WO2023020421A1 - Régulation des gènes à empreinte inactivés par l'expression de smn1 et snrpn et utilisations associées - Google Patents

Régulation des gènes à empreinte inactivés par l'expression de smn1 et snrpn et utilisations associées Download PDF

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WO2023020421A1
WO2023020421A1 PCT/CN2022/112441 CN2022112441W WO2023020421A1 WO 2023020421 A1 WO2023020421 A1 WO 2023020421A1 CN 2022112441 W CN2022112441 W CN 2022112441W WO 2023020421 A1 WO2023020421 A1 WO 2023020421A1
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protein
disorder
gene
disease
amino acid
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Peixin ZHU
Guojie YE
Zhenhua Wu
Lijun Wang
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Hangzhou Jiayin Biotech Ltd.
Hangzhou Exegenesis Bio Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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

Definitions

  • the present disclosure relates to nucleic acid constructs, gene therapies based on such constructs, and methods of use thereof.
  • Angelman syndrome (AS) , Prader Willi syndrome (PWS) , Beckwith-Wiedemann syndrome (BWS) , and Silver-Russell syndrome (SRS) are complex genetic disorders that primarily affects the nervous system. These genetic disorders cause developmental disabilities and nerve-related symptoms. These disorders are not detected until developmental delays become noticeable, usually during a child’s early developmental stages. Characteristic features of these disorders include delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia) . Most affected children also have recurrent seizures (epilepsy) and a small head size. Currently treatment includes anti-seizure medication and therapies to help manage medical and developmental concerns.
  • the epigenetic regulation preserves the cell-or tissue-specific expression profile of the gene.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN.
  • the gene is NPAP1. In some embodiments, the gene is SNRPN. In some embodiments, the gene is HDAC1. In some embodiments, the gene is HDAC6. In some embodiments, the gene is HDAC8. In some embodiments, the gene is KAT6. In some embodiments, the gene is KAT8. In some embodiments, the gene is TET1. In some embodiments, the gene is TET2. In some embodiments, the gene is TET3.
  • a method for epigenetically modifying a gene in a cell comprising introducing into the cell a SMN1 protein or a SNRPN protein or variants thereof, or a nucleic acid encoding the SMN1 protein or the SNRPN protein or variants thereof, wherein the gene is selected from a group consisting of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, and SNRPN.
  • the epigenetic modification preserves the cell-or tissue-specific expression profile of the gene.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN. In some embodiments, the gene is NPAP1. In some embodiments, the gene is SNRPN.
  • a method for epigenetically regulating the expression of a gene in a cell comprising introducing into the cell a SMN1 protein or a SNRPN protein or variants thereof, or a nucleic acid encoding the SMN1 protein or the SNRPN protein or variants thereof, wherein the gene is selected from a group consisting of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2, and TET3, wherein the cell is in a subject having a disease or disorder associated with UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN,
  • the disease or disorder is an UBE3A associated disease or disorder. In some embodiments, the disease or disorder is a GABRB3 associated disease or disorder. In some embodiments, the disease or disorder is a GABRA5 associated disease or disorder. In some embodiments, the disease or disorder is a GABRG3 associated disease or disorder. In some embodiments, the disease or disorder is an ATP10A associated disease or disorder. In some embodiments, the disease or disorder is an OCA2 associated disease or disorder. In some embodiments, the disease or disorder is an HERC2 associated disease or disorder. In some embodiments, the disease or disorder is a NIPA1 associated disease or disorder. In some embodiments, the disease or disorder is a NIPA2 associated disease or disorder.
  • the disease or disorder is a CYFIP1 associated disease or disorder. In some embodiments, the disease or disorder is a TUBGCP5 associated disease or disorder. In some embodiments, the disease or disorder is a MKRN3 associated disease or disorder. In some embodiments, the disease or disorder is a MAGEL2 associated disease or disorder. In some embodiments, the disease or disorder is a NDN associated disease or disorder. In some embodiments, the disease or disorder is a NPAP1 associated disease or disorder. In some embodiments, the disease or disorder is a SNRPN associated disease or disorder. In some embodiments, the disease or disorder is a HDAC1 associated disease or disorder. In some embodiments, the disease or disorder is a HDAC6 associated disease or disorder.
  • the disease or disorder is a HDAC8 associated disease or disorder. In some embodiments, the disease or disorder is a KAT6 associated disease or disorder. In some embodiments, the disease or disorder is a KAT8 associated disease or disorder. In some embodiments, the disease or disorder is a TET1 associated disease or disorder. In some embodiments, the disease or disorder is a TET2 associated disease or disorder. In some embodiments, the disease or disorder is a TET3 associated disease or disorder. In some embodiments, the disease or disorder is Angelman syndrome, Prader Willi syndrome, Beckwith-Wiedemann syndrome, Silver-Russell syndrome, or other imprinting diseases.
  • the SMN1 protein or the SNRPN protein or variants thereof is introduced into the cell.
  • the nucleic acid encoding the SMN1 protein or the SNRPN protein or variants thereof is introduced into the cell.
  • the nucleic acid is in a vector.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a combination or variant thereof.
  • the AAV vector is in a recombinant AAV (rAAV) particle comprising a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.
  • rAAV recombinant AAV
  • the nucleic acid encoding the SMN protein or variant thereof comprising an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 1.
  • the nucleic acid encoding the SNRPN protein or variant thereof comprising an amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 30.
  • a method for treating a disease or disorder in a subject comprising administering to the subject a nucleic acid encoding a SMN1 protein or a SNRPN protein or variants thereof, wherein the disease or disorder is associated with UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2, and TET3.
  • the nucleic acid is in an AAV vector.
  • the nucleic acid is administered after birth but before 2 years old of the subject.
  • FIG. 1 schematically illustrates the epigenetic regulation of the paternally imprinted UBE3A, as well as its neighboring genes (ATP10A, GABRB3, GABRA5, GABRG3, OCA2, and HERC2) , through overexpressing SMN1 or SNRPN via rAAV vectors.
  • FIGs. 2A-2C illustrate the changed mRNA expression of certain genes after overexpressing SMN1 using various rAAV vectors in the SMN ⁇ 7 -/- mouse model.
  • FIG. 2A illustrates the mRNA expression of paternally imprinted UBE3A in spinal cord, as well as its neighboring genes (GABRG3, GABRA5, and GABRB3) in brain, after overexpressing SMN1.
  • FIG. 2A illustrates the mRNA expression of paternally imprinted UBE3A in spinal cord, as well as its neighboring genes (GABRG3, GABRA5, and GABRB3) in brain, after overexpressing SMN1.
  • FIG. 2B illustrates the mRNA expression of HDAC1 (encoding histone deacetylase 1) , HDAC6 (encoding histone deacetylase 6) , HDAC8 (encoding histone deacetylase 8) , and KAT8 (encoding lysine acetyltransferase 8) in brain after overexpressing SMN1.
  • FIG. 2C illustrates the mRNA expression of TET1 (encoding Tet methylcytosine dioxygenase 1) , TET2 (encoding Tet methylcytosine dioxygenase 2) , and TET3 (encoding Tet methylcytosine dioxygenase 3) in both spinal cord and brain after overexpressing SMN1.
  • FIGs. 3A-3B quantitatively illustrate the increased mRNA expression of paternally imprinted UBE3A, as well as its neighboring genes (GABRG3 and GABRB3) , after overexpressing SMN1 in the SMN ⁇ 7 -/- mouse model (FIG. 3A) and the Angelman +/- mouse model (i.e., the Ube3A m-/p+ mouse model) (FIG. 3B) .
  • Wt stands for wildtype C57BL/6 mice
  • Het stands for Angelman +/- mice
  • Het + EXG340 stands for Angelman +/- mice treated with a SMN1 expression rAAV vector, EXG340.
  • FIG. 4 quantitatively illustrates the decreased mRNA expression of PTPA (encoding PTPA protein that is a substrate of UBE3A) after overexpressing SMN1 in the SMN ⁇ 7 -/- mouse model (left panel) and the Angelman +/- mouse model (right panel) .
  • FIG. 5 quantitatively illustrates the increased mRNA expression of KAT6 (encoding lysine acetyltransferase 6) after overexpressing SMN1 in the SMN ⁇ 7 -/- mouse model (left panel) and the Angelman +/- mouse model (right panel) .
  • FIGs. 6A-6B quantitatively illustrate the increased mRNA expression of TET1, TET2, and TET3 after overexpressing SMN1 in the SMN ⁇ 7 -/- mouse model (FIG. 6A) and the Angelman +/- mouse model (FIG. 6B) .
  • FIG. 7 quantitatively illustrates the increased mRNA expression of OCA2 (encoding melanocyte-specific transporter protein) , ATP10A (encoding ATPase class V type 10A) , and HERC2 (encoding E3 ubiquitin-protein ligase HERC2) after overexpressing SMN1 in the Angelman +/- mouse model.
  • OCA2 encoding melanocyte-specific transporter protein
  • ATP10A encoding ATPase class V type 10A
  • HERC2 encoding E3 ubiquitin-protein ligase HERC2
  • FIG. 8 quantitatively illustrates the mRNA expression of UBE3A (left panel) and ATP10A (right panel) in liver and spinal cord after overexpressing SMN1 in the Angelman +/- mouse model.
  • the present disclosure is based, in part, on the surprising finding that expression (or overexpression) of a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein epigenetically regulates the expression of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2, and TET3, thereby providing novel therapeutic options for treating Angelman syndrome (AS) , Prader Willi syndrome (PWS) , Beckwith-Wiedemann syndrome (BWS) , Silver-Russell syndrome (SRS) , and other imprinting diseases among other
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • Polynucleotide or “nucleic acid, ” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the polypeptides have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences. ”
  • an “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is usually substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • an “isolated” nucleic acid molecule encoding a polypeptide described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • epigenetic regulation or “epigenetic modification” as used herein refers to the process by which the expression of a particular gene is regulated by modulating the frequency, rate, or extent of gene expression in a mitotically or meiotically heritable way that does not entail a change in the DNA sequence.
  • the expression of the gene is upregulated.
  • the expression of the gene is downregulated.
  • the expression of the gene is silenced.
  • impringing refers to the phenomenon in which genes are expressed in a parent-of-origin specific manner, due to epigenetic modifications.
  • the impringing is cell-or tissue-specific.
  • substantially homologous refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Methods for determining percent identity are well known in the art. For example, percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
  • percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif. ) . From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six) .
  • BLAST BLAST
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease (s) , and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, for example, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • the term “vector” includes cloning and expression vehicles, as well as viral vectors.
  • the vector provided herein is a recombinant AAV vector.
  • rAAV vector refers to a polynucleotide vector comprising a nucleic acid sequence from an AAV and one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) .
  • the one or more heterologous sequences are flanked by at least one, in certain embodiments two, AAV inverted terminal repeat sequences (ITRs) .
  • such rAAV vectors can be replicated and packaged into infectious viral capsid particles, e.g., when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins) .
  • An rAAV vector may be incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection) , and can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.
  • An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral capsid particle, particularly an AAV particle.
  • An rAAV vector can be packaged into an AAV capsid to generate a “recombinant adeno-associated viral capsid particle (rAAV particle) . ”
  • heterologous refers to sequences that are not normally joined together, and/or are not normally associated with a particular cell.
  • a “heterologous” region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene) .
  • flanking indicates the presence of one or more the flanking elements upstream and/or downstream, i.e., 5’ and/or 3’, relative to the sequence.
  • flanking is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element.
  • a sequence e.g., a transgene
  • TRs two other elements
  • inverted terminal repeat refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.
  • An “AAV inverted terminal repeat (ITR) ” sequence is well known in the art, and is usually an approximately 145-nucleotide sequence that is present at both termini of the native single-stranded AAV genome.
  • the outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
  • the outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A′, B, B′, C, C′ and D regions) , allowing intrastrand base-pairing to occur within this portion of the ITR.
  • a “coding sequence” or a sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus.
  • a transcription termination sequence may be located 3′ to the coding sequence.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • operatively linked, ” and similar phrases when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other.
  • an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA) .
  • operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame) .
  • an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.
  • promoter refers to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3′-direction) coding sequence.
  • Transcription promoters can include “inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc. ) , “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc. ) , and “constitutive promoters. ”
  • transgene as used herein in a broad sense means any heterologous nucleotide sequence incorporated in a viral vector, e.g., for expression in a target cell and it can be associated with expression control sequences, such as promoters. It is appreciated by those of skill in the art that expression control sequences will be selected based on ability to promote expression of the transgene in the target cell.
  • An example of a transgene is a nucleic acid encoding a therapeutic polypeptide or a detectable marker.
  • AAV capsid or “AAV capsid protein” or “AAV cap” as used herein refers to a protein encoded by an AAV capsid (cap) gene (e.g., VPI, VP2, and VP3) or a variant thereof.
  • AAV capsid e.g., VPI, VP2, and VP3
  • the term includes but not limited to a capsid protein derived from any AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 , AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV-2/1, AAV 2/6, AAV 2/7, AAV 2/8, AAV 2/9, AAV LK03, AAVrh10, AAVrh74, AAV44-9, or a variant thereof.
  • the term also includes a capsid protein expressed by or derived from a recombinant AAV such as a chimeric AAV.
  • AAV capsid particle or “AAV particle” as used herein includes at least one AAV capsid protein (e.g., a VP1 protein, a VP2 protein, a VP3 protein, or variant thereof) and optionally encapsulates a nucleic acid from an AAV genome or a nucleic acid derived from an AAV genome.
