Novel gene therapy constructs for stxbp1 haploinsufficiency

Abstract

Provided herein are methods of expressing syntaxin-binding protein 1 (STXBP1) in a neuron. Also provided herein is a modified adeno-associated virus (AAV) encoding a STXBP1 under the control of a promoter operable in a brain cell or neuronal cell.

Description

DESCRIPTION NOVEL GENE THERAPY CONSTRUCTS FOR STXBP1 HAPLOINSUFFICIENCY PRIORITY CLAIM

[0001] This application claims benefit of priority to U.S. Provisional 5 Application Serial No. 63 / 575,062, filed April 5, 2024, the entire contents of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted 10 electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on March 28, 2025, is named CHOPP0077WO.xml and is 80,891 bytes in size. BACKGROUND 1. Field 15

[0003] The present disclosure relates generally to the fields of medicine, virology, and neurology. More particularly, it concerns targeting peptides that target delivery of viral vectors to distinct structures in the brain, particularly in the treatment of STXBP1-related epileptic encephalopathy. 2. Description of Related Art 20

[0004] The syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1) plays an essential role in synaptic vesicle fusion and is required for neurotransmitter release. Mutations in STXBP1 are a leading cause of developmental epileptic encephalopathy. Multiple mutations in STXBP1 result in premature termination codons, causing transcript nonsense mediated decay and STXBP1 haploinsufficiency. The ability to modulate STXBP1 25 expression would potentially serve to treat related diseases, such as epileptic encephalopathy.

[0005] Adeno-associated viruses (AAVs) represent strong therapeutic candidates for the treatment of neurodegenerative disease. AAVs are non-enveloped, single-stranded DNA viruses that can infect both dividing and non-dividing cells. Following infection, the virus does not exhibit robust integration within the host genome but persists as an episome in the 30 cell nucleus. Expression of AAV cargoes is controlled spatially at the level of the packaging 1 4932-3429-7135, v.1capsid and by the transgene promoter. Because use of AAV for the treatment of disease may necessitate intervention in diseased tissue, a problem can arise in that target tissue that contains a different gene expression profile than its healthy counterpart. Finding the correct promoter sequence to drive therapeutic transgene expression is an important goal. 5

[0006] Different strategies have been developed to generate AAV vector variants including rational design and directed evolution. The rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, such as tyrosine mutations on the capsid surface for increasing transduction efficiency. The directed evolution approach does not require any knowledge of 10 capsid structure and is done through random mutagenesis, capsid shuffling, or random peptide insertions. These strategies generally use in vitro systems or mice, which are ideal for cell-based or mouse studies, but do not imply translation to the clinic. In fact, no AAV variants target distinct brain structures specifically or efficiently. As such, AAV variants that are capable of targeting distinct brain structures are needed. 15 2 4932-3429-7135, v.1SUMMARY

[0007] Thus, in accordance with the present disclosure, there is provided a method of expressing a syntaxin-binding protein 1 (STXBP1) in a mammalian cell, a brain cell, a mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron of a 5 subject, comprising administering to the subject a modified adeno-associated virus (AAV) encoding a STXBP1 under the control of a promoter operable in said brain cell, mammalian cell, mammalian brain cell, mammalian neuron, mouse neuron human neuron. The AAV may comprise a modified capsid protein comprising or consisting of the targeting sequence of SEQ ID NO: 4 or 6. The promoter may be a constitutive promoter or a tissue specific 10 promoter. The method promoter may be an Mecp2 promoter or an iCAG promoter. The Mecp2 promoter may comprise the sequence of SEQ ID NO: 9 and the iCAG promoter may comprise the sequence of SEQ ID NO: 10. The modified AAV may be derived from AAV1 and AAV2. The STXBP1 sequence may be operably linked to a poly-adenylation signal. The modified AAV may further comprise a 3’ UTR, such as native STXBP1 UTR, adjacent to a 15 coding sequence for STXBP1.

[0008] The targeting sequence may be inserted near or after position 590 of SEQ ID NO: 1 or after position 587 of SEQ ID NO: 2. The targeting sequence may consist essentially of SEQ ID NO: 4 or 6. The targeting sequence may consist of SEQ ID NO: 4 or 6. The STXBP1 may comprise the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 8. 20 The STXBP1 may consist of the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 8. The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting sequence and AS on the C- terminal side of the targeting sequence, or wherein the linker sequences may be AAA on the 25 N-terminal side of the targeting sequence and AA on the C-terminal side of the targeting sequence. The modified capsid protein may comprise the sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 5.

[0009] The administering may be direct intracerebroventricular or intracisternal magna or intrathecal delivery. The administering may occur more than once, such as 2, 3, 4, 30 5, 6, 7, 8, 9, 10 or more administrations. The administering may occur monthly, every other month, every two months, every three months, every four months, every six months, annually every other year, every three years, every four year or every five years. A plurality of viral 3 4932-3429-7135, v.1particles may be administered. The administering may comprise dosing at about 1×106to about 1×1014vector genomes per kilogram (vg / kg). The administering may comprise dosing from about 1x107-1x1014, about 1x108-1x1014, about 1x109-1x1014, about 1x1010-1x1014, about 1x1010-1x1013, about 1x1010-1x1013, about 1x1010-1x1011, about 1x1011-1x1012, or 5 about 1x1012-1x1013vector genomes per kilogram (vg / kg) of the patient. The subject may suffer from STXBP1-related epileptic encephalopathy. The subject may be less than 4 years old, or is between 4 and 25 years old.

[0010] Also provide is a modified adeno-associated virus (AAV) encoding a STXBP1 under the control of a promoter operable in a mammalian cell, a mammalian brain cell brain 10 cell, such as a mammalian neuron, a mouse neuron or human neuron. The AAV may comprise a modified capsid protein comprising the targeting sequence of SEQ ID NO: 4 or 6. The promoter may be a constitutive promoter or a tissue specific promoter. The promoter may be an Mecp2 promoter or an iCAG promoter. The Mecp2 promoter may comprise the sequence of SEQ ID NO: 9 and the iCAG promoter may comprise the sequence of SEQ ID 15 NO: 10. The modified AAV may be AAV1 or AAV2. The modified AAV may further comprise a 3’ UTR, such as native STXBP1 UTR, adjacent to a coding sequence for STXBP1.

[0011] The targeting sequence may be inserted near or after position 590 of SEQ ID NO: 1. The targeting sequence may consist essentially of 587 of SEQ ID NO: 2 or may 20 comprise or consist of SEQ ID NO: 4 or 6. The STXBP1 may comprise the sequence of SEQ ID NO: 7 or SEQ ID NO: 8 may consists of the sequence of SEQ ID NO: 7 or SEQ ID NO: 8. The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting sequence and AS on the C- 25 terminal side of the targeting sequence, or wherein the linker sequences may be AAA on the N-terminal side of the targeting sequence and AA on the C-terminal side of the targeting sequence. The modified capsid protein may comprise the sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0012] In a further aspect, there is provided pharmaceutical composition comprising a 30 modified AAV as described herein and a pharmaceutically acceptable carrier, and a kit comprising a modified AAV as described herein. 4 4932-3429-7135, v.1

[0013] Further provided are: uses of a modified adeno-associated virus (AAV) encoding a syntaxin-binding protein 1 (STXBP1) under the control of a promoter operable in a mammalian cell, a brain cell, a mammalian brain cell, such as a neuron, a human neuron or a mouse neuron, 5 such as wherein said AAV comprises a modified capsid protein comprising the targeting sequence of SEQ ID NO: 4 or 6, for expressing STXBP1 in a mammalian cell, a mammalian brain cell brain cell, such as a mammalian neuron, mouse neuron or human neuron; uses of a modified adeno-associated virus (AAV) encoding a syntaxin binding protein 10 1 (STXBP1) under the control of a promoter operable in a brain cell, a mammalian cell, a mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron, such as wherein said AAV comprises a modified capsid protein comprising the targeting sequence SEQ ID NO: 4 or 6, for treating STXBP1-related epileptic encephalopathy in a subject afflicted therewith; and 15 uses of a modified adeno-associated virus (AAV) encoding a syntaxin-binding protein 1 (STXBP1) under the control of a promoter operable in a brain cell, a mammalian cell, such as mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron, such as wherein said AAV comprises a modified capsid protein comprising the targeting sequence SEQ ID NO: 4 or 6, in the preparation of a 20 medicament for treating STXBP1-related epileptic encephalopathy in a subject afflicted therewith.

[0014] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word 25 “about” means plus or minus 5% of the stated number.

[0015] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description 30 and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and 5 4932-3429-7135, v.1scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings form part of the present specification and are included 5 to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] FIG. 1. Image of transduced iPSC-N. Excitatory neurons were generated, as previously described (Waxman et al., 2023, PMID: 38148714), from STXBP1 patient 10 (STXBP1 with c.663+1G>C mutation) and WT10 iPSC lines, and resulting neurons were transduced on Day 40-45 of differentiation with the indicated viruses expressing a fluorescent reporter at an MOI of 5E+03. Representative images were taken at day 5 and day 12 post- transduction.

[0018] FIGS. 2A-B. Transduction of iPSC (STXBP1 c.663 +5G>A) using 15 BLD004 and BLD005 Capsid – Day 13. iPSC-derived excitatory neurons from patient STXBP1 line (STXBP1 c.663+1G>C) were transduced on day 40-45 of differentiation by indicated viruses (Mecp2-STXBP1 and Mecp2-STXBP1-Flag at an MOI 1E5 and iCAG- STXBP1 and iCAG-STXBP1-Flag at an MOI of 5E4), each containing cDNA of wild-type STXBP1. Samples were harvested for RNA and protein extraction 13 days post-transduction. 20 (FIG. 2A) Quantitative PCR measurement of STXBP1 mRNA normalized to GAPDH and is presented relative to untransduced cells. (FIG. 2B) Western blot analysis of STXBP1 and ATP5F1 (loading control) levels. The graph on the right represents the quantification of STXBP1 protein normalized to ATP5F1 and is presented relative to untransduced cells.

[0019] FIG. 3. STXBP1 expression in neurons by qPCR. Neurons were generated 25 from STXBP1 patient (STXBP1 with c.663+1G>C mutation) iPSC lines or original STXBP1 patient line with a CRISPR generated correction of the c.663+1G>C mutation for an isogenic control (“corrected”) into excitatory or inhibitory neurons, as previously described (Waxman et al., 2023, PMID: 38148714). Resulting neurons were harvested for RNA Day 40-50 of differentiation as assayed by qPCR for STXBP1, STX1A, VGLUT1, VGLUT2, GAD67 30 (GAD1), or GAD65 (GAD2). 6 4932-3429-7135, v.1

[0020] FIGS. 4A-C. STXBP1 patient vs isogenic control. Multielectrode array (MEA) analysis of STXBP1 patient and isogenic control (“Corrected”) generated excitatory neurons. Different densities of both iPSC-N were plated on MEA plates for terminal differentiation. Electrical activity was recorded every 2-3 days and mean burst duration (FIG. 5 4A), burst count (FIG. 4B), and network burst duration (FIG. 4C) was plotted for both the lines at different days of differentiation.

[0021] FIGS. 5A-C. Transduction of STXBP1 patient line with AAV containing the cDNA of STXBP1 under different promoters. Multi-electrode array (MEA) analysis of AAV transduced STXBP1 patient neurons. Neurons were generated from STXBP1 patient 10 (STXBP1 with c.663+1G>C mutation) iPSC lines and were plated on MEA plates. They were then transduced at Day 47 of transduction using 4 viruses at indicated MOI. 1. pkAAV-Mecp2-STXBP1-BLD004 (MOI = 1E5) 2. pkAAV-Mecp2-GFP-BLD004 (MOI = 1E5) 3. pkAAV-iCAG-STXBP1-BLD005 (MOI = 5E4) 15 4. pkAAV-iCAG-TFP-BLD005 (MOI = 5E4) Electrical activity was recorded every 2-3 days and burst count (FIG 5A), Network Duration (FIG.5B) and Network Burst count (FIG.5C) was plotted.

[0022] FIG. 6. mRNA -Fold Change in Treated Mice. Postnatal day 1 STXBP1 haploinsufficient mice received bilateral ICV injection of AAV-DB3 expressing iCAG- 20 STXBP1-Flag at a total dose of 2E+10 vg per animal. Levels of STXBP1 overexpression at the mRNA level were evaluated 6 weeks post-injection in different regions of the brain and compared to vehicle treated mice (VSB) mice.

[0023] FIGS. 7A-C. STXBP1 protein upregulation. Postnatal day 1 STXBP1 haploinsufficient mice received bilateral ICV injection of AAV DB-3 expressing iCAG- 25 STXBP1-Flag at a total dose of 2E+10 vg per animal. Levels of STXBP1 overexpression at the protein level were evaluated 6 weeks post-injection in different regions of the brain and compared to vehicle treated haploinsufficient mice. Respective quantification is shown on the right of the blot (FIG.7A) Cortex, (FIG.7B) Striatum, (FIG.7C) Hippocampus.

[0024] FIG. 8. Comparison of iCAG-STXBP1 / iCAG-STXBP1_UTR / iCAG- 30 STXBP1_WPRE (woodchuck hepatitis post-transcriptional regulatory element) – 7 4932-3429-7135, v.1mRNA level. iPSC-derived excitatory neurons from patient STXBP1 line (STXBP1 c.663+1G>C) were transduced on day 40-45 of differentiation by indicated viruses (iCAG- STXBP1 / iCAG-STXBP1_UTR and iCAG-STXBP1_WPRE at an MOI of 5E4). Samples were harvested for RNA extraction 13 days post-transduction. Quantitative PCR 5 measurement of STXBP1 mRNA normalized to GAPDH and is presented relative to vehicle transduced cells in 2 independent experiments.

[0025] FIG. 9. Comparison of iCAG-STXBP1 / iCAG-STXBP1_UTR / iCAG- STXBP1_WPRE – Protein level. iPSC-derived excitatory neurons from patient STXBP1 line (STXBP1 c.663+1G>C) were transduced on day 40-45 of differentiation by indicated 10 viruses (iCAG-STXBP1 / iCAG-STXBP1_UTR and iCAG-STXBP1_WPRE at an MOI of 5E4). Samples were harvested for protein extraction 13 days post-transduction. Western blot analysis of STXBP1 and ATP5F1 (loading control) levels. The graph on the bottom represents the quantification of STXBP1 protein normalized to ATP5F1 and is presented relative to vehicle transduced cells in 3 independent experiments. 15 8 4932-3429-7135, v.1DETAILED DESCRIPTION

[0026] Provided herein are methods of expressing a syntaxin-binding protein 1 (STXBP1) in a the ependyma and neurons of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) capsid encapsidating a transgene encoding 5 a STXBP1 under the control of a promoter operable in said neuron / ependyma, wherein said AAV comprises a modified capsid protein comprising a targeting sequence.

