, starting around expected therapeutic levels in the clinical population.

[0295] F: female; IV: intravenous; M: male; N: number; ROA: route of administration NHPs were selected as a closely related species, phylogenetically and physiologically, to humans and an accepted non-rodent species for prcclinical toxicity evaluations by regulatory agencies. The candidate capsid was engineered in NHPs, and use of NHPs is expected to mimic the expected clinical biodistribution of the candidate in humans, maximizing the likelihood of identifying responses that are quantitatively and qualitatively similar to those that may be expected in humans. The total number of animals is the minimum needed to properly characterize responses related to test article administration and, thus, to meet experimental objectives.

[0296] Assigned animals may be replaced with spare animals before initiation of dose administration based on evaluation of the data collected during the acclimation phase (e.g, serostatus, pre-dose health).

[0297] All groups receive an immunomodulatory agent. The final immunosuppression regimen used in this study is determined by the results of the non-GLP DRF Study in NHPs.

[0298] The dose formulations are administered to designated groups of animals by IV infusion over 30 minutes (± 3 minutes) once on Day 1. The infusion rate is not to exceed 1 mL / min. Dose formulation is administered using a temporary catheter inserted into a saphenous or cephalic vein connected to an infusion line. The appropriate volume is delivered using an infusion pump. Dose administration is initiated within 180 minutes from the time of removal of bulk test article from frozen storage. This administration route is consistent with the route of administration in humans.

[0299] To ensure dose administration accuracy, pre- and post-dose retains are taken from each study group and tested for titer using the transgene-specific ddPCR assay used for clinical material.

[0300] Clinical observations and examinations are performed pre- and post-dose on all animals. In the immediate 5-day period post-dose, clinical observations are performed and include twice- daily monitoring for mortality and morbidity, daily monitoring of food consumption and body temperature, and 48 hours post-dose blood sampling for neutralizing antibody, hematology, and coagulation parameters. Subsequent monitoring includes twice-daily mortality and morbidity checks, daily food consumption checks, and weekly clinical examination, including body weight and temperature measurements. Blood samples are collected at pre-dose and post-dose intervals for hematology and coagulation parameters and serum chemistries. Blood is also collected at scheduled intervals to evaluate the immune response (neutralizing antibody) to the administered product.

[0301] NHPs are euthanized and analyzed after 3-months or 6-months, based on pre-assigned terminal timepoints. Study readouts include vector DNA and RNA biodistribution in central nervous system (CNS) and peripheral organs, membrane-bound total STXBP1 protein expression in brain and liver, clinical observations, and histopathology. Analyses were performed for the detection of cargo DNA and RNA levels in the blood (DNA only), major perfused organs (liver, heart, kidney, spleen, lung), as well as multiple regions of interest in the brain (brainstem, cerebellar cortex, globus pallidus, basal ganglia, substantial nigra) and spinal cord (multiple levels). Additionally, comprehensive gross pathology and histopathology assessments were made for all animals in the following tissues:

[0302] Multiple regions of the brain (brainstem [represented by pons and medulla] , cerebellar cortex, globus pallidus, basal ganglia [represented by corpus striatum and ventral tegmental area], substantial nigra);

[0303] Spinal cord (cervical, thoracic, and lumbar areas) and vagus nerve;

[0304] Dorsal root ganglion (cervical, thoracic, and lumbar areas) and meninges;

[0305] Major peripheral organs (adrenal glands, heart, kidneys, liver, lungs, sex organs [testes or ovaries], pancreas, skeletal muscle), spleen, and eyes;

[0306] All gross lesions.

[0307] Modified sequences and percent identity

[0308] For sequences disclosed throughout this application, it is understood that nucleic acid molecules and peptides may comprise one or more substitutions, for example conservative substitutions, that allow sequences to continue to function. Accordingly, sequences may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence disclosed.

[0309] “Percent (%) sequence identity” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, or other software appropriate for nucleic acid sequences. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0310] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a some % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0311] As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin & Altschul 1990, modified as in Karlin & Altschul 1993. Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul 1990. Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Homologous sequences described herein include sequences having the same percentage identity as the indicated percentage homology. Sequences sharing a percentage identity are understood in the art to mean those sequences sharing the indicated percentage of same residues over the length of the reference sequence (e.g., the linker or leader sequences disclosed herein and in the sequence listing).