  • AAV capsid protein e.g., a VP1 protein, a VP2 protein, a VP3 protein, or variant thereof.
  • serotype used with respect to vector or virus capsid is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure.
  • chimeric as used herein means, with respect to a viral capsid or particle, that the capsid or particle includes sequences from different parvoviruses, preferably different AAV serotypes, as described in Rabinowitz et al., U.S. Pat. No. 6,491,907 the disclosure of which is incorporated in its entirety herein by reference.
  • polynucleotide means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature.
  • recombinant virus refers to a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
  • recombinant AAV particle or “rAAV” as used herein refers to an AAV that has been genetically altered, e.g., by the deletion or other mutation of an endogenous AAV gene and/or the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of the AAV particle.
  • specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.
  • stringent hybridization conditions or “stringent conditions” refers to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52 : 456, Sambrook et al.
  • Transduction of a cell by a virus means that there is transfer of a nucleic acid such as DNA or RNA from the virus particle to the cell.
  • host cell refers to a particular cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell.
  • Host cells may be bacterial cells, yeast cells, insect cells or mammalian cell.
  • purified refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance of interest comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, 80%-85%, 90-99%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • treat, ” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • Treating includes both managing and ameliorating the disease.
  • the terms “manage, ” “managing, ” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.
  • Treatment includes: (1) preventing the disease, i.e., preventing the development of the disease or causing the disease to occur with less intensity in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting the development, preventing or retarding progression, or reversing the disease state, (3) relieving symptoms of the disease i.e., decreasing the number of symptoms experienced by the subject, and (4) reducing, preventing or retarding progression of the disease or a symptom thereof.
  • the terms “prevent, ” “preventing, ” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom (s) .
  • administer refers to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or a patient (e.g., human) , such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery and/or any other method of physical delivery described herein or known in the art. In a particular embodiment, administration is by intravenous infusion.
  • a conjugate or a composition provided herein may be delivered systemically or to a specific tissue.
  • the terms “effective amount” or “therapeutically effective amount” refer to an amount of a therapeutic (e.g., a conjugate or pharmaceutical composition provided herein) which is sufficient to treat, diagnose, prevent, delay the onset of, reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease and/or a symptom related thereto. These terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect (s) of another therapy or to serve as a bridge to another therapy. In some embodiments, “effective amount” as used herein also refers to the amount of a conjugate described herein to achieve a specified result. As used herein, the terms “subject” and “patient” are used interchangeably.
  • a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc. ) or a primate (e.g., monkey and human) , for example a human.
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein.
  • the subject is human.
  • the terms “therapies” and “therapy” can refer to any protocol (s) , method (s) , compositions, formulations, and/or agent (s) that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or symptom thereof (e.g., a disease or disorder provided herein or one or more symptoms or condition associated therewith) .
  • the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or disorder or one or more symptoms thereof.
  • the term “therapy” refers to a therapy other than a conjugate described herein or pharmaceutical composition thereof.
  • disease or disorder associated with UBE3A refers to a disease or disorder that involves UBE3A (including abnormal expression level of a UBE3A protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with UBE3A includes, but not limited to, a disease or disorder associated with decreased expression of UBE3A gene or with a mutant UBE3A gene.
  • disease or disorder associated with GABRG3 refers to a disease or disorder that involves GABRG3 (including abnormal expression level of a GABRG3 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with GABRG3 includes, but not limited to, a disease or disorder associated with decreased expression of GABRG3 gene or with a mutant GABRG3 gene.
  • disease or disorder associated with GABRA5 refers to a disease or disorder that involves GABRA5 (including abnormal expression level of a GABRA5 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with GABRA5 includes, but not limited to, a disease or disorder associated with decreased expression of GABRA5 gene or with a mutant GABRA5 gene.
  • disease or disorder associated with GABRB3 refers to a disease or disorder that involves GABRB3 (including abnormal expression level of a GABRB3 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with GABRB3 includes, but not limited to, a disease or disorder associated with decreased expression of GABRB3 gene or with a mutant GABRB3 gene.
  • disease or disorder associated with ATP10A refers to a disease or disorder that involves ATP10A (including abnormal expression level of a ATP10A protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with ATP10A includes, but not limited to, a disease or disorder associated with decreased expression of ATP10A gene or with a mutant ATP10A gene.
  • disease or disorder associated with OCA2 refers to a disease or disorder that involves OCA2 (including abnormal expression level of a OCA2 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with OCA2 includes, but not limited to, a disease or disorder associated with decreased expression of OCA2 gene or with a mutant OCA2 gene.
  • disease or disorder associated with HERC2 refers to a disease or disorder that involves HERC2 (including abnormal expression level of a HERC2 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with HERC2 includes, but not limited to, a disease or disorder associated with decreased expression of HERC2 gene or with a mutant HERC2 gene.
  • disease or disorder associated with NIPA1 refers to a disease or disorder that involves NIPA1 (including abnormal expression level of a NIPA1 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with NIPA1 includes, but not limited to, a disease or disorder associated with decreased expression of NIPA1 gene or with a mutant NIPA1 gene.
  • disease or disorder associated with NIPA2 refers to a disease or disorder that involves NIPA2 (including abnormal expression level of a NIPA2 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with NIPA2 includes, but not limited to, a disease or disorder associated with decreased expression of NIPA2 gene or with a mutant NIPA2 gene.
  • disease or disorder associated with CYFIP1 refers to a disease or disorder that involves CYFIP1 (including abnormal expression level of a CYFIP1 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with CYFIP1 includes, but not limited to, a disease or disorder associated with decreased expression of CYFIP1 gene or with a mutant CYFIP1 gene.
  • disease or disorder associated with TUBGCP5 refers to a disease or disorder that involves TUBGCP5 (including abnormal expression level of a TUBGCP5 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with TUBGCP5 includes, but not limited to, a disease or disorder associated with decreased expression of TUBGCP5 gene or with a mutant TUBGCP5 gene.
  • disease or disorder associated with MKRN3 refers to a disease or disorder that involves MKRN3 (including abnormal expression level of a MKRN3 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with MKRN3 includes, but not limited to, a disease or disorder associated with decreased expression of MKRN3 gene or with a mutant MKRN3 gene.
  • disease or disorder associated with MAGEL2 refers to a disease or disorder that involves MAGEL2 (including abnormal expression level of a MAGEL2 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with MAGEL2 includes, but not limited to, a disease or disorder associated with decreased expression of MAGEL2 gene or with a mutant MAGEL2 gene.
  • disease or disorder associated with NDN refers to a disease or disorder that involves NDN (including abnormal expression level of a NDN protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with NDN includes, but not limited to, a disease or disorder associated with decreased expression of NDN gene or with a mutant NDN gene.
  • disease or disorder associated with NPAP1 refers to a disease or disorder that involves NPAP1 (including abnormal expression level of a NPAP1 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with NPAP1 includes, but not limited to, a disease or disorder associated with decreased expression of NPAP1 gene or with a mutant NPAP1 gene.
  • disease or disorder associated with SNRPN refers to a disease or disorder that involves SNRPN (including abnormal expression level of a SNRPN protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with SNRPN includes, but not limited to, a disease or disorder associated with decreased expression of SNRPN gene or with a mutant SNRPN gene.
  • disease or disorder associated with HDAC1 refers to a disease or disorder that involves HDAC1 (including abnormal expression level of a HDAC1 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with HDAC1 includes, but not limited to, a disease or disorder associated with decreased expression of HDAC1 gene or with a mutant HDAC1 gene.
  • disease or disorder associated with HDAC6 refers to a disease or disorder that involves HDAC6 (including abnormal expression level of a HDAC6 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with HDAC6 includes, but not limited to, a disease or disorder associated with decreased expression of HDAC6 gene or with a mutant HDAC6 gene.
  • disease or disorder associated with HDAC8 refers to a disease or disorder that involves HDAC8 (including abnormal expression level of a HDAC8 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with HDAC8 includes, but not limited to, a disease or disorder associated with decreased expression of HDAC8 gene or with a mutant HDAC8 gene.
  • disease or disorder associated with KAT6 refers to a disease or disorder that involves KAT6 (including abnormal expression level of a KAT6 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with KAT6 includes, but not limited to, a disease or disorder associated with decreased expression of KAT6 gene or with a mutant KAT6 gene.
  • disease or disorder associated with KAT8 refers to a disease or disorder that involves KAT8 (including abnormal expression level of a KAT8 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with KAT8 includes, but not limited to, a disease or disorder associated with decreased expression of KAT8 gene or with a mutant KAT8 gene.
  • disease or disorder associated with TET1 refers to a disease or disorder that involves TET1 (including abnormal expression level of a TET1 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with TET1 includes, but not limited to, a disease or disorder associated with decreased expression of TET1 gene or with a mutant TET1 gene.
  • disease or disorder associated with TET2 refers to a disease or disorder that involves TET2 (including abnormal expression level of a TET2 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with TET2 includes, but not limited to, a disease or disorder associated with decreased expression of TET2 gene or with a mutant TET2 gene.
  • disease or disorder associated with TET3 refers to a disease or disorder that involves TET3 (including abnormal expression level of a TET3 protein) , e.g., either as a symptom or direct or indirect cause.
  • a disease or disorder associated with TET3 includes, but not limited to, a disease or disorder associated with decreased expression of TET3 gene or with a mutant TET3 gene.
  • the present disclosure provides methods and uses of SMN1, SNRPN, Ascl1, Banp, Foxg1, Klf4, NeuroD1, NeuroD4, Pou5F1, Sox2, Zfp36l1, or CamKIIa-T286D for modulating expression levels of certain genes among other apsects.
  • the methods or uses provided herein comprise introducing a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof into a cell including both in vitro or in vivo.
  • the methods or uses provided herein comprise introducing a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof into a cell including both in vitro or in vivo.
  • the SMN1 protein is a human SMN1 protein. In some embodiments, the SMN1 protein is a variant of human SMN1 protein, but retains at least one of its biological activities or functions. In some embodiments, the SMN1 protein comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 1.
  • the SMN1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 90% sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 1.
  • the SMN1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 1. In some embodiments, the SMN1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 1.
  • the SNRPN protein is a human SNRPN protein. In some embodiments, the SNRPN protein is a variant of human SNRPN protein, but retains at least one of its biological activities or functions. In some embodiments, the SNRPN protein comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 30.
  • the SNRPN protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 30.
  • the SNRPN protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 30. In some embodiments, the SNRPN protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 30.
  • the Ascl1 protein is a human Ascl1 protein. In some embodiments, the Ascl1 protein is a variant of human Ascl1 protein, but retains at least one of its biological activities or functions. In some embodiments, the Ascl1 protein comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 31.
  • the Ascl1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 31.
  • the Ascl1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 31.
  • the Ascl1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 31. In some embodiments, the Ascl1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 31.
  • the Banp protein is a human Banp protein. In some embodiments, the Banp protein is a variant of human Banp protein, but retains at least one of its biological activities or functions. In some embodiments, the Banp protein comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 32.
  • the Banp protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 32.
  • the Banp protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 32. In some embodiments, the Banp protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 32.
  • the Foxg1 protein is a human Foxg1 protein. In some embodiments, the Foxg1 protein is a variant of human Foxg1 protein, but retains at least one of its biological activities or functions. In some embodiments, the Foxg1 protein comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 33.
  • the Foxg1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 94% sequence identity of SEQ ID NO: 33.
  • the Foxg1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 33. In some embodiments, the Foxg1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 33.
  • the Klf4 protein is a human Klf4 protein. In some embodiments, the Klf4 protein is a variant of human Klf4 protein, but retains at least one of its biological activities or functions. In some embodiments, the Klf4 protein comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 34.
  • the Klf4 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 34.
  • the Klf4 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 34.
  • the Klf4 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 34. In some embodiments, the Klf4 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 34.
  • the NeuroD1 protein is a human NeuroD1 protein. In some embodiments, the NeuroD1 protein is a variant of human NeuroD1 protein, but retains at least one of its biological activities or functions. In some embodiments, the NeuroD1 protein comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 35.
  • the NeuroD1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 35.
  • the NeuroD1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 35. In some embodiments, the NeuroD1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 35.
  • the NeuroD4 protein is a human NeuroD4 protein. In some embodiments, the NeuroD4 protein is a variant of human NeuroD4 protein, but retains at least one of its biological activities or functions. In some embodiments, the NeuroD4 protein comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 36.
  • the NeuroD4 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 36.
  • the NeuroD4 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 36. In some embodiments, the NeuroD4 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 36.
  • the Pou5F1 protein is a human Pou5F1 protein. In some embodiments, the Pou5F1 protein is a variant of human Pou5F1 protein, but retains at least one of its biological activities or functions. In some embodiments, the Pou5F1 protein comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 37.
  • the Pou5F1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 37.
  • the Pou5F1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 37.
  • the Pou5F1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 37. In some embodiments, the Pou5F1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 37.
  • the Sox2 protein is a human Sox2 protein. In some embodiments, the Sox2 protein is a variant of human Sox2 protein, but retains at least one of its biological activities or functions. In some embodiments, the Sox2 protein comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 38.
  • the Sox2 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 38.
  • the Sox2 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 38. In some embodiments, the Sox2 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 38.
  • the Zfp36l1 protein is a human Zfp36l1 protein. In some embodiments, the Zfp36l1 protein is a variant of human Zfp36l1 protein, but retains at least one of its biological activities or functions. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 39.