[0027] In some embodiments, viral vectors each comprise a modified capsid, wherein the modified capsid comprises an amino acid sequence that targets the viral vector to a distinct brain structure. In certain embodiments, the brain structure is the brainstem, caudate, 10 cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nuclei, thalamus or ependymal cells. In certain embodiments, the targeting peptide is ERDRTRG (SEQ ID NO: 4) or GRGAPGG (SEQ ID NO: 6).

[0028] In certain embodiments, the viral vector is an adeno associated viral vector 15 (AAV). In certain embodiments, the AAV is derived from AAV1. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 1. In certain aspects, a targeting peptide is inserted at position 590 of the AAV1 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 3, which shows the targeting peptide insertion after position 590 as SSAX7AS (SEQ ID NO: 18), where the leading SSA 20 and the trailing AS are linker sequences and X7 represents the targeting peptide.

[0029] In certain embodiments, the viral vector is an adeno associated viral vector (AAV). In certain embodiments, the AAV is derived from AAV2. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO: 2. In certain aspects, a targeting peptide is inserted at position 587 of the AAV2 capsid. An exemplary modified 25 AAV2 capsid protein sequence is provided in SEQ ID NO: 5, which shows the targeting peptide insertion after position 587 as AAAX7AA (SEQ ID NO: 19), where the leading AAA and the trailing AA are linker sequences and X7represents the targeting peptide. I. STXBP1 and Epileptic Encephalopathy

[0030] Syntaxin-binding protein 1 (also known as Munc18-1) is a protein that in 30 humans is encoded by the STXBP1 gene. This gene encodes a syntaxin-binding protein. The encoded protein appears to play a role in release of neurotransmitters via regulation of 9 4932-3429-7135, v.1syntaxin, a transmembrane attachment protein receptor. Mutations in this gene have been associated with neurological disorders including epilepsy, intellectual disability, and movement disorders.

[0031] The STXBP1 gene is located on the q arm of chromosome 9 in position 34.11 5 and has 19 exons spanning 80,510 base pairs. The encoded protein is a peripheral membrane protein located in the cytosol. In the retina and cerebellum, an alternatively spliced transcript variant is expressed, containing an additional exon and totaling 594 amino acids. Alternative splicing can produce an isoform with exon 20 and an isoform without.

[0032] The encoded protein may participate in the regulation of synaptic 10 vesicle docking and fusion, possibly through interaction with GTP-binding proteins. It is essential for neurotransmission and binds syntaxin, a component of the synaptic vesicle fusion machinery, probably in a 1:1 ratio. It can interact with syntaxins 1, 2, and 3 but not syntaxin 4 and may play a role in determining the specificity of intracellular fusion reactions. This protein functions in a late stage of the intracellular membrane fusion process 15 of exocytosis. Dissociation of this protein from syntaxin determines the kinetics of postfusion events. This protein is essential for presynpatic vesicle release and is rapidly phosphorylated by protein kinase C upon neuronal depolarization. The protein participates in the secretory pathway between the Golgi apparatus and cell membrane.

[0033] Mutations in the STXBP1 cause early infantile epileptic encephalopathy type 4 20 (EIEE4), a severe form of epilepsy characterized by frequent tonic seizures or spasms beginning in infancy with a specific EEG finding of suppression-burst patterns, characterized by high-voltage bursts alternating with almost flat suppression phases. Affected individuals have neonatal or infantile onset of seizures, profound intellectual disability, and MRI evidence of brain hypomyelination. Inheritance of EIEE4 is autosomal dominant, 25 but due to the severity of the condition most cases are de novo.

[0034] This gene was initially discovered in 2008 as cause for this severe form of epilepsy also called Ohtahara syndrome. Since then, it has become one of the most prominent genes for epileptic encephalopathies, and is increasingly being associated with other forms of epilepsy. STXBP1 variants are increasingly being identified in people with wider neurological 30 problems, including intellectual disability or movement disorders without epilepsy. 10 4932-3429-7135, v.1

[0035] In melanocytic cells STXBP1 gene expression may be regulated by MITF. The STXBP1 gene is expressed in the brain and spinal cord and highly enriched in axons. Expression of this protein is highest in the retina and cerebellum. The encoded protein binds SYTL4. STXBP1 has been shown to interact with STX2, STX4 and STX1A. 5 II. Adeno-Associated Virus (AAV) Vectors

[0036] Adeno-associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV is distinct from other members of this family by its dependence upon a helper virus for replication. 10

[0037] AAV genomes can exist in an extrachromosomal state without integrating into host cellular genomes; possess a broad host range; transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes. AAV viral particles are heat stable; resistant to solvents, detergents, changes in pH, and temperature; and can be column purified and / or concentrated on CsCl gradients or by other 15 means. The AAV genome comprises a single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The approximately 4.7 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. The ends of the genome are short-inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as the origin of viral DNA replication. 20

[0038] An AAV “genome” refers to a recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. An AAV particle often comprises an AAV genome packaged with AAV capsid proteins. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the AAV vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome 25 sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for plasmid propagation and production but is not itself packaged or encapsulated into viral particles. Thus, an AAV vector “genome” refers to nucleic acid that is packaged or encapsulated by AAV capsid proteins. 30

[0039] The AAV virion (particle) is a non-enveloped, icosahedral particle approximately 25 nm in diameter that comprises an AAV capsid. The AAV particle 11 4932-3429-7135, v.1comprises an icosahedral symmetry comprised of three related capsid proteins, VP1, VP2 and VP3, which interact together to form the capsid. The genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins 5 are often found in a ratio of 1:1:10 respectively, but may be in varied ratios, and are all derived from the right-hand ORF. The VP1, VP2 and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1 which is translated from an alternatively spliced message results in a reduced yield of infectious particles. Mutations within the VP3 coding 10 region result in the failure to produce any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of an AAV particle encodes one, two or all three VP1, VP2 and VP3 polypeptides.

[0040] The left ORF often encodes the non-structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in regulation of replication and transcription in 15 addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68 / 78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities. Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. In certain embodiments the genome 20 of an AAV (e.g., an rAAV) encodes some or all of the Rep proteins. In certain embodiments the genome of an AAV (e.g., an rAAV) does not encode the Rep proteins. In certain embodiments one or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a polypeptide.

[0041] The ends of the AAV genome comprise short-inverted terminal repeats (ITR) 25 which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Accordingly, the genome of an AAV comprises one or more (e.g., a pair of) ITR sequences that flank a single stranded viral DNA genome. The ITR sequences often have a length of about 145 bases each. Within the ITR region, two elements have been described which are believed to be central to the function of the ITR, a GAGC repeat motif 30 and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs which occurs in a site- and strand-specific manner. In addition to their 12 4932-3429-7135, v.1role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration.

[0042] The term “recombinant,” as a modifier of vector, such as recombinant viral, 5 e.g., lentivirus or parvovirus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector would be where a nucleic acid sequence that is not normally present in the wild-type viral 10 genome is inserted within the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into a vector, with or without 5ʹ, 3ʹ and / or intron regions that the gene is normally associated within the viral genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” 15 forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission.

[0043] A recombinant viral “vector” is derived from the wild-type genome of a virus by using molecular methods to remove part of the wild-type genome from the virus, and replacing it with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for 20 example, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A “recombinant” viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome, since part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic 25 acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”

[0044] In certain embodiments, an AAV (e.g., a rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs. In certain 30 embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs that flank (i.e., are at each 5ʹ and 3ʹ end) of a nucleic acid sequence that at least encodes a polypeptide having function or activity. 13 4932-3429-7135, v.1

[0045] An AAV vector (e.g., rAAV vector) can be packaged and is referred to herein as an “AAV particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle can also be referred to as a “rAAV particle.” In certain embodiments, an AAV 5 particle is a rAAV particle. A rAAV particle often comprises a rAAV vector, or a portion thereof. A rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles). rAAV particles typically comprise proteins that encapsulate or package the rAAV vector genome (e.g., capsid proteins). It is noted that reference to a rAAV vector can also be used to reference a rAAV particle. 10

[0046] Any suitable AAV particle (e.g., rAAV particle) can be used for a method or use herein. A rAAV particle, and / or genome comprised therein, can be derived from any suitable serotype or strain of AAV. A rAAV particle, and / or genome comprised therein, can be derived from two or more serotypes or strains of AAV. Accordingly, a rAAV can comprise proteins and / or nucleic acids, or portions thereof, of any serotype or strain of AAV, 15 wherein the AAV particle is suitable for infection and / or transduction of a mammalian cell. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV- 2i8.

[0047] In certain embodiments a plurality of rAAV particles comprises particles of, or 20 derived from, the same strain or serotype (or subgroup or variant). In certain embodiments a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains).

[0048] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic 25 distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other 30 AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype. 14 4932-3429-7135, v.1

[0049] In certain embodiments, a rAAV vector based upon a first serotype genome corresponds to the serotype of one or more of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that comprises the AAV vector genome corresponds to the serotype of a capsid that comprises the rAAV particle. 5

[0050] In certain embodiments, a rAAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from the serotype of one or more of the AAV capsid proteins that package the vector. For example, a rAAV vector genome can comprise AAV1 derived nucleic acids (e.g., ITRs), whereas at least one or more of the three capsid proteins are derived from a different serotype, e.g., an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, 10 AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype or variant thereof.

[0051] In certain embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 15 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 particle. In particular embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence at least 60% 20 or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype.

[0052] In certain embodiments, a method herein comprises use, administration or 25 delivery of an rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74 or rAAV-2i8 particle.

[0053] In certain embodiments, a method herein comprises use, administration or delivery of a rAAV1 particle. In certain embodiments a rAAV1 particle comprises an AAV1 capsid. In certain embodiments a rAAV1 particle comprises one or more capsid proteins 30 (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 15 4932-3429-7135, v.199.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV1 particle. In certain embodiments a rAAV1 particle comprises VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 5 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to a corresponding capsid protein of a native or wild-type AAV1 particle. In certain embodiments, a rAAV2 particle is a variant of a native or wild-type AAV1 particle. In some aspects, one or more capsid proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to capsid protein(s) of a native or wild-type AAV1 particle. 10

[0054] In certain embodiments, a rAAV particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, 15 AAV-rh74, AAV-rh10 or AAV-2i8, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired).

[0055] In certain embodiments, a rAAV1 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 20 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to corresponding ITRs of a native or wild-type AAV1 particle, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired). 25

[0056] A rAAV particle can comprise an ITR having any suitable number of “GAGC” repeats. In certain embodiments an ITR of an AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR comprising three “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR which has less than four “GAGC” repeats. In certain embodiments 30 a rAAV2 particle comprises an ITR which has more than four “GAGC” repeats. In certain embodiments an ITR of a rAAV2 particle comprises a Rep binding site wherein the fourth nucleotide in the first two “GAGC” repeats is a C rather than a T. 16 4932-3429-7135, v.1

[0057] Exemplary suitable length of DNA can be incorporated in rAAV vectors for packaging / encapsidation into a rAAV particle can about 5 kilobases (kb) or less. In particular, embodiments, length of DNA is less than about 5kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb. 5

[0058] rAAV vectors that include a nucleic acid sequence that directs the expression of an RNAi or polypeptide can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transduction competent AAV particles and propagated using an AAV viral packaging system. A transduction competent AAV particle is capable of binding to and entering a 10 mammalian cell and subsequently delivering a nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, an intact rAAV particle that is transduction-competent is configured to transduce a mammalian cell. A rAAV particle configured to transduce a mammalian cell is often not replication competent and requires additional protein machinery to self-replicate. Thus, a rAAV particle that is configured to transduce a mammalian cell is 15 engineered to bind and enter a mammalian cell and deliver a nucleic acid to the cell, wherein the nucleic acid for delivery is often positioned between a pair of AAV ITRs in the rAAV genome.

[0059] Suitable host cells for producing transduction competent AAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells 20 that can be, or have been, used as recipients of a heterologous rAAV vectors. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. In certain embodiments a modified human embryonic kidney cell line (e.g., HEK293), which is transformed with adenovirus type-5 DNA fragments and expresses the adenoviral E1a and E1b genes is used to 25 generate recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform in which to produce rAAV particles. Methods of generating high titer AAV particles capable of transducing mammalian cells are known in the art.

[0060] In certain embodiments, AAV helper functions are introduced into the host 30 cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to 17 4932-3429-7135, v.1complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used 5 plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. A number of other vectors are known which encode Rep and / or Cap expression products. III. AAV-STXBP1 as a Therapeutic Agent

[0061] In some embodiments, viral gene transfer methods can be used to introduce nucleic acids in mammalian cells. Such methods can be used to administer nucleic acids 10 encoding therapeutic proteins to cells in culture or in a host organism. In some embodiments, the therapeutic proteins may be useful for the treatment of STXBP1-related epileptic encephalopathy. Some embodiments may concern the expression of a polypeptide comprising syntaxin-binding protein 1 (STXBP1) activity.

[0062] The rAAV vector(s) may further comprise a modified adeno-associated virus 15 (AAV) Cap gene encoding a modified AAV capsid protein comprising a targeting peptide. The modified AAV capsid protein may be a modified AAV1 capsid protein, a modified AAV2 capsid protein. The targeting peptide may be three to ten amino acids in length. The targeting peptide may be 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. As referred to here, AAV construct BLD004 is GRGAPGG (SEQ ID NO: 6) (targeting peptide) modified AAV 20 serotype 2. BLD005 is ERDRTRG (SEQ ID NO: 4) (targeting) modified AAV serotype 1.

[0063] If the modified AAV capsid protein is derived from an AAV1 capsid protein, then the targeting peptide may be inserted after residue 590 of the AAV1 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. The linker sequences may be 25 SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. The modified AAV1 capsid protein may have a sequence identical to, at least 90% identical to, or at least 95% identical to SEQ ID NO: 3.

[0064] If the modified AAV capsid protein is derived from an AAV2 capsid protein (see SEQ ID NO: 2), then the targeting peptide may be inserted after residue 587 of the 30 AAV2 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. The 18 4932-3429-7135, v.1linker sequences may be AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. The modified AAV2 capsid protein may have a sequence identical to, at least 90% identical to, or at least 95% identical to SEQ ID NO: 5.