[0312] A “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally, or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. Variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure can, in certain embodiments, comprise one or more conservative substitutions relative to a reference amino acid sequence.

[0313] A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom, or functional group is different, added, or removed; or one or more amino acids are substituted, mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency of activity of the parent polypeptide.

[0314] As used herein, a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, motif, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion, or fragment of the parent or reference compound.

[0315] In certain embodiments, a functional variant or functional portion or functional fragment each refers to a “signaling portion” of an effector molecule, effector domain, costimulatory molecule, or costimulatory domain. In other aspects, a functional variant or functional portion or functional fragment each refers to a linking function or a leader peptide function as disclosed herein. In certain aspects, a functional variant / portion / fragment refers to a linking function or a leader peptide function as described herein. In specific aspects, variant linkers and leader peptides are at least 60% as efficient, at least 70% as efficient, at least 80% as efficient, at least 90% as efficient, at least 95% as efficient, or at least 99% as efficient as the reference / parent polypeptides disclosed herein.

[0316] Incorporation by Reference

[0317] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0318] Equivalents

[0319] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. Select Sequences

[0320] SEQ ID NO: 1 - Capsid insert

[0321] ATRNGEVF1AQ

[0322] SEQ ID NO: 2 - AAV9

[0323] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKY LGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTS FGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAK KRLNFGQTGDTESVPDPQP1GEPPAAPSGVGSETMASGGGAPVADNNEGADGVGSSSGN WHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFD FNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQV FTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLK FSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNP GPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQ VATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMG GFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0324] SEQ ID NO: 3 - STXBP1 isoform a

[0325] MAP1GLKAVV GEK1MHDV1K KVKKKGEWKV LVVDQLSMRM LSSCCKMTD1 MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDSC PDALFNELVK SRAAKVIKTL TEINIAFLPY ESQVYSLDSA DSFQSFYSPH KAQMKNPILE RLAEQIATLC ATLKEYPAVR YRGEYKDNAL LAQLIQDKLD AYKADDPTMG

[0326] EGPDKARSQL LILDRGFDPS SPVLHELTFQ AMSYDLLPIE NDVYKYETSG

[0327] IGEARVKEVL LDEDDDLWIA LRHKHIAEVS QEVTRSLKDF SSSKRMNTGE

[0328] KTTMRDLSQM LKKMPQYQKE LSKYSTHLHL AEDCMKHYQG TVDKLCRVEQ

[0329] DLAMGTDAEG EKIKDPMRAI VPILLDANVS TYDKIRIILL YIFLKNGITE ENLNKLIQHA QIPPEDSEII TNMAHLGVPI VTDSTLRRRS KPERKERISE QTYQLSRWTP IIKDIMEDTI EDKLDTKHYP YTSTRSSASF STTAVSARYG HWHKNKAPGE YRSGPRLIIF

[0330] ILGGVSLNEM RCAYEVTQAN GKWEVLIGST HILTPTKFLM DLRHPDFRES

[0331] SRVSFEDQAP TME

[0332] SEQ ID NO: 4 - STXBP1 isoform b

[0333] MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMTDIMT EGITIVEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTDSCPDAL FNELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKNPILERLAEQ IATLCATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGPDKARSQLLIL DRGFDPSSPVLHELTFQAMSYDLLP1ENDVYKYETSG1GEARVKEVLLDEDDDLW1ALR HKHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQYQKELSKYSTHLHL AEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPILLDANVSTYDKIRIIL LYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDSTLRRRSKPERKERISEQT YQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTAVSARYGHWHKNKAPGEYR SGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGSTHILTPQKLLDTLKKLNKTDEEIS S

[0334] SEQ ID NO: 5 - STXBP1 isoform c

[0335] MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDYA LFNELVKSRA AKVIKTLTEI NIAFLPYESQ VYSLDSADSF QSFYSPHKAQ MKNPILERLA EQ1ATLCATL KEYPAVRYRG EYKDNALLAQ L1QDKLDAYK ADDPTMGEGP

