WO2024030930A2 - Compositions et méthodes de modification de cellules gliales de müller - Google Patents

Compositions et méthodes de modification de cellules gliales de müller Download PDF

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WO2024030930A2
WO2024030930A2 PCT/US2023/071472 US2023071472W WO2024030930A2 WO 2024030930 A2 WO2024030930 A2 WO 2024030930A2 US 2023071472 W US2023071472 W US 2023071472W WO 2024030930 A2 WO2024030930 A2 WO 2024030930A2
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nucleic acid
vector
acid sequence
aav
encoding
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WO2024030930A3 (fr
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Seth Blackshaw
Thanh Hoang
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The Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 1 encoding the human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 2 encoding the human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence encoding the human Yes1 associated transcriptional regulator (hYAP) protein.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence encoding the human octamer-binding transcription factor 4 (hOCT-4) protein.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • hRBPJ human recombination signal binding protein
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human recombination signal binding protein (hRBPJ) protein operably linked to one or more regulatory elements; and a polyadenylation tail signal, wherein the nucleic acid sequence capable of encoding hRBPJ has a nucleic acid sequence of SEQ ID NO: 2.
  • hRBPJ human recombination signal binding protein
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human nuclear factor 1 X-type (hNFIX) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • hNFIX human nuclear factor 1 X-type
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human Yes1 associated transcriptional regulator (hYAP) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence encoding human octamer-binding transcription factor 4 (hOCT-4) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • methods of inducing reprogramming of a Muller glial cell in a subject the methods comprising inhibiting Notch signaling in a Muller glial cell in the subject.
  • FIG.1A shows the schematic of generation of Muller glia-specific loss of function mutant of Rbpj, and analysis of gene expression in Rbpj-deficient glia. Tamoxifen induction simultaneously labels glia with Sun1-GFP while inactivating conditional allele of Rbpj.
  • FIG. 1A shows the schematic of generation of Muller glia-specific loss of function mutant of Rbpj, and analysis of gene expression in Rbpj-deficient glia. Tamoxifen induction simultaneously labels glia with Sun1-GFP while inactivating conditional allele of Rbpj.
  • FIG.1B shows ScRNA-Seq analysis revealing that Rbpj-deficient Muller glia show reduced expression of genes specific to resting glia and induction of genes specific to neurogenic retinal progenitors.
  • FIG.1C shows a UMAP plot of cells following analysis of retina in whole control and Rbpj-deficient retina. Muller glia are indicated.
  • FIG.1D shows a UMAP plot of expression of the neurogenic bHLH factors Ascl1 and Neurog2 in control and Rbpj- deficient Muller glia.
  • FIG.2A shows that a limited number of Sun1-GFP-positive cells are labeled with the amacrine/retinal ganglion cell marker HuC/D four weeks following tamoxifen induction (inset shows high magnification image).
  • FIG.2B shows the quantification of the relative fraction of Sun1-GFP-positive cells that express either HuC/D or the bipolar cell marker Otx2 in uninjured and NMDA-treated retina. Excitotoxic injury strongly stimulates neurogenesis.
  • FIG.2C shows representative images Rpbj-deficient retinas stained with Otx2, HuC/D and the amacrine cell-specific marker glycine.
  • FIG.3A is a schematic showing injection and analysis of AAV overexpression constructs. Tamoxifen-dependent, GlastCreER-mediated Sun1-GFP expression is used to irreversibly label Muller glia prior to AAV injection.
  • FIG.3B shows representative immunohistochemical images showing lack of co-expression of neuronal markers in Gfap- mCherry control viruses, but induction of both bipolar and amacrine cell-specific markers following Muller glia-specific overexpression of Insm1 and Atoh7/Neurog2.
  • FIG.4 shows the results of reprogramming mammalian Muller glia for retinal repair.
  • FIG.5 shows integrated regulatory network analysis (IReNA).
  • FIG.6 shows that the loss of Rbpj leads to injury-independent mouse Muller glia reprogramming.
  • FIGS.7A-D show that Muller glia-specific loss of function of Rbpj induces neurogenesis.
  • FIGS.8A-D show that NMDA-mediated excitotoxic injury enhances neurogenesis in Rbpj-deficient Muller glia.