[0065] A polypeptide comprising STXBP1 activity may comprise a truncated, 5 mutated, chimeric, or modified form of a STXBP1 polypeptide that retains at least partial STXBP1 activity. A polypeptide comprising STXBP1 activity may comprise a STXBP1 protein, or a portion thereof, obtained from any suitable organism (e.g., from a mammal, from a human, from a non-human mammal, e.g., from a dog, pig, cow, or the like). In certain embodiments a polypeptide comprising STXBP1 activity has at least 60% identity, at least 10 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the STXBP1 encoded by the sequence set forth in SEQ ID NO: 7.

[0066] In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid encoding a polypeptide comprising STXBP1 activity. In certain 15 embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid that directs the expression and / or secretion of a polypeptide comprising STXBP1 activity. In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid encoding a STXBP1 polypeptide, or enzymatically active portion thereof. In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid that 20 directs the expression and / or secretion of a STXBP1 polypeptide, or enzymatically active portion thereof. In certain embodiments, an AAV particle comprises a polypeptide having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8. In 25 certain embodiments, a nucleic acid being administered encodes STXBP1, a STXBP1 that has substantial identity to wild-type STXBP1, and / or a variant, mutant or fragment of a STXBP1. In certain embodiments a nucleic acid encoding a STXBP1 activity or encoding or directing the expression of a STXBP1 polypeptide is a nucleic acid having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% 30 identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the nucleic acid set forth in SEQ ID NO: 7 or SEQ ID NO: 8. 19 4932-3429-7135, v.1

[0067] Recombinant STXBP1 polypeptides may possess deletions and / or substitutions of amino acids, i.e., a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments, these proteins may further include insertions or added amino acids, such as with fusion proteins or 5 proteins with linkers, for example. A “modified deleted protein” lacks one or more residues of the native protein but may possess the specificity and / or activity of the native protein.

[0068] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or 10 bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; 15 isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0069] In addition to a deletion or substitution, a modified protein may possess an 20 insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue.

[0070] The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and 25 about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of a control polypeptide are included, provided the biological activity of the protein is maintained. A recombinant protein may be biologically functionally equivalent to its native counterpart in certain aspects. 30

[0071] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ 20 4932-3429-7135, v.1sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include 5 various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. IV. Methods of Administration

[0072] Viral vectors, in some aspects, may be administered directly to patients (in vivo) or they can be used to treat cells in vitro or ex vivo, and then administered to patients. 10 The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells. 15

[0073] Any suitable cell or mammal can be administered or treated by a method or use described herein. Typically, a mammal in need of methods described herein is suspected of having or expressing an abnormal or aberrant protein that is associated with a disease state. Alternatively, the mammalian recipient may have a condition that is amenable to gene replacement therapy. As used herein, “gene replacement therapy” refers to administration to 20 the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase “condition amenable to gene replacement therapy” embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition which is not attributable to an inborn defect), cancers and prophylactic 25 processes (i.e., prevention of a disease or of an undesired medical condition). Accordingly, as used herein, the term “therapeutic agent” refers to any agent or material which has a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” embraces both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0074] Non-limiting examples of mammals include humans, non-human primates 30 (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In certain embodiments a mammal 21 4932-3429-7135, v.1is a human. In certain embodiments a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like). In certain embodiments a non-rodent mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In certain embodiments a 5 mammal can be an animal disease model, for example, animal models having or expressing an abnormal or aberrant protein that is associated with a disease state or animal models with insufficient expression of a protein, which causes a disease state.

[0075] Mammals (subjects) treated by a method or composition described herein include adults (18 years or older) and children (less than 18 years of age). Adults include the 10 elderly. Representative adults are 50 years or older. Children range in age from 1-2 years old, or from 2–4, 4–6, 6–18, 8–10, 10–12, 12–15 and 15–18 years old. Children also include infants. Infants typically range from 1–12 months of age.

[0076] In certain embodiments, a method includes administering a plurality of viral particles to a mammal as set forth herein, where severity, frequency, progression or time of 15 onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, decreased, reduced, prevented, inhibited or delayed. In certain embodiments, a method includes administering a plurality of viral particles to a mammal to treat an adverse symptom of a disease state, such as a neuro-degenerative disease. In certain embodiments, a method includes administering a plurality of viral particles to a mammal to stabilize, delay or prevent 20 worsening, or progression, or reverse and adverse symptom of a disease state, such as a neuro-degenerative disease.

[0077] In certain embodiments a method includes administering a plurality of viral particles to the central nervous system, or portion thereof as set forth herein, of a mammal and severity, frequency, progression or time of onset of one or more symptoms of a disease 25 state, such as a neuro-degenerative disease, are decreased, reduced, prevented, inhibited or delayed by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days.

[0078] In certain embodiments, a symptom or adverse effect comprises an early stage, middle or late-stage symptom; a behavior, personality or language symptom; swallowing, 30 movement, seizure, tremor or fidgeting symptom; ataxia; and / or a cognitive symptom such as memory, ability to organize. 22 4932-3429-7135, v.1

[0079] In certain embodiments, a method includes administering or delivering AAV- STXBP1 particles to a mammal and administering one or more immunosuppressive agents to the mammal. In certain embodiments a method includes administering or delivering AAV- STXBP1 particles to a mammal and administering 2, 3, 4 or more immunosuppressive agents 5 to the mammal. In certain embodiments a method includes administering or delivering AAV- STXBP1 particles to a mammal and administering two immunosuppressive agents to the mammal. In one representative embodiment, a method of treating a mammal includes administering or delivering AAV-STXBP1 particles to a mammal and administering first and second immunosuppressive agents to the mammal. 10

[0080] Where two or more immunosuppressive agents are administered, each immunosuppressive agent is distinct and / or different (e.g., each agent differs in structure and / or mechanism of action). An "agent" refers to an active pharmaceutical ingredient. In certain embodiments, an immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, an immunosuppressive agent is mycophenolate, or a derivative thereof. An 15 example of such a mycophenolate derivative is mycophenolate mofetil (MMF). In certain embodiments, an immunosuppressive agent is cyclosporine or a derivative thereof. In certain embodiments a first immunosuppressive agent comprises cyclosporine and a second immunosuppressive agent comprises mycophenolate, or a derivative thereof (e.g., MMF). In certain embodiments a first immunosuppressive agent comprises cyclosporine and a second 20 immunosuppressive agent comprises MMF.

[0081] In certain embodiments, an immunosuppressive agent is administered before, during and / or after administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered concurrently with administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive 25 agent is administered after administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1 to about 60 minutes after, about 1 to about 24 hours after, about 1 to about 100 days after, about 1 to about 12 months after, or about 1 to about 5 years after administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered before 30 administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1 to about 60 minutes before, about 1 to about 24 hours before, about 1 to about 100 days before, or about 1 to about 3 months before 23 4932-3429-7135, v.1administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 days before administration of AAV-STXBP1 particles to a mammal. In certain embodiments, an immunosuppressive agent is administered at 5 predetermined intervals before, during and / or after administration of AAV-STXBP1 particles to a mammal (e.g., once a day, twice a day, three times a day, every other day, weekly, biweekly, bi-monthly, combination thereof or the like).

[0082] In certain embodiments a first immunosuppressive agent is administered to a mammal at least about 1 to about 7 days before, or about 1, about 2, about 3, about 4 or about 10 5 weeks before administration of AAV-STXBP1 particles to a mammal and a second immunosuppressive agent is administered about 1 to about 7 days before, about 1, about 2, about 3, about 4 or about 5 weeks before, during and / or within about 10, about 20, about 30, about 40, 19 about 50, about 100, about 200, about 300, about 350, about 400 or about 500 days after administration of AAV-STXBP1 particles to the mammal. In certain embodiments 15 cyclosporine is administered to a mammal at least about I to about 7 days before, or about 1, about 2, about 3, about 4 or about 5 weeks before administration of AAV-STXBP1 particles to a mammal, and mycophenolate or a derivative thereof (e.g., MMF) is administered about 1 to about 7 days before, about 1, about 2, about 3, about 4 or about 5 weeks before, during and / or within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 20 300, about 350, about 400 or about 500 days after administration of AAV-STXBP1 particles to the mammal. In certain embodiments, cyclosporine is administered about 1 to about 7 days before, or about 1, about 2, about 3, about 4 or about 5 weeks before administration of AAV- STXBP1 particles and at regular intervals after treatment, and mycophenolate or a derivative thereof (e.g., MMF) is administered once at about 1 to about 7 days before, about 1, about 2, 25 about 3, about 4 or about 5 weeks before, during and / or within about 10 to about 40 days after administration of AAV-STXBP1 particles to the mammal.

[0083] An immunosuppressive agent can be administered at any suitable dose. In certain embodiments, cyclosporine is administered at a dosage of about 1 to about 50 mg / kg, about 1 to about 20 mg / kg, or about 5 to about 10 mg / kg at a frequency of once, twice or 30 three times a day, to once every other day. In certain embodiments cyclosporine is administered at about 10 mg / kg twice a day. In certain embodiments, cyclosporine is administered at about 10 mg / kg twice a day for a period of at least about 1, about 2, about 3, 24 4932-3429-7135, v.1about 4 or about 5 months. In certain embodiments, a dosage of cyclosporine is tapered down to a dose of less than about 5 mg / kg, or less than about 2 mg / kg about 1 to about 2 months after the administration of AAV-STXBP1 particles to a mammal.

[0084] In certain embodiments, mycophenolate or a derivative thereof (e.g., MMF), is 5 administered at a dosage of about 1 to about 100 mg / kg, about 1 to about 50 mg / kg, about 1 to about 25 mg / kg, or about 5 to about 20 mg / kg at a frequency of once, twice or three times a day, to once every other day. In certain embodiments, mycophenolate or a derivative thereof (e.g., MMF) is administered at about 10 to about 20 mg / kg once a day. In certain embodiments, a dosage of mycophenolate or a derivative thereof (e.g., MMF) is reduced 10 down to a dose of less than about 5 mg / kg, or less than about 2 mg / kg about 1 to about 2 months after the administration of AAV-STXBP1 particles to a mammal. An immunosuppressive agent can be formulated in any suitable formulation suitable for a particular route of administration. Various pharmaceutically acceptable formulations of immunosuppressive agents are commercially available and readily obtainable by a medical 15 practitioner.

[0085] An immunosuppressive agent can be administered by any suitable route. In certain embodiments, an immunosuppressive agent is administered orally. In certain embodiments, mycophenolate or a derivative thereof, such as Mycophenolate Mofetil (MMF), is administered orally. In certain embodiments, cyclosporine is administered orally. 20 An immunosuppressive agent can also be administered parenterally (e.g., intramuscularly, intravenously, subcutaneously), or administered by injection to the brain, spinal cord, or a portion thereof (e.g., injected into the CSF).

[0086] In certain embodiments, a method includes administering one or more (e.g., a plurality of) AAV-STXBP1 particles to the central nervous system of a mammal (e.g., a 25 mammal having a LSD). In certain embodiments, the central nervous system includes brain, spinal cord and cerebral spinal fluid (CSF). In certain embodiments, a method includes administering one or more AAV-STXBP1 particles to the brain or spinal cord or CSF of a mammal. In certain embodiments AAV-STXBP1 particles are administered to a portion of brain or spinal cord. In certain embodiments, a composition including AAV-STXBP1 30 particles, and an immunosuppressive agent are administered to a mammal's cisterna magna and / or to the mammal's brain ventricle, subarachnoid space, and / or intrathecal space, and / or ependyma. For example, AAV-STXBP1 particles can be delivered directly to the cisterna 25 4932-3429-7135, v.1magna, intraventricular space, a brain ventricle, subarachnoid space, intrathecal space or ependyma. In certain embodiments a method includes administering one or more AAV- STXBP1 particles to the ependyma of a mammal.

[0087] In certain embodiments, AAV-STXBP1 particles are administered to one or 5 more cells that contact the CSF in a mammal, for example by contacting cells with AAV- STXBP1 particles. Nonlimiting examples of cells that contact the CSF include ependymal cells, pial cells, endothelial cells and / or meningeal cells. In certain embodiments AAV- STXBP1 particles are administered to ependymal cells. In certain embodiments AAV- STXBP1 particles are delivered to ependymal cells, for example by contacting ependymal 10 cells with AAV-STXBP1 particles.

[0088] In certain embodiments, AAV-STXBP1 particles are delivered locally. "Local delivery" refers to delivery of an active agent directly to a target site within a mammal (e.g., directly to a tissue or fluid). For example, an agent can be locally delivered by direct injection into an organ, tissue or specified anatomical location. In certain embodiments one or more 15 AAV-STXBP1 particles are delivered or administered by direct injection to the brain, spinal cord, or a tissue or fluid thereof (e.g., CSF, such as ependymal cells, pial cells, endothelial cells and / or meningeal cells). For example, AAV-STXBP1 particles can be directly delivered, by way of direct injection, to the CSF, cisterna magna, intraventricular space, a brain ventricle, subarachnoid space and / or intrathecal space and / or ependyma. In certain 20 embodiments AAV-STXBP1 particles are contacted with a tissue, fluid or cell of the brain or spinal cord by direct injection into a tissue or fluid of the brain or spinal cord. In certain embodiments AAV-TPPl particles are not delivered systemically by, for example, intravenous, subcutaneous, or intramuscular injection, or by intravenous infusion. In certain embodiments AAV-STXBP1 particles are delivered to a tissue or fluid of the brain or spinal 25 cord by stereotactic injection.

[0089] In certain embodiments, one or more AAV-STXBP1 particles are delivered or administered by direct injection of AAV-STXBP1 particles to the brain, spinal cord, or a tissue or fluid thereof (e.g., CSF such as ependyma). In a particular aspect, AAV-TPP particles transduce ependymal cells, pial cells, endothelial cells and / or meningeal cells. 30

[0090] As is apparent to those skilled in the art in view of the teachings herein, such as the dose ranges provided herein, an effective amount of AAV-STXBP1 particles can be 26 4932-3429-7135, v.1empirically determined. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Effective doses of administration can be determined by those of skill in the art and may vary according to the AAV serotype, viral titer and the weight, condition and species of mammal being treated. Single and multiple 5 administrations can be carried out with the dose level, target and timing being selected by the treating physician.