[0336] DKARSQLLIL DRGFDPSSPV LHELTFQAMS YDLLPIENDV YKYETSGIGE

[0337] ARVKEVLLDE DDDLWIALRH KHIAEVSQEV TRSLKDFSSS KRMNTGEKTT

[0338] MRDLSQMLKK MPQYQKELSK YSTHLHLAED CMKHYQGTVD KLCRVEQDLA

[0339] MGTDAEGEKI KDPMRAIVPI LLDANVSTYD KIRIILLYIF LKNGITEENL NKLIQHAQIP PEDSEIITNM AHLGVPIVTD STLRRRSKPE RKERISEQTY QLSRWTPIIK DIMEDTIEDK LDTKHYPYIS TRSSASFSTT AVSARYGHWH KNKAPGEYRS GPRLIIFILG GVSLNEMRCA YEVTQANGKW EVLIGSTHIL TPQKLLDTLK KLNKTDEEIS S SEQ ID NO: 6 - STXBP1 isoform d

[0340] MHDVIKKVKK KGEWKVLVVD QLSMRMLSSC CKMTDIMTEG ITIVEDINKR REPLPSLEAV YLITPSEKSV HSLISDFKDP PTAKYRAAHV FFTDSCPDAL FNELVKSRAA KVIKTLTEIN IAFLPYESQV YSLDSADSFQ SFYSPHKAQM KNPILERLAE QIATLCATLK EYPAVRYRGE YKDNALLAQL IQDKLDAYKA DDPTMGEGPD KARSQLLILD

[0341] RGFDPSSPVL HELTFQAMSY DLLPIENDVY KYETSGIGEA RVKEVLLDED

[0342] DDLWIALRHK HIAEVSQEVT RSLKDFSSSK RMNTGEKTTM RDLSQMLKKM

[0343] PQYQKELSKY STHLHLAEDC MKHYQGTVDK LCRVEQDLAM GTDAEGEKIK

[0344] DPMRAIVPIL LDANVSTYDK IRIILLYIFL KNGITEENLN KLIQHAQIPP EDSEIITNMA HLGVPIVTDS TLRRRSKPER KERISEQTYQ LSRWTPIIKD IMEDTIEDKL DTKHYPYIST RSSASFSTTA VSARYGHWHK NKAPGEYRSG PRLIIFILGG VSLNEMRCAY EVTQANGKWE VLIGSTHILT PTKFLMDLRH PDFRESSRVS FEDQAPTME

[0345] SEQ ID NO: 7 - AAV vector capsid sequence comprising ATRNGEVFIAQ

[0346] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKY LGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTS FGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAK KRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGN WHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFD FNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQV FTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLK FSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNP GPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQ VATNHQSATRNGEVFIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQK ENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0347] SEQ ID NO: 8 Nucleic acid sequence encoding capsid ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAG

[0348] GAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAA

[0349] CAACATCAAGACAACGCTaGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCC

[0350] GGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCG

[0351] AGCACGACAAaGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAG

[0352] TACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGG

[0353] GGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTG

[0354] GTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAG

[0355] TCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCT

[0356] AAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCA

[0357] ACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTC

[0358] AGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTT

[0359] CCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACC

[0360] AGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCC

[0361] AACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCC

[0362] TGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAG

[0363] CGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTt

[0364] AACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAA

[0365] CCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCT

[0366] CGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCC

[0367] TCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTT

[0368] TTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTT

[0369] CAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCT

[0370] GGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCTAGAACTAT

[0371] TAACGGCAGCGGCCAAAACCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCA

[0372] ACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGT

[0373] GTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCT

[0374] TCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCtctCA

[0375] CAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACA

[0376] AGGTACtGGCAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAG AAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAAC

[0377] CACCAGAGTGCCACCAGAAACGGCGAAGTCTTCATCGCCCAAGCCCAAACCGGTTG

[0378] GGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACC

[0379] TGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTC

[0380] CGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAAC

[0381] ACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTC

[0382] ATCACCCAGTATTCTACTGGtCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAG

[0383] GAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTC

[0384] TAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCAT

[0385] TGGCACCAGATACCTGACTCGTAATCTGTAA

[0386] SEQ ID NO: 9 - DNA sequence encoding hSTXBPlb cargo

[0387] ATGGCCCCCATTGGCCTCAAAGCTGTTGTCGGAGAGAAGATTATGCATGATG

[0388] TGATAAAGAAGGTCAAGAAGAAGGGGGAATGGAAGGTGCTGGTGGTGGATCAGTT

[0389] AAGCATGAGGATGCTGTCCTCCTGCTGCAAGATGACAGACATCATGACCGAGGGCA

[0390] TAACGATTGTGGAAGATATCAATAAGCGCAGAGAGCCGCTCCCCAGCCTGGAGGCT

[0391] GTGTATCTCATCACTCCATCCGAGAAGTCCGTCCACTCTCTCATCAGTGACTTTAAGG

[0392] ACCCGCCGACTGCTAAATACCGGGCTGCACACGTCTTCTTCACTGACTCTTGTCCAG

[0393] ATGCCCTGTTTAATGAACTGGTAAAATCCCGAGCAGCCAAAGTCATCAAAACTCTGA

[0394] CGGAAATCAATATTGCATTTCTCCCGTATGAATCCCAGGTCTATTCCTTGGACTCTGC

[0395] TGACTCTTTCCAAAGCTTCTACAGTCCCCACAAGGCTCAGATGAAGAATCCTATACT

[0396] GGAGCGCCTGGCAGAGCAGATCGCGACCCTTTGTGCCACCCTGAAGGAGTACCCGG

[0397] CTGTGCGGTATCGGGGGGAATACAAGGACAATGCCCTGCTGGCTCAGCTAATCCAG

[0398] GACAAGCTCGATGCCTATAAAGCTGATGATCCAACAATGGGGGAGGGCCCAGACAA

[0399] GGCACGCTCCCAGCTCCTGATCCTGGATCGAGGCTTTGACCCCAGCTCCCCTGTGCT

[0400] CCATGAATTGACTTTTCAGGCTATGAGTTATGATCTGCTGCCTATCGAAAATGATGT

[0401] ATACAAGTATGAGACCAGCGGCATCGGGGAGGCACGGGTGAAGGAGGTGCTCCTGG

[0402] ACGAGGACGACGACCTGTGGATAGCACTGCGCCACAAGCACATCGCAGAGGTGTCC

[0403] CAGGAAGTCACCCGGTCTCTGAAAGATTTTTCTTCTAGCAAGAGAATGAATACTGGA

[0404] GAGAAGACCACCATGCGGGACCTGTCCCAGATGCTGAAGAAGATGCCTCAGTACCA GAAAGAGCTCAGCAAGTACTCCACCCACCTGCACCTTGCTGAGGACTGTATGAAGC

[0405] ATTACCAAGGCACCGTAGACAAACTCTGCCGAGTGGAGCAGGACCTGGCCATGGGC

[0406] ACAGATGCTGAGGGAGAGAAGATCAAGGACCCTATGCGAGCCATCGTCCCCATTCT

[0407] GCTGGATGCCAATGTCAGCACTTATGACAAAATCCGCATCATCCTTCTCTACATCTTT

[0408] TTGAAGAATGGCATCACGGAGGAAAACCTGAACAAACTGATCCAGCACGCCCAGAT

[0409] ACCCCCGGAGGATAGTGAGATCATCACCAACATGGCTCACCTCGGCGTGCCCATCGT

[0410] CACCGATTCCACGCTGCGTCGCCGGAGCAAGCCGGAGCGGAAGGAACGCATCAGCG

[0411] AGCAGACCTACCAGCTCTCACGGTGGACTCCGATTATCAAGGACATCATGGAGGAC

[0412] ACTATTGAGGACAAACTTGACACCAAACACTACCCTTATATCTCTACCCGTTCCTCT

[0413] GCCTCCTTCAGCACCACCGCCGTCAGCGCCCGCTATGGGCACTGGCATAAGAACAA

[0414] GGCCCCAGGCGAGTACCGCAGTGGCCCCCGCCTCATCATTTTCATCCTTGGGGGTGT

[0415] GAGCCTGAATGAGATGCGCTGCGCCTACGAGGTGACCCAGGCCAACGGAAAGTGGG

[0416] AGGTGCTGATAGGATCCACACACATCCTCACCCCACAGAAACTGCTGGACACACTG

[0417] AAGAAACTGAATAAAACAGATGAAGAAATAAGCAGTTAG

[0418] SEQ ID NO: 10 - Engineered 3’ ITR

[0419] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGAC

[0420] CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT

[0421] CCATCACTAGGGGTTCCT

[0422] SEQ ID NO: 11 - Engineered 5’ ITR

[0423] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG

[0424] AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG

[0425] AGCGAGCGAGCGCGCAGCTGCCTGCAGG

Claims

Claims1. An adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising the amino acid sequence having at least 80% shared sequence identity to ATRNGEVFTAQ (SEQ ID NO: 1); and a nucleic acid encoding: a promoter; and an STXBP1 transgene.

2. The AAV of claim 1, wherein the AAV capsid protein includes the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1).

3. The AAV of claim 1, wherein the capsid protein is engineered from an AAV9 capsid protein.

4. The AAV of claim 3, wherein the capsid protein is engineered, relative to an AAV9 capsid protein, by substitution of amino acid position 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 of the amino acids RNGEVFI, wherein parental AAV9 amino acids AQ are present at positions 589 and 590.

5. The AAV of claim 4, wherein the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS).

6. The AAV of claim 5, wherein the AAV capsid protein is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

7. The AAV of claim 1, wherein the transgene is a wild-type human STXBP1 transgene.

8. The AAV of claim 7, wherein the STXBP1 transgene encodes a peptide having at least 95% identity to the sequence of STXBP1 isoform b.

9. The AAV of claim 1 , wherein the promoter is a CAG promoter.

10. The AAV of claim 9, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken 0-actin promoter, and a chimeric rabbit 0- globin (rBG) intron.

11. The AAV of claim 10, wherein the AAV further comprises at least one regulatory element and / or a poly(A) signal.

12. The AAV of claim 1, wherein the regulatory element is a Woodchuck hepatitis virus Post- transcriptional Regulatory Element (WPRE).

13. The AAV of claim 11, wherein the poly(A) signal is hGH polyA.

14. A method of treatment of a disease, the method comprising administering a composition comprising adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1); a nucleic acid encoding: a promoter; and an STXBP1 transgene.

15. The method of claim 14, wherein the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1).

16. The method of claim 14, wherein the capsid protein is engineered from an AAV9 capsid protein.

17. The method of claim 16, wherein the capsid protein is engineered, relative to an AAV9 capsid protein, by substitution of amino acid position 588 with the amino acid T, peptideinsertion between amino acid positions 588 and 589 of the amino acids RNGEVFI, wherein parental AAV9 amino acids AQ arc present at positions 589 and 590.

18. The method of claim 17, wherein the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS).

19. The method of claim 18, wherein the AAV capsid protein is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

20. The method of claim 14, wherein the transgene is a wild-type human STXBP1 transgene.

21. The method of claim 20, wherein the STXBP1 transgene encodes a peptide having at least 95% identity to the sequence of STXBP1 isoform b.

22. The method of claim 14, wherein the promoter is a CAG promoter.

23. The method of claim 22, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken P-actin promoter, and a chimeric rabbit 0- globin (rBG) intron.

24. The method of claim 14, wherein the AAV further comprises at least one regulatory element and / or a poly(A) signal.

25. The method of claim 24, wherein the regulatory element is a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE).

26. The method of claim 24, wherein the poly(A) signal is hGH polyA.

27. The method of claim 14, wherein the disease is a disease caused by an STXBP1 mutation.

28. The method of claim 27, wherein the disease is an STXBP1 encephalopathy.

29. The method of claim 14, wherein the composition is administered as an intravenous injection.

30. The AAV vector of claim 1, wherein the transgene is flanked by inverted terminal repeats (ITRs), wherein: the 5’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 10, and / or the 3’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 11.

31. The method of claim 14, wherein the transgene is flanked by inverted terminal repeats (ITRs), wherein: the 5’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 10, and / or the 3’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 11.

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