  • the Zfp36l1 protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 39.
  • the Zfp36l1 protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 39.
  • the Zfp36l1 protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 39. In some embodiments, the Zfp36l1 protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 39.
  • the CamKIIa-T286D protein is a human CamKIIa-T286D protein. In some embodiments, the CamKIIa-T286D protein is a variant of human CamKIIa-T286D protein, but retains at least one of its biological activities or functions. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity of SEQ ID NO: 40.
  • the CamKIIa-T286D protein comprises an amino acid sequence having about 80%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 85%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 90%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 91%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 92%sequence identity of SEQ ID NO: 40.
  • the CamKIIa-T286D protein comprises an amino acid sequence having about 93%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 94%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 95%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 96%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 97%sequence identity of SEQ ID NO: 40.
  • the CamKIIa-T286D protein comprises an amino acid sequence having about 98%sequence identity of SEQ ID NO: 40. In some embodiments, the CamKIIa-T286D protein comprises an amino acid sequence having about 99%sequence identity of SEQ ID NO: 40.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or a variant thereof.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a SMN1 protein having an amino acid sequence of SEQ ID NO: 1.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a SMN1 protein having an amino acid sequence of SEQ ID NO: 1.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a SNRPN protein having an amino acid sequence of SEQ ID NO: 30.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a SNRPN protein having an amino acid sequence of SEQ ID NO: 30.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Ascl1 protein having an amino acid sequence of SEQ ID NO: 31.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Ascl1 protein having an amino acid sequence of SEQ ID NO: 31.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Banp protein having an amino acid sequence of SEQ ID NO: 32.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Banp protein having an amino acid sequence of SEQ ID NO: 32.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Foxg1 protein having an amino acid sequence of SEQ ID NO: 33.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Foxg1 protein having an amino acid sequence of SEQ ID NO: 33.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Klf4 protein having an amino acid sequence of SEQ ID NO: 34.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Klf4 protein having an amino acid sequence of SEQ ID NO: 34.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a NeuroD1 protein having an amino acid sequence of SEQ ID NO: 35.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a NeuroD1 protein having an amino acid sequence of SEQ ID NO: 35.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a NeuroD4 protein having an amino acid sequence of SEQ ID NO: 36.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a NeuroD4 protein having an amino acid sequence of SEQ ID NO: 36.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Pou5F1 protein having an amino acid sequence of SEQ ID NO: 37.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Pou5F1 protein having an amino acid sequence of SEQ ID NO: 37.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Sox2 protein having an amino acid sequence of SEQ ID NO: 38.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Sox2 protein having an amino acid sequence of SEQ ID NO: 38.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a Zfp36l1 protein having an amino acid sequence of SEQ ID NO: 39.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a Zfp36l1 protein having an amino acid sequence of SEQ ID NO: 39.
  • the nucleic acid constructs provided herein comprises a nucleic acid sequence encoding a CamKIIa-T286D protein having an amino acid sequence of SEQ ID NO: 40.
  • the nucleic acid encoding a protein of interest provided herein comprises at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the nucleic acid sequence encoding a CamKIIa-T286D protein having an amino acid sequence of SEQ ID NO: 40.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87: 2264 2268 (1990) , modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873 5877 (1993) .
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215: 403 (1990) .
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25: 3389 3402 (1997) .
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • sequences described herein can further comprise one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
  • the nucleic acid is a human nucleic acid (i.e., a nucleic acid that is derived from a human SMN1, SNRPN, Ascl1, Banp, Foxg1, Klf4, NeuroD1, NeuroD4, Pou5F1, Sox2, Zfp36l1, or CamKIIa-T286D gene) .
  • the nucleic acid is a non-human nucleic acid (i.e., a nucleic acid that is derived from a non-human SMN1, SNRPN, Ascl1, Banp, Foxg1, Klf4, NeuroD1, NeuroD4, Pou5F1, Sox2, Zfp36l1, or CamKIIa-T286D gene) .
  • the nucleic acid provided herein comprises one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acid construct provided herein comprises a nucleic acid region which has been codon optimized.
  • the nucleic acid encoding SMN1 is codon optimized.
  • the nucleic acid encoding SMN1 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding SMN1 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding SNRPN is codon optimized.
  • the nucleic acid encoding SNRPN is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding SNRPN is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding Ascl1 is codon optimized.
  • the nucleic acid encoding Ascl1 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding Ascl1 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding Bamp is codon optimized. According to one embodiment, the nucleic acid encoding Bamp is codon optimized for expression in a eukaryote, e.g., humans. According to some embodiments, a coding sequence encoding Bamp is codon optimized for expression in particular cells, such as eukaryotic cells. According to one embodiment, the nucleic acid encoding Foxg1 is codon optimized. According to one embodiment, the nucleic acid encoding Foxg1 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding Foxg1 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding Klf4 is codon optimized.
  • the nucleic acid encoding Klf4 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding Klf4 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding NeuroD1 is codon optimized.
  • the nucleic acid encoding NeuroD1 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding NeuroD1 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding NeuroD4 is codon optimized.
  • the nucleic acid encoding NeuroD4 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding NeuroD4 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding Pou5F1 is codon optimized. According to one embodiment, the nucleic acid encoding Pou5F1 is codon optimized for expression in a eukaryote, e.g., humans. According to some embodiments, a coding sequence encoding Pou5F1 is codon optimized for expression in particular cells, such as eukaryotic cells. According to one embodiment, the nucleic acid encoding Sox2 is codon optimized. According to one embodiment, the nucleic acid encoding Sox2 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding Sox2 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding Zfp36l1 is codon optimized.
  • the nucleic acid encoding Zfp36l1 is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding Zfp36l1 is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the nucleic acid encoding CamKIIa-T286D is codon optimized.
  • the nucleic acid encoding CamKIIa-T286D is codon optimized for expression in a eukaryote, e.g., humans.
  • a coding sequence encoding CamKIIa-T286D is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database"available at www. kazusa. orjp/codon/and these tables can be adapted in a number of ways. See Nakamura, Y., et al., Nucl. Acids Res. 28: 292 (2000) . Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa. ) , are also available.
  • a nucleic acid molecule (including, for example, a SMN1, SNRPN, Ascl1, Banp, Foxg1, Klf4, NeuroD1, NeuroD4, Pou5F1, Sox2, Zfp36l1, or CamKIIa-T286D nucleic acid) of the present disclosure can be isolated using standard molecular biology techniques. Using all or a portion of a nucleic acid sequence of interest as a hybridization probe, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) .
  • a nucleic acid molecule for use in the methods of the disclosure can also be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of a nucleic acid molecule of interest.
  • a nucleic acid molecule used in the methods of the disclosure can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • oligonucleotides corresponding to nucleotide sequences of interest can also be chemically synthesized using standard techniques. Numerous methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which has been automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein) . Automated methods for designing synthetic oligonucleotides are available. See e.g., Hoover, D.M. &Lubowski, J. Nucleic Acids Research, 30 (10) : e43 (2002) .
  • variants of the protein described herein can be prepared.
  • peptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired polypeptide.
  • amino acid changes may alter post-translational processes of the peptide.
  • Variations may be a substitution, deletion, or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence as compared with the original polypeptide.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parental peptides.
  • Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include a polypeptide with an N-terminal methionyl residue.
  • Proteins generated by conservative amino acid substitutions are included in the present disclosure.
  • a conservative amino acid substitution an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • families of amino acid residues having side chains with similar charges have been defined in the art.
  • 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
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed and the activity of the protein can be determined.
  • Conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties. Exemplary substitutions are shown in the table below.
  • Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975) ) : (1) non-polar: Ala (A) , Val (V) , Leu (L) , Ile (I) , Pro (P) , Phe (F) , Trp (W) , Met (M) ; (2) uncharged polar: Gly (G) , Ser (S) , Thr (T) , Cys (C) , Tyr (Y) , Asn (N) , Gln (Q) ; (3) acidic: Asp (D) , Glu (E) ; and (4) basic: Lys (K) , Arg (R) , His (H) .
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • any cysteine residue not involved in maintaining the proper conformation of the polypeptide provided herein also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking.
  • another amino acid such as alanine or serine
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include a polypeptide with an N-terminal methionyl residue.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis see, e.g., Carter, Biochem J. 237: 1-7 (1986) ; and Zoller et al., Nucl. Acids Res. 10: 6487-500 (1982)
  • cassette mutagenesis see, e.g., Wells et al., Gene 34: 315-23 (1985)
  • other known techniques can be performed on the cloned DNA to produce the polypeptide variant DNA.
  • the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof provided herein can be delivered into a cell (e.g., in a subject) via nucleic acid construct encoding same.
  • the nucleic acid construct can be a vector.
  • the vector is a recombinant viral vector.
  • the vetor is a AAV vectors.
  • virus based gene therapies for delivering the nucleic acid provided herein.
  • viral vectors e.g., rAAV vectors
  • viral particles e.g., rAAVs or rAAV particles
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, vaccinia vector, herpes simplex viral vector, and derivatives thereof.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.
  • the viral vector or viral particle provided herein is derived from an adenovirus.
  • exemplary vectors are based on or derived from HAd5, ChAd3, HAd26, HAd6, AdCH3NSmut, HAd35, ChAd63, HAd4, rcAd26.
  • Recombinant adenovirus vectors can be constructed according to known methods in the art.
  • third-generation adenoviral vectors also called “high capacity adenoviral vectors” (HCAds) , helper-dependent or “gutless” adenoviral vectors
  • HCAds high capacity adenoviral vectors
  • helper-dependent or “gutless” adenoviral vectors can be used herein to deliver longer sequences.
  • the polynucleotide of interest e.g., a transgene is cloned into an adenoviral vector that only contains the ITRs and a packaging signal.
  • a helper adenoviral vector may be co-transfected into HEK cells to generate the adenoviral particle. See Lee et al., Genes and Diseases, 4 (2) : 43-63 (2007) .
  • the viral vector or viral particle provided herein is derived from a lentivirus.
  • Exemplary vectors are based on or derived from HIV-1, HIV-2, SIVSM, SIVAGM, EIAV, FIV, VNV, CAEV, or BIV.
  • Lentiviral vectors can be produced according to the known methods in the art, e.g., as described in Cribbs et al., BMC Biotechnology, 13: 98 (2003) ; Merten et al., Mol Ther Methods Clin Dev., 13 (3) : 16017 (2016) ; Durand and Cimarelli, Viruses, 3: 132-159 (2011) .
  • third-generation self-inactivating lentiviral vectors are used herein.
  • the viral vector or viral particle provided herein is derived from a herpes simplex virus (HSV) .
  • the herpes simplex virus is a herpes simplex type 1 virus (HSV-1) , a herpes simplex type 2 virus (HSV-2) , or any derivative thereof.
  • Exemplary vectors are based on or derived from HSV-1, HSV-2, CMV, VZV, EBV, and KSHV.
  • HSV-based vectors can be constructed according the methods known in the art, e.g., as described in U.S. Pat. Nos.
  • the HSV-based vector provided herein is an amplicon vector. In other embodiments, the HSV-based vector provided herein is a replication-defective vector. In yet other embodiments, the HSV-based vector provided herein is a replication-competent vector.
  • amplicons are plasmid-derived vectors engineered to contain both the origin of HSV DNA replication (ori) and HSV cleavage–packaging recognition sequences (pac) .
  • amplicons When amplicons are transfected into mammalian cells with HSV helper functions, they are replicated, form head-to-tail linked concatamers and are then packaged into viral particles.
  • There are two major methods currently used for producing amplicon particles one based on infection with defective helper HSVs and the other based on transfection of HSV-1 genes, such as a set of pac-deleted overlapping cosmids or a pac-deleted and ICP27-deleted BAC-HSV-1.
  • amplicons used herein can accommodate large fragments of foreign DNA (e.g., up to 152 kb) , including multiple copies of the transgene (e.g., up to 15 copies) , and are non-toxic.
  • an HSV-based vector used herein is deficient in at least one essential HSV gene, and the HSV-based vector may also comprise one or more deletions of non-essential genes.
  • the HSV-based vector is replication-deficient. Most replication-deficient HSV-based vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV-based vector is deficient in an immediate early gene selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47, and a combination thereof. In a specific embodiment, the HSV-based vector is deficient for all of ICP0, ICP4, ICP22, ICP27, and ICP47.
  • Exemplary replication-competent vectors include NV-1020 (HSV-1) , RAV9395 (HSV-2) , AD-472 (HSV-2) , NS-gEnull (HSV-1) , and ImmunoVEX (HSV2) .
  • Exemplary replication-defective vectors include dl5-29 (HSV-2) , dl5-29-41L (HSV-1) , DISC-dH (HSV-1 and HSV-2) , CJ9gD (HSV-1) , TOH-OVA (HSV-1) , d106 (HSV-1) , d81 (HSV-1) , HSV-SIV d106 (HSV-1) , and d106 (HSV-1) .
  • Replication-deficient HSV-based vectors are typically produced in complementing cell lines that provide gene functions not present in the replication-deficient HSV-based vectors, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock.
  • An exemplary cell line complements for at least one and, in some embodiments, all replication-essential gene functions not present in a replication-deficient HSV-based vector.
  • a HSV-based vector deficient in ICP0, ICP4, ICP22, ICP27, and ICP47 can be complemented by the human osteosarcoma line U2OS.
  • the cell line can also complement non-essential genes that, when missing, reduce growth or replication efficiency (e.g., UL55) .