[0091] In certain embodiments, a plurality of AAV-STXBP1 particles are administered. As used herein, a plurality of AAV particle refers to about 1x105to about 1x108particles. 10

[0092] In certain embodiments, AAV-STXBP1 particles are administered at a dose of about 1x105to about 1x1016vg / ml in about 1 to about 5 ml; at a dose of about 1 to about 3 ml of 1x107to about 1x1014vg / ml; or at a dose of about 1 to about 2 ml of 1x108to about 1x1013vg / ml In certain embodiments, AAV-STXBP1 particles are administered at a dose of about 1x108to about 1x1015vg / kg body weight of the mammal being treated. For example, AAV- 15 STXBP1 particles can be administered at a dose of about 1x108vg / kg, about 5x108vg / kg, about 1x109vg / kg, about 5x109vg / kg, about 1x1010vg / kg, about 5x1010vg / kg, about 1x1011vg / kg, about 5x1011vg / kg, about 1x 1012vg / kg, about 5x1012vg / kg, about 1x1013vg / kg, about 5x1013vg / kg, about 1x1014vg / kg, about 5x1014vg / kg, or about 1x1015vg / kg body weight of the mammal being treated. 20

[0093] Administration of AAV-STXBP1 particles may be in one or more doses. Multiple doses may be administered as is required to maintain adequate enzyme activity, for example. V. Pharmaceutical Compositions

[0094] As used herein the term “pharmaceutically acceptable” and “physiologically 25 acceptable” mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Such 30 composition, “pharmaceutically acceptable” and “physiologically acceptable” formulations 27 4932-3429-7135, v.1and compositions can be sterile. Such pharmaceutical formulations and compositions may be used, for example in administering a viral particle to a subject.

[0095] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in- 5 oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated 10 into the formulations and compositions.

[0096] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but 15 are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering 20 substances, and the like, may be present in such vehicles.

[0097] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes. 25

[0098] Pharmaceutical forms suitable for injection or infusion of AAV-STXBP1 particles can include sterile aqueous solutions or dispersions which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate form should be a sterile fluid and stable under the conditions of manufacture, use and storage. The liquid carrier or vehicle 30 can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), 28 4932-3429-7135, v.1vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Isotonic agents, for example, sugars, buffers or salts (e.g., sodium chloride) can be included. Prolonged 5 absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0099] Solutions or suspensions of AAV-STXBP1 particles can optionally include the following components: a sterile diluent such as water for injection, saline solution, such as phosphate buffered saline (PBS), artificial CSF, fixed oils, a polyol (for example, glycerol, 10 propylene glycol, and liquid polyethylene glycol, and the like), glycerin, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. 15

[0100] Pharmaceutical formulations, compositions and delivery systems appropriate for the compositions, methods and uses of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20thed., Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12thed., Merck Publishing Group, 20 Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11thed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., 2004).

[0101] AAV-STXBP1 particles and compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as 25 used herein refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms are dependent upon the amount of AAV-STXBP1 particles necessary to produce the desired effect(s). The amount necessary can be formulated in a single dose or30 can be formulated in multiple dosage units. The dose may be adjusted to a suitable AAV- STXBP1 particles concentration, optionally combined with an anti-inflammatory agent, and packaged for use. 29 4932-3429-7135, v.1

[0102] In one embodiment, pharmaceutical compositions will include sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate symptoms of a disease state in question or an amount sufficient to confer the desired benefit. Pharmaceutical compositions typically contain a 5 pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, 10 for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0103] A “unit dosage form” as used herein refers to physically discrete units 15 suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a 20 freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Thus, for example, viral particles, and pharmaceutical compositions thereof, can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage. 25

[0104] Formulations containing AAV-STXBP1 particles will contain an effective amount of the rAAV particles in a vehicle, the effective amount being readily determined by one skilled in the art. The AAV-STXBP1 particles may typically range from about 1% to about 95% (w / w) of the composition, or even higher if suitable. The quantity to be administered depends upon factors such as the age, weight and physical condition of the 30 mammal or the human subject considered for treatment. Effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose response curves. 30 4932-3429-7135, v.1VI. Definitions

[0105] A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper 5 transcription. In some embodiments, the promoter is an Mecp2 promoter or an iCAG promoter. In some embodiments, the promoter comprises a sequence having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the nucleic acid set forth in SEQ ID NO: 9 or SEQ ID NO: 10. 10

[0106] An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5’->3’ or 3’->5’) and may be capable of functioning even when positioned either upstream or downstream of the promoter. 15

[0107] Promoters and / or enhancers may be derived in their entirety from a native gene or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological 20 or developmental conditions.

[0108] A “transgene” is used herein to conveniently refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes an inhibitory RNA polypeptide or protein (e.g., STXBP1) and are generally heterologous with respect to 25 naturally occurring genomic sequences.

[0109] The term “transduce” refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (e.g., a viral particle). Introduction of a transgene into a cell by a viral particle can therefore be referred to as “transduction” of the cell. The transgene may or may not be integrated into genomic nucleic acid of a transduced 30 cell. If an introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further 31 4932-3429-7135, v.1passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extra chromosomally, or only transiently. A “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction. Thus, a “transduced” cell is a cell into 5 which, or a progeny thereof in which a transgene has been introduced. A transduced cell can be propagated, a transgene transcribed and the encoded protein expressed. For gene therapy uses and methods, a transduced cell can be in a mammal.

[0110] As used herein, the terms “modify” or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from 10 a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, e.g., a missense or nonsense mutation. 15

[0111] A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein, e.g., a variant STXBP1 protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence 20 which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of a STXBP1 protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes 25 the amino acids of a protein encoded thereby.

[0112] The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptides” encoded by a “nucleic acid” or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, 30 and sequence variants thereof, so long as the polypeptide (e.g., STXBP1) retains some degree of function or activity. Accordingly, in methods and uses of the disclosure, such polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein 32 4932-3429-7135, v.1that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal.

[0113] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., about 1 to about 3, about 3 to about 5, about 5 to 5 about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 or more nucleotides or residues).

[0114] An example of an amino acid modification is a conservative amino 10 acid substitution or a deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (e.g., wild-type sequence). Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors, to the central nervous system, such as to distinct brain regions. 15

[0115] A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase 20 chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 25 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type). 30

[0116] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 33 4932-3429-7135, v.176%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these 5 values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%. 10

[0117] The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two 15 polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

[0118] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, inhibit, reduce, 20 or decrease an undesired physiological change or disorder, such as the development, progression or worsening of the disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect of disease, delay or slowing of disease progression, amelioration or palliation of the 25 disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed (e.g., as determined by a genetic assay). VII. Kits 30

[0119] The disclosure provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components 34 4932-3429-7135, v.1therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, and / or viral particles.

[0120] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material 5 commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

[0121] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include 10 information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or 15 treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.

[0122] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information 20 on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol 25 or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

[0123] Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer30 readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD- ROM / RAM, DVD, MP3, or an electrical storage media such as RAM and ROM or hybrids of 35 4932-3429-7135, v.1these such as magnetic / optical storage media, FLASH memory, hybrids and memory type cards. VIII. Examples

[0124] The following examples are included to demonstrate preferred embodiments 5 of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are 10 disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Methods

[0125] iPSC-derived neurons were generated from iPSCs as previously described (Waxman et al., 2023, PMID: 38148714). STXBP1 patient and isogenic control 15 corrected iPSC lines were differentiated as indicated and stocks of neural progenitor cells (NPC) were cryopreserved at Day 14 of differentiation. For continuing experimentation, cryopreserved NPC were re-expanded in Expansion Media for 6 days, followed by continuation of the protocol to generate cortical, excitatory neurons.

[0126] Multi-electrode array (MEA) 24-well plates (Harvard Biosciences 20 Multichannel Systems Cat # 24W300 / 30G-288) were prepared with poly-D-lysine, followed by growth factor reduced matrigel, as described for replating for the terminal differentiation phase. At Day 22 of differentiation, neurons were replated directly on the MEA plate, seeded at 1X105– 3X105cells per well. Five to seven days after replating, the neuron media was exchanged into BrainPhys media (StemCell Technologies Cat # 05793) containing BDNF 25 and GDNF (20ng / ml each). Half media changes were performed 3 times per week with BrainPhys media containing BDNF and GDNF for the life of the culture.

[0127] Starting approximately Day 35 of differentiation, recordings were performed on a Multichannel Systems Multiwell-MEA System (Harvard Biosciences Multichannel Systems) with the system maintained at 37°C. Plates were acclimated to the 30 MEA system for 10min prior to recording, after which the plate was recorded for 10min. 36 4932-3429-7135, v.1Plates were recorded 3 times a week prior to the half-media changes for the remaining life of the culture.

[0128] Virus treatments were performed during a scheduled half-media change with the virus added to the half-media addition, performed at Day 39, Day 47, or 5 between 40-45 days of differentiation.

[0129] Prior to recording bad electrodes were reviewed by assessing high background noise. After recording, 1-3 times per week, wells were reviewed for morphology and a lack of neuron coverage near the electrodes.

[0130] Analysis was performed using the Multiwell-Screen Analyzer 10 Software (Harvard Biosciences Multichannel Systems) setting the Butterworth Highpass Filter to 100Hz, the Notch Filter to 60Hz, and the threshold detection to 3 X 3s. Afterwards data output was further processed in R (R studio) such that the electrodes with high background and / or lack of coverage were omitted from analysis and all of the datapoints in 1 well were averaged to produce a single datapoint per well. 15 Example 3 - Results

[0131] FIG. 1 shows images iPSC lines transduced with AAV vectors expression fluorescent reporters. The results show that the variant capsids transduce both patient and WT iPSC-N at higher efficiency AAV1.

[0132] FIGS.2A-B show iPSC-derived neurons from a patient line transduced 20 with various viruses. The results show that both STXBP1 RNA and proteins can be upregulated using AAV mediated delivery of STXBP1 coding regions.

[0133] FIG. 3 shows iPSC-derived neurons from a patient line and an original patient line corrected by CRISPR. The results show that STXBP1 isogenic control restored STXBP1 expression. The patient and the isogenic control expressed similar amounts of 25 STX1A, VGLUT1, VGLUT2, GAD67, and GAD65, indicating similar differentiation efficiency into the desired neuron type.

[0134] FIGS. 4A-C MEA analysis of STXBP1 patient and isogenic control (“corrected”) generated excitatory neurons. The STXBP1 patient line resulted in neurons with a shorter burst duration, higher burst count and shorter network burst duration. 37 4932-3429-7135, v.1

[0135] FIGS. 5A-C show MEA analysis of AAV transduced STXBP1 patient neurons. The STXBP1 patients neurons transduced with Mecp2-STXBP1 and iCAG- STXBP1 resulted in lower burst count, longer network duration and lower network burst count as compared to the neurons treated with vehicle or CAG-TFP. 5

[0136] FIG. 6 shows mRNA -Fold change in mice treated with bilateral ICV injections of AAV-DB3 expressing iCAG-STXBP1-Flag. Levels of STXBP1 mRNA overexpression were evaluated 6 weeks post-injection in different regions of the brain and compared to WT mice. The inventors observed a 2-fold over expression of STXBP1 mRNA in the cortex, 1.8-fold in striatum and around 3-fold in the hippocampus of injected mice 10 compared to vehicle injected haploinsufficient mice (VSB).

[0137] FIG. 7A shows STXBP1 protein upregulation following bilateral ICV injection of AAV DB-3 expressing iCAG-STXBP1-Flag into STXBP1 haploinsufficient mice. Levels of STXBP1 overexpression at the protein level were evaluated 6 weeks post- injection in different regions of the brain and compared to vehicle treated haploinsufficient 15 mice. Respective quantification is shown on the right of the blot (a, Cortex; b. Striatum; c, Hippocampus). The inventors observed a 1.8-fold over expression of STXBP1 protein in the cortex of injected mice compared to VSB treated mice.

[0138] FIGS. 7B-C show STXBP1 protein upregulation on the right of the blot (b, Striatum; c, Hippocampus) and show a 1.5-fold over expression of STXBP1 protein 20 in the striatum and hippocampus of injected mice compared to VSB mice.

[0139] FIG. 8 shows a comparison of iCAG-STXBP1 / iCAG- STXBP1_UTR / iCAG-STXBP1_WPRE – mRNA level. iPSC-derived excitatory neurons from patient STXBP1 line were transduced by the indicated viruses and samples harvested for RNA extraction. Quantitative PCR measurement of STXBP1 mRNA normalized to 25 GAPDH and is presented relative to vehicle transduced cells in 2 independent experiments. The inventors observed up regulation of STXBP1 mRNA in transduced cells.

