[go: up one dir, main page]

WO2025122548A1 - Compositions et méthodes pour le traitement de troubles liés à une déficience en cdkl5 - Google Patents

Compositions et méthodes pour le traitement de troubles liés à une déficience en cdkl5 Download PDF

Info

Publication number
WO2025122548A1
WO2025122548A1 PCT/US2024/058361 US2024058361W WO2025122548A1 WO 2025122548 A1 WO2025122548 A1 WO 2025122548A1 US 2024058361 W US2024058361 W US 2024058361W WO 2025122548 A1 WO2025122548 A1 WO 2025122548A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
cdkl5
aav
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/058361
Other languages
English (en)
Inventor
Mathieu Emmanuel NONNENMACHER
Jing Lin
Hongxing Wang
Jinzhao Hou
Wei Wang
Jiangyu LI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Voyager Therapeutics Inc
Original Assignee
Voyager Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Voyager Therapeutics Inc filed Critical Voyager Therapeutics Inc
Publication of WO2025122548A1 publication Critical patent/WO2025122548A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11022—Cyclin-dependent kinase (2.7.11.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2857—Seizure disorders; Epilepsy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • compositions and methods relating to adeno-associated virus (AAV) viral particles for the delivery of polynucleotides, e.g., polynucleotides encoding cyclin-dependent kinase-like 5 (CDKL5) proteins and peptides for use in the treatment of a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome (collectively, CDKL5- related disorders)).
  • a CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome (collectively, CDKL5- related disorders)
  • compositions described herein may be used to treat a subject in need thereof, such as a human subject diagnosed with a CDKL5-related disorder or other condition resulting from a deficiency in the quantity and/or function of CDKL5 protein, or as a research tool in the study of diseases or conditions in cells or animal models of a CDKL5-related disorder.
  • a subject in need thereof such as a human subject diagnosed with a CDKL5-related disorder or other condition resulting from a deficiency in the quantity and/or function of CDKL5 protein
  • CDKL5 Cyclin-dependent kinase-like 5
  • CDKL5 Endoclin-dependent kinase-like 5
  • CDKL5 protein is an enzyme and is thought to play an important role in brain development and regulation of response to oxidative stress. CDKL5 is thought to be expressed throughout the cell, including in the nucleus and cytoplasm of soma and dendrites. [06] CDKL5 is responsible for phosphorylation of a number of targets. CDKL5 may target and phosphorylate the gene MECP2, which has been characterized as important in the function of neurons and other brain cells, and in the maintenance of neuronal synapses, and is thought to be the causative agent for Rett syndrome. CDKL5 may also target and phosphorylate CEP131, which is thought to play a role in cell proliferation.
  • CDKL5 may also target and phosphorylate MAP1S, DLG5, EB2, and/or ARHGEF, which are microtubule associated proteins, thought to play roles in neuronal division, differentiation, migration, and neurite growth. CDKL5 may also target and phosphorylate AKT and mTOR, which are thought to play roles in cell proliferation, migration, and development. CDKL5 may Attorney Docket No.14640.0106-00304 act on targets in both the cytoplasm and in the nucleus of the cell. In the cytoplasm, CDKL5 may affect RAC1 signaling and dendritic arborization.
  • CDKL5 activity may mediate effects on gene expression, DNA methylation, mRNA splicing, and more, through targets such as MeCP2, DNM1, or SC35.
  • CDKL5 may be involved in the formation, growth, and migration of neurons. It may also play a role in cell division and/or transmission of chemical signals at neuronal synapses.
  • Mutations in CDKL5 are known to cause disease in subjects, e.g., human subjects. For example, CDKL5 mutations are one of the most common causes for genetic epilepsies, including epileptic encephalopathies.
  • CDK5-related epilepsies are often early-onset epilepsies that manifest in infants before 3 months of age.
  • Epilepsy may occur in three typical stages: (i) early epilepsy with focal seizures at the age of 4 weeks to 6 months, (ii) epileptic encephalopathy with infantile spasms in infancy and early childhood and (iii) tonic seizures and late myoclonic epilepsy in childhood. Movement disorders (hand-wringing stereotypy, chorea and ballisms) are also a reported feature.
  • Developmental epileptic encephalopathies associated with CDKL5 mutations may be severe and treatment-resistant.
  • CDKL5 mutations have been associated with disorders such as infantile spasms (also called West syndrome), Lennox Gastaut syndrome, Rett syndrome, autism, early-onset encephalopathy, and X- linked mental retardation. CDKL5 mutations may also cause a clinical phenotype that overlaps with Rett syndrome. [010] In addition, CDKL5 mutations may lead to CDKL5 deficiency disorder (CDD), a neurodevelopmental disorder characterized by nervous system symptoms including epilepsy (e.g., early- onset epilepsy), low muscle tone, and developmental challenges. CDD may be characterized by autism, deficits in cognition, limited motor skills, sleep difficulties, visual impairment, and/or gastrointestinal reflux.
  • CDD CDKL5 deficiency disorder
  • CDD may be characterized by autism, deficits in cognition, limited motor skills, sleep difficulties, visual impairment, and/or gastrointestinal reflux.
  • CDD can manifest with a broad array of clinical manifestations.
  • Clinical manifestations of CDD comprise behavioral symptoms such as episodes of laughing or crying that occur for what appears to be no reason, hypersensitivity to touch, and disrupted sleep.
  • Clinical manifestations also comprise facial appearance changes including microcephaly; a high, broad forehead; large, deep-set eyes; smaller- than normal space between the nose and upper lip; an upturned nose; full lips; and widely spaced teeth.
  • Further clinical manifestations include difficulties standing and walking; small, cold feet; lack of or poor eye contact; frequent sideways glances; and cortical visual impairment or cortical blindness.
  • CDD has an incidence of 1 in 42000 births in the USA, and 85% of cases occur in females. Typically, CDD patients are fully reliant on caregivers for the duration of their lives. [013] CDD is typically caused by de novo mutations in CDKL5. CDKL5 mutations in CDD patients result in a reduced amount of functional CDKL5 protein. Attorney Docket No.14640.0106-00304 [014] Existing treatments for patients with CDD focus on alleviating symptoms such as seizures.
  • Second line treatment for refractory patients comprises first line treatment plus additional anti-epileptic drugs such as clobazam and/or lamotrigine in combination with ganaxolone.
  • Adeno-associated viruses have emerged as 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).
  • 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 CDKL5, to a target cell or tissue, e.g., a CNS cell or tissue.
  • compositions and methods directed to AAV-based gene delivery of CDKL5 to ameliorate loss-of-function as well as compositions and methods directed to AAV-based gene delivery of CDKL5 to improve neurotransmitter release.
  • compositions and methods are useful to improve presynaptic vesicle release, and to slow, halt, or reverse neurodegenerative and/or other symptoms of CDKL5 deficiency disorder (CDD) or a CDKL5-related disorder in a subject (e.g., a subject having one or more mutations in CDKL5).
  • CDD CDKL5 deficiency disorder
  • a CDKL5-related disorder in a subject
  • the present disclosure provides an AAV particle comprising an AAV capsid and a nucleotide sequence encoding a CDKL5 protein, also referred to herein as a CDKL5-encoding sequence.
  • the nucleotide sequence encoding a CDKL5 protein is comprised in a viral genome.
  • the CDKL5 protein is a human CDKL5 protein. In some embodiments, the CDKL5 protein is a wildtype human CDKL5 protein. In some embodiments, the AAV Attorney Docket No.14640.0106-00304 particle comprises a viral genome encoding the CDKL5 protein (e.g., a human CDKL5 protein) and the AAV capsid is an AAV capsid variant. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.
  • 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 cyclin- dependent kinase-like 5 (CDKL5)-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 X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q, K, E, S, P, R, N, or H; and/or [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I, V, T, M, S, N, L, or F.
  • AAV adeno-associated virus
  • the AAV capsid variant is an AAV9 capsid variant.
  • 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.
  • loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981.
  • X 1 is T, S, R, A, I, C, N, K, L, or Q
  • X 2 is N, T, G, V, S, Y, K, I, H, D, or F
  • X 3 is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C
  • X 6 is A, Y, P, N, S, T, G, E, V, W, F, or Q
  • X 7 is Q, G, N, K, H, R, E, L, P, or M.
  • [N1] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688).
  • [N3] comprises the amino acid sequence of IAQ.
  • [N1]-N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).
  • [N1] is present at amino acids corresponding to positions 582-585 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
  • [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 981.
  • the AAV capsid variant further comprises [N4] comprising amino acids X 8 , X 9 , X 10 , and X 11 , wherein: X 8 is T, S, N, P, A, or I; X 9 is G, N, D, R, V, A, S, or Q; X 10 is W, S, C, R, L, or G; and/or X 11 is V, A, S, I, C, G, D, F, L, or T.
  • [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066).
  • [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 981. [025] In some embodiments, [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).
  • the present disclosure provides an AAV particle comprising a viral genome comprising a CDKL5-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 amino acid sequence of SEQ ID NO: 981, W at an amino acid corresponding to position 587 of the amino acid Attorney Docket No.14640.0106-00304 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.
  • the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981.
  • the present disclosure provides an AAV particle comprising a viral genome comprising a CDKL5-encoding sequence and an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to postitions 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:
  • 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.
  • the present disclosure provides an AAV particle comprising a viral genome comprising a CDKL5-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
  • the present disclosure provides an AAV particle comprising a viral genome comprising a CDKL5-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.
  • the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).
  • 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). 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 Attorney Docket No.14640.0106-00304 of SEQ ID NO: 981.
  • 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.
  • 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.
  • the viral genome encodes a wildtype CDKL5 protein or a fragment thereof. In some embodiments, the viral genome encodes a human CDKL5 protein. In some embodiments, the CDKL5 protein comprises the amino acid sequence of SEQ ID NO: 6413. [033] In some embodiments, viral genome comprises a CDKL5-encoding 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 6414.
  • CDKL5-encoding 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ
  • the CDKL5-encoding sequence may be at least 95% identical to the nucleotide sequence of SEQ ID NO: 6414 or the CDKL5-encoding sequence may be at least 99% identical to the nucleotide sequence of SEQ ID NO: 6414.
  • the CDKL5-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6414.
  • the CDKL5- encoding sequence consists of the nucleotide sequence of SEQ ID NO: 6414.
  • the viral genome comprises a promoter operably linked to the CDKL5-encoding sequence.
  • the viral genome further comprises an inverted terminal repeat (ITR) sequence.
  • the viral genome comprises an ITR sequence positioned 5’ relative to the CDKL5-encoding sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 3’ relative to the CDKL5-encoding sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative to the CDKL5- encoding sequence, and an ITR sequence positioned 3’ relative to the CDKL5-encoding sequence. [035] In some aspects, the present disclosure provides a cell comprising the AAV particle of any one of the preceding embodiments.
  • the cell is a mammalian cell, e.g., an HEK293 cell, an insect cell, e.g., an Sf9 cell, or a bacterial cell.
  • 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 a CDKL5-encoding sequence provided herein and a nucleic acid encoding an AAV capsid variant provided herein; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the 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 (i) the amino acid sequence of SEQ ID NO: 981 or an amino acid Attorney Docket No.14640.0106-00304 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
  • 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 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.
  • 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.
  • 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.
  • the method of making an AAV particle further comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell.
  • the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.
  • the present disclosure provides a pharmaceutical composition comprising an AAV particle provided herein and a pharmaceutically acceptable excipient.
  • the present disclosure provides a method of delivering an AAV particle encoding a CDKL5 protein to a cell, comprising administering an effective amount of a pharmaceutical composition or AAV particle provided herein.
  • the cell is in a subject.
  • the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder.
  • the CDKL5-related disorder is a CDKL5-related neurodegenerative or neuromuscular disorder.
  • the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • Attorney Docket No.14640.0106-00304 [040]
  • the present disclosure provides a method of treating a CDKL5-related disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein.
  • the subject has, has been diagnosed with having, or is at risk of having a
  • the CDKL5-related disorder is a CDKL5-related neurodegenerative or neuromuscular disorder.
  • the CDKL5-related neurodegenerative or neuromuscular disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 e.g., West syndrome
  • Lennox Gastaut syndrome e.g., Rett syndrome
  • atypical Rett syndrome e.g., autism
  • early-onset encephalopathy e.g., autism
  • X-linked mental retardation e.g., X-linked mental retardation.
  • the subject has, has been diagnosed with having, or is at risk of having CDD. [042] In some embodiments, the subject has one or more mutations in the CDKL5 gene. [043] In some embodiments, the subject has lower CDKL5 activity as compared to CDKL5 activity in an individual who does not have a CDKL5-related disorder. [044] In some embodiments, the treating results in prevention of progression of the disorder in the subject. In some embodiments, the treating results in amelioration of at least one symptom of the disorder, as indicated by one or more biomarkers.
  • the one or more biomarkers comprises a marker of CDKL5 activity, e.g., as measured by phosphorylation levels of substrate proteins (e.g., MECP2) or as measured by mass spectrometry.
  • the at least one symptom comprises epilepsy (e.g., early-onset epilepsy); autism; deficits in cognition; limited motor skills; sleep difficulties; visual impairment; low muscle tone; gastrointestinal reflux; behavioral symptoms (including episodes of laughing or crying that occur for what appears to be no reason, hypersensitivity to touch, and/or disrupted sleep); facial appearance changes (including microcephaly; a high, broad forehead; large, deep-set eyes; smaller-than normal space between the nose and upper lip; an upturned nose; and/or full lips and widely-spaced teeth); difficulties standing and walking; small, cold feet; lack of or poor eye contact; frequent sideways glances; cortical visual impairment or cortical blindness; bruxism; limited or absent speech; difficulties eating; stereotypies; limited ability to make
  • epilepsy e
  • the subject is a human.
  • the AAV particle or pharmaceutical composition is delivered to a cell, tissue, or region of the central nervous system (CNS) and/or the peripheral nervous system (PNS) in the subject.
  • CNS central nervous system
  • PNS peripheral nervous system
  • the cell, tissue, or region of the CNS and/or PNS is a cell (e.g., a 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), dorsal root ganglion, external cuneate nucleus, geniculate nucleus, globus pallidus, Attorney Docket No.14640.0106-00304 gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguous, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or a spinal cord (
  • the AAV particle or the pharmaceutical composition is delivered to the subject via intravenous administration.
  • a method of delivery or treating provided herein further comprises evaluating, e.g., measuring, the level of CDKL5 expression, e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject.
  • the level of CDKL5 protein expression is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.
  • the level of CDKL5 expression (e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression) is performed prior to and/or subsequent to administration of the pharmaceutical composition or AAV particle.
  • the subject’s level of CDKL5 expression e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression
  • prior to administration is compared to the subject’s level of CDKL5 expression subsequent to administration (e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression).
  • the level of CDKL5 expression (e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression) may be evaluated in a cell or tissue of the CNS and/or the PNS.
  • the cell or tissue of the CNS and/or PNS is a cell (e.g., a 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), dorsal root ganglion, external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguous, oculomotor nucleus, putamen, retina, 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).
  • cerebellum e.g., Purkinje cell
  • the subject’s level of CDKL5 protein expression subsequent to administration is increased relative to the subject’s level of CDKL5 protein expression prior to administration.
  • the method of delivery or treating further comprises evaluating, e.g., measuring, the level of CDKL5 activity in the subject, e.g., in a cell or tissue of the subject.
  • administering a pharmaceutical composition or AAV particle provided herein to the subject results in an increase in: (i) the level of CDKL5 activity 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, Attorney Docket No.14640.0106-00
  • a method of delivery or treatment provided herein further comprises administering to the subject at least one additional agent and/or therapy.
  • the at least one additional agent and/or therapy comprises an agent and/or therapy suitable for treating a CDKL5-related disorder.
  • the at least one additional agent and/or therapy comprises one or more anti-epileptic drugs (e.g., valproate, levetiracetam, clobazam, lamotrigine, ganaxolone, topiramate, bromide, felbamate, phenobarbital, or a combination thereof).
  • a method of delivery or treatment provided herein further comprises administering an immunosuppressant to the subject.
  • the immunosuppressant comprises a corticosteroid (for example, without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxychloroquine.
  • the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in a method of treating a disorder provided herein.
  • the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in the treatment of a CDKL5-related disorder.
  • the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder, optionally wherein the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • infantile spasms e.g., West syndrome
  • Lennox Gastaut syndrome e.g., Rett syndrome
  • atypical Rett syndrome
  • the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X- linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X- linked mental retardation.
  • the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder, optionally wherein the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, early-onset encephalopathy, or X-linked mental retardation.
  • An adeno-associated virus (AAV) particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)-encoding sequence (e.g., encoding a human CDKL5 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 X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q, K, E, S, P, R, N, H; and/or (ii) [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I, V, T, M, S
  • 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. 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.
  • Attorney Docket No.14640.0106-00304 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.
  • an amino acid other than T at position 582 e.g., S, R, A, I, C, N, K
  • 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 embodiment 15, wherein [N1] is or comprises TNTQ (SEQ ID NO: 4688). 17.
  • 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 (S
  • [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 ID 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: 4840), GKQDWHR (SEQ
  • [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: 4910), TTSQDWHR (SEQ ID NO: 4911),
  • 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
  • an amino acid other than Q at position 592 e.g., G, N,
  • the AAV capsid variant comprises the amino acid I at position 590, as numbered according to SEQ ID NO: 138 or 981.
  • the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981.
  • X 6 is A, Y, P, N, S, T, G, E, V, W, F, or Q; and/or (ii) X 7 is Q, G, N, K, H, R, E, L, P, or M. 32.
  • 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.
  • 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
  • an amino acid other than T at position 593 e.g., S, N, P, A, or I
  • 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,
  • 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.
  • 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.
  • 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 cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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 X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q, P, or a conservative substitution thereof; and/or (ii) [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I, V, or a conservative substitution thereof.
  • CDKL5 cyclin-dependent kinase-like 5
  • 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.
  • AAV particle of any one of embodiments 51-53, wherein [N1] comprises amino acids X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q or P.
  • 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 51-62, wherein [N1]-[N2] comprises: (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
  • 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), or VTQDWHR (SEQ ID NO: 4897); (ii) an amino acids
  • 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: 4898
  • AAV particle of embodiment 65 wherein [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898).
  • an amino acid other than Q at position 590 e.g., I or V
  • an amino acid other than A at Attorney Docket No.14640.0106-00304 position 591 e.g., P, S, Y, or N
  • an amino acid other than Q at position 592 e.g., G or N