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene function encoded by the early regions, immediate-early regions, late regions, viral packaging regions, virus-associated regions, or combinations thereof, including all HSV functions (e.g., to enable propagation of HSV amplicons, which comprise minimal HSV sequences, such as only inverted terminal repeats and the packaging signal or only ITRs and an HSV promoter) .
  • the cell line is further characterized in that it contains the complementing genes in a non-overlapping fashion with the HSV-based vector, which minimizes, and practically eliminates, the possibility of the HSV-based vector genome recombining with the cellular DNA. Accordingly, the presence of replication competent HSV is minimized, if not avoided in the vector stock, which, therefore, is suitable for certain therapeutic purposes, especially gene therapy purposes.
  • the construction of complementing cell lines involves standard molecular biology and cell culture techniques well known in the art.
  • the viral vector or viral particle provided herein is derived from an adeno-associated virus (AAV) . More detailed description related to AAV is provided in Sections 5.3.2-5.3.4 below.
  • AAV adeno-associated virus
  • the nucleic acid of interest can be cloned into the vector using any known molecular cloning methods in the art, including, for example, using restriction endonuclease sites and one or more selectable markers.
  • the nucleic acid is operably linked to a promoter.
  • Varieties of promoters have been explored for gene expression in mammalian cells, and any of the promoters known in the art may be used in the present disclosure. Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.
  • the nucleic acid provided herein is operably linked to a constitutive promoter.
  • Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells.
  • Exemplary constitutive promoters contemplated herein include, but are not limited to, Cytomegalovirus (CMV) promoters, human elongation factors-1 alpha (hEF1 ⁇ ) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , and chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAGG) .
  • CMV Cytomegalovirus
  • hEF1 ⁇ human elongation factors-1 alpha
  • UbiC ubiquitin C promoter
  • PGK phosphoglycerokinase promoter
  • SV40 simian virus 40 early promoter
  • CAGG chicken ⁇
  • the nucleic acid provided herein is operably linked to an inducible promoter.
  • Inducible promoters belong to the category of regulated promoters.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered immune effector cell, or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducing agent) , or a combination thereof.
  • the inducing condition does not induce the expression of endogenous genes in the engineered mammalian cell, and/or in the subject that receives the pharmaceutical composition.
  • the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light) , temperature (such as heat) , redox state, tumor environment, and the activation state of the engineered mammalian cell.
  • a target cell may require 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 its entirety.
  • the promoter is a promoter deemed to be efficient to drive the expression of the polynucleotides described herein.
  • Promoters for which promote expression in most tissues include, for example, but are not limited to, human elongation factor 1 ⁇ -subunit (EF1 ⁇ ) , immediate-early cytomegalovirus (CMV) , the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter and a CK6 promoter, a transthyretin promoter (TTR) , a TK promoter, a tetracycline responsive promoter (TRE) , an HBV promoter, an hAAT promoter, a LSP promoter, chimeric liver-specific promoters (LSPs) , the telomerase (hTERT) promoter, chicken ⁇ -actin (CBA) and its derivative CAG and miniCBA, the ⁇ glucuronidase (GUSB) , or ubiquitin C (UBC) .
  • EF1 ⁇ human
  • Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.
  • tissue-specific expression elements for neurons include neuron-specific enolase (NSE) , platelet-derived growth factor (PDGF) , platelet-derived growth factor B-chain (PDGF- ⁇ . ) , the synapsin (Syn) , the methyl-CpG binding protein 2 (MeCP2) , CaMKII, mGluR2, NFL, NFH, n ⁇ 2, PPE, Enk and EAAT2 promoters.
  • NSE neuron-specific enolase
  • PDGF platelet-derived growth factor
  • PDGF- ⁇ . platelet-derived growth factor B-chain
  • Syn the synapsin
  • MeCP2 methyl-CpG binding protein 2
  • CaMKII methyl-CpG binding protein 2
  • the promoter is capable of expressing the heterologous nucleic acid in a neuronal cell. In some embodiments, the promoter is capable of expressing the heterologous nucleic acid in a motor neuron cell. In some embodiments, the promoter is capable of expressing the heterologous nucleic acid in astrocytes. According to some embodiments, the promoter is a human Synapsin 1 (hSyn) promoter, or hSyn combined with synthetic regulatory element that is specific for neuronal cells. According to some embodiments, the promoter is a glial fibrillary acidic protein (GFAP) or EAAT2 promoter or GFAP or EAAT2 combined with synthetic regulatory element, that are specific for astrocytes.
  • GFAP glial fibrillary acidic protein
  • the nucleic acid construct comprises a promoter such as, but not limited to, CMV or U6, or CMV or U6 combined with synthetic regulatory element.
  • the promoter in the present rAAV vector is a CBA or a miniCBA promoter.
  • the promoter in the present rAAV vector is a modified miniCBA promoter.
  • the rAAV vector has an engineered promoter.
  • the rAAV vector further comprises a synthetic enhancer element.
  • the vector 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 its entirety) such as an intron, or a synthetic intron with modified sequences from mammalian genomes.
  • introns include, MVM (67-97 bps) , F.
  • IX truncated intron 1 (300 bps) , ⁇ -globin SD/immunoglobulin heavy chain splice acceptor (250 bps) , adenovirus splice donor/immunoglobin splice acceptor (500 bps) , SV40 late splice donor/splice acceptor (19S/16S) (180 bps) , and hybrid adenovirus splice donor/IgG splice acceptor (230 bps) .
  • the intron may be 100-500 nucleotides in length.
  • the intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500.
  • the promoter may have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500.
  • the vector also contains a selectable marker gene or a reporter gene to select cells expressing the protein from the population of host cells transfected through vectors.
  • selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells.
  • the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.
  • the nucleic acid provided herein is delivered by a AAV based system, and thus is included in a recombinant AAV vector.
  • AAV serotypes may include, but not limited to, AAV1 (Genbank Accession No. NC_002077.1; HC000057.1) , AAV2 (Genbank Accession No. NC_001401.2, JC527779.1) , AAV2i8 (Asokan, A., 2010, Discov. Med. 9: 399) , AAV3 (Genbank Accession No. NC_001729.1) , AAV3-B (Genbank Accession No. AF028705.1) , AAV4 (Genbank Accession No. NC_001829.1) , AAV5 (Genbank Accession No.
  • NC_006152.1; JC527780.1) AAV6 (Genbank Accession No. AF028704.1; JC527781.1) , AAV7 (Genbank Accession No. NC_006260.1; JC527782.1) , AAV8 (Genbank Accession No. NC_006261.1; JC527783.1) , AAV9 (Genbank Accession No AX753250.1; JC527784.1) , AAV10 (Genbank Accession No AY631965.1) , AAVrh10 (Genbank Accession No.
  • AAV11 Genbank Accession No AY631966.1
  • AAV12 Genbank Accession No DQ813647.1
  • AAV13 Genebank Accession No EU285562.1
  • AAV LK03 AAVrh74
  • AAV DJ Wang Z, et al., J Virol. 80: 11393–7 (2006)
  • AAVAnc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127 Zin, E. et al., Cell. Rep. 12: 1056 (2016)
  • AAV_go. 1 Arbetum, A.E. et al., J. Virol.
  • AAV variants include, but not limited to, AAV1 variants (e.g., AAV comprising AAV1 variant capsid proteins) , AAV2 variants (e.g., AAV comprising AAV2 variant capsid proteins) , AAV3 variants (e.g., AAV comprising AAV3 variant capsid proteins) , AAV3-B variants (e.g., AAV comprising AAV3-B variant capsid proteins) , AAV4 variants (e.g., AAV comprising AAV4 variant capsid proteins) , AAV5 variants (e.g., AAV comprising AAV5 variant capsid proteins) , AAV6 variants (e.g., AAV comprising AAV6 variant capsid proteins) , AAV7 variants (e.g., AAV comprising AAV7 variant capsid proteins) , AAV8 variants (e.g., AAV comprising AAV8 variant capsid proteins) , AAVrh8,
  • Recombinant AAV (rAAV) vectors used in the present disclosure can be constructed according to known techniques.
  • the rAAV vector is constructed to include operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the polynucleotide provided herein and a transcriptional termination region.
  • the control elements can be selected based on the cell of interest.
  • the resulting rAAV vector construct comprising the operatively linked components is franked (5′ and 3′) with functional AAV ITR sequences.
  • the polypeptide (e.g., encoding a SMN protein or a SNRPN protein) provided herein is operatively linked to at least one regulatory sequence.
  • regulatory sequences may, for example, include promoter sequences, enhancer sequences, e.g., upstream enhancer sequences (USEs) , RNA processing signals, e.g., splicing signals, polyadenylation signal sequences, sequences that stabilize cytoplasmic mRNA, post-transcriptional regulatory elements (PREs) and/or microRNA (miRNA) target sequences.
  • regulatory sequences may include sequences that enhance translation efficiency (e.g., Kozak sequences) , sequences that enhance protein stability, and/or sequences that enhance protein processing and/or secretion.
  • the polynucleotide may encode regulatory miRNAs.
  • a regulatory sequence comprises a constitutive promoter and/or regulatory control element. In certain embodiments, a regulatory sequence comprises a regulatable promoter and/or regulatory control element. In certain embodiments, a regulatory sequence comprises a ubiquitous promoter and/or regulatory control element. In certain embodiments, a regulatory sequence comprises a cell-or tissue-specific promoter and/or regulatory control element. In certain embodiments, the regulatory control element is 5’ of the coding sequence of the protein (that is, is present in ‘5 untranslated regions; 5’ UTRs) . In other embodiments, the regulatory control element is 3’ of the coding sequence of the protein (that is, is present in ‘3 untranslated regions; 3’ UTRs) .
  • the polynucleotide comprises more than one regulatory control element, for example may comprise two, three, four or five control elements. In instances wherein the polynucleotide comprises more than one control element, each control element may independently be 5’ of, 3’ of, flank, or within the coding sequence of the protein.
  • control element is an enhancer.
  • control elements included direct the transcription or expression of the polynucleotide of the protein in the subject in vivo.
  • Control elements can comprise control sequences normally associated with the selected polynucleotide of interest or alternatively heterologous control sequences.
  • control sequences include those derived from sequences encoding mammalian or viral genes, such as neuron-specific enolase promoter, a GFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP) ; a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE) , a rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters.
  • mammalian or viral genes such as neuron-specific enolase promoter, a GFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP) ; a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early
  • a promoter is not cell-or tissue-specific., e.g., the promoter is considered a ubiquitous promoter.
  • promoter sequences that may promote expression in multiple cell or tissue types include, for example, human elongation factor 1a-subunit (EFla) , cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chicken beta-actin (CBA) and its derivatives, e.g., CAG, for example, a CBA promoter with an S40 intron, beta glucuronidase (GUSB) , or ubiquitin C (UBC) .
  • EFla human elongation factor 1a-subunit
  • CMV cytomegalovirus
  • CBA chicken beta-actin
  • CAG for example, a CBA promoter with an S40 intron, beta glucuronidase (GUSB) , or ubiquitin C (UBC) .
  • a promoter sequence can promote expression in particular cell types or tissues.
  • a promoter may be a muscle-specific promoter, e.g., may be a mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, or a mammalian skeletal alpha-actin (ASKA) promoter.
  • MCK mammalian muscle creatine kinase
  • DES mammalian desmin
  • TNNI2 mammalian troponin I
  • ASKA mammalian skeletal alpha-actin
  • a promoter sequence may be able to promote expression in neural cells or cell types, e.g., may be a neuron-specific enolase (NSE) , synapsin (Syn) , methyl-CpG binding protein 2 (MeCP2) , Ca2+/calmodulin-dependent protein kinase II (CaMKII) , metabotropic glutamate receptor 2 (mGluR2) , neurofilament light (NFL) or heavy (NFH) , beta-globin minigene hb2, preproenkephalin (PPE) , enkephalin (Enk) or excitatory amino acid transporter 2 (EAAT2) promoter.
  • NSE neuron-specific enolase
  • Syn synapsin
  • MeCP2 methyl-CpG binding protein 2
  • CaMKII Ca2+/calmodulin-dependent protein kinase II
  • mGluR2 metabotropic glutamate receptor 2
  • NFL
  • a promoter sequence may promote expression in the liver, e.g., may be an alpha-1-antitrypsin (hAAT) or thyroxine binding globulin (TBG) promoter.
  • a promoter sequence may promote expression in cardiac tissue, e.g., may be a cardiomyocyte-specific promoter such as an MHC, cTnT, or CMV-MUC2k promoter.
  • the polynucleotide may comprise at least one polyadenylation (polyA) signal sequence, which are well known in the art. In instances where a polyadenylation sequence is present, it is generally located between the 3'end of the transgene coding sequence and the 5'end of the 3'ITR. In certain embodiments, the polynucleotide further comprises a polyA upstream enhancer sequence 5’ of the polyA signal sequence. In certain instances, the regulatory sequence is a sequence that increases translation efficiency, e.g., a Kozak sequence.
  • the polynucleotide comprises an intron.
  • the intron is present within the coding sequence of the protein provided herein.
  • the intron is 5’ or 3’ of the coding sequence of the protein.
  • the intron flanks the 5’ or 3’ terminus of the coding sequence of the protein.
  • the polynucleotide comprises two introns.
  • one intron is 5’ of and one intron is 3’ of the coding sequence of the protein.
  • one intron flanks the 5’ terminus of the coding sequence of the protein and the second intron flanks the 3’ terminus of the coding sequence of the protein.