[0140] FIG. 9 shows a comparison of iCAG-STXBP1 / iCAG- STXBP1_UTR / iCAG-STXBP1_WPRE – protein level. iPSC-derived excitatory neurons from patient STXBP1 line were transduced by indicated viruses and samples were harvested 30 for protein extraction. Western blot analysis of STXBP1 and ATP5F1 show that STXBP1 protein levels can be upregulated in iPSC derived patient line. Also, addition of a 3’UTR 38 4932-3429-7135, v.1element in the expression cassette increases the expression of STXBP1 protein; however, whether endogenous 3’UTR is more effective or WPRE cannot be determined. SEQUENCES 5 SEQ ID NO: 1 - AAV1 capsid protein MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLG PFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTS FGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQ 10 QPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGA DGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHY FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTND GVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG SQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLID 15 QYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKT DNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAG ASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGM VWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAE FSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDN 20 NGLYTEPRPIGTRYLTRPL SEQ ID NO: 2 - AAV2 capsid protein MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLG 25 PFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTS FGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQ QPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEG ADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQN 30 DGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSA DNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGS EKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPG 35 MVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPST TFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVD TNGVYSEPRPIGTRYLTRNL SEQ ID NO: 3 - BLD005 capsid protein (ERDRTRG_PMAAV1) 40 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLG PFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTS FGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQ QPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGA 45 DGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHY FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTND GVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG SQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLID 39 4932-3429-7135, v.1QYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKT DNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAG ASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDSSAERDRTRGASPAT GDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQI 5 LIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTS NYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL SEQ ID NO: 4: Targeting Peptide 10 ERDRTRG SEQ ID NO: 5 - BLD004 capsid protein (GRGAPGG_PMAAV2) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLG 15 PFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTS FGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQ QPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEG ADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQN 20 DGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSA DNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGS EKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAAAGRGAPGGAARQAA 25 TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQI LIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN YNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 6 - Targeting Peptide 30 GRGAPGG SEQ ID NO: 7 - STXBP1 Protein (Variant 2) 35 MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMTDIMTEGITI VEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTDSCPDALF NELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKNPILERLA EQIATLC ATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGPDKARSQLLILDRG 40 FDPSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLLDEDDDLWIALRH KHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQYQKELSKYSTHL HLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPILLDANVSTYDKI RIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDSTLRRRSKPERKE RISEQTYQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTAVSARYGHWHK 45 NKAPGEYRSGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGSTHILTPQKLLDTL KKLNKTDEEISS 40 4932-3429-7135, v.1SEQ ID NO: 8 - STXBP1 cDNA (Variant 2) atggcccccattggcctcaaagctgttgtcggagagaagattatgcatgatgtgataaagaaggtcaagaagaagg gggaatggaaggtgctggtggtggatcagttaagcatgaggatgctgtcctcctgctgcaagatgacagacatcatg 5 accgagggcataacgattgtggaagatatcaataagcgcagagagccgctccccagcctggaggctgtgtatctcat cactccatccgagaagtccgtccactctctcatcagtgactttaaggacccgccgactgctaaataccgggctgcaca cgtcttcttcactgactcttgtccagatgccctgtttaatgaactggtaaaatcccgagcagccaaagtcatcaaaactct gacggaaatcaatattgcatttctcccgtatgaatcccaggtctattccttggactctgctgactctttccaaagcttctaca gtccccacaaggctcagatgaagaatcctatactggagcgcctggcagagcagatcgcgaccctttgtgccaccctg10 aaggagtacccggctgtgcggtatcggggggaatacaaggacaatgccctgctggctcagctaatccaggacaag ctcgatgcctataaagctgatgatccaacaatgggggagggcccagacaaggcacgctcccagctcctgatcctgg atcgaggctttgaccccagctcccctgtgctccatgaattgacttttcaggctatgagttatgatctgctgcctatcgaaaa tgatgtatacaagtatgagaccagcggcatcggggaggcacgggtgaaggaggtgctcctggacgaggacgacg acctgtggatagcactgcgccacaagcacatcgcagaggtgtcccaggaagtcacccggtctctgaaagatttttctt15 ctagcaagagaatgaatactggagagaagaccaccatgcgggacctgtcccagatgctgaagaagatgcctcagt accagaaagagctcagcaagtactccacccacctgcaccttgctgaggactgtatgaagcattaccaaggcaccgt agacaaactctgccgagtggagcaggacctggccatgggcacagatgctgagggagagaagatcaaggaccct atgcgagccatcgtccccattctgctggatgccaatgtcagcacttatgacaaaatccgcatcatccttctctacatcttttt gaagaatggcatcacggaggaaaacctgaacaaactgatccagcacgcccagatacccccggaggatagtgag20 atcatcaccaacatggctcacctcggcgtgcccatcgtcaccgattccacgctgcgtcgccggagcaagccggagc ggaaggaacgcatcagcgagcagacctaccagctctcacggtggactccgattatcaaggacatcatggaggaca ctattgaggacaaacttgacaccaaacactacccttatatctctacccgttcctctgcctccttcagcaccaccgccgtc agcgcccgctatgggcactggcataagaacaaggccccaggcgagtaccgcagtggcccccgcctcatcattttca tccttgggggtgtgagcctgaatgagatgcgctgcgcctacgaggtgacccaggccaacggaaagtgggaggtgct25 gataggatccacacacatcctcaccccacagaaactgctggacacactgaagaaactgaataaaacagatgaag aaataagcagttaa SEQ ID NO: 9 - Mecp2 30 agctgaatggggtccgcctcttttccctgcctaaacagacaggaactcctgccaattgagggcgtcaccgctaaggct ccgccccagcctgggctccacaaccaatgaagggtaatctcgacaaagagcaaggggtggggcgcgggcgcgc aggtgcagcagcacacaggctggtcgggagggcggggcgcgacgtctgccgtgcggggtcccggcatcggttgc gcgc 35 SEQ ID NO: 10 – iCAG cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggca40 gtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccc agtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccc cacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatg ggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcg gagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcg45 gccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgc cgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctc cgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggaggg ccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgc tgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcgg50 ccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtggg 41 4932-3429-7135, v.1ggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagca cggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggc aggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccc cggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcag 5 ggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcgggg cgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtcccctt ctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggct tctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacag 10 SEQ ID NO: 11 - pkAAV_CAG_STXBP1_SA (11,053 bp) aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactca15 agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcct ggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctg20 cgcgctcgctcgctcactgaggccgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg gagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatc tccctaggactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataactt acggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagt aacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtg25 tatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt atgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttc actctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggg gggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcg gcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaa30 aagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgc cgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaa ttagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcg gggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcg gctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggc35 ggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagc agggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggct tcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggt gccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccg gcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgt40 cccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgc ggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagc ctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgac cggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgct gtctcatcattttggcaaagaattaaactcgaggccaccatggcccccattggcctcaaagctgttgtcggagagaag45 attatgcatgatgtgataaagaaggtcaagaagaagggggaatggaaggtgctggtggtggatcagttaagcatga ggatgctgtcctcctgctgcaagatgacagacatcatgaccgagggcataacgattgtggaagatatcaataagcgc agagagccgctccccagcctggaggctgtgtatctcatcactccatccgagaagtccgtccactctctcatcagtgact ttaaggacccgccgactgctaaataccgggctgcacacgtcttcttcactgactcttgtccagatgccctgtttaatgaac tggtaaaatcccgagcagccaaagtcatcaaaactctgacggaaatcaatattgcatttctcccgtatgaatcccaggt50 ctattccttggactctgctgactctttccaaagcttctacagtccccacaaggctcagatgaagaatcctatactggagcg 42 4932-3429-7135, v.1cctggcagagcagatcgcgaccctttgtgccaccctgaaggagtacccggctgtgcggtatcggggggaatacaag gacaatgccctgctggctcagctaatccaggacaagctcgatgcctataaagctgatgatccaacaatgggggagg gcccagacaaggcacgctcccagctcctgatcctggatcgaggctttgaccccagctcccctgtgctccatgaattga cttttcaggctatgagttatgatctgctgcctatcgaaaatgatgtatacaagtatgagaccagcggcatcggggaggc 5 acgggtgaaggaggtgctcctggacgaggacgacgacctgtggatagcactgcgccacaagcacatcgcagagg tgtcccaggaagtcacccggtctctgaaagatttttcttctagcaagagaatgaatactggagagaagaccaccatgc gggacctgtcccagatgctgaagaagatgcctcagtaccagaaagagctcagcaagtactccacccacctgcacct tgctgaggactgtatgaagcattaccaaggcaccgtagacaaactctgccgagtggagcaggacctggccatgggc acagatgctgagggagagaagatcaaggaccctatgcgagccatcgtccccattctgctggatgccaatgtcagca10 cttatgacaaaatccgcatcatccttctctacatctttttgaagaatggcatcacggaggaaaacctgaacaaactgatc cagcacgcccagatacccccggaggatagtgagatcatcaccaacatggctcacctcggcgtgcccatcgtcaccg attccacgctgcgtcgccggagcaagccggagcggaaggaacgcatcagcgagcagacctaccagctctcacgg tggactccgattatcaaggacatcatggaggacactattgaggacaaacttgacaccaaacactacccttatatctcta cccgttcctctgcctccttcagcaccaccgccgtcagcgcccgctatgggcactggcataagaacaaggccccagg15 cgagtaccgcagtggcccccgcctcatcattttcatccttgggggtgtgagcctgaatgagatgcgctgcgcctacgag gtgacccaggccaacggaaagtgggaggtgctgataggatccacacacatcctcaccccacagaaactgctgga cacactgaagaaactgaataaaacagatgaagaaataagcagttaaggtacccaattcgccctatagtgagtcgtg ctagctgcagggtgtcgatatcagcggccgcactagacctcgactgtgccttctagttgccagccatctgttgtttgcccc tcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgt20 ctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagca ggcatgctggggaatctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctg cgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtg agcgagcgagcgcgcagctgcctgcaggggcagcttgaaggaaatactaaggcaaaggtactgcaagtgctcgc aacattcgcttatgcggattattgccgtagtgccgcgacgccgggggcaagatgcagagattgccatggtacaggcc25 gtgcggttgatattgccaaaacagagctgtgggggagagttgtcgagaaagagtgcggaagatgcaaaggcgtcg gctattcaaggatgccagcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctgg tcacgcactgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtcaatcgcagacaacattttg aatgcggtcacacgttagcagcatgattgccacggatggcaacatattaacggcatgatattgacttattgaataaaatt gggtaaatttgactcaacgatgggttaattcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgattcc30 gcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctgcaaaatgcaatcccga aacagttcgcaggtaatagttagagcctgcataacggtttcgggattttttatatctgcacaacaggtaagagcattgag tcgataatcgtgaagagtcggcgagcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagca gaaccggatcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacccaaactgagccgtagcca ctgtctgtcctgaattcattagtaatagttacgctgcggccttttacacatgaccttcgtgaaagcgggtggcaggaggtc35 gcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaatctgattactaaacacagtagcctggattt gttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaa aacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtac cttcgcggcagatataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctagttca ttcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcggtactga40 ctcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgta aggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgac gtcattgtaggcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaat caacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttct ctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattgatcgtggtgatat45 ccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggct gacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcga ttatctccgctctggttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaa gcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcg tgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgatgat50 gttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtg 43 4932-3429-7135, v.1gataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactat gcaaaaacaactggaaggaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaact catgcaattattgtgagcaatacacacgcgcttccagcggagtataaatgcctaaagtaataaaaccgagcaatcca tttacgaatgtttgctgggtttctgttttaacaacattttctgcgccgccacaaattttggctgcatcgacagttttcttctgccc 5 aattccagaaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaacgtctgt tgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcatggtgttattcccgatgctttttgaag ttcgcagaatcgtatgtgtagaaaattaaacaaaccctaaacaatgagttgaaatttcatattgttaatatttattaatgtat gtcaggtgcgatgaatcgtcattgtattcccggattaactatgtccacagccctgacggggaacttctctgcgggagtgt ccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtattgcattatgccaacgccccggtgctgaca10 cggaagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagtaaatagtaatgaatta tcaaaggtatagtaatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagattttccctgtatt gctgaaatgtgatttctcttgatttcaacctatcataggacgtttctataagatgcgtgtttcttgagaatttaacatttacaac ctttttaagtccttttattaacacggtgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataatgtg agacgttgtgacgttttagttcagaataaaacaattcacagtctaaatcttttcgcacttgatcgaatatttctttaaaaatg15 gcaacctgagccattggtaaaaccttccatgtgatacgagggcgcgtagtttgcattatcgtttttatcgtttcaatctggtc tgacctccttgtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgcgtaacaaagtgcggt cctgctggcattctggagggaaatacaaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaaga tttgaagtaatattttaaccgctagatgaagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgat gatccctccgtggatctgattcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgatgttgtaattgcatgt20 atagaacataaggtgtctctggaagcattcagagcaattgaggcagcgttggtgaagcacgataataatatgaagga ttattccctggtggttgactgatcaccataactgctaatcattcaaactatttagtctgtgacagagccaacacgcagtctg tcactgtcaggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatcgtcctgt tcggagggaagaacgcgggatgttcattcttcatcacttttaattgatgtatatgctctcttttctgacgttagtctccgacgg caggcttcaatgacccaggctgagaaattcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttag25 gaaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttgacatgaggttgccccgtattcagtgtcgctgatttgt attgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttga tggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacggg gcggcgacctgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagt acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc30 cttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggct ccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggcc atcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaac aacactcaactctatctcgggctattcttttgatttagacctgcaggcatgcaagcttggcactggccgtcgttttacaa cgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaata35 gcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgcgatttattcaacaaagc cgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgag catcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggaga aaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaataca acctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaat40 ggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaac caaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacag gaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaata cctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggt cggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccat45 gtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcg agcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcttcgagcaagacgtttcccgttgaatatg gctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaaca tcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcc cgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatc50 aaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgag 44 4932-3429-7135, v.1gcagacctctcgacggagttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct gcgcgtaatctgctgcttgcaaac SEQ ID NO: 12 - pkAAV_Mecp2_STXBP1_SA (9535 bp) 5 aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactca agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg10 aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcct ggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctg cgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtga15 gcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgta gagcgagtgttccgatactctaatctccctagactagtgaagcttagctgaatggggtccgcctcttttccctgcctaaac agacaggaactcctgccaattgagggcgtcaccgctaaggctccgccccagcctgggctccacaaccaatgaagg gtaatctcgacaaagagcaaggggtggggcgcgggcgcgcaggtgcagcagcacacaggctggtcgggaggg cggggcgcgacgtctgccgtgcggggtcccggcatcggttgcgcgcaccggtgcacgtggccaccatggcccccat20 tggcctcaaagctgttgtcggagagaagattatgcatgatgtgataaagaaggtcaagaagaagggggaatggaa ggtgctggtggtggatcagttaagcatgaggatgctgtcctcctgctgcaagatgacagacatcatgaccgagggcat aacgattgtggaagatatcaataagcgcagagagccgctccccagcctggaggctgtgtatctcatcactccatccg agaagtccgtccactctctcatcagtgactttaaggacccgccgactgctaaataccgggctgcacacgtcttcttcact gactcttgtccagatgccctgtttaatgaactggtaaaatcccgagcagccaaagtcatcaaaactctgacggaaatc25 aatattgcatttctcccgtatgaatcccaggtctattccttggactctgctgactctttccaaagcttctacagtccccacaa ggctcagatgaagaatcctatactggagcgcctggcagagcagatcgcgaccctttgtgccaccctgaaggagtac ccggctgtgcggtatcggggggaatacaaggacaatgccctgctggctcagctaatccaggacaagctcgatgcct ataaagctgatgatccaacaatgggggagggcccagacaaggcacgctcccagctcctgatcctggatcgaggctt tgaccccagctcccctgtgctccatgaattgacttttcaggctatgagttatgatctgctgcctatcgaaaatgatgtatac30 aagtatgagaccagcggcatcggggaggcacgggtgaaggaggtgctcctggacgaggacgacgacctgtggat agcactgcgccacaagcacatcgcagaggtgtcccaggaagtcacccggtctctgaaagatttttcttctagcaaga gaatgaatactggagagaagaccaccatgcgggacctgtcccagatgctgaagaagatgcctcagtaccagaaa gagctcagcaagtactccacccacctgcaccttgctgaggactgtatgaagcattaccaaggcaccgtagacaaact ctgccgagtggagcaggacctggccatgggcacagatgctgagggagagaagatcaaggaccctatgcgagcc35 atcgtccccattctgctggatgccaatgtcagcacttatgacaaaatccgcatcatccttctctacatctttttgaagaatg gcatcacggaggaaaacctgaacaaactgatccagcacgcccagatacccccggaggatagtgagatcatcacc aacatggctcacctcggcgtgcccatcgtcaccgattccacgctgcgtcgccggagcaagccggagcggaaggaa cgcatcagcgagcagacctaccagctctcacggtggactccgattatcaaggacatcatggaggacactattgagg acaaacttgacaccaaacactacccttatatctctacccgttcctctgcctccttcagcaccaccgccgtcagcgcccg40 ctatgggcactggcataagaacaaggccccaggcgagtaccgcagtggcccccgcctcatcattttcatccttgggg gtgtgagcctgaatgagatgcgctgcgcctacgaggtgacccaggccaacggaaagtgggaggtgctgataggat ccacacacatcctcaccccacagaaactgctggacacactgaagaaactgaataaaacagatgaagaaataagc agttaatctagactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgcc actcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgggg45 tggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggaagatctgtgtgttggttttttg tgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaag gtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcagctt gaaggaaatactaaggcaaaggtactgcaagtgctcgcaacattcgcttatgcggattattgccgtagtgccgcgac gccgggggcaagatgcagagattgccatggtacaggccgtgcggttgatattgccaaaacagagctgtgggggag50 agttgtcgagaaagagtgcggaagatgcaaaggcgtcggctattcaaggatgccagcaagcgcagcatatcgcgc 45 4932-3429-7135, v.1tgtgacgatgctaatcccaaaccttacccaacccacctggtcacgcactgttaagccgctgtatgacgctctggtggtg caatgccacaaagaagagtcaatcgcagacaacattttgaatgcggtcacacgttagcagcatgattgccacggat ggcaacatattaacggcatgatattgacttattgaataaaattgggtaaatttgactcaacgatgggttaattcgctcgttg tggtagtgagatgaaaagaggcggcgcttactaccgattccgcctagttggtcacttcgacgtatcgtctggaactcca 5 accatcgcaggcagagaggtctgcaaaatgcaatcccgaaacagttcgcaggtaatagttagagcctgcataacg gtttcgggattttttatatctgcacaacaggtaagagcattgagtcgataatcgtgaagagtcggcgagcctggttagcc agtgctctttccgttgtgctgaattaagcgaataccggaagcagaaccggatcaccaaatgcgtacaggcgtcatcgc cgcccagcaacagcacaacccaaactgagccgtagccactgtctgtcctgaattcattagtaatagttacgctgcggc cttttacacatgaccttcgtgaaagcgggtggcaggaggtcgcgctaacaacctcctgccgttttgcccgtgcatatcg10 gtcacgaacaaatctgattactaaacacagtagcctggatttgttctatcagtaatcgaccttattcctaattaaatagag caaatccccttattgggggtaagacatgaagatgccagaaaaacatgacctgttggccgccattctcgcggcaaagg aacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaa cagtaatcgacgcaacgatgtgcgccattatcgcctagttcattcgtgaccttctcgacttcgccggactaagtagcaat ctcgcttatataacgagcgtgtttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaa15 aagccggagtagaagatggtagaaatcaataatcaacgtaaggcgttcctcgatatgctggcgtggtcggagggaa ctgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagagctatttactgattactccga tcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttg gtgggatgcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcaga ttaaggagcgtggcgctttacctatgattgatcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggctt20 cactgccgggcgctggttatggtcagttcgagcataaggctgacagcctgattgcaaaattcaaagaagcgggcgg aacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctggttatctgcatcatcgtctgcctgtca tgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagct ggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggag ttagctgatgctaaagctgaaaatgatgctctgcgtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagt25 ctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctccccccgactggcagacaccgct gaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggaaggaacccagaagtatatta atgagcagtgcagatagagttgcccatatcgatgggcaactcatgcaattattgtgagcaatacacacgcgcttccag cggagtataaatgcctaaagtaataaaaccgagcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctg cgccgccacaaattttggctgcatcgacagttttcttctgcccaattccagaaacgaagaaatgatgggtgatggtttcct30 ttggtgctactgctgccggtttgttttgaacagtaaacgtctgttgagcacatcctgtaataagcagggccagcgcagta gcgagtagcatttttttcatggtgttattcccgatgctttttgaagttcgcagaatcgtatgtgtagaaaattaaacaaaccct aaacaatgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcgatgaatcgtcattgtattcccggattaact atgtccacagccctgacggggaacttctctgcgggagtgtccgggaataattaaaacgatgcacacagggtttagcg cgtacacgtattgcattatgccaacgccccggtgctgacacggaagaaaccggacgttatgatttagcgtggaaaga35 tttgtgtagtgttctgaatgctctcagtaaatagtaatgaattatcaaaggtatagtaatatcttttatgttcatggatatttgta acccatcggaaaactcctgctttagcaagattttccctgtattgctgaaatgtgatttctcttgatttcaacctatcataggac gtttctataagatgcgtgtttcttgagaatttaacatttacaacctttttaagtccttttattaacacggtgttatcgttttctaaca cgatgtgaatattatctgtggctagatagtaaatataatgtgagacgttgtgacgttttagttcagaataaaacaattcaca gtctaaatcttttcgcacttgatcgaatatttctttaaaaatggcaacctgagccattggtaaaaccttccatgtgatacga40 gggcgcgtagtttgcattatcgtttttatcgtttcaatctggtctgacctccttgtgttttgttgatgatttatgtcaaatattagga atgttttcacttaatagtattggttgcgtaacaaagtgcggtcctgctggcattctggagggaaatacaaccgacagatgt atgtaaggccaacgtgctcaaatcttcatacagaaagatttgaagtaatattttaaccgctagatgaagagcaagcgc atggagcgacaaaatgaataaagaacaatctgctgatgatccctccgtggatctgattcgtgtaaaaaatatgcttaat agcaccatttctatgagttaccctgatgttgtaattgcatgtatagaacataaggtgtctctggaagcattcagagcaattg45 aggcagcgttggtgaagcacgataataatatgaaggattattccctggtggttgactgatcaccataactgctaatcatt caaactatttagtctgtgacagagccaacacgcagtctgtcactgtcaggaaagtggtaaaactgcaactcaattact gcaatgccctcgtaattaagtgaatttacaatatcgtcctgttcggagggaagaacgcgggatgttcattcttcatcacttt taattgatgtatatgctctcttttctgacgttagtctccgacggcaggcttcaatgacccaggctgagaaattcccggacc ctttttgctcaagagcgatgttaatttgttcaatcatttggttaggaaagcggatgttgcgggttgttgttctgcgggttctgttc50 ttcgttgacatgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaat 46 4932-3429-7135, v.1taatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcatt atcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctgcctgatgcggtattttctccttacgcatc tgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtg tggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgcc 5 acgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggag tccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttagacctg caggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcg ccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgc10 gcagcctgaatggcgaatgcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttac aaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaat accatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctg gtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtga gaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggc15 cagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaa atacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcat caacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaacc atgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccat ctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcg20 atagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaat cgcggcttcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttat tgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaac ttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaat caccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcag25 gcctggtatgagtcagcaacaccttcttcacgaggcagacctctcgacggagttccactgagcgtcagaccccgtag aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac SEQ ID NO: 13 - STXP1 UTR Seq – truncated (771bp) 30 AAAAATAAGTCGCCCCTCCAAAACACGCCCCCATCCCACAGCGCTCCGCAGCT TCCCACCACCGCCCGCCTCAGTTCCTTTGCGTCTGTTGCCTCCCCAGCCCTGC ACGCCCTGGCTGGCACTGTTGCCGCTGCATTCTCGTGTTCAGTGATGCCCTCTT CTTGTTTGAAACAAAAGAAAATAATGCATTGTGTTTTTTAAAAAGAGTATCTTATA CATGTATCCTAAAAAGAGAAGCTCATGTGCAATTGGTGCACAGCAGGAGAAATT 35 TCTGGACTGTTAGGATGAATGGACGCCTTCTCCCCGTTATTTAAGATTTGTGAC CTTGTACATAACCCTGGGTGACGTGCACATTGCTTGGGTATGGAACGGTAGAAA TTTGGGTGTTTTTAAAACCTTGTTTGGGGTTGTTCCTGTCCTTGTTGAGAATCAT AGAGATGTCTGTGTTCTTGGAGTATTTCACACTGAGGACTAATCTGCTATCTTCA TTCCAGTCCCTACCCCTCAGTGCCTGCTCTCATCCAAATAACCTGGGAGGTGAC 40 AATCAGGATATCTCAGGAGGTCCAAGGTGGAACAGACCTCTTTGCCTTTCCCAG CGTCTCATACCCCCGGTAGTGCAGCTGTGGGTGGAGGCTGGGGTGTCTGCAC GAAGTCAGGCCAGCGTCCTCCTCCACAGCCTGTCACTGCCCCCTCCCCAGCCT GTGTCCACAGTGCTGTGATCCCGAGGGAAGTCCTCCAGTCTAAGTCACAGTGC CCTGACAGGTGAGAAG 45 SEQ ID NO: 14 - pkAAV_iCAG_STXBP1_UTR (11576) ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat caagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc50 gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg 47 4932-3429-7135, v.1ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcta tgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc 5 gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggc cttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgggcgacctttggtcgc ccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgt ctgcacatttcgtagagcgagtgttccgatactctaatctccctaggactagttattaatagtaatcaattacggggtcatt agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc10 ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagt atttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggt aaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc gctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtattt atttattttttaattattttgtgcagcgatgggggcgggggggggggggggggcgcgcgccaggcggggcggggcgg15 ggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagttt ccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcg ctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccac aggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctg cgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtg20 cgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgc gctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaa caaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccc cctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcgggg ctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggag25 ggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgc cttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgcc gccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggc cttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcggg ggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgcct30 tcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattaaactcgaggccac catggcccccattggcctcaaagctgttgtcggagagaagattatgcatgatgtgataaagaaggtcaagaagaag ggggaatggaaggtgctggtggtggatcagttaagcatgaggatgctgtcctcctgctgcaagatgacagacatcat gaccgagggcataacgattgtggaagatatcaataagcgcagagagccgctccccagcctggaggctgtgtatctc atcactccatccgagaagtccgtccactctctcatcagtgactttaaggacccgccgactgctaaataccgggctgca35 cacgtcttcttcactgactcttgtccagatgccctgtttaatgaactggtaaaatcccgagcagccaaagtcatcaaaac tctgacggaaatcaatattgcatttctcccgtatgaatcccaggtctattccttggactctgctgactctttccaaagcttcta cagtccccacaaggctcagatgaagaatcctatactggagcgcctggcagagcagatcgcgaccctttgtgccacc ctgaaggagtacccggctgtgcggtatcggggggaatacaaggacaatgccctgctggctcagctaatccaggaca agctcgatgcctataaagctgatgatccaacaatgggggagggcccagacaaggcacgctcccagctcctgatcct40 ggatcgaggctttgaccccagctcccctgtgctccatgaattgacttttcaggctatgagttatgatctgctgcctatcgaa aatgatgtatacaagtatgagaccagcggcatcggggaggcacgggtgaaggaggtgctcctggacgaggacga cgacctgtggatagcactgcgccacaagcacatcgcagaggtgtcccaggaagtcacccggtctctgaaagattttt cttctagcaagagaatgaatactggagagaagaccaccatgcgggacctgtcccagatgctgaagaagatgcctc agtaccagaaagagctcagcaagtactccacccacctgcaccttgctgaggactgtatgaagcattaccaaggcac45 cgtagacaaactctgccgagtggagcaggacctggccatgggcacagatgctgagggagagaagatcaaggac cctatgcgagccatcgtccccattctgctggatgccaatgtcagcacttatgacaaaatccgcatcatccttctctacatc tttttgaagaatggcatcacggaggaaaacctgaacaaactgatccagcacgcccagatacccccggaggatagtg agatcatcaccaacatggctcacctcggcgtgcccatcgtcaccgattccacgctgcgtcgccggagcaagccgga gcggaaggaacgcatcagcgagcagacctaccagctctcacggtggactccgattatcaaggacatcatggagga50 cactattgaggacaaacttgacaccaaacactacccttatatctctacccgttcctctgcctccttcagcaccaccgccg 48 4932-3429-7135, v.1tcagcgcccgctatgggcactggcataagaacaaggccccaggcgagtaccgcagtggcccccgcctcatcatttt catccttgggggtgtgagcctgaatgagatgcgctgcgcctacgaggtgacccaggccaacggaaagtgggaggt gctgataggatccacacacatcctcaccccacagaaactgctggacacactgaagaaactgaataaaacagatga agaaataagcagttaaggtaccaaaaataagtcgcccctccaaaacacgcccccatcccacagcgctccgcagct 5 tcccaccaccgcccgcctcagttcctttgcgtctgttgcctccccagccctgcacgccctggctggcactgttgccgctg cattctcgtgttcagtgatgccctcttcttgtttgaaacaaaagaaaataatgcattgtgttttttaaaaagagtatcttatac atgtatcctaaaaagagaagctcatgtgcaattggtgcacagcaggagaaatttctggactgttaggatgaatggacg ccttctccccgttatttaagatttgtgaccttgtacataaccctgggtgacgtgcacattgcttgggtatggaacggtagaa atttgggtgtttttaaaaccttgtttggggttgttcctgtccttgttgagaatcatagagatgtctgtgttcttggagtatttcaca10 ctgaggactaatctgctatcttcattccagtccctacccctcagtgcctgctctcatccaaataacctgggaggtgacaat caggatatctcaggaggtccaaggtggaacagacctctttgcctttcccagcgtctcatacccccggtagtgcagctgt gggtggaggctggggtgtctgcacgaagtcaggccagcgtcctcctccacagcctgtcactgccccctccccagcct gtgtccacagtgctgtgatcccgagggaagtcctccagtctaagtcacagtgccctgacaggtgagaaggtctaataa ataaagaaatcagcacccctgtcgactgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctc15 tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt gagcgagcgagcgcgcagctgcctgcaggggcagcttgaaggaaatactaaggcaaaggtactgcaagtgctcg caacattcgcttatgcggattattgccgtagtgccgcgacgccgggggcaagatgcagagattgccatggtacaggc cgtgcggttgatattgccaaaacagagctgtgggggagagttgtcgagaaagagtgcggaagatgcaaaggcgtc ggctattcaaggatgccagcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctg20 gtcacgcactgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtcaatcgcagacaacatttt gaatgcggtcacacgttagcagcatgattgccacggatggcaacatattaacggcatgatattgacttattgaataaaa ttgggtaaatttgactcaacgatgggttaattcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgatt ccgcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctgcaaaatgcaatcccg aaacagttcgcaggtaatagttagagcctgcataacggtttcgggattttttatatctgcacaacaggtaagagcattga25 gtcgataatcgtgaagagtcggcgagcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagc agaaccggatcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacccaaactgagccgtagcc actgtctgtcctgaattcattagtaatagttacgctgcggccttttacacatgaccttcgtgaaagcgggtggcaggaggt cgcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaatctgattactaaacacagtagcctggatt tgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaa30 aaacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgta ccttcgcggcagatataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctagttc attcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcggtactg actcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgt aaggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatga35 cgtcattgtaggcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaa tcaacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttct ctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattgatcgtggtgatat ccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggct gacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcga40 ttatctccgctctggttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaa gcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcg tgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgatgat gttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtg gataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactat45 gcaaaaacaactggaaggaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaact catgcaattattgtgagcaatacacacgcgcttccagcggagtataaatgcctaaagtaataaaaccgagcaatcca tttacgaatgtttgctgggtttctgttttaacaacattttctgcgccgccacaaattttggctgcatcgacagttttcttctgccc aattccagaaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaacgtctgt tgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcatggtgttattcccgatgctttttgaag50 ttcgcagaatcgtatgtgtagaaaattaaacaaaccctaaacaatgagttgaaatttcatattgttaatatttattaatgtat 49 4932-3429-7135, v.1gtcaggtgcgatgaatcgtcattgtattcccggattaactatgtccacagccctgacggggaacttctctgcgggagtgt ccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtattgcattatgccaacgccccggtgctgaca cggaagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagtaaatagtaatgaatta tcaaaggtatagtaatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagattttccctgtatt 5 gctgaaatgtgatttctcttgatttcaacctatcataggacgtttctataagatgcgtgtttcttgagaatttaacatttacaac ctttttaagtccttttattaacacggtgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataatgtg agacgttgtgacgttttagttcagaataaaacaattcacagtctaaatcttttcgcacttgatcgaatatttctttaaaaatg gcaacctgagccattggtaaaaccttccatgtgatacgagggcgcgtagtttgcattatcgtttttatcgtttcaatctggtc tgacctccttgtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgcgtaacaaagtgcggt10 cctgctggcattctggagggaaatacaaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaaga tttgaagtaatattttaaccgctagatgaagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgat gatccctccgtggatctgattcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgatgttgtaattgcatgt atagaacataaggtgtctctggaagcattcagagcaattgaggcagcgttggtgaagcacgataataatatgaagga ttattccctggtggttgactgatcaccataactgctaatcattcaaactatttagtctgtgacagagccaacacgcagtctg15 tcactgtcaggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatcgtcctgt tcggagggaagaacgcgggatgttcattcttcatcacttttaattgatgtatatgctctcttttctgacgttagtctccgacgg caggcttcaatgacccaggctgagaaattcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttag gaaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttgacatgaggttgccccgtattcagtgtcgctgatttgt attgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttga20 tggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacggg gcggcgacctgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagt acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggct ccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggcc25 atcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaa cactcaactctatctcgggctattcttttgatttagacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtc gtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcg aagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgcgatttattcaacaaagccgc cgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcat30 caaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaa ctcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacct attaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggc aaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaa accgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaat35 cgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctg gaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttca gaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagccc atttatacccatataaatcagcatccatgttggaatttaatcgcggcttcgagcaagacgtttcccgttgaatatggctcat40 aacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcaga gattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgaca acgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccg tggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcaga cctctcgacggagttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc 45 SEQ ID NO: 15 - WPRE (589bp) aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag50 gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgc 50 4932-3429-7135, v.1caccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgc ccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggct gctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcc ttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggc 5 cgcctccccgc SEQ ID NO: 16 - pkAAV_iCAG_STXBP1_WPRE (11600bp) ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat10 caagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcta tgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga15 gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggc cttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgggcgacctttggtcgc ccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgt ctgcacatttcgtagagcgagtgttccgatactctaatctccctaggactagttattaatagtaatcaattacggggtcatt20 agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagt atttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggt aaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc gctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtattt25 