  • 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. 76.
  • 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); Attorney Docket No.14640.0106-00304 (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 (
  • 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
  • T e.g., S or N
  • G e.g., N
  • V amino acid other than V
  • 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; (
  • 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.
  • the AAV capsid variant further comprises [N4], wherein [N4] comprises amino acids X 8 , X 9 , X 10 , and X 11 , wherein X 10 is W. 88.
  • the AAV particle of embodiment 87 wherein: (i) X 8 is T, S, or N; (ii) X 9 is G or N; and/or (iv) X 11 is V, A, I, or S. 89.
  • the AAV particle of any one of embodiments 87-89, wherein [N4] comprises TGW, SNW, NNW, GWV, GWA, NWV, GWI, or GWS. 91.
  • [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).
  • 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
  • [N1]-[N2] corresponds to amino acids 582-589 (e.g., T582, N583, T584, Q585, D586, W587, H588, R589) of SEQ ID NO: 981.
  • Attorney Docket No.14640.0106-00304 110 is a group of amino acids 590-592 (e.g., I590, A591, and Q592) of SEQ ID NO: 981.
  • amino acids 582-596 e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596
  • 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]. 129.
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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
  • 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 130-134 and 140, wherein the at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688).
  • 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).
  • Attorney Docket No.14640.0106-00304 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.
  • SEQ ID NOs 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.
  • 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).
  • SEQ ID NOs 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,
  • 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.
  • 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.
  • amino acids 584-590 e.g., T584, Q585, D586, W587, H588, R589, and/or I590
  • 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
  • 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, G59
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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)
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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.
  • CDKL5-encoding sequence e.g., encoding a human CDKL5 protein
  • an AAV capsid variant e.g., an AAV9 capsid variant
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 protein) and an AAV capsid variant (e.g., an AAV9 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.
  • CDKL5-encoding sequence e.g., encoding a human CDKL5 protein
  • an AAV capsid variant e.g., an AAV9 capsid variant
  • Attorney Docket No.14640.0106-00304 177 e.g., T
  • 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
  • an amino acid other than Q at position 590 e.
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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. 183.
  • CDKL5-encoding sequence e.g., encoding a human CDKL5 protein
  • an AAV capsid variant e.g., an AAV9 capsid variant
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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 capsid variant comprises the substitutions A581T or A581V, as numbered according to SEQ ID NO: 138 or 981.
  • 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.
  • 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.
  • amino acids 138-736 e.g., a VP2, of SEQ ID NO: 138. 200.
  • Attorney Docket No.14640.0106-00304 201 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.
  • 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 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.
  • AAV particle of embodiment 207 wherein the AAV capsid variant is encoded by a codon- optimized nucleotide sequence.
  • 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 cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981. 212.
  • CDKL5 cyclin-dependent kinase-like 5
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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. 213.
  • CDKL5-encoding sequence e.g., encoding a human CDKL5 protein
  • an AAV capsid variant e.g., an AAV9 capsid variant
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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 Attorney Docket No.14640.0106-00304 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.
  • CDKL5 cyclin-dependent kinase-like 5
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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.
  • CDKL5 cyclin-dependent kinase-like 5 (
  • 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 cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981. 219.
  • CDKL5 cyclin-dependent kinase-like 5
  • An AAV particle comprising a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)- encoding sequence (e.g., encoding a human CDKL5 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 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.
  • CDKL5 cyclin-dependent kinase-like 5
  • 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.
  • 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.
  • 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, 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 Attorney Docket No.14640.0106-00304 comprising an A
  • 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 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 CDKL5 to a brain region, optionally wherein the CDKL5 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).
  • an assay e.g., a qRT-PCR, a dd
  • 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.
  • an assay e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2).
  • 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. 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.
  • 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, 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).
  • a Rep protein e.g., a non-structural protein
  • 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 cell of embodiment 319, wherein the cell is a mammalian cell or an insect cell. 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.
  • Attorney Docket No.14640.0106-00304 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 CDKL5 to a cell or 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.
  • a cell or tissue e.g., a CNS cell or CNS tissue
  • 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.
  • the method of embodiment 327 or 328, wherein the cell is a neuron, a sensory neuron, and/or a motor neuron.
  • 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.
  • 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.
  • Attorney Docket No.14640.0106-00304 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.
  • 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 CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic
  • 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.
  • 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.
  • ICM intra-cisterna magna injection
  • intracerebrally intrathecally, intracerebroventricularly
  • intraparenchymal administration intraarterially, or intramuscularly.
  • 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.
  • FUS focused ultrasound
  • FUS-MB microbubbles
  • MRI-guided FUS coupled with intravenous administration.
  • 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).
  • ICM intra-cisterna magna injection
  • 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 activity of a CDKL5 gene, mRNA, protein, or a combination thereof.
  • 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.
  • 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 cyclin-dependent kinase-like 5 (CDKL5)-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 X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q, K, E, S, P, R, N, or H; and/or (ii) [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I, V, T, M, S, N, L, or F.
  • AAV adeno-associated virus
  • 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.
  • loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981. 358.
  • X 1 is T, S, R, A, I, C, N, K, L, or Q
  • X 2 is N, T, G, V, S, Y, K, I, H, D, or F
  • X 3 is 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) X6 is A, Y, P, N, S, T, G, E, V, W, F, or Q; and (ii) X 7 is Q, G, N, K, H, R, E, L, P, or M. 360.
  • 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 embodiment 364, wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066). 366.
  • 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 cyclin-dependent kinase-like 5 (CDKL5)-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.
  • An AAV particle comprising: (i) a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)-encoding sequence; and Attorney Docket No.14640.0106-00304 (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.
  • An AAV particle comprising: (i) a viral genome comprising a cyclin-dependent kinase-like 5 (CDKL5)-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.
  • Attorney Docket No.14640.0106-00304 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 embodiment 382 wherein the CDKL5-encoding sequence is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 6414. 384.
  • the AAV particle of embodiment 384, wherein the CDKL5-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6414. 386.
  • the AAV particle of embodiment 385, wherein the CDKL5-encoding sequence consists of the nucleotide sequence of SEQ ID NO: 6414. 387.
  • ITR inverted terminal repeat
  • 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 comprising: (i) providing a cell comprising a viral genome comprising a CDKL5-encoding sequence and a nucleic acid encoding an AAV capsid variant; and Attorney Docket No.14640.0106-00304 (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the recombinant AAV particle. 394.
  • 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. 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.
  • any one of embodiments 393-395 further comprising, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell.
  • the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.
  • 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 a CDKL5 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 CDKL5 protein.
  • the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder, optionally wherein the CDKL5-related disorder is a CDKL5- related neurodegenerative or neuromuscular disorder.
  • CDKL5 deficiency disorder CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X- linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms
  • Lennox Gastaut syndrome e.g., West syndrome
  • Rett syndrome e.g., atypical Rett syndrome
  • autism developmental and epileptic encephalopathy 2
  • developmental and epileptic encephalopathy 2 e.g., X- linked mental retardation.
  • a method of treating a CDKL5-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 disorder. 403.
  • the method of embodiment 402 wherein the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder.
  • the CDKL5-related disorder is a CDKL5-related neurodegenerative or neuromuscular disorder.
  • CDKL5-related neurodegenerative or neuromuscular disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation. 406.
  • CDKL5 deficiency disorder CDKL5 deficiency disorder
  • a method of treating CDKL5 deficiency disorder (CDD) 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 CDD. 407.
  • the at least one symptom comprises epilepsy (e.g., early-onset epilepsy); autism; deficits in cognition; limited motor skills; sleep difficulties; visual impairment; low muscle tone; gastrointestinal reflux; behavioral symptoms (including episodes of laughing or crying that occur for what appears to be no reason, hypersensitivity to touch, and/or disrupted sleep); facial appearance changes (including microcephaly; a high, broad forehead; large, deep-set eyes; smaller-than normal space between the nose and upper lip; an upturned nose; and/or full lips and widely- spaced teeth); difficulties standing and walking; small, cold feet; lack of or poor eye contact; frequent sideways glances; cortical visual impairment or cortical blindness; bruxism; limited or absent speech; difficulties eating; stereotypies; limited ability to make small, focused hand movements; gastroesophageal reflux; constipation; or a combination thereof.
  • epilepsy e.g., early-onset epilepsy
  • autism deficits in cognition
  • limited motor skills sleep difficulties
  • visual impairment low muscle tone
  • the cell, tissue, or region of the CNS and/or the PNS is a cell (e.g., a 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), dorsal root ganglion, external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguous, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoraxa cell, or a cell, e.g.
  • any one of embodiments 399-416 further comprising evaluating, e.g., measuring, the level of CDKL5 expression, e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. 418.
  • Attorney Docket No.14640.0106-00304 419 Attorney Docket No.14640.0106-00304 419.
  • the cell or tissue of the CNS and/or PNS is a cell (e.g., a 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), dorsal root ganglion, external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguous, oculomotor nucleus, putamen, retina, 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).
  • cerebellum e.g., Purkinje cell
  • a cell 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 deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), dorsal root ganglion, external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguous, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., a cell or tissue of the CNS and/or the PNS, e.g., the amygdala, brainstem, cau
  • the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxychloroquine.
  • a corticosteroid for example, and without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone
  • rapamycin for example, and without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone
  • rapamycin for example, and without limitation, prednisone, prednisolone, methylprednisolone, and
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms
  • Lennox Gastaut syndrome e.g., West syndrome
  • CDKL5 deficiency disorder CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation. 433.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation. 434.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation. 434.
  • embodiment 433 wherein the subject has, has been diagnosed with having, or is at risk of having the CDKL5-related disorder; optionally wherein the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e
  • compositions comprising an AAV capsid variant comprising a sequence encoding a CDKL5 protein, e.g., a human CDKL5 protein.
  • the present disclosure provides a method of delivering the AAV capsid variant comprising the sequence encoding the CDKL5 protein to a cell or tissue in a subject.
  • the present disclosure provides a method for delivering the AAV capsid variant, thereby providing a CDKL5 protein, e.g., a human CDKL5 protein to a cell or tissue in a subject.
  • a CDKL5 protein e.g., a human CDKL5 protein
  • 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.
  • 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.
  • 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 CDKL5.
  • the genome of the virus may encode a CDKL5 protein, and the viral particle comprising said genome may be delivered to a target cell, tissue, or organism.
  • the genome encodes a human CDKL5 protein, e.g., a wildtype human CDKL5 protein.
  • the target cell is a CNS cell.
  • the target tissue is a CNS tissue.
  • the target CNS tissue is brain tissue.
  • CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome.
  • CDD CDKL5 deficiency disorder
  • AAVs are commonly used in gene therapy approaches as a result of a number of advantageous features.
  • an AAV particle described herein can be used to administer and/or deliver a gene encoding CDKL5 protein (e.g., human CDKL5 protein) preferentially to the CNS.
  • an AAV particle described herein can be used to administer and/or deliver a gene encoding CDKL5 protein (e.g., human CDKL5 protein) preferentially to the brain.
  • an AAV particle described herein can be used to administer and/or deliver a gene encoding CDKL5 protein (e.g., human CDKL5 protein) preferentially to the spinal cord.
  • compositions and methods which may provide for improved features compared to prior AAV-mediated enzyme replacement 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, retina, 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
  • spinal cord e.g
  • AAV capsid variants e.g., AAV9 capsid variants
  • 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, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and
  • the AAV capsid variants described herein enhance the delivery of a CDKL5 to multiple regions of the CNS, such as the brain, and/or 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord (e
  • the AAV capsid variants described herein enhance the delivery of CDKL5 to the forebrain. In some embodiments, the AAV capsid variants described herein enhance the delivery of CDKL5 to the spinal cord. In some embodiments, the AAV capsid variants described herein enhance the delivery of CDKL5 to the dentate nucleus. In some embodiments, the AAV capsid variants described herein enhance the delivery of CDKL5 to the somatosensory cortex. In some embodiments, the AAV capsid variants with enhanced brain tropism described herein increase CDKL5 mRNA expression in the brain.
  • the AAV capsid variants increase CDKL5 mRNA expression in the forebrain.
  • a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental
  • the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome.
  • 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 a CDKL5 protein for use in treating a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation; optionally, CDKL5 deficiency disorder (CDD), developmental and epileptic
  • AAVs Adeno-associated viral Particles
  • 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.
  • ITR Inverted Terminal Repeat
  • 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 four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame.
  • VP1 refers to amino acids 1-736
  • VP2 refers to amino acids 138-736
  • VP3 refers to amino acids 203-736.
  • VP1 comprises amino acids 1-736
  • VP2 comprises amino acids 138-736
  • VP3 comprises amino acids 203-736.
  • VP1 is the full-length capsid protein sequence
  • VP2 and VP3 are shorter components of the whole.
  • 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.
  • the nucleic acid sequence encoding these proteins can be similarly described.
  • the three capsid proteins assemble to create the AAV capsid.
  • the AAV capsid typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3.
  • 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. [065] AAV particles have been investigated for delivery of gene therapeutics because of several unique features.
  • a co-helper e.g., adenovirus
  • 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).
  • AAV particles for CDKL5 delivery may be recombinant viral particles which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome.
  • 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 a CDKL5 protein.
  • the AAV particles of the present disclosure may be introduced into mammalian cells.
  • 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.
  • AAV particles of the present disclosure may be used to deliver CDKL5 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.
  • compositions described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
  • Attorney Docket No.14640.0106-00304 [071]
  • 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.
  • 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.
  • loop e.g., loop VIII
  • variable region e.g., variable region VIII
  • VR e.g., VR-VIII
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • loop VIII or variable region 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.
  • loop VIII is present at amino acids comprising those corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138.
  • the AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms.
  • the AAV particles demonstrate enhanced tropism for a target cell type, tissue, or organ.
  • the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively).
  • an AAV particle may, in addition, or alternatively, have decreased tropism for a cell-type, tissue or organ.
  • 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 Attorney Docket No.14640.0106-00304 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.
  • the AAV particle is a recombinant AAV particle.
  • the wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length.
  • inverted terminal repeats cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome.
  • an AAV viral genome comprises two ITR sequences.
  • 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.
  • 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.
  • AAV particles of the present disclosure may be produced recombinantly and may be based on AAV reference sequences.
  • 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.
  • the AAV particle of the present disclosure is an scAAV. In some embodiments, the AAV particle of the present disclosure is an ssAAV. [076] 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).
  • 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 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).
  • 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.
  • 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.
  • the modification (e.g., comprising the one or more substitutions relative to a wildtype AAV capsid) is in loop VIII.
  • the AAV capsid variant is an AAV9 capsid variant.
  • the one or more substitutions in the AAV capsid 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, retina, 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 neurons.
  • cerebellum e.g., Purkinje cell layer and/or deep cerebellar nuclei
  • 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. [082] 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.
  • the CNS e.g., the cortex
  • 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, retina, 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 neurons) following focused ultrasound (FUS), e.g., coupled with
  • FUS
  • the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g., DRG) after intravenous administration.
  • 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.
  • the one or more 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.
  • the one or more substitutions may Attorney Docket No.14640.0106-00304 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.
  • 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.
  • the one or more substitutions may increase distribution of an AAV particle to a heart cell, region, or tissue after intravenous administration.
  • the one or more modifications may increase distribution of an AAV particle to a cell, region, or tissue of a muscle.
  • the muscle is a heart muscle (e.g., a heart atrium or a heart ventricle) or a quadriceps muscle.
  • the one or more substitutions may increase distribution of an AAV particle to a muscle cell, region, or tissue after intravenous administration.
  • the one or more modifications, e.g., substitutions may increase distribution an AAV particle to a cell, region, or tissue of the kidney.
  • 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.
  • 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.
  • 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).