  • the intron is an SV40 intron, e.g., a 5’ UTR SV40 intron.
  • sequences of AAV ITR known in the art can be used in the present rAAV vector.
  • the AAV ITR used in the present vectors has a wild-type nucleotide sequence.
  • the AAV ITR sequence used in the present vectors is not wild-type sequence, and instead it comprises, e.g., the insertion, deletion or substitution of nucleotides.
  • AAV ITRs provided herein may be derived from any AAV serotypes, including but not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.
  • the 5′and 3′ITRs which flank a nucleotide sequence in a rAAV vector provided herein are identical and derived from the same AAV serotype. In other embodiments, the 5′and 3′ITRs which flank a nucleotide sequence in a rAAV vector provided herein are different and/or derived from different AAV serotypes.
  • the rAAV vector comprising the polynucleotide of the protein flanked by AAV ITRs can be constructed by directly inserting the polynucleotide of interest into an AAV genome, e.g., into an excised AAV open reading frames, and certain portions of the AAV genome can optionally be deleted, as described in, e.g., WO 1993/003769; Kotin (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med. 179: 1867-1875.
  • AAV ITRs are excised from an AAV genome or from an AAV vector containing such ITRs, and then are fused to 5′and 3′of a polynucleotide sequence of the protein that is present in another vector using standard ligation techniques.
  • the rAAV vector provided herein comprises a recombinant self-complementing genome.
  • a rAAV comprising a self-complementing genome can usually quickly form a double stranded DNA molecule by its partially complementing sequences (e.g., complementing coding and non-coding strands of a transgene) .
  • an rAAV vector provided herein comprises an rAAV genome that comprises a first heterologous polynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heterologous polynucleotide sequence (e.g., the noncoding or antisense strand of the therapeutic transgene) , and the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence.
  • the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrastrand base-pairing, e.g., a hairpin DNA structure.
  • the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a mutated ITR, so that the rep proteins do not cleave the viral genome at the mutated ITR.
  • rAAV vectors comprising self-complementing genomes can be made using the methods known in the art, e.g., as described in U.S. Pat. Nos. 7,125,717; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457.
  • the polynucleotide molecules in the rAAV vectors provided herein is less than about 5 kilobases (kb) in size. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein is less than about 4.5 kb in size. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein is less than about 4.0 kb in size. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein is less than about 3.5 kb in size. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein is less than about 3.0 kb in size. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein is less than about 2.5 kb in size.
  • a recombinant AAV (rAAV) vector comprising a nucleic acid encodes SMN or SNRPN or a variant thereof, wherein the rAAV vector comprises an inverted terminal repeat (ITR) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, or AAV44-9.
  • ITR inverted terminal repeat
  • the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7.
  • the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13.
  • the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.
  • recombinant AAVs or rAAV particles comprising a nucleic acid provided herein, and at least an AAV capsid protein.
  • the nucleic acid includes any nucleic acids and rAAV vectors described in Section 5.2 and 5.3.2 above.
  • the capsid protein may be derived from the same serotype as the ITRs, or a derivative thereof.
  • the capsid may also be of a different serotype than the ITR.
  • an AAV particle comprises AAV2 ITRs and an AAV6 capsid (AAV 2/6) , AAV2 ITRs and an AAV7 capsid (AAV 2/7) , AAV2 ITRs and an AAV8 capsid (AAV 2/8) , or AAV2 ITRs and an AAV9 capsid (AAV 2/9) .
  • Naturally occurring AAV capsids comprise AAV VP1, VP2 and VP3 capsid proteins, which are each encoded by splice variants of the AAV cap gene.
  • an AAV particle comprises three proteins, VP1, VP2 and VP3, with VP2 and VP3 being truncated version of VP1 so having sequences that are also comprised by VP1.
  • the amino acid sequence of VP1 defines the serotype of the capsid.
  • the VP1 capsid protein encodes for an AAV2 VP1 protein
  • AAV will be of the AAV2 serotype
  • the VP1 capsid protein encodes an AAV8 VP1 protein
  • the AAV will be of the AAV8 serotype
  • an AAV capsid protein (e.g., VP1, VP2 and/or VP3) in the present rAAV particle is not a naturally occurring capsid protein.
  • an AAV capsid protein (e.g., VP1, VP2 and/or VP3) is derived from a naturally occurring capsid protein.
  • the AAV capsid protein is a VP1 capsid protein. In other embodiments, the AAV capsid protein is a VP2 capsid protein. In other embodiments, the AAV capsid protein is a VP3 capsid protein. In some embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein and/or a VP3 capsid protein. In other embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein and a VP3 capsid protein.
  • the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein and/or a VP3 capsid protein, wherein the capsid proteins of the rAAV particle are of the same serotype.
  • the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein and a VP3 capsid protein, wherein the capsid proteins of the AAV particle are of the same serotype.
  • the capsid protein is a variant capsid protein.
  • a variant capsid protein may comprise one or more mutations, e.g. amino acid substitutions, amino acid deletions, and heterologous peptide insertions, compared to a corresponding reference capsid protein such as the naturally occurring parental capsid protein, i.e. the capsid protein from which it was derived.
  • the amino acid sequence of the AAV capsid protein is identical to the amino acid sequence of the wild type, or reference, or parent AAV capsid protein except for 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 or 30 amino acid residues, e.g., except for 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 or 30 amino acid residue substitutions.
  • the capsid protein or AAV particle described herein may be a chimeric capsid protein or AAV particle, respectively, comprising a protein sequence of two or more AAV serotype capsid proteins or particles, respectively.
  • the capsid protein in the rAAV particle provided herein is derived from an AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9 capsid protein.
  • the capsid protein in the rAAV particle provided herein has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%identical to the amino acid sequence of an AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9 capsid protein.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV1.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV2.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV2i8.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV3.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV3-B.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV4.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV5.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV6.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV7.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV8.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAVrh8.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAVrh8R.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV9.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV10.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAVrh10.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV11.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV12.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV13.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV-DJ.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV LK03.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAVrh74.
  • an AAV particle provided herein comprises VP1, VP2 and/or VP3 capsid proteins that comprises a VPl, VP2 and/or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%or 100%sequence identity, to any of the VP1, VP2 or VP3 amino acid sequences of AAV44-9.
  • the rAAV particle provided herein comprises a nucleic acid encoding the protein of interest provided herein and VP1 of an AAV comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the rAAV particles described herein may be produced using any suitable method known in the art.
  • a host cell e.g., a mammalian cell
  • a host cell e.g., a mammalian cell
  • a plasmid or multiple plasmids
  • a selectable marker such as an antibiotic (e.g., neomycin or ampicillin) resistance gene into the genome of the cell.
  • the cell can be, e.g., an insect or mammalian cell which can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the rAAV vector comprising the 5' and 3' AAV ITR.
  • a helper virus e.g., adenovirus or baculovirus providing the helper functions
  • the rAAV vector comprising the 5' and 3' AAV ITR.
  • a selectable marker allows for large-scale production of the rAAV.
  • adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells.
  • both the viral vector containing the 5' and 3' AAV ITRs and the rep and cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the rAAV.
  • a helper virus for AAV refers to a virus that allows AAV to be replicated and packaged by a host cell.
  • a helper virus provides helper functions that allow for the replication of AAV.
  • helper viruses have been identified, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.
  • Ad5 Adenovirus type 5 of subgroup C
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC.
  • Viruses of the herpes family which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV) , Epstein-Barr viruses (EBV) , cytomegaloviruses (CMV) and pseudorabies viruses (PRV) .
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • adenovirus helper functions for the replication of AAV include E1A functions, E1B functions, E2A functions, VA functions and E4orf6 functions.
  • a preparation of AAV is said to be substantially free of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 102: 1; at least about 104: 1, at least about 106: 1; or at least about 108: 1.
  • Preparations can also be free of equivalent amounts of helper virus proteins (i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form) .
  • helper virus proteins i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form
  • Viral and/or cellular protein contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2 and VP3) .
  • host cells containing the rAAV vectors described above is rendered capable of providing AAV helper functions to replicate and encapsulate the polynucleotide encoding the protein of interest provided herein flanked by the AAV ITRs to produce rAAV particles.
  • AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions are used herein to complement necessary AAV functions that are missing from the rAAV vectors.
  • AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.
  • AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the rAAV vector.
  • AAV helper constructs can be used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection.
  • AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves.
  • the AAV helper constructs can be in the form of, e.g., a plasmid, phage, transposon, cosmid, virus, or virion.
  • the host cell is also capable of providing or is provided with non AAV-derived functions or “accessory functions” to produce rAAV particles.
  • Accessory functions are non AAV-derived viral and/or cellular functions upon which AAV is dependent for its replication, such as non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
  • viral-based accessory functions can be derived from a known helper virus.
  • a recombinant AAV particle is produced, and the produced rAAV particle is infectious, replication-defective virus, and includes an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs.
  • rAAV particles can be purified from the host cell using a purification method known in the art, such as chromatography, CsCl gradients, and other methods as described, for example, in U.S. Pat. Nos. 6,989,264 and 8,137,948 and WO 2010/148143.
  • residual helper virus can be inactivated using known methods, e.g., by heating.
  • a variety of host cells can be used to produce rAAV particles described herein.
  • Suitable host cells for producing AAV particles from the polynucleotides and AAV vectors provided herein include microorganisms, yeast cells, insect cells, and mammalian cells. Typically, such cells can be, or have been, used as recipients of a heterologous nucleic acid molecule and can grow in, e.g., suspension culture and a bioreactor.
  • the cell is a mammalian host cell, for example, a HEK293, HEK293-T, A549 , WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • a mammalian host cell for example, a HEK293, HEK293-T, A549 , WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the cell is an insect cell, for example an Sf9, SF21, SF900+, or a drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyxmori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines.
  • insect cell for example an Sf9, SF21, SF900+, or a drosophila cell lines
  • mosquito cell lines e.g., Aedes albopictus derived cell lines
  • domestic silkworm cell lines e.g. Bombyxmori cell lines
  • Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines.
  • insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.
  • AAV recombinant AAV in cells, including Sf9 insect cells, has been described by Kotin RM.
  • Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith.
  • the present disclosure further provides pharmaceutical compositions comprising the vector or viral particle of the present disclosure.
  • a pharmaceutical composition comprises a therapeutically effective amount of the vectors or viral particles provided herein and a pharmaceutically acceptable excipient.
  • provided herein is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the rAAV vectors provided herein and a pharmaceutically acceptable excipient.
  • provided herein is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the rAAV particles provided herein and a pharmaceutically acceptable excipient.
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) , carrier or vehicle.
  • adjuvant e.g., Freunds’ adjuvant (complete or incomplete)
  • Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the choice of excipient is determined in part by the particular cell, viral particle, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.
  • Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent.
  • Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof.
  • buffers may comprise histidine and trimethylamine salts such as Tris.
  • Preservatives may be added to retard microbial growth.
  • Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide) , benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride
  • benzalkonium halides e.g., chloride, bromide, iodide
  • benzethonium chloride thimerosal, phenol, butyl or
  • Tonicity agents can be present to adjust or maintain the tonicity of liquid in a composition.
  • stabilizers When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions.
  • exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall.
  • excipients include: polyhydric sugar alcohols (enumerated above) ; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol) , polyethylene glycol; sulfur
  • Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein.
  • Suitable non-ionic surfactants include, e.g., polysorbates (20, 40, 60, 65, 80, etc. ) , polyoxamers (184, 188, etc. ) , polyols, polyoxyethylene sorbitan monoethers ( -20, -80, etc.
  • lauromacrogol 400 lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride.
  • compositions are preferably sterile.
  • the pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes.
  • the pharmaceutical compositions herein generally can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, intravitreal, subretinal injection, topical administration, inhalation or by sustained release or extended-release means.
  • a pharmaceutical composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987) ; Buchwald et al., Surgery 88: 507-16 (1980) ; and Saudek et al., N. Engl. J. Med. 321: 569-74 (1989) ) .
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984) ; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61-126 (1983) ; Levy et al., Science 228: 190-92 (1985) ; During et al., Ann. Neurol. 25: 351-56 (1989) ; Howard et al., J. Neurosurg.
  • polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxy ethyl methacrylate) , poly (methyl methacrylate) , poly (acrylic acid) , poly (ethylene-co-vinyl acetate) , poly (methacrylic acid) , polyglycolides (PLG) , polyanhydrides, poly (N-vinyl pyrrolidone) , poly (vinyl alcohol) , polyacrylamide, poly (ethylene glycol) , polylactides (PLA) , poly (lactide-co-glycolides) (PLGA) , and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984) ) .
  • Controlled release systems are discussed, for example, by Langer, Science 249: 1527-33 (1990) .
  • Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos.
  • compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated.
  • the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent.
  • cytotoxic agent chemotherapeutic agent
  • cytokine cytokine
  • immunosuppressive agent or growth inhibitory agent.
  • growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecule provided herein, construction of a nucleic acid as part of a viral vector or other vector, etc.
  • the pharmaceutical composition provided herein contains the binding molecules and/or viral particles in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.
  • a method for epigenetically regulating the expression of a gene in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the gene is selected from a group consisting of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1,
  • the epigenetic regulation preserves the cell-or tissue-specific expression profile of the gene. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in a cell-or tissue-specificy way. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in neurons.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN. In some embodiments, the gene is NPAP1. In some embodiments, the gene is SNRPN.