atttattttttaattattttgtgcagcgatgggggcggggggggggggggggggcgcgcgccaggcggggcggggcg gggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagt ttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgc gctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactccca caggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggct30 gcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgt gcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtg cgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgagggga acaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaacccc ccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggg35 gctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccgggga gggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattg ccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgc cgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggaggg ccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgg40 gggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgc cttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattaaactcgaggcc accatggcccccattggcctcaaagctgttgtcggagagaagattatgcatgatgtgataaagaaggtcaagaagaa gggggaatggaaggtgctggtggtggatcagttaagcatgaggatgctgtcctcctgctgcaagatgacagacatca tgaccgagggcataacgattgtggaagatatcaataagcgcagagagccgctccccagcctggaggctgtgtatctc45 atcactccatccgagaagtccgtccactctctcatcagtgactttaaggacccgccgactgctaaataccgggctgca cacgtcttcttcactgactcttgtccagatgccctgtttaatgaactggtaaaatcccgagcagccaaagtcatcaaaac tctgacggaaatcaatattgcatttctcccgtatgaatcccaggtctattccttggactctgctgactctttccaaagcttcta cagtccccacaaggctcagatgaagaatcctatactggagcgcctggcagagcagatcgcgaccctttgtgccacc ctgaaggagtacccggctgtgcggtatcggggggaatacaaggacaatgccctgctggctcagctaatccaggaca50 agctcgatgcctataaagctgatgatccaacaatgggggagggcccagacaaggcacgctcccagctcctgatcct 51 4932-3429-7135, v.1ggatcgaggctttgaccccagctcccctgtgctccatgaattgacttttcaggctatgagttatgatctgctgcctatcgaa aatgatgtatacaagtatgagaccagcggcatcggggaggcacgggtgaaggaggtgctcctggacgaggacga cgacctgtggatagcactgcgccacaagcacatcgcagaggtgtcccaggaagtcacccggtctctgaaagattttt cttctagcaagagaatgaatactggagagaagaccaccatgcgggacctgtcccagatgctgaagaagatgcctc 5 agtaccagaaagagctcagcaagtactccacccacctgcaccttgctgaggactgtatgaagcattaccaaggcac cgtagacaaactctgccgagtggagcaggacctggccatgggcacagatgctgagggagagaagatcaaggac cctatgcgagccatcgtccccattctgctggatgccaatgtcagcacttatgacaaaatccgcatcatccttctctacatc tttttgaagaatggcatcacggaggaaaacctgaacaaactgatccagcacgcccagatacccccggaggatagtg agatcatcaccaacatggctcacctcggcgtgcccatcgtcaccgattccacgctgcgtcgccggagcaagccgga10 gcggaaggaacgcatcagcgagcagacctaccagctctcacggtggactccgattatcaaggacatcatggagga cactattgaggacaaacttgacaccaaacactacccttatatctctacccgttcctctgcctccttcagcaccaccgccg tcagcgcccgctatgggcactggcataagaacaaggccccaggcgagtaccgcagtggcccccgcctcatcatttt catccttgggggtgtgagcctgaatgagatgcgctgcgcctacgaggtgacccaggccaacggaaagtgggaggt gctgataggatccacacacatcctcaccccacagaaactgctggacacactgaagaaactgaataaaacagatga15 agaaataagcagttaaggtaccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgc tccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtata aatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgc aacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggc ggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcg20 gggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttc ggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctc agacgagtcggatctccctttgggccgcctccccgcgcggccgcactagacctcgactgtgccttctagttgccagcc atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgg25 gaagacaatagcaggcatgctggggaatctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttg gccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccg ggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcagcttgaaggaaatactaaggcaaaggta ctgcaagtgctcgcaacattcgcttatgcggattattgccgtagtgccgcgacgccgggggcaagatgcagagattgc catggtacaggccgtgcggttgatattgccaaaacagagctgtgggggagagttgtcgagaaagagtgcggaagat30 gcaaaggcgtcggctattcaaggatgccagcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttac ccaacccacctggtcacgcactgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtcaatcg cagacaacattttgaatgcggtcacacgttagcagcatgattgccacggatggcaacatattaacggcatgatattga cttattgaataaaattgggtaaatttgactcaacgatgggttaattcgctcgttgtggtagtgagatgaaaagaggcggc gcttactaccgattccgcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctgca35 aaatgcaatcccgaaacagttcgcaggtaatagttagagcctgcataacggtttcgggattttttatatctgcacaacag gtaagagcattgagtcgataatcgtgaagagtcggcgagcctggttagccagtgctctttccgttgtgctgaattaagcg aataccggaagcagaaccggatcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacccaaac tgagccgtagccactgtctgtcctgaattcattagtaatagttacgctgcggccttttacacatgaccttcgtgaaagcgg gtggcaggaggtcgcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaatctgattactaaaca40 cagtagcctggatttgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatg aagatgccagaaaaacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgt ttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgcc attatcgcctagttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcgg ctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaat45 caataatcaacgtaaggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccaga aatcatggttatgacgtcattgtaggcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaac ccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttgg cctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgatt gatcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagtt50 cgagcataaggctgacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagc 52 4932-3429-7135, v.1agagtcaccgcgattatctccgctctggttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacg ccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaattactgacatg cagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgat gctctgcgtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccacca 5 ccgcctccggcgtggataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagaga gaggctgatcactatgcaaaaacaactggaaggaacccagaagtatattaatgagcagtgcagatagagttgccca tatcgatgggcaactcatgcaattattgtgagcaatacacacgcgcttccagcggagtataaatgcctaaagtaataa aaccgagcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctgcgccgccacaaattttggctgcatcg acagttttcttctgcccaattccagaaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtttgttttga10 acagtaaacgtctgttgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcatggtgttattc ccgatgctttttgaagttcgcagaatcgtatgtgtagaaaattaaacaaaccctaaacaatgagttgaaatttcatattgtt aatatttattaatgtatgtcaggtgcgatgaatcgtcattgtattcccggattaactatgtccacagccctgacggggaact tctctgcgggagtgtccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtattgcattatgccaacg ccccggtgctgacacggaagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagta15 aatagtaatgaattatcaaaggtatagtaatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagc aagattttccctgtattgctgaaatgtgatttctcttgatttcaacctatcataggacgtttctataagatgcgtgtttcttgaga atttaacatttacaacctttttaagtccttttattaacacggtgttatcgttttctaacacgatgtgaatattatctgtggctagat agtaaatataatgtgagacgttgtgacgttttagttcagaataaaacaattcacagtctaaatcttttcgcacttgatcgaa tatttctttaaaaatggcaacctgagccattggtaaaaccttccatgtgatacgagggcgcgtagtttgcattatcgtttttat20 cgtttcaatctggtctgacctccttgtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgcgt aacaaagtgcggtcctgctggcattctggagggaaatacaaccgacagatgtatgtaaggccaacgtgctcaaatct tcatacagaaagatttgaagtaatattttaaccgctagatgaagagcaagcgcatggagcgacaaaatgaataaag aacaatctgctgatgatccctccgtggatctgattcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctga tgttgtaattgcatgtatagaacataaggtgtctctggaagcattcagagcaattgaggcagcgttggtgaagcacgat25 aataatatgaaggattattccctggtggttgactgatcaccataactgctaatcattcaaactatttagtctgtgacagagc caacacgcagtctgtcactgtcaggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatt tacaatatcgtcctgttcggagggaagaacgcgggatgttcattcttcatcacttttaattgatgtatatgctctcttttctgac gttagtctccgacggcaggcttcaatgacccaggctgagaaattcccggaccctttttgctcaagagcgatgttaatttgt tcaatcatttggttaggaaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttgacatgaggttgccccgtattc30 agtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgatt atttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatc cgacaggttacggggcggcgacctgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtc aaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgct acacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagct35 ctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggtt cacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttc caaactggaacaacactcaactctatctcgggctattcttttgatttagacctgcaggcatgcaagcttggcactggccg tcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc tggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgcgatttatt40 caacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaa actcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaat gaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaac atcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatcc ggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcact45 cgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaatt acaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatat tcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatg cttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctac ctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacat50 tatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcttcgagcaagacgtttcccgtt 53 4932-3429-7135, v.1gaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgca atgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcac gcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaa gctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttct 5 tcacgaggcagacctctcgacggagttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc SEQ ID NO: 17 - pkAAC_iCAG_STXBP1-Flag (11145 bp) ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat10 caagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcta tgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga15 gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggc cttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagccc gggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccat cactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggactagttatt20 aatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccg cctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggac tttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttg gcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctccccc25 ccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggggggggggggcgc gcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatc agagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgc ggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggct ctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaat30 gacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctc ggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgc gggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtg cggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggc gcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggc35 tccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggc ggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgag gcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgc ggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcag gaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtc40 cgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctcta gagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttg gcaaagaattaaactcgaggccaccatggcccccattggcctcaaagctgttgtcggagagaagattatgcatgatg tgataaagaaggtcaagaagaagggggaatggaaggtgctggtggtggatcagttaagcatgaggatgctgtcctc ctgctgcaagatgacagacatcatgaccgagggcataacgattgtggaagatatcaataagcgcagagagccgct45 ccccagcctggaggctgtgtatctcatcactccatccgagaagtccgtccactctctcatcagtgactttaaggacccgc cgactgctaaataccgggctgcacacgtcttcttcactgactcttgtccagatgccctgtttaatgaactggtaaaatccc gagcagccaaagtcatcaaaactctgacggaaatcaatattgcatttctcccgtatgaatcccaggtctattccttggac tctgctgactctttccaaagcttctacagtccccacaaggctcagatgaagaatcctatactggagcgcctggcagagc agatcgcgaccctttgtgccaccctgaaggagtacccggctgtgcggtatcggggggaatacaaggacaatgccct50 gctggctcagctaatccaggacaagctcgatgcctataaagctgatgatccaacaatgggggagggcccagacaa 54 4932-3429-7135, v.1ggcacgctcccagctcctgatcctggatcgaggctttgaccccagctcccctgtgctccatgaattgacttttcaggctat gagttatgatctgctgcctatcgaaaatgatgtatacaagtatgagaccagcggcatcggggaggcacgggtgaag gaggtgctcctggacgaggacgacgacctgtggatagcactgcgccacaagcacatcgcagaggtgtcccagga agtcacccggtctctgaaagatttttcttctagcaagagaatgaatactggagagaagaccaccatgcgggacctgtc 5 ccagatgctgaagaagatgcctcagtaccagaaagagctcagcaagtactccacccacctgcaccttgctgaggac tgtatgaagcattaccaaggcaccgtagacaaactctgccgagtggagcaggacctggccatgggcacagatgctg agggagagaagatcaaggaccctatgcgagccatcgtccccattctgctggatgccaatgtcagcacttatgacaaa atccgcatcatccttctctacatctttttgaagaatggcatcacggaggaaaacctgaacaaactgatccagcacgccc agatacccccggaggatagtgagatcatcaccaacatggctcacctcggcgtgcccatcgtcaccgattccacgctg10 cgtcgccggagcaagccggagcggaaggaacgcatcagcgagcagacctaccagctctcacggtggactccgat tatcaaggacatcatggaggacactattgaggacaaacttgacaccaaacactacccttatatctctacccgttcctct gcctccttcagcaccaccgccgtcagcgcccgctatgggcactggcataagaacaaggccccaggcgagtaccgc agtggcccccgcctcatcattttcatccttgggggtgtgagcctgaatgagatgcgctgcgcctacgaggtgacccag gccaacggaaagtgggaggtgctgataggatccacacacatcctcaccccacagaaactgctggacacactgaa15 gaaactgaataaaacagatgaagaaataagcagtgactacaaagaccatgacggtgattataaagatcatgacat cgattacaaggatgacgatgacaagtgaggtacccaattcgccctatagtgagtcgtgctagctgcagggtgtcgata tcagcggccgcactagacctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgacc ctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggaatctag20 agatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactga ggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagc tgcctgcaggggcagcttgaaggaaatactaaggcaaaggtactgcaagtgctcgcaacattcgcttatgcggattat tgccgtagtgccgcgacgccgggggcaagatgcagagattgccatggtacaggccgtgcggttgatattgccaaaa cagagctgtgggggagagttgtcgagaaagagtgcggaagatgcaaaggcgtcggctattcaaggatgccagca25 agcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctggtcacgcactgttaagccgct gtatgacgctctggtggtgcaatgccacaaagaagagtcaatcgcagacaacattttgaatgcggtcacacgttagc agcatgattgccacggatggcaacatattaacggcatgatattgacttattgaataaaattgggtaaatttgactcaacg atgggttaattcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgattccgcctagttggtcacttcga cgtatcgtctggaactccaaccatcgcaggcagagaggtctgcaaaatgcaatcccgaaacagttcgcaggtaata30 gttagagcctgcataacggtttcgggattttttatatctgcacaacaggtaagagcattgagtcgataatcgtgaagagt cggcgagcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagcagaaccggatcaccaaat gcgtacaggcgtcatcgccgcccagcaacagcacaacccaaactgagccgtagccactgtctgtcctgaattcatta gtaatagttacgctgcggccttttacacatgaccttcgtgaaagcgggtggcaggaggtcgcgctaacaacctcctgc cgttttgcccgtgcatatcggtcacgaacaaatctgattactaaacacagtagcctggatttgttctatcagtaatcgacct35 tattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaaaacatgacctgttggccg ccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatg gcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctagttcattcgtgaccttctcgacttc gccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcggtactgactcgattggttcgcttatca aacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgtaaggcgttcctcgatatgct40 ggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggaga gctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacg ctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggac gctgtggcattgcagcagattaaggagcgtggcgctttacctatgattgatcgtggtgatatccgtcaggcaatcgacc gttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgacagcctgattgcaaa45 attcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctggttatc tgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaa atgccagagaactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctgcgctcgat gcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgatgatgttgccgctggtcgtcgtc ggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctccccc50 cgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggaag 55 4932-3429-7135, v.1gaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaactcatgcaattattgtgagca atacacacgcgcttccagcggagtataaatgcctaaagtaataaaaccgagcaatccatttacgaatgtttgctgggtt tctgttttaacaacattttctgcgccgccacaaattttggctgcatcgacagttttcttctgcccaattccagaaacgaaga aatgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaacgtctgttgagcacatcctgtaataa 5 gcagggccagcgcagtagcgagtagcatttttttcatggtgttattcccgatgctttttgaagttcgcagaatcgtatgtgta gaaaattaaacaaaccctaaacaatgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcgatgaatcgt cattgtattcccggattaactatgtccacagccctgacggggaacttctctgcgggagtgtccgggaataattaaaacg atgcacacagggtttagcgcgtacacgtattgcattatgccaacgccccggtgctgacacggaagaaaccggacgtt atgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagtaaatagtaatgaattatcaaaggtatagtaatatct10 tttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagattttccctgtattgctgaaatgtgatttctcttg atttcaacctatcataggacgtttctataagatgcgtgtttcttgagaatttaacatttacaacctttttaagtccttttattaaca cggtgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataatgtgagacgttgtgacgttttagttc agaataaaacaattcacagtctaaatcttttcgcacttgatcgaatatttctttaaaaatggcaacctgagccattggtaa aaccttccatgtgatacgagggcgcgtagtttgcattatcgtttttatcgtttcaatctggtctgacctccttgtgttttgttgatg15 atttatgtcaaatattaggaatgttttcacttaatagtattggttgcgtaacaaagtgcggtcctgctggcattctggaggga aatacaaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaagatttgaagtaatattttaaccgct agatgaagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgatgatccctccgtggatctgattc gtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgatgttgtaattgcatgtatagaacataaggtgtctctg gaagcattcagagcaattgaggcagcgttggtgaagcacgataataatatgaaggattattccctggtggttgactgat20 caccataactgctaatcattcaaactatttagtctgtgacagagccaacacgcagtctgtcactgtcaggaaagtggta aaactgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatcgtcctgttcggagggaagaacgcgg gatgttcattcttcatcacttttaattgatgtatatgctctcttttctgacgttagtctccgacggcaggcttcaatgacccagg ctgagaaattcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttaggaaagcggatgttgcgggtt gttgttctgcgggttctgttcttcgttgacatgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgtt25 aagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttgtg atatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctgcctgatgc ggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcg cattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcg ctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttag30 tgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttt tcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggc tattcttttgatttagacctgcaggcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcg cccttcccaacagttgcgcagcctgaatggcgaatgcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaa35 tgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattc atatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccatag gatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaa taaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttcca gacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcg40 cctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcagg aacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatc gcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcag ccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcg ggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagc45 atccatgttggaatttaatcgcggcttcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttat gtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggct ttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaa agcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctgg atgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctctcgacggagttccactga 50 gcgtcagaccccgtagaaaagatcaaaggatcttc 56 4932-3429-7135, v.1* * *

[0141] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred 5 embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be 10 achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. 57 4932-3429-7135, v.1

Claims

WHAT IS CLAIMED IS:

1. A method of expressing a syntaxin-binding protein 1 (STXBP1) in a brain cell, mammalian cell, mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron, of a subject, comprising administering to the subject a modified adeno- 5 associated virus (AAV) encoding a STXBP1 under the control of a promoter operable in said brain cell, mammalian cell, mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron.

2. The method of claim 1, wherein said AAV comprises a modified capsid protein comprising the targeting sequence of SEQ ID NO: 4 or 6. 10 3. The method of claim 1, wherein the promoter may be a constitutive promoter or a tissue specific promoter.

4. The method of claim 1, wherein the promoter is an Mecp2 promoter or an iCAG promoter.

5. The method of claim 4, wherein the Mecp2 promoter comprises the sequence of SEQ 15 ID NO: 9 and wherein the iCAG promoter comprises the sequence of SEQ ID NO:

10.

6. The method of any one of claims 1-5, wherein the modified AAV is derived from AAV1 and AAV2.

7. The method of any one of claims 2-6, wherein the targeting sequence is inserted near or after position 590 of SEQ ID NO: 1 or after position 587 of SEQ ID NO:

2. 20 8. The method of any one of claims 2-7, wherein the targeting sequence comprises of SEQ ID NO: 4 or 6.

9. The method of any one of claims 2-7, wherein the targeting sequence consists or consists essentially of SEQ ID NO: 4 or 6.

10. The method of any one of claims 1-9, wherein the STXBP1 comprises the sequence 25 of SEQ ID NO: 7 or SEQ ID NO:

8.

11. The method of any one of claims 1-9, wherein the STXBP1 consists of the sequence of SEQ ID NO: 7 or SEQ ID NO:

8. 58 4932-3429-7135, v.

112. The method of any one of claims 2-11, wherein the targeting sequence is flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long, such as wherein the linker sequences are SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence, or 5 wherein the linker sequences are AAA on the N-terminal side of the targeting sequence and AA on the C-terminal side of the targeting sequence.

13. The method of any one of claims 1-12, wherein a coding sequence for STXBP1 further comprises a 3’ UTR, such as native STXBP1 UTR.

14. The method of any one of claims 2-13, wherein the modified capsid protein comprises 10 the sequence of SEQ ID NO:

3.

15. The method of any one of claims 2-13, wherein the modified capsid protein consists of the sequence of SEQ ID NO:

5.

16. The method of any one of claims 1-15, wherein administering is direct intracerebroventricular or intracisternal magna or intrathecal delivery. 15 17. The method of any one of claims 1-16, wherein the modified AAV administering occurs more than once.

18. The method of claim 17, wherein the modified AAV administering comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations.

19. The method of claim 17, wherein the modified AAV administering occurs monthly, 20 every other month, every two months, every three months, every four months, every six months, annually every other year, every three years, every four year or every five years.

20. The method of any one of claims 1-19, wherein a plurality of viral particles is administered.

21. The method of claim 20, wherein administering comprises dosing at about 1×106to 25 about 1×1014vector genomes per kilogram (vg / kg).

22. The method of claim 21, wherein administering comprises dosing from about 1x107- 1x1014, about 1x108-1x1014, about 1x109-1x1014, about 1x1010-1x1014, about 1x1010-1x1013, 59 4932-3429-7135, v.1about 1x1010-1x1013, about 1x1010-1x1011, about 1x1011-1x1012, or about 1x1012-1x1013vector genomes per kilogram (vg / kg) of the patient.

23. The method of any one of claims 1-22, wherein the STXBP1 sequence is operably linked to a poly-adenylation signal. 5 24. The method of any one of claims 1-23, wherein said subject suffers from STXBP1- related epileptic encephalopathy.

25. The method of any one of claims 1-24, wherein said subject is less than 4 years old, or is between 4 and 25 years old.

26. A modified adeno-associated virus (AAV) encoding a STXBP1 under the control of a 10 promoter operable in a brain cell, mammalian cell, mammalian brain cell, such as a neuron, a human neuron or a mouse neuron.

27. The AAV of claim 26, wherein said AAV comprises a modified capsid protein comprising, consisting of or consisting essentially of the targeting sequence of SEQ ID NO: 4 or 6. 15 28. The modified AAV of claim 26, wherein the promoter may be a constitutive promoter or a tissue specific promoter.

29. The modified AAV of claim 26, wherein the promoter is an Mecp2 promoter or an iCAG promoter.

30. The modified AAV of claim 29, wherein the Mecp2 promoter comprises the sequence 20 of SEQ ID NO: 9 and wherein the iCAG promoter comprises the sequence of SEQ ID NO:

10.

31. The modified AAV of any one of claims 26-29, wherein the modified AAV is AAV1 or AAV2.

32. The modified AAV of any one of claims 27-31, wherein the targeting sequence is 25 inserted near or after position 590 of SEQ ID NO:

1.

33. The modified AAV of any one of claims 27-32, wherein the targeting sequence consists essentially of 587 of SEQ ID NO:

2. 60 4932-3429-7135, v.

134. The modified AAV of any one of claims 27-32, wherein the targeting sequence consists of SEQ ID NO: 4 or 6.

35. The modified AAV of any one of claims 26-34, wherein the STXBP1 comprises the sequence of SEQ ID NO: 7 or SEQ ID NO:

8. 5 36. The modified AAV of any one of claims 26-34, wherein the STXBP1 consists of the sequence of SEQ ID NO: 7 or SEQ ID NO:

8.

37. The modified AAV of any one of claims 27-36, wherein the targeting sequence is flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long, such as wherein the linker sequences are SSA on 10 the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence, or wherein the linker sequences are AAA on the N-terminal side of the targeting sequence and AA on the C-terminal side of the targeting sequence.

38. The modified AAV of any one of claim s 27-37, wherein a coding sequence for STXBP1 further comprises a 3’ UTR, such as native STXBP1 UTR. 15 39. The modified AAV of any one of claims 27-38, wherein the modified capsid protein comprises the sequence of SEQ ID NO:

3.

40. The modified AAV of any one of claims 27-38, wherein the modified capsid protein consists of the sequence of SEQ ID NO:

5.

41. A pharmaceutical composition comprising the modified AAV of any one of claims 20 26-40 and a pharmaceutically acceptable carrier.

42. A kit comprising the modified AAV of any one of claims 26-40.

43. Use of a modified adeno-associated virus (AAV) encoding a syntaxin-binding protein 1 (STXBP1) under the control of a promoter operable in a brain cell, a mammalian cell, a 25 mammalian brain cell, such as a neuron, such as a mouse neuron or human neuron.

44. The use of claim 32, wherein said AAV comprises a modified capsid protein comprising the targeting sequence of SEQ ID NO: 4 or 6, for expressing STXBP1 in a brain cell, mammalian cell, mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron. 61 4932-3429-7135, v.

145. Use of a modified adeno-associated virus (AAV) encoding a syntaxin binding protein 1 (STXBP1) under the control of a promoter operable in brain cell, a mammalian cell, a mammalian brain cell, such as a mammalian neuron, a mouse neuron or human neuron.

46. The use of claim 45, wherein said AAV comprises a modified capsid protein 5 comprising the targeting sequence SEQ ID NO: 4 or 6, for treating STXBP1-related epileptic encephalopathy in a subject afflicted therewith.

47. Use of a modified adeno-associated virus (AAV) encoding a syntaxin-binding protein 1 (STXBP1) under the control of a promoter operable in a brain cell, a mammalian cell, a mammalian brain cell, such as a mammalian neuron, mouse neuron or human neuron. 10 48. The use of claim 47, wherein said AAV comprises a modified capsid protein comprising the targeting sequence SEQ ID NO: 4 or 6, in the preparation of a medicament for treating STXBP1-related epileptic encephalopathy in a subject afflicted therewith. 62 4932-3429-7135, v.1

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