  • the AAV capsid variant comprises (e.g., in loop VIII) a sequence set forth in Table 9.
  • the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 24.
  • the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 25. In some embodiments, the AAV capsid variant is an AAV9 capsid variant. Table 1. Exemplary Sequences Amino Acid SEQ Amino Acid SEQ Amino Ac SEQ SEQ ID ID id ID Amino Acid ID O: 0 1 2 3 4 5 6 7 8 9 0 1 Attorney Docket No.14640.0106-00304 T TSQDWHRIAQ 355 TNTKDWHRVAQ 404 TNHQDWHRIAQ 453 TFSQDWHRIAQ 502 T TDQDWHRIAQ 356 TITKDWHRIAQ 405 STTQDWHRIAQ 454 RNSQDWHRIAQ 503 T NPSDWHRIAQ 357 INTKDWHRIAQ 406 TNNQDWHRTAQ 455 INTQDWHRIAQ 504 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5
  • [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682), [N1] comprises X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q, K, E, S, P, R, N, or H, and/or [N3] comprises X 5 , X 6 , and X 7 , wherein X 5 is I, V, T, M, S, N, L, or F.
  • X 4 of [N1] is Q.
  • X 4 of [N1] is K.
  • X 5 of [N3] is I.
  • X 1 of [N1] is T, S, R, A, I, C, N, K, L, or Q.
  • X 2 of [N1] is N, T, G, V, S, Y, K, I, H, D, or F.
  • X 3 of [N1] is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C.
  • [N1] is or comprises TNTQ (SEQ ID NO: 4688).
  • [N1] is or comprises TNTK (SEQ ID NO: 4689).
  • [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments, [N1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899).
  • X 6 of [N3] is A, Y, P, N, S, T, G, E, V, W, F, or Q.
  • X 7 of [N3] is Q, G, N, K, H, R, E, L, P, or M.
  • [N3] is or comprises IAQ.
  • [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027).
  • [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).
  • the amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X 8 , X 9 , X 10 , and X 11 , wherein X 8 is T, S, N, P, A, or I; X 9 is G, N, D, R, V, A, S, or Q; X 10 is W, S, C, R, L, or G; and/or X 11 is V, A, S, I, C, G, D, F, L, or T.
  • [N4] is or comprises TGWV (SEQ ID NO: 5066).
  • [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
  • [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).
  • 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 X 1 , X 2 , X 3 , and Attorney Docket No.14640.0106-00304 X 4 , wherein X 4 is Q, P, or a conservative substitution thereof; and/or [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I, V, or a conservative substitution thereof.
  • [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); [N1] comprises amino acids X 1 , X 2 , X 3 , and X 4 , wherein X 4 is Q or P; and/or [N3] comprises amino acids X 5 , X 6 , and X 7 , wherein X 5 is I or V.
  • X 4 of [N1] is Q.
  • X 5 of [N3] is I.
  • X 5 of [N3] is V.
  • X 1 of [N1] is T or S.
  • X 2 of [N1] is N, T, G, S, I, or V.
  • X 3 of [N1] is T, N, I, S, A, V, or L.
  • [N1] is or comprises TNTQ (SEQ ID NO: 4688).
  • [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898).
  • X 6 of [N3] is A, P, S, Y, or N.
  • X 7 of [N3] is Q, G, or N.
  • [N3] is or comprises IAQ.
  • [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343).
  • the amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X 8 , X 9 , X 10 , and X 11 , wherein X 10 is W.
  • X 8 of [N4] is T, S, or N.
  • X 9 of [N4] is G or N.
  • X 11 of [N4] is V, A, I, or S.
  • [N4] is or comprises TGWV (SEQ ID NO: 5066).
  • [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.
  • [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201). [091] 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.
  • 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.
  • [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.
  • X 1 of [N1] is present at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 2 of [N1] is present at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 3 of [N1] is present at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 4 of [N1] is present at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 or 981.
  • [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.
  • [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.
  • [N2] replaces Attorney Docket No.14640.0106-00304 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).
  • [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.
  • [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).
  • [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.
  • X 5 of [N3] is present at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X6 of [N3] is present at an amino acid corresponding to position 591 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 7 of [N3] is present at an amino acid corresponding to position 592 of the amino acid sequence of SEQ ID NO: 138 or 981.
  • [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. [096] 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.
  • [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).
  • [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.
  • [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).
  • [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.
  • X 8 of [N4] is present at an amino acid corresponding to position 593 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 9 of [N4] is present at an amino acid corresponding to position 594 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 10 of [N4] is present at an amino acid corresponding to position 595 of the amino acid sequence of SEQ ID NO: 138 or 981
  • X 11 of [N4] is present at an amino acid corresponding to position 596 of the amino acid sequence of SEQ ID NO: 138 or 981.
  • [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. [099] 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.
  • [N2]-[N3]-[N4] replaces amino acids corresponding to positions 586-596 of the amino acid sequence of Attorney Docket No.14640.0106-00304 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).
  • 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.
  • [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.
  • [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).
  • 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].
  • 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, 24, 25, or 26.
  • 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.
  • the at least 3 consecutive amino acids comprise TQD.
  • 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). [0104] 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).
  • 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 some 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).
  • 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, Attorney Docket No.14640.0106-00304 2B, 9, 24, 25, or 26.
  • 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, 24, 25, or 26.
  • 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.
  • 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.
  • 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). [0107] 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, 24, 25, or 26.
  • 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.
  • the amino acid sequence is present in loop VIII.
  • loop VIII comprises amino acids corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138.
  • loop VIII comprises amino acids corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 138.
  • 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.
  • 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., 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.
  • position(s) 582 e.g., T582
  • 583 e.g.
  • 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.
  • the AAV capsid variant Attorney Docket No.14640.0106-00304 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.
  • position(s) 582 e.g., T582
  • 583
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid comprises a nucleotide sequence described herein, e.g., as described in Table 2A.
  • the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid is codon optimized.
  • the nucleotide sequence is an isolated nucleotide sequence. In some embodiments, the nucleotide sequence is a recombinant nucleotide sequence. [0111] 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.
  • the nucleotide sequence comprises 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 substitutions, but no more than ten substitutions, relative to the nucleotide sequence of SEQ ID NO: 942.
  • 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.
  • 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.
  • the AAV capsid variant comprises the substitution A581T or A581V, wherein the substitution is at a position that corresponds to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981.
  • 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 (
  • 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:
  • 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 Attorney Docket No.14640.0106-00304 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.
  • 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.
  • 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.
  • 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.
  • the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, wherein the substitution(s) is/are at position(s) correspond to position(s) 584, 586, 587, 588, 589, and/or 590 of the amino acid sequence of SEQ ID NO: 138 or 981.
  • 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.
  • 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. [0118] 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the AAV capsid variant comprises an R at an amino acid corresponding to position 449 of the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises a modification in loop I, II, IV, and/or VI.
  • 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.
  • 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.
  • 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.
  • 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%, 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),
  • 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 TQD
  • 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. [0125] 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.
  • the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 941 in a loop VIII region.
  • 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.
  • 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.
  • 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 e VP1 D VP1 (amino Peptide Nam NA acid) SEQ ID (amino acid) Peptide DNA SEQ .
  • 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.
  • 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.
  • the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV capsid protein comprises Attorney Docket No.14640.0106-00304 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.
  • 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 Attorney Docket No.14640.0106-00304 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
  • 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.
  • an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region.
  • 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.
  • the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and/or cerebellum.
  • an AAV capsid variant delivers an increased level of a payload to a brain region.
  • 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.
  • the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and/or cerebellum.
  • 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.
  • an AAV capsid variant of the present disclosure has decreased tropism for the liver.
  • an AAV capsid variant comprises a modification that results in reduced tropism (e.g., de-targeting) and/or activity in the liver.
  • the reduced tropism in the liver is compared to an otherwise similar capsid that does not comprise the modification, e.g., a wild-type capsid polypeptide.
  • 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 Attorney Docket No.14640.0106-00304 galactose.
  • 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; Pueuerla 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.
  • 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
  • N470A at an amino
  • 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
  • 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: 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 Attorney Docket No.14640.0106-00304 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.
  • N470 e
  • the AAV capsid variant comprises a modification 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.
  • 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.
  • the AAV capsid variant is an isolated capsid variant. In some embodiments, the AAV capsid variant is a recombinant capsid variant.
  • a polynucleotide encoding an AAV capsid polypeptide e.g., an AAV capsid variant is an isolated and/or a recombinant AAV capsid polypeptide.
  • polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.
  • AAV serotypes and capsids [0148]
  • 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.
  • the AAV capsid protein e.g., an AAV capsid variant
  • the AAV capsid variant allows for blood brain barrier penetration following intravenous administration.
  • 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.
  • the AAV capsid variant allows for increased distribution to a brain region.
  • 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, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, and/or vestibular nucleus.
  • 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 e.g.,
  • 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 Attorney Docket No.14640.0106-00304 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).
  • a non-neuronal cell e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof).
  • 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).
  • a glial cell e.g., an astrocyte, an oligodendrocyte, or a combination thereof.
  • an AAV capsid variant allows for increased distribution to a spinal cord region.
  • the spinal region comprises a cervical spinal cord region, thoracic spinal cord region, and/or lumbar spinal cord region.
  • the AAV capsid variant allows for increased distribution to a heart region.
  • the AAV capsid variant is suitable for intramuscular administration and/or transduction of muscle fibers.
  • the AAV capsid variant allows for increased distribution to a muscle region.
  • the muscle region comprises a heart muscle, quadriceps muscle, a diaphragm muscle region, or a combination thereof.
  • the muscle region comprises a heart muscle region, e.g., a heart atrium muscle region or a heart ventricle muscle region.
  • the AAV capsid variant is suitable for increased distribution to a kidney.
  • the AAV capsid variant is suitable for increased distribution to a pancreas.
  • the AAV capsid variant is suitable for increased distribution to a retina.
  • 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.
  • 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.
  • 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.
  • 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-).
  • Met/AA-clipping in capsid proteins see Jin, et al.
  • references to capsid proteins 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 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-).
  • 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).
  • 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.
  • 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.
  • references to viral capsids formed from VP capsid proteins 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-).
  • 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 VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met- /AA1-).
  • 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: Attorney Docket No.14640.0106-00304 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, 24, 25, or 26.
  • 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, 2
  • 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, 24, 25, or 26 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,
  • 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).
  • AAV serotype e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7,
  • 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, 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).
  • AAV serotype
  • 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 Attorney Docket No.14640.0106-00304 polypeptide, an AAV9 K449R capsid polypeptide, or an AAVrh10 capsid polypeptide.
  • 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.
  • 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 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.
  • the AAV particle of the present disclosure serves as an expression vector comprising a viral genome that encodes a CDKL5 protein (e.g., a human CDKL5 protein).
  • a CDKL5 protein e.g., a human CDKL5 protein
  • an AAV particle e.g., an AAV particle for the vectorized delivery of CDKL5 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.
  • 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 a CDKL5 protein, a polyA region, or a combination thereof.
  • ITR inverted terminal repeat
  • the viral genome e.g., the AAV viral genome, further comprises at least one miRNA binding site.
  • Viral Genome Component Inverted Terminal Repeats (ITRs) [0167]
  • 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 a CDKL5-encoding sequence.
  • the viral genome has two ITRs. These two ITRs flank the region comprising the CDKL5-encoding sequence at the 5’ and Attorney Docket No.14640.0106-00304 3’ ends.
  • the ITR functions as an origin of replication comprising a recognition site for replication.
  • the ITR comprises a sequence region which can be complementary and symmetrically arranged.
  • the ITR incorporated into a viral genome described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.
  • the ITR is of the same serotype as the capsid, selected from any one of the serotypes described herein, or a derivative thereof.
  • the ITR is of a different serotype than the capsid.
  • the AAV particle has more than one ITR.
  • the AAV particle comprises a viral genome comprising two ITRs.
  • 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 [0169]
  • 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.
  • 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).
  • the viral genome comprises a promoter that is sufficient for expression, e.g., in a target cell, of a CDKL5 protein (e.g., a human CDKL5 protein) encoded by a transgene.
  • the promoter is deemed to be efficient when it drives expression of the CDKL5 encoded in the viral genome of the AAV particle.
  • the promoter is a promoter deemed to be efficient when it drives expression in the cell or tissue being targeted.
  • Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters.
  • the promoters may be human promoters. In some embodiments, the promoters may be truncated. [0174] 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.
  • a promoter that drives or promotes expression in most mammalian tissues comprises a human elongation factor 1 ⁇ -subunit (EF1 ⁇ ) promoter, a cytomegalovirus (CMV) immediate-early Attorney Docket No.14640.0106-00304 enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter, a CAG promoter, a ⁇ glucuronidase (GUSB) promoter, or a ubiquitin C (UBC) promoter.
  • EF1 ⁇ human elongation factor 1 ⁇ -subunit
  • CMV cytomegalovirus immediate-early Attorney Docket No.14640.0106-00304 enhancer and/or promoter
  • CBA chicken ⁇ -actin
  • CAG CAG promoter
  • GUSB ⁇ glucuronidase
  • UBC ubiquitin C
  • the viral genome comprises a nervous system specific promoter, i.e., a promoter that results in expression of CDKL5 in a neuron, an astrocyte, and/or an oligodendrocyte.
  • tissue-specific expression elements for neurons include synapsin (Syn) or synapsin 1 (Syn1), e.g., human synapsin or synapsin 1.
  • the promoter may be less than 1 kb.
  • the promoter may be a combination of two or more components of the same or different starting or parental promoters.
  • the viral genome comprises an enhancer.
  • the viral genome comprises an engineered promoter.
  • Viral Genome Component Intron and Exon Sequences
  • the AAV viral genome comprises at least one intron or a fragment or derivative thereof.
  • the AAV viral genome comprises at least one exon or a fragment or derivative thereof.
  • the intron may be 100-600 nucleotides in length.
  • the CDKL5-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.
  • the CDKL5-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.
  • a promoter comprising an intron e.g., 3’ relative to the promoter comprising an intron
  • upstream of the polyadenylation sequence e.g., 5’ relative to the polyadenylation sequence
  • the CDKL5-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.
  • the CDKL5-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.
  • the CDKL5-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.
  • the intron sequence is not an enhancer sequence.
  • the intron sequence is not a sub-component of a promoter sequence.
  • the intron sequence is a sub-component of a promoter sequence.
  • UTRs Viral Genome Component: Untranslated Regions (UTRs)
  • UTRs Untranslated Regions
  • 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.
  • UTRs Untranslated Regions
  • the viral genome encoding a transgene described herein comprises a Kozak sequence.
  • wild-type 5′ untranslated regions 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'.
  • the 5’UTR in the viral genome includes a Kozak sequence.
  • the 5’UTR in the viral genome does not include a Kozak sequence.
  • 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.
  • AU rich elements 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.
  • AREs 3′ UTR AU rich elements
  • the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • Any UTR from any gene known in the art may be incorporated into the viral genome of the Attorney Docket No.14640.0106-00304 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.
  • 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.
  • 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.
  • 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.
  • the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild type UTR.
  • 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
  • the viral genome of the AAV particles of the present disclosure comprises at least one polyadenylation (polyA) sequence.
  • the polyA signal region is positioned 3’ relative to the nucleic acid comprising the CDKL5-encoding sequence.
  • the AAV particle viral genome comprises at least one filler sequence.
  • 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.
  • 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.
  • the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 2.3 kb.
  • 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 CDKL5-encoding sequence
  • the disclosure provides an AAV particle comprising a viral genome encoding a CDKL5 protein, e.g., a CDKL5 protein encoded by the nucleotide sequence of SEQ ID NO: 6414.
  • the viral genome comprises a promoter operably linked to a nucleotide sequence encoding a CDKL5 protein, e.g., SEQ ID NO: 6414.
  • the disclosure herein provides constructs that allow for improved expression of CDKL5 protein delivered by gene therapy vectors. [0201] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of CDKL5 protein delivered by gene therapy vectors. Attorney Docket No.14640.0106-00304 [0202] In some embodiments, the disclosure provides constructs that allow for improved sub-cellular distribution or trafficking of CDKL5 protein delivered by gene therapy vectors. [0203] In some embodiments, the disclosure provides constructs that allow for improved trafficking of CDKL5 protein to lysosomal membranes delivered by gene therapy vectors.
  • the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding a CDKL5 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 CDKL5.