  • the gene is HDAC1. In some embodiments, the gene is HDAC6. In some embodiments, the gene is HDAC8. In some embodiments, the gene is KAT6. In some embodiments, the gene is KAT8. In some embodiments, the gene is TET1. In some embodiments, the gene is TET2. In some embodiments, the gene is TET3.
  • SMN1 protein SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof for epigenetically regulating the expression of a gene in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKI
  • the epigenetic regulation preserves the cell-or tissue-specific expression profile of the gene. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in a cell-or tissue-specificy way. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in neurons.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN. In some embodiments, the gene is NPAP1. In some embodiments, the gene is SNRPN.
  • the gene is HDAC1. In some embodiments, the gene is HDAC6. In some embodiments, the gene is HDAC8. In some embodiments, the gene is KAT6. In some embodiments, the gene is KAT8. In some embodiments, the gene is TET1. In some embodiments, the gene is TET2. In some embodiments, the gene is TET3.
  • the expression of UBE3A is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of GABRB3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of GABRA5 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of GABRG3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of ATP10A is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of OCA2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of HERC2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of NIPA1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of NIPA2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of CYFIP1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of TUBGCP5 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of MKRN3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of MAGEL2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of NDN is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of NPAP1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of SNRPN is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of HDAC1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of HDAC6 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of HDAC8 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the transduced cells.
  • the expression of KAT6 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of KAT8 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of TET1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the transduced cells.
  • the expression of TET2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of TET3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells.
  • the expression of UBE3A is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of GABRB3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of GABRA5 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of GABRG3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of ATP10A is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of OCA2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of HERC2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of NIPA1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of NIPA2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of CYFIP1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of TUBGCP5 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of MKRN3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of MAGEL2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of NDN is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a nucleic acid,
  • the expression of NPAP1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or
  • the expression of SNRPN is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of HDAC1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of HDAC6 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of HDAC8 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the transduced cells.
  • the expression of KAT6 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of KAT8 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of TET1 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the transduced cells.
  • the expression of TET2 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • the expression of TET3 is enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%or 95-100%, 100-500%in the cells after introducing into the cell the SMN1 protein or variants thereof, or a
  • a method for epigenetic modifying a gene in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the gene is selected from a group consisting of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP
  • the epigenetic modification preserves the cell-or tissue-specific expression profile of the gene. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in a cell-or tissue-specificy way. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in neurons.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN. In some embodiments, the gene is NPAP1. In some embodiments, the gene is SNRPN.
  • SMN1 protein SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof for epigenetic modifying a gene in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T2
  • the epigenetic modification preserves the cell-or tissue-specific expression profile of the gene. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in a cell-or tissue-specificy way. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in neurons.
  • the gene is UBE3A. In some embodiments, the gene is GABRB3. In some embodiments, the gene is GABRA5. In some embodiments, the gene is GABRG3. In some embodiments, the gene is ATP10A. In some embodiments, the gene is OCA2. In some embodiments, the gene is HERC2. In some embodiments, the gene is NIPA1. In some embodiments, the gene is NIPA2. In some embodiments, the gene is CYFIP1. In some embodiments, the gene is TUBGCP5. In some embodiments, the gene is MKRN3. In some embodiments, the gene is MAGEL2. In some embodiments, the gene is NDN. In some embodiments, the gene is NPAP1. In some embodiments, the gene is SNRPN.
  • the method or use increases the expression of UBE3A. In some embodiments, the method or use increases the expression of GABRB3. In some embodiments, the method or use increases the expression of GABRA5. In some embodiments, the method or use increases the expression of GABRG3. In some embodiments, the method or use increases the expression of ATP10A. In some embodiments, the method or use increases the expression of OCA2. In some embodiments, the method or use increases the expression of HERC2. In some embodiments, the method or use increases the expression of NIPA1. In some embodiments, the method or use increases the expression of NIPA2. In some embodiments, the method or use increases the expression of CYFIP1. In some embodiments, the method or use increases the expression of TUBGCP5.
  • the method or use increases the expression of MKRN3. In some embodiments, the method or use increases the expression of MAGEL2. In some embodiments, the method or use increases the expression of NDN. In some embodiments, the method or use increases the expression of NPAP1. In some embodiments, the method or use decreases the expression of SNRPN.
  • a method for epigenetically regulating the expression of a gene in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the gene is selected from a group consisting of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1,
  • the epigenetic regulation preserves the cell-or tissue-specific expression profile of the gene. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in a cell-or tissue-specificy way. In some embodiments, the epigenetic regulation specifically rescues the expression of the gene that has been imprinting silenced in neurons.
  • the disease or disorder is an UBE3A associated disease or disorder. In some embodiments, the disease or disorder is a GABRB3 associated disease or disorder. In some embodiments, the disease or disorder is a GABRA5 associated disease or disorder. In some embodiments, the disease or disorder is a GABRG3 associated disease or disorder. In some embodiments, the disease or disorder is an ATP10A associated disease or disorder. In some embodiments, the disease or disorder is an OCA2 associated disease or disorder. In some embodiments, the disease or disorder is an HERC2 associated disease or disorder. In some embodiments, the disease or disorder is a NIPA1 associated disease or disorder. In some embodiments, the disease or disorder is a NIPA2 associated disease or disorder.
  • the disease or disorder is a CYFIP1 associated disease or disorder. In some embodiments, the disease or disorder is a TUBGCP5 associated disease or disorder. In some embodiments, the disease or disorder is a MKRN3 associated disease or disorder. In some embodiments, the disease or disorder is a MAGEL2 associated disease or disorder. In some embodiments, the disease or disorder is a NDN associated disease or disorder. In some embodiments, the disease or disorder is a NPAP1 associated disease or disorder. In some embodiments, the disease or disorder is a SNRPN associated disease or disorder. In some embodiments, the disease or disorder is a HDAC1 associated disease or disorder. In some embodiments, the disease or disorder is a HDAC6 associated disease or disorder.
  • the disease or disorder is a HDAC8 associated disease or disorder. In some embodiments, the disease or disorder is a KAT6 associated disease or disorder. In some embodiments, the disease or disorder is a KAT8 associated disease or disorder. In some embodiments, the disease or disorder is a TET1 associated disease or disorder. In some embodiments, the disease or disorder is a TET2 associated disease or disorder. In some embodiments, the disease or disorder is a TET3 associated disease or disorder.
  • a method for epigenetically regulating the expression of UBE3A in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with UBE3A (or an UBE3A associated disease or disorder) .
  • a method for epigenetically regulating the expression of GABRB3 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with GABRB3 (or a GABRB3 associated disease or disorder) .
  • a method for epigenetically regulating the expression of GABRA5 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with GABRA5 (or a GABRA5 associated disease or disorder) .
  • a method for epigenetically regulating the expression of GABRG3 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with GABRG3 (or a GABRG3 associated disease or disorder) .
  • a method for epigenetically regulating the expression of ATP10A in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with ATP10A (or a ATP10A associated disease or disorder) .
  • a method for epigenetically regulating the expression of OCA2 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with OCA2 (or a OCA2 associated disease or disorder) .
  • a method for epigenetically regulating the expression of HERC2 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with HERC2 (or a HERC2 associated disease or disorder) .
  • a method for epigenetically regulating the expression of NIPA1 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with NIPA1 (or a NIPA1 associated disease or disorder) .
  • a method for epigenetically regulating the expression of NIPA2 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with NIPA2 (or a NIPA2 associated disease or disorder) .
  • a method for epigenetically regulating the expression of CYFIP1 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with CYFIP1 (or a CYFIP1 associated disease or disorder) .
  • a method for epigenetically regulating the expression of TUBGCP5 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with TUBGCP5 (or a TUBGCP5 associated disease or disorder) .
  • a method for epigenetically regulating the expression of MKRN3 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with MKRN3 (or a MKRN3 associated disease or disorder) .
  • a method for epigenetically regulating the expression of MAGEL2 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with MAGEL2 (or a MAGEL2 associated disease or disorder) .
  • a method for epigenetically regulating the expression of NDN in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with NDN (or a NDN associated disease or disorder) .
  • a method for epigenetically regulating the expression of NPAP1 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with NPAP1 (or a NPAP1 associated disease or disorder) .
  • a method for epigenetically regulating the expression of SNRPN in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with SNRPN (or a SNRPN associated disease or disorder) .
  • a method for epigenetically regulating the expression of HDAC1 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with HDAC1 (or a HDAC1 associated disease or disorder) .
  • a method for epigenetically regulating the expression of HDAC6 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with HDAC6 (or a HDAC6 associated disease or disorder) .
  • a method for epigenetically regulating the expression of HDAC8 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with HDAC8 (or a HDAC8 associated disease or disorder) .
  • a method for epigenetically regulating the expression of KAT6 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with KAT6 (or a KAT6 associated disease or disorder) .
  • a method for epigenetically regulating the expression of KAT8 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with KAT8 (or a KAT8 associated disease or disorder) .
  • a method for epigenetically regulating the expression of TET1 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with TET1 (or a TET1 associated disease or disorder) .
  • a method for epigenetically regulating the expression of TET2 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with TET2 (or a TET2 associated disease or disorder) .
  • a method for epigenetically regulating the expression of TET3 in a cell comprising introducing into the cell a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, or a nucleic acid encoding the SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the cell is in a subject having a disease or disorder associated with TET3 (or a TET3 associated disease or disorder) .
  • the disease or disorder is an imprinting diesease.
  • the disease or disorder is Angelman syndrome.
  • the disease or disorder is Prader Willi syndrome.
  • the disease or disorder is Beckwith-Wiedemann syndrome.
  • the disease or disorder is Silver-Russell syndrome.
  • a method for treating a disease or disorder in a subject comprising administering to the subject a nucleic acid encoding a SMN1 protein, SNRPN protein, Ascl1 protein, Banp protein, Foxg1 protein, Klf4 protein, NeuroD1 protein, NeuroD4 protein, Pou5F1 protein, Sox2 protein, Zfp36l1 protein, or CamKIIa-T286D protein or variants thereof, wherein the disease or disorder is associated with UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2, and TET3.
  • the nucleic acid is in an AAV vector.
  • the nucleic acid is administered after birth but before 2 years old of the subject.
  • kits for using and uses of the vectors, or the viral particles (rAAV) provided herein are provided herein.
  • Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, rAAV or compositions containing the same, to a subject having a disease or disorder.
  • the molecule, viral particle, and/or composition is administered in an effective amount to effect treatment of the disease or disorder.
  • Uses include uses of viral particles in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods.
  • the methods are carried out by administering the viral particles, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.
  • the treatment provided herein cause complete or partial amelioration or reduction of a disease or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the terms include, but do not imply, complete curing of a disease or complete elimination of any symptom or effect (s) on all symptoms or outcomes.
  • the treatment provided herein delay development of a disease or disorder, e.g., defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as AS) .
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder.
  • the method or the use provided herein prevents a disease or disorder.
  • the disease or disorder is associated with UBE3A. In some embodiments, the disease or disorder is associated with insufficient expression of UBE3A protein. In some embodiments, the disease or disorder is associated with a deficient UBE3A protein (such as a mutant UBE3A protein) .
  • the disease or disorder is associated with GABRB3. In some embodiments, the disease or disorder is associated with insufficient expression of GABRB3 protein. In some embodiments, the disease or disorder is associated with a deficient GABRB3 protein (such as a mutant GABRB3 protein) .
  • the disease or disorder is associated with GABRA5. In some embodiments, the disease or disorder is associated with insufficient expression of GABRA5 protein. In some embodiments, the disease or disorder is associated with a deficient GABRA5 protein (such as a mutant GABRA5 protein) .
  • the disease or disorder is associated with GABRG3. In some embodiments, the disease or disorder is associated with insufficient expression of GABRG3 protein. In some embodiments, the disease or disorder is associated with a deficient GABRG3 protein (such as a mutant GABRG3 protein) .
  • the disease or disorder is associated with ATP10A. In some embodiments, the disease or disorder is associated with insufficient expression of ATP10A protein. In some embodiments, the disease or disorder is associated with a deficient ATP10A protein (such as a mutant ATP10A protein) .
  • the disease or disorder is associated with OCA2. In some embodiments, the disease or disorder is associated with insufficient expression of OCA2 protein. In some embodiments, the disease or disorder is associated with a deficient OCA2 protein (such as a mutant OCA2 protein) .
  • the disease or disorder is associated with HERC2. In some embodiments, the disease or disorder is associated with insufficient expression of HERC2 protein. In some embodiments, the disease or disorder is associated with a deficient HERC2 protein (such as a mutant HERC2 protein) .
  • the disease or disorder is associated with NIPA1. In some embodiments, the disease or disorder is associated with insufficient expression of NIPA1 protein. In some embodiments, the disease or disorder is associated with a deficient NIPA1 protein (such as a mutant NIPA1 protein) .
  • the disease or disorder is associated with NIPA2. In some embodiments, the disease or disorder is associated with insufficient expression of NIPA2 protein. In some embodiments, the disease or disorder is associated with a deficient NIPA2 protein (such as a mutant NIPA2 protein) .
  • the disease or disorder is associated with CYFIP1. In some embodiments, the disease or disorder is associated with insufficient expression of CYFIP1 protein. In some embodiments, the disease or disorder is associated with a deficient CYFIP1 protein (such as a mutant CYFIP1 protein) .
  • the disease or disorder is associated with TUBGCP5. In some embodiments, the disease or disorder is associated with insufficient expression of TUBGCP5 protein. In some embodiments, the disease or disorder is associated with a deficient TUBGCP5 protein (such as a mutant TUBGCP5 protein) .