  • FIGS.9A-D show that Notch1/2 deletion phenocopies effects of loss of function of Rbpj in Muller glia.
  • FIGS.10A-F show that loss of function of Rbpj in Müller glia activates expression of neurogenic genes while downregulating Notch target genes.
  • FIGS.11A-C show that deletion of both Nfia/b/x and Rbpj converts >80% of Müller glial into neurons.
  • FIGS.12A-B show that overexpression of dominant-active Yap5SA induces proliferation in Rbpj-deficient Müller glia.
  • FIGS.13A-D show that overexpression of Oct4 selectively enhances neurogenesis in Rbpj-deficient Müller glia.
  • FIG.14 shows that deletion of both Nfia/b/x and Rbpj generates small numbers of Nrl-positive Müller glial-derived rod photoreceptors.
  • a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value.
  • transgene refers to a gene or genetic material that has been transferred or artificially introduced into the genome by a genetic engineering technique from one organism to another, i.e., the host organism.
  • transgene expression relates to the control of the amount and timing of appearance of the functional product of a transgene in a host organism.
  • endogenous refers to substances and processes originating from within an organism, tissue or cell. “Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease gene expression, activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease.
  • the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to wild-type or control levels.
  • the inhibition or reduction is 0- 25, 25-50, 50-75, or 75-100% as compared to wild-type or control levels.
  • “Promote,” “promotion,” and “promoting” refer to an increase in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 10% increase in the activity, response, condition, or disease as compared to the wild-type or control level. Thus, in some aspects, the increase or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion in between compared to native or control levels. In some aspects, the increase or promotion is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to wild-type or control levels.
  • the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, such as, for example, 200, 300, 500, or 1000% more as compared to wild-type or control levels. In some aspects, the increase or promotion can be greater than 100 percent as compared to wild- type or control levels, such as 100, 150, 200, 250, 300, 350, 400, 450, 500% or more as compared to the wild-type or control levels.
  • the term “operatively linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences linked to other sequences in order confer functional activity of the construct as a whole.
  • operative linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • promoter refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • promoter refers to a DNA sequence which when operatively linked to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA.
  • a promoter is located 5' (i.e., upstream) of a nucleotide sequence of interest (e.g., proximal to the transcriptional start site of a structural gene), although not necessarily immediately upstream because of the optional inclusion of intervening sequences between the promoter and the sequence to be transcribed, whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • sample is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
  • the term “subject” refers to the target of administration, e.g., a human.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • a subject is a mammal.
  • a subject is a human.
  • the term does not denote a particular age or sex.
  • the term “patient” refers to a subject afflicted with a disease or disorder.
  • the term “patient” includes human and veterinary subjects.
  • the “patient” has been diagnosed with a need for treatment for an inherited blinding disease (e.g., macular degeneration, retinitis pigmentosa or glaucoma), such as, for example, prior to the administering step.
  • an inherited blinding disease e.g., macular degeneration, retinitis pigmentosa or glaucoma
  • the term “comprising” can include the aspects “consisting of” and “consisting essentially of.”
  • the term “normal” refers to an individual, a sample or a subject that does not inherited blinding disease (e.g., macular degeneration, retinitis pigmentosa or glaucoma) or does not have an increased susceptibility of developing inherited blinding disease (e.g., macular degeneration, retinitis pigmentosa or glaucoma).
  • the term “treat” or “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, (e.g., an inherited blinding disease).
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) inhibiting the disease, i.e., arresting its development; or (ii) relieving the disease, i.e., causing regression of the disease (e.g., inherited blinding disease).
  • a mammal e.g., a human
  • the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
  • prevent is meant to mean minimize the chance that a subject who has an increased susceptibility for developing inherited blinding disease will develop inherited blinding disease. In the context as used herein, preventing does not need to eliminate completely all sequela associated with an inherited blinding disease and would encompass any reduction in the expression of one or more symptoms associated or disease conditions associated with an inherited blinding disease.
  • vector or “construct” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).
  • Plasmid and “vector” are used interchangeably, as a plasmid is a commonly used form of vector.
  • the invention is intended to include other vectors which serve equivalent functions.