  • 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 CDKL5-encoding sequence, a polyA sequence, and a 3’ ITR sequence.
  • the CDKL5 protein encoded by the nucleotide sequence has an amino acid sequence that is 100% identical to a wildtype CDKL5 protein.
  • the AAV genome comprises a payload construct that comprises a combination of coding and non-coding nucleic acid sequences.
  • 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.
  • the CDKL5-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.
  • 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.
  • 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.
  • a gene therapy product may comprise a polynucleotide encoding a CDKL5 protein to treat CDKL5 deficiency or CDKL5-related disorders.
  • the gene therapy product comprises a polynucleotide sequence encoding a CDKL5 protein.
  • the payload construct encodes a messenger RNA (mRNA).
  • mRNA messenger RNA
  • 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.
  • a CDKL5-encoding sequence may comprise or encode a selectable marker.
  • 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, Attorney Docket No.14640.0106-00304 selection, and/or purification of the host cell from a population of cells that may or may not express the selectable marker.
  • the selectable marker provides resistance to survive a selection process that would otherwise kill the host cell, such as treatment with an antibiotic.
  • 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.
  • a CDKL5-encoding sequence may comprise a selectable marker including, but not limited to, ⁇ -lactamase, luciferase, ⁇ -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.
  • NGFR truncated nerve growth factor
  • a CDKL5-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).
  • CDKL5-encoding sequence may encode a selectable marker comprising a human influenza hemagglutinin (HA) tag.
  • a nucleic acid for expression of CDKL5 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
  • the viral genome encoding a CDKL5 protein further comprises a nucleotide sequence encoding a signal sequence.
  • the nucleotide sequence encoding the signal sequence is located 5’ relative to the nucleotide sequence encoding the CDKL5 protein.
  • the encoded CDKL5 protein comprises a signal sequence at the N-terminus, wherein the signal sequence is optionally cleaved during cellular processing and/or localization of the CDKL5 protein.
  • Exemplary CDKL5 Payload [0216] In some embodiments, the encoded CDKL5 protein is a wildtype CDKL5 protein. [0217] Tables 7 and 8 provide exemplary polynucleotide sequences encoding a CDKL5 protein and polypeptide sequences of exemplary CDKL5 proteins that may be used in the viral genomes disclosed herein and which may constitute a CDKL5 protein payload.
  • the CDKL5 protein suitable for delivery in an AAV particle disclosed herein is encoded by the nucleotide sequence of SEQ ID NO: 6414. In some embodiments, the CDKL5 protein suitable for delivery in an AAV particle disclosed herein is encoded by the nucleotide sequence of SEQ ID NO: 6416 or 6418. Table 7.
  • CDKL5 Sequences SEQ ID NO: Type Species Description Attorney Docket No.14640.0106-00304 sapiens]” National Center for Biotechnology Information, Feb 12, 2023 6419 DNA Homo sapiens CDKL5 mRNA transcript variant NM_001323289.2 ), ), ), Tabl Description Sequence SEQ ID NO: Attorney Docket No.14640.0106-00304 ATGTATGTGACCCGTGACAAAGTGAGAGCCAAGGGCTTGGATGGAAGCTTGAGC ATAGGGCAAGGGATGGCAGCTAGAGCCAACAGCCTGCAACTCTTGTCACCCCAG CCTGGAGAACAGCTCCCTCCAGAGATGACTGTGGCAAGATCTTCGGTCAAAGAG e exemp ary sequence n orma on rom e a ona ener or o ec noogy Information cited in Tables 7 and 8 is hereby incorporated by reference in its entirety.
  • the present disclosure provides a viral genome comprising a CDKL5- 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.
  • the CDKL5-encoding sequence is at least 95% identical to SEQ ID NO: 6414.
  • the CDKL5-encoding sequence is at least 99% identical to SEQ ID NO: 6414.
  • the CDKL5-encoding sequence comprises SEQ ID NO: 6414. In some embodiments, the CDKL5-encoding sequence consists of SEQ ID NO: 6414.
  • the AAV viral genome further comprises a nucleic acid encoding a capsid protein, e.g., a structural protein.
  • the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and/or a VP3 polypeptide.
  • the VP1 polypeptide, the VP2 polypeptide, and/or the VP3 polypeptide are encoded by at least one Cap gene.
  • the AAV viral genome further comprises a nucleic acid encoding a Rep protein, e.g., a non-structural protein.
  • the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and/or a Rep40 protein.
  • the Rep78 protein, the Rep68 protein, the Rep52 protein, and/or the Rep40 protein are encoded by at least one Rep gene.
  • the capsid comprises the amino acid sequence of SEQ ID NO: 981.
  • 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.
  • 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 least 96%, at least 97%, at least 97%, at least 98%
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Attorney Docket No.14640.0106-00304 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides in some embodiments, vectors, cells, and/or AAV particles comprising the above identified viral genomes.
  • the AAV viral genome used in the present disclosure is single- stranded (ssAAV).
  • the AAV viral genome is capable of forming double-stranded DNA.
  • 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.
  • AAV production 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 CDKL5.
  • an AAV particle of the present disclosure e.g., an AAV particle comprising an AAV capsid variant disclosed herein
  • the method comprises: (i) providing a cell comprising a viral genome described herein (e.g., a viral genome comprising a CDKL5-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.
  • a viral genome described herein e.g., a viral genome comprising a CDKL5-encoding sequence and a nucleic acid encoding an AAV capsid variant disclosed herein
  • incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant, e.g.
  • the viral genome comprises a CDKL5-encoding sequence comprising SEQ ID NO: 6414.
  • the AAV capsid variant comprises an amino acid sequence of SEQ ID NO: 981.
  • the viral genome comprises a CDKL5-encoding sequence comprising SEQ ID NO: 6414 and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.
  • the method comprises, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell.
  • the method comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell.
  • the AAV particle described herein is an isolated AAV particle.
  • the AAV particle described herein is a recombinant AAV particle.
  • Any method known in the art may be used for the preparation of AAV particles.
  • AAV particles are produced in mammalian cells (e.g., HEK293 cells).
  • AAV particles are produced in insect cells (e.g., Sf9 cells).
  • Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos.
  • 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. Attorney Docket No.14640.0106-00304 III.
  • Pharmaceutical Compositions [0237]
  • the present disclosure provides pharmaceutical compositions of an adeno-associated virus (AAV) particle comprising a viral genome comprising a CDKL5-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.
  • a composition described herein comprises an AAV polynucleotide or AAV genome or AAV particle, and at least one excipient.
  • compositions provided herein 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.
  • compositions 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.
  • compositions are administered to humans, e.g., human patients or human subjects.
  • the AAV particle formulations described herein may contain a nucleic acid encoding at least one CDKL5-encoding sequence.
  • the formulations may contain a nucleic acid encoding 1, 2, 3, 4, or 5 CDKL5-encoding sequences.
  • the formulation may contain CDKL5-encoding sequences encoding CDKL5 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.
  • the AAV formulation comprises at least one sequence encoding human CDKL5.
  • the AAV formulation comprises at least one sequence encoding wildtype human CDKL5.
  • 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.
  • 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.
  • Attorney Docket No.14640.0106-00304 IV Formulations
  • 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.
  • 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.
  • the composition may comprise about 0.1% to about 99% (w/w) of the active ingredient.
  • 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.
  • 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 CDKL5.
  • 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. [0248] In some embodiments, the viral vectors encoding CDKL5 may be formulated to optimize baricity and/or osmolality.
  • 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 [0249]
  • 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.
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • 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 Attorney Docket No.14640.0106-00304 United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United States Food and Drug Administration
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • Excipients 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).
  • AAV formulations may comprise at least one excipient which is an inactive ingredient.
  • active ingredient refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in formulations.
  • 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).
  • Formulations of AAV particles may include cations or anions.
  • the formulations include metal cations such as, but not limited to, Zn 2+ , Ca 2+ , Cu 2+ , Mg + , or combinations thereof.
  • 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.
  • compositions of the disclosure may be administered to a subject, e.g., to deliver CDKL5, e.g., to a subject who has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism,
  • compositions may similarly be used in the manufacture of a medicament for administration to a subject having a CDKL5- related disorder (e.g., a CDKL5-related neurodegenerative or neuromuscular disorder).
  • a CDKL5- related disorder e.g., a CDKL5-related neurodegenerative or neuromuscular disorder.
  • the disclosure provides a method of delivering a CDKL5 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 a CDKL5 protein, thereby delivering the CDKL5 protein.
  • the subject has, has been diagnosed with having, Attorney Docket No.14640.0106-00304 or is at risk of having a CDKL5-related disorder.
  • the CDKL5-related disorder may be a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation).
  • the disclosure provides an AAV particle or pharmaceutical composition according to any one of the embodiments disclosed herein for treating a CDKL5-related disorder, such as CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDKL5-related disorder such as CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • 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 CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDKL5 deficiency disorder CDKL5 deficiency disorder (CDD)
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDKL5 deficiency disorder CDKL5 deficiency disorder
  • a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding CDKL5 may be administered to a subject to treat a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder, thereby treating the CDKL5-related disorder.
  • the subject has, has been diagnosed with having, or is at risk of having a CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X- linked mental retardation).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X- linked mental retardation).
  • the CDKL5-related disorder is seizures, e.g., early-onset seizures.
  • the seizures may occur in one or more of three stages: (i) early epilepsy with focal seizures (e.g., in infants aged about 4 weeks to about 6 months), (ii) epileptic encephalopathy with infantile spasms (e.g., in infancy and early childhood), and (iii) tonic seizures and late myoclonic epilepsy (e.g, in childhood).
  • the CDKL5-related disorder may be characterized by movement disorders such as hand-wringing stereotypy, chorea, and ballisms (also called ballismus).
  • the CDKL5-related disorder is CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut Attorney Docket No.14640.0106-00304 syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 e.g., West syndrome
  • Lennox Gastaut Attorney Docket No.14640.0106-00304 syndrome e.g., Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • the disclosure provides a method of treating CDKL5 deficiency disorder (CDD) in a subject.
  • a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding CDKL5 may be administered to a subject to treat CDD.
  • the subject has, has been diagnosed with having, or is at risk of having CDD.
  • a subject may have one or more mutations in the CDKL5 gene.
  • the subject has lower CDKL5 activity as compared to CDKL5 activity in an individual who does not have a CDKL5-related disorder.
  • the treatment may result in prevention of progression of the CDKL5- related disorder.
  • the treatment may result in in amelioration of at least one symptom of the disorder, as indicated by one or more biomarkers.
  • the one or more biomarkers comprises a marker of CDKL5 activity, e.g., as measured by phosphorylation levels of substrate proteins (e.g., MECP2) or as measured by mass spectrometry.
  • the treatment improves at least one symptom of a CDKL5-related disorder, e.g., (e.g., early-onset epilepsy); autism; deficits in cognition; limited motor skills; sleep difficulties; visual impairment; low muscle tone; gastrointestinal reflux; behavioral symptoms (including episodes of laughing or crying that occur for what appears to be no reason, hypersensitivity to touch, and/or disrupted sleep); facial appearance changes (including microcephaly; a high, broad forehead; large, deep-set eyes; smaller-than normal space between the nose and upper lip; an upturned nose; and/or full lips and widely-spaced teeth); difficulties standing and walking; small, cold feet; lack of or poor eye contact; frequent sideways glances; cortical visual impairment or cortical blindness; bru
  • the methods disclosed herein further comprise evaluating, e.g., measuring, the level of CDKL5 expression, e.g., CDKL5 gene expression, CDKL5 mRNA expression, and/or CDKL5 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject.
  • CDKL5 protein expression may be measured by an ELISA, a Western blot, or an immunohistochemistry assay.
  • evaluating the level of CDKL5 expression is performed prior to and/or subsequent to administration of the AAV particle, optionally wherein the level of CDKL5 expression prior to treatment is compared to the level of CDKL5 expression subsequent to administration.
  • the level of CDKL5 expression may be evaluated in a cell or tissue of the central nervous system. In some embodiments, the level of CDKL5 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 CDKL5 protein expression subsequent to administration is increased relative to the subject’s level of CDKL5 protein expression prior to administration.
  • the administration of the effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a CDKL5 protein may be a treatment that results in an increase in: (i) the level of CDKL5 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus
  • a cell, tissue e.g., a cell or tissue of the C
  • At least one additional agent suitable for treatment of a CDKL5-related disorder may be administered together with the effective amount of a pharmaceutical composition or AAV particle disclosed herein (comprising a viral genome encoding a CDKL5 protein).
  • the at least one additional agent may comprise one or more anti-epileptic drugs (e.g., valproate, levetiracetam, clobazam, lamotrigine, ganaxolone, topiramate, bromide, felbamate, phenobarbital, or a combination thereof).
  • 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.
  • 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.
  • 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 one of the viral particles, e.g., AAV, AAV particles, or AAV genome that produces CDKL5 protein described herein (i.e., viral genomes or “VG”), administering to the subject a composition or formulation comprising said AAV particle.
  • the viral particles e.g., AAV, AAV particles, or AAV genome that produces CDKL5 protein described herein (i.e., viral genomes or “VG”)
  • VG viral genomes
  • Delivery of a payload construct comprising a CDKL5-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.
  • a gene product e.g., an absence or defect in a protein
  • 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.
  • the AAV particles are delivered to a cell, tissue, or region of the CNS.
  • the AAV particles are delivered to a cell or tissue of 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palla
  • cerebellum e.g
  • the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.
  • a subject including a mammalian subject
  • any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.
  • Attorney Docket No.14640.0106-00304 [0273]
  • the AAV particles may be delivered to bypass anatomical blockages (e.g., the blood brain barrier).
  • the AAV particles may be formulated and delivered to a subject by a route which increases the speed of drug effect as compared to oral delivery.
  • the AAV particles may be delivered using intrathecal infusion.
  • a subject may be administered the AAV particles described herein using a bolus infusion.
  • the AAV particles may be delivered in a continuous and/or bolus infusion.
  • Each site of delivery may use a different dosing regimen or the same dosing regimen may be used for each site of delivery.
  • the sites of delivery may be in the cervical and the lumbar region.
  • the sites of delivery may be in the cervical region.
  • the sites of delivery may be in the lumbar region.
  • the AAV particles may be delivered to a subject via a single route of administration.
  • the AAV particles may be delivered to a subject via a multi-site route of administration.
  • a subject may be administered the AAV particles at 2, 3, 4, 5, or more than 5 sites.
  • a subject may be administered the AAV particles described herein using sustained delivery over a period of minutes, hours, or days. The infusion rate may be changed depending on the subject, distribution, formulation, or another delivery parameter known to those in the art.
  • the continuous infusion may be for 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more than 24 hours.
  • the intracranial pressure may be evaluated prior to administration. The route, volume, AAV particle concentration, infusion duration and/or vector titer may be optimized based on the intracranial pressure of a subject.
  • the AAV particles may be delivered by systemic delivery.
  • the systemic delivery may be by intravascular administration. In some embodiments, the systemic delivery may be by intravenous administration.
  • the AAV particles may be delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration.
  • an AAV particle described herein is administered intravenously.
  • the AAV particles may be delivered by direct (intraparenchymal) injection into the substance of an organ, e.g., one or more regions of the brain. Attorney Docket No.14640.0106-00304 [0287] In some embodiments, the AAV particles may be delivered by subpial injection into the spinal cord.
  • subjects may be placed into a spinal immobilization apparatus.
  • a dorsal laminectomy may be performed to expose the spinal cord.
  • Guiding tubes and XYZ manipulators may be used to assist catheter placement.
  • Subpial catheters may be placed into the subpial space by advancing the catheter from the guiding tube and AAV particles may be injected through the catheter (Miyanohara et al., Mol Ther Methods Clin Dev.2016; 3: 16046).
  • the AAV particles may be injected into the cervical subpial space.
  • the AAV particles may be injected into the thoracic subpial space.
  • the AAV particles may be delivered by direct injection to the CNS of a subject.
  • direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cisterna magna injection, or any combination thereof.
  • direct injection to the CNS of a subject comprises convection enhanced delivery (CED).
  • administration comprises peripheral injection.
  • peripheral injection is intravenous injection.
  • the AAV particles may be delivered to a subject in order to increase a CDKL5 protein level in the CNS and/or 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and
  • the AAV particles may be delivered to a subject in order to increase a CDKL5 protein level in the CNS and/or 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and
  • Transduction may also be referred to as the number of cells that are positive for CDKL5 protein.
  • delivery of AAV particles comprising a viral genome encoding CDKL5 as described herein to neurons in the brain 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 Attorney Docket No.14640.0106-00304 nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, substantia nigra, thalamus, ventral palladium, and/or vestibular nucleus) may lead
  • the increased CDKL5 protein expression may lead to improved survival and/or function of various cell types in these CNS regions and/or improvement of at least one symptom of a CDKL5-related disorder, such as a CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2 infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation.
  • CDD CDKL5 deficiency disorder
  • developmental and epileptic encephalopathy 2
  • the AAV particles may be delivered to a subject in order to establish widespread distribution of CDKL5 throughout the CNS, e.g., by administering the AAV particles to the thalamus of the subject.
  • the increased expression of CDKL5 protein may lead to a reduction in at least one symptom of a CDKL5-related disorder such as epilepsy (e.g., early-onset epilepsy); autism; deficits in cognition; limited motor skills; sleep difficulties; visual impairment; low muscle tone; gastrointestinal reflux; behavioral symptoms (including episodes of laughing or crying that occur for what appears to be no reason, hypersensitivity to touch, and/or disrupted sleep); facial appearance changes (including microcephaly; a high, broad forehead; large, deep-set eyes; smaller-than normal space between the nose and upper lip; an upturned nose; and/or full lips and widely-spaced teeth); difficulties standing and walking; small, cold feet; lack of or poor eye contact; frequent sideways glances; cortical visual impairment or cort
  • epilepsy e.g.,
  • the present disclosure provides methods comprising administering viral vectors in accordance with the disclosure to a subject in need thereof.
  • Viral vector pharmaceutical, diagnostic, or prophylactic compositions thereof may be administered to a subject using any amount and any route of administration effective for treating, or diagnosing a disease, disorder, and/or condition associated with decreased CDKL5 expression or CDKL5 deficiency.
  • the disease, disorder, and/or condition is a CDKL5-related disorder, such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation; optionally CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic
  • compositions in accordance with the disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope Attorney Docket No.14640.0106-00304 of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific protein employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • split dosing regimens such as those described herein may be used.
  • a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any therapeutic composition administered in one dose/at one time/single route/single point of contact, e.g., single administration event.
  • a single unit dose is provided as a discrete dosage form (e.g., a tablet, capsule, patch, loaded syringe, vial, etc.).
  • a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose.
  • the viral particles may be formulated in buffer only or in a formulation described herein.
  • a pharmaceutical composition described herein can be formulated into a topical, intranasal, pulmonary, intratracheal, or injectable dosage form.
  • a pharmaceutical composition described herein can be formulated in a dosage form suitable for intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and/or subcutaneous administration.
  • an AAV particle described herein is administered via intravenous administration.
  • an AAV particle described herein is formulated for intravenous administration.
  • delivery of the AAV particles described herein results in minimal serious adverse events (SAEs) as a result of the delivery of the AAV particles.
  • SAEs minimal serious adverse events
  • the AAV particles may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
  • the phrase “in combination with,” is not intended to require that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
  • Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may Attorney Docket No.14640.0106-00304 improve their bioavailability, reduce and/or modify their metabolism, and/or modify their distribution within the body.
  • the therapeutic agents may be approved by the US Food and Drug Administration or may be in clinical trial or at the preclinical research stage.
  • the therapeutic agents may utilize any therapeutic modality known in the art, with non-limiting examples including gene silencing or interference (e.g., miRNA, siRNA, RNAi, shRNA), gene editing (e.g., TALEN, CRISPR/Cas9 systems, zinc finger nucleases), and gene, protein, or enzyme replacement.
  • gene silencing or interference e.g., miRNA, siRNA, RNAi, shRNA
  • gene editing e.g., TALEN, CRISPR/Cas9 systems, zinc finger nucleases
  • gene, protein, or enzyme replacement e.g., TALEN, CRISPR/Cas9 systems, zinc finger nucleases
  • the at least one additional therapeutic agent and/or therapy comprises an agent and/or therapy for treating an CDKL5-related disorder such as a CDKL5-related neurodegenerative or neuromuscular disorder (e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome, autism, developmental and epileptic encephalopathy 2, early-onset encephalopathy, or X-linked mental retardation; optionally CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, or atypical Rett syndrome).
  • CDKL5-related neurodegenerative or neuromuscular disorder e.g., CDKL5 deficiency disorder (CDD), developmental and epileptic encephalopathy 2, infantile spasms (e.g., West syndrome), Lennox Gastaut syndrome, Rett syndrome, atypical Rett syndrome
  • the at least one additional therapeutic agent and/or therapy comprises one or more anti-epileptic drugs (e.g., levetiracetam, phenobarbital, clobazam, topiramate, felbamate, bromide), adrenocorticotropic hormone, or a combination thereof.
  • the at least one additional therapeutic agent and/or therapy comprises an immunosuppressant.
  • the immunosuppressant may be administered to the subject prior to administration of an AAV particle or pharmaceutical composition described herein.
  • the immunosuppressant may be administered to the subject simultaneously with administration of an AAV particle or pharmaceutical composition described herein.
  • the immunosuppressant may be administered to the subject after administration of an AAV particle or pharmaceutical composition described herein.
  • the AAV particle or pharmaceutical composition is administered to a subject who is receiving or has received an immunosuppressant.
  • the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxychloroquine.
  • the corticosteroid comprises prednisone, prednisolone, methylprednisolone, and/or dexamethasone.
  • Measurement of Expression may be determined using various methods known in the art such as, but not limited to immunochemistry (e.g., IHC), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT, flow cytometry, immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography-mass spectrometry (LCMS), high-performance Attorney Docket No.14640.0106-00304 liquid chromatography (HPLC), BCA assay, immunoelectrophoresis, Western blot, SDS-PAGE, protein immunoprecipitation, PCR, and/or in situ hybridization (ISH).
  • immunochemistry e.g., IHC
  • ELISA enzyme-linked immunosorbent assay
  • affinity ELISA affinity ELISA
  • ELISPOT enzyme-linked immunosorbent assay
  • transgenes encoding CDKL5 delivered in different AAV capsid variants may have different expression levels in dorsal root ganglion (DRG).
  • the CDKL5 protein is detectable by an enzyme-linked immunosorbent assay (ELISA).
  • the CDKL5 protein is detectable by an immunohistochemistry assay.
  • the CDKL5 protein is detectable by Western blot.
  • expression of an CDKL5 gene, mRNA, and/or protein is measured in a cell or tissue of a subject who is receiving or has received an AAV particle described herein.
  • the CDKL5 gene, mRNA, and/or protein expression is measured in a cell or tissue of the CNS and/or PNS 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), dorsal root ganglion (DRG), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, retina, 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 (e.g
  • kits for conveniently and/or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • Any of the vectors, constructs, or CDKL5 sequences (polypeptides or nucleotides) of the present disclosure may be comprised in a kit.
  • kits may further include reagents and/or instructions for creating and/or synthesizing compounds and/or compositions of the present disclosure.
  • kits may also include one or more buffers.
  • kits of the disclosure may include components for making protein or nucleic acid arrays or libraries and thus, may include, for example, solid supports.
  • kit components may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and suitably aliquoted.
  • kits Attorney Docket No.14640.0106-00304 may also generally contain second, third or other additional containers into which additional components may be separately placed.
  • kits may also comprise second container means for containing sterile, pharmaceutically acceptable buffers and/or other diluents.
  • various combinations of components may be comprised in one or more vial.
  • Kits of the present disclosure may also typically include means for containing compounds and/or compositions of the present disclosure, e.g., proteins, nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which desired vials are retained.
  • kit components are provided in one and/or more liquid solutions.
  • liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly used.
  • kit components may be provided as dried powder(s). When reagents and/or components are provided as dry powders, such powders may be reconstituted by the addition of suitable volumes of solvent. In some embodiments, it is envisioned that solvents may also be provided in another container means. In some embodiments, labeling dyes are provided as dried powders.
  • kits may include instructions for employing kit components as well the use of any other reagent not included in the kit. Instructions may include variations that may be implemented.
  • devices may include, but are not limited to, dental implants, stents, bone replacements, artificial joints, valves, pacemakers and/or other implantable therapeutic device.
  • the present disclosure provides for devices which may incorporate viral vectors that encode one or more CDKL5 molecules. These devices contain in a stable formulation the viral vectors which may be immediately delivered to a subject in need thereof, such as a human patient.
  • Devices for administration may be employed to deliver the viral vectors encoding CDKL5 of the present disclosure according to single, multi- or split-dosing regimens taught herein.
  • the articles “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • Adeno-associated virus As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependovirus genus or a functional variant thereof.
  • AAV may refer to wildtype (i.e., naturally occurring) AAV or recombinant AAV.
  • AAV Particle refers to a particle comprising an AAV capsid, e.g., an AAV capsid variant (such as a parent capsid sequence with at least one peptide insertion and/or with at least one substitution), and a polynucleotide, e.g., a viral genome or a vector genome.
  • the AAV particle may be capable of delivering a CDKL5 polynucleotide to cells.
  • the cells may be mammalian cells, e.g., human cells.
  • an AAV particle of the present disclosure may be produced recombinantly.
  • an AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary).
  • the AAV particle may be replication defective and/or targeted.
  • the AAV particle may comprise a peptide present in, e.g., inserted into and/or replacing a wildtype amino acid of, the capsid to enhance tropism for a desired target tissue.
  • Administering refers to providing a pharmaceutical agent or composition to a subject.
  • Amelioration refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of a neurodegenerative disorder, amelioration includes the reduction or stabilization of neuron loss.
  • Baseline refers to a measurement made before starting the treatment.
  • capsid refers to the exterior, e.g., a protein shell, of a virus particle, e.g., an AAV particle, that is substantially (e.g., >50%, >60%, >70%, >80%, >90%, >95%, >99%, or 100%) protein.
  • the capsid is an AAV capsid comprising an AAV capsid protein described herein, e.g., a VP1, VP2, and/or VP3 polypeptide.
  • the AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and/or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.”
  • an AAV capsid variant may refer to at least a VP1 protein, a VP2 protein, or a VP3 protein (e.g., all of the VP1, VP2, and VP3 proteins forming the AAV capsid) as will be clear from context.
  • the AAV capsid variant described herein may comprise a peptide and/or amino acid insertion and/or substitution.
  • the AAV capsid variant described herein has the ability to encapsulate a viral genome and/or is capable of entry into a cell, e.g., a mammalian cell.
  • the AAV capsid variant described herein may have modified tropism compared to that of a wild-type AAV capsid, e.g., the corresponding wild-type capsid.
  • CDKL5-related disorder refers to a disease, disorder, or condition in which one or more symptoms is caused by or associated with a deficiency of cyclin-dependent kinase-like 5 (CDKL5) in a subject.
  • CNS cells refers to cells of the central nervous system and sub- structures thereof. Non-limiting examples of CNS cells include neurons and sub-types thereof, glia, microglia, oligodendrocytes, ependymal cells, and astrocytes.
  • Non-limiting examples of neurons include sensory neurons, motor neurons, interneurons, unipolar cells, bipolar cells, multipolar cells, pseudounipolar cells, pyramidal cells, basket cells, stellate cells, Purkinje cells, Betz cells, amacrine cells, granule cell, ovoid cell, medium aspiny neurons and large aspiny neurons, GABAergic neurons and/or glutamatergic neurons.
  • Codon optimization refers to a process of changing codons of a given gene in such a manner that the polypeptide sequence encoded by the gene remains the same.
  • Complementary and substantially complementary refers to the ability of polynucleotides to form base pairs with one another. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand Attorney Docket No.14640.0106-00304 can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other.
  • the polynucleotide strands exhibit 10% complementarity.
  • the polynucleotide strands exhibit 90% complementarity.
  • complementary as used herein can encompass fully complementary or partially (e.g., substantially) complementary.
  • “Fully complementary”, “perfect complementarity”, or “100% complementarity” refers to the situation in which each nucleotide unit of one polynucleotide or oligonucleotide strand can base-pair with a nucleotide unit of a second polynucleotide or oligonucleotide strand.
  • the term “substantially complementary” means that >50% of the nucleotide units of a first polynucleotide strand can base pair with nucleotide units on a second polynucleotide strand.
  • substantially complementary refers to an siRNA that has a sequence (e.g., in the antisense strand) that is sufficient to bind the desired target mRNA and to trigger the RNA silencing of the target mRNA.
  • Conservative substitution As used herein, a conservative substitution, as applied to an amino acid sequence, also referred to as a “conservative amino acid substitution,” is one in which the amino acid residue is replaced with an amino acid residue having similar biochemical properties.
  • conservative substitution refers to a nucleotide replacement that results in an amino acid residue having similar biochemical properties compared to a reference sequence.
  • Families of amino acid residues having similar biochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g
  • an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 refers to the amino acid at the 585th position from the N- terminus in the amino acid sequence of SEQ ID NO: 138 or the equivalent position in an aligned sequence.
  • an amino acid at a position corresponding to that in a designated sequence may also be referred to as an amino acid at a particular position, numbered according or numbered relative to the designated sequence.
  • an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 may also be referred to as an amino acid at position 585 (or amino acid 585), numbered according to the amino acid sequence of SEQ ID NO: 138 or as numbered according to a Attorney Docket No.14640.0106-00304 sequence corresponding to the amino acid sequence of SEQ ID NO: 138 (i.e., relative to a reference sequence of the amino acid sequence of SEQ ID NO: 138).
  • Derivative refers to a composition (e.g., sequence, compound, formulation, etc.) that is derived from, or finds its basis in, a parent composition.
  • Non- limiting examples of a parent composition include a wild-type or original amino acid or nucleic acid sequence, or an undiluted formulation.
  • a derivative is a variant of a parent composition.
  • a derivative may differ from the parent composition by less than about 1%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, or less than about 50%.
  • a derivative may differ from a parent composition by more than about 50%.
  • a derivative may differ from a parent composition by more than about 75%.
  • a derivative may be a fragment or truncation of a parent amino acid or nucleotide sequence.
  • a derivative may be a sequence with a nucleotide, amino acid, or peptide substitution and/or insertion as compared to a parent nucleic acid or amino acid sequence (e.g., as compared to AAV9).
  • Effective amount As used herein, the term “effective amount” or “therapeutically effective amount” of an agent is that amount sufficient to effect beneficial or desired results. An effective amount is provided in a single dose or multiple doses to treat, improve symptoms of, delay progression of symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • fragment refers to a contiguous portion of a reference sequence.
  • a fragment may comprise a functional fragment that retains at least one activity of the reference sequence.
  • fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • a fragment may also refer to a truncation (e.g., an N- terminal and/or C-terminal truncation) of a protein or a truncation (e.g., at the 5’ and/or 3’ end) of a nucleic acid.
  • a protein fragment may be obtained by expression of a truncated nucleic acid, such that the nucleic acid encodes a portion of the full-length protein.
  • Healthy individual As used herein, the term “healthy individual” refers to an individual who does not have a disease or disorder associated with CDKL5 protein deficiency, e.g., an individual who does not have a CDKL5-related disorder.
  • Identity As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of the percent identity of two polynucleotide sequences may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second Attorney Docket No.14640.0106-00304 sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Myers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference in its entirety.
  • sequence identity may be determined using BLAST, Clustal Omega, or EMBOSS Needle.
  • Isolated refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of component with which it is associated in the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in Attorney Docket No.14640.0106-00304 which it is found in nature.
  • an isolated nucleic acid is recombinant, e.g., incorporated into a vector.
  • miRNA binding site refers either to a DNA sequence corresponding to an RNA sequence that is bound by a microRNA, or to the RNA sequence that is bound by the microRNA.
  • the miR binding site is capable of binding, or binds, in whole or in part to a microRNA (miRNA, miR) through complete or partial hybridization.
  • a miR binding site may be encoded or transcribed in series, also referred to as a “miR binding site series” or “miR BSs”, which includes two or more miR binding sites having the same or a different nucleic acid sequence.
  • Modification refers to any substance, compound, or molecule that has been changed in any way.
  • a modification in an amino acid sequence may comprise a substitution (e.g., a conservative substitution), an insertion, and/or a deletion of one or more amino acids in the sequence.
  • Neurological disease As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).
  • Operably linked As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
  • Payload As used herein, “payload,” “payload sequence,” or “payload region” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide.
  • Pharmaceutical Composition As used herein, the term “pharmaceutical composition” or pharmaceutically acceptable composition” comprises AAV polynucleotides, AAV genomes, or AAV particle and one or more pharmaceutically acceptable excipients, solvents, adjuvants, and/or the like.
  • Position refers to the location of a particular amino acid or set of amino acids relative to a larger sequence. A position or positions of amino acids may interchangeably be referred to by an amino acid number or numbers of a reference sequence.
  • positions 1-736, as numbered according to SEQ ID NO: 981 is interchangeable with “amino acids 1-736, as numbered according to SEQ ID NO: 981” and “one or more modifications at one or more of positions 580-599, as numbered according to SEQ ID NO: 138” is interchangeable with “one or more modifications at one or more of amino acids 580-599, as numbered according to SEQ ID NO: 138.”
  • an amino acid position is counted from the N-terminus.
  • the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression Attorney Docket No.14640.0106-00304 from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
  • Region refers to a zone or general area.
  • a region when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area.
  • regions comprise terminal regions.
  • terminal region refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini.
  • N-termini refer to the end of a protein comprising an amino acid with a free amino group.
  • C-termini refer to the end of a protein comprising an amino acid with a free carboxyl group.
  • N- and/or C-terminal regions may comprise the N- and/or C-termini as well as surrounding amino acids.
  • a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure.
  • regions comprise terminal regions.
  • the term “terminal region” refers to regions located at the ends or termini of a given agent.
  • sample refers to a subset of tissues, cells, nucleic acids, or a component or part of the body (e.g., a body fluid, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
  • a body fluid including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
  • Self-complementary AAV As used herein, the term “self-complementary AAV” refers to an AAV comprising at least a protein capsid and a self-complementary viral genome.
  • Serotype As used herein, the term “serotype” refers to distinct variations in a capsid of an AAV based on surface antigens which allow epidemiologic classifications of the AAVs at the sub- species level.
  • Silent Mutation As used herein, a “silent mutation” or “silent substitution” refers to a nucleotide replacement that results in the same amino acid residue as a reference sequence.
  • Signal Sequence As used herein, the phrase “signal sequence” refers to a sequence which can direct the transport or localization.
  • Similarity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
  • Spacer is generally any selected nucleic acid sequence of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive miR binding site sequences. In some embodiments, spacers may also be more than 10 nucleotides in length, e.g., 20, 30, 40, or 50 or more than 50 nucleotides.
  • Subject As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • subject or patient refers to an organism who may seek, who may require, who is receiving, or who will receive treatment or who is under care by a trained professional for a particular disease or condition.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).
  • a subject or patient may be susceptible to, suspected of having, or have a deficiency in CDKL5 protein, e.g., human CDKL5 protein, and, e.g., may be susceptible to, suspected of having, or have a CDKL5-related disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Suffering from An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms.
  • an individual who is susceptible to a disease, disorder, and/or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition.
  • Target cells refers to any one or more cells of interest.
  • the cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
  • the organism may be an animal, preferably a mammal, more preferably a human and most preferably a human patient.
  • Target Tissue refers to a tissue of interest that may be found in vitro, in situ, or as part of an animal, preferably a mammal, more preferably a human and most preferably a human patient.
  • Therapeutic Agent refers to any agent that, when administered to a subject, elicits a desired biological and/or pharmacological effect.
  • therapeutically effective outcome means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, delay progression of symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • Treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, reducing incidence of, and/or preventing one or more symptoms or features of a particular infection, disease, disorder, and/or condition.
  • Unmodified refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild-type or native form of a biomolecule or entity. Molecules or entities may undergo a series of modifications whereby each modified product may serve as the “unmodified” starting molecule or entity for a subsequent modification.
  • variant refers to a polypeptide or polynucleotide that has an amino acid or a nucleotide sequence that has at least 90% (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 a reference sequence.
  • the variant may be a functional variant.
  • functional variant refers to a polypeptide variant or a polynucleotide variant that has at least one activity of the reference sequence.
  • Vector As used herein, a “vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequence(s).
  • AAV adeno-associated virus
  • Viral genome As used herein, a “viral genome,” “vector genome,” or “VG” is a polynucleotide comprising at least one inverted terminal repeat (ITR) and at least one nucleic acid sequence encoding a payload. A viral genome encodes at least one copy of the payload.