  • the disease or disorder is associated with MKRN3. In some embodiments, the disease or disorder is associated with insufficient expression of MKRN3 protein. In some embodiments, the disease or disorder is associated with a deficient MKRN3 protein (such as a mutant MKRN3 protein) .
  • the disease or disorder is associated with MAGEL2. In some embodiments, the disease or disorder is associated with insufficient expression of MAGEL2 protein. In some embodiments, the disease or disorder is associated with a deficient MAGEL2 protein (such as a mutant MAGEL2 protein) .
  • the disease or disorder is associated with NDN. In some embodiments, the disease or disorder is associated with insufficient expression of NDN protein. In some embodiments, the disease or disorder is associated with a deficient NDN protein (such as a mutant NDN protein) .
  • the disease or disorder is associated with NPAP1. In some embodiments, the disease or disorder is associated with insufficient expression of NPAP1 protein. In some embodiments, the disease or disorder is associated with a deficient NPAP1 protein (such as a mutant NPAP1 protein) .
  • the disease or disorder is associated with SNRPN. In some embodiments, the disease or disorder is associated with insufficient expression of SNRPN protein. In some embodiments, the disease or disorder is associated with a deficient SNRPN protein (such as a mutant SNRPN protein) .
  • the disease or disorder is associated with HDAC1. In some embodiments, the disease or disorder is associated with insufficient expression of HDAC1 protein. In some embodiments, the disease or disorder is associated with a deficient HDAC1 protein (such as a mutant HDAC1 protein) .
  • the disease or disorder is associated with HDAC6. In some embodiments, the disease or disorder is associated with insufficient expression of HDAC6 protein. In some embodiments, the disease or disorder is associated with a deficient HDAC6 protein (such as a mutant HDAC6 protein) .
  • the disease or disorder is associated with HDAC8. In some embodiments, the disease or disorder is associated with insufficient expression of HDAC8 protein. In some embodiments, the disease or disorder is associated with a deficient HDAC8 protein (such as a mutant HDAC8 protein) .
  • the disease or disorder is associated with KAT6. In some embodiments, the disease or disorder is associated with insufficient expression of KAT6 protein. In some embodiments, the disease or disorder is associated with a deficient KAT6 protein (such as a mutant KAT6 protein) .
  • the disease or disorder is associated with KAT8. In some embodiments, the disease or disorder is associated with insufficient expression of KAT8 protein. In some embodiments, the disease or disorder is associated with a deficient KAT8 protein (such as a mutant KAT8 protein) .
  • the disease or disorder is associated with TET1. In some embodiments, the disease or disorder is associated with insufficient expression of TET1 protein. In some embodiments, the disease or disorder is associated with a deficient TET1 protein (such as a mutant TET1 protein) .
  • the disease or disorder is associated with TET2. In some embodiments, the disease or disorder is associated with insufficient expression of TET2 protein. In some embodiments, the disease or disorder is associated with a deficient TET2 protein (such as a mutant TET2 protein) .
  • the disease or disorder is associated with TET3. In some embodiments, the disease or disorder is associated with insufficient expression of TET3 protein. In some embodiments, the disease or disorder is associated with a deficient TET3 protein (such as a mutant TET3 protein) .
  • the vectors or viral particles described herein are used to treat a subject with Angelman syndrome, Prader Willi syndrome, Beckwith-Wiedemann syndrome, Silver-Russell syndrome, or other imprinting diseases.
  • administration of the composition of the disclosure to a subject may enhance UBE3A mRNA transcription in the transduced cells of a subject.
  • the transcription of UBE3A may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance GABRB3 mRNA transcription in the transduced cells of a subject.
  • the transcription of GABRB3 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance GABRA5 mRNA transcription in the transduced cells of a subject.
  • the transcription of GABRA5 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance GABRG3 mRNA transcription in the transduced cells of a subject.
  • the transcription of GABRG3 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance ATP10A mRNA transcription in the transduced cells of a subject.
  • the transcription of ATP10A may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance OCA2 mRNA transcription in the transduced cells of a subject.
  • the transcription of OCA2 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance HERC2 mRNA transcription in the transduced cells of a subject.
  • the transcription of HERC2 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance NIPA1 mRNA transcription in the transduced cells of a subject.
  • the transcription of NIPA1 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance NIPA2 mRNA transcription in the transduced cells of a subject.
  • the transcription of NIPA2 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance CYFIP1 mRNA transcription in the transduced cells of a subject.
  • the transcription of CYFIP1 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80
  • administration of the composition of the disclosure to a subject may enhance TUBGCP5 mRNA transcription in the transduced cells of a subject.
  • the transcription of TUBGCP5 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80
  • administration of the composition of the disclosure to a subject may enhance MKRN3 mRNA transcription in the transduced cells of a subject.
  • the transcription of MKRN3 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance MAGEL2 mRNA transcription in the transduced cells of a subject.
  • the transcription of MAGEL2 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance NDN mRNA transcription in the transduced cells of a subject.
  • the transcription of NDN may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%
  • administration of the composition of the disclosure to a subject may enhance NPAP1 mRNA transcription in the transduced cells of a subject.
  • the transcription of NPAP1 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%
  • administration of the composition of the disclosure to a subject may enhance SNRPN mRNA transcription in the transduced cells of a subject.
  • the transcription of SNRPN may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance HDAC1 mRNA transcription in the transduced cells of a subject.
  • the transcription of HDAC1 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance HDAC6 mRNA transcription in the transduced cells of a subject.
  • the transcription of HDAC6 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance HDAC8 mRNA transcription in the transduced cells of a subject.
  • the transcription of HDAC8 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance KAT6 mRNA transcription in the transduced cells of a subject.
  • the transcription of KAT6 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance KAT8 mRNA transcription in the transduced cells of a subject.
  • the transcription of KAT8 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance TET1 mRNA transcription in the transduced cells of a subject.
  • the transcription of TET1 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30- 70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-9
  • administration of the composition of the disclosure to a subject may enhance TET2 mRNA transcription in the transduced cells of a subject.
  • the transcription of TET2 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • administration of the composition of the disclosure to a subject may enhance TET3 mRNA transcription in the transduced cells of a subject.
  • the transcription of TET3 may be enhanced by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90
  • compositions described herein can be administered to an individual by any route, e.g., intravascularly (e.g., intravenously (IV) or intraarterially) , directly into arteries, systemically (for example by intravenous injection) , or locally (for example by intraarterial or intraocular injection) .
  • intravascularly e.g., intravenously (IV) or intraarterially
  • IV intravenously
  • intraarterially directly into arteries
  • systemically for example by intravenous injection
  • locally for example by intraarterial or intraocular injection
  • Non-limiting exemplary administration methods include intravenous (e.g., by infusion pumps) , intraperitoneal, intraocular, intra-arterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarterial, topical, inhalational (e.g., as mists of sprays) , mucosal (such as via nasal mucosa) , subcutaneous, transdermal, gastrointestinal, intraarticular, intracisternal, intraventricular, intracranial, intraurethral, intrahepatic, intratumoral, intravitreal and subretinal injection.
  • intravenous e.g., by infusion pumps
  • intraperitoneal intraocular, intra-arterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarte
  • the composition of the present disclosure for treating Angelman syndrome, Prader Willi syndrome, Beckwith-Wiedemann syndrome, Silver-Russell syndrome, or other imprinting diseases is administered to the subject in need thereof intravenously, intramuscularly, subcutaneously, intraperitoneally, intrathecally and/or intraventricularly, allowing the present nucleic acid to pass through one or both the blood-brain barrier and the blood spinal cord barrier.
  • the method includes administering (e.g., intraventricularly administering and/or intrathecally administering) directly to the nervous system (e.g., CNS) of a subject (using, e.g., an infusion pump and/or a delivery scaffold) a therapeutically effective amount of a composition of the present disclosure.
  • administering e.g., intraventricularly administering and/or intrathecally administering directly to the nervous system (e.g., CNS) of a subject (using, e.g., an infusion pump and/or a delivery scaffold) a therapeutically effective amount of a composition of the present disclosure.
  • compositions of AAV vector or AAV particle comprising a nucleic acid sequence described herein may be administered in a way which facilitates the composition to enter the central nervous system and penetrate into motor neurons.
  • the AAV vector or AAV particle of the present disclosure may be administered by muscular injection.
  • the AAV vector or AAV particle that comprises the nucleic acid of the present disclosure is administered to a subject by peripheral injections and/or intranasal delivery.
  • the AAV vector or AAV particle that comprises the nucleic acid of the present disclosure is administered to a subject by intracranial delivery (e.g. intrathecal or intracerebroventricular administration, see e.g., U.S. Pat. No. 8,119,611; the content of which is incorporated herein by reference in its entirety) .
  • intracranial delivery e.g. intrathecal or intracerebroventricular administration, see e.g., U.S. Pat. No. 8,119,611; the content of which is incorporated herein by reference in its entirety
  • the composition comprising the AAV vector or particle of the present disclosure is administered intravenous or intracranial to the central nervous system (CNS) of the subject.
  • CNS central nervous system
  • the composition comprising the AAV vector or particle of the present disclosure is administered to CNS, such as neurons, motor neurons, microglia and astrocytes.
  • the composition comprising the AAV vector or particle of the present disclosure is administered to astrocytes.
  • composition comprising the AAV vector or particle of the present disclosure may be delivered into specific types of targeted cells, including neurons, motor neurons; glial cells including oligodendrocyte, astrocyte and microglia; and/or other cells surrounding neurons such as T cells.
  • the AAV vector or AAV particle of the present disclosure may be administered in a therapeutically effective amount, e.g., an amount that is sufficient to alleviate and/or prevent at least one symptom associated with the disease, or provide improvement in the condition of the subject.
  • the AAV vector or AAV particle of the present disclosure may be administered to the cisterna magna in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
  • the composition may be administered intrathecally.
  • the AAV vector or AAV particle of the present disclosure may be administered using intrathecal infusion in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
  • the composition may be administered intrathecally.
  • the AAV vector or AAV particle of the present disclosure may be administered using a bolus infusion.
  • the AAV vector or AAV particle of the present disclosure may be administered 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.
  • the catheter may be located at more than one site in the spine for multi-site delivery.
  • the AAV vector or AAV particle of the present disclosure may be delivered in a continuous and/or bolus infusion.
  • Each site of delivery may be 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.
  • a subject may be analyzed for spinal anatomy and pathology prior to delivery of the AAV vector or AAV particle described herein.
  • a subject with scoliosis may have a different dosing regimen and/or catheter location compared to a subject without scoliosis.
  • the orientation of the spine of the subject during delivery of the present AAV vector or particle may be vertical to the ground. In other embodiments, the orientation of the spine of the subject during delivery of the AAV vector or AAV particle may be horizontal to the ground.
  • the spine of the subject may be at an angle as compared to the ground during the delivery of the present AAV vector or AAV particle.
  • the angle of the spine of the subject as compared to the ground may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 180 degrees.
  • the delivery method and duration is chosen to provide broad transduction in the spinal cord.
  • intrathecal delivery is used to provide broad transduction along the rostral-caudal length of the spinal cord.
  • multi-site infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.
  • prolonged infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.
  • compositions of the present disclosure may be administered to a subject using any amount effective for reducing, preventing and/or treating a disease or disorder associated with UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2, and TET3.
  • the disease or disorder is an imprinting diesease.
  • the disease or disorder is Angelman syndrome.
  • the disease or disorder is Prader Willi syndrome.
  • the disease or disorder is Beckwith-Wiedemann syndrome. In a specific embodiment, the disease or disorder is Silver-Russell syndrome.
  • the exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the AAV vector or AAV particle of the present disclosure may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution, and may be formulated with any appropriate and pharmaceutically acceptable excipient.
  • the AAV vector or particle is formulated.
  • the basicity 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.
  • the pharmaceutical composition provided herein is a suspension, e.g., a refrigerated suspension.
  • the method further comprises agitating the suspension to ensure even distribution of the suspension prior to the administration step.
  • the method further comprises warming the pharmaceutical composition to room temperature prior to the administration step.
  • the compositions may also be administered in a sustained release formulation.
  • the sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, and the like) may be administered by injection or surgical implanted in various locations.
  • compositions of the present disclosure are typically 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 of sound medical judgment.
  • the specific therapeutic effectiveness 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; 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 age and sex of a subject may be used to determine the dose of the compositions of the present disclosure.
  • a subject who is older may receive a larger dose (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more) of the composition as compared to a younger subject.
  • a subject who is younger may receive a larger dose (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more) of the composition as compared to an older subject.
  • a larger dose e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more
  • a subject who is female may receive a larger dose (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more) of the composition as compared to a male subject.
  • a larger dose e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more
  • a subject who is male may receive a larger dose (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more) of the composition as compared to a female subject.
  • a larger dose e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more than 90%more
  • the doses of AAV vectors or AAV particles for delivering the nucleic acid of the present disclosure may be adapted dependent on the disease condition, the subject and the treatment strategy.
  • the concentration of vector or viral particle that is administered may differ depending on production method and may be chosen or optimized based on concentrations determined to be therapeutically effective for the particular route of administration.
  • the concentration in vector genomes per milliliter is selected from the group consisting of about 10 8 vg/ml -10 15 vg/ml.
  • the vector or viral particle provided herein is delivered by intravenous, intracranial injection, or intra cisterna magna injection, or intrathecal injection, or intramuscular injection, or intravitreal injection.
  • the vector or viral particle provided herein is injected in a volume between about 0.1 ml and about 20 ml.
  • a rAAV comprising the nucleic acid described herein can be administered to a subject at a dose of 1x10 8 to 1x10 17 vector genomes (vg) .
  • a rAAV comprising the nucleic acid described herein can be administered to a subject at a dose of 1x10 8 to 1x10 17 vector genomes/kg (vg/kg) .
  • one or more additional therapeutic agents may be administered to the subject.
  • compositions described herein can be monitored by several criteria. For example, after treatment in a subject using methods of the present disclosure, the subject may be assessed for e.g., an improvement and/or stabilization and/or delay in the progression of one or more signs or symptoms of the disease state by one or more clinical parameters including those described herein. Examples of such tests are known in the art, and include objective as well as subjective (e.g., subject reported) measures.
  • the AAV vector or AAV particle of the present disclosure may be delivered to a subject via a single route administration. In other embodiments, the AAV vector or AAV particle of the present disclosure may be delivered to a subject via a multi-site route of administration, e.g., at 2, 3, 4, 5 or more than 5 sites.
  • compositions comprising the present AAV vector or AAV particle may be administered in a single daily dose, or the daily dose may be administered in divided dosages of two, three, or four times daily.
  • Compositions provided herein can also be administered multiple times (e.g., twice, three times, four times, or five times) within a time period (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years) .
  • composition comprising the AAV vector or particle of the present disclosure is administered as solo therapeutics or combination therapeutics for the treatment of Angelman syndrome, Prader Willi syndrome, Beckwith-Wiedemann syndrome, Silver-Russell syndrome, or other imprinting diseases.
  • compositions comprising the AAV vector or particle of the present disclosure may be used in combination with one or more other therapeutic agents.
  • agents By “in combination with, ” it is not intended to imply 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. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • therapeutic agents that may be used in combination with the AAV vector or particle of the present disclosure can be small molecule compounds including, e.g., immunosuppressants, antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, and compounds involved in metal ion regulation.
  • small molecule compounds including, e.g., immunosuppressants, antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, and compounds involved in metal ion regulation.
  • therapeutic agents that may be used in combination therapy with the vectors or viral particles described herein may be hormones or variants that can protect neuronal loss, such as adrenocorticotropic hormone (ACTH) or fragments thereof (e.g., U.S. Patent Publication No. 20130259875) ; Estrogen (e.g., U.S. Pat. Nos. 6,334,998 and 6,592,845) ; the content of each of which is incorporated herein by reference in their entirety.
  • ACTH adrenocorticotropic hormone
  • Estrogen e.g., U.S. Pat. Nos. 6,334,998 and 6,592,845
  • neurotrophic factors may be used in combination therapy with the AAV vector or AAV particle of the present disclosure.
  • a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron.
  • the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment.
  • Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.
  • Enhancement of levels or expression of a gene can be assayed in a variety of ways known in the art.
  • a gene e.g., a UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 nucleic acid
  • a gene e.g., a UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 nucleic acid
  • mRNA sequence of a gene can be used to prepare a probe that is at least partially complementary to the mRNA sequence.
  • the probe can then be used to detect the mRNA in a sample, using any suitable assay, such as PCR based methods, northern blotting, a dipstick assay, and the like.
  • the assay method can be varied depending on the type of mRNA information desired.
  • Exemplary methods include but are not limited to northern blots and PCR-based methods (e.g., qRT-PCR) .
  • Methods such as qRT-PCR can also accurately quantitate the amount of the mRNA in a sample.
  • an assay may be in the form of a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multi-well plate, or an optical fiber.
  • An assay system may have a solid support on which a nucleic acid corresponding to the mRNA is attached.
  • the solid support may comprise, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide.
  • the assay components can be prepared and packaged together as a kit for detecting an mRNA.
  • the nucleic acid can be labeled, if desired, to make a population of labeled mRNAs.
  • a sample can be labeled using methods that are well known in the art (e.g., using DNA ligase, terminal transferase, or by labeling the RNA backbone, etc. ) . See, e.g., Ausubel et al., Short Protocols in Molecular Biology (Wiley &Sons, 3rd ed. 1995) ; Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 3rd ed. 2001) .
  • the sample is labeled with fluorescent label.
  • Exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes (e.g., fluorescein isothiocyanate (FITC) , 6-carboxyfluorescein (FAM) , 6 carboxy-2’, 4’, 7’, 4, 7-hexachlorofluorescein (HEX) , 6-carboxy-4’, 5’-dichloro-2’, 7’-dimethoxyfluorescein (JOE) ) , rhodamine dyes (e.g., rhodamine 110 (R110) , N, N, N’, N’-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 5 carboxyrhodamine 6G (R6G5 or G5) , 6-carboxyrhodamine 6G (R6G6 or G6) ) , cyanine dyes (
  • a typical mRNA assay method can contain the steps of 1) obtaining surface-bound subject probes; 2) hybridizing a population of mRNAs to the surface-bound probes under conditions sufficient to provide for specific binding; (3) post-hybridization washing to remove nucleic acids not specifically bound to the surface-bound probes; and (4) detecting the hybridized mRNAs.
  • the reagents used in each of these steps and their conditions for use may vary depending on the particular application.
  • Hybridization can be carried out under suitable hybridization conditions, which may vary in stringency as desired. Typical conditions are sufficient to produce probe/target complexes on a solid surface between complementary binding members, i.e., between surface-bound subject probes and complementary mRNAs in a sample. In certain embodiments, stringent hybridization conditions may be employed.
  • Hybridization is typically performed under stringent hybridization conditions.
  • Standard hybridization techniques e.g., under conditions sufficient to provide for specific binding of target mRNAs in the sample to the probes
  • Several guides to general techniques are available, e.g., Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993) .
  • the surface bound polynucleotides are typically washed to remove unbound nucleic acids. Washing may be performed using any convenient washing protocol, where the washing conditions are typically stringent, as described above. The hybridization of the target mRNAs to the probes is then detected using standard techniques.
  • PCR-based methods can also be used to detect the expression of a gene.
  • PCR methods can be found in U.S. Patent No. 6,927,024, which is incorporated by reference herein in its entirety.
  • RT-PCR methods can be found in U.S. Patent No. 7,122,799, which is incorporated by reference herein in its entirety.
  • a method of fluorescent in situ PCR is described in U.S. Patent No. 7,186,507, which is incorporated by reference herein in its entirety.
  • qRT-PCR quantitative Reverse Transcription-PCR
  • RNA targets Bustin et al., Clin. Sci. 2005, 109: 365-379. Quantitative results obtained by qRT-PCR are generally more informative than qualitative data.
  • qRT-PCR-based assays can be useful to measure mRNA levels during cell-based assays. The qRT-PCR method is also useful to monitor patient therapy. Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7,101,663, which is incorporated by reference herein in its entirety.
  • qRT PCR In contrast to regular reverse transcriptase-PCR and analysis by agarose gels, qRT PCR gives quantitative results.
  • An additional advantage of qRT-PCR is the relative ease and convenience of use. Instruments for qRT-PCR, such as the Applied Biosystems 7500, are available commercially, so are the reagents, such as Sequence Detection Chemistry. For example, Gene Expression Assays can be used, following the manufacturer’s instructions. These kits are pre-formulated gene expression assays for rapid, reliable detection and quantification of human, mouse, and rat mRNA transcripts.
  • the data can be analyzed, for example, using 7500 Real-Time PCR System Sequence Detection software vs. using the comparative CT relative quantification calculation method. Using this method, the output is expressed as a fold-change of expression levels.
  • the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but sufficiently low to be within the exponential growth region of an amplification curve.
  • a target RNA can be detected or quantified by Next
  • NGS Generation Sequencing
  • Enhanced protein expression of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 nucleic acids can be assessed by measuring UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 protein levels.
  • Protein levels can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting) , enzyme-linked immunosorbent assay (ELISA) , quantitative protein assays, protein activity assays (for example, caspase activity assays) , immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS) , LC-MS (Liquid-chromatography-MassSpec) , and other methods.
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.
  • Antibodies useful for the detection of mouse, rat, monkey, and human UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 are commercially available.
  • protein levels can be measure using labeled or unlabeled methods.
  • In vivo assays can be used to assess enhanced expression of UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3, such as, improved motor function and respiration, meanwhile reducing off-target toxicity in liver and/or heart.
  • motor function is measured by righting, open field performance in the animal.
  • respiration is measured by whole body plethysmograph, invasive resistance, and compliance measurements in the animal.
  • OS overall survival
  • DFS disease free survival
  • oligonucleotides can be formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline.
  • Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight.
  • RNA can be isolated from tissues of interest, including liver, heart, spleen, CNS tissue or CSF and changes in UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT 6, KAT8, TET1, TET2, or TET3 nucleic acid expression are measured, e.g., using NGS.
  • kits, unit dosages, and articles of manufacture comprising any of the compositions described herein.
  • a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder (such as Angelman syndrome) described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • the label or package insert indicates that the composition is used for treating the particular condition in an individual.
  • the label or package insert will further comprise instructions for administering the composition to the individual.
  • the label may indicate directions for reconstitution and/or use.
  • the container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • the disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the disclosure also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • the disclosure is generally not expressed herein in terms of what the disclosure does not include, aspects that are not expressly included in the disclosure are nevertheless disclosed herein.
  • SMN1 The epigenetic regulation of imprinting silenced genes through overexpressing SMN1 using rAAV vectors were tested in the SMN ⁇ 7 -/- mouse model and the Angelman +/- mouse model (i.e., the Ube3A m-/p+ mouse model) .
  • the amino acid sequence of SMN1 is
  • the mRNA expression of paternally imprinted UBE3A, neighboring genes of UBE3A (ATP10A, GABRB3, GABRA5, GABRG3, OCA2, and HERC2) , genes encoding proteins related to epigenetic regulation such as HDAC1 (encoding histone deacetylase 1) , HDAC6 (encoding histone deacetylase 6) , HDAC8 (encoding histone deacetylase 8) , KAT6 (encoding lysine acetyltransferase 6) , KAT8 (encoding lysine acetyltransferase 8) , TET1 (encoding Tet methylcytosine dioxygenase 1) , TET2 (encoding Tet methylcytosine dioxygenase 2) , and TET3 (encoding Tet methylcytosine dioxygenase 3) , as well as PTPA (encoding PTPA protein that is a substrate of UBE3A
  • the mRNA expression of paternally imprinted UBE3A in spinal cord increased after overexpressing SMN1; and the mRNA expression of neighboring genes (GABRB3, GABRA5, and GABRG3) increased in brain after overexpressing SMN1.
  • the mRNA expression of HDAC1, HDAC6, HDAC8, and KAT8 generally increased in brain;
  • the mRNA expression of TET1, TET2, and TET3 generally increased in both spinal cord and brain.
  • the protein expression of the above described genes is tested via western blot and immunofluorescence. Moreover, the DNA methylation level at the imprinting control region of UBE3A is measured via BS-seq, and the histone acetylation level at the imprinting control region of UBE3A is measured via ChIP-seq. Methods described herein are well known to those skilled in the art.
  • nucleotide sequences of exemplary rAAV vectors expressing proteins capable of epigenetically regulating imprinting silenced genes are provided in Table 3 below.
  • the components of exemplary rAAV vectors (101-02, 101-03A, and 101-05ME) are provided in Table 4 below.
  • the amino acid sequences of exemplary proteins capable of epigenetically regulating imprinting silenced genes are provided in Table 5 below.
  • iPSC cells derived from children with Angelman Syndrome and healthy children are plated on Matrigel coated surface of 6-well plates.
  • the N2B27 medium are supplemented with 5 ⁇ M SB43152 and 1 ⁇ M dorsomorphin for neural induction.
  • SB431542 and dorsomorphin are withdrawn. Formation of neural rosettes can be observed after around another eight days. The newly formed neural rosettes are selected and transferred to suspension culture for continued incubation.
  • the induced neural stem cells are further validated in two appraoches.
  • total RNA of the neural stem cells is extracted using Trizol followed by real-time PCR using Premix Ex Taq TM kit.
  • the targets of the real-time PCR include stem cell markers (OCT4, SOX2, and NANOG) , neural stem cell markers (PAX6, NES, SOX1, SOX2, and VIM) , and an internal control (GAPDH) .
  • immunofluorescence is used to detect the expression of neuron-specific antigens (NSE, nestin, GFAP, Tau, NCAM1, and NF-1) .
  • the induced neural stem cells are divided into two groups.
  • Cells in the experimental group are transfected with an exemplary rAAV vector that expressing human SMN1.
  • Cells in the control group are treated with empty vectors.
  • the protein expression of the above described genes is tested via western blot and immunofluorescence. Moreover, the DNA methylation level at the imprinting control region of UBE3A is measured via BS-seq, and the histone acetylation level at the imprinting control region of UBE3A is measured via ChIP-seq. Methods described herein are well known to those skilled in the art.

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Abstract

Procédé de régulation épigénétique de l'expression d'un gène dans une cellule comprenant l'introduction dans la cellule d'une protéine SMN1 ou d'une protéine SNRPN ou de leurs variants, le gène étant choisi dans un groupe constitué par UBE3A, GABRB3, GABRA5, GABRG3, ATP10A, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, TUBGCP5, MKRN3, MAGEL2, NDN, NPAP1, SNRPN, HDAC1, HDAC6, HDAC8, KAT6, KAT8, TET1, TET2 et TET3.
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