Compositions and methods for the treatment of disorders related to syntaxin-binding protein 1 deficiency

Abstract

The disclosure relates to compositions and methods for altering, e.g., enhancing, the level of STXBP1 protein via delivery using an adeno-associated viral (AAV) capsid variant. The compositions and methods of the present disclosure are useful in the treatment of an STXBP1-related disorder, e.g., an STXBP1-related neurodegenerative or neuromuscular disorder.

Description

Attorney Docket No.14640.0105-00304 COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISORDERS RELATED TO SYNTAXIN-BINDING PROTEIN 1 DEFICIENCY RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to US Provisional Application Serial No. 63 / 606,423, filed December 5, 2023, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 14640_0105-00304_SL.xml, was created on September 18, 2024, and is 2,039,437 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety. FIELD

[0003] Described herein are compositions and methods relating to adeno-associated virus (AAV) viral particles for the delivery of polynucleotides, e.g., polynucleotides encoding syntaxin-binding protein-1 (STXBP1) proteins and peptides for use in the treatment of an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). In some embodiments, compositions described herein may be used to treat a subject in need thereof, such as a human subject diagnosed with an STXBP1-related disorder or other condition resulting from a deficiency in the quantity and / or function of STXBP1 protein, or as a research tool in the study of diseases or conditions in cells or animal models of an STXBP1-related disorder. BACKGROUND

[0004] Syntaxin-binding protein 1 (STXBP1), also known as Munc18-1, is part of the synaptic fusion machinery that enables vesicles to fuse with the plasma membrane. Other names for STXBP1 include P67, DEE4, NSEC1, UNC18, N-Sec1, RBSEC1, unc-18A, and unc18-1. STXBP1 regulates neurotransmitter transmission by interacting with the SNARE complex. The SNARE complex is primarily composed of SNAP-25, vesicular associated membrane protein and syntaxin1. SNAP-25 and syntaxin-1 form the target membrane vesicle protein (T-SNARE), which binds to synaptic vesicle protein (VAMP). STXBP1 has a complex, arched tertiary structure. The arch comprises four closely connected domains, 1, 2, 3a, and 3b. Domains 1 and 3a form an arched gap. STXBP1 primarily regulates vesicle fusion by interacting with syntaxin-1. Domain 3a of STXBP1 is in close contact with the Habc domain of syntaxin-1, and domain 1 of STXBP1 binds to the N-terminal domain of syntaxin-1. STXBP1 regulatesAttorney Docket No.14640.0105-00304 vesicle docking and fusion by interacting with the SNARE complex. STXBP1 affects the released vesicles and participates in the transmission of neurotransmitters. STXBP1 is prominently involved in the early process of neurotransmitter release.

[0005] STXBP1 is essential for presynaptic vesicle release. It is rapidly phosphorylated by protein kinase C upon neuronal depolarization.

[0006] STXBP1 is encoded by the STXBP1 gene (Ensembl Gene ID No. ENSG00000136854), which is located on chromosome 9. It is expressed in the brain and spinal cord, and highly enriched in axons. Expression of STXBP1 is highest in the retina and cerebellum. STXBP1 is also found outside the brain.

[0007] Mutations in the STXBP1 gene are known to cause disease in human subjects. Abnormal expression of STXBP1 plays a role in the pathogenesis of a variety of neurological diseases, including STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), and Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

[0008] STXBP1 expression is also abnormal in STXBP1 encephalopathy. There are an estimated 750 known cases of STXBP1 encephalopathy worldwide, and STXBP1 encephalopathy has an estimated incidence of 3.3-3.8 per 100,000 births.

[0009] Patients with STXBP1 mutations and / or STXBP1 encephalopathy often present with epilepsy. Some patients with STXBP1 mutations present with autistic features, including aggressive behavior, self- mutilation, hyperactivity, compulsive symptoms, episodes of psychosis and / or auditory hallucinations. Phenotypes range from severe neonatal epilepsy to infantile-onset epilepsy.

[0010] Many patients with STXBP1 mutations also present with non-epileptic movement disorders, including truncal and limb ataxia, generalized tremors, and dystonia. Patients often present with unremitting epileptic activity.

[0011] Patients with STXBP1 encephalopathy are reliant on caregivers for the duration of their lives. Moreover, 40% of patients become non-ambulatory and lifespan is expected to be significantly reduced to about 30 years.

[0012] STXBP1 encephalopathy is caused by haploinsufficiency. Thus, disease may occur where there is a mutation to only a single functional copy of the STXBP1 gene. Disease-causing mutations include missense, nonsense, frameshift, splice-site, and whole gene deletions. There are about 135 known pathogenic variants of the STXBP1 gene.

[0013] Interneurons may be more affected by haploinsufficiency than excitatory neurons.

[0014] Studies have demonstrated that heterozygous STXBP1 knock-out mice display impaired glutamate and GABA transmission, increased anxiety, increased aggression, and impaired emotional learning, in addition to modest seizure phenotype.Attorney Docket No.14640.0105-00304

[0015] Studies in mice demonstrated that normalizing the excitatory synaptic transmission in STXBP1 heterozygotic knockout mice reduces aggression. This indicated a therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

[0016] Existing therapies target the symptoms of STXBP1 encephalopathy. Existing first line treatment comprises anti-epileptic drugs such as levetiracetam and phenobarbital. Existing second line treatment comprises further anti-epileptic drugs such as clobazam, topiramate. Existing third line treatment comprises further anti-epileptic drugs and / or interventions. Known interventions for existing third line treatment include adrenocorticotropic hormone, ketogenic diet, and vagal nerve stimulation.

[0017] No approved therapies target the underlying cause of STXBP1 mutations. There is a high unmet need for such treatments driven by the high seizure rate and shortened lifespan of patients with STXBP1 mutations.

[0018] Thus, there remains a long-felt need to develop pharmaceutical compositions and methods that can be delivered to the CNS for the treatment of diseases associated with STXBP1 mutations. In particular, a need exists for treatments targeting neurons, including GABAergic and glutamatergic neurons.

[0019] Adeno-associated viruses (AAVs) have emerged as a widely studied and utilized viral particles for delivery of therapeutically effective polypeptides to mammalian cells. See, e.g., Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999).

[0020] The present disclosure provides improved pharmaceutical compositions and methods. In some embodiments, the disclosure provides methods of treatment using AAV capsid variants that are capable of delivering a payload of interest, e.g., human STXBP1, to a target cell or tissue, e.g., a CNS cell or tissue. SUMMARY

[0021] The present disclosure addresses these challenges by providing AAV-based compositions, AAV-based compositions for use in methods for treating a syntaxin-binding protein-1 (STXBP1)-related disorder in subjects, and methods for treating a STXBP1-related disorder in subjects. Disclosed herein are compositions and methods directed to AAV-based gene delivery of STXBP1 to ameliorate loss-of- function. Disclosed herein are compositions and methods directed to AAV-based gene delivery of STXBP1 to improve neurotransmitter release. The compositions and methods are useful to improve presynaptic vesicle release, and to slow, halt, or reverse neurodegenerative and other symptoms of STXBP1 encephalopathy or a STXBP1-related disorder in a subject (e.g., a subject having one or more mutations in STXBP1).

[0022] In some aspects, the present disclosure provides an AAV particle comprising an AAV capsid and a nucleotide sequence encoding an STXBP1 protein, also referred to herein as an STXBP1-encoding sequence. In some embodiments, the nucleotide sequence encoding an STXBP1 protein is comprised in a viral genome. In some embodiments, the STXBP1 protein is a human STXBP1 protein. In someAttorney Docket No.14640.0105-00304 embodiments, the STXBP1 protein is a wildtype human STXBP1 protein. In some embodiments, the AAV particle comprises a viral genome encoding the STXBP1 protein (e.g., a human STXBP1 protein) and the AAV capsid is an AAV capsid variant. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.

[0023] In some aspects, the present disclosure provides an adeno-associated virus (AAV) particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises a syntaxin- binding protein-1 (STXBP1)-encoding sequence and the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII, wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H; and / or [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, or F. In some embodiments, the AAV capsid variant is an AAV9 capsid variant. In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981.

[0024] In some embodiments, X1is T, S, R, A, I, C, N, K, L, or Q; X2is N, T, G, V, S, Y, K, I, H, D, or F; and X3is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C. In some embodiments, X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and X7 is Q, G, N, K, H, R, E, L, P, or M.

[0025] In some embodiments, [N1] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688). In some embodiments, [N3] comprises the amino acid sequence of IAQ. In some embodiments, [N1]-N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).

[0026] In some embodiments, [N1] is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981, [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 981, and [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 981.

[0027] In some embodiments, the AAV capsid variant further comprises [N4] comprising amino acids X8, X9, X10, and X11, wherein: X8is T, S, N, P, A, or I; X9is G, N, D, R, V, A, S, or Q; X10is W, S, C, R, L, or G; and / or X11is V, A, S, I, C, G, D, F, L, or T. In some embodiments, [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066). In some embodiments, [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 981.

[0028] In some embodiments, [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).

[0029] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1-encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the aminoAttorney Docket No.14640.0105-00304 acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

[0030] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1-encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981. In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1-encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

[0031] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1-encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981. In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1- encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

[0032] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising an STXBP1-encoding sequence and an AAV capsid variant, wherein the AAV capsid variant comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 981; (ii) an amino acid sequence that is at least 99% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) an amino acid sequence that is at least 99% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).Attorney Docket No.14640.0105-00304

[0033] In some embodiments, the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO: 981.

[0034] In some embodiments, the AAV capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 981; (ii) the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

[0035] In some embodiments, the viral genome encodes a wildtype STXBP1 protein or a fragment thereof. In some embodiments, the viral genome encodes a human STXBP1 protein. In some embodiments, the STXBP1 protein comprises the amino acid sequence of SEQ ID NO: 6413.

[0036] In some embodiments, the STXBP1-encoding sequence is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence is at least 99% identical to the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence consists of the nucleotide sequence of SEQ ID NO: 6414.

[0037] In some embodiments, the viral genome comprises a promoter operably linked to the STXBP1-encoding sequence. In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR) sequence. In some embodiments, viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1-encoding sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1- encoding sequence, and an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence.

[0038] In some aspects, the present disclosure provides a cell comprising an AAV particle provided herein. In some embodiments, the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

[0039] In some aspects, the present disclosure provides a method of making an AAV particle provided herein, wherein the method comprises: (i) providing a cell comprising a viral genome comprising an STXBP1-encoding sequence and a nucleic acid encoding an AAV capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the AAV particle.

[0040] In some embodiments of the method of making an AAV particle, the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 90% (e.g., at leastAttorney Docket No.14640.0105-00304 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0041] In some embodiments of the method of making an AAV particle, the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

[0042] In some embodiments of the method of making an AAV particle, the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

[0043] In some embodiments, the method of making an AAV particle further comprises, prior to step (i), introducing a nucleic acid molecule comprising the viral genome into the cell. In some embodiments, the method further comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell. In some embodiments, the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

[0044] In some aspects, the present disclosure provides a pharmaceutical composition comprising an AAV particle provided herein and a pharmaceutically acceptable excipient.

[0045] In some aspects, the present disclosure provides a method of delivering an AAV particle encoding an STXBP1 protein to a cell, comprising administering an effective amount of a pharmaceutical composition or AAV particle provided herein. In some embodiments, the cell is in a subject. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder. In some embodiments, the STXBP1-related disorder is a STXBP1-related neurodegenerative or neuromuscular disorder. In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, WestAttorney Docket No.14640.0105-00304 syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

[0046] In some aspects, the present disclosure provides a method of treating an STXBP1-related disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein, thereby treating the STXBP1-related disorder. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having a STXBP1-related disorder. In some embodiments, the STXBP1-related disorder is a STXBP1-related neurodegenerative or neuromuscular disorder. In some embodiments, the STXBP1-related neurodegenerative or neuromuscular disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox- Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

[0047] In some aspects, the present disclosure provides a method of treating an STXBP1-related disorder in a subject, wherein the disorder is STXBP1 encephalopathy, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having STXBP1 encephalopathy.

[0048] In some embodiments, the subject has one or more mutations in the STXBP1 gene.

[0049] In some embodiments, the subject has lower STXBP1 activity as compared to STXBP1 activity in an individual who does not have an STXBP1-related disorder.

[0050] In some embodiments, treating results in prevention of progression of an STXBP1-related disorder (e.g., STXBP1 encephalopathy) in the subject. In some embodiments, treating results in amelioration of at least one symptom of an STXBP1-related disorder (e.g., STXBP1 encephalopathy), as indicated by one or more biomarkers. In some embodiments, one or more biomarkers comprise increased release of the neurotransmitters glutamate and / or GABA or reduction in abnormal electroencephalographic activity as evidence of improved STXBP1 activity. In some embodiments, the at least one symptom comprises epilepsy, autistic features, ataxia, generalized tremors, dystonia, or a combination thereof.

[0051] In some embodiments, the subject is a human.

[0052] In some embodiments, the AAV particle or pharmaceutical composition is delivered to a cell tissue, or region of the central nervous systems (CNS) of the subject. In some embodiments, the cell, tissue, or region of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron), tissue, or region of the of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus,Attorney Docket No.14640.0105-00304 gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region). In some embodiments, the AAV particle or the pharmaceutical composition is delivered to the subject via intravenous administration.

[0053] In some embodiments, a method of delivery or treating provided herein further comprises evaluating, e.g., measuring, the level of STXBP1 expression, e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the level of STXBP1 protein expression is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay. In some embodiments, evaluating the level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) is performed prior to and / or subsequent to administration of the AAV particle or pharmaceutical composition. In some embodiments, the subject’s level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) prior to administration is compared to the subject’s level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) subsequent to administration. In some embodiments, the method comprises evaluating the level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) in a cell or tissue of the CNS. In some embodiments, the cell or tissue of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron) or tissue of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region). In some embodiments, the subject’s level of STXBP1 protein expression subsequent to administration is increased relative to the subject’s level of STXBP1 protein expression prior to administration. In some embodiments, a method of delivery or treating provided herein further comprises evaluating, e.g., measuring, the level of STXBP1 activity in the subject, e.g., in a cell or tissue of the subject.

[0054] In some embodiments, administering a pharmaceutical composition or AAV particle provided herein to the subject results an increase in: (i) the level of STXBP1 activity in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinalAttorney Docket No.14640.0105-00304 cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)), and / or fluid (e.g., CSF and / or serum) of the subject, relative to baseline and / or relative to the level of STXBP1 activity in a cell, tissue, or fluid of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle; (ii) the number and / or level of viral genomes (VG) per cell level in a tissue of the CNS (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject, relative to the number and / or level of VG per cell in a peripheral tissue of the subject; and / or (iii) the level of STXBP1 protein or STXBP1 gene expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject relative to baseline and / or relative to the level of STXBP1 protein or STXBP1 gene in a cell or tissue of an individual with a STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle.

[0055] In some embodiments, a method of delivery or treating provided herein further comprises administering to the subject at least one additional agent and / or therapy. In some embodiments, the at least one additional agent and / or therapy comprises an agent and / or therapy suitable for treating the STXBP1-related disorder. In some embodiments, the at least one additional agent and / or therapy comprises one or more anti-epileptic drugs (e.g., bromide, clobazam, felbamate, ganaxolone, lamotrigine, levetiracetam, phenobarbital, topiramate, valproate, or a combination thereof).

[0056] In some embodiments, a method of delivery or treating provided herein further comprises administering an immunosuppressant to the subject. In some embodiments, the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.

[0057] In some aspects, the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in a method of treating a disorder provided herein.Attorney Docket No.14640.0105-00304

[0058] In some aspects, the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in the treatment of an STXBP1-related disorder in a subject. In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

[0059] In some aspects, the present disclosure provides a use of a pharmaceutical composition or AAV particle provided herein in the manufacture of a medicament for the treatment of an STXBP1- related disorder. In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). Enumerated Embodiments 1. An adeno-associated virus (AAV) particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)-encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant), wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N1]-[N2]-[N3] (SEQ ID NO: 4681), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein: (i) [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, H; and / or (ii) [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, F. 2. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises the amino acid Q at position 585, as numbered according to SEQ ID NO: 138 or 981.Attorney Docket No.14640.0105-00304 3. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises an amino acid other than Q at position 585, as numbered according to SEQ ID NO: 138 or 981. 4. The AAV particle of embodiment 1 or 3, wherein the AAV capsid variant comprises the amino acid K at position 585, as numbered according to SEQ ID NO: 138 or 981. 5. The AAV particle of any one of embodiments 1-4, wherein the AAV capsid variant comprises one, two, three, or all of: (i) an amino acid other than T at position 582 (e.g., S, R, A, I, C, N, K, L, or Q); (ii) an amino acid other than N at position 583 (e.g., T, G, V, S, Y, K, I, H, D, or F); (iii) an amino acid other than H at position 584 (e.g., T, N, K, D, I, S, P, A, Y, E, V, L, M, R, Q, or C); and / or (iv) an amino acid other than Q at position 585 (e.g., K, E, S, P, R, N, or H); wherein the amino acids are numbered according to SEQ ID NO: 138. 6. The AAV particle of any one of embodiments 1-5, wherein [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H. 7. The AAV particle of embodiment 6, wherein X4is Q or K. 8. The AAV particle of any one of embodiment 7, wherein X4is Q. 9. The AAV particle of embodiment 7, wherein X4is K. 10. The AAV particle of any one of embodiments 1-9, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), as numbered according to SEQ ID NO: 138. 11. The AAV particle of any one of embodiments 1-10, wherein the AAV capsid variant comprises the amino acid T at position 584, as numbered according to SEQ ID NO: 138 or 981. 12. The AAV particle of any one of embodiments 1-11, wherein: (i) X1is T, S, R, A, I, C, N, K, L, or Q; (ii) X2is N, T, G, V, S, Y, K, I, H, D, or F; and / or (iii) X3is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C. 13. The AAV particle of any one of embodiments 1-12, wherein [N1] comprises TN, NT, NK, SN, TT, RN, TG, TV, ST, TS, TY, AN, TK, TI, IN, TH, TD, CN, NN, KN, LN, SG, TF, RT, SY, SS, QN, ND,Attorney Docket No.14640.0105-00304 NP, GK, TA, VK, NY, TE, SK, NI, YN, GT, TL, TM, YT, TR, NS, IT, NA, KT, GN, HT, DT, NE, NH, YI, HN, NQ, FS, NM, NL, SM, NC, VT, KQ, TQ, DQ, IQ, SQ, PS, KE, AQ, YQ, TP, EQ, VQ, LQ, MQ, KS, IE, RQ, IK, AK, PK, NR, HQ, QQ, or CQ. 14. The AAV particle of any one of embodiments 1-13, wherein [N1] comprises TNT, TNK, TNN, SNN, SNK, SNT, TTN, TND, TTI, RNT, TTK, TTS, TTD, TNP, TTT, TGK, TTA, TVK, TNY, STK, TTE, TSK, TNI, TYN, STI, TTV, TGT, TTL, TTM, ANN, SNI, TKN, TYT, TTR, TNS, TST, TIT, INT, TNA, TKT, STN, ANT, RNN, TGN, TSN, THT, TDT, TNE, CNT, INN, NNN, KNN, LNN, TIN, TNH, STT, SNS, STS, TYI, SGT, THN, TNQ, RNI, TFS, RNS, TNM, RTT, KNT, TNL, TSM, SYT, TNC, SST, TVT, QNT, NTK, NNQ, NKQ, NNE, NTQ, NDQ, TIQ, TKQ, TSQ, TDQ, NPS, NKE, TTQ, GKQ, TAQ, VKQ, NYQ, NTP, TEQ, SKQ, NIQ, YNQ, TVQ, GTQ, NTR, TLQ, TMQ, KNQ, YTQ, NKS, NTE, NIE, TRQ, NSQ, YTK, NIK, NNK, NSK, ITK, NAK, KTK, GNQ, SNQ, HTK, DTK, NEQ, NPK, YTE, NNR, INQ, NHQ, YIQ, HNQ, ITQ, STQ, NSN, NQQ, NNP, ITE, NTN, FSQ, NNH, NMQ, NTS, NLQ, SMQ, NCQ, or VTQ 15. The AAV particle of any one of embodiments 1-14, wherein [N1] is or comprises TNTQ (SEQ ID NO: 4688), TNTK (SEQ ID NO: 4689), TNNQ (SEQ ID NO: 4690), SNNQ (SEQ ID NO: 4691), TNKQ (SEQ ID NO: 4692), TNNE (SEQ ID NO: 4693), SNKQ (SEQ ID NO: 4694), SNTQ (SEQ ID NO: 4695), TTNQ (SEQ ID NO: 4696), TNDQ (SEQ ID NO: 4697), TTIQ (SEQ ID NO: 4698), RNTQ (SEQ ID NO: 4699), TTKQ (SEQ ID NO: 4700), TTSQ (SEQ ID NO: 4701), TTDQ (SEQ ID NO: 4702), TNPS (SEQ ID NO: 4703), TNKE (SEQ ID NO: 4704), TTTQ (SEQ ID NO: 4705), TGKQ (SEQ ID NO: 4706), TTAQ (SEQ ID NO: 4707), TVKQ (SEQ ID NO: 4708), TNYQ (SEQ ID NO: 4709), TNTP (SEQ ID NO: 4710), STKQ (SEQ ID NO: 4711), TTEQ (SEQ ID NO: 4712), TSKQ (SEQ ID NO: 4713), TNIQ (SEQ ID NO: 4714), TYNQ (SEQ ID NO: 4715), STIQ (SEQ ID NO: 4716), TTVQ (SEQ ID NO: 4717), TGTQ (SEQ ID NO: 4718), TNTR (SEQ ID NO: 4719), TTLQ (SEQ ID NO: 4720), TTMQ (SEQ ID NO: 4721), ANNQ (SEQ ID NO: 4722), SNIQ (SEQ ID NO: 4723), TKNQ (SEQ ID NO: 4724), TYTQ (SEQ ID NO: 4725), TNKS (SEQ ID NO: 4726), SNTE (SEQ ID NO: 4727), TNTE (SEQ ID NO: 4728), TNIE (SEQ ID NO: 4729), TTRQ (SEQ ID NO: 4730), TNSQ (SEQ ID NO: 4731), TYTK (SEQ ID NO: 4732), TTTK (SEQ ID NO: 4733), TNIK (SEQ ID NO: 4734), SNTK (SEQ ID NO: 4735), TNNK (SEQ ID NO: 4736), TNSK (SEQ ID NO: 4737), TSTK (SEQ ID NO: 4738), TITK (SEQ ID NO: 4739), INTK (SEQ ID NO: 4740), TNAK (SEQ ID NO: 4741), TKTK (SEQ ID NO: 4742), STNQ (SEQ ID NO: 4743), ANTK (SEQ ID NO: 4744), RNNQ (SEQ ID NO: 4745), TGNQ (SEQ ID NO: 4746), TSNQ (SEQ ID NO: 4747), THTK (SEQ ID NO: 4748), TDTK (SEQ ID NO: 4749), TNEQ (SEQ ID NO: 4750), CNTQ (SEQ ID NO: 4751), TNPK (SEQ ID NO: 4752), INNQ (SEQ ID NO: 4753), TYTE (SEQ ID NO: 4754), NNNQ (SEQ ID NO: 4755), KNNQ (SEQ ID NO: 4756), TNNR (SEQ ID NO: 4757), LNNQ (SEQ ID NO: 4758), TINQ (SEQ ID NO: 4759), TNHQ (SEQ ID NO: 4760), STTQ (SEQ ID NO: 4761), SNSQ (SEQ ID NO: 4762), STSQ (SEQ ID NO: 4763), TYIQAttorney Docket No.14640.0105-00304 (SEQ ID NO: 4764), SGTQ (SEQ ID NO: 4765), THNQ (SEQ ID NO: 4766), TITQ (SEQ ID NO: 4767), TSTQ (SEQ ID NO: 4768), TNSN (SEQ ID NO: 4769), TNQQ (SEQ ID NO: 4770), RNIQ (SEQ ID NO: 4771), TNNP (SEQ ID NO: 4772), TITE (SEQ ID NO: 4773), TNTN (SEQ ID NO: 4774), TFSQ (SEQ ID NO: 4775), RNSQ (SEQ ID NO: 4776), INTQ (SEQ ID NO: 4777), RNTE (SEQ ID NO: 4778), TNNH (SEQ ID NO: 4779), TNMQ (SEQ ID NO: 4780), RTTQ (SEQ ID NO: 4781), SNIE (SEQ ID NO: 4782), TNTS (SEQ ID NO: 4783), KNTQ (SEQ ID NO: 4784), TNLQ (SEQ ID NO: 4785), TSMQ (SEQ ID NO: 4786), SYTQ (SEQ ID NO: 4787), TNCQ (SEQ ID NO: 4788), SSTQ (SEQ ID NO: 4789), TVTQ (SEQ ID NO: 4790), or QNTQ (SEQ ID NO: 4791). 16. The AAV particle of embodiment 15, wherein [N1] is or comprises TNTQ (SEQ ID NO: 4688). 17. The AAV particle of embodiment 15, wherein [N1] is or comprises TNTK (SEQ ID NO: 4689). 18. The AAV particle of any one of embodiments 1-17, wherein [N1]-[N2] comprises: (i) TQDWHR (SEQ ID NO: 4686), TKDWHR (SEQ ID NO: 4792), NQDWHR (SEQ ID NO: 4793), KQDWHR (SEQ ID NO: 4794), NEDWHR (SEQ ID NO: 4795), DQDWHR (SEQ ID NO: 4796), IQDWHR (SEQ ID NO: 4797), SQDWHR (SEQ ID NO: 4798), PSDWHR (SEQ ID NO: 4799), KEDWHR (SEQ ID NO: 4800), AQDWHR (SEQ ID NO: 4801), YQDWHR (SEQ ID NO: 4802), TPDWHR (SEQ ID NO: 4803), EQDWHR (SEQ ID NO: 4804), VQDWHR (SEQ ID NO: 4805), TRDWHR (SEQ ID NO: 4806), LQDWHR (SEQ ID NO: 4807), MQDWHR (SEQ ID NO: 4808), KSDWHR (SEQ ID NO: 4809), TEDWHR (SEQ ID NO: 4810), IEDWHR (SEQ ID NO: 4811), RQDWHR (SEQ ID NO: 4812), IKDWHR (SEQ ID NO: 4813), NKDWHR (SEQ ID NO: 4814), SKDWHR (SEQ ID NO: 4815), AKDWHR (SEQ ID NO: 4816), PKDWHR (SEQ ID NO: 4817), NRDWHR (SEQ ID NO: 4818), HQDWHR (SEQ ID NO: 4819), SNDWHR (SEQ ID NO: 4820), QQDWHR (SEQ ID NO: 4821), NPDWHR (SEQ ID NO: 4822), TNDWHR (SEQ ID NO: 4823), NHDWHR (SEQ ID NO: 4824), TSDWHR (SEQ ID NO: 4825), or CQDWHR (SEQ ID NO: 4826); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 19. The AAV particle of any one of embodiments 1-18, wherein [N1]-[N2] comprises: (i) NTQDWHR (SEQ ID NO: 4827), NTKDWHR (SEQ ID NO: 4828), NNQDWHR (SEQ ID NO: 4829), NKQDWHR (SEQ ID NO: 4830), NNEDWHR (SEQ ID NO: 4831), TNQDWHR (SEQ ID NO: 4832), NDQDWHR (SEQ ID NO: 4833), TIQDWHR (SEQ ID NO: 4834), TKQDWHR (SEQ IDAttorney Docket No.14640.0105-00304 NO: 4835), TSQDWHR (SEQ ID NO: 4836), TDQDWHR (SEQ ID NO: 4837), NPSDWHR (SEQ ID NO: 4838), NKEDWHR (SEQ ID NO: 4839), TTQDWHR (SEQ ID NO: 4840), GKQDWHR (SEQ ID NO: 4841), TAQDWHR (SEQ ID NO: 4842), VKQDWHR (SEQ ID NO: 4843), NYQDWHR (SEQ ID NO: 4844), NTPDWHR (SEQ ID NO: 4845), TEQDWHR (SEQ ID NO: 4846), SKQDWHR (SEQ ID NO: 4847), NIQDWHR (SEQ ID NO: 4848), YNQDWHR (SEQ ID NO: 4849), TVQDWHR (SEQ ID NO: 4850), GTQDWHR (SEQ ID NO: 4851), NTRDWHR (SEQ ID NO: 4852), TLQDWHR (SEQ ID NO: 4853), TMQDWHR (SEQ ID NO: 4854), KNQDWHR (SEQ ID NO: 4855), YTQDWHR (SEQ ID NO: 4856), NKSDWHR (SEQ ID NO: 4857), NTEDWHR (SEQ ID NO: 4858), NIEDWHR (SEQ ID NO: 4859), TRQDWHR (SEQ ID NO: 4860), NSQDWHR (SEQ ID NO: 4861), YTKDWHR (SEQ ID NO: 4862), TTKDWHR (SEQ ID NO: 4863), NIKDWHR (SEQ ID NO: 4864), NNKDWHR (SEQ ID NO: 4865), NSKDWHR (SEQ ID NO: 4866), STKDWHR (SEQ ID NO: 4867), ITKDWHR (SEQ ID NO: 4868), NAKDWHR (SEQ ID NO: 4869), KTKDWHR (SEQ ID NO: 4870), GNQDWHR (SEQ ID NO: 4871), SNQDWHR (SEQ ID NO: 4872), HTKDWHR (SEQ ID NO: 4873), DTKDWHR (SEQ ID NO: 4874), NEQDWHR (SEQ ID NO: 4875), NPKDWHR (SEQ ID NO: 4876), YTEDWHR (SEQ ID NO: 4877), NNRDWHR (SEQ ID NO: 4878), INQDWHR (SEQ ID NO: 4879), NHQDWHR (SEQ ID NO: 4880), YIQDWHR (SEQ ID NO: 4881), HNQDWHR (SEQ ID NO: 4882), ITQDWHR (SEQ ID NO: 4883), STQDWHR (SEQ ID NO: 4884), NSNDWHR (SEQ ID NO: 4885), NQQDWHR (SEQ ID NO: 4886), NNPDWHR (SEQ ID NO: 4887), ITEDWHR (SEQ ID NO: 4888), NTNDWHR (SEQ ID NO: 4889), FSQDWHR (SEQ ID NO: 4890), NNHDWHR (SEQ ID NO: 4891), NMQDWHR (SEQ ID NO: 4892), NTSDWHR (SEQ ID NO: 4893), NLQDWHR (SEQ ID NO: 4894), SMQDWHR (SEQ ID NO: 4895), NCQDWHR (SEQ ID NO: 4896), or VTQDWHR (SEQ ID NO: 4897); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5 or 6 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 20. The AAV particle of any one of embodiments 1-19, wherein [N1]-[N2] is or comprises: (i) TNTQDWHR (SEQ ID NO: 4898), TNTKDWHR (SEQ ID NO: 4899), TNNQDWHR (SEQ ID NO: 4900), SNNQDWHR (SEQ ID NO: 4901), TNKQDWHR (SEQ ID NO: 4902), TNNEDWHR (SEQ ID NO: 4903), SNKQDWHR (SEQ ID NO: 4904), SNTQDWHR (SEQ ID NO: 4905), TTNQDWHR (SEQ ID NO: 4906), TNDQDWHR (SEQ ID NO: 4907), TTIQDWHR (SEQ ID NO: 4908), RNTQDWHR (SEQ ID NO: 4909), TTKQDWHR (SEQ ID NO: 4910), TTSQDWHR (SEQ ID NO: 4911), TTDQDWHR (SEQ ID NO: 4912), TNPSDWHR (SEQ ID NO: 4913), TNKEDWHR (SEQ ID NO: 4914), TTTQDWHR (SEQ ID NO: 4915), TGKQDWHR (SEQ ID NO: 4916), TTAQDWHR (SEQ ID NO: 4917), TVKQDWHR (SEQ ID NO: 4918), TNYQDWHR (SEQ ID NO: 4919),Attorney Docket No.14640.0105-00304 TNTPDWHR (SEQ ID NO: 4920), STKQDWHR (SEQ ID NO: 4921), TTEQDWHR (SEQ ID NO: 4922), TSKQDWHR (SEQ ID NO: 4923), TNIQDWHR (SEQ ID NO: 4924), TYNQDWHR (SEQ ID NO: 4925), STIQDWHR (SEQ ID NO: 4926), TTVQDWHR (SEQ ID NO: 4927), TGTQDWHR (SEQ ID NO: 4928), TNTRDWHR (SEQ ID NO: 4929), TTLQDWHR (SEQ ID NO: 4930), TTMQDWHR (SEQ ID NO: 4931), ANNQDWHR (SEQ ID NO: 4932), SNIQDWHR (SEQ ID NO: 4933), TKNQDWHR (SEQ ID NO: 4934), TYTQDWHR (SEQ ID NO: 4935), TNKSDWHR (SEQ ID NO: 4936), SNTEDWHR (SEQ ID NO: 4937), TNTEDWHR (SEQ ID NO: 4938), TNIEDWHR (SEQ ID NO: 4939), TTRQDWHR (SEQ ID NO: 4940), TNSQDWHR (SEQ ID NO: 4941), TYTKDWHR (SEQ ID NO: 4942), TTTKDWHR (SEQ ID NO: 4943), TNIKDWHR (SEQ ID NO: 4944), SNTKDWHR (SEQ ID NO: 4945), TNNKDWHR (SEQ ID NO: 4946), TNSKDWHR (SEQ ID NO: 4947), TSTKDWHR (SEQ ID NO: 4948), TITKDWHR (SEQ ID NO: 4949), INTKDWHR (SEQ ID NO: 4950), TNAKDWHR (SEQ ID NO: 4951), TKTKDWHR (SEQ ID NO: 4952), STNQDWHR (SEQ ID NO: 4953), ANTKDWHR (SEQ ID NO: 4954), RNNQDWHR (SEQ ID NO: 4955), TGNQDWHR (SEQ ID NO: 4956), TSNQDWHR (SEQ ID NO: 4957), THTKDWHR (SEQ ID NO: 4958), TDTKDWHR (SEQ ID NO: 4959), TNEQDWHR (SEQ ID NO: 4960), CNTQDWHR (SEQ ID NO: 4961), TNPKDWHR (SEQ ID NO: 4962), INNQDWHR (SEQ ID NO: 4963), TYTEDWHR (SEQ ID NO: 4964), NNNQDWHR (SEQ ID NO: 4965), KNNQDWHR (SEQ ID NO: 4966), TNNRDWHR (SEQ ID NO: 4967), LNNQDWHR (SEQ ID NO: 4968), TINQDWHR (SEQ ID NO: 4969), TNHQDWHR (SEQ ID NO: 4970), STTQDWHR (SEQ ID NO: 4971), SNSQDWHR (SEQ ID NO: 4972), STSQDWHR (SEQ ID NO: 4973), TYIQDWHR (SEQ ID NO: 4974), SGTQDWHR (SEQ ID NO: 4975), THNQDWHR (SEQ ID NO: 4976), TITQDWHR (SEQ ID NO: 4977), TSTQDWHR (SEQ ID NO: 4978), TNSNDWHR (SEQ ID NO: 4979), TNQQDWHR (SEQ ID NO: 4980), RNIQDWHR (SEQ ID NO: 4981), TNNPDWHR (SEQ ID NO: 4982), TITEDWHR (SEQ ID NO: 4983), TNTNDWHR (SEQ ID NO: 4984), TFSQDWHR (SEQ ID NO: 4985), RNSQDWHR (SEQ ID NO: 4986), INTQDWHR (SEQ ID NO: 4987), RNTEDWHR (SEQ ID NO: 4988), TNNHDWHR (SEQ ID NO: 4989), TNMQDWHR (SEQ ID NO: 4990), RTTQDWHR (SEQ ID NO: 4991), SNIEDWHR (SEQ ID NO: 4992), TNTSDWHR (SEQ ID NO: 4993), KNTQDWHR (SEQ ID NO: 4994), TNLQDWHR (SEQ ID NO: 4995), TSMQDWHR (SEQ ID NO: 4996), SYTQDWHR (SEQ ID NO: 4997), TNCQDWHR (SEQ ID NO: 4998), SSTQDWHR (SEQ ID NO: 4999), TVTQDWHR (SEQ ID NO: 5000), or QNTQDWHR (SEQ ID NO: 5001); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions, relative to any one of the amino acid sequences in (i).Attorney Docket No.14640.0105-00304 21. The AAV particle of embodiment 20, wherein [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). 22. The AAV particle of embodiment 20, wherein [N1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899). 23. The AAV particle of any one of embodiments 1-22, wherein the AAV capsid variant comprises one, two, or all of: an amino acid other than Q at position 590 (e.g., I, V, T, M, S, N, L, or F), an amino acid other than A at position 591 (e.g., Y, P, N, S, T, G, E, V, W, F, Q), and / or an amino acid other than Q at position 592 (e.g., G, N, K, H, R, E, L, P, or M), as numbered according to SEQ ID NO: 138 or 981. 24. The AAV particle of any one of embodiments 1-23, wherein the AAV capsid variant comprises an amino acid other than Q at position 590 (e.g., I, V, T, M, S, N, L, or F), as numbered according to SEQ ID NO: 138 or 981. 25. The AAV particle of any one of embodiments 1-24, wherein the AAV capsid variant comprises the amino acid I at position 590, as numbered according to SEQ ID NO: 138 or 981. 26. The AAV particle of any one of embodiments 1-24, wherein the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981. 27. The AAV particle of any one of embodiments 1-26, wherein the AAV capsid variant comprises the amino acid A at position 591 and / or the amino acid Q at position 592, as numbered according to SEQ ID NO: 138 or 981. 28. The AAV particle of any one of embodiments 1-27, wherein [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, or F. 29. The AAV particle of any one of embodiments 1-28, wherein X5is I or V. 30. The AAV particle of embodiment 29, wherein X5is I. 31. The AAV particle of any one of embodiments 1-30, wherein: (i) X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and / or (ii) X7is Q, G, N, K, H, R, E, L, P, or M.Attorney Docket No.14640.0105-00304 32. The AAV particle of any one of embodiments 1-31, wherein [N3] comprises IA, IY, VP, IN, VN, VY, VA, IS, IT, TA, MA, SA, IG, IE, IV, NA, LA, IP, FA, VS, VT, IW, IF, IQ, VQ, AQ, AG, YQ, PQ, AN, NQ, SG, SQ, TQ, GQ, EQ, AK, AH, AR, AE, AL, AP, TM, SM, WQ, FQ, QQ, FM, AM, or SN. 33. The AAV particle of any one of embodiments 1-32, wherein [N3] is or comprises IAQ, IAG, IYQ, VPQ, IAN, INQ, VNQ, VYQ, VAN, ISG, ISQ, VAQ, ITQ, TAQ, MAQ, SAQ, IGQ, IEQ, IVQ, NAQ, LAQ, IAK, IAH, IPQ, IAR, IAE, IAL, IAP, FAQ, VSQ, VTM, ISM, IWQ, IFQ, IQQ, VQQ, IFM, IAM, or ISN. 34. The AAV particle of any one of embodiments 1-33, wherein [N3] is or comprises IAQ. 35. The AAV particle of any one of embodiments 1-34, wherein [N2]-[N3] comprises: (i) DWHRIA (SEQ ID NO: 5002), DWHRIY (SEQ ID NO: 5003), DWHRVP (SEQ ID NO: 5004), DWHRIN (SEQ ID NO: 5005), DWHRVN (SEQ ID NO: 5006), DWHRVY (SEQ ID NO: 5007), DWHRVA (SEQ ID NO: 5008), DWHRIS (SEQ ID NO: 5009), DWHRIT (SEQ ID NO: 5010), DWHRTA (SEQ ID NO: 5011), DWHRMA (SEQ ID NO: 5012), DWHRSA (SEQ ID NO: 5013), DWHRIG (SEQ ID NO: 5014), DWHRIE (SEQ ID NO: 5015), DWHRIV (SEQ ID NO: 5016), DWHRNA (SEQ ID NO: 5017), DWHRLA (SEQ ID NO: 5018), DWHRIP (SEQ ID NO: 5019), DWHRFA (SEQ ID NO: 5020), DWHRVS (SEQ ID NO: 5021), DWHRVT (SEQ ID NO: 5022), DWHRIW (SEQ ID NO: 5023), DWHRIF (SEQ ID NO: 5024), DWHRIQ (SEQ ID NO: 5025), or DWHRVQ (SEQ ID NO: 5026); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 36. The AAV particle of any one of embodiments 1-35, wherein [N2]-[N3] is or comprises: (i) DWHRIAQ (SEQ ID NO: 5027), DWHRIAG (SEQ ID NO: 5028), DWHRIYQ (SEQ ID NO: 5029), DWHRVPQ (SEQ ID NO: 5030), DWHRIAN (SEQ ID NO: 5031), DWHRINQ (SEQ ID NO: 5032), DWHRVNQ (SEQ ID NO: 5033), DWHRVYQ (SEQ ID NO: 5034), DWHRVAN (SEQ ID NO: 5035), DWHRISG (SEQ ID NO: 5036), DWHRISQ (SEQ ID NO: 5037), DWHRVAQ (SEQ ID NO: 5038), DWHRITQ (SEQ ID NO: 5039), DWHRTAQ (SEQ ID NO: 5040), DWHRMAQ (SEQ ID NO: 5041), DWHRSAQ (SEQ ID NO: 5042), DWHRIGQ (SEQ ID NO: 5043), DWHRIEQ (SEQ ID NO: 5044), DWHRIVQ (SEQ ID NO: 5045), DWHRNAQ (SEQ ID NO: 5046), DWHRLAQ (SEQ ID NO: 5047), DWHRIAK (SEQ ID NO: 5048), DWHRIAH (SEQ ID NO: 5049), DWHRIPQ (SEQ IDAttorney Docket No.14640.0105-00304 NO: 5050), DWHRIAR (SEQ ID NO: 5051), DWHRIAE (SEQ ID NO: 5052), DWHRIAL (SEQ ID NO: 5053), DWHRIAP (SEQ ID NO: 5054), DWHRFAQ (SEQ ID NO: 5055), DWHRVSQ (SEQ ID NO: 5056), DWHRVTM (SEQ ID NO: 5057), DWHRISM (SEQ ID NO: 5058), DWHRIWQ (SEQ ID NO: 5059), DWHRIFQ (SEQ ID NO: 5060), DWHRIQQ (SEQ ID NO: 5061), DWHRVQQ (SEQ ID NO: 5062), DWHRIFM (SEQ ID NO: 5063), DWHRIAM (SEQ ID NO: 5064), or DWHRISN (SEQ ID NO: 5065); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 37. The AAV particle of any one of embodiments 1-36, wherein [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). 38. The AAV particle of any one of embodiments 1-37, wherein [N1]-[N2]-[N3] is or comprises: (i) the amino acid sequence of any one of SEQ ID NOs: 343-538; (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 39. The AAV particle of embodiment 38, wherein [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). 40. The AAV particle of embodiment 38, wherein [N1]-[N2]-[N3] is or comprises TNTKDWHRIAQ (SEQ ID NO: 344). 41. The AAV particle of any one of embodiments 1-40, wherein the AAV capsid variant comprises one, two, three, or all of: an amino acid other than T at position 593 (e.g., S, N, P, A, or I), an amino acid other than G at position 594 (e.g., N, D, R, V, A, S, or Q), an amino acid other than W at position 595 (e.g., S, C, R, L, or G), and / or an amino acid other than V at position 596 (e.g., A, S, I, C, G, D, F, L, or T), as numbered according to SEQ ID NO: 138 or 981.Attorney Docket No.14640.0105-00304 42. The AAV particle of any one of embodiments 1-40, wherein the AAV capsid variant comprises the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981. 43. The AAV particle of any one of embodiments 1-42, wherein the AAV capsid variant further comprises [N4], wherein [N4] comprises amino acids X8, X9, X10, and X11, wherein: (i) X8is T, S, N, P, A, or I; (ii) X9is G, N, D, R, V, A, S, or Q; (iii) X10is W, S, C, R, L, or G; and / or (iv) X11is V, A, S, I, C, G, D, F, L, or T. 44. The AAV particle of embodiment 43, wherein [N4] comprises TG, TN, SN, NN, SG, PG, TD, AG, IG, NG, TR, TV, TA, TS, SV, TQ, WV, WA, WS, WI, WC, WG, CV, RV, LV, GV, WD, WF, WL, WT, GW, NW, GS, DW, GC, GR, GL, GG, RW, VW, AW, SW, or QW. 45. The AAV particle of embodiment 43 or 44, wherein [N4] comprises TGW, TNW, SNW, NNW, SGW, PGW, TGS, TDW, TGC, TGR, TGL, TGG, AGW, IGW, NGW, TRW, TVW, TAW, TSW, SVW, TQW, GWV, GWA, NWS, NWV, NWI, GWS, GWI, GWC, GWG, GSV, DWV, GCV, GRV, GLV, GGV, GWD, GWF, RWV, VWV, GWL, AWV, SWV, GWT, or QWV. 46. The AAV particle of any one of embodiments 43-45, wherein [N4] is or comprises TGWV (SEQ ID NO: 5066), TGWA (SEQ ID NO: 5067), TNWS (SEQ ID NO: 5068), SNWV (SEQ ID NO: 5069), TNWV (SEQ ID NO: 5070), TNWI (SEQ ID NO: 5071), NNWV (SEQ ID NO: 5072), TGWS (SEQ ID NO: 5073), TGWI (SEQ ID NO: 5074), TGWC (SEQ ID NO: 5075), TGWG (SEQ ID NO: 5076), SGWV (SEQ ID NO: 5077), PGWV (SEQ ID NO: 5078), TGSV (SEQ ID NO: 5079), TDWV (SEQ ID NO: 5080), TGCV (SEQ ID NO: 5081), TGRV (SEQ ID NO: 5082), TGLV (SEQ ID NO: 5083), TGGV (SEQ ID NO: 5084), AGWV (SEQ ID NO: 5085), IGWV (SEQ ID NO: 5086), TGWD (SEQ ID NO: 5087), NGWV (SEQ ID NO: 5088), TGWF (SEQ ID NO: 5089), TRWV (SEQ ID NO: 5090), TVWV (SEQ ID NO: 5091), TGWL (SEQ ID NO: 5092), TAWV (SEQ ID NO: 5093), TSWV (SEQ ID NO: 5094), TGWT (SEQ ID NO: 5095), SVWV (SEQ ID NO: 5096), TQWV (SEQ ID NO: 5097), or PGWG (SEQ ID NO: 5098). 47. The AAV particle of any one of embodiments 43-46, wherein [N4] is or comprises TGWV (SEQ ID NO: 5066). 48. The AAV particle of any one of embodiments 43-47, wherein [N1]-[N2]-[N3]-[N4] is or comprises:Attorney Docket No.14640.0105-00304 (i) the amino acid sequence of any one of SEQ ID NOs: 201-245, 247-250, 253-255, 257-265, 268-274, 276-286, 288, 290-297, 299-303, 305-309, 311, 313-319, 323-328, 330-337, 339-342, 539-542, 544, 546, 547, 549-557, 559-589, 592, 593, 595, 596, 598, 599, 601-608, 610-614, 616-622, 625, 628, 630, 631, 633, 636, 638, 639-646, 649, 651-657, 667, 669, 670, 672, 673, 679-683, 685-690, 692, 693, 695, 697, 699-701, 703-705, 708-710, 712-717, 719-723, 728-731, 733-738, 740, and 742; (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 49. The AAV particle of embodiment 48, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201). 50. The AAV particle of any one of embodiment 48, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTKDWHRIAQTGWV (SEQ ID NO: 202). 51. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising an amino acid sequence having the following formula: [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein: (i) [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, P, or a conservative substitution thereof; and / or (ii) [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, or a conservative substitution thereof. 52. The AAV particle of embodiment 51, wherein the AAV capsid variant comprises the amino acid Q at position 585, as numbered according to SEQ ID NO: 138 or 981. 53. The AAV particle of embodiment 51 or 52, wherein the AAV capsid variant comprises one, two, three, or all of: (i) an amino acid other than T at position 582 (e.g., S); (ii) an amino acid other than N at position 583 (e.g., T, G, S, I, or V); (iii) an amino acid other than H at position 584 (e.g., N, I, S, A, V, or L); and / or (iv) an amino acid other than Q at position 585 (e.g., P),Attorney Docket No.14640.0105-00304 wherein the amino acids are as numbered according to SEQ ID NO: 138 or 981. 54. The AAV particle of any one of embodiments 51-53, wherein [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q or P. 55. The AAV particle of any one of embodiments 51-54, wherein X4is Q. 56. The AAV particle of any one of embodiments 51-55, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), as numbered according to SEQ ID NO: 138 or 981. 57. The AAV particle of any one of embodiments 51-56, wherein the AAV capsid variant comprises the amino acid T at position 584, as numbered according to SEQ ID NO: 138 or 981. 58. The AAV particle of any one of embodiments 51-57, wherein: (i) X1is T or S; (ii) X2is N, T, G, S, I, or V; and / or (iii) X3is T, N, I, S, A, V, or L. 59. The AAV particle of any one of embodiments 51-58, wherein [N1] comprises TN, TT, TG, ST, TS, TI, TV, TQ, NQ, IQ, SQ, AQ, VQ, TP, LQ, NT, TA, NI, GT, IT, NN, TL, NS, or VT. 60. The AAV particle of any one of embodiments 51-59, wherein [N1] comprises TNT, TTN, TTI, TTS, TTT, TTA, TNI, TTV, TGT, STT, TST, TIT, TNN, TTL, TNS, TVT, NTQ, TNQ, TIQ, TSQ, TTQ, TAQ, NIQ, TVQ, GTQ, STQ, ITQ, NTP, NNQ, TLQ, NSQ, or VTQ. 61. The AAV particle of any one of embodiments 51-60, wherein [N1] is or comprises TNTQ (SEQ ID NO: 4688), TTNQ (SEQ ID NO: 4696), TTIQ (SEQ ID NO: 4698), TTSQ (SEQ ID NO: 4701), TTTQ (SEQ ID NO: 4705), TTAQ (SEQ ID NO: 4707), TNIQ (SEQ ID NO: 4714), TTVQ (SEQ ID NO: 4717), TGTQ (SEQ ID NO: 4718), STTQ (SEQ ID NO: 4761), TSTQ (SEQ ID NO: 4768), TITQ (SEQ ID NO: 4767), TNTP (SEQ ID NO: 4710), TNNQ (SEQ ID NO: 4690), TTLQ (SEQ ID NO: 4720), TNSQ (SEQ ID NO: 4731), or TVTQ (SEQ ID NO: 4790). 62. The AAV particle of embodiment 61, wherein [N1] is or comprises TNTQ (SEQ ID NO: 4688). 63. The AAV particle of any one of embodiments 51-62, wherein [N1]-[N2] comprises:Attorney Docket No.14640.0105-00304 (i) TQDWHR (SEQ ID NO: 4686), NQDWHR (SEQ ID NO: 4793), IQDWHR (SEQ ID NO: 4797), SQDWHR (SEQ ID NO: 4798), AQDWHR (SEQ ID NO: 4801), VQDWHR (SEQ ID NO: 4805), TPDWHR (SEQ ID NO: 4803), or LQDWHR (SEQ ID NO: 4807); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 64. The AAV particle of any one of embodiments 51-63, wherein [N1]-[N2] comprises: (i) NTQDWHR (SEQ ID NO: 4827), TNQDWHR (SEQ ID NO: 4832), TIQDWHR (SEQ ID NO: 4834), TSQDWHR (SEQ ID NO: 4836), TTQDWHR (SEQ ID NO: 4840), TAQDWHR (SEQ ID NO: 4842), NIQDWHR (SEQ ID NO: 4848), TVQDWHR (SEQ ID NO: 4850), GTQDWHR (SEQ ID NO: 4851), STQDWHR (SEQ ID NO: 4884), ITQDWHR (SEQ ID NO: 4883), NTPDWHR (SEQ ID NO: 4845), NNQDWHR (SEQ ID NO: 4829), TLQDWHR (SEQ ID NO: 4853), NSQDWHR (SEQ ID NO: 4861), VTQDWHR (SEQ ID NO: 4897); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5 or 6 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 65. The AAV particle of any one of embodiments 51-64, wherein [N1]-[N2] is or comprises: (i) TNTQDWHR (SEQ ID NO: 4898), TTNQDWHR (SEQ ID NO: 4906), TTIQDWHR (SEQ ID NO: 4908), TTSQDWHR (SEQ ID NO: 4911), TTTQDWHR (SEQ ID NO: 4915), TTAQDWHR (SEQ ID NO: 4917), TNIQDWHR (SEQ ID NO: 4924), TTVQDWHR (SEQ ID NO: 4927), TGTQDWHR (SEQ ID NO: 4928), STTQDWHR (SEQ ID NO: 4971), TSTQDWHR (SEQ ID NO: 4978), TITQDWHR (SEQ ID NO: 4977), TNTPDWHR (SEQ ID NO: 4920), TNNQDWHR (SEQ ID NO: 4900), TTLQDWHR (SEQ ID NO: 4930), TNSQDWHR (SEQ ID NO: 4941), TVTQDWHR (SEQ ID NO: 5000); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 56, or 7 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); orAttorney Docket No.14640.0105-00304 (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 66. The AAV particle of embodiment 65, wherein [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). 67. The AAV particle of any one of embodiments 51-66, wherein the AAV capsid variant comprises one, two, or all of: an amino acid other than Q at position 590 (e.g., I or V), an amino acid other than A at position 591 (e.g., P, S, Y, or N), and / or an amino acid other than Q at position 592 (e.g., G or N), as numbered according to SEQ ID NO: 138 or 981. 68. The AAV particle of any one of embodiments 51-67, wherein the AAV capsid variant comprises an amino acid other than Q at position 590 (e.g., I or V), as numbered according to SEQ ID NO: 138 or 981. 69. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid I at position 590, as numbered according to SEQ ID NO: 138 or 981. 70. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981. 71. The AAV particle of any one of embodiments 51-70, wherein the AAV capsid variant comprises the amino acid A at position 591 and / or the amino acid Q at position 592, as numbered according to SEQ ID NO: 138 or 981. 72. The AAV particle of any one of embodiments 51-71, wherein [N3] comprises amino acids X5, X6, and X7, wherein X5is I or V. 73. The AAV particle of embodiment 72, wherein X5is I. 74. The AAV particle of any one of embodiments 51-73, wherein: (i) X6is A, P, S, Y, or N; and / or (ii) X7is Q, G, or N. 75. The AAV particle of any one of embodiments 51-74, wherein [N3] comprises IA, VP, VA, VS, IY, IN, IS, AQ, AG, PQ, SQ, AN, YQ, or NQ.Attorney Docket No.14640.0105-00304 76. The AAV particle of any one of embodiments 51-75, wherein [N3] is or comprises IAQ, IAG, VPQ, VAQ, VSQ, IAN, IYQ, INQ, or ISQ. 77. The AAV particle of any one of embodiments 51-76, wherein [N3] is or comprises IAQ. 78. The AAV particle of any one of embodiments 51-77, wherein [N2]-[N3] comprises: (i) DWHRIA (SEQ ID NO: 5002), DWHRVP (SEQ ID NO: 5004), DWHRVA (SEQ ID NO: 5008), DWHRVS (SEQ ID NO: 5021), DWHRIY (SEQ ID NO: 5003), DWHRIN (SEQ ID NO: 5005), or DWHRIS (SEQ ID NO: 5009); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 79. The AAV particle of any one of embodiments 51-78, wherein [N2]-[N3] is or comprises: (i) DWHRIAQ (SEQ ID NO: 5027), DWHRIAG (SEQ ID NO: 5028), DWHRVPQ (SEQ ID NO: 5030), DWHRVAQ (SEQ ID NO: 5038), DWHRVSQ (SEQ ID NO: 5056), DWHRIAN (SEQ ID NO: 5031), DWHRIYQ (SEQ ID NO: 5029), DWHRINQ (SEQ ID NO: 5032), or DWHRISQ (SEQ ID NO: 5037); (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 80. The AAV particle of any one of embodiments 51-79, wherein [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). 81. The AAV particle of any one of embodiments 51-80, wherein [N1]-[N2]-[N3] is or comprises: (i) the amino acid sequence of any one of SEQ ID NOs: 343, 350, 352, 355, 359, 361, 364, 367, 370, 371, 373, 374, 376, 377, 378, 381, 395, 420, 454, 457, 460, 464, 481, 482, 488, 493, 494, 516, 525, 536; (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids, thereof;Attorney Docket No.14640.0105-00304 (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 82. The AAV particle of any one of embodiments 51-81, wherein [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). 83. The AAV particle of any one of embodiments 51-82, wherein the AAV capsid variant comprises the amino acid W at position 595, as numbered according to SEQ ID NO: 138 or 981. 84. The AAV particle of any one of embodiments 51-83, wherein the AAV capsid variant comprises one, two or all of: an amino acid other than T (e.g., S or N) at position 593, an amino acid other than G (e.g., N) a position 594, and / or an amino acid other than V (e.g., A, I, or S) at position 596, as numbered according to SEQ ID NO: 138 or 981. 85. The AAV particle of any one of embodiments 51-84, wherein the AAV capsid variant comprises: (i) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981; (ii) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid A at position 596, as numbered according to SEQ ID NO: 138 or 981; (iii) the amino acid S at position 593, the amino acid N at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981; (iv) the amino acid N at position 593, the amino acid N at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981; (v) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid I at position 596, as numbered according to SEQ ID NO: 138 or 981; or (vi) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid S at position 596, as numbered according to SEQ ID NO: 138 or 981. 86. The AAV particle of any one of embodiments 51-85, wherein the AAV capsid variant comprises the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981. 87. The AAV particle of any one of embodiments 51-86, wherein the AAV capsid variant further comprises [N4], wherein [N4] comprises amino acids X8, X9, X10, and X11, wherein X10is W.Attorney Docket No.14640.0105-00304 88. The AAV particle of embodiment 87, wherein: (i) X8is T, S, or N; (ii) X9is G or N; and / or (iv) X11is V, A, I, or S. 89. The AAV particle of embodiment 87 or 88, wherein [N4] comprises TG, SN, NN, WV, WA, WI, WS, GW, or NW. 90. The AAV particle of any one of embodiments 87-89, wherein [N4] comprises TGW, SNW, NNW, GWV, GWA, NWV, GWI, or GWS. 91. The AAV particle of any one of embodiments 87-90, wherein [N4] is or comprises TGWV (SEQ ID NO: 5066), TGWA (SEQ ID NO: 5067), SNWV (SEQ ID NO: 5069), NNWV (SEQ ID NO: 5072), TGWI (SEQ ID NO: 5074), or TGWS (SEQ ID NO: 5073). 92. The AAV particle of any one of embodiments 87-91, wherein [N4] is or comprises TGWV (SEQ ID NO: 5066). 93. The AAV particle of any one of embodiments 87-92, wherein [N1]-[N2]-[N3]-[N4] is or comprises: (i) the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336; (ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i). 94. The AAV particle of any one of embodiments 87-93, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201). 95. The AAV particle of any one of embodiments 1-94, wherein [N1]-[N2]-[N3] is present in loop VIII, optionally wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 138 or 981.Attorney Docket No.14640.0105-00304 96. The AAV particle of any one of embodiments 43-50 and 87-95, wherein [N4] is present in loop VIII, optionally wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 138 or 981. 97. The AAV particle of any one of embodiments 1-96, wherein [N1] replaces amino acids 582-585 (e.g., T582, N583, H584, Q585) of SEQ ID NO: 138. 98. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, and 85-97, wherein [N1] corresponds to amino acids 582-585 (e.g., T582, N583, T584, Q585) of SEQ ID NO: 981. 99. The AAV particle of any one of embodiments 1-98, wherein [N1] is present at amino acids 582-585, as numbered according to SEQ ID NO: 138 or 981. 100. The AAV particle of any one of embodiments 1-99, wherein [N1] corresponds to positions 582-585 (e.g., T582, N583, T584, Q585) of SEQ ID NO: 981. 101. The AAV particle of any one of embodiments 1-100, wherein [N2] replaces amino acids 586-589 (e.g., S586, A587, Q588, A589) of SEQ ID NO: 138. 102. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101, wherein [N2] corresponds to amino acids 586-589 (e.g., D586, W587, H588, R589) of SEQ ID NO: 981. 103. The AAV particle of any one of embodiments 1-102, wherein [N2] is present at amino acids 586- 589, as numbered according to SEQ ID NO: 138 or 981. 104. The AAV particle of any one of embodiments 1-103, wherein [N1]-[N2] replaces amino acids 582- 589 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589) of SEQ ID NO: 138. 105. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-104, wherein [N1]-[N2] corresponds to amino acids 582-589 (e.g., T582, N583, T584, Q585, D586, W587, H588, R589) of SEQ ID NO: 981. 106. The AAV particle of any one of embodiments 1-105, wherein [N1]-[N2] is present at amino acids 582-589, as numbered according to SEQ ID NO: 138 or 981.Attorney Docket No.14640.0105-00304 107. The AAV particle of any one of embodiments 1-106, wherein [N3] replaces amino acids 590-592 (e.g., Q590, A591, and Q592) of SEQ ID NO: 138. 108. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-107, wherein [N3] corresponds to amino acids 590-592 (e.g., I590, A591, and Q592) of SEQ ID NO: 981. 109. The AAV particle of any one of embodiments 1-108, wherein [N3] is present at amino acids 590- 592, as numbered according to SEQ ID NO: 138 or 981. 110. The AAV particle of any one of embodiments 1-109, wherein [N2]-[N3] replaces amino acids 586- 592 (e.g., S586, A587, Q588, A589, Q590, A591, and Q592) of SEQ ID NO: 138. 111. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-110, wherein [N2]-[N3] corresponds to amino acids 586-592 (e.g., D586, W587, H588, R589, I590, A591, and Q592) of SEQ ID NO: 981. 112. The AAV particle of any one of embodiments 1-111, wherein [N2]-[N3] is present at amino acids 586-592, as numbered according to SEQ ID NO: 138 or 981. 113. The AAV particle of any one of embodiments 1-112, wherein [N1]-[N2]-[N3] replaces amino acids 582-592 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592) of SEQ ID NO: 138. 114. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-113, wherein [N1]-[N2]-[N3] corresponds to amino acids 582-592 (e.g., T582, N583, T584, Q585, D586, W587, H588, R589, I590, A591, Q592) of SEQ ID NO: 981. 115. The AAV particle of any one of embodiments 1-114, wherein [N1]-[N2]-[N3] is present at amino acids 582-592, as numbered according to SEQ ID NO: 138 or 981. 116. The AAV particle of any one of embodiments 43-50 and 87-115, wherein [N4] replaces amino acids 593-596 (e.g., T593, G594, W595, and V596) of SEQ ID NO: 138. 117. The AAV particle of any one of embodiments 43-49 and 87-115, wherein [N4] corresponds to amino acids 593-596 (e.g., T593, G594, W595, and V596) of SEQ ID NO: 138 or 981.Attorney Docket No.14640.0105-00304 118. The AAV particle of any one of embodiments 43-50 and 87-117, wherein [N4] is present at amino acids 593-596, as numbered according to SEQ ID NO: 138 or 981. 119. The AAV particle of any one of embodiments 43-50 and 87-118, wherein [N2]-[N3]-[N4] replaces amino acids 586-596 (e.g., S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596) of SEQ ID NO: 138. 120. The AAV particle of any one of embodiments 43-49 and 87-119, wherein [N2]-[N3]-[N4] corresponds to amino acids 586-596 (e.g., D586, W587, H588, R589, I590, A591, Q592, T593, G594, W595, and V596) of SEQ ID NO: 981. 121. The AAV particle of any one of embodiments 43-50 and 87-120, wherein [N2]-[N3]-[N4] is present at amino acids 586-596, as numbered according to SEQ ID NO: 138 or 981. 122. The AAV particle of any one of embodiments 43-50 and 87-121, wherein [N1]-[N2]-[N3]-[N4] replaces amino acids 582-596 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596), as numbered according to SEQ ID NO: 138. 123. The AAV particle of any one of embodiments 43-49 and 87-122, wherein [N1]-[N2]-[N3]-[N4] corresponds to amino acids 582-596 (e.g., T582, N583, T584, Q585, D586, W587, H588, R589, I590, A591, Q592, T593, G594, W595, and V596) of SEQ ID NO: 981. 124. The AAV particle of any one of embodiments 43-50 and 87-123, wherein [N1]-[N2]-[N3]-[N4] is present at amino acids 582-596, as numbered according to SEQ ID NO: 138 or 981. 125. The AAV particle of any one of embodiments 1-124, wherein [N2] is present immediately subsequent to [N1]. 126. The AAV particle of any one of embodiments 1-125, wherein [N3] is present immediately subsequent to [N2]. 127. The AAV particle of any one of embodiments 43-50 and 87-126, wherein [N4] is present immediately subsequent to [N3]. 128. The AAV particle of any one of embodiments 1-127, wherein the AAV capsid variant comprises, from N-terminus to C-terminus, [N1]-[N2]-[N3].Attorney Docket No.14640.0105-00304 129. The AAV particle of any one of embodiments 43-50 and 87-128, wherein the AAV capsid variant comprises, from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4]. 130. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising: (a) the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25; (b) an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25; or (c) an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25; or (d) an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25. 131. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising: (a) the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336; (b) an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336; (c) an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336; or (d) an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.Attorney Docket No.14640.0105-00304 132. The AAV particle of embodiment 130 or 131, wherein the AAV capsid variant does not comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive amino acids from TNHQSAQAQ (SEQ ID NO: 5100), optionally wherein the TNHQSAQAQ (SEQ ID NO: 5100) corresponds to amino acids 582-592 of SEQ ID NO: 138. 133. The AAV particle of any one of embodiments 130-132, wherein the AAV capsid variant does not comprise: TNH, TNHQ (SEQ ID NO: 4760), TNHQS (SEQ ID NO: 5101), TNHQSA (SEQ ID NO: 5102), TNHQSAQ (SEQ ID NO: 5103), TNHQSAQA (SEQ ID NO: 5104), TNHQSAQAQ (SEQ ID NO: 5100), NHQ, NHQS (SEQ ID NO: 5105), NHQSA (SEQ ID NO: 5106), NHQSAQ (SEQ ID NO: 5107), NHQSAQA (SEQ ID NO: 5108), NHQSAQAQ (SEQ ID NO: 5109), HQS, HQSA (SEQ ID NO: 5110), HQSAQ (SEQ ID NO: 5111), HQSAQA (SEQ ID NO: 5112), HQSAQAQ (SEQ ID NO: 5113), QSA, QSAQ (SEQ ID NO: 5114), QSAQA (SEQ ID NO: 5115), QSAQAQ (SEQ ID NO: 5116), SAQA (SEQ ID NO: 5117), or SAQAQ (SEQ ID NO: 5118). 134. The AAV particle of any one of embodiments 130-124, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. 135. The AAV particle of any one of embodiments 130-134, wherein the at least 3 consecutive amino acids comprise TQD. 136. The AAV particle of any one of embodiments 130-135, wherein the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684). 137. The AAV particle of any one of embodiments 130-136, wherein the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685). 138. The AAV particle of any one of embodiments 130-137, wherein the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686). 139. The AAV particle of any one of embodiments 130-138, wherein the at least 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941). 140. The AAV particle of any one of embodiments 130-134, wherein the at least 3 consecutive amino acids comprise TNT.Attorney Docket No.14640.0105-00304 141. The AAV particle of any one of embodiments 130-134 and 140, wherein the at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688). 142. The AAV particle of any one of embodiments 130-134, 140, and 141, wherein the at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119). 143. The AAV particle of any one of embodiments 130-134 and 140-142, wherein the at least 6 consecutive amino acids comprise TNTQDW (SEQ ID NO: 5120). 144. The AAV particle of any one of embodiments 130-134 and 140-143, wherein the at least 7 consecutive amino acids comprise TNTQDWH (SEQ ID NO: 5121). 145. The AAV particle of any one of embodiments 130-134 and 140-144, wherein the at least 8 consecutive amino acids comprise TNTQDWHR (SEQ ID NO: 4898). 146. The AAV particle of any one of embodiments 130-134 and 140-145, wherein the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746). 147. The AAV particle of any one of embodiments 130-146, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. 148. The AAV particle of any one of embodiments 130-147, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). 149. The AAV particle of any one of embodiments 130-147, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746). 150. The AAV particle of any one of embodiments 130-149, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744).Attorney Docket No.14640.0105-00304 151. The AAV particle of any one of embodiments 130-150, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. 152. The AAV particle of any one of embodiments 130-149 and 151, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). 153. The AAV particle of any one of embodiments 130-149, 151, and 152, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746). 154. The AAV particle of any one of embodiments 130-153, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744). 155. The AAV particle of any one of embodiments 1-154, wherein the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. 156. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-155, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence replaces amino acids 584-590 of SEQ ID NO: 138. 157. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-156, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence corresponds to amino acids 584-590 of SEQ ID NO: 981. 158. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, 101-149, 151-153, and 155-157, wherein the AAV capsid variant comprises an amino acid sequence encoded by: (i) the nucleotide sequence of SEQ ID NO: 942;Attorney Docket No.14640.0105-00304 (ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942; or (iii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 942. 159. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-158, wherein the AAV capsid variant comprises an amino acid sequence encoded by: (i) the nucleotide sequence of SEQ ID NO: 747; (ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 747; or (iii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 747. 160. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-149, 151-153, and 155-158, wherein the nucleotide sequence encoding the AAV capsid variant comprises: (i) the nucleotide sequence of SEQ ID NO: 942; (ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942; or (iii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 942. 161. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-160, wherein the nucleotide sequence encoding the AAV capsid variant comprises: (i) the nucleotide sequence of SEQ ID NO: 747; (ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 747; or (iii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 747.Attorney Docket No.14640.0105-00304 162. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 is present in loop VIII, optionally wherein loop VIII comprises amino acids 580-599, as numbered according to SEQ ID NO: 138 or 981. 163. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 replaces amino acids 584, 585, 586, 587, 588, 589, and / or 590 (e.g., H584, Q585, S586, A587, Q588, A589, and / or Q590), as numbered according to the amino acid sequence of SEQ ID NO: 138. 164. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 is present at amino acids 584, 585, 586, 587, 588, 589, and / or 590, as numbered according to the amino acid sequence of SEQ ID NO: 981. 165. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 corresponds to amino acids 584-590 (e.g., T584, Q585, D586, W587, H588, R589, and / or I590), numbered according to the amino acid sequence of SEQ ID NO: 981. 166. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 replaces amino acids 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, and / or Q590) of SEQ ID NO: 138. 167. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 is present at amino acids 582, 583, 584, 585, 586, 587, 588, 589, and / or 590, as numbered according to the amino acid sequence of SEQ ID NO: 981. 168. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 replaces amino acids 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, and / or 596 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and / or V5965) of SEQ ID NO: 138. 169. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 is present at amino acids 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, and / or 596, as numbered according to the amino acid sequence of SEQ ID NO: 981.Attorney Docket No.14640.0105-00304 170. The AAV particle of any one of embodiments 1-169, wherein the AAV capsid variant comprises one, two, three, four, five, or all of: (i) an amino acid other than H at position 584 (e.g., T); (ii) an amino acid other than S at position 586 (e.g., D); (iii) an amino acid other than A at position 587 (e.g., W); (iv) an amino acid other than Q at position 588 (e.g., H); (v) an amino acid other than A at position 589 (e.g., R); and / or (vi) an amino acid other than Q at position 590 (e.g., I); wherein the amino acids in (i)-(vi) are numbered according to SEQ ID NO: 138. 171. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising one, two, three, four, five, or all of: (i) an amino acid other than H at position 584 (e.g., T); (ii) an amino acid other than S at position 586 (e.g., D); (iii) an amino acid other than A at position 587 (e.g., W); (iv) an amino acid other than Q at position 588 (e.g., H); (v) an amino acid other than A at position 589 (e.g., R); and / or (vi) an amino acid other than Q at position 590 (e.g., I); wherein the amino acids in (i)-(vi) are numbered according to SEQ ID NO: 138. 172. The AAV particle of any one of embodiments 1-171, wherein the AAV capsid variant comprises one, two, three, four, five, or all of: T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and / or I at position 590, as numbered according to SEQ ID NO: 981. 173. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising one, two, three, four, five, or all of: T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and / or I at position 590, as numbered according to SEQ ID NO: 981. 174. The AAV particle of any one of embodiments 1-173, wherein the AAV capsid variant comprises one, two, three, four, five, or all of the substitutions H584T, S586D, A587W, Q588H, A589R, and / or Q590I, as numbered according to SEQ ID NO: 138. 175. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9Attorney Docket No.14640.0105-00304 capsid variant) comprising one, two, three, four, five or all of the substitutions H584T, S586D, A587W, Q588H, A589R, and / or Q590I, as numbered according to SEQ ID NO: 138. 176. The AAV particle of any one of embodiments 1-175, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), an amino acid other than S at position 586 (e.g., D), an amino acid other than A at position 587 (e.g., W), an amino acid other than Q at position 588 (e.g., H), an amino acid other than A at position 589 (e.g., R), and an amino acid other than Q at position 590 (e.g., I), as numbered according to SEQ ID NO: 138. 177. The AAV particle of any one of embodiments 1-176, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981. 178. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-98, and 100-164, wherein the AAV capsid variant comprises an amino acid at each of positions 584, 586, 587, 588, 589, and 590 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138. 179. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-178, wherein the AAV capsid variant comprises an amino acid at each of positions 582, 583, 584, 586, 587, 588, and 589 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138. 180. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-179, wherein the AAV capsid variant comprises an amino acid at each of positions 582, 583, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, and 596 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138. 181. The AAV particle of any one of embodiments 1-181, wherein the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, as numbered according to SEQ ID NO: 138. 182. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981.Attorney Docket No.14640.0105-00304 183. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, as numbered according to SEQ ID NO: 138. 184. The AAV particle of any one of embodiments 1-183, wherein the AAV capsid variant further comprises an amino acid other than A position 581, as numbered according to SEQ ID NO: 138 or 981. 185. The AAV particle of any one of embodiments 1-184, wherein the AAV capsid variant further comprises a T at position 581 or a V at position 581, as numbered according to SEQ ID NO: 138 or 981. 186. The AAV particle of any one of embodiments 1-185, wherein the AAV capsid variant comprises the substitutions A581T or A581V, as numbered according to SEQ ID NO: 138 or 981. 187. The AAV particle of any one of embodiments 1, 3-7, 9-15, 17-20, 22-38, 40-48, 50, 130-134, 147, 151, 155, 162, 163, 166-177, and 181-186, wherein the AAV capsid variant comprises an amino acid other than Q at position 585, as numbered according to SEQ ID NO: 138. 188. The AAV particle of any one of embodiments 1, 3-7, 9-15, 17-20, 22-38, 40-48, 50, 130-134, 147, 151, 155, 162, 163, 166-177, and 181-187, wherein the AAV capsid variant comprises the amino acid K at position 585, as numbered according to SEQ ID NO: 138. 189. The AAV particle of any one of embodiments 1-189, wherein the AAV capsid variant further comprises: (i) a modification in loop I, II, IV, and / or VI; and / or (ii) a substitution at position K449, e.g., a K449R substitution, as numbered according to SEQ ID NO: 138 or 981. 190. The AAV particle of any one of embodiments 1-189, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications, relative to the amino acid sequence of SEQ ID NO: 138. 191. The AAV particle of any one of embodiments 1-190, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three, but no more than not more than 30, not more than 20, or not more than 10 substitutions relative to the amino acid sequence of SEQ ID NO: 138.Attorney Docket No.14640.0105-00304 192. The AAV particle of any one of embodiments 1-191, wherein the AAV capsid variant an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 138. 193. The AAV particle of embodiment 192, wherein the AAV capsid variant an amino acid sequence with at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 138. 194. The AAV particle of any one of embodiments 1-193, wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 137. 195. The AAV particle of embodiment 194, wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 137. 196. The AAV particle of any one of embodiments 1-195, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, and a VP3 protein in an about 1:1:10 ratio. 197. The AAV particle of any one of embodiments 1-196, wherein the AAV capsid variant comprises amino acids 138-736, e.g., a VP2, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. 198. The AAV particle of any one of embodiments 1-197, wherein the AAV capsid variant comprises amino acids 203-736, e.g., a VP3, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. 199. The AAV particle of any one of embodiments 1-198, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736, e.g., a VP2, of SEQ ID NO: 138.Attorney Docket No.14640.0105-00304 200. The AAV particle of any one of embodiments 1-199, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736, e.g., a VP3, of SEQ ID NO: 138. 201. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, 101-186, and 189-200, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, or all 7 consecutive amino acids from the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein: (i) the at least 3 consecutive amino acids comprise TQD; (ii) the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684); (iii) the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685); (iv) the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686); or (v) the 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941); wherein the AAV capsid variant comprises: (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 981; (b) a VP2 protein comprising amino acids 138-736 of SEQ ID NO: 981; (c) a VP3 protein comprising amino acids 203-736 of SEQ ID NO: 981; or (d) an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to any one of the amino acid sequences in (a)-(c). 202. The AAV particle of embodiment 201, wherein the AAV capsid variant comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to any one of the amino acid sequences in (a)-(c). 203. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, 101-186, and 189-202, wherein the AAV capsid variant comprises 1, 2, or 3 substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein the AAV capsid variant comprises an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. 204. The AAV particle of any one of embodiments 1-203, wherein the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NO: 981, or an amino acid sequence with at least 90% (e.g.,Attorney Docket No.14640.0105-00304 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. 205. The AAV particle of any one of embodiments 1-204, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20 or not more than 10 modifications relative to the amino acid sequence of SEQ ID NO: 981. 206. The AAV particle of any one of embodiments, 1-205, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two or at least three, but not more than 30, not more than 20 or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 981. 207. The AAV particle of any one of embodiments 1-206, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. 208. The AAV particle of embodiment 207, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983. 209. The AAV particle of embodiment 207, wherein the AAV capsid variant is encoded by a codon- optimized nucleotide sequence. 210. An AAV particle comprising an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85- 99, 101-186, and 189, and further comprising an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 981. 211. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981. 212. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.Attorney Docket No.14640.0105-00304 213. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981, or an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981. 214. The AAV particle of embodiment 213, wherein the AAV capsid variant comprises amino acids 203- 736 of SEQ ID NO: 981. 215. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981. 216. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981. 217. The AAV particle any one of embodiments 213-216, wherein the AAV capsid variant comprises amino acids 138-736 of SEQ ID NO: 981. 218. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981. 219. An AAV particle comprising a viral genome comprising a syntaxin-binding protein-1 (STXBP1)- encoding sequence (e.g., encoding a human STXBP1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical thereto, wherein theAttorney Docket No.14640.0105-00304 AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981. 220. The AAV particle any one of embodiments 213-219, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981. 221. The AAV particle of any one of embodiments 1-220, wherein the AAV capsid variant has increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. 222. The AAV particle of any one of embodiments 1-221, wherein the AAV capsid variant transduces a brain region, e.g., sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum; optionally wherein the level of transduction is at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold greater as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR or ddPCR assay, e.g., as described in Example 2. 223. The AAV particle of any one of embodiments 1-222, which is enriched at least 10, at least 14, at least 20, at least 24, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 3. 224. The AAV particle of any one of embodiments 1-223, which is enriched at least 200, at least 300, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1. 225. The AAV particle of any one of embodiments 1-224, which is enriched in the brain of at least two or at least three species, e.g., a non-human primate and rodent (e.g., mouse), e.g., as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. 226. The AAV particle of any one of embodiments 1-225, which is enriched at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50,Attorney Docket No.14640.0105-00304 at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 190, at least 200, at least 205, or at least 210-fold in the brain of at least two or at least three species, e.g., a non-human primate and rodent (e.g., mouse), compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 4. 227. The AAV particle of embodiment 225 or 226, wherein the at least two or at least three species comprise Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, and / or mouse (e.g., outbred mice). 228. The AAV particle of any one of embodiments 1-227, which is enriched at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 17, at least 20, at least 50, at least 75, at least 100, at least 103, at least 107, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1200-fold in the brain compared to an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981, e.g., when measured by an assay as described in Example 3. 229. The AAV particle of any one of embodiments 1-228, which delivers an increased level of STXBP1 to a brain region, optionally wherein the STXBP1 level is increased by at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR, a ddPCR, or a qPCR assay (e.g., as described in Example 2). 230. The AAV particle of any one of embodiments 1-229, which delivers an increased level of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 2, at least 5, at least 7, at least 10, at least 15, at least 19, at least 20, at least 22, or at least 25-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2). 231. The AAV particle of embodiment 229 or 230, wherein the brain region is a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.Attorney Docket No.14640.0105-00304 232. The AAV particle of any one of embodiments 1-231, which is enriched at least 5, at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 2. [Embodiments 233-311 are intentionally absent.] 312. The AAV particle of any one of embodiments 1-232, wherein the viral genome is single-stranded. 313. The AAV particle of any one of embodiments 1-232, wherein the viral genome is self- complementary. 314. The AAV particle of any one of embodiments 1-232, embodiment 312, or embodiment 313, wherein the viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein (e.g., a Rep78 protein and a Rep52 protein). 315. The AAV particle of any one of embodiments 1-232, embodiment 312, or embodiment 313, wherein the AAV particle further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein (e.g., a Rep78 protein and a Rep52 protein). 316. The AAV particle of embodiment 314 or 315, wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein is encoded by at least one Rep gene. 317. The AAV particle of any one of embodiments 1-232 and 312-316, wherein the AAV particle is an isolated AAV particle. 318. The AAV particle of any one of embodiments 1-232 and 312-317, wherein the AAV particle is a recombinant AAV particle. 319. A cell, e.g., a host cell, comprising the AAV particle of any one of embodiments 1-232 and 312- 318. 320. The cell of embodiment 319, wherein the cell is a mammalian cell or an insect cell.Attorney Docket No.14640.0105-00304 321. The cell of embodiment 319 or 320, wherein the cell is a cell of a brain region or a spinal cord region, optionally wherein the cell is a cell of the sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, or cerebellum. [Embodiments 322-325 are intentionally absent] 326. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-232 and 312-318, and a pharmaceutically acceptable excipient. 327. A method of delivering STXBP1 to a cell or tissue (e.g., a CNS cell or CNS tissue), comprising administering an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318. 328. The method of embodiment 327, wherein the cell is a cell of a brain region or a spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate, cerebellar cortex, cerebral cortex, brain stem, hippocampus, or thalamus. 329. The method of embodiment 327 or 328, wherein the cell is a neuron, a sensory neuron, and / or a motor neuron. 330. The method of any one of embodiments 327-329, wherein the cell or tissue is within a subject. 331. The method of embodiment 330, wherein the subject has, has been diagnosed with having, or is at risk of having a genetic disorder. 332. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having a neurological disorder. 333. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having a neurodegenerative disorder. 334. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having a muscular disorder or a neuromuscular disorder. 335. A method of treating a subject having or diagnosed with having a genetic disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.Attorney Docket No.14640.0105-00304 336. A method of treating a subject having or diagnosed with having a neurological disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318. 337. A method of treating a subject having or diagnosed with having a muscular disorder or a neuromuscular disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318. 338. A method of treating a subject having or diagnosed with having a neurodegenerative disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318. 339. The method of any one of embodiments 327-338, wherein the genetic disorder, neurological disorder, neurodegenerative disorder, muscular disorder, or neuromuscular disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). 340. The method of any one of embodiments 335-339, where treating comprises prevention of progression of the disorder in the subject. 341. The method of any one of embodiments 330-340, wherein the subject is a human. 342. The method of any one of embodiments 330-341, wherein the AAV particle or the pharmaceutical composition is administered to the subject intravenously, via intra-cisterna magna injection (ICM), intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, intraarterially, or intramuscularly. 343. The method of any one of embodiments 330-342, wherein the AAV particle or pharmaceutical composition is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.Attorney Docket No.14640.0105-00304 344. The method of any one of embodiments 330-343, wherein the AAV particle or pharmaceutical composition is administered to the subject intravenously. 345. The method of any one of embodiments 330-344, wherein the AAV particle or pharmaceutical composition is administered to the subject via intra-cisterna magna injection (ICM). 346. The method of any one of embodiments 330-345, wherein the AAV particle or pharmaceutical composition is administered to the subject intraarterially. 347. The method of any one of embodiments 342-346, wherein administration of the AAV particle or pharmaceutical composition results in an increased expression of an STXBP1 gene, mRNA, protein, or combination thereof. 348. The method of any one of embodiments 342-346, wherein administration of the AAV particle or pharmaceutical composition results in an increased activity of an STXBP1 gene, mRNA, protein, or a combination thereof. 349. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in a method of delivering STXBP1 to a cell or tissue. 350. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in a method of treating a genetic disorder, a neurological disorder, a neurodegenerative disorder, a muscular disorder, or a neuromuscular disorder. 351. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in the manufacture of a medicament. 352. Use of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318 in the manufacture of a medicament. 353. Use of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318 in the manufacture of a medicament for treating a genetic disorder, a neurological disorder, a neurodegenerative disorder, a muscular disorder, or a neuromuscular disorder. 354. An adeno-associated virus (AAV) particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises a syntaxin-binding protein 1 (STXBP1)-encoding sequence and theAttorney Docket No.14640.0105-00304 AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII; wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein: (i) [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H; and / or (ii) [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, or F. 355. The AAV particle of embodiment 354, wherein the AAV capsid variant is an AAV9 capsid variant. 356. The AAV particle of embodiment 354 or embodiment 355, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981. 357. The AAV particle of any one of embodiments 354-356, wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981. 358. The AAV particle of any one of embodiments 354-357, wherein: (i) X1is T, S, R, A, I, C, N, K, L, or Q; (ii) X2is N, T, G, V, S, Y, K, I, H, D, or F; and (iii) X3is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C. 359. The AAV particle of any one of embodiments 354-358, wherein: (i) X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and (ii) X7is Q, G, N, K, H, R, E, L, P, or M. 360. The AAV particle of any one of embodiments 354-359, wherein [N1] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688). 361. The AAV particle of any one of embodiments 354-360, wherein [N3] comprises the amino acid sequence of IAQ. 362. The AAV particle of any one of embodiments 354-361, wherein [N1]-N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343). 363. The AAV particle of any one of embodiments 354-362, wherein: (i) [N1] is present at amino acids 582-585;Attorney Docket No.14640.0105-00304 (ii) [N2] is present at amino acids 586-589; and (iii) [N3] is present at amino acids 590-592; wherein the amino acids are numbered according to SEQ ID NO: 981. 364. The AAV particle of any one of embodiments 354-363, further comprising [N4] comprising amino acids X8, X9, X10, and X11, wherein: (i) X8is T, S, N, P, A, or I; (ii) X9is G, N, D, R, V, A, S, or Q; (iii) X10is W, S, C, R, L, or G; and / or (iv) X11is V, A, S, I, C, G, D, F, L, or T. 365. The AAV particle of embodiment 364, wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066). 366. The AAV particle of embodiment 364 or embodiment 365, wherein [N4] is present at amino acids 593-596 as numbered according to SEQ ID NO: 981. 367. The AAV particle of any one of embodiments 354-366, wherein [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201). 368. An AAV particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at position 584, D at position 586, W position 587, H at position 588, R at position 589, and I at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981. 369. The AAV particle of any one of embodiments 354-368, wherein the AAV capsid variant comprises an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to amino acids 138-736 of SEQ ID NO: 981.Attorney Docket No.14640.0105-00304 370. An AAV particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at position 584, D at position 586, W position 587, H at position 588, R at position 589, and I at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981. 371. The AAV particle of any one of embodiments 354-370, wherein the AAV capsid variant comprises an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 981. 372. An AAV particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at position 584, D at position 586, W position 587, H at position 588, R at position 589, and I at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981. 373. The AAV particle of embodiment 372, wherein the AAV capsid variant comprises: (i) a VP1 protein comprising an amino acid sequence having at least 99% identity to SEQ ID NO:981; (ii) a VP2 protein comprising an amino acid sequence having at least 99% identity to amino acids138-736 of SEQ ID NO: 981; and / or (iii) a VP3 protein comprising an amino acid sequence having at least 99% identity to amino acids 203-736 of SEQ ID NO: 981.Attorney Docket No.14640.0105-00304 374. The AAV particle of any one of embodiments 368-373, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941). 375. The AAV particle of any one of embodiments 368-374, wherein the AAV capsid variant comprises at least 8, at least 9, or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343). 376. The AAV particle of any one of embodiments 368-375, wherein the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) at amino acids 582-592 as numbered according to SEQ ID NO: 981. 377. The AAV particle of any one of embodiments 374-376, wherein the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981. 378. The AAV particle of any one of embodiments 354-377, wherein the AAV capsid variant comprises: (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981; and / or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981. 379. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome encodes a wildtype STXBP1 protein. 380. The AAV particle of any one of embodiments 1-232, 312-318, and 354-379, wherein the viral genome encodes a human STXBP1 protein. 381. The AAV particle of embodiment 379 or embodiment 380, wherein the STXBP1 protein comprises the amino acid sequence of SEQ ID NO: 6413. 382. The AAV particle of any one of embodiments 1-232, 312-318, and 354-381, wherein the STXBP1- encoding sequence is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 6414.Attorney Docket No.14640.0105-00304 383. The AAV particle of embodiment 382, wherein the STXBP1-encoding sequence is at least 95% identical to SEQ ID NO: 6414. 384. The AAV particle of embodiment 383, wherein the STXBP1-encoding sequence is at least 99% identical to SEQ ID NO: 6414. 385. The AAV particle of embodiment 384, wherein the STXBP1-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6414. 386. The AAV particle of embodiment 385, wherein the STXBP1-encoding sequence consists of the nucleotide sequence of SEQ ID NO: 6414. 387. The AAV particle of any one of embodiments 1-232, 312-318, and 354-386, wherein the viral genome comprises a promoter operably linked to the STXBP1-encoding sequence. 388. The AAV particle of any one of embodiments 1-232, 312-318, and 354-387, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence. 389. The AAV particle of embodiment 388, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1-encoding sequence. 390. The AAV particle of embodiment 388 or embodiment 389, wherein the viral genome comprises an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence. 391. The AAV particle of any one of embodiments 388-390, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1-encoding sequence, and an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence. 392. A cell comprising the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell. 393. A method of making the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, wherein the method comprises: (i) providing a cell comprising a viral genome comprising an STXBP1-encoding sequence and a nucleic acid encoding an AAV capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAVAttorney Docket No.14640.0105-00304 capsid variant; thereby making the AAV particle. 394. The method of embodiment 393, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto; and the capsid variant comprises the amino acid sequence of SEQ ID NO: 981. 395. The method of embodiment 393 or embodiment 394, further comprising, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell. 396. The method of any one of embodiments 393-395, further comprising, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell. 397. The method of any one of embodiments 393-396, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell. 398. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-232, 312- 318, and 354-391 and a pharmaceutically acceptable excipient. 399. A method of delivering an STXBP1 protein to a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, thereby delivering the STXBP1 protein. 400. The method of embodiment 399, wherein the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder, optionally wherein the STXBP1-related disorder is an STXBP1-related neurodegenerative or neuromuscular disorder. 401. The method of embodiment 400, wherein the STXBP1-related neurodegenerative or neuromuscular disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).Attorney Docket No.14640.0105-00304 402. A method of treating an STXBP1-related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, thereby treating the STXBP1-related disorder. 403. The method of embodiment 402, wherein the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder. 404. The method of embodiment 403, wherein the STXBP1-related disorder is an STXBP1-related neurodegenerative or neuromuscular disorder. 405. The method of embodiment 404, wherein the STXBP1-related neurodegenerative or neuromuscular disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). 406. A method of treating an STXBP1-related disorder in a subject, wherein the disorder is STXBP1 encephalopathy, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, thereby treating STXBP1 encephalopathy. 407. The method of embodiment 406, wherein the subject has, has been diagnosed with having, or is at risk of having STXBP1 encephalopathy. 408. The method of any one of embodiments 402-407, wherein the subject has one or more mutations in the STXBP1 gene. 409. The method of any one of embodiments 402-408, wherein the subject has lower STXBP1 activity as compared to STXBP1 activity in an individual who does not have an STXBP1-related disorder. 410. The method of any one of embodiments 402-409, wherein the treating results in prevention of progression of the disorder in the subject. 411. The method of any one of embodiments 402-410, wherein the treating results in amelioration of at least one symptom of the disorder, as indicated by one or more biomarkers.Attorney Docket No.14640.0105-00304 412. The method of embodiment 411, wherein the one or more biomarkers comprise increased release of the neurotransmitters glutamate and / or GABA or reduction in abnormal electroencephalographic activity as evidence of improved STXBP1 activity. 413. The method of embodiment 411 or embodiment 412, wherein the at least one symptom comprises epilepsy, autistic features, ataxia, generalized tremors, reduced STXBP1 activity, accumulation of glucocerebroside and other glycolipids, e.g., within immune cells (e.g., macrophages), build-up of synuclein aggregates (e.g., Lewy bodies), developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor dysfunction, bulbar palsy, generalized weakness, trembling of a limb, depression, visual hallucinations, cognitive decline, dystonia, or a combination thereof. 414. The method of any one of embodiments 399-413, wherein the subject is a human subject. 415. The method of any one of embodiments 399-414, wherein the AAV particle is delivered to a cell, tissue, or region of the CNS. 416. The method of embodiment 415, wherein the cell, tissue, or region of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron), tissue, or region of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region). 417. The method of any one of embodiments 399-416, further comprising evaluating, e.g., measuring, the level of STXBP1 expression, e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. 418. The method of embodiment 417, wherein the level of STXBP1 protein expression is measured by an ELISA, a Western blot, or an immunohistochemistry assay. 419. The method of embodiment 417 or embodiment 418, wherein evaluating the level of STXBP1 expression is performed prior to and / or subsequent to administration of the AAV particle, optionally wherein the level of STXBP1 expression prior to treatment is compared to the level of STXBP1 expression subsequent to administration.Attorney Docket No.14640.0105-00304 420. The method of any one of embodiments 417-419, comprising evaluating the level of STXBP1 expression in a cell or tissue of the CNS. 421. The method of embodiment 420, wherein the cell or tissue of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron) or tissue of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region). 422. The method of any one of embodiments 417-421, wherein the subject’s level of STXBP1 protein expression subsequent to administration is increased relative to the subject’s level of STXBP1 protein expression prior to administration. 423. The method of any one of embodiments 399-422, further comprising evaluating, e.g., measuring, the level of STXBP1 activity in the subject, e.g., in a cell or tissue of the subject. 424. The method of any one of embodiments 399-423, wherein the administration results in an increase in: (i) the level of STXBP1 activity in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)), and / or fluid (e.g., CSF and / or serum) of the subject, relative to baseline and / or relative to the level of STXBP1 activity in a cell, tissue, or fluid of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle; (ii) the number and / or level viral genomes (VG) per cell level in a tissue of the CNS (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbarAttorney Docket No.14640.0105-00304 spinal cord region, or thoracic spinal cord region)), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject, relative to the number and / or level of VG per cell in a peripheral tissue of the subject; and / or (iii) the level of STXBP1 protein or STXBP1 gene expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject relative to baseline and / or relative to the level of STXBP1 protein or STXBP1 gene in a cell or tissue of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle. 425. The method of any one of embodiments 399-424, further comprising administering to the subject at least one additional therapeutic agent and / or therapy. 426. The method of embodiment 425, wherein the at least one additional therapeutic agent and / or therapy comprises an agent and / or therapy for treating the STXBP1-related disorder. 427. The method of embodiment 426, wherein the at least one additional therapeutic agent and / or therapy comprises one or more anti-epileptic drugs (e.g., bromide, clobazam, felbamate, ganaxolone, lamotrigine, levetiracetam, phenobarbital, topiramate, valproate, or a combination thereof). 428. The method of any one of embodiments 399-427, further comprising administering an immunosuppressant to the subject. 429. The method of embodiment 428, wherein the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxychloroquine. 430. The pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391 for use in a method of treating a disorder according to any one of embodiments 402-429.Attorney Docket No.14640.0105-00304 431. The pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391, for use in the treatment of an STXBP1-related disorder in a subject; optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). 432. The pharmaceutical composition or AAV particle for use of embodiment 431, wherein the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, optionally wherein the STXBP-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). 433. Use of an effective amount of the pharmaceutical composition of embodiment 398 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-391 in the manufacture of a medicament for the treatment of an STXBP1-related disorder in a subject; optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). 434. The use of embodiment 433, wherein the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

[0060] The details of various aspects or embodiments of the present disclosure are set forth below. Other features, objects, and advantages of the disclosure will be apparent from the description and the claims. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the sameAttorney Docket No.14640.0105-00304 meaning as commonly understood by one of ordinary skill in the art in the field of this disclosure. In the case of conflict, the present description will control. DETAILED DESCRIPTION Overview

[0061] Described herein, inter alia, are compositions comprising an AAV capsid variant comprising a sequence encoding a an STXBP1 protein, e.g., a human STXBP1 protein. In some embodiments, the present disclosure provides a method of delivering the AAV capsid variant comprising the sequence encoding the STXBP1 protein to a cell or tissue in a subject. In some embodiments, the present disclosure provides a method for delivering the AAV capsid variant, thereby providing an STXBP1 protein, e.g., a human STXBP1 protein to a cell or tissue in a subject. In some embodiments, the AAV capsid variants described herein have enhanced CNS tropism compared to other cells or tissues in the body, e.g., liver and / or the DRG.

[0062] AAVs have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. Engineered adeno-associated virus (AAV) capsids with improved brain tropism represent an attractive solution to the limitations of CNS delivery. AAV-derived vectors are promising tools for clinical gene transfer because of their non-pathogenic nature, their low immunogenic profile, low rate of integration into the host genome, and long-term transgene expression in non-dividing cells. However, the transduction efficiency of naturally occurring AAVs in certain organs is too low for clinical applications, and capsid neutralization by pre-existing neutralizing antibodies may prevent treatment of a large proportion of patients. For these reasons, considerable efforts have been devoted to obtaining capsid variants with enhanced properties. Of many approaches tested so far, significant advances have resulted from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by capsid sequence randomization using either error-prone PCR, shuffling of various parent serotypes, or insertion of fully randomized short peptides at defined positions.

[0063] The genome of the virus may be modified to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver STXBP1. The genome of the virus may encode an STXBP1 protein, and the viral particle comprising said genome may be delivered to a target cell, tissue, or organism. In some embodiments, the genome encodes a human STXBP1 protein, e.g., a wildtype human STXBP1 protein. In some embodiments, the target cell is a CNS cell. In some embodiments, the target tissue is a CNS tissue. In some embodiments, the target CNS tissue is brain tissue.

[0064] Gene therapy presents an alternative approach for treating an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1Attorney Docket No.14640.0105-00304 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). AAVs are commonly used in gene therapy approaches as a result of a number of advantageous features. Without being bound by theory, it is believed, in some embodiments, an AAV particle described herein can be used to administer and / or deliver a gene encoding STXBP1 protein (e.g., human STXBP1 protein) preferentially to the CNS. In some embodiments, an AAV particle described herein can be used to administer and / or deliver a gene encoding STXBP1 protein (e.g., human STXBP1 protein) preferentially to the brain. In some embodiments, an AAV particle described herein can be used to administer and / or deliver a gene encoding STXBP1 protein (e.g., human STXBP1 protein) preferentially to the spinal cord.

[0065] Provided herein are compositions and methods which may provide for improved features compared to prior AAV-mediated approaches, including (i) increased biodistribution throughout the CNS (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)), (ii) elevated STXBP1 expression in multiple brain regions (e.g., cortex, thalamus, and brain stem); and (iii) preferential biodistribution in the CNS over the liver and / or DRG, of the subject.

[0066] Also provided herein are AAV capsid variants (e.g., AAV9 capsid variants) with improved properties compared to wildtype AAV9, such as (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron), (iii) elevated STXBP1 expression in multiple brain regions, (iv) wider distribution in one or more peripheral tissues, e.g., the heart, kidney, muscle (e.g., quadriceps muscle), pancreas, and / or liver, and / or (v) elevated STXBP1 expression in one or more peripheral tissues. In some embodiments, the AAV capsid variants described herein enhance the delivery of an STXBP1 to multiple regions of the CNS, such as the brain, and / or the PNS. Exemplary regions may comprise the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracileAttorney Docket No.14640.0105-00304 nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron). In some embodiments, the AAV capsid variants described herein enhance the delivery of STXBP1 to the forebrain. In some embodiments, the AAV capsid variants described herein enhance the delivery of STXBP1 to the spinal cord. In some embodiments, the AAV capsid variants described herein enhance the delivery of STXBP1 to the dentate nucleus. In some embodiments, the AAV capsid variants described herein enhance the delivery of STXBP1 to the somatosensory cortex. In some embodiments, the AAV capsid variants with enhanced brain tropism described herein increase STXBP1 mRNA expression in the brain. In some embodiments, the AAV capsid variants increase STXBP1 mRNA expression in the forebrain.

[0067] Thus, the compositions and methods described herein can be used in the treatment of an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy. In some embodiments, the disclosure provides an AAV particle comprising one of the AAV capsid variants disclosed herein and an AAV viral genome comprising a nucleotide sequence comprising a promoter and a sequence encoding an STXBP1 protein for use in treating an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). I. Compositions Adeno-associated viral (AAV) Particles

[0068] AAVs have a genome of about 5,000 nucleotides in length and contains two open reading frames encoding the proteins responsible for replication (Rep) and the structural protein of the capsid (Cap). The open reading frames are flanked by two Inverted Terminal Repeat (ITR) sequences, which serve as the origin of replication of the viral genome. The wild-type AAV viral genome comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid. Alternative splicing and alternate initiation codons and promoters result in the generation of fourAttorney Docket No.14640.0105-00304 different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV serotype, as a non-limiting example, for AAV9 / hu.14 (SEQ ID NO: 123 of US 7,906,111, the contents of which are herein incorporated by reference in their entirety), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, with reference to the amino acid sequence of SEQ ID NO: 981, VP1 comprises amino acids 1-736, VP2 comprises amino acids 138-736, and VP3 comprises amino acids 203-736. In other words, VP1 is the full-length capsid protein sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid. Without being bound by theory, the AAV capsid typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3.

[0069] The AAV particle typically requires a co-helper (e.g., adenovirus) to undergo productive infection in cells. In the absence of such helper functions, the AAV virions essentially enter host cells but do not integrate into the cells’ genome.

[0070] AAV particles have been investigated for delivery of gene therapeutics because of several unique features. Non-limiting examples of the features include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range for infectivity, including human cells; (iii) wild-type AAV has not been associated with any disease and has not been shown to replicate in infected cells; (iv) the lack of cell-mediated immune response against the particle, and (v) the non-integrative nature in a host chromosome thereby reducing potential for long-term genetic alterations. Moreover, infection with AAV particles has minimal influence on changing the pattern of cellular gene expression (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148, the contents of which are herein incorporated by reference in their entirety).

[0071] Typically, AAV particles for STXBP1 delivery may be recombinant viral particles which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, the replication defective AAV particles may lack most or all coding sequences and essentially only contain one or two AAV ITR sequences and a nucleic acid sequence encoding an STXBP1 protein.

[0072] In some embodiments, the AAV particles of the present disclosure may be introduced into mammalian cells.

[0073] AAV particles may be modified to enhance the efficiency of delivery. Such modified AAV particles of the present disclosure can be packaged efficiently and can be used to successfully infect the target cells at high frequency and with minimal toxicity.Attorney Docket No.14640.0105-00304

[0074] In other embodiments, AAV particles of the present disclosure may be used to deliver STXBP1 to the central nervous system (see, e.g., U.S. Pat. No.6,180,613; the contents of which are herein incorporated by reference in their entirety) or to specific tissues of the CNS.

[0075] It is understood that the compositions described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

[0076] In some embodiments, an AAV capsid variant comprises one or more modifications in loop VIII of AAV9, e.g., at one or more amino acids corresponding to positions 580-599 of VP1 of AAV9. In some embodiments, an AAV capsid variant comprises a substitution at one, two, three, four, five, or all of amino acids corresponding to position(s) 584, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the term loop (e.g., loop VIII) is used interchangeably herein with the term variable region (e.g., variable region VIII), or VR (e.g., VR-VIII). In some embodiments, loop VIII comprises amino acids corresponding to positions 580-599 (e.g., the positions of amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present (is located) at amino acids comprising those corresponding to positions 580-599 (e.g., the positions of amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 582-593 (e.g., the positions of amino acids TNHQSAQAQAQT (SEQ ID NO: 5123)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 587-593 (e.g., the positions of amino acids AQAQAQT (SEQ ID NO: 4687)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 587-590 (e.g., the positions of amino acids AQAQ (SEQ ID NO: 5099)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 587-590 (e.g., the positions of amino acids AQAQ (SEQ ID NO: 5099)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII or variable region VIII (VR- VIII) is as described in DiMattia et al. “Structural Insights into the Unique Properties of the Adeno- Associated Virus Serotype 9,” Journal of Virology, 12(86):6947-6958 (the contents of which are hereby incorporated by reference in their entirety), e.g., comprising amino acids corresponding to positions 581- 593 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138.

[0077] The AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue, or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, an AAV particle may, in addition, or alternatively, have decreased tropism for a cell-type, tissue or organ.Attorney Docket No.14640.0105-00304

[0078] In some embodiments, AAV particles are used as a biological tool due to a relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. The genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload.

[0079] In some embodiments, the AAV particle is a recombinant AAV particle. In some embodiments, the wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome. In some embodiments, an AAV viral genome comprises two ITR sequences. In some embodiments, the ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. In some embodiments, the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0080] AAV particles of the present disclosure may be produced recombinantly and may be based on AAV reference sequences. In addition to single-stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) viral genomes. scAAV viral genomes contain DNA strands that anneal together to form double-stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell. In some embodiments, the AAV particle of the present disclosure is an scAAV. In some embodiments, the AAV particle of the present disclosure is an ssAAV.

[0081] Methods for producing and / or modifying AAV particles are disclosed in the art such as pseudotyped AAV particles (PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO2005005610; and WO2005072364, the content of each of which is incorporated herein by reference in its entirety).

[0082] As described herein, the AAV particles of the disclosure comprising an AAV capsid variant, and a viral genome, have enhanced tropism for a cell-type or a tissue, e.g., a CNS cell-type, region, or tissue. AAV Capsid Variants

[0083] Disclosed herein are AAV particles comprising an AAV capsid variant comprising one or more modifications (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) for enhanced or improved transduction of a target tissue (e.g., cells, regions, and / or tissues of the CNS and / or PNS). In some embodiments, the peptide (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) is present in VP1, VP2, and / or VP3 proteins of the AAV capsid variant. In someAttorney Docket No.14640.0105-00304 embodiments, the modification (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) is present in VP1, VP2, and VP3 proteins of the AAV capsid variant.

[0084] In some embodiments, the modification (e.g., comprising the one or more substitutions relative to a wildtype AAV capsid) is in loop VIII. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.

[0085] In some embodiments, the one or more substitutions in the AAV capsid variant may increase distribution of an AAV particle to a cell, region, or tissue of the CNS. The cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and / or supporting cells of the brain such as immune cells (e.g., T cells). The tissue of the CNS may be, but is not limited to, the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron).

[0086] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a cell, region, or tissue of the CNS. In some embodiments, the one or more substitutions may decrease distribution of an AAV particle to the DRG.

[0087] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the CNS (e.g., the cortex) after intravenous administration. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the CNS (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0088] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g., DRG) after intravenous administration. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to non-DRG cells of the PNS following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the one or moreAttorney Docket No.14640.0105-00304 substitutions may decrease distribution of an AAV particle to the DRG following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g., DRG) following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0089] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution of an AAV particle to a cell, region, or tissue of a heart, e.g., a heart atrium or a heart ventricle. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a heart cell, region, or tissue after intravenous administration.

[0090] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution of an AAV particle to a cell, region, or tissue of a muscle. In some embodiments, the muscle is a heart muscle (e.g., a heart atrium or a heart ventricle) or a quadriceps muscle. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a muscle cell, region, or tissue after intravenous administration.

[0091] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution an AAV particle to a cell, region, or tissue of the kidney. In some embodiments, the one or more substitutions may increase distribution an AAV particle to a cell, region, or tissue of the pancreas. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a cell, region, or tissue of the retina.

[0092] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 1. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 2A. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 2B (e.g., a sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336). In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence set forth in Table 9. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 23. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 24. In some embodiments, the AAV capsid variant is an AAV9 capsid variant. Table 1. Exemplary Sequences Amino Acid SEQ Amino Ac SEQ SEQ SEQ ID id ID Amino Acid ID Amino Acid ID O: 012 345 6Attorney Docket No.14640.0105-00304 TTNQDWHRIAQ350TNTKDWHRISQ399TINQDWHRIAQ448TNNPDWHRIAQ497TNDQDWHRIAQ351SNTKDWHRIAQ400TNNQDWHRIAH449TITEDWHRIAQ498TTIQDWHRIAQ352TNNKDWHRIAQ401TNNQDWHRIGQ450TNTNDWHRIAQ499012345678901234567 8901234 5678901 2345678SEQ ID Amino Acid Sequence SEQ ID Nucleotide Sequence NO: NO: C. p y q Amino Acid SEQ ID Amin A id S n SEQ ID Amin A id S n SEQ IDAttorney Docket No.14640.0105-00304 TTNQDWHRIAQTGWV205STTQDWHRIAQTGWV230TTTQDWHRVAQTGWV290TTIQDWHRIAQTGWV206TNTQDWHRVAQTGWV232TNSQDWHRIAQTGWA291TTSQDWHRIAQTGWV207TNTQDWHRVSQTGWV237TNSQDWHRVAQTGWV293idsequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, H, or a conservative substitution thereof and / or [N3] comprises X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, F, or a conservative substitution thereof. In some embodiments, [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682), [N1] comprises X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H, and / or [N3] comprises X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, or F. In some embodiments, X4of [N1] is Q. In some embodiments, X4of [N1] is K. In some embodiments, X5of [N3] is I. In some embodiments, X1of [N1] is T, S, R, A, I, C, N, K, L, or Q. In some embodiments, X2of [N1] is N, T, G, V, S, Y, K, I, H, D, or F. In some embodiments, X3of [N1] is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C. In some embodiments, [N1] is or comprises TNTQ (SEQ ID NO: 4688). In some embodiments, [N1] is or comprises TNTK (SEQ ID NO: 4689). In some embodiments, [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments, [N1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899). In some embodiments, X6of [N3] is A, Y, P, N, S, T, G, E, V, W, F, or Q. In some embodiments, X7of [N3] is Q, G, N, K, H, R, E, L, P, or M. In some embodiments, [N3] is or comprises IAQ. In some embodiments, [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTKDWHRIAQ (SEQ ID NO: 344).

[0094] In some embodiments, the amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X8, X9, X10, and X11, wherein X8is T, S, N, P, A, or I; X9is G, N, D, R, V, A, S, or Q; X10is W, S, C, R, L, or G; and / or X11is V, A, S, I, C, G, D, F, L, or T. In some embodiments, [N4] is or comprises TGWV (SEQ ID NO: 5066). In some embodiments, [N1]- [N2]-[N3]-[N4] is or comprises any one of SEQ ID NOs: 201-245, 247-250, 253-255, 257-265, 268-274, 276-286, 288, 290-297, 299-303, 305-309, 311, 313-319, 323-328, 330-337, 339-342, 539-542, 544, 546, 547, 549-557, 559-589, 592, 593, 595, 596, 598, 599, 601-608, 610-614, 616-622, 625, 628, 630, 631, 633, 636, 638, 639-646, 649, 651-657, 667, 669, 670, 672, 673, 679-683, 685-690, 692, 693, 695, 697, 699-701, 703-705, 708-710, 712-717, 719-723, 728-731, 733-738, 740, and 742. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTKDWHRIAQTGWV (SEQ ID NO: 202).Attorney Docket No.14640.0105-00304

[0095] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, P, or a conservative substitution thereof; and / or [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, or a conservative substitution thereof. In some embodiments, [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q or P; and / or [N3] comprises amino acids X5, X6, and X7, wherein X5is I or V. In some embodiments, X4of [N1] is Q. In some embodiments, X5of [N3] is I. In some embodiments, X5of [N3] is V. In some embodiments, X1of [N1] is T or S. In some embodiments, X2of [N1] is N, T, G, S, I, or V. In some embodiments, X3of [N1] is T, N, I, S, A, V, or L. In some embodiments, [N1] is or comprises TNTQ (SEQ ID NO: 4688). In some embodiments, [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments, X6of [N3] is A, P, S, Y, or N. In some embodiments, X7of [N3] is Q, G, or N. In some embodiments, [N3] is or comprises IAQ. In some embodiments, [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, the amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X8, X9, X10, and X11, wherein X10is W. In some embodiments, X8of [N4] is T, S, or N. In some embodiments, X9of [N4] is G or N. In some embodiments, X11 of [N4] is V, A, I, or S. In some embodiments, [N4] is or comprises TGWV (SEQ ID NO: 5066). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201).

[0096] In some embodiments, [N1]-[N2]-[N3] is present in loop VIII of the AAV capsid variant. In some embodiments, [N4] is present in loop VIII of the AAV capsid variant. In some embodiments, [N1]- [N2]-[N3]-[N4] is present in loop VIII of the AAV capsid variant. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981.

[0097] In some embodiments, [N1] is present at amino acids corresponding to positions 582-585 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, X1of [N1] is present at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 138 or 981, X2of [N1] is present at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 138 or 981, X3of [N1] is present at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, and X4of [N1] is present at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 or 981. In someAttorney Docket No.14640.0105-00304 embodiments, [N1] replaces amino acids corresponding to positions 582-585 (e.g., T582, N583, H584, and Q585) of the amino acid sequence of SEQ ID NO: 138.

[0098] In some embodiments, [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2] replaces amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 138 (e.g., S586, A587, Q588, and A589 may be replaced with D586, W587, H588, and R589).

[0099] In some embodiments, [N1]-[N2] is present at amino acids corresponding to positions 582- 589 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N1]- [N2] replaces amino acids corresponding to positions 582-589 of the amino acid sequence of SEQ ID NO: 138 (e.g., T582, N583, H584, Q585, S586, A587, Q588, and A589 may be replaced with T582, N583, T584, Q585, D586, W587, H588, and R589).

[0100] In some embodiments, [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, X5of [N3] is present at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981, X6of [N3] is present at an amino acid corresponding to position 591 of the amino acid sequence of SEQ ID NO: 138 or 981, and X7of [N3] is present at an amino acid corresponding to position 592 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, [N3] replaces amino acids corresponding to positions 590-592 (e.g., I590, A591, and Q592) of the amino acid sequence of SEQ ID NO: 138.

[0101] In some embodiments, [N2]-[N3] is present at amino acids corresponding to positions 586- 592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2]- [N3] replaces amino acids corresponding to positions 586-592 of the amino acid sequence of SEQ ID NO: 138 (e.g., S586, A587, Q588, A589, Q590, A591, and Q592 may be replaced with D586, W587, H588, R589, I590, A591, and Q592).

[0102] In some embodiments, [N1]-[N2]-[N3] is present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N1]-[N2]-[N3] replaces amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 138 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, and Q592 may be replaced with T582, N583, T584, Q585, D586, W587, H588, R589, I590, A591, and Q592).

[0103] In some embodiments, [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, X8of [N4] is present at an amino acid corresponding to position 593 of the amino acid sequence of SEQ ID NO: 138 or 981, X9of [N4] is present at an amino acid corresponding to position 594 of the amino acid sequence of SEQ ID NO: 138 or 981, X10of [N4] is present at an amino acid corresponding to position 595 of the amino acid sequence of SEQ ID NO: 138 or 981, and X11of [N4] is present at an amino acid corresponding to position 596 of the amino acid sequence of SEQ ID NO: 138 or 981. In someAttorney Docket No.14640.0105-00304 embodiments, [N4] replaces amino acids corresponding to positions 593-596 (e.g., T593, G594, W595, and V596) of the amino acid sequence of SEQ ID NO: 138.

[0104] In some embodiments, [N2]-[N3]-[N4] is present at amino acids corresponding to positions 586-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2]-[N3]-[N4] replaces amino acids corresponding to positions 586-596 of the amino acid sequence of SEQ ID NO: 138 (e.g., S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596 may be replaced with D586, W587, H588, R589, I590, A591, Q592, T593, G594, W595, and V596).

[0105] In some embodiments, [N1]-[N2]-[N3]-[N4] is present at amino acids corresponding to positions 582-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N1]-[N2]-[N3]-[N4] replaces amino acids corresponding to positions 582-596 of the amino acid sequence of SEQ ID NO: 138 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596 may be replaced with T582, N583, T584, Q585, D586, W587, H588, R589, I590, A591, Q592, T593, G594, W595, and V596).

[0106] In some embodiments, [N2] is present immediately subsequent to [N1]. In some embodiments, [N3] is present immediately subsequent to [N2]. In some embodiments, [N4] is present immediately subsequent to [N3]. In some embodiments, loop VIII of the AAV capsid variant comprises an amino acid sequence comprising, from N-terminus to C-terminus, [N1]-[N2]-[N3]. In some embodiments, loop VIII of the AAV capsid variant comprises an amino acid sequence comprising, from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4].

[0107] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.

[0108] In some embodiments, the at least 3 consecutive amino acids comprise TQD. In some embodiments, the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684). In some embodiments, the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685). In some embodiments, the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686). In some embodiments, the at least 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941).

[0109] In some embodiments, the at least 3 consecutive amino acids comprise TNT. In some embodiments, the at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688). In some embodiments, the at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119). In some embodiments, the at least 6 consecutive amino acids comprise TNTQDW (SEQ ID NO: 5120). In some embodiments, the at least 7 consecutive amino acids comprise TNTQDWH (SEQ ID NO: 5121). In someAttorney Docket No.14640.0105-00304 embodiments, the at least 8 consecutive amino acids comprise TNTQDWHR (SEQ ID NO: 4898). In some embodiments, the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746).

[0110] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three modifications, but no more than four modifications, relative to the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more than four substitutions, relative to the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three modifications, but no more than four modifications, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more than four substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211- 214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.

[0111] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three modifications, but no more than four modifications, relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more than four substitutions, relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

[0112] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.

[0113] In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, loop VIII comprises amino acids corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present (is located) at amino acids comprising those corresponding to positions 580-599 (e.g., the positions of amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of amino acids corresponding to position(s) 582 (e.g., T582), 583 (e.g., N583), 584 (e.g., H584), 585 (e.g., Q585), 586 (e.g., S586), 587 (e.g., A587), 588 (e.g., Q588), 589 (e.g.,Attorney Docket No.14640.0105-00304 A589), 590 (e.g., Q590), 591 (e.g., A591), 592 (e.g., Q592), 593 (e.g., T593), 594 (e.g., G594), 595 (e.g., W595), and / or 596 (e.g., V596) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of amino acids corresponding to position(s) 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, and / or 596 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises one or more substitutions at amino acids corresponding to position(s) 582 (e.g., T582), 583 (e.g., N583), 584 (e.g., H584), 585 (e.g., Q585), 586 (e.g., S586), 587 (e.g., A587), 588 (e.g., Q588), 589 (e.g., A589), 590 (e.g., Q590), 591 (e.g., A591), 592 (e.g., Q592), 593 (e.g., T593), 594 (e.g., G594), 595 (e.g., W595), and / or 596 (e.g., V596) of the amino acid sequence of SEQ ID NO: 138.

[0114] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by a nucleotide sequence described herein, e.g., a nucleotide sequence of Table 2A. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven different substitutions, but not more than ten substitutions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity) thereto.

[0115] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid (e.g., comprising one or more substitutions relative to a wildtype AAV capsid), comprises a nucleotide sequence described herein, e.g., as described in Table 2A. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid (e.g., comprising one or more substitutions relative to a wildtype AAV capsid), is codon optimized. In some embodiments, the nucleotide sequence is an isolated nucleotide sequence. In some embodiments, the nucleotide sequence is a recombinant nucleotide sequence.

[0116] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid (e.g., comprising one or more substitutions relative to a wildtype AAV capsid), comprises the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the nucleotide sequence comprises a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at leastAttorney Docket No.14640.0105-00304 six, or at least seven substitutions, but no more than ten substitutions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity) thereto.

[0117] In some embodiments, the AAV capsid variant further comprises an amino acid other than A at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant further comprises the amino acid T at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant further comprises the amino acid V at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the substitution A581T or A581V, wherein the substitution is at position that corresponds to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0118] In some embodiments, an AAV capsid variant described herein comprises one, two, three, four, five, or all of an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g., comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and / or an amino acid other than Q at a position corresponding to position 590 (e.g., comprises I at an amino acid corresponding to position 590) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g., comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than Q at a position corresponding to position 590 (e.g., comprises I at an amino acid corresponding to position 590) of the amino acid sequence of SEQ ID NO: 138. In someAttorney Docket No.14640.0105-00304 embodiments, an AAV capsid variant described herein comprises the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid W at a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid R at a position corresponding to position 589 of the amino acid sequence of SEQ ID NO: 138 or 981, and amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0119] In some embodiments, an AAV capsid variant described herein comprises one, two, three, four, five, or all of the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid W at a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid R at a position corresponding to position 589 of the amino acid sequence of SEQ ID NO: 138 or 981, and / or amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid W at a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid R at a position corresponding to position 589 of the amino acid sequence of SEQ ID NO: 138 or 981, and amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0120] In some embodiments, an AAV capsid variant described herein comprises one, two, three, four, five, or all of the substitutions H584T, S586D, A587W, Q588H, A589R, and / or Q590I wherein the substitution(s) is / are at position(s) that correspond to position(s) 584, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I wherein the substitution(s) is / are at position(s) that correspond to position(s) 584, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0121] In some embodiments, an AAV capsid variant described herein comprises the amino acid Q at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0122] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid K at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138.Attorney Docket No.14640.0105-00304

[0123] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid V at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138.

[0124] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein TQDWHRI (SEQ ID NO: 941) is present in the AAV capsid variant at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, TQDWHRI (SEQ ID NO: 941) is present at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 (e.g., H584, S586, A587, Q588, A589, and Q590 may be replaced with T584, D586, W587, H588, R589, and I590).

[0125] In some embodiments, an AAV capsid variant described herein comprises the amino acid W at an amino acid corresponding to position 595 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0126] In some embodiments, the AAV capsid variant further comprises a substitution corresponding to amino acid K449, (e.g., K449R) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an amino acid other than K at a position corresponding to position 449 (e.g., comprises an R corresponding to position 449) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an R at amino acid corresponding to position 449 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification in loop I, II, IV, and / or VI.

[0127] In some embodiments, the AAV capsid variant further comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications, relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence does not comprise more than 10 modifications. In some embodiments, the AAV capsid variant further comprises an amino acid sequence comprising at least one, at least two, or at least three, but not more than 30, not more than 20, or not more than 10 amino acids that differ from the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0128] In some embodiments, the AAV capsid variant comprises (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 981 (i.e., comprising amino acids 1-736 of SEQ ID NO: 981); (b) a VP2 protein comprising amino acids 138-736 of SEQ ID NO: 981; (c) a VP3 protein comprising amino acids 203-736 of SEQ ID NO: 981; or (d) an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,Attorney Docket No.14640.0105-00304 or at least 99%) sequence identity to any one of the amino acid sequences in (a)-(c), an amino acid sequence comprising at least one, at least two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions relative to any one of the amino acid sequences in (a)-(c), or an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 such modifications relative to any one of the amino acid sequences in (a)-(c).

[0129] In some embodiments, the AAV capsid variant comprises (a) a VP1 protein comprising an amino acid sequence that is at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 981; (b) a VP2 protein comprising an amino acid sequence that is at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736 of SEQ ID NO: 981; and / or (c) a VP3 protein comprising an amino acid sequence that is at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or a sequence comprising one, two, or three but not more than three substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises the amino acid sequence comprising one, two, or three but not more than three substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941) in loop VIII. In some embodiments, the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941) in loop VIII.

[0130] In some embodiments, the AAV capsid variant comprises an amino acid sequence as described herein, e.g., an amino acid sequence of TTJ-001, e.g., as described in Tables 3 and 4. In some embodiments, the AAV capsid variant is or comprises TTJ-001. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 941 in a loop VIII region.

[0131] In some embodiments, the AAV capsid variant comprises a VP1, VP2, and / or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of TTJ-001, e.g., as described in Tables 3 and 4.

[0132] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., a nucleotide sequence encoding TTJ-001, e.g., as described in Tables 3 and 5.

[0133] In some embodiments, the polynucleotide or nucleic acid encoding the AAV capsid variant comprises a nucleotide sequence described herein, e.g., a nucleotide sequence encoding TTJ-001, e.g., as described in Tables 3 and 5. Table 3. Exemplary full length capsid sequences VP1 DN VP1 (amino Peptide Name A acid) SEQ ID (amino acid) Peptide DNA SEQAttorney Docket No.14640.0105-00304 Table 4. Exemplary full length capsid amino acid sequences Name and SEQ Amino Acid Sequence Annotation ID NO: PGGG AK SS TP AN RS LS WP MI QG IT IGName and SEQ Nucleotide Sequence Annotation ID NO: T C C A A C T C A C G C G A C A T T T A G T C A G C T C A A A A A T G C AAttorney Docket No.14640.0105-00304 CAGCAAGCGGTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATA ATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGC ACCAGATACCTGACTCGTAATCTGTAAnucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0135] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, at least two, or at least three modifications but not more than 30, not more than 20, or not more than 10 modifications, relative to the nucleotide sequence of SEQ ID NO: 983. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, at least two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.

[0136] In some embodiments, the AAV capsid variant comprises one or more substitutions in loop VIII and comprises the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications, relative to the amino acid sequence of SEQ ID NO: 981. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, at least two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 981.

[0137] In some embodiments, the AAV capsid variant comprises one or more substitutions in loop VIII and comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications, relative to the nucleotide sequence of SEQ ID NO: 983.

[0138] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, VP3 protein, wherein at least one of the VP1, VP2, and VP3 comprise one or more substitutions in loop VIII.Attorney Docket No.14640.0105-00304 In some embodiments, the AAV capsid variant comprises amino acids 138-736, e.g., a VP2, of the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises amino acids 203-736, e.g., a VP3, of the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises amino acids 1-736, e.g., a VP1, of the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0139] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981, or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0140] In some embodiments, an AAV capsid variant described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0141] In some embodiments, an AAV capsid variant described herein transduces a brain region, e.g., a midbrain region (e.g., the hippocampus, or thalamus) or the brain stem. In some embodiments, the level of transduction is at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550- fold greater as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0142] In some embodiments, an AAV capsid variant described herein is enriched at least 10, at least 14, at least 20, at least 24, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0143] In some embodiments, an AAV capsid variant described herein is enriched in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse) species, compared toAttorney Docket No.14640.0105-00304 enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 190, at least 200, at least 205, or at least 210-fold in the brain of at least two to three species, e.g., a non- human primate and rodent (e.g., mouse) species, compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the at least two to three species are Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, and / or mouse (e.g., outbred mice).

[0144] In some embodiments, an AAV capsid variant described herein is enriched at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 17, at least 20, at least 50, at least 75, at least 100, at least 103, at least 107, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, or at least 1200-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 981.

[0145] In some embodiments, an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region. In some embodiments, the level of viral genomes is increased by at least 2, at least 5, at least 7, at least 10, at least 15, at least 19, at least 20, at least 22, or at least 25-fold, as compared to the level of viral genomes delivered by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.

[0146] In some embodiments, an AAV capsid variant delivers an increased level of a payload to a brain region. In some embodiments, the level of the payload is increased by at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold, as compared to the level of a payload delivered by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.

[0147] In some embodiments, an AAV capsid variant described herein is enriched at least 5, at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to enrichment in the spinal cord of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0148] In some embodiments, an AAV capsid variant of the present disclosure has decreased tropism for the liver. In some embodiments, an AAV capsid variant comprises a modification that results in reduced tropism (e.g., de-targeting) and / or activity in the liver. In some embodiments, the reducedAttorney Docket No.14640.0105-00304 tropism in the liver is compared to an otherwise similar capsid that does not comprise the modification, e.g., a wild-type capsid polypeptide. In some embodiments, an AAV capsid variant comprises a modification that results in one or more of the following properties: (1) reduced tropism in the liver; (2) de-targeted expression in the liver; (3) reduced activity in the liver; and / or (4) reduced binding to galactose. In some embodiments, the reduction in any one or all of properties (1)-(3) is compared to an otherwise similar AAV capsid variant that does not comprise the modification. Exemplary modifications are provided in WO 2018 / 119330; Pulicherla et al. (2011) Mol. Ther.19(6): 1070-1078; Adachi et al. (2014) Nature Communications 5(3075), DOI: 10.1038 / ncomms4075; and Bell et al. (2012) J. Virol. 86(13): 7326-33; the contents of which are hereby incorporated by reference in their entirety.

[0149] In some embodiments, the AAV capsid variant comprises a modification at an amino acid corresponding to N470 (e.g., comprises N470A at an amino acid corresponding to N470) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to D271 (e.g., comprises D271A at an amino acid corresponding to D271) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to N272 (e.g., comprises N272A at an amino acid corresponding to N272) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to Y446 (e.g., comprises Y446A at an amino acid corresponding to Y446) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to N498 (e.g., comprises N498Y or N498I at an amino acid corresponding to N498) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to W503 (e.g., comprises W503R or W503A at an amino acid corresponding to W503) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to L620 (e.g., comprises L620F at an amino acid corresponding to L620) of the amino acid sequence of SEQ ID NO: 138; or a combination thereof. In some embodiments, the AAV capsid variant comprises one, two, three, four, five, or all of an amino acid other than N at a position corresponding to position 470 (e.g., comprises A at an amino acid corresponding to position 470) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than D at a position corresponding to position 271 (e.g., comprises A at an amino acid corresponding to position 271) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than N at a position corresponding to position 272 (e.g., comprises A at an amino acid corresponding to position 272) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Y at a position corresponding to position 446 (e.g., comprises A at an amino acid corresponding to position 446) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than N at a position corresponding to position 498 (e.g., comprises Y or I at an amino acid corresponding to position 498) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than W at a position corresponding to position 503 (e.g., comprises R or A at an amino acid corresponding to position 503) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than L at a position corresponding to position 620 (e.g., comprises F at an amino acid corresponding to position 620) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification at positions corresponding to amino acid N470 (e.g., comprises N470A at an amino acid corresponding to N470) of the amino acid sequence of SEQ ID NO:Attorney Docket No.14640.0105-00304 138; corresponding to amino acid D271 (e.g., comprises D271A at an amino acid corresponding to D271) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid N272 (e.g., comprises N272A at an amino acid corresponding to N272) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid Y446 (e.g., comprises Y446A at an amino acid corresponding to Y446) of the amino acid sequence of SEQ ID NO: 138; and corresponding to amino acid W503 (e.g., comprises W503R or W503A at an amino acid corresponding to W503) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification at a position corresponding to amino acid N498 (e.g., comprises N498Y at an amino acid corresponding to N498) of the amino acid sequence of SEQ ID NO: 138 and corresponding to amino acid L620 (e.g., comprises L620F at an amino acid corresponding to L620) of the amino acid sequence of SEQ ID NO: 138.

[0150] In some embodiments, the AAV capsid variant comprises a modification as described in Adachi et al. (2014) Nature Communications 5(3075), DOI: 10.1038 / ncomms4075, the contents of which are hereby incorporated by reference in its entirety. Exemplary modifications that alter or do not alter tissue transduction in at least the brain, liver, heart, lung, and / or kidney can be found in Supplementary Data 2 showing the AAV Barcode-Seq data obtained with AAV9-AA-VBCLib of Adachi et al. (supra), the contents of which are hereby incorporated by reference in their entirety.

[0151] In some embodiments, the AAV capsid variant is an isolated capsid variant. In some embodiments, the AAV capsid variant is a recombinant capsid variant. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant is an isolated and / or a recombinant AAV capsid polypeptide.

[0152] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same. AAV serotypes and capsids

[0153] In some embodiments, the AAV particle may comprise a capsid protein or variant of any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profiles. Without being bound by theory, it is believed in some embodiments, that the AAV capsid protein, e.g., an AAV capsid variant can modulate AAV particle tropism in a particular tissue.

[0154] In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the AAV capsid variant allows for increased distribution to a brain region. In some embodiments, the brain region comprises the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferiorAttorney Docket No.14640.0105-00304 olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, and / or vestibular nucleus. In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the liver. In some embodiments, the AAV capsid variant allows for transduction in neuronal cells. In some embodiments, the AAV capsid variant allows for transduction in a non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof). In some embodiments, the AAV capsid variant allows for transduction in both neuronal cells and non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof).

[0155] In some embodiments, an AAV capsid variant allows for increased distribution to a spinal cord region. In some embodiments, the spinal region comprises a cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region.

[0156] In some embodiments, the AAV capsid variant allows for increased distribution to a heart region.

[0157] In some embodiments, the AAV capsid variant is suitable for intramuscular administration and / or transduction of muscle fibers. In some embodiments, the AAV capsid variant allows for increased distribution to a muscle region. In some embodiments, the muscle region comprises a heart muscle, quadriceps muscle, a diaphragm muscle region, or a combination thereof. In some embodiments, the muscle region comprises a heart muscle region, e.g., a heart atrium muscle region or a heart ventricle muscle region.

[0158] In some embodiments, the AAV capsid variant is suitable for increased distribution to a kidney. In some embodiments, the AAV capsid variant is suitable for increased distribution to a pancreas. In some embodiments, the AAV capsid variant is suitable for increased distribution to a retina.

[0159] In some embodiments, the initiation codon for translation of the AAV VP1 capsid protein, e.g., a capsid variant, described herein may be CTG, TTG, or GTG as described in US Patent No. US8163543, the contents of which are herein incorporated by reference in its entirety.

[0160] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (e.g., capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Met1), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Met1) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met / AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.Attorney Docket No.14640.0105-00304

[0161] Where the Met / AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Met1 / AA1 amino acid (Met+ / AA+) and some of which may lack a Met1 / AA1 amino acid as a result of Met / AA- clipping (Met- / AA-). For further discussion regarding Met / AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods.2017 Oct.28(5):255- 267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science.2010 February 19.327(5968): 973–977; the contents of which are each incorporated herein by reference in its entirety.

[0162] According to the present disclosure, references to capsid proteins, e.g., AAV capsid variants, is not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and / or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a capsid protein or capsid polypeptide (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Met1 / AA1 amino acid (Met+ / AA+) as well as corresponding VP capsid proteins which lack the Met1 / AA1 amino acid as a result of Met / AA-clipping (Met- / AA-).

[0163] Further according to the present disclosure, a reference to a specific SEQ ID NO (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Met1 / AA1 amino acid (Met+ / AA+) should be understood to teach the VP capsid proteins which lack the Met1 / AA1 amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Met1 / AA1).

[0164] As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Met1” amino acid (Met+) encoded by the AUG / ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Met1” amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.

[0165] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Met1 / AA1 amino acid (Met+ / AA1+), corresponding VP capsid proteins which lack the Met1 / AA1 amino acid as a result of Met / AA1-clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0166] As a non-limiting example, an AAV capsid serotype can include VP1 (Met+ / AA1+), VP1 (Met- / AA1-), or a combination of VP1 (Met+ / AA1+) and VP1 (Met- / AA1-). An AAV capsid serotype can also include VP3 (Met+ / AA1+), VP3 (Met- / AA1-), or a combination of VP3 (Met+ / AA1+) and VP3Attorney Docket No.14640.0105-00304 (Met- / AA1-); and can also include similar optional combinations of VP2 (Met+ / AA1) and VP2 (Met- / AA1-). Additional AAV Capsid Sequences

[0167] In some embodiments, the AAV capsid variant comprises at amino acids corresponding to positions 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25.

[0168] In some embodiments, the AAV capsid variant comprises, immediately subsequent to an amino acid corresponding to position 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021 / 230987 (the contents of which are hereby incorporated by reference in their entirety))), at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1, 2A, 2B, 9, 14, 15, or 16. In some embodiments, the at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1, 2A, 2B, 9, 23, 24, or 25 replaces at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of amino acid(s) corresponding to position(s) 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, and / or Q590) of the amino acid sequence of SEQ ID NO: 138 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021 / 230987, which is hereby incorporated by reference in its entirety)). In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type amino acid, at one, two, three, four, five, six, seven, eight, or all of amino acid(s) corresponding to position(s) 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021 / 230987, which is hereby incorporated by reference in its entirety). In some embodiments, the AAV capsid variant comprises a modification replacing one, two, three, four, five, six, seven, eight, or all amino acid(s) corresponding to position(s) 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, and / or Q590) of the amino acid sequence of SEQ ID NO: 138 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8,Attorney Docket No.14640.0105-00304 AAVrh10, AAVrh32.33, AAVrh74, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021 / 230987, which is hereby incorporated by reference in its entirety).

[0169] In some embodiments, the AAV capsid variant may comprise a variant of a VOY101 capsid polypeptide, an AAVPHP.B (PHP.B) capsid polypeptide, a AAVPHP.N (PHP.N) capsid polypeptide, an AAV1 capsid polypeptide, an AAV2 capsid polypeptide, an AAV5 capsid polypeptide, an AAV9 capsid polypeptide, an AAV9 K449R capsid polypeptide, or an AAVrh10 capsid polypeptide. In some embodiments, the AAV capsid polypeptide, e.g., AAV capsid variant, comprises an amino acid sequence of any one of the AAV capsid polypeptides in Table 6, or an amino acid sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity) thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid polypeptide comprises any one of the nucleotide sequences in Table 6, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity) thereto. Table 6. AAV Sequences Serotype SEQ Sequence ID D Q E I R H V P S T P F D Q E I R H V P S G T V D Q E I R H V PAttorney Docket No.14640.0105-00304 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG ILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT V C C C C C G T C G T A C C C A T C C G C A C G T A T T A G A C G G C G AAAV Viral Genome

[0170] In some embodiments, the AAV particle of the present disclosure serves as an expression vector comprising a viral genome that encodes an STXBP1 protein (e.g., a human STXBP1 protein).

[0171] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of STXBP1 described herein, comprises a viral genome, e.g., an AAV viral genome, also referred to as an AAV genome, a vector genome, or an AAV vector genome. In some embodiments, the viral genome, e.g., the AAV viral genome, further comprises an inverted terminal repeat (ITR) region, an enhancer, a promoter, an intron region, an exon region, a nucleic acid encoding a transgene encoding an STXBP1 protein, a polyA region, or a combination thereof. In some embodiments, the viral genome, e.g., the AAV viral genome, further comprises a nucleotide sequence encoding at least one miRNA binding site.Attorney Docket No.14640.0105-00304 Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0172] In some embodiments, the viral genome may comprise at least one inverted terminal repeat (ITR) region. The AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a region comprising an STXBP1-encoding sequence. In some embodiments, the viral genome has two ITRs. These two ITRs flank the region comprising the STXBP1-encoding sequence at the 5’ and 3’ ends. In some embodiments, the ITR functions as an origin of replication comprising a recognition site for replication. In some embodiments, the ITR comprises a sequence region which can be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into a viral genome described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.

[0173] In some embodiments, the ITR is of the same serotype as the capsid, selected from any one of the serotypes described herein, or a derivative thereof. In some embodiments, the ITR is of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In some embodiments, the AAV particle comprises a viral genome comprising two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one, or both of the ITRs having the same serotype as the capsid. Viral Genome Component: Promoters and Enhancers

[0174] In some embodiments, the viral genome comprises at least one element to enhance the transgene target specificity and expression. See, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety. Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) region, upstream enhancers (USEs), CMV enhancers, and introns.

[0175] In some embodiments, expression of the polypeptides in a target cell may be driven by a specific promoter, including but not limited to, a promoter that is species specific, inducible, tissue- specific, or cell cycle-specific (Parr et al., Nat. Med.3:1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).

[0176] In some embodiments, the viral genome comprises a promoter that is sufficient for expression, e.g., in a target cell, of an STXBP1 protein (e.g., a human STXBP1 protein) encoded by a transgene. In some embodiments, the promoter is deemed to be efficient when it drives expression of the STXBP1 encoded in the viral genome of the AAV particle.

[0177] In some embodiments, the promoter is a promoter deemed to be efficient when it drives expression in the cell or tissue being targeted.Attorney Docket No.14640.0105-00304

[0178] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the promoters may be truncated.

[0179] In some embodiments, the viral genome comprises a promoter that results in expression in one or more cells and / or tissues. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, a promoter that drives or promotes expression in most mammalian tissues comprises a human elongation factor 1α-subunit (EF1α) promoter, a cytomegalovirus (CMV) immediate-early enhancer and / or promoter, a chicken β-actin (CBA) promoter, a CAG promoter, a β glucuronidase (GUSB) promoter, or a ubiquitin C (UBC) promoter.

[0180] In some embodiments, the viral genome comprises a nervous system specific promoter, i.e., a promoter that results in expression of STXBP1 in a neuron, an astrocyte, and / or an oligodendrocyte. Non-limiting examples of tissue-specific expression elements for neurons include synapsin (Syn) or synapsin 1 (Syn1), e.g., human synapsin or synapsin 1.

[0181] In some embodiments, the promoter may be less than 1 kb.

[0182] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters.

[0183] In some embodiments, the viral genome comprises an enhancer.

[0184] In some embodiments, the viral genome comprises an engineered promoter. Viral Genome Component: Intron and Exon Sequences

[0185] In some embodiments, the AAV viral genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, the AAV viral genome comprises at least one exon or a fragment or derivative thereof.

[0186] In some embodiments, the intron may be 100-600 nucleotides in length.

[0187] In some embodiments, the STXBP1-encoding sequence may be located downstream of an intron in an expression vector such as a beta globin intron or others known in the art. In some embodiments, the STXBP1-encoding sequence may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides downstream from a promoter comprising an intron (e.g., 3’ relative to the promoter comprising an intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the polyadenylation sequence) in an expression vector. In some embodiments, the STXBP1-encoding sequence may be located within 1-5, 1- 10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream from the intron (e.g., 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the polyadenylation sequence) in an expression vector. In some embodiments, the STXBP1-encoding sequence may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of the nucleotides downstream from the intron (e.g., 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the polyadenylation sequence) in an expression vector. In someAttorney Docket No.14640.0105-00304 embodiments, the STXBP1-encoding sequence may be located within the first 1-5%, 1-10%, 1-15%, 1- 20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the sequence downstream from the intron (e.g., 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the polyadenylation sequence) in an expression vector.

[0188] In certain embodiments, the intron sequence is not an enhancer sequence. In some embodiments, the intron sequence is not a sub-component of a promoter sequence. In some embodiments, the intron sequence is a sub-component of a promoter sequence. Viral Genome Component: Untranslated Regions (UTRs)

[0189] In some embodiments, a wildtype untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0190] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.

[0191] In some embodiments, the viral genome encoding a transgene described herein (e.g., a transgene encoding an STXBP1 protein) comprises a Kozak sequence. Without being bound by theory, wild-type 5′ untranslated regions (UTRs) include features that play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G'.

[0192] In some embodiments, the 5’UTR in the viral genome includes a Kozak sequence.

[0193] In some embodiments, the 5’UTR in the viral genome does not include a Kozak sequence.

[0194] Without being bound by theory, wild-type 3′ UTRs are known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0195] Introduction, removal or modification of 3′ UTR AU rich elements (AREs) can be used toAttorney Docket No.14640.0105-00304 modulate the stability of polynucleotides. When engineering specific polynucleotides, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0196] In some embodiments, the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

[0197] Any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location. In some embodiments, the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made with one or more other 5′ UTRs or 3′ UTRs known in the art. As used herein, the term “altered,” as it relates to a UTR, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ or 5′ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.

[0198] In some embodiments, the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild type UTR.

[0199] In some embodiments, the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property. Viral Genome Component: Polyadenylation Region

[0200] In some embodiments, the viral genome of the AAV particles of the present disclosure comprises at least one polyadenylation (polyA) sequence. In some embodiments, the polyA signal region is positioned 3’ relative to the nucleic acid comprising the STXBP1-encoding sequence. Viral Genome Component: Filler (Stuffer) Sequence

[0201] As used herein, the terms “stuffer sequence” and “filler sequence” are used interchangeably. In some embodiments, the AAV particle viral genome comprises at least one filler sequence.

[0202] In some embodiments, the viral genome comprises one or more filler sequences. The filler sequence may be a wild-type sequence or an engineered sequence. A filler sequence may be a variant of a wild-type sequence.

[0203] In some embodiments, the viral genome comprises one or more filler sequences in order to have the length of the viral genome be the optimal size for packaging. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 2.3 kb. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 4.6 kb. Viral Genome Component: STXBP1-encoding sequence

[0204] In some embodiments, the disclosure provides an AAV particle comprising a viral genomeAttorney Docket No.14640.0105-00304 encoding an STXBP1 protein, e.g., an STXBP1 protein encoded by the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the viral genome comprises a promoter operably linked to a nucleotide sequence encoding an STXBP1 protein, e.g., SEQ ID NO: 6414.

[0205] In some embodiments, the disclosure herein provides constructs that allow for improved expression of STXBP1 protein delivered by gene therapy vectors.

[0206] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of STXBP1 protein delivered by gene therapy vectors.

[0207] In some embodiments, the disclosure provides constructs that allow for improved sub-cellular distribution or trafficking of STXBP1 protein delivered by gene therapy vectors.

[0208] In some embodiments, the disclosure provides constructs that allow for improved trafficking of STXBP1 protein to lysosomal membranes delivered by gene therapy vectors.

[0209] In some embodiments, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding an STXBP1 protein or a functional fragment or variant thereof and methods of administering the composition in vitro or in vivo in a subject, e.g., a human subject and / or an animal model of disease, e.g., a disease related to expression of STXBP1.

[0210] In some embodiments, the nucleotide sequence comprises one or more, e.g., all of, a 5’ ITR sequence, an enhancer sequence, a promoter sequence, an intron sequence, a signal sequence, an STXBP1-encoding sequence, a polyA sequence, and a 3’ ITR sequence. In some embodiments, the STXBP1 protein encoded by the nucleotide sequence has an amino acid sequence that is 100% identical to a wildtype STXBP1 protein.

[0211] In some embodiments, the AAV genome comprises a payload construct that comprises a combination of coding and non-coding nucleic acid sequences.

[0212] In some embodiments, the viral genome encodes more than one payload. As a non-limiting example, a viral genome encoding more than one payload may be replicated and packaged into a viral particle. A target cell transduced with a viral particle comprising more than one payload may express each of the payloads in a single cell.

[0213] In some embodiments, the STXBP1-encoding sequence comprises a gene therapy product including, but not limited to, a polypeptide, protein, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In some embodiments, a gene therapy product may comprise a substitute for a non-functional gene or a gene that is absent, expressed in insufficient amounts, or mutated. In some embodiments, a gene therapy product may comprise a substitute for a non-functional protein or polypeptide or a protein or polypeptide that is absent, expressed in insufficient amounts, misfolded, degraded too rapidly, or mutated. For example, a gene therapy product may comprise a polynucleotide encoding an STXBP1 protein to treat STXBP1 deficiency or STXBP1-related disorders. In some embodiments, the gene therapy product comprises a polynucleotide sequence encoding an STXBP1 protein.

[0214] In some embodiments, the payload construct encodes a messenger RNA (mRNA). As usedAttorney Docket No.14640.0105-00304 herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and that is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Certain embodiments provide the mRNA as encoding STXBP1 or a variant thereof.

[0215] In some embodiments, an STXBP1-encoding sequence may comprise or encode a selectable marker. In some embodiments, a selectable marker may comprise a gene sequence or a protein or polypeptide encoded by a gene sequence expressed in a host cell that allows for the identification, selection, and / or purification of the host cell from a population of cells that may or may not express the selectable marker. In some embodiments, the selectable marker provides resistance to survive a selection process that would otherwise kill the host cell, such as treatment with an antibiotic. In some embodiments, an antibiotic selectable marker may comprise one or more antibiotic resistance factors, including but not limited to neomycin resistance (e.g., neo), hygromycin resistance, kanamycin resistance, and / or puromycin resistance.

[0216] In some embodiments, an STXBP1-encoding sequence may comprise a selectable marker including, but not limited to, β-lactamase, luciferase, β-galactosidase, or any other reporter gene as that term is understood in the art, including cell-surface markers, such as CD4 or the truncated nerve growth factor (NGFR) (for GFP, see WO 96 / 23810; Heim et al., Current Biology 2:178-182 (1996); Heim et al., Proc. Natl. Acad. Sci. USA (1995); or Heim et al., Science 373:663-664 (1995); for β-lactamase, see WO 96 / 30540); the contents of each of which are herein incorporated by reference in their entirety.

[0217] In some embodiments, an STXBP1-encoding sequence may encode a selectable marker comprising a fluorescent protein. A fluorescent protein as herein described may comprise any fluorescent marker including but not limited to green, yellow, and / or red fluorescent protein (GFP, YFP, and / or RFP). In some embodiments, STXBP1-encoding sequence may encode a selectable marker comprising a human influenza hemagglutinin (HA) tag.

[0218] In certain embodiments, a nucleic acid for expression of STXBP1 protein in a target cell as described herein will be incorporated into the viral genome and located between two ITR sequences. Viral Genome Component: Signal Sequence

[0219] In some embodiments, the viral genome encoding an STXBP1 protein further comprises a nucleotide sequence encoding a signal sequence.

[0220] In some embodiments, the nucleotide sequence encoding the signal sequence is located 5’ relative to the nucleotide sequence encoding the STXBP1 protein. In some embodiments, the encoded STXBP1 protein comprises a signal sequence at the N-terminus, wherein the signal sequence is optionally cleaved during cellular processing and / or localization of the STXBP1 protein. Exemplary STXBP1 Payload

[0221] In some embodiments, the encoded STXBP1 protein is a wildtype STXBP1 protein.

[0222] Tables 7 and 8 provide exemplary polynucleotide sequences encoding an STXBP1 protein and polypeptide sequences of exemplary STXBP1 proteins that may be used in the viral genomesAttorney Docket No.14640.0105-00304 disclosed herein and which may constitute an STXBP1 protein payload. In some embodiments, the STXBP1 protein suitable for delivery in an AAV disclosed herein is encoded by the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the STXBP1 protein suitable for delivery in an AAV disclosed herein is encoded by the nucleotide sequence of SEQ ID NO: 6416, 6418, 6420, 6422, 6424, 6426, 6428, 6430, 6432, 6434, or 6436. Table 7. Exemplary STXBP1 Sequences SEQ ID NO: Type Species Description 6413 Protein Homo sapiens STXBP1 protein NP_001027392.1 “Attorney Docket No.14640.0105-00304 6423 Protein Homo sapiens STXBP1 protein NP_001361239.1 “syntaxin-binding protein 1 isoform e [Homo sapiens]”, National Center forAttorney Docket No.14640.0105-00304 “syntaxin-binding protein 1 isoform a [Homo sapiens]”, National Center for Biotechnology Information, Mar 18, 2023 Table 8Description Sequence SEQ ID NO: TXBP1 tAT ATT T AAA T TT T A A AA ATTAT AT414Attorney Docket No.14640.0105-00304 HIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQYQKELSK YSTHLHLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVP ILLDANVSTYDKIRIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITInformation cited in Tables 7 and 8 is hereby incorporated by reference in its entirety.

[0224] In some embodiments, the present disclosure provides a viral genome comprising an STXBP1-encoding sequence comprising SEQ ID NO: 6414 or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence is at least 95% identical to SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence is at least 99% identical to SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence comprises SEQ ID NO: 6414. In some embodiments, the STXBP1-encoding sequence consists of SEQ ID NO: 6414.

[0225] In some embodiments, the AAV viral genome further comprises a nucleic acid encoding a capsid protein, e.g., a structural protein. In some embodiments, the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and / or a VP3 polypeptide. In some embodiments, the VP1 polypeptide, the VP2 polypeptide, and / or the VP3 polypeptide are encoded by at least one Cap gene. In some embodiments, the AAV viral genome further comprises a nucleic acid encoding a Rep protein, e.g., a non-structural protein. In some embodiments, the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and / or a Rep40 protein. In some embodiments, the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein are encoded by at least one Rep gene.

[0226] In some embodiments, the capsid comprises the amino acid sequence of SEQ ID NO: 981.

[0227] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 6414, or a sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity thereto, comprises a capsid comprising an amino acid sequence selected from Table 3 or Table 4.

[0228] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 6414, or a sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity thereto, comprises a capsid comprising (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at leastAttorney Docket No.14640.0105-00304 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and / or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises T at amino acid 584, D at amino acid 586, W at amino acid 587, H at amino acid 588, R at amino acid 589, and / or I at amino acid 590 of the amino acid sequence of SEQ ID NO: 981.

[0229] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, numbered according to SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0230] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid T at a position corresponding to position 584 of SEQ ID NO: 981, D at a position corresponding to position 586 of SEQ ID NO: 981, W at a position corresponding to position 587 of SEQ ID NO: 981, H at a position corresponding to position 588 of SEQ ID NO: 981, R at a position corresponding to position 589 of SEQ ID NO: 981, and I at a position corresponding to position 590 of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence SEQ ID NO: 981, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0231] In some embodiments, the present disclosure provides an AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 6414 and an AAV capsid variant comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 981.

[0232] In some embodiments, the present disclosure provides an AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 6414 and an AAV capsid variant comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981.Attorney Docket No.14640.0105-00304

[0233] In some embodiments, the present disclosure provides an AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 6414 and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981.

[0234] The present disclosure provides in some embodiments, vectors, cells, and / or AAV particles comprising the above identified viral genomes. Self-Complementary and Single Stranded Viral Genomes

[0235] In some embodiments, the AAV viral genome used in the present disclosure is single- stranded (ssAAV).

[0236] In some embodiments, the AAV viral genome is capable of forming double-stranded DNA. In some embodiments, the AAV viral genome is self-complementary. See, e.g., US Patent No.7,465,583. scAAV particles contain both DNA strands that anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[0237] Methods for producing and / or modifying AAV viral genome and particles are disclosed in the art such as pseudotyped AAV particles (International Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO 2005005610 and WO 2005072364, the content of each of which are incorporated herein by reference in their entirety). II. AAV Production

[0238] Viral production disclosed herein describes processes and methods for producing AAV particles (with enhanced, improved and / or increased tropism for a target tissue), e.g., an AAV particle comprising an AAV capsid variant that may be used to contact a target cell to deliver STXBP1.

[0239] In some embodiments, disclosed herein is a method of making an AAV particle of the present disclosure, e.g., an AAV particle comprising an AAV capsid variant disclosed herein, wherein the method comprises: (i) providing a cell comprising a viral genome described herein (e.g., a viral genome comprising an STXBP1-encoding sequence and a nucleic acid encoding an AAV capsid variant disclosed herein) and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant, e.g., an AAV capsid variant described herein (e.g., an AAV capsid variant listed in Tables 3, 4, or 5), thereby making the AAV particle. In some embodiments, the viral genome comprises an STXBP1-encoding sequence comprising SEQ ID NO: 6414. In some embodiments, the AAV capsid variant comprises an amino acid sequence of SEQ ID NO: 981. In some embodiments, the viral genome comprises an STXBP1-encoding sequence comprising SEQ ID NO: 6414 and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981. In some embodiments, the method comprises, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell. In some embodiments, the method comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell. In some embodiments, the AAV particle described herein is an isolated AAV particle. In some embodiments, the AAV particle described herein is a recombinant AAV particle.Attorney Docket No.14640.0105-00304

[0240] Any method known in the art may be used for the preparation of AAV particles. In some embodiments, AAV particles are produced in mammalian cells (e.g., HEK293 cells). In some embodiments, AAV particles are produced in insect cells (e.g., Sf9 cells).

[0241] Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353 and WO2001023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir.63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir.66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir.272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety. III. Pharmaceutical Compositions

[0242] In some embodiments, the present disclosure provides pharmaceutical compositions of an adeno-associated virus (AAV) particle comprising a viral genome comprising an STXBP1-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 6414 and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981.

[0243] In some embodiments, a composition described herein comprises an AAV polynucleotide or AAV genome or AAV particle, and at least one excipient.

[0244] Although pharmaceutical compositions provided herein, e.g., comprising AAV particles comprising an STXBP1-encoding sequence, are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions may be suitable for administration to any other animal, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various non-human animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0245] In some embodiments, compositions are administered to humans, e.g., human patients or human subjects.Attorney Docket No.14640.0105-00304

[0246] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one STXBP1-encoding sequence. In some embodiments, the formulations may contain a nucleic acid encoding 1, 2, 3, 4, or 5 STXBP1-encoding sequences. In some embodiments, the formulation may contain STXBP1-encoding sequences encoding STXBP1 proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic proteins, cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, and / or proteins associated with non-human diseases. In some embodiments, the AAV formulation comprises at least one sequence encoding human STXBP1. In some embodiments, the AAV formulation comprises at least one sequence encoding wildtype human STXBP1.

[0247] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. IV. Formulations

[0248] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0249] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0250] For example, the composition may comprise about 0.1% to about 99% (w / w) of the active ingredient. By way of example, the composition may comprise about 0.1% to about 100%, e.g., about 0.5% to about 50%, about 1% to about 30%, about 5% to about 80%, or at least 80% (w / w) active ingredient.

[0251] The AAV particles of the disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; (6) alter the release profile of encoded protein in vivo and / or (7) allow for regulatable expression of STXBP1.Attorney Docket No.14640.0105-00304

[0252] Formulations of the present disclosure can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present disclosure may be formulated using self- assembled nucleic acid nanoparticles.

[0253] In some embodiments, the viral vectors encoding STXBP1 may be formulated to optimize baricity and / or osmolality. In some embodiments, the baricity and / or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system. Excipients

[0254] The formulations of the disclosure can include one or more excipients, each in an amount that together increases the stability of the AAV particle, increases cell transfection or transduction by the viral particle, increases the expression of viral particle encoded protein, and / or alters the release profile of AAV particle encoded proteins. In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0255] Excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the contents of which are herein incorporated by reference in their entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Inactive Ingredients

[0256] In some embodiments, AAV formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in formulations. In some embodiments, all, none, or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).Attorney Docket No.14640.0105-00304

[0257] Formulations of AAV particles may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, or combinations thereof. In some embodiments, formulations may include polymers or polynucleotides complexed with a metal cation (See, e.g., U.S. Pat. Nos.6,265,389 and 6,555,525, the contents of each of which are herein incorporated by reference in their entirety). V. Uses and Applications

[0258] The compositions of the disclosure may be administered to a subject, e.g., to deliver STXBP1, e.g., to a subject who has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder. A STXBP-related disorder refers to any disease, disorder, or condition associated with or caused by STXBP1 protein deficiency, such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). The compositions may similarly be used in the manufacture of a medicament for administration to a subject having an STXBP1-related disorder (e.g., an STXBP1-related neurodegenerative or neuromuscular disorder). In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy.

[0259] In some embodiments, the disclosure provides a method of delivering an STXBP1 protein to a subject comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding an STXBP1 protein, thereby delivering the STXBP1 protein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder. The STXBP1-related disorder may be an STXBP1- related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy.

[0260] In some embodiments, the disclosure provides a method for treating an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder. In some embodiments, the disclosure provides an AAV particle or pharmaceutical composition according to any one of the embodiments disclosed herein for treating an STXBP1-related disorder, such as STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In someAttorney Docket No.14640.0105-00304 embodiments, the present disclosure provides the pharmaceutical composition or the AAV particle of any one the embodiments disclosed herein for use in a method of treating a disorder as disclosed herein, such as STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In some embodiments, the disorder is STXBP1 encephalopathy.

[0261] In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding STXBP1 may be administered to a subject to treat an STXBP1- related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder, thereby treating the STXBP1-related disorder. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder such as an STXBP1-related neurodegenerative or neuromuscular disorder (e.g., STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox- Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5)). In some embodiments, the treatment results in an increase in the subject’s STXBP1 protein level as compared to baseline.

[0262] In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gaustaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5). In some embodiments, the STXBP1-related disorder is STXBP1 encephalopathy.

[0263] In some embodiments, the disclosure provides a method of treating STXBP1 encephalopathy in a subject. In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding STXBP1 may be administered to a subject to treat STXBP1 encephalopathy. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having STXBP1 encephalopathy.

[0264] A subject may have one or more mutations in the STXBP1 gene. In some embodiments, the subject has lower STXBP1 activity as compared to STXBP1 activity in an individual who does not have an STXBP1-related disorder.

[0265] In some embodiments, the treatment may result in prevention of progression of the STXBP1- related disorder. For example, the treatment may result in in amelioration of at least one symptom of the disorder, as indicated by one or more biomarkers. In some embodiments, the one or more biomarkers comprises increased release of the neurotransmitters glutamate and / or GABA or reduction in abnormal electroencephalographic activity as evidence of improved STXBP1 activity.Attorney Docket No.14640.0105-00304

[0266] In certain embodiments, the treatment improves at least one symptom of an STXBP1-related disorder, e.g., epilepsy, autistic features, ataxia, generalized tremors, reduced STXBP1 activity, accumulation of glucocerebroside and other glycolipids, e.g., within immune cells (e.g., macrophages), build-up of synuclein aggregates (e.g., Lewy bodies), developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor dysfunction, bulbar palsy, generalized weakness, trembling of a limb, depression, visual hallucinations, cognitive decline, dystonia, or a combination thereof.

[0267] In some embodiments, the methods disclosed herein further comprise evaluating, e.g., measuring, the level of STXBP1 expression, e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. STXBP1 protein expression may be measured by an ELISA, a Western blot, or an immunohistochemistry assay. In some embodiments, evaluating the level of STXBP1 expression is performed prior to and / or subsequent to administration of the AAV particle, optionally wherein the level of STXBP1 expression prior to treatment is compared to the level of STXBP1 expression subsequent to administration. In some embodiments, the level of STXBP1 expression may be evaluated in a cell or tissue of the central nervous system. In some embodiments, the level of STXBP1 expression may be evaluated in a cell of a peripheral tissue (e.g., liver, heart, muscle, or spleen). In some embodiments, the cell of a peripheral tissue is a muscle cell. In some embodiments, the subject’s level of STXBP1 protein expression subsequent to administration is increased relative to the subject’s level of STXBP1 protein expression prior to administration.

[0268] In some embodiments, the administration of the effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding an STXBP1 protein may be a treatment that results in an increase in: (i) the level of STXBP1 activity in a cell, tissue, (e.g., a cell or tissue of the CNS and / or the PNS, e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron), and / or fluid (e.g., CSF and / or serum), of the subject, relative to baseline and / or relative to the level of STXBP1 activity in a cell, tissue, or fluid of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or (ii) the level and / or number of viral genomes (VG) per cell in a tissue of the CNS and / or the PNS, e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferiorAttorney Docket No.14640.0105-00304 olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron) of the subject relative to the number and / or level of VG per cell in a peripheral tissue of the subject; and / or (iii) the level of STXBP1 mRNA expression in a cell or tissue (e.g., a cell or tissue of the CNS and / or the PNS, e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject relative to baseline and / or relative to the level of STXBP1 mRNA expression in a cell or tissue of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle.

[0269] In some embodiments, at least one additional agent suitable for treatment of an STXBP1- related disorder may be administered together with the effective amount of a pharmaceutical composition or AAV particle disclosed herein (comprising a viral genome encoding an STXBP1 protein). The at least one additional agent may comprise one or more anti-epileptic drugs (e.g., bromide, clobazam, felbamate, ganaxolone, lamotrigine, levetiracetam, phenobarbital, topiramate, valproate, or a combination thereof).

[0270] In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and / or modify their metabolism, and / or modify their distribution within the body.

[0271] In certain embodiments, the pharmaceutical compositions described herein are used as research tools, particularly in in vitro investigations using human cell lines such as HEK293T and in vivo testing in nonhuman primates which will occur prior to human clinical trials.

[0272] The present disclosure provides a method for treating a disease, disorder and / or condition in a mammalian subject, including a human subject, comprising administering to the subject any of the viral particles, e.g., AAV, AAV particles, or AAV genome that produces STXBP1 protein described herein (i.e., viral genomes or “VG”), administering to the subject a composition or formulation comprising said AAV particle.

[0273] Delivery of a payload construct comprising an STXBP1-encoding sequence may alleviate or reduce symptoms that result from abnormal level and / or function of a gene product (e.g., an absence or defect in a protein) in a subject in need thereof or that otherwise confers a benefit to a CNS disorder in a subject in need thereof.Attorney Docket No.14640.0105-00304 VI. Delivery of AAV Particles Delivery to Cells

[0274] In some aspects, the present disclosure provides a method of delivering to a cell or tissue any of the above-described AAV particles, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions. The method of delivering the AAV particle to a cell or tissue can be accomplished in vitro, ex vivo, or in vivo.

[0275] In some embodiments, the AAV particles are delivered to a cell, tissue, or region of the CNS. In some embodiments, the AAV particles are delivered to a cell or tissue of the amygdala, brainstem, caudate, ce...

Claims

Attorney Docket No.14640.0105-00304 CLAIMS 1. An adeno-associated virus (AAV) particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises a syntaxin-binding protein 1 (STXBP1)-encoding sequence and the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII; wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein: (i) [N1] comprises amino acids X1, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H; and / or (ii) [N3] comprises amino acids X5, X6, and X7, wherein X5is I, V, T, M, S, N, L, or F.

2. The AAV particle of claim 1, wherein the AAV capsid variant is an AAV9 capsid variant.

3. The AAV particle of claim 1 or claim 2, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to position 203-736 of the amino acid sequence of SEQ ID NO:

981.

4. The AAV particle of any one of claims 1-3, wherein loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO:

981.

5. The AAV particle of any one of claims 1-4, wherein: (i) X1is T, S, R, A, I, C, N, K, L, or Q; (ii) X2is N, T, G, V, S, Y, K, I, H, D, or F; and (iii) X3is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C.

6. The AAV particle of any one of claims 1-5, wherein: (i) X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and (ii) X7is Q, G, N, K, H, R, E, L, P, or M.

7. The AAV particle of any one of claims 1-6, wherein [N1] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688).

8. The AAV particle of any one of claims 1-7, wherein [N3] comprises the amino acid sequence of IAQ.

9. The AAV particle of any one of claims 1-8, wherein [N1]-[N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).Attorney Docket No.14640.0105-00304 10. The AAV particle of any one of claims 1-9, wherein: (i) [N1] is present at amino acids corresponding to positions 582-585 of the amino acid sequence of SEQ ID NO: 981; (ii) [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 981; and (iii) [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO:

981.

11. The AAV particle of any one of claims 1-10, wherein the AAV capsid variant further comprises [N4] comprising amino acids X8, X9, X10, and X11, wherein: (i) X8 is T, S, N, P, A, or I; (ii) X9is G, N, D, R, V, A, S, or Q; (iii) X10is W, S, C, R, L, or G; and / or (iv) X11is V, A, S, I, C, G, D, F, L, or T.

12. The AAV particle of claim 11, wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066).

13. The AAV particle of claim 11 or claim 12, wherein [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO:

981.

14. The AAV particle of any one of claims 11-13, wherein [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).

15. An adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981;Attorney Docket No.14640.0105-00304 R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO:

981.

16. The AAV particle of any one of claims 1-15, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% to positions 138-736 of the amino acid sequence of SEQ ID NO:

981.

17. An adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO:

981.

18. The AAV particle of any one of claims 1-17, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

981.

19. An adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a syntaxin-binding protein 1 (STXBP1)-encoding sequence; and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981;Attorney Docket No.14640.0105-00304 H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO:

981.

20. The adeno-associated virus (AAV) particle of claim 19, wherein the AAV capsid variant comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 981; (ii) an amino acid sequence that is at least 99% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) an amino acid sequence that is at least 99% identical to positions 203-736 of the amino acid sequence of SEQ ID NO:

981.

21. The AAV particle of any one of claims 15-20, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

22. The AAV particle of any one of claims 15-21, wherein the AAV capsid variant comprises at least 8, at least 9, or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).

23. The AAV particle of any one of claims 15-22, wherein the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO:

981.

24. The AAV particle of any one of claims 21-23, wherein the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO:

981.

25. The AAV particle of any one of claims 1-24, wherein the AAV capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 981; (ii) the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.Attorney Docket No.14640.0105-00304 26. The AAV particle of any one of claims 1-25, wherein the viral genome encodes a wildtype STXBP1 protein or a fragment thereof.

27. The AAV particle of any one of claims 1-26, wherein the viral genome encodes a human STXBP1 protein.

28. The AAV particle of claim 26 or claim 27, wherein the STXBP1 protein comprises the amino acid sequence of SEQ ID NO: 6413.

29. The AAV particle of any one of claims 1-28, wherein the STXBP1-encoding sequence is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 6414.

30. The AAV particle of claim 29, wherein the STXBP1-encoding sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO: 6414.

31. The AAV particle of claim 30, wherein the STXBP1-encoding sequence is at least 99% identical to the nucleotide sequence of SEQ ID NO: 6414.

32. The AAV particle of claim 31, wherein the STXBP1-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6414.

33. The AAV particle of claim 32, wherein the STXBP1-encoding sequence consists of the nucleotide sequence of SEQ ID NO: 6414.

34. The AAV particle of any one of claims 1-33, wherein the viral genome comprises a promoter operably linked to the STXBP1-encoding sequence.

35. The AAV particle of any one of claims 1-34, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence.

36. The AAV particle of claim 35, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1-encoding sequence.

37. The AAV particle of claim 35 or claim 36, wherein the viral genome comprises an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence.Attorney Docket No.14640.0105-00304 38. The AAV particle of any one of claims 35-37, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the STXBP1-encoding sequence, and an ITR sequence positioned 3’ relative to the STXBP1-encoding sequence.

39. A cell comprising the AAV particle of any one of claims 1-38, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

40. A method of making the AAV particle of any one of claims 1-38, the method comprising: (i) providing a cell comprising the viral genome comprising an STXBP1-encoding sequence and a nucleic acid encoding the AAV capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the AAV particle.

41. The method of claim 40, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto; and wherein the AAV capsid variant comprises (i) the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and / or (iii) the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

42. The method of claim 41, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ IDAttorney Docket No.14640.0105-00304 NO: 981, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO:

981.

43. The method of claim 41, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 6414 and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO:

981.

44. The method of any one of claims 40-43, further comprising, prior to step (i), introducing a nucleic acid molecule comprising the viral genome into the cell.

45. The method of any one of claims 40-44, further comprising, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell.

46. The method of any one of claims 40-45, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

47. A pharmaceutical composition comprising the AAV particle of any one of claims 1-38 and a pharmaceutically acceptable excipient.

48. A method of delivering an AAV particle encoding an STXBP1 protein to a cell, comprising administering an effective amount of the pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38.

49. The method of claim 48, wherein the cell is in a subject, optionally wherein the subject has, has been diagnosed with having, or is at risk of having an STXBP1-related disorder, further optionally wherein the STXBP1-related disorder is an STXBP1-related neurodegenerative or neuromuscular disorder.

50. The method of claim 49, wherein the subject has, has been diagnosed with having, or is at risk of having STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).Attorney Docket No.14640.0105-00304 51. A method of treating an STXBP1-related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38.

52. The method of claim 51, wherein the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder.

53. The method of claim 52, wherein the STXBP1-related disorder is an STXBP1-related neurodegenerative or neuromuscular disorder.

54. The method of claim 53, wherein the STXBP1-related neurodegenerative or neuromuscular disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

55. A method of treating an STXBP1-related disorder in a subject, wherein the disorder is STXBP1 encephalopathy, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38.

56. The method of claim 55, wherein the subject has, has been diagnosed with having, or is at risk of having STXBP1 encephalopathy.

57. The method of any one of claims 51-56, wherein the subject has one or more mutations in the STXBP1 gene.

58. The method of any one of claims 51-57, wherein the subject has lower STXBP1 activity as compared to STXBP1 activity in an individual who does not have an STXBP1-related disorder.

59. The method of any one of claims 51-58, wherein the treating results in prevention of progression of the disorder in the subject.

60. The method of any one of claims 51-59, wherein the treating results in amelioration of at least one symptom of the disorder in the subject, as indicated by one or more biomarkers.Attorney Docket No.14640.0105-00304 61. The method of claim 60, wherein the one or more biomarkers comprises (i) increased release of the neurotransmitters glutamate and / or GABA or (ii) reduction in abnormal electroencephalographic activity as evidence of improved STXBP1 activity.

62. The method of claim 61, wherein the at least one symptom comprises epilepsy, autistic features, ataxia, generalized tremors, dystonia, or a combination thereof.

63. The method of any one of claims 49-62, wherein the subject is a human.

64. The method of any one of claims 49-63, wherein the AAV particle is delivered to a cell, tissue, or region of the central nervous systems (CNS) of the subject.

65. The method of claim 64, wherein the cell, tissue, or region of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron), tissue, or region of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region).

66. The method of any one of claims 49-65, wherein the AAV particle or the pharmaceutical composition is delivered to the subject via intravenous administration.

67. The method of any one of claims 49-66, further comprising evaluating, e.g., measuring, the level of STXBP1 expression, e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject.

68. The method of claim 67, wherein the level of STXBP1 protein expression is measured by an enzyme- linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.

69. The method of claim 67 or claim 68, wherein the evaluating the level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) prior to administration is compared to theAttorney Docket No.14640.0105-00304 subject’s level of STXBP1 expression (e.g., STXBP1 gene expression, STXBP1 mRNA expression, and / or STXBP1 protein expression) subsequent to administration.

70. The method of any one of claims 67-69, comprising evaluating the level of STXBP1 expression in a cell or tissue of the CNS.

71. The method of claim 70, wherein the cell or tissue of the CNS is a cell (e.g., a neuron, e.g., a GABAergic neuron and / or a glutamatergic neuron) or tissue of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region).

72. The method of any one of claims 67-71, wherein the subject’s level of STXBP1 protein expression subsequent to administration is increased relative to the subject’s level of STXBP1 protein expression prior to administration.

73. The method of any one of claims 49-72, further comprising evaluating, e.g., measuring, the level of STXBP1 activity in the subject, e.g., in a cell or tissue of the subject.

74. The method of any one of claims 49-73, wherein administering the pharmaceutical composition or AAV particle to the subject results in an increase in: (i) the level of STXBP1 activity in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) and / or fluid (e.g., CSF and / or serum) of the subject, relative to baseline and / or relative to the level of STXBP1 activity in a cell, tissue, or fluid of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle; (ii) the number and / or level of viral genomes (VG) per cell level in a tissue of the CNS (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex),Attorney Docket No.14640.0105-00304 external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject, relative to the number and / or level of VG per cell in a peripheral tissue of the subject; and / or (iii) the level of STXBP1 protein or STXBP1 gene expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), and / or a neuron (e.g., a GABAergic neuron and / or a glutamatergic neuron)) of the subject relative to baseline and / or relative to the level of STXBP1 protein or STXBP1 gene in a cell or tissue of an individual with an STXBP1-related disorder who has not been administered the pharmaceutical composition or AAV particle.

75. The method of any one of claims 49-74, further comprising administering to the subject at least one additional agent and / or therapy.

76. The method of claim 75, wherein the at least one additional agent and / or therapy comprises an agent and / or therapy suitable for treating the STXBP1-related disorder.

77. The method of claim 76, wherein the at least one additional agent and / or therapy comprises one or more anti-epileptic drugs (e.g., bromide, clobazam, felbamate, ganaxolone, lamotrigine, levetiracetam, phenobarbital, topiramate, valproate, or a combination thereof).

78. The method of any one of claims 49-77, further comprising administering an immunosuppressant to the subject.

79. The method of claim 78, wherein the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.

80. The pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38 for use in a method of treating a disorder according to any one of claims 51-79.Attorney Docket No.14640.0105-00304 81. The pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38, for use in the treatment of an STXBP1-related disorder in a subject; optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5) 82. The pharmaceutical composition or AAV particle for use of claim 81, wherein the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, optionally wherein the STXBP-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox- Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

83. Use of the pharmaceutical composition of claim 47 or the AAV particle of any one of claims 1-38 in the manufacture of a medicament for the treatment of an STXBP1-related disorder in a subject; optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

84. The use of claim 83, wherein the subject has, has been diagnosed with having, or is at risk of having the STXBP1-related disorder, optionally wherein the STXBP1-related disorder is STXBP1 encephalopathy, epileptic encephalopathy, Ohtahara syndrome, developmental encephalopathy, West syndrome, early myoclonic epileptic encephalopathy, Lennox-Gastaut syndrome, autism (e.g., autism with STXBP1 mutations and optionally further mutations), Dravet syndrome (not caused by mutations in SCN1A), or Rett syndrome phenotype (not caused by mutation of MECP2 or CDKL5).

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