  • the library of AAV capsid variants was generated using a sliding window approach, where amino acid modifications, e.g., substitutions, were randomly introduced into various positions across loop VIII of AAV9, including between positions 581-593, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • nervous tissue e.g., brain, spinal cord, and DRG of the NHPs.
  • RNA recovery and RT-PCR amplification a systematic NGS enrichment analysis was performed, and the peptides comprised within the variants were identified and the capsid enrichment ratio for each variant compared to the wild-type AAV9 control was calculated (fold enrichment relative to wild-type AAV9). Values above 1 indicate an increase in expression relative to AAV9. All animals were dosed intravenously at 2e13 VG/kg across the screen. [0375] Following these three passages, approximately 1542 variants were identified with an average fold change greater than wild-type AAV9 in the brain of the NHPs (Macaca fascicularis).
  • the capsid variant that demonstrated the greatest fold-change in expression relative to AAV9 in the brain of the NHP comprised the amino acid sequence TQDWHRI (SEQ ID NO: 941), resulting in a fold-change in expression of 460 relative to the AAV9 control (Table 9).
  • Table 9 The capsid variant that demonstrated the greatest fold-change in expression relative to AAV9 in the brain of the NHP comprised the amino acid sequence TQDWHRI (SEQ ID NO: 941), resulting in a fold-change in expression of 460 relative to the AAV9 control (Table 9). Table 9.
  • NGS fold-enrichment of AAV capsid variants in NHPs (Macaca fascicularis)
  • BBB blood brain barrier
  • the goal of these experiments was to determine the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of a capsid variant selected from the study described in Example 1 relative to AAV9 following intravenous injection in NHPs (cynomolgus macaques (Macaca fascicularis)) and marmosets (Callithrix jacchus).
  • the capsid variant was TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201), as outlined in Table 3 above.
  • TTJ-001 The amino acid and DNA sequences of TTJ-001 are provided, e.g., in Tables 4 and 5, respectively.
  • AAV particles were generated with this capsid variant encapsulating a sequence encoding a cynomolgus frataxin protein tagged with hemagglutinin (cyno-FXN-HA) driven by a CBA promoter.
  • the in-life period was 14 days and the brain, spinal cord, and peripheral tissues including the heart, liver, and quadriceps were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution).
  • the AAV particles administered to the NHPs comprised single stranded viral genomes.
  • the brains isolated from the NHPs injected with the AAV particles encapsulated in the TTJ- 001 capsid variant (AAV_TTJ-001) were assayed by ddPCR for the presence of transgene RNA as a measure of transgene expression, and the presence of viral DNA as a measure of viral genome levels.
  • mRNA transgene expression from the TTJ-001 capsid variant which is an AAV9 capsid variant
  • mRNA transgene expression from the TTJ-001 capsid variant was significantly higher in the brain of NHPs relative to the wild-type AAV9 control. More specifically, mRNA expression was approximately 40-280-fold higher from the TTJ-001 capsid variant compared to wild-type AAV9 in the brain of NHPs. Additionally, mRNA expression was approximately 3-274-fold higher from the TTJ-001 capsid variants compared to wild-type AAV9 in the spinal cord of the NHPs.
  • biodistribution of the AAV9 capsid variant, TTJ-001 was significantly higher in the NHP brain relative to the wild-type AAV9 control. Biodistribution of TTJ-001 was lower in the NHP liver relative to the wild-type AAV9 control. Comparable results were obtained when average DNA levels were measured by qPCR. Table 11.
  • TTJ-001 demonstrated moderately to strongly positive staining in the globus pallidus, thalamus, substantia nigra, oculomotor nucleus, geniculate nucleus, central grey, inferior colliculus, external cuneate nucleus, gracile nucleus, nucleus ambiguus, ventral horn of the spinal cord, vestibular nucleus, inferior olivary complex, deep cerebellar nuclei, and the DRG.
  • TTJ-001 demonstrated weakly positive to moderately positive staining in the ventral pallidum, amygdala, caudate, putamen, hippocampus, temporal cortex, auditory cortex, and somatosensory cortex.
  • TTJ-001 resultsed in widespread transduction of multiple peripheral tissues, as compared to the AAV9 control.
  • TTJ-001 demonstrated weakly positive to moderately positive staining in the pancreas and quadriceps and moderately to strongly positive staining in the heart, kidney, and liver.
  • TTJ-001 is an enhanced CNS-tropic capsid variant in NHPs. Additionally, this capsid variant was able to successfully penetrate the blood brain barrier following intravenous injection.
  • AAV particles were generated with the TTJ-001 capsid variant or the AAV9 capsid control which comprised a self-complementary viral genome encoding a histone H2b protein with a VSV tag (TTJ-001 capsid variant) or HA tag (AAV9 control capsid) driven by a ubiquitous CAG promoter.
  • the in-life period was 28 days and then various CNS and peripheral tissues were collected for measuring transgene mRNA (expression) by RT-qPCR, protein expression by IHC, and viral DNA (biodistribution) by ddPCR. Data were then normalized to the dose of each viral vector in the dosing solution. Table 12.
  • biodistribution and transgene expression were increased over 5-7 fold and 15-17 fold, respectively, for TTJ-001 in the brain of marmosets relative to AAV9. Similar expression and biodistribution was observed by immunohistochemistry. More specifically, staining for TTJ-001 was detected in the mid-brain, caudate, putamen, thalamus, and cerebellum, and this staining was increased relative to AAV9. Staining for TTJ-001 was also observed in the molecular and granule layer of the cerebellum. [0386] Biodistribution and transgene expression were also measured in the peripheral tissues of the liver, heart, and quadriceps.
  • the TTJ-001 capsid variant In the liver, the TTJ-001 capsid variant exhibited lower biodistribution (Table 13) and transgene expression (Table 14) relative to AAV9, indicating that the TTJ-001 capsid variant was detargeted in the liver relative to AAV9 in marmosets. Additionally, the TTJ-001 capsid variant resulted in increased transduction of the heart and muscle, as compared to the AAV9 control (Table 13 and Table 14). Table 13.
  • TTJ-001 in Cynomolgus Macaques (Macaca fascicularis)
  • AAV particles were generated with the TTJ-001 capsid variant comprising a single-stranded viral genome encapsulating codon-optimized nucleotide sequence encoding GBA1, which is tagged with hemagglutinin (GBA1-HA).
  • GBA1-HA hemagglutinin
  • the GBA1-HA payload is driven by a CBA promoter.
  • the in-life period was 28 days and the brain, spinal cord, and peripheral tissues including the heart, liver, and quadriceps were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution).
  • the TTJ-001 capsid variant demonstrated increased biodistribution and transduction as well as transgene expression in the brain (putamen, motor cortex, frontal cortex, substantia nigra, and dentate nucleus) and spinal cord (cervical C3 ventral horn) relative to AAV9.
  • TTJ-001 When dosed at 1e13 vg/kg, TTJ-001 led to at least a 6-9-fold increase in biodistribution in the brain and at least an 8-fold increase in biodistribution in the spinal cord relative to AAV9. With respect to transgene expression, when dosed at 1e13 kg/vg, TTJ-001 demonstrated at least a 25-61-fold increase in expression in the brain and at least a 20-fold increase in the spinal cord relative to AAV9. TTJ-001 also demonstrated comparable biodistribution and transgene expression in the DRG relative to AAV9 (Tables 15, 16, and 17).
  • TTJ-001 By immunohistochemistry (IHC), when dosed at 1e13 vg/kg, TTJ-001 demonstrated largely neuronal tropism, as evidenced by positive co-staining for HA/SMI 331 (neuronal marker) at least in the putamen and substantia nigra. No staining was observed in astrocytes in the putamen and substantia nigra.
  • IHC immunohistochemistry
  • TTJ- Attorney Docket No.14640.0106-00304 001 was capable of transducing greater than 0.9 to 5% cells in the brain regions tested (Table 18). The percent HA+ cells in Table 18 may underestimate true positivity for the TTJ-001 capsid variant, as it demonstrated neuronal tropism. [0392] Biodistribution and transgene expression were also measured in the peripheral tissues of the liver and heart.
  • the TTJ-001 capsid variant In the liver, at a dose of 1e13 vg/kg, the TTJ-001 capsid variant exhibited lower biodistribution (Table 15) and transgene expression (Tables 16 and 17) relative to AAV9, indicating that the TTJ-001 capsid variant was detargeted in the liver relative to AAV9.
  • the TTJ-001 demonstrated comparable biodistribution but decreased transgene expression in the heart relative to AAV9 (Tables 15, 16, and 17). Similar transduction and expression were observed in the heart by IHC following transduction with AAV particles comprising the TTJ-001 capsid variant and AAV particles comprising the AAV9 capsid control at a dose of 1e13 vg/kg.
  • AAV particles were generated with the TTJ-001 capsid variant or the AAV9 capsid control, each of which comprised a self-complementary viral genome encoding a histone H2b protein driven by a ubiquitous CAG promoter.
  • the AAV particles comprising the TTJ-001 capsid control were administered to a first group of male cynomolgus macaques (Macaca fascicularis; 4-6 kg body weight; over 2 years old) intravenously at a dose per capsid of 4e12 VG/kg.
  • the AAV particles comprising the AAV9 capsid control were administered to a second group of male cynomolgus macaques (Macaca fascicularis; 4-6 kg body weight; over 2 years old) intravenously at a dose per capsid of 4e12 VG/kg.
  • the in-life period was 28 days for both groups, and then various CNS and peripheral tissues were collected for measuring transgene mRNA (expression) by RT-qPCR; protein expression by IHC/chromogenic staining (e.g., DAB staining for percent of DAB+ cells indicating the percent of cells transduced); percent positive cells (e.g., Attorney Docket No.14640.0106-00304 neurons, motor neurons, and astrocytes) in brain and spinal cord regions by immunofluorescence microscopy; and viral DNA (biodistribution) by ddPCR.
  • IHC/chromogenic staining e.g., DAB staining for percent of DAB+ cells indicating the percent of cells transduced
  • percent positive cells e.g., Attorney Docket No.14640.0106-00304 neurons, motor neurons, and astrocytes
  • viral DNA biodistribution
  • TTJ-001 demonstrated increased CNS transduction and/or biodistribution in several regions of the brain (greater than 10-20% of cells transduced observed in several regions) and spinal cord of the cynomolgus macaques after intravenous administration at a relatively low dose of 4e12 vg/kg. More specifically, TTJ-001 was capable of transducing up to 11% of cells in the putamen; up to 19% of cells in the caudate, and up to 23% of cells in the putamen. TTJ-001 also showed improved delivery to the spinal cord relative to AAV9, at a dose of 4e12 vg/kg.
  • TTJ-001 also demonstrated increased transduction and/or biodistribution in the peripheral tissues of the muscle, specifically the vastus lateralis muscle and the gastrocnemius muscle relative to AAV9, and comparable transduction and/or biodistribution in the heart relative to AAV9 (Table 20 and Table 21). TTJ-001 also appeared to exhibit partial de-targeting in the liver (Table 20 and Table 21). Table 19.
  • Example 3 Evaluation of TTJ-001 AAV9 capsid variant in Diverse Primate Species [0398]
  • This Example evaluates the tropism and cross-species compatibility of the TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201) capsid variant in two diverse primate species, marmosets (Callithrix jacchus) and African green monkeys (Chlorocebus sabaeus), as compared to their tropism in cynomolgus macaques (Macaca fascicularis) provided in Example 1 and 2.
  • the brains and tissues liver quadriceps, and Attorney Docket No.14640.0106-00304 heart
  • RNA was extracted.
  • a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV9 wild-type control.
  • the TTJ-001 capsid variant demonstrated increased CNS tropism in diverse primate species relative to the AAV9 control.
  • the TTJ-001 capsid variant demonstrated a 460.2-fold increase in expression relative to AAV9 in the brain of cynomolgus macaques (Table 9, Example 1), a 14.67-fold increase in expression relative to AAV9 in the brain of African green monkeys, and a 24.2-fold increase in expression relative to AAV9 in the brain of marmosets. Table 22.
  • NGS fold-enrichment of TTJ-001 AAV capsid variant comprising SEQ ID NO: 941 in African green monkeys Fold enrichment relative to AAV9 Sequence SEQ ID NO: Brain DRG Heart Liver DNA Liver RNA Muscle DNA T DWHRI 941 14670 005099 14567 01935 04688 12547 S equence SEQ ID o d enr c ment re at ve to 9 NO: Brain Heart Liver DNA Liver RNA Muscle DNA ated increased CNS tropism relative to the AAV9 control in the CNS across three diverse primate species, providing evidence of strong cross-species capacity.
  • This Example describes maturation of the AAV9 capsid variant, TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 (encoded by SEQ ID NO: 942)) or 201) in two species of NHPs, cynomolgus macaques (Macaca fascicularis) and marmosets (Callithrix jacchus) to further enhance their transduction and biodistribution in the central nervous system as well as other tissues, and evolve the AAV capsid variants to provide further cross-species compatibility.
  • the TTJ-001 capsid variant was also matured in mice.
  • Two approaches were used to mature the TTJ-001 capsid sequence in order to randomize and mutate within and around the modifications comprised within loop VIII of the capsid variant.
  • sets of three contiguous amino acids were randomized across the mutagenesis region in the TTJ-001 sequence, which spanned from position 582 to position 593, numbered according to SEQ ID NO: 981.
  • mutagenic primers were used to introduce point mutations at a low frequency, scattered across the mutagenesis region in the TTJ-001 sequence ranging from position 582 to position 593, numbered according to SEQ ID NO: 981.
  • the library of pooled matured AAV capsid variants generated from TTJ-001 using the first maturation approach and the library of pooled matured AAV capsid variants generated from TTJ-001 using the second maturation approach were each injected into two cynomolgus macaques (Macaca fascicularis), two marmosets (Callithrix jacchus), and three outbred mice. After a 14- or 28-day period in Attorney Docket No.14640.0106-00304 life, the brains of the NHPs and mice were isolated and RNA was extracted.
  • RNA recovery and RT-PCR amplification a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the TTJ-001 non-matured control or an AAV9 control, and the peptides comprised within the variants were identified.
  • the matured capsid variants were filtered based on their coefficient of variation (CV), which was calculated for each peptide across the brain samples taken from the NHPs and mice. Those that had a CV value ⁇ 1 were identified, as these were the peptides that were reliably detected in the majority of samples isolated from the brains of the two NHPs.
  • CV coefficient of variation
  • Table 24 provides the peptide sequences of the matured capsid variants from the first maturation approach having an RPM greater than or equal to 1 in the original virus stock, a CV of less than 1 for the brain samples isolated, and that also demonstrated a 24-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of cynomolgus macaques (Macaca fascicularis) and/or a 50-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of marmosets (Callithrix jacchus), that were also detected in the maturation screen in mice, as these variants demonstrate cross-species compatibility.
  • RPM read per million
  • the TTJ-001 capsid variants comprising SEQ ID NOs: 205, 208, 212, 219, 286, or 307 generated using the first maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and mice.
  • TTJ-001 capsid variants comprising any one of SEQ ID NOs: 262-265 generated using the first maturation approach demonstrated a fold change greater than the non- matured TTJ-001 control and the AAV9 control in both species of NHPs. Table 24.
  • the TTJ-001 capsid variants comprising SEQ ID NOs: 283, 286, 290, 291, and 293 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and mice.
  • the TTJ-001 capsid variants comprising SEQ ID NOs: 205, 208, 212, 213, or 307 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control in cynomolgus macaques and mice and a fold-change greater than the AAV9 control in both species of NHPs and mice.
  • the TTJ-001 capsid variants comprising SEQ ID NOs: 219 and 265 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and the TTJ-001 capsid variant comprising SEQ ID NO: 263 generated using the second maturation approach lead to a fold-change greater than AAV9 in both species of NHPs and a fold-change greater than TTJ-001 in marmosets.
  • Table 25 The TTJ-001 capsid variants comprising SEQ ID NOs: 219 and 265 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and the TTJ-001 capsid variant comprising SEQ ID NO: 263 generated using the second maturation approach lead to a fold-change greater than AAV9 in both species of NHPs and a fold-change greater than TTJ-
  • Example 5 Individual Capsid Characterization in Mice
  • the goal of these experiments was to determine the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of a capsid variant selected from the study described in Example 1 relative to AAV9 following intravenous injection in BALB/c mice.
  • the capsid variant was TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201), as outlined in Table 3 above.
  • the amino acid and DNA sequences of TTJ-001 are provided, e.g., in Tables 4 and 5, respectively.
  • AAV particles were generated with this capsid variant encapsulating a luciferase-EGFP-HA tagged reporter gene driven by a CBA promoter encoded by a single stranded viral genome.
  • the in-life period was 28 days and the brain and the liver were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution).
  • mice were injected with luciferin and imaged on the ventral and dorsal side by the IVIS imager. Robust luciferase signal was observed in mice injected with AAV particles encapsulated in the AAV9 control Attorney Docket No.14640.0106-00304 capsid but less luciferase signal was observed in mice injected with the AAV particles encapsulated in the TTJ-001 capsid variant.
  • the brains and livers were isolated on day 28 and subsequently assayed by qPCR for the presence of transgene mRNA, as a measure of transgene expression.
  • mRNA expression from the TTJ-001 capsid variant was slightly increased in the brain of the mice relative to the AAV9 control. More specifically, mRNA expression was approximately 1.3-fold higher from the TTJ-001 capsid variant compared to wild- type AAV9 in the brain of the mice. In the liver, mRNA expression from the TTJ-001 capsid variant was decreased relative to the AAV9 control. Table 27.
  • Transgene mRNA expression with the TTJ-001 capsid variant in the brain and liver of mice Capsid Variant Tissue AVG transgene mRNA fold change Fold change relative to AAV9 relative to housekeeping gene AAV9 Brain 0544 10 esence of viral DNA by qPCR as a measure of viral genome levels.
  • Data are provided in Table 28 as average DNA (viral genome (VG) copies per diploid genome (dg) as well as fold change relative to the AAV9 control.
  • VG viral genome
  • dg diploid genome
  • TTJ-001 Viral DNA biodistribution with the TTJ-001 capsid variant in the brain and liver of mice Capsid Variant Tissue AVG VG per Fold change relative to AAV9 diploid cell bjected to immunohistochemistry staining to evaluate overall tropism in the CNS and peripheral tissues and transgene expression.
  • TTJ-001 showed comparable staining in the brain, muscle, and liver, as compared to the AAV9 control. Additionally, TTJ-001 demonstrated decreased staining in the heart relative to AAV9.
  • Example 6 Example 6
  • TTJ-001 capsid variants were injected into cynomolgus macaques (Macaca fascicularis), marmosets (Callithrix jacchus), or mice.
  • the matured AAV capsid variants generated from the TTJ-001 non-matured capsid variant (SEQ ID NO: 981 (amino acid) and SEQ ID NO: 983 (DNA), comprising SEQ ID NO: 941 or Attorney Docket No.14640.0106-00304 SEQ ID NO: 201)) were injected into two NHPs (cynomolgus macaques or marmosets) or mice. After a period in life, the brains, heart, muscles, and/or livers of the NHPs or mice were isolated and DNA/RNA was extracted.
  • NHPs cynomolgus macaques or marmosets
  • Table 29 provides the sequences of the matured capsid variants having a counts per million greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in the brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in cynomolgus macaques. These variants demonstrated increased tropism in the brain of cynomolgus macaques. Table 29.
  • AAV capsid variants comprising SEQ ID NO: 216 or SEQ ID NO: 943, were able to cross the blood brain barrier and demonstrate increased expression in the brains of both cynomolgus macaques (Table 29) and marmosets (Table 30) relative to AAV9, and comparable or increased expression relative to TTJ-001 in the same tissues. Table 30.
  • Table 32 provides data that was filtered by a counts per million greater than or equal to 10, a CV across the brain tissues of less than or equal to 1, and an average fold-change in cDNA levels in the brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in mice. Table 32.
  • NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the brains, hearts, and muscles of mice (changes in the peptide sequence relative to TTJ-001 are bolded and underlined)
  • Mutational Approach 1 SE Fold change relative to TTJ-001 Fold change relative to AAV9 Sequence Q ID 9 n greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in the heart relative to the cDNA levels of AAV9 that is greater than or equal to 2 in cynomolgus macaques.
  • These variants demonstrated increased tropism in the heart and muscles (intercostal, diaphragm, and quadriceps) relative to AAV9 and comparable expression to TTJ-001 in these same tissues in cynomolgus macaques.
  • the AAV capsid variants comprising SEQ ID NO: 241 or SEQ ID NO: 984 also demonstrated detargeting in the liver relative to AAV9 and TTJ-001 (Table 34).
  • Table 34 provides data that was filtered by a counts per million greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in the heart relative to the cDNA levels of AAV9 that is greater than or equal to 2 in marmosets. Table 34.
  • compositions of the disclosure e.g., any, composition, therapeutic or active ingredient; any method of production; any method of use; etc.
  • any particular embodiment of the compositions of the disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physiology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions et des procédés pour modifier, par exemple, améliorer, le niveau de protéine CDKL5 par l'intermédiaire d'une administration à l'aide d'un variant de capside de virus adéno-associé (AAV). Les compositions et les procédés de la présente invention sont utiles dans le traitement d'un trouble lié à CDKL5, par exemple, un trouble neurodégénératif ou neuromusculaire lié à CDKL5.
PCT/US2024/058361 2023-12-05 2024-12-04 Compositions et méthodes pour le traitement de troubles liés à une déficience en cdkl5 Pending WO2025122548A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363606438P 2023-12-05 2023-12-05
US63/606,438 2023-12-05

Publications (1)

Publication Number Publication Date
WO2025122548A1 true WO2025122548A1 (fr) 2025-06-12

Family

ID=94083417

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/058361 Pending WO2025122548A1 (fr) 2023-12-05 2024-12-04 Compositions et méthodes pour le traitement de troubles liés à une déficience en cdkl5

Country Status (1)

Country Link
WO (1) WO2025122548A1 (fr)

Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5064764A (en) 1988-12-20 1991-11-12 Commissariat A L'energie Atomique Mineral hollow fiber bioreactor for the cultivation of animal cells
WO1996023810A1 (fr) 1994-11-10 1996-08-08 The Regents Of The University Of California Proteines fluorescentes vertes modifiees
WO1996030540A2 (fr) 1995-03-20 1996-10-03 The Regents Of The University Of California Substrats pour beta-lactamase et utilisations de ces substrats
WO1996039530A2 (fr) 1995-06-05 1996-12-12 The Trustees Of The University Of Pennsylvania Adenovirus et virus adeno-associe de recombinaison, lignees cellulaires et leurs procedes de production et d'utilisation
WO1998010088A1 (fr) 1996-09-06 1998-03-12 Trustees Of The University Of Pennsylvania Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7
US5756283A (en) 1995-06-05 1998-05-26 The Trustees Of The University Of Pennsylvania Method for improved production of recombinant adeno-associated viruses for gene therapy
WO1999014354A1 (fr) 1997-09-19 1999-03-25 The Trustees Of The University Of The Pennsylvania Procedes et produits genetiques vectoriels utiles pour obtenir un virus adeno-associe (aav)
WO1999015685A1 (fr) 1997-09-19 1999-04-01 The Trustees Of The University Of Pennsylvania Procedes et lignee cellulaire utiles pour la production de virus adeno-associes recombines
WO1999047691A1 (fr) 1998-03-20 1999-09-23 Trustees Of The University Of Pennsylvania Compositions et methodes de production de virus adeno-associes recombines sans auxiliaire
WO2000028004A1 (fr) 1998-11-10 2000-05-18 The University Of North Carolina At Chapel Hill Vecteurs viraux et leurs procedes d'elaboration et d'administration
WO2000055342A1 (fr) 1999-03-18 2000-09-21 The Trustees Of The University Of Pennsylvania Compositions et techniques de production sans auxiliaire de virus adeno-associes de recombinaison
WO2000075353A1 (fr) 1999-06-02 2000-12-14 Trustees Of The University Of Pennsylvania Compositions et methodes pour la fabrication de virus recombines necessitant des virus auxiliaires
US6180613B1 (en) 1994-04-13 2001-01-30 The Rockefeller University AAV-mediated delivery of DNA to cells of the nervous system
US6194191B1 (en) 1996-11-20 2001-02-27 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
WO2001023597A2 (fr) 1999-09-29 2001-04-05 The Trustees Of The University Of Pennsylvania Lignees de cellules et produits d'assemblage servant a l'obtention d'adenovirus a deletion e-1 en l'absence d'adenovirus a capacite de replication
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
WO2001023001A3 (fr) 1999-09-29 2002-03-14 Univ Pennsylvania Procedes de modification rapide du peg de vecteurs viraux, compositions servant a ameliorer la transduction de genes, compositions presentant une stabilite physique augmentee, et leurs utilisations
US6485966B2 (en) 1999-03-18 2002-11-26 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
WO2004112727A2 (fr) 2003-06-19 2004-12-29 Avigen, Inc. Virions aav presentant une immunoreactivite reduite et utilisations
WO2005005610A2 (fr) 2003-06-30 2005-01-20 The Regents Of The University Of California Virions de virus adeno-associes mutants et procedes d'utilisation
WO2005072364A2 (fr) 2004-01-27 2005-08-11 University Of Florida Systeme d'expression baculovirus modifie utilise pour la production d'un vecteur raav pseudotype
US6953690B1 (en) 1998-03-20 2005-10-11 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
US7291498B2 (en) 2003-06-20 2007-11-06 The Trustees Of The University Of Pennsylvania Methods of generating chimeric adenoviruses and uses for such chimeric adenoviruses
US7465583B2 (en) 2000-06-01 2008-12-16 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US7491508B2 (en) 2003-06-20 2009-02-17 The Trustees Of The University Of Pennsylvania Methods of generating chimeric adenoviruses and uses for such chimeric adenoviruses
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
US8137948B2 (en) 2003-05-21 2012-03-20 Genzyme Corporation Methods for producing preparations of recombinant AAV virions substantially free of empty capsids
US8163543B2 (en) 2005-10-20 2012-04-24 Amsterdam Molecular Therapeutics B.V. AAV vectors produced in insect cells
WO2015191508A1 (fr) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Capsides chimériques
WO2018071831A1 (fr) * 2016-10-13 2018-04-19 University Of Massachusetts Conceptions de capsides de vaa
WO2018119330A2 (fr) 2016-12-22 2018-06-28 Oregon Health & Science University Vecteurs viraux adéno-associés
WO2020072683A1 (fr) 2018-10-02 2020-04-09 Voyager Therapeutics, Inc. Redirection de tropisme de capsides aav
WO2021202651A1 (fr) 2020-04-01 2021-10-07 Voyager Therapeutics, Inc. Redirection de tropisme de capsides de vaa
WO2021230987A1 (fr) 2020-05-13 2021-11-18 Voyager Therapeutics, Inc. Redirection de tropisme de capsides de vaa
WO2022098699A1 (fr) * 2020-11-04 2022-05-12 Voyager Therapeutics, Inc. Compositions et procédés pour le traitement de la tauopathie
US20230054144A1 (en) * 2020-02-14 2023-02-23 Ultragenyx Pharmaceutical Inc. Gene therapy for treating cdkl5 deficiency disorder
WO2023060269A1 (fr) * 2021-10-07 2023-04-13 Regenxbio Inc. Virus adéno-associés recombinants pour administration ciblée
WO2023091948A1 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Variants de capsides d'aav et leurs utilisations
WO2023201207A1 (fr) * 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
WO2023235791A1 (fr) * 2022-06-02 2023-12-07 Voyager Therapeutics, Inc. Variants de capside de vaa et leurs utilisations

Patent Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5064764A (en) 1988-12-20 1991-11-12 Commissariat A L'energie Atomique Mineral hollow fiber bioreactor for the cultivation of animal cells
US6180613B1 (en) 1994-04-13 2001-01-30 The Rockefeller University AAV-mediated delivery of DNA to cells of the nervous system
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
WO1996023810A1 (fr) 1994-11-10 1996-08-08 The Regents Of The University Of California Proteines fluorescentes vertes modifiees
WO1996030540A2 (fr) 1995-03-20 1996-10-03 The Regents Of The University Of California Substrats pour beta-lactamase et utilisations de ces substrats
WO1996039530A2 (fr) 1995-06-05 1996-12-12 The Trustees Of The University Of Pennsylvania Adenovirus et virus adeno-associe de recombinaison, lignees cellulaires et leurs procedes de production et d'utilisation
US5756283A (en) 1995-06-05 1998-05-26 The Trustees Of The University Of Pennsylvania Method for improved production of recombinant adeno-associated viruses for gene therapy
US6281010B1 (en) 1995-06-05 2001-08-28 The Trustees Of The University Of Pennsylvania Adenovirus gene therapy vehicle and cell line
US6270996B1 (en) 1995-06-05 2001-08-07 The Trustees Of The University Of Pennsylvania Recombinant adenovirus and adeno-associated virus, cell lines and methods of production and use thereof
US6261551B1 (en) 1995-06-05 2001-07-17 The Trustees Of The University Of Pennsylvania Recombinant adenovirus and adeno-associated virus, cell lines, and methods of production and use thereof
US6555525B2 (en) 1995-08-31 2003-04-29 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
WO1998010088A1 (fr) 1996-09-06 1998-03-12 Trustees Of The University Of Pennsylvania Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7
US6194191B1 (en) 1996-11-20 2001-02-27 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6482634B1 (en) 1997-09-19 2002-11-19 The Trustees Of The University Of Pennsylvania Methods and vector constructs useful for production of recombinant AAV
US7238526B2 (en) 1997-09-19 2007-07-03 The Trustees Of The University Of Pennsylvania Methods and cell line useful for production of recombinant adeno-associated viruses
US6943019B2 (en) 1997-09-19 2005-09-13 The Trustees Of The University Of Pennsylvania Methods and vector constructs useful for production of recombinant AAV
WO1999014354A1 (fr) 1997-09-19 1999-03-25 The Trustees Of The University Of The Pennsylvania Procedes et produits genetiques vectoriels utiles pour obtenir un virus adeno-associe (aav)
WO1999015685A1 (fr) 1997-09-19 1999-04-01 The Trustees Of The University Of Pennsylvania Procedes et lignee cellulaire utiles pour la production de virus adeno-associes recombines
US6475769B1 (en) 1997-09-19 2002-11-05 The Trustees Of The University Of Pennsylvania Methods and cell line useful for production of recombinant adeno-associated viruses
US6953690B1 (en) 1998-03-20 2005-10-11 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
WO1999047691A1 (fr) 1998-03-20 1999-09-23 Trustees Of The University Of Pennsylvania Compositions et methodes de production de virus adeno-associes recombines sans auxiliaire
WO2000028004A1 (fr) 1998-11-10 2000-05-18 The University Of North Carolina At Chapel Hill Vecteurs viraux et leurs procedes d'elaboration et d'administration
US7022519B2 (en) 1999-03-18 2006-04-04 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
US6485966B2 (en) 1999-03-18 2002-11-26 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
US6258595B1 (en) 1999-03-18 2001-07-10 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
WO2000055342A1 (fr) 1999-03-18 2000-09-21 The Trustees Of The University Of Pennsylvania Compositions et techniques de production sans auxiliaire de virus adeno-associes de recombinaison
WO2000075353A1 (fr) 1999-06-02 2000-12-14 Trustees Of The University Of Pennsylvania Compositions et methodes pour la fabrication de virus recombines necessitant des virus auxiliaires
WO2001023597A2 (fr) 1999-09-29 2001-04-05 The Trustees Of The University Of Pennsylvania Lignees de cellules et produits d'assemblage servant a l'obtention d'adenovirus a deletion e-1 en l'absence d'adenovirus a capacite de replication
US6365394B1 (en) 1999-09-29 2002-04-02 The Trustees Of The University Of Pennsylvania Cell lines and constructs useful in production of E1-deleted adenoviruses in absence of replication competent adenovirus
WO2001023001A3 (fr) 1999-09-29 2002-03-14 Univ Pennsylvania Procedes de modification rapide du peg de vecteurs viraux, compositions servant a ameliorer la transduction de genes, compositions presentant une stabilite physique augmentee, et leurs utilisations
US7465583B2 (en) 2000-06-01 2008-12-16 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US8137948B2 (en) 2003-05-21 2012-03-20 Genzyme Corporation Methods for producing preparations of recombinant AAV virions substantially free of empty capsids
WO2004112727A2 (fr) 2003-06-19 2004-12-29 Avigen, Inc. Virions aav presentant une immunoreactivite reduite et utilisations
US7291498B2 (en) 2003-06-20 2007-11-06 The Trustees Of The University Of Pennsylvania Methods of generating chimeric adenoviruses and uses for such chimeric adenoviruses
US7491508B2 (en) 2003-06-20 2009-02-17 The Trustees Of The University Of Pennsylvania Methods of generating chimeric adenoviruses and uses for such chimeric adenoviruses
WO2005005610A2 (fr) 2003-06-30 2005-01-20 The Regents Of The University Of California Virions de virus adeno-associes mutants et procedes d'utilisation
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
WO2005072364A2 (fr) 2004-01-27 2005-08-11 University Of Florida Systeme d'expression baculovirus modifie utilise pour la production d'un vecteur raav pseudotype
US8163543B2 (en) 2005-10-20 2012-04-24 Amsterdam Molecular Therapeutics B.V. AAV vectors produced in insect cells
WO2015191508A1 (fr) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Capsides chimériques
WO2018071831A1 (fr) * 2016-10-13 2018-04-19 University Of Massachusetts Conceptions de capsides de vaa
WO2018119330A2 (fr) 2016-12-22 2018-06-28 Oregon Health & Science University Vecteurs viraux adéno-associés
WO2020072683A1 (fr) 2018-10-02 2020-04-09 Voyager Therapeutics, Inc. Redirection de tropisme de capsides aav
US20230054144A1 (en) * 2020-02-14 2023-02-23 Ultragenyx Pharmaceutical Inc. Gene therapy for treating cdkl5 deficiency disorder
WO2021202651A1 (fr) 2020-04-01 2021-10-07 Voyager Therapeutics, Inc. Redirection de tropisme de capsides de vaa
WO2021230987A1 (fr) 2020-05-13 2021-11-18 Voyager Therapeutics, Inc. Redirection de tropisme de capsides de vaa
WO2022098699A1 (fr) * 2020-11-04 2022-05-12 Voyager Therapeutics, Inc. Compositions et procédés pour le traitement de la tauopathie
WO2023060269A1 (fr) * 2021-10-07 2023-04-13 Regenxbio Inc. Virus adéno-associés recombinants pour administration ciblée
WO2023091948A1 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Variants de capsides d'aav et leurs utilisations
WO2023201207A1 (fr) * 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
WO2023235791A1 (fr) * 2022-06-02 2023-12-07 Voyager Therapeutics, Inc. Variants de capside de vaa et leurs utilisations

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Methods In Molecular Biology", 1995, HUMANA PRESS
"Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
ADACHI ET AL., NATURE COMMUNICATIONS, vol. 5, no. 3075, 2014
ALTSCHUL, S. F. ET AL., J. MOLECULAR BIOL., vol. 215, 1990, pages 403
BELL ET AL., J. VIROL., vol. 86, no. 13, 2012, pages 7326 - 33
CABIOS, vol. 4, 1989, pages 11 - 17
CARILLO, H.LIPMAN, D., SIAM J APPLIED MATH., vol. 48, 1988, pages 1073
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
DIMATTIA ET AL.: "Structural Insights into the Unique Properties of the Adeno-Associated Virus Serotype 9", JOURNAL OF VIROLOGY, vol. 12, no. 86, pages 6947 - 6958
GRIMM ET AL., HUM. GENE THER., vol. 10, no. 15, 1999, pages 2445 - 2450
HEIM ET AL., CURRENT BIOLOGY, vol. 2, 1996, pages 178 - 182
HEIM ET AL., PROC. NATL. ACAD. SCI. USA, 1995
HEIM ET AL., SCIENCE, vol. 373, 1995, pages 663 - 664
HWANG ET AL.: "N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals", SCIENCE, vol. 327, no. 5968, 19 February 2010 (2010-02-19), pages 973 - 977, XP055369420, DOI: 10.1126/science.1183147
JIN ET AL.: "Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins", HUM GENE THER METHODS, no. 5, 28 October 2017 (2017-10-28), pages 255 - 267
KAJIGAYA ET AL., PROC. NAT'L. ACAD. SCI. USA, vol. 88, 1991, pages 4646 - 50
KIMBAUER ET AL., VIR.,, vol. 219, pages 37 - 44
MEDICI GIORGIO ET AL: "EXPRESSION OF A SECRETABLE, CELL-PENETRATING CDKL5 PROTEIN ENHANCES THE EFFICACY OF AAV VECTOR-MEDIATED GENE THERAPY FOR CDKL5 DEFICIENCY DISORDER", BIORXIV, 17 December 2021 (2021-12-17), pages 1 - 25, XP093224097, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.07.26.453746v2.full.pdf> DOI: 10.1101/2021.07.26.453746 *
MIYANOHARA ET AL., MOL THER METHODS CLIN DEV, vol. 3, 2016, pages 16046
O'REILLY ET AL.: "Baculovirus Expression Vectors, A Laboratory Manual", 1994, OXFORD UNIV. PRESS
PARR ET AL., NAT. MED., vol. 3, 1997, pages 1145 - 9
POWELL ET AL.: "Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression", GENE THERAPY, 2015
PULICHERLA ET AL., MOL. THER., vol. 19, no. 6, 2011, pages 1070 - 1078
REMINGTON: "The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS & WILKINS
RUFFING ET AL., J. VIR., vol. 66, 1992, pages 6922 - 30
SAMULSKI ET AL., J. VIR., vol. 63, 1989, pages 3822 - 8
TRATSCHIN ET AL., MOL. CELL BIOL., vol. 5, no. 11, 1985, pages 3251 - 3260
ZHAO ET AL., VIR, vol. 272, 2000, pages 382 - 93

Similar Documents

Publication Publication Date Title
CN119487051A (zh) Aav衣壳变体及其用途
AU2020282352B2 (en) Compositions and methods for selective gene regulation
JP2022523766A (ja) Ｇｒｎ関連成人発症性神経変性の治療のための組換えアデノ随伴ウイルス
JP2023501897A (ja) Ｃ９ｏｒｆ７２関連疾患の治療のための三重機能アデノ随伴ウイルス（ａａｖ）ベクター
WO2025038796A1 (fr) Compositions et méthodes pour le traitement de troubles liés à un déficit en cdkl5
WO2025038802A1 (fr) Compositions et méthodes pour le traitement de troubles liés à l&#39;ataxine-2
WO2025038795A1 (fr) Compositions et méthodes pour le traitement de troubles liés à la protéine kinase de la dystrophie myotonique
WO2025038805A1 (fr) Compositions et méthodes pour le traitement de troubles liés à une déficience en glucosylcéramidase bêta 1
EP3737423B1 (fr) Compositions et méthodes de traitement de troubles de la rétine
TW202523846A (zh) 用於治療與共濟蛋白(frataxin)不足有關之疾病之組合物及方法
TW202502803A (zh) 衣殼多肽及其使用方法
AU2023364350A1 (en) Recombinant aavs with improved tropism and specificity
TW202449167A (zh) 用於治療肌肉萎縮性脊髓側索硬化症之組成物及方法
KR20260020501A (ko) 신탁신 결합 단백질 1 결핍과 관련된 장애의 치료를 위한 조성물 및 방법
KR20260021096A (ko) Cdkl5 결핍과 관련된 장애의 치료를 위한 조성물 및 방법
TW202509226A (zh) 用於治療與ataxin-2相關之病症的組合物及方法
TW202112807A (zh) 用於ａｒｓａ基因轉移之腺相關病毒組成物及其使用方法
JP2023544068A (ja) Ｎｅｕｒｏｄ１ベクター
WO2025122548A1 (fr) Compositions et méthodes pour le traitement de troubles liés à une déficience en cdkl5
JP2023543362A (ja) Ｎｅｕｒｏｄ１組み合わせベクター
WO2025122543A1 (fr) Compositions et méthodes pour le traitement de troubles liés à une déficience en protéine 1 de liaison à la syntaxine
WO2025122532A1 (fr) Compositions et méthodes pour le traitement de troubles liés à l&#39;ataxine 2
WO2025122536A1 (fr) Compositions et méthodes pour le traitement de troubles liés à la protéine kinase de la dystrophie myotonique
WO2025122531A1 (fr) Compositions et méthodes pour le traitement de troubles liés à une déficience en frataxine
WO2025122530A1 (fr) Compositions et méthodes pour le traitement de troubles liés à une déficience en glucosylcéramidase bêta 1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24828643

Country of ref document: EP

Kind code of ref document: A1