  • expression vector is herein to refer to vectors that are capable of directing the expression of genes to which they are operatively-linked. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid as disclosed herein in a form suitable for expression of the acid in a host cell.
  • the recombinant expression vectors can include one or more regulatory elements or promoters, which can be selected based on the host cells used for expression that is operatively linked to the nucleic acid sequence to be expressed.
  • “Modulate”, “modulating” and “modulation” as used herein mean a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.
  • alter or “modulate” can be used interchangeable herein referring, for example, to the expression of a nucleotide sequence in a cell means that the level of expression of the nucleotide sequence in a cell after applying a method as described herein is different from its expression in the cell before applying the method.
  • disease or “disorder” or “condition” are used interchangeably referring to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person.
  • a disease or disorder or condition can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, or affection.
  • Suitable promoters can be derived from genes of the host cells where expression should occur or from pathogens for this host cells (e.g., tissue promoters or pathogens like viruses). If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • the promoter may be regulated in a tissue-specific or tissue preferred manner such that it is only active in transcribing the associated coding region in a specific tissue type(s) such as leaves, roots or meristem.
  • tissue specific refers to a promoter that is capable of directing selective expression of a nucleotide sequence or gene of interest to a specific type of tissue in the relative absence of expression of the same nucleotide sequence or gene of interest in a different type of tissue.
  • Muller glia-derived neurons is typically limited to bipolar and amacrine cells, although recent studies have successfully generated retinal ganglion-like cells. Therefore, identifying positive and negative regulators of neurogenic competence is urgently needed.
  • Transcriptional effectors of the Notch pathway have been identified as important regulators of Muller glial quiescence in both zebrafish and mice.
  • Notch signaling plays an important role in controlling vertebrate retinal development, where it maintains progenitor status, promotes gliogenesis and inhibits neurogenesis, particularly photoreceptor specification.
  • compositions and method for inducing generation of retinal neurons from endogenous glia cells thereby allowing replacement of neurons lost due to inherited blinding diseases such as macular degeneration, retinitis pigmentosa, and glaucoma.
  • compositions and methods that convert glia-to- neurons that can be efficiently induced by viral over-expression of either Insm1 or Atoh7 in combination with Neurog2. Furthermore, disclosed herein is that loss of function of the common Notch pathway effector Rbpj is also effective at inducing glia-to-neuron conversion, demonstrating that small molecule-based inhibition of Notch signaling can enhance the efficiency of glia-to-neuron conversion.
  • nucleic acids comprising at least one transgene operably linked to a promoter.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 1 encoding the human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain.
  • hRBPJ human recombination signal binding protein
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 2 encoding the human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation.
  • the expression cassette can further comprise a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 3 encoding the human nuclear factor 1 X-type (hNFIX) protein.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence encoding the human Yes1 associated transcriptional regulator (hYAP) protein. Further disclosed herein are polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence encoding the human octamer-binding transcription factor 4 (hOCT-4) protein.
  • hYAP human Yes1 associated transcriptional regulator
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain.
  • the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1 capable of encoding the human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human recombination signal binding protein (hRBPJ) protein or a human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation.
  • the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human recombination signal binding protein (hRBPJ) protein or a human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 2 capable of encoding the human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation.
  • the expression cassette further comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human nuclear factor 1 X-type (hNFIX) protein.
  • polynucleotides comprising an expression cassettes, wherein the expression cassette further comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 3 encoding the human nuclear factor 1 X- type (hNFIX) protein.
  • the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human Yes1 associated transcriptional regulator (hYAP) protein.
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence capable of encoding the human Yes1 associated transcriptional regulator (hYAP) protein.
  • hYAP human Yes1 associated transcriptional regulator
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence capable of encoding the human octamer-binding transcription factor 4 (hOCT-4) protein.
  • hOCT-4 human octamer-binding transcription factor 4
  • polynucleotides comprising an expression cassette, wherein the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence capable of encoding the human octamer-binding transcription factor 4 (hOCT-4) protein.
  • the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence having a sequence at least 90% identical to the sequence capable of encoding the human octamer-binding transcription factor 4 (hOCT-4) protein.
  • the RBPJ gene can encode a protein having the amino acid sequence: MDHTEGSPAEEPPAHAPSPGKFGERPPPKRLTREAMRNYLKERGDQTVLILHAKVAQ KSYGNEKRFFCPPPCVYLMGSGWKKKKEQMERDGCSEQESQPCAFIGIGNSDQEMQ QLNLEGKNYCTAKTLYISDSDKRKHFMLSVKMFYGNSDDIGVFLSKRIKVISKPSKK KQSLKNADLCIASGTKVALFNRLRSQTVSTRYLHVEGGNFHASSQQWGAFFIHLLDD DESEGEEFTVRDGYIHYGQTVKLVCSVTGMALPRLIIRKVDKQTALLDADDPVSQLH KCAFYLKDTERMYLCLSQERIIQFQATPCPKEPNKEMINDGASWTIISTDKAEYTFYE GMGPVLAPVTPVPVVESLQLNGGGDVAMLELTGQNFTPNLRVWFGDVEAETMYRC
  • the RBPJ gene can encode a protein having the amino acid sequence: MDHTEGSPAEEPPAHAPSPGKFGERPPPKRLTREAMRNYLKERGDQTVLILHAKVAQ KSYGNEKRFFCPPPCVYLMGSGWKKKKEQMERDGCSEQESQPCAFIGIGNSDQEMQ QLNLEGKNYCTAKTLYISDSDKRKHFMLSVKMFYGNSDDIGVFLSKRIKVISKPSKK KQSLKNADLCIASGTKVALFNRLRSQTVSTRYLHVEGGNFHASSQQWGAFFIHLLDD DESEGEEFTVRDGYIHYGQTVKLVCSVTGMALPHLIIRKVDKQTALLDADDPVSQLH KCAFYLKDTERMYLCLSQERIIQFQATPCPKEPNKEMINDGASWTIISTDKAEYTFYE GMGPVLAPVTPVPVVESLQLNGGGDVAMLELTGQNFTPNLRVWFGDVEAETMYRC
  • the NFIX gene can encode a protein having the amino acid sequence: MLPACRLQDEFHPFIEALLPHVRAFSYTWFNLQARKRKYFKKHEKRMSKDEERAVK DELLGEKPEIKQKWASRLLAKLRKDIRPEFREDFVLTITGKKPPCCVLSNPDQKGKIR RIDCLRQADKVWRLDLVMVILFKGIPLESTDGERLYKSPQCSNPGLCVQPHHIGVTIK ELDLYLAYFVHTPESGQSDSSNQQGDADIKPLPNGHLSFQDCFVTSGVWNVTELVRV SQTPVATASGPNFSLADLESPSYYNINQVTLGRRSITSPPSTSTTKRPKSIDDSEMESPV DDVFYPGTGRSPAAGSSQSSGWPNDVDAGPASLKKSGKLDFCSALSSQGSSPRMAFT HHPLPVLAGVRPGSPRATASALHFPSTSIIQQSSPYFTHPTIRYHHHHGQDSLK
  • the YAP gene can encode a protein having the amino acid sequence MDPGQQPPPQPAPQGQGQPPSQPPQGQGPPSGPGQPAPAATQAAPQAPPAGHQIVHV RGDSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSRQASTDAGT AGALTPQHVRAHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLRQSSFEIPDDVP LPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRKAMLSQMNVTAPTSPPVQQNMMNS ASGPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRFAMNQRISQSAPVKQPPP LAPQSPQGGVMGGSNSNQQQQMRLQQLQMEKERLRLKQQELLRQAMRNINPSTAN SPKCQELALRSQLPTLEQDGGTQNPVSSPGMSQELRTMTTNSSDPFLNSGTYHS
  • the OCT-4 gene can encode a protein having the amino acid sequence MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEV WGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEP CTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTL GVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQ ARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKG KRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGE AFPPVSVTTLGSPMHSN (SEQ ID NO: 5).
  • any of the genes disclosed herein can be codon-optimized, for example, for expression in a mammal, such as a human.
  • DNA sequences provided herein may also include the reverse complement to form the double stranded DNA sequence or may be a reverse complement of the sequences disclosed herein.
  • the nucleic acid sequence encoding RBPJ can be SEQ ID NO: 1.
  • the nucleic acid sequence encoