Gene therapy for stxbp1 encephalopathy

Abstract

The disclosure concerns compositions and methods for treatment of encephalopathies, including encephalopathies caused by, or associated with, and STXBP1 haploinsufficiency or mutation. Compositions and methods provided herein encompass AAV particles that encapsidate the STXBP1 transgene and allow for STXBP1 expression, including expression of STXBP1 in central and / or peripheral nervous system cells.

Description

GENE THERAPY FOR STXBP1 ENCEPHALOPATHYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under R01NS 100893 awarded by the National Institute of Neurological Disorders and Stroke (NINDS), and under P50HD103555 awarded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] Embodiments of the disclosure concern at least the fields of cellular biology, molecular biology, physiology, and medicine.INCORPORATION OF SEQUENCE LISTING

[0003] The present application includes a Sequence Listing filed herewith as an ST.26 compliant Sequence Listing XML, “CAPS-037-01WO-Sequence-Listing” created on April 4, 2025 and having a size of 7 KB . The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.BACKGROUND

[0004] STXBP1 haploinsufficiency causes STXBP1 encephalopathy, which is one of the top five causes of pediatric epilepsies, including Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, and Dravet syndrome. STXBP1 is one of the most frequently mutated genes in sporadic intellectual disabilities. STXBP1 mutations are often truncating mutations. STXBP1 mutations are typically heterozygous and de novo. There are currently no treatments for the underlying disorder with limited therapies being focused on diminishing symptoms of the disease.

[0005] Individuals with STXBP1 mutations have intellectual disabilities and the vast majority have epilepsy. STXBP1 encephalopathy leads to motor deficits, including dystonia, ataxia, hypotonia, and tremor. Individuals often have psychiatric conditions, including hyperactivity, anxiety, stereotypies, aggression, and autistic features.

[0006] The STXBP1 gene encodes the syntaxin-binding protein 1 (also known as MUNC18-1). It is a core component of neurotransmitter release machinery in all neurons. Absence of STXBP1 inmodel organisms (including worms, fruit flies, and mice) abolishes neurotransmitter release and causes embryonic lethality. Therapies that restore STXBP1 in neurons are needed.SUMMARY

[0007] The present disclosure is directed to compositions and methods for treating encephalopathies caused by one or more mutations in the STXBP1 gene. In some cases, 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.

[0008] Specifically, aspects of the invention provide a formulation comprising: an adeno- associated virus (AAV) particle comprising (e.g., encapsidating) a nucleic acid comprising a promoter and a sequence encoding an STXBP1 gene product, wherein the formulation comprises at least 1 x 1011vector genomes (vg) / kilogram (kg). In preferred aspects, the formulation comprises between 1 x 1011vg / kg and 5 x 1013. In certain embodiments, the formulation comprises between 5 x 1011vg / kg to 2 x 1013vg / kg. In certain embodiments, the formulation comprises at least 5 x I011vg / kg, or dose with similar biological impact. In certain embodiments, the formulation comprises at least 1 x 1012vg / kg, or dose with similar biological impact. In certain embodiments, the formulation comprises least 2 x 1012vg / kg, or dose with similar biological impact. In certain embodiments, the formulation comprises at least 5 x 1012vg / kg, or dose with similar biological impact. In certain embodiments, the formulation comprises at least 1 x 1013vg / kg, or dose with similar biological impact. In certain embodiments, the formulation comprises at least 2 x 1013vg / kg, or dose with similar biological impact.

[0009] The AAV particle may transduce cells of the central and / or peripheral nervous system, including neurons, in specific cases. In particular embodiments, the viral particle may transduce any neuron, including for example, glutamatergic neurons, GABAergic neurons, glycinergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, adrenergic neurons, and / or noradrenergic neurons. In some embodiments, the viral particle is an AAV-PHP.eB particle, AAV-PHP.B particle, AAV-PHP.S particle, AAV-PHP.B4 particle, AAV-PHP.B5 particle, AAV- PHP.N particle, AAV-CAP-B1 particle, AAV-CAP-B10 particle, AAV-CAP-B22 particle, AAVtype 1 particle, AAV type 5 particle, AAV type 8 particle, or AAV type 9 particle. In some embodiments, the viral particle is an AAV-PHP.eB particle. In some embodiments, the viral particle is derived from cells transfected with an AAV-PHP.eB construct, AAV-PHP.B construct, AAV-PHP.S construct, AAV-PHP.B4 construct, AAV- PHP.B5 construct, AAV-PHP.N construct, AAV-CAP-B1 construct, AAV-CAP-B10 construct, AAV-CAP-B22 construct, AAV type 1 construct, AAV type 5 construct, AAV type 8 construct, or AAV type 9 construct. In certain embodiments, the promoter is a CAG promoter and the nucleic acid further includes a woodchuck hepatitis virus (WPRE).

[0010] The viral particle may comprise (e.g., encapsidate) one or more nucleic acids. The nucleic acids may encode for one or more genes that are mutated or haploinsufficient in individuals with an encephalopathy. In some embodiments, the nucleic acid encodes for an isoform of STXBP1. The isoform may be isoform a of STXBP1 or isoform b of STXBP1. In some embodiments, the nucleic acid encodes isoform a of STXBP1. In some embodiments, the nucleic acid encodes isoform b of STXBP1. In some embodiments, the nucleic acid encodes isoform c of STXBP1. In some embodiments, the nucleic acid encodes isoform d of STXBP1.

[0011] In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising an amino acid sequence having at least 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 99.9% sequence identity with SEQ ID NO:1, or any range or value derivable therein. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising SEQ ID NO:1. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising an amino acid sequence having at least 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 99.9% sequence identity with SEQ ID NO:2, or any range or value derivable therein. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising SEQ ID NO:2. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising an amino acid sequence having at least 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 99.9% sequence identity with SEQ ID NOG, or any range or value derivable therein. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising SEQ ID NOG. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising an amino acid sequence having at least 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 99.9% sequence identity with SEQ ID NO:4, or any range or value derivable therein. In some embodiments, the nucleic acid encodes an STXBP1 gene product comprising SEQ ID NO:4.

[0012] In some embodiments, the nucleic acids encoding a gene encode exons, and the nucleic acid sequence may lack introns. The nucleic acid sequence may comprise at least one regulatory sequence. In some embodiments, the regulatory sequence, including a promoter, induces constitutive expression, including constitutive expression in neurons. In some embodiments, the viral particle comprises (e.g., encapsidate) nucleic acids encoding for at least one regulatory sequence and at least one transgene. The regulatory sequence may be capable of inducing constitutive expression of the transgene(s) in any cell (including neurons) when the cell is transduced by the viral particle. In some embodiments, the viral particle comprises (e.g., encapsidates) nucleic acids encoding one or more transgenes and comprises one or more regulatory sequences that induce expression of the transgene(s), including specifically in neurons, in some cases. The neurons may be glutamatergic neurons, GABAergic neurons, glycinergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, adrenergic neurons, and / or noradrenergic neurons. The viral particle may comprise one or more surface markers that cause the specific transduction of the nucleic acid into neurons, including glutamatergic neurons, GABAergic neurons, glycinergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, adrenergic neurons, and / or noradrenergic neurons. The viral particle may comprise one or more surface markers or other proteins that specifically induce trafficking of the viral particle to the central and / or peripheral nervous system, in some cases.

[0013] In certain embodiments, a composition, such as any viral particle encompassed herein, is administered to an individual with STXBP1 encephalopathy. The administration may be by any suitable route or delivery regimen.

[0014] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized bythose skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features which are believed to be characteristic of the designs disclosed herein, both as to the organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows genomic structures of STXBP1 wild type (WT) and tmld (KO) alleles.

[0016] FIG. 2 shows an overview of an experimental timeline of testing STXBP1 gene supplementation in Stxbpl haploinsufficient mice.

[0017] FIG. 3 shows a summary of the experimental study design.

[0018] FIG. 4A-4H show graphs of STXBP1 expression levels in various brain regions of mice 3 months post-dosing

[0019] FIG. 5A-5H show graphs of STXBP1 expression levels in various brain regions of mice 7 months post-dosing.

[0020] FIG. 6 shows a hindlimb clasping (dystonia) score guide.

[0021] FIG. 7A-7B show graphs of hindlimb clasping (dystonia) score in male and female mice, respectively, post-dosing.

[0022] FIG. 8A-8B show graphs of body weight in male and female mice, respectively, postdosing.

[0023] FIG. 9A-9C show an example of spike-wave discharges (SWDs) (A) and line graphs of individual animal trajectories (B) and bar graphs of treatment group (B) changes in SWDs in mice post-dosing.

[0024] FIG. 10A-10H show line graphs (10A-10D) of individual animal trajectories of changes in myoclonic jumps and bar graphs of treatment group changes in myoclonic jumps (10E-10H) in mice post-dosing.

[0025] FIG. 11A-11H show line graphs (11A-11D) of individual animal trajectories of changes in myoclonic jerks and bar graphs of treatment group changes in myoclonic jerks (11E-11H) in mice post-dosing.

[0026] FIG. 12A-12B show graphs measuring fear memory tests in mice at 6 weeks post-dosing.

[0027] FIG. 13A-13B show graphs measuring fear memory tests in mice at 6 months post-dosing.

[0028] FIG. 14A shows the familiar and novel object used at 6 weeks in a recognition test in mice.

[0029] FIG. 14B show graphs measuring novel object recognition in mice at 6 weeks post-dosing.

[0030] FIG. 15A shows the familiar and novel object used at 6 months in a recognition test in mice.

[0031] FIG. 15B show graphs measuring novel object recognition in mice at 6 months postdosing.

[0032] FIG. 16A-16B show graphs measuring vertical pole test results in mice at 6 weeks (A) and 6 months (B) post-dosing.

[0033] FIG. 17A-17B show graphs measuring foot slip test results in mice at 6 weeks (17 A) and 6 months (17B) post-dosing.

[0034] FIG. 18A-18B show graphs measuring marble bury test results in mice at 6 weeks (18A) and 6 months (18B) post-dosing.

[0035] FIG. 19A-19D show graphs measuring open field assay tests in mice at 6 weeks postdosing.

[0036] FIG. 20A-20D show graphs measuring open field assay tests in mice at 6 months postdosing.

[0037] FIG. 21A-21D show graphs measuring DNA and RNA biodistribution at 3 months and 7 months post-dosing.

[0038] FIG. 22 shows the experimental overview and timeline in treating StxbplR406H / +mice, which are a knock-in model for a debilitating STXBP1 missense variant.

[0039] FIG. 23 provides graphs of the body weight in male and female mice, respectively, from 2 weeks before to 26 weeks after dosing across the 5 groups for Cohort A (EEG Cohort).

[0040] FIG. 24 provides graphs that provide the body weight in male and female mice, respectively, from 2 weeks before to 34 weeks after dosing across the 5 groups for Cohort B (BEH cohort).

[0041] FIG. 25 shows a graph of hindlimb clasping score in all mice of Cohort A from 2 weeks before dosing until 28 weeks after dosing.

[0042] FIG. 26 shows a graph of hindlimb clasping score in all mice of Cohort B from 2 weeks before dosing until 34 weeks after dosing.

[0043] FIGS. 27A-27B show graphs of EPM performance at 6weeks post-dose in Cohort B mice across all five groups, including entries, time spent within, and distance travelled for both open and closed arms.

[0044] FIGS. 28A-28B show graphs of EPM performance at 6 months post dose in Cohort B mice across all five groups. As shown, compared to 6-weeks, the treatment provided a partial phenotype rescue, with the high dose providing the greatest corrective effect.

[0045] FIGS. 29A-29B show graphs of light-dark assay results at six weeks post-dose for Cohort B mice from all five groups, showing measures of light / dark duration, light / dark entry, total distance traveled in light / dark, movement time in light / dark, and time resting in light / dark.

[0046] FIGS. 30A-30B show graphs of light-dark assay results at six-months post-dose for Cohort B mice from all five groups showing measures of light / dark duration, light / dark entry, total distance traveled in light / dark, movement time in light / dark, and time resting in light / dark.

[0047] FIGS. 31A-C show graphs measuring open field assay tests measured at 6 weeks post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0048] FIGS. 32A-33C show graphs measuring open field assay tests measured at 4 months post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0049] FIGS. 33A-33D show graphs measuring open field assay tests measured at 6 months post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0050] FIG. 34 shows graphs measuring vertical pole test results in mice post AAV injection as measured by time on pole at 6 weeks and 6 months, respectively.

[0051] FIG. 35 shows graphs measuring foot slip test results in Cohort B mice post AAV injection as measured by number of normalized foot slips at 6 weeks and 6 months, respectively.

[0052] FIG. 36 shows graphs measuring marble bury test results in Cohort B mice across all groups post AAV injection as measured by number of buried marbles, at 6 weeks and 6 months, respectively.

[0053] FIG. 37 shows graphs measuring long term memory in the contextual and cued fear conditioning tests in Cohort B mice across all five groups measured 6 weeks post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0054] FIG. 38 show graphs measuring long term memory in the contextual and cued fear conditioning tests in Cohort B mice across all five groups measured 6 months post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0055] FIG. 39 shows graphs that provide results of the hot plate test in Cohort B mice across all five groups at six-weeks and six-months post-dose.

[0056] FIG. 40 shows graphs that provide results of the mouse tail flick test in Cohort B mice across all five groups at six-weeks and six-months post-dose.

[0057] FIG. 41 shows graphs that provide results of the nesting test in Cohort A and Cohort B mice across all five groups at six-weeks and six-months post-dose.

[0058] FIG. 42 shows graphs that provide changes in myoclonic jumps as measured in all jumps in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0059] FIG. 43 shows graphs that provide changes in myoclonic jumps as measured in jumps during rapid eye movement (REM) sleep in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0060] FIG. 44 shows graphs that provide changes in myoclonic jumps as measured in jumps during non-rapid eye movement sleep (NREM) in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0061] FIG. 45 shows graphs that provide changes in myoclonic jumps as measured in jumps during wakefulness (wake) in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0062] FIG. 46 shows graphs that provide changes in myoclonic jerks as measured in jerks in total in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0063] FIG. 47 shows graphs that provide changes in myoclonic jerks as measured in jerks during REM sleep in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0064] FIG. 48 shows graphs that provide changes in myoclonic jerks as measured in jerks during NREM sleep in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0065] FIG. 49 shows graphs that provide changes in myoclonic jerks as measured in jerks during wakefulness (wake) in mice from Cohort A from all five groups measured from pre-dose and six- weeks post-dose.

[0066] FIGS. 50A-50B show graphs of STXBP1 expression levels in mice from all five groups at 6-weeks post-dosing as measured by western blot, in the anterior cortex, posterior cortex, olfactory bulb, cervical spinal cord, hippocampus, cerebellum, thalamus+hypothalamus, striatum, and brainstem, respectively.

[0067] FIG. 51A-51B shows graphs of STXBP1 protein expression levels in mice from all five groups at 6-months post-dosing as measured by western blot, in the anterior cortex, posterior cortex, hippocampus, striatum, brainstem, cerebellum, olfactory bulb, thalamus, and cervical spinal cord, respectively. FIG. 51B shows STXBP1 protein expression from a subcohort of animals assessed for STXBP1 protein in anterior cortex only at 6 months post-dosing.DETAILED DESCRIPTION

[0068] The present invention provides an AAV product that delivers a STXBP1 gene replacement strategy throughout the human CNS at levels of DNA Biodistribution, RNA expression, and hSTXBPl protein levels expected to restore function in STXBP1 encephalopathy patients.STXBP1 and Encephalopathies

[0069] Embodiments of the disclosure concern compositions, including gene therapies, viral particles, and nucleic acids, that are useful for restoring 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. In some embodiments, the composition comprises a viral particle. The viral particle may comprise (e.g. encapsidate) 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 a STXBP1 gene. A 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 acid that encodes an STXBP1 gene product may be an STXBP1 gene with one or more silent mutations. In some embodiments, the nucleic acid encodes for isoform a of STXBP1 (also known as STXBPla). In some embodiments, the nucleic acid encodes for isoform b of STXBP1 (also known as STXBPlb). In some embodiments, the nucleic acidencodes for isoform c of STXBP1 (also known as STXBPlc). In some embodiments, the nucleic acid encodes for isoform d of STXBP1 (also known as STXBPld).

[0070] In some embodiments, the viral particle comprises (e.g. encapsidates) 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 1, or any range or value derivable therein. In certain embodiments, the viral particle comprises (e.g. encapsidates) a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:1 (isoform a).

[0071] In some embodiments, the viral particle comprises (e.g. encapsidates) 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 2, or any range or value derivable therein. In certain embodiments, the viral particle comprises (e.g. encapsidates) a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:2 (isoform b).

[0072] In some embodiments, the viral particle comprises (e.g. encapsidates) 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 (e.g. encapsidates) a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:3 (isoform c).

[0073] In some embodiments, the viral particle comprises (e.g. encapsidates) 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 (e.g. encapsidates) a nucleic acid that encodes for the STXBP1 amino acid sequence of SEQ ID NO:4 (isoform d).

[0074] Particular sequences of STXBP1 isoform a, STXBP1 isoform b, STXBP1 isoform c, and STXBP1 isoform d are provided below.

[0075] (SEQ ID NO: 1, STXBP1 isoform a):MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDSCPDALFNELVK SRAAKVIKTL TEINIAFLPY ESQVYSLDSA DSFQSFYSPH KAQMKNPILE RLAEQIATLC ATLKEYPAVR YRGEYKDNAL LAQLIQDKLD AYKADDPTMGEGPDKARSQL LILDRGFDPS SPVLHELTFQ AMSYDLLPIE NDVYKYETSGIGEARVKEVL LDEDDDLWIA LRHKHIAEVS QEVTRSLKDF SSSKRMNTGEKTTMRDLSQM LKKMPQYQKE LSKYSTHLHL AEDCMKHYQG TVDKLCRVEQDLAMGTDAEG EKIKDPMRAI VPILLDANVS TYDKIRIILL YIFLKNGITE ENLNKLIQHA QIPPEDSEII TNMAHLGVPI VTDSTLRRRS KPERKERISE QTYQLSRWTP IIKDIMEDTI EDKLDTKHYP YISTRSSASF STTAVSARYG HWHKNKAPGE YRSGPRLIIF ILGGVSLNEM RCAYEVTQAN GKWEVLIGST HILTPTKFLM DLRHPDFRES SRVSFEDQAP TME

[0076] (SEQ ID NO: 2, STXBP1 isoform b):MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDSC PDALFNELVK SRAAKVIKTL TEINIAFLPY ESQVYSLDSA DSFQSFYSPH KAQMKNPILE RLAEQIATLC ATLKEYPAVR YRGEYKDNAL LAQLIQDKLD AYKADDPTMGEGPDKARSQL LILDRGFDPS SPVLHELTFQ AMSYDLLPIE NDVYKYETSGIGEARVKEVL LDEDDDLWIA LRHKHIAEVS QEVTRSLKDF SSSKRMNTGEKTTMRDLSQM LKKMPQYQKE LSKYSTHLHL AEDCMKHYQG TVDKLCRVEQDLAMGTDAEG EKIKDPMRAI VPILLDANVS TYDKIRIILL YIFLKNGITE ENLNKLIQHA QIPPEDSEII TNMAHLGVPI VTDSTLRRRS KPERKERISE QTYQLSRWTP IIKDIMEDTI EDKLDTKHYP YISTRSSASF STTAVSARYG HWHKNKAPGE YRSGPRLIIF ILGGVSLNEM RCAYEVTQAN GKWEVLIGST HILTPQKLLD TLKKLNKTDE EISS

[0077] (SEQ ID NO: 3, STXBP1 isoform c):MAPIGLKAVV GEKIMHDVIK KVKKKGEWKV LVVDQLSMRM LSSCCKMTDI MTEGITIVED INKRREPLPS LEAVYLITPS EKSVHSLISD FKDPPTAKYR AAHVFFTDYA LFNELVKSRA AKVIKTLTEI NIAFLPYESQ VYSLDSADSF QSFYSPHKAQ MKNPILERLA EQIATLCATL KEYPAVRYRG EYKDNALLAQ LIQDKLDAYK ADDPTMGEGPDKARSQLL1L DRGFDPSSPV LHELTFQAMS YDLLP1ENDV YKYETSG1GEARVKEVLLDE DDDLWIALRH KHIAEVSQEV TRSLKDFSSS KRMNTGEKTTMRDLSQMLKK MPQYQKELSK YSTHLHLAED CMKHYQGTVD KLCRVEQDLAMGTDAEGEKI KDPMRAIVPI LLDANVSTYD KIRIILLYIF LKNGITEENL NKLIQHAQIP PEDSEIITNM AHLGVPIVTD STLRRRSKPE RKERISEQTY QLSRWTPIIK DIMEDTIEDK LDTKHYPYIS TRSSASFSTT AVSARYGHWH KNKAPGEYRS GPRLIIFILG GVSLNEMRCA YEVTQANGKW EVLIGSTHIL TPQKLLDTLK KLNKTDEEIS S

[0078] (SEQ ID NO: 4, STXBP1 isoform d):MHDVIKKVKK KGEWKVLVVD QLSMRMLSSC CKMTDIMTEG ITIVEDINKR REPLPSLEAV YLITPSEKSV HSLISDFKDP PTAKYRAAHV FFTDSCPDAL FNELVKSRAA KVIKTLTEIN IAFLPYESQV YSLDSADSFQ SFYSPHKAQM KNPILERLAE QIATLCATLK EYPAVRYRGE YKDNALLAQL IQDKLDAYKA DDPTMGEGPD KARSQLLILDRGFDPSSPVL HELTFQAMSY DLLP1ENDVY KYETSG1GEA RVKEVLLDEDDDLWIALRHK HIAEVSQEVT RSLKDFSSSK RMNTGEKTTM RDLSQMLKKM PQYQKELSKY STHLHLAEDC MKHYQGTVDK LCRVEQDLAM GTDAEGEKIKDPMRAIVPIL 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 acid encodes for part or all of one of SEQ ID NOs:l- 4. In some embodiments, the viral particle comprises (e.g. encapsidates) 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:l-4. In some embodiments, the viral particle comprises (e.g. encapsidates) a nucleic acid sequence encoding one of SEQ ID NOs:l-4, 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:l-4 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.

[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 (e.g. encapsidates), AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased enrichment or specificity for the target in vivo environment.Transgene delivery

[0083] 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 (e.g. encapsidates) 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 motorfunctions in the individual. In some embodiments, the compositions restore psychiatric functions in the individual.

[0084] 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.

[0085] 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.

[0086] 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 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 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.

[0087] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intra-cisterna magna (ICM), 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.

[0088] 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 bedelivered 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.

[0089] 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 blood brain barrier (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 intraparenchymal injection.

[0090] 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 (“i.v.”) 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.

[0091] The term “CNS” or “central nervous system” means a tissue selected from brain, thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brain stem, and spinal cord. The brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital and parietal lobes.

[0092] 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 takeplace via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes both blood and 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.

[0093] In some embodiments, routes for administration include administration into the CSF, for example via an intracerebroventricular (ICV), intrathecal cisternal, intra-cisterna magna (ICM), 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 ccrcbrovcinricular administration, intranasal administration, endovascular administration, and intraparenchymal administration.

[0094] 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). In instances wherein the therapeutic rAAV composition is not therapeutically effective or is not providing a sufficient alleviation of the disease or condition, or symptom of the disease or condition, then 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.

[0095] In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases, the concentration of infectious particles is 2xl07, 2xl08, 2xl09, 2xlO10, 2xlOu,2xl012, 2xlO13, 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, 5xl08, 5xl09, 5xlO10, SxlO11, 5xl012, 5xl013, 5xl014, 5xl015, 5xl016, or 5xl017. In some cases, the concentration of the infectious particles is 6xl07, 6xl08, 6xl09, 6xlO10, 6xlOu, 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, 8xlO9, 8xlO10, 8xl011, 8xl012, 8xl013, 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.

[0096] 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 either intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intra-cisterna magna (ICM), intramuscularly, intrathecally, intraperitoneally, by nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.

[0097] 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 aslecithin, 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 mono stearate and gelatin.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] For example, the number of infectious particles administered to a mammal may be on the order of about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or even higher, infectious particles / 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.

[0104] 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.

[0105] 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.Viral Vectors

[0106] 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.

[0107] Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). Upon delivery to a host in vivo environment, a rAAV will (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 (“transduction”). rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell) and transduction efficiency (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0108] 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 upof three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1 : 1 : 10 ratio to form the viral capsid. VP 1 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 (VP 1 / 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).

[0109] 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.

[0110] 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 form the top of the loops. These VRs, such as the AA588 loop, are found on the capsid surface and can be associated with specific functional roles in the AAV life cycle including receptor binding, transduction, and antigenic specificity.

[0111] In some embodiments, the viral particle comprises an AAV particle, such as any AAV particle encompassed herein. The viral particle may be an AAV-PHP.eB particle, AAV-PHP.B particle, AAV-PHP.S particle, AAV-PHP.B4 particle, AAV-PHP.B 5 particle, AAV-PHP.N particle, AAV-CAP-B1 particle, AAV-CAP-B10 particle, AAV-CAP-B22 particle, AAV type 1 particle, AAV type 5 particle, AAV type 8 particle, or AAV type 9 particle. In some embodiments, the viral particle is an AAV-PHP.eB particle.

[0112] In some embodiments, the viral particle is a particle described in one or more of PCT Patent Application Publications W02020206189, W02020028751, WO2020168145, W02020077165, WO2021025995, WO2017197355, W02018022905, W0201700671, WO2017218842, WO2016154344, WO2017192750; W02016081811, WO2019222329 and WO2019028306, all of which are incorporated by reference herein in their entirety.

[0113] In some embodiments, the composition comprises at least one nucleic acid molecule that encodes for one or more gene products capable of generating one or more viral particles, including any viral particle encompassed herein. The nucleic acid(s) may comprise one or more plasmids,including any viral plasmids. The plasmid(s) may be an AAV plasmid, including an AAV-PHP.eB, AAV-PHP.B, AAV-PHP.S, AAV-PHP.B4, AAV-PHP.B5, AAV-PHP.N, AAV- CAP-B1, AAV- CAP-B10, AAV-CAP-B22, AAV type 1, AAV type 5, AAV type 8, or AAV type 9 plasmid. The plasmid(s) may be an AAV plasmid as described in one or more of PCT Patent Application Publications W02020206189, W02020028751, WO2020168145,

[0114] W02020077165, WO2021025995, WO2017197355, W02018022905, W0201700671, WO2017218842, WO2016154344, WO2017192750; W02016081811, WO2019222329 and WO20 19028306, all of which are incorporated by reference herein in their entirety. The nucleic acid may comprise a sequence that encodes for a transgene. The transgene may encode any gene product encompassed herein, including any STXBP1 gene product encompassed herein. The nucleic acid molecules may comprise any regulatory sequence encompassed herein.

[0115] 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.

[0116] Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased transduction enrichment 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).Promoter / Enhancers

[0117] 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.

[0118] 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 / enhancer element may be constitutive or inducible.

[0119] 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. See 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 1 ) :509- 16; the content of each of which is incorporated herein by reference.

[0120] 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-sub unit (EFl ), 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),

[0121] Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The vector may comprise a tissuespecific 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.

[0122] 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.Pharmaceutical Compositions and Delivery

[0123] The disclosure concerns compositions comprising, consisting essentially of, or consisting of an adeno-associated vims (AAV) particle comprising a nucleic acid comprising a sequence encoding part or all of an STXBP1 gene product, including wherein the AAV particle comprises one or more elements that imparts activity of the particle to transduce cells of the central and / or peripheral nervous system of an individual in need thereof. In cases wherein the nucleic acid encodes part of the STXBP1 gene product, the part will be functional in treating a medical condition associated with defective STXBP1, as addressed elsewhere herein.

[0124] In particular embodiments, any composition encompassed herein comprises a pharmaceutically acceptable (e.g., physiologically acceptable) earner. When the composition consists essentially of the inventive particle and a pharmaceutically acceptable carrier, additional components can be included that do not materially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.). When the compositionconsists of the inventive particle and the pharmaceutically acceptable carrier, the composition does not comprise any additional components. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally can be sterile with the exception of the composition components described herein. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).

[0125] Suitable formulations for the composition include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. In one embodiment, the carrier is a buffered saline solution. In one embodiment, the inventive particle is administered in a composition formulated to protect the composition and its contents from damage prior to administration. For example, the composition can be formulated to reduce damage from devices used to prepare, store, or administer the particle, such as glassware, syringes, or needles. The composition can be formulated to decrease the light sensitivity and / or temperature sensitivity of the nucleic acid. To this end, the composition may comprise a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such a composition extends the shelf life of the gene transfer vector, facilitates administration, and increases the efficiency of the inventive method. Formulations for gene transfer vector-containing compositions are further described in, for example, Wright et al,, Curr. Opin. Drug Discov. Devek, 6(2): 174-178 (2003) and Wright et al., Molecular Therapy, 12: 171-178 (2005))

[0126] The composition also can be formulated to enhance transduction efficiency. In addition, one of ordinary skill in the art will appreciate that the inventive gene transfer composition can be present in a composition with other therapeutic or biologically-active agents. For example, immunosuppressants or factors that control inflammation, such as ibuprofen, steroids, cytokine and complement inhibitors, and T- and B-cell inhibitors, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene transfer composition. Immune system stimulators or adjuvants, e.g., interleukins, lipopolysaccharide, and doublestranded RNA, can be administered to enhance or modify the immune response. Antibiotics, i.e., microbicides and fungicides, can be present to treat existing infection and / or reduce the risk of future infection, such as infection associated with gene transfer procedures.

[0127] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

[0128] In certain embodiments, a formulation of the present disclosure comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, poly alkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, poly glycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, poly anhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0129] The composition can be administered in or on a device (such as a mechanical reservoir) that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants, devices, such as an implantable device, e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition, are particularly useful for administration of the inventive gene transfer vector. The composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Pat. No. 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, apolyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and / or a polylactic-glycolic acid.

[0130] Delivery of the compositions comprising the inventive gene transfer compositions may be intracerebral (including but not limited to intracerebroventricular, intraparenchymal, intraventricular, or intracistemal), intrathecal (including but not limited to lumbar or cisterna magna), or systemic, including but not limited to intravenous, or any combination thereof, using devices known in the art. Delivery may also be via surgical implantation of an implanted device.

[0131] The dose of the gene transfer composition administered to a mammal will depend on a number of factors, including the size (mass) of the mammal, the extent of any side-effects, the particular route of administration, and the like. In one embodiment, the inventive method comprises administering a "therapeutically effective amount" of the composition comprising the inventive gene transfer composition described herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the extent of the disease, age, gender, and / or weight of the individual, and the ability of the gene transfer composition to elicit a desired response in the individual. The dose of gene transfer composition required to achieve a particular therapeutic effect may be determined by standard means. An example of a range of doses includes IxlO11vg / kg to IxlO14vg / kg. In preferred aspects, a dose comprises a range of doses that includes IxlO11vg / kg to 2xl013vg / kg.

[0132] In one embodiment of the disclosure, the composition is administered once to the mammal. However, in certain cases, it may be appropriate to administer the composition multiple times during a therapeutic period to ensure sufficient exposure of cells to the composition. For example, the composition may be administered to the mammal two or more times (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 or more times) during a therapeutic period.

[0133] The present disclosure provides pharmaceutically acceptable compositions that comprise a therapeutically-effective amount of composition comprising a nucleic acid sequence that encodes a functional STXBP1.Subjects

[0134] The subject (or individual or patient) that is the recipient of methods and compositions of the disclosure may be any subject with a defective STXBP1 for any reason. The subject may beany animal, including a human and non-human animal. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are envisioned as subjects, such as non-human primates, sheep, dogs, cats, cows and horses. The subject may also be livestock such as, cattle, swine, sheep, poultry, and horses, or pets, such as dogs and cats.

[0135] Particular subjects include human subjects suffering from or at risk for the medical diseases and conditions described herein. The subject may be generally diagnosed with the condition of the disclosure by skilled artisans, such as a medical practitioner. In some cases, the methods of the disclosure include steps of determining the presence of defective STXBP1 in a suitable sample from the subject.

[0136] The methods of the disclosure described herein can be employed for subjects of any species, gender, sex, age, ethnic population, and / or genotype across the genome (e.g., genetic background). Accordingly, the term subject includes males and females, and it includes elderly, elderly-to-adult transition age subjects, adults, adult-to-pre-adult transition age subjects, and preadults, including adolescents, children, and infants. Examples of human ethnic populations include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semites, and Pacific Islanders. The term subject also includes subjects of any genotype or phenotype as long as they are in need of the disclosed methods and compositions, as described above. In addition, the subject can have the genotype or phenotype for any hair color, eye color, skin color or any combination thereof. The term subject includes a subject of any height, body weight, or any organ or body part size or shape.Kits of the Disclosure

[0137] Any of the viral and / or non-viral compositions described herein or similar thereto may be comprised in a kit. In a non-limiting example, one or more reagents for use in methods for preparing viral particles may be comprised in a kit. Such reagents may include cells, vectors, one or more growth factors, one or more costimulatory factors, media, enzymes, buffers, nucleotides, salts, primers, compounds, and so forth. The kit components are provided in suitable container means.

[0138] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle,syringe or other container means into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0139] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and / or other such like apparatus, or may be a substrate with multiple compartments for a desired reaction.

[0140] Some components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent. In specific embodiments, reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases, the reagents include apparatus or reagents for isolation of a particular desired cell(s).EXAMPLESExample 1: AAV-mediated gene therapy in a mouse model of STXBP1 related developmental and epileptic encephalopathy

[0141] Developmental and epileptic encephalopathies (DEEs) are a group of devastating pediatric neurological disorders, manifesting with aggressive seizures and significant neurological comorbidities. De novo heterozygous pathogenic variants in STXBP1 encoding syntaxin-binding protein 1 are one of the most frequent genetic causes of DEEs. The abnormal brain activity during early development is believed to contribute to the pathogenesis of STXBP1 encephalopathy, presenting a great challenge for developing clinical treatments that can remain effective later in life. To develop a therapeutic approach with translational potential, intravenous delivery of AAV expressing hSTXBPl in rescuing adult Stxbpl haploinsufficient mice was tested. The vectorbiodistribution and mRNA / protein levels were quantified and correlated with the outcomes examined by health monitoring, video-electroencephalogram (EEG) recording and neurobehavioral tests. It was found that gene replacement therapy in Stxpbl haploinsufficient mice could rescue different phenotypes in a dose-dependent manner. Thus, our results indicated that gene replacement is a promising gene therapy for STXBP 1 -related developmental and epileptic encephalopathy.Study Design & Overview of Mouse Model

[0142] FIG. 1 shows genomic structures of Stxbpl wild type (WT) and tmld (KO) alleles. Exon (E); FRT (left arrows), and loxP (right arrows) sites are identified.

[0143] By sequentially crossing with Flp and Cre germline deleted mice, removing both the trapping cassette and exon 7 from the tm la heterozygous mice leads to a premature stop codon in exon 8 and generates a conventional knockout (KO) allele (tmld). Knockout mice are described in Chen et al. (2020) “ Stxbpl IMuncl 8-1 haploinsufficiency impairs inhibition and mediates key neurological features of STXBP1 encephalopathy” eLife 9:e4876, which is incorporated by reference herein.

[0144] Heterozygous KO (Stxbpltmldl+', pink text; Stxbpl+ / '’, “Het mice”; Stxbpl haploinsufficient mice) are shown with exon 7 removed. All mice were maintained on the C57BL / 6J isogenic background for all experiments. Stxbpltm,dl+Het mice were given retro-orbital (RO) intravenous injections of B Q-hSTXBP 1 once at 2-months of age. Control WT and Het mice were given a RO injection of vehicle once at 2-months of age.

[0145] FIG. 2 shows an overview of the experimental timeline in mice. Mice were injected with AAV at 2-months of age (week 8; WK8). A subset of mice (“EEG”) were implanted with EEG electrodes prior to IV dosing with B Q-hSTXBPl to establish baseline seizure activity. EEGs were measured 3-weeks (WK11), 6-weeks (WK14), and 6-months post-dosing. Following EEG recordings at 6-months post-dosing EEG mice were taken down at 7-months post-dosing to assess STXBP1 protein levels using western blot (WB) and cargo DNA and RNA levels using qPCR. A separate group of mice received behavioral testing beginning 6-weeks (WK14) post-dosing and a subset (6 / gp; N-30 total) were taken down 3-months post dosing (WK20) to assess STXBP1 protein levels using WB and cargo DNA and RNA levels using qPCR. The remaining animals were re-tested for behavior 6-months post-dosing.

[0146] FIGS. 21A-D show graphs measuring DNA and RNA biodistribution at 3 months and 7 months post-dosing. DNA and RNA Biodistribution were measured by qPCR to detect transgene DNA / RNA from tissue lysates. STXBP1 cargo DNA biodistributed and RNA expressed widely throughout key regions of interest (ROIs) of the MUR CNS, including cortical regions as early as 3-months post-dosing. DNA biodistribution and RNA expression throughout the MUR CNS are sustained through to 7-months post-dosing. Across all timepoints and ROIs, a significant dosedependent increase in DNA biodistribution and RNA biodistribution was observed across all three dose groups (Low Dose (Light); Medium Dose; High Dose (Dark)).

[0147] FIG. 3 shows a summary of treatment in mice, including assignment and number of animals per dose, per group, per cohort. A total of 280 WT and Stxbpl,mldl+(Stxbpl+I') mice (8-9 weeks old, 140 male and 140 female) were assigned to 5 main groups to evaluate correction of key disease features following intravenous (IV) administration of a surrogate capsid (B10) enclosing the clinical cargo (z.e., CAG-hSTXBPlb-WPRE-hGHpA).

[0148] Each group was divided into 2 cohorts for an evaluation of either EEG or behavior (BEH). Animals in the EEG cohort (N=12 / group, 6 male and 6 female) were assessed for spike-wave discharges and myoclonic seizures. Animals in the behavior (BEH) cohort (N=46 / group, 23 male and 23 female) were tested for 12 behavioral features using previously established measures of motor, cognitive, emotional, and sensory behaviors.

[0149] The Table below further shows a brief study design. Vector constructions utilize a surrogate capsid to enable CNS transductions following IV administration.STXBP1 Protein Expression

[0150] Total STXBP1 protein levels were assessed using western blot. Because human and mouse STXBP1 proteins are identical, this method detects endogenous STXBP1 and cargo STXBP1.

[0151] Antibody staining against STXBP1 was normalized to GAPDH, a housekeeping gene used as a loading control. Summary data of normalized STXBP1 expression levels from different brain regions was assessed. STXBP1 levels were first normalized by the GAPDH levels and then by the average STXBP1 levels of all WT mice from the same blot.

[0152] FIG. 4A-H show graphs of STXBP1 expression levels as measured by western blot, in the cortex, hippocampus, striatum, thalamus+hypothalamus, cerebellum, midbrain+hindbrain, olfactory bulb, and cervical spinal cord, respectively, at 3 months post AAV injection.

[0153] FIG. 5A-H show graphs of STXBP1 expression levels as measured by western blot in the cortex, hippocampus, striatum, thalamus+hypothalamus, cerebellum, midbrain+hindbrain, olfactory bulb, and cervical spinal cord, respectively, at 7 months post AAV injection.

[0154] Each filled (male) or open (female) circle represents one mouse.

[0155] STXBP1 protein levels were surveyed in different brain regions of Stxbpltmldl+mice at 3 months of age. STXBP1 was reduced by 40-50% in most brain areas except the cerebellum and olfactory bulb where the reduction was 20-30%. These results demonstrate that Stxbpltmldl+is indeed a Stxbpl haploinsufficient mouse model.

[0156] STXBP1 protein was reduced by 40-50% in most brain regions and in spinal cord (Het + vehicle (VEH)). There was a dose-dependent increase in STXBP1 protein 3-months post-dosing (N=6 per group, 3 males and 3 females) and 7-months post-dosing (N=12-14 per group, half male). In the majority of brain regions, the low dose does not achieve a significant increase in STXBP1 protein compared to the Het+VEH; exceptions include spinal cord and midbrain-hindbrain at 7- months. In the majority of regions, the medium and high dose groups have significant increases in STXBP1 protein. Dose-dependent increases are seen in all regions and there are no sex differences.Correction of Dystonia

[0157] Motor impairments are frequently observed in STXBP1 encephalopathy patients and previous work has shown that Stxbpl heterozygous mice show deficits in several tasks that are commonly used to assess motor function in mice. The effect of gene therapy was examined in four different tasks six to twenty-four weeks post-dosing.

[0158] The effect of gene therapy on hindlimb clasping was examined, a task that is commonly used as a measure for dystonia. Hindlimb clasping is first observed at around 4 weeks of age and by 3 months almost all Stxbpl heterozygous mice develop this phenotype.

[0159] FIG. 6 shows the hindlimb clasping score guide used. The images show what the phenotype looks like on a scale of 0-4 as 0 = no stiffness or clasping, 1 = stiffness in hindlimb, 2 = clasping of one hindlimb, 3 = clasping of both hindlimbs, 4 = tight clasping of both hindlimbs.

[0160] FIG. 7A-B show graphs of hindlimb clasping score in male and female mice, respectfully, from 5 weeks before to 24 weeks after treatment.

[0161] The medium and high doses correct hindlimb clasping within two weeks of administration and this correction is maintained for at least 24 weeks post-dosing at levels similar to WT controls. The low dose generates a significant partial correction of clasping with a similar time course. The gene therapy was sufficient to correct a physical manifestation of dystonia. Similar magnitudes of dystonia and correction are observed in both males and females.Body weights in Het mice

[0162] FIG. 8A-B show graphs of body weight in male and female mice, respectfully, from 5 weeks before to 24 weeks after treatment.

[0163] Reductions in body-weight observed in Stxbpl haploinsufficient mice are present before dosing (Time 0; males and females) and are not corrected in either sex by gene therapy (measured up to 24- weeks post-dosing). This result is not surprising because treatment at 2-months of age is after the key development events determining body size and composition have occurred.Reduction of spike-wave discharges (SWDs), myoclonic jumps and jerks

[0164] FIG. 9A-C show graphs of changes in SWD in mice measured from pre-treatment to 6 months post-treatment.

[0165] FIG. 10A-H show graphs of changes in myoclonic jumps in mice measured from pretreatment to 6 months post-treatment, as measured in all jumps, jumps in rapid eye movement sleep (REM), jumps in non-rapid eye movement sleep (NREM), and jumps during wakefulness (wake).

[0166] FIG. 11A-H show graphs of changes in myoclonic jerks in mice measured from pretreatment to 6 months post-treatment, as measured in all jumps, jumps in REM, jumps in NREM, and jumps in wake.

[0167] Overall outcomes show that low, medium, and high doses reduce both types of seizures rapidly (within 3-weeks) and with long-lasting correction present at 6-weeks and 6-months postdosing.Correction of contextual and cued fear memory

[0168] Intellectual disability is a core feature of STXBP1 encephalopathy, and Stxbpl heterozygous mice show significant impairments in several forms of learning and memory.

[0169] Mice were tested for two forms of fear conditioning to evaluate contextual and cued associative learning and memory. Learning in these tasks depends on activity in the hippocampus, prefrontal cortex, and amygdala. Briefly, mice are placed into a chamber that is defined by a unique set of sensory features (i.e., the context) and are presented with an auditory tone that co-terminates with the delivery of a mild aversive event i.e., foot-shock). Through the conditioning process, mice learn to associate both the context and the discrete cue with the aversive event and will display freezing responses when subsequently exposed to the context (or presented with the discrete cue) in the absence of any shock. In this case, the magnitude of freezing during testing is used to infer the strength of the associative memory, with higher levels of freezing indicative of a “stronger” memory.

[0170] The effects of gene therapy at low, medium, and high doses were examined in two different learning and memory tasks 6-weeks and 6-months post-dosing.

[0171] FIG. 12A-B show graphs measuring fear memory tests in mice measured 6 weeks post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0172] FIG. 13A-B show graphs measuring fear memory tests in mice measured 6 months post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0173] Stxbpl heterozygous mice exhibit significant impairments in freezing responses to both contextual and auditory cues, and this was corrected to levels comparable to WT controls in mice treated with the high dose (contextual + cued fear) at 6-weeks and 6-months post-dosing.

[0174] Low and medium doses did not show the same levels of significant correction.Correction of novel object recognition memory

[0175] Mice were tested in a novel object recognition task which is thought to depend on interactions between the hippocampus and prefrontal cortex, two regions that are critically involved in a variety of types of learning and memory.

[0176] During training (Days 1-3) mice are placed in an arena and allowed to freely explore and familiarize themselves with two identical objects. On test day (Day 4), one of the familiar objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familial- object, it will spend most of its time interacting with the novel object.

[0177] FIG. 14A shows the familiar and novel object used at 6 weeks post AAV injection.

[0178] FIG. 14B shows graphs measuring novel object recognition in mice measured 6 weeks post AAV injection as measured by exploratory preference.

[0179] FIG. 15A shows the familiar and novel object used at 6 months post AAV injection.

[0180] FIG. 15B shows graphs measuring novel object recognition in mice measured 6 months post AAV injection as measured by exploratory preference.

[0181] Stxbpl heterozygous mice show significantly impaired object memory and significant correction is observed at low, medium, and high doses, with the high dose correcting at levels comparable to WT controls.Impact on climbing ability in vertical pole test and foot slip test

[0182] Mice were tested in two tasks that are commonly employed to assess fine motor coordination, balance, and agility.

[0183] The vertical pole test, which is commonly employed to assess basal ganglia related movement disorders in mice, involves placing mice head downward on the top of a vertical pole and the time it takes them to descend is recorded.

[0184] FIG. 16A-B show graphs measuring vertical pole test results in mice post AAV injection as measured by time on pole at 6 weeks and 6 months, respectively.

[0185] Stxbpl heterozygous mice exhibit significant impairments in this task which is corrected in a dose-dependent manner, with animals receiving the high dose performing similar to WT controls at 6 weeks post-dosing.

[0186] FIG. 17A-B show graphs measuring foot slip test results in mice post AAV injection as measured by number of normalized footslips at 6 weeks and 6 months, respectively.

[0187] During the foot slip task, mice are placed onto an elevated wire grid and allowed to freely move for 5 minutes. Stxbpl heterozygous mice show impairments in this task, which is manifested as an increased number of foot slips. All Stxbpl+ / ' mice displayed significantly more foot slips during the task relative to WT controls and this phenotype was not corrected at any of the doses administered.Impact on marble burying

[0188] How many marbles Stxbpl+ / ' mice will bury was measured in a marble burying task. This task is a complex task that relies upon the natural tendency of mice to bury objects in their environments, which involves placing 20 black marbles in grid-like pattern on top of an abundance of fluffy bedding. Behavior in this task is highly variable, but well-powered experiments can examine differences between groups.

[0189] The motivation for burying has been hypothesized to come from 3 diverse and overlapping sources: (1) an innate desire to move and dig, which is elicited by repetitive small and visually salient marbles in the environment, (2) an anxiety-provoking feature of the marbles, where mice are burying the marbles out of a desire not to see many unfamiliar objects in their environment, and (3) the marbles elicit a repetitive digging response. Studies in mice carrying mutations in autism-risk genes have observed increased marble burying and some have interpreted this task as a measure of autistic-like repetitive behavior. However, this task has also been hypothesized to reflect an innate behavior in mice that may not have to do with repetitive behavior.

[0190] FIG. 18A-B show graphs measuring marble bury test results in mice post AAV injection as measured by number of buried marbles at 6 weeks and 6 months, respectively.

[0191] A dose-dependent increase in marble burying was demonstrated in the Stxbpl+ / ~ mice, which is fully corrected at the low dose and exceeds WT levels at the high dose. Because Stxbpl+ / ' mice have differences in baseline motor ability, which is corrected with treatment, it is possible that increased burying is due to improved motor function. It is also possible that increased digging reflects increased anxiety in response to the marbles, increased repetitive behavior, or increased engagement with the environment as a function of the marbles being salient and novel to mice that are beginning to show new cognitive abilities.Locomotor activity in an open field

[0192] Locomotor activity was examined in an open field at 6-weeks and 6-months post-dosing. Center distance in the open field is used to assess anxiety-like behavior, where anxious mice spend less time in the center of the open field.

[0193] FIG. 19A-D show graphs measuring open field assay tests measured at 6 weeks post- AAV injection as measured in distance, moving time, moving speed, and center duration, respectively.

[0194] FIG. 20A-D show graphs measuring open field assay tests measured at 6 months post- AAV injection as measured in distance, moving time, moving speed, and center duration, respectively.

[0195] Unlike previous analyses (Chen et al., 2020), Stxbpl+I~ mice did not show changes in activity compared to WT controls at either time point, most likely due to the difference in ages.

[0196] Significant reductions in activity (distance, moving time, and moving speed) were found in Stxbpl+ / ' mice treated with low, medium and high doses compared to WT-VEH and at the medium and high doses compared to Het- VEH treated mice at 6-weeks post-dosing. There are no differences in activity in dosed mice compared to WT-VEH at 6-months post-dosing; the medium and high dose groups continue to differ from Het-VEH at 6-months post-dosing in some measures.

[0197] Het-VEH mice also show reduced center time compared to WT-VEH at 6-weeks postdosing, suggesting they have increased anxiety. The low and medium doses show increased center time compared to Het-VEH suggesting reduced anxiety, but they also show significantly less center time compared to WT-VEH, suggesting only partial correction of this phenotype. At 6- months post-dosing there is no difference between WT-VEH and Het-VEH in center time; the slightly lower level of center time in Het-VEH treated animals is no longer significantly different. The high dose group shows increased center time compared to Het-VEH and Low dose groups providing additional evidence that center time is normalized in all groups, with the high-dose fully restoring center exploration time to WT levels.Example 2: A AV-mediated gene therapy in a missense mouse model of STXBP1 related developmental and epileptic encephalopathyHeterozygous pathogenic valiants of the syntaxin binding protein 1 (STXBP1, also known as MUNC18-1) are known to cause STXBP1 encephalopathy, which are among the most frequent causes of developmental and epileptic encephalopathies and intellectual disabilities. Haploinsufficiency has been considered as the primary disease mechanism for STXBP1 encephalopathy. Thus, gene replacement represents a promising therapeutic strategy for treating STXBP1 encephalopathy. Indeed, as set forth in Example 1 herein, restoring expression of the Stxbpl protein to the wildtype level in adult Stxbpl haploinsufficient mice rescued most of the phenotypes including epileptic seizures, motor, cognitive, and psychiatric impairments.

[0198] However, overexpression studies showed that a subset of missense mutations, including R406H, caused the formation of insoluble aggregates with wildtype Stxbpl proteins in vitro, which exerted dominant negative-like effects. While in in vivo mouse models and the human patient population missense mutations are expected to function similarly to haploinsufficiency, previous work in vitro indicated a potential limit on the efficacy of gene replacement in treating STXBP1 encephalopathy.

[0199] To assess the efficacy of gene replacement in a mouse model of STXBP1 missense variant, a StxbplR406H / +knock-in model was used to test intravenous delivery of an engineered AAV capsid expressing human STXBP1 with low, medium, and high dosages in rescuing adult StxbplR406H / +mice. As shown in the present Example, AAV mediated gene replacement therapy rescues most of the phenotypes of adult StxbplR406H / +mice, in a dose dependent manner and with long lasting effects, including abnormal hindlimb clasping, epileptic seizures, intellectual disability, motor deficits, and impaired innate mouse behaviors. Results from the present example show that the high dose rescues the most phenotypes, compared to the medium and low doses, which rescue or partially improve some of the phenotypes. Thus, gene replacement shows efficacy as a therapy for STXBP1 encephalopathy, which may help treat individuals with either loss of function or missense pathogenic variants.Study Design & Overview of Mouse ModelFIG. 22 shows an overview of the experimental overview and timeline in treating StxbplR406H / +mice, which are a knock-in model for a debilitating STXBP1 missense variant, which has been implicated in causing aggregates with wildtype STXBP1 in vitro and in C. elegans models. As shown in FIG. 22, the mice were divided into 5 groups. The five different groups were used to evaluate the correction of key disease features following administration of a surrogate capsid (BIO) enclosing the human STXBP1 cargo (i.e., CAG-hSTXBPlb-WPRE-hGHpA).

[0200] One group was Stxbpl wildtype (“WT”) mice that were injected with a pharmaceutical formulation vehicle. A second group was StxbplR406H / +knock-in mice that received the pharmaceutical vehicles (“MV”). Three groups of StxbplR406II / +mice received retro-orbital (RO) intravenous injections of the BlQ-hSTXBPl AAV vector once at 8-9 weeks of age. The first group “MLD” received a low dose of 5xl0nvg / kg of the vector, a second group “MMD” received a medium dose of IxlO12vg / kg, while the third group “MHD” received a high dose of 5xl012vg / kg. A subset of mice, n=16 from each group were placed in Cohort A. Mice in this cohort, wereimplanted with EEG electrodes prior to dosing to establish baseline seizure activity. EEGs were measured before dosing, then at 6-weeks post-dosing, and 6-months post-dosing. Following EEG recordings at 6-months post-dosing EEG mice were taken down at 7-months post-dosing to assess STXBP1 protein levels using western blot (WB) and to assess biodistribution.

[0201] A separate Cohort, “Cohort B”, from each group (n=51 / group) of mice received behavioral testing beginning 6-weeks post-dosing and a subset (6 / gp; N=30 total) were taken down 3-months post dosing to assess STXBP1 protein levels using WB and biodistribution. The remaining animals were re-tested for behavior 6-months post-dosing. Following testing at 6-months post-dosing the remaining mice were taken down at 7-months post-dosing to assess STXBP1 protein levels using western blot (WB) and to assess biodistribution. Animals in the behavior (BEH) cohort (Cohort B) were tested for behavioral features using previously established measures of motor, cognitive, emotional, and sensory behaviors.Body weights in StxbplR406H / +mice

[0202] FIG. 23 provides graphs that provide the body weight in male and female mice, respectfully, from 2 weeks before to 26 weeks after dosing across the 5 groups for Cohort A, theEEG cohort.

[0203] FIG. 24 provides graphs that provide the body weight in male and female mice, respectfully, from 2 weeks before to 34 weeks after dosing across the 5 groups for Cohort B, theBEH cohort.

[0204] Reductions in body-weight observed in StxbplR406H / +mice are present before dosing (Time -2; males and females) and are not corrected in either sex by gene therapy (measured up to 26 / 34- weeks post-dosing). This result is not surprising because treatment at 2-months of age is after the key development events determining body size and composition have occurred.Correction of Dystonia

[0205] Motor impairments are frequently observed in STXBP1 encephalopathy patients, and StxbplR406H / +mice show deficits in several tasks that are commonly used to assess motor function in mice. The effect of gene therapy was examined in four different tasks 4 / 6 weeks to twenty-four weeks post-dosing.

[0206] The effect of gene therapy on hindlimb clasping in StxbplR406H / +mice was examined, a task that is commonly used as a measure for dystonia. Hindlimb clasping is first observed at around 4 weeks of age (-2 weeks) and by 3 months almost all StxbplR406H / +mice develop this phenotype.

[0207] FIG. 6 shows the hindlimb clasping score guide used. The images show what the phenotype looks like on a scale of 0-4 as 0 = no stiffness or clasping, 1 = stiffness in hindlimb, 2 = clasping of one hindlimb, 3 = clasping of both hindlimbs, 4 = tight clasping of both hindlimbs.

[0208] FIG. 25 shows a graph of hindlimb clasping score in all mice of Cohort A from 2 weeks before dosing until 28 weeks after dosing. FIG. 26 shows a graph of hindlimb clasping score in all mice of Cohort B from 2 weeks before dosing until 34 weeks after dosing.

[0209] In both Cohorts, the high dose corrects hindlimb clasping within two weeks of administration and this correction is maintained for at least 28 / 34 weeks post-dosing at levels similar to WT controls. The medium and low doses generate a significant partial correction of clasping with a similar time course. The gene therapy was sufficient to correct a physical manifestation of dystonia.Correction of anxiety (Elevated Plus Maze & Light-Dark Box)

[0210] Intellectual disability is a core feature of STXBP1 encephalopathy, and StxbplR406H / +mice show significant neural impairments as evidenced by changes in instinctual behavior.

[0211] StxbplR406II / +mice were tested for changes in instinctual behavior using an Elevated Plus Maze (EPM). The EPM is a behavioral test used to assess anxiety-like behaviors in mice, based on their natural preference for enclosed areas and aversion to open, elevated spaces. The EPM includes a four-armed platform resembling a plus sign, with two arms having walls (closed arms) and two arms without walls (open arms). Rodents instinctually prefer to explore novel environments and avoid open and elevated areas. Anxious rodents tend to spend less time in the open arms and more time in the closed arms, while rodents with lower anxiety levels will explore the open arms more. During the test, each mouse is monitored to quantify: the number of entries into each arm, the time spent in each arm, and distance travelled along each arm. A higher percentage of time spent in the open arms, a further distance travelled in an open arm, and / or a high number of entries into an open arm are indicative of less anxiety and a willingness to explore novel spaces. Conversely, more entries, time spent, and distance in closed arms represents an anxious state. The EPM can also be used to study other behaviors such as exploration, locomotion, and spatial memory

[0212] FIGS. 27A-27B show graphs of Cohort B mice across all five groups, from 6 weeks postdose, from the EPM, including entries, time spent within, and distance travelled for both open and closed arms. As shown, at 6 weeks, the StxbplR406H / +mice showed a more anxious phenotype relative to the wildtype mice.

[0213] FIGS. 28A-28B show graphs of Cohort B mice across all five groups, from 6-months postdose. As shown, compared to 6- weeks, the treatment provided a partial phenotype rescue, with the high dose providing the greatest corrective effect.

[0214] StxbplR406H / +mice were also tested for changes in instinctual behavior using a Light-Dark Box Assay. The mouse Light-Dark Box Assay is a behavioral assay used to assess anxiety-like behavior in mice, where mice are placed in a box with a bright and a dark compartment, and their time spent in each area, as well as their transitions between them, are measured. Mice with anxietylike behavior tend to spend less time in the light compartment and make fewer transitions between the compartments.

[0215] FIGS. 29A-29B show graphs at six weeks post-dose for Cohort B mice from all five groups from the light-dark box assay showing measures of light / dark duration, light / dark entry, total distance traveled in light / dark, movement time in light / dark, and time resting in light / dark. FIGS. 30A-30B show graphs at six-months post-dose for Cohort B mice from all five groups from the light-dark box assay showing measures of light / dark duration, light / dark entry, total distance traveled in light / dark, movement time in light / dark, and time resting in light / dark.

[0216] StxbplR406H / +mice exhibit anxious behaviors relative to WT mice in the light-dark box assay. Some partial rescue occurred at six weeks post-dosing, with greater effects attributed to the high-dose. Anxious behaviors improved in treated mice at 6-months post dosing, with the high dose providing the most significant correction.Locomotor activity in an open field

[0217] Locomotor activity was examined in an open field at 6-weeks, 4-months, and 6-months post-dosing. Center distance in the open field is used to assess anxiety-like behavior, where anxious mice spend less time in the center of the open field.

[0218] FIGS. 31A-C show graphs measuring open field assay tests measured at 6 weeks post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0219] FIGS. 32A-C show graphs measuring open field assay tests measured at 4 months post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0220] FIGS. 33A-D show graphs measuring open field assay tests measured at 6 months post- AAV injection as measured in distance, moving time, moving speed, and center duration for mice of Cohort B across all five groups.

[0221] StxbplR406H / +mice showed changes in activity compared to WT controls reductions in activity as measured by distance, moving time, and moving speed.

[0222] Significant reductions in activity (distance, moving time, and moving speed) were found in StxbplR406H / +mice treated with low, medium and high doses compared to StxbplR406H / +vehicle- only (MV) mice, specifically at the high dose. These changes continued through the month 4 and month 6 measures.Impact on fine motor coordination and sensitivity climbing ability in vertical pole test and foot slip test

[0223] Mice were tested in three tasks that are commonly employed to assess fine motor coordination, balance, and agility.

[0224] Mice from all five groups of Cohort B were assessed in the vertical pole test. The vertical pole test is commonly employed to assess basal ganglia related movement disorders in mice, involves placing mice head downward on the top of a vertical pole and the time it takes them to descend is recorded.

[0225] FIG. 34 shows graphs measuring vertical pole test results in mice post AAV injection as measured by time on pole at 6 weeks and 6 months, respectively.

[0226] StxbplR406H / +mice exhibit significant impairments in this task which is partially corrected in a dose-dependent manner, with animals receiving the high dose performing similar to WT controls at 6 months post-dosing.

[0227] FIG. 35 shows graphs measuring foot slip test results in Cohort B mice post AAV injection as measured by number of normalized foot slips at 6 weeks and 6 months, respectively.

[0228] During the foot slip task, mice are placed onto an elevated wire grid and allowed to freely move for 5 minutes. StxbplR406H / +mice show impairments in this task, which is manifested as an increased number of foot slips. All StxbplR406H / +mice displayed significantly more foot slipsduring the task relative to WT controls and this phenotype was not corrected at any of the doses administered.Impact on marble burying

[0229] How many marbles StxbplR406H / +mice will bury was measured in a marble burying task. This task is a complex task that relies upon the natural tendency of mice to bury objects in their environments, which involves placing 20 black marbles in grid-like pattern on top of an abundance of fluffy bedding. Behavior in this task is highly variable, but well-powered experiments can examine differences between groups.

[0230] FIG. 36 shows graphs measuring marble bury test results in Cohort B mice across all groups post AAV injection as measured by number of buried marbles, at 6 weeks and 6 months, respectively.

[0231] A dose-dependent increase in marble burying was demonstrated in the StxbplR406H / +mice, which is fully corrected at the medium dose and exceeds WT levels at the high dose by six-months post-dose. Because StxbplR406H / Jrmice have differences in baseline motor ability, which is corrected with treatment, it is possible that increased burying is due to improved motor function. It is also possible that increased digging reflects increased anxiety in response to the marbles, increased repetitive behavior, or increased engagement with the environment as a function of the marbles being salient and novel to mice that are beginning to show new cognitive abilities.Correction of contextual and cued fear memory

[0232] Intellectual disability is a core feature of STXBP1 encephalopathy, and StxbplR406H / +mice show significant impairments in several forms of learning and memory.

[0233] Mice were tested for forms of fear conditioning to evaluate contextual and cued associative learning and memory. Learning in these tasks depends on activity in the hippocampus, prefrontal cortex, and amygdala. Briefly, mice are placed into a chamber that is defined by a unique set of sensory features (z.e., the context) and are presented with an auditory tone that co-terminates with the delivery of a mild aversive event (z.e., foot-shock). Through the conditioning process, mice learn to associate both the context and the discrete cue with the aversive event and will display freezing responses when subsequently exposed to the context (or presented with the discrete cue) in the absence of any shock. In this case, the magnitude of freezing during testing is used to inferthe strength of the associative memory, with higher levels of freezing indicative of a “stronger” memory.

[0234] The effects of gene therapy at low, medium, and high doses were examined in two different learning and memory tasks 6-weeks and 6-months post-dosing.

[0235] FIG. 37 shows graphs measuring fear memory tests in Cohort B mice across all five groups measured 6 weeks post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0236] FIG. 38 show graphs measuring fear memory tests in Cohort B mice across all five groups measured 6 months post AAV injection as measured by freezing percentage in contextual tests and cue tests, respectively.

[0237] StxbplR406H / +mice exhibit significant impairments in freezing responses to both contextual and auditory cues, and this was corrected to levels comparable to WT controls in mice treated with the high dose (contextual + cued fear) at 6-weeks and 6-months post-dosing.

[0238] Low and medium doses did not show the same levels of significant correction.Correction of pain response

[0239] Research suggests that STXBP1 plays a crucial role in synaptic function, and disruptions in this function could potentially impact pain processing or the ability to properly respond to such stimuli, e.g., due to problems with locomotive control. StxbplR406II / +mice show impairments in pain response.

[0240] FIG. 39 shows graphs that provide results of the hot plate test in Cohort B mice across all five groups at six-weeks and six-months post-dose.

[0241] The mouse hot plate test is a method to assess thermal pain sensitivity by measuring the latency (time) it takes for a mouse to show a nociceptive response (like licking or jumping) after being placed on a heated plate. As shown in FIG. 39, particularly at 6-months post dose, the wildtype mice have a faster response time relative to the StxbplR406H / +(fAN mice. There was no significant different between WT controls and treated mice at 6-weeks or 6-months post-dosing.

[0242] FIG. 40 shows graphs that provide results of the mouse tail flick test in Cohort B mice across all five groups at six-weeks and six-months post-dose.

[0243] The mouse tail flick response test is a behavioral test used to assess pain sensitivity in rodents, measuring the time it takes for a mouse to remove its tail from a heat source, indicating a pain threshold. As shown in FIG. 40, the WT mice have a faster response time relative to theStxbplR406H / +(fAN mice. There was no significant difference between WT controls and treated mice at 6-weeks and 6-months post-dosing.Impact on Nest Building

[0244] Mouse nestbuilding requires a complex set of behaviors that are often impacted by neurological diseases that affect hippocampal function.

[0245] FIG. 41 shows graphs that provide results of the nesting test in Cohort B mice across all five groups at six- weeks and six-months post-dose. StxbplR406H / +mice show significant disruptions in nestbuilding activity relative to wildtype mice. This was nearly corrected to levels comparable to WT controls in mice treated with the high dose at 6-months post-dosing.Reduction of myoclonic jumps and jerks

[0246] FIG. 42 shows graphs that provide changes in myoclonic jumps as measured in all jumps in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0247] FIG. 43 shows graphs that provide changes in myoclonic jumps as measured in jumps during REM in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0248] FIG. 44 shows graphs that provide changes in myoclonic jumps as measured in jumps during non-rapid eye movement sleep (NREM) in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0249] FIG. 45 shows graphs that provide changes in myoclonic jumps as measured in jumps during wakefulness (wake) in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0250] FIG. 46 shows graphs that provide changes in myoclonic jerks as measured in jerks in total in mice from Cohort A from all five groups measured from pre-dose and six-weeks post-dose.

[0251] FIG. 47 shows graphs that provide changes in myoclonic jerks as measured in jerks during REM in mice from Cohort A from all five groups measured from pre-dose and six-weeks postdose.

[0252] FIG. 48 shows graphs that provide changes in myoclonic jerks as measured in jerks during NREM in mice from Cohort A from all five groups measured from pre-dose and six-weeks postdose.

[0253] FIG. 49 shows graphs that provide changes in myoclonic jerks as measured in jerks during wakefulness (wake) in mice from Cohort A from all five groups measured from pre-dose and six- weeks post-dose.

[0254] Overall outcomes show that low, medium, and high doses reduce both types of seizures rapidly and with long-lasting correction present at 6-weeks.STXBP1 Protein Expression

[0255] Total STXBP1 protein levels were assessed using western blot. Because human and mouse STXBP1 proteins are identical, this method detects endogenous STXBP1 and cargo STXBP1.

[0256] Antibody staining against STXBP1 was normalized to GAPDH, a housekeeping gene used as a loading control. Summary data of normalized STXBP1 expression levels from different brain regions was assessed. STXBP1 levels were first normalized by the GAPDH levels and then by the average STXBP1 levels of all WT mice from the same blot.

[0257] FIGS. 50A-50B show graphs of STXBP1 expression levels in mice from all five groups at 6-weeks post-dosing as measured by western blot, in the cortex, hippocampus, striatum, thalamus+hypothalamus, cerebellum, brainstem, olfactory bulb, and cervical spinal cord, respectively.

[0258] FIG. 51A-51B shows graphs of STXBP1 expression levels in mice from all five groups at 6-weeks post-dosing as measured by western blot, in the cortex, hippocampus, striatum, thalamus+hypothalamus, cerebellum, brainstem, olfactory bulb, and cervical spinal cord, respectively. FIG. 51B specifically shows STXBP1 protein expression from a subcohort of animals assessed for STXBP1 protein in anterior cortex only at 6 months post-dosing.

[0259] Each filled (male) or open (female) circle represents one mouse.

[0260] STXBP1 protein levels were surveyed in different brain regions of StxbplR406IR+mice at 3 months of age. STXBP1 was reduced by 50-70% in most brain areas except the cerebellum and olfactory bulb where the reduction was 30-40%. These results demonstrate that StxbplR406H / +mimics a Stxbpl haploinsufficient mouse model.

[0261] STXBP1 protein was reduced by 50-70% in most brain regions and in spinal cord in StxbplR4 6T4 / +mice. There was a dose-dependent increase in STXBP1 protein 6-weeks post-dosing (N=5-6 per group) and 7-months post-dosing (N=12-14 per group). In the majority of brain regions, the low and medium doses did not achieve a significant increase in STXBP1 protein compared to the StxbplR406H / +mice (MV) without treatment. In the majority of regions, the highdose groups have significant increases in STXBP1 protein. Dose-dependent increases are seen in all regions and there are no sex differences.Discussion

[0262] AAV mediated gene replacement therapy rescues most of the phenotypes of adult Stxbpl missense mice, including abnormal hindlimb clasping, epileptic seizures, intellectual disability, motor deficits, and impaired innate mouse behaviors. The highest dose rescues the most phenotypes while lower doses either partly improve or rescue some of the phenotypes. Gene supplementation through AAV-delivered therapeutic cargo resulted in phenotypic correction across the major disease domains, including: seizure (e.g., myoclonic jumps and jerks); motor (e.g., correction observed in hindlimb clasping and pole test); and cognitive (e.g., fear conditioning). Further, as shown, hSTXBPl gene supplementation through the therapeutic cargo restored STXBP1 protein levels throughout the brain.Incorporation by Reference

[0263] 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.Equivalents

[0264] 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.

Claims

CLAIMSWhat is claimed is:1 . A formulation comprising: an adeno-associated virus (AAV) particle comprising a nucleic acid comprising a promoter and a sequence encoding an STXBP1 gene product, wherein the formulation comprises at least 1 x 1011vector genomes (vg) / kilogram (kg) or dose with similar biological impact.

2. The formulation of claim 1, wherein the formulation comprises at least 5 x 1011vg / kg, or dose with similar biological impact.

3. The formulation of claim 1, wherein the formulation comprises at least 1 x 1012vg / kg, or dose with similar biological impact.

4. The formulation of claim 1, wherein the formulation comprises at least 2 x 1012vg / kg, or dose with similar biological impact.

5. The formulation of claim 1, wherein the formulation comprises at least 5 x 1012vg / kg, or dose with similar' biological impact.

6. The formulation of claim 1, wherein the formulation comprises at least 1 x 1013vg / kg, or dose with similar- biological impact.

7. The formulation of claim 1, wherein the formulation comprises at least 2 x 1013vg / kg, or dose with similar' biological impact.

8. The formulation of claim 1, wherein: the AAV particle is an AAV-PHP.eB particle, AAV-PHP.B particle, AAV-PHP.S particle, AAV-PHP.B4 particle, AAV-PHP.B5 particle, AAV-PHP.N particle, AAV-CAP-B1 particle, AAV-CAP-B10 particle, AAV-CAP-B22 particle, AAV type 1 particle, AAV type 5 particle, AAV type 8 particle, or AAV type 9 particle; the promoter is a CAG promoter; andthe nucleic acid further comprises a woodchuck hepatitis virus (WPRE).

9. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1.

10. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises the amino acid sequence of SEQ ID NO: 1.

11. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2.

12. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises the amino acid sequence of SEQ ID NO: 2.

13. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3.

14. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises the amino acid sequence of SEQ ID NO: 3.

15. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4.

16. The formulation of any one of claims 1-8, wherein the STXBP1 gene product comprises the amino acid sequence of SEQ ID NO:4.

17. The formulation of any one of claims 1-16, wherein the sequence encoding the STXBP1 gene product lacks an intron.

18. The formulation of any one of claims 1-17, wherein the sequence encoding the STXBP1 gene product is operably linked to one or more regulatory sequences.

19. The formulation of claim 18, wherein one or more regulatory sequences induce expression of the STXBP1 gene product in cells of the central and / or peripheral nervous system.

20. The formulation of claim 19, wherein the cells of the central and / or peripheral nervous system comprise glutamatergic neurons, GABAergic neurons, glycinergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, adrenergic neurons, and / or noradrenergic neurons.

21. A method of treating an individual with an encephalopathy comprising administering a formulation of any one of claims 1-20 to the individual.

22. The method of claim 21, wherein the encephalopathy is STXBP1 encephalopathy or early infantile epileptic encephalopathy type 4.

23. The method of claim 22, wherein the encephalopathy is STXBP1 encephalopathy.

24. The method of any of claims 21-22, wherein the individual has Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, or Rett syndrome with mutated STXBP1.

25. The method of any one of claims 21-24, wherein the individual has one or more mutations in a STXBP1 gene.

26. The method of any one of claims 21-25, wherein the individual has one or more mis sense mutations, nonsense mutations, deletions, inversions, insertions, duplications, frameshift mutations, repeat expansions, haploinsufficiencies, or a combination thereof in the STXBP1 gene.

27. The method of any one of claims 21-26 wherein the individual is an infant, child, or adolescent.

28. The method of any one of claims 21-26, wherein the individual is an adult.

29. The method of any one of claims 21-28, wherein the AAV particle is administered by intravenous delivery.

30. The method of any one of claims 21-28, wherein the AAV particle is administered by injection into brain tissue.

31. The method of any one of claims 21-28, wherein the AAV particle is administered by injection outside of the brain.

32. The method of any of claims 21-31, further comprising, prior to administering the AAV particle to the individual, detecting a mutation in STXBP1 in the individual.

33. An AAV particle comprising a nucleic acid encoding an STXBP1 gene product having at least 90% sequence identity with SEQ ID NO:2.

34. The AAV particle of claim 33, wherein the STXBP1 gene product has at least 95% sequence identity with SEQ ID NO:2.

35. The AAV particle of claim 33, wherein the STXBP1 gene product comprises SEQ ID NO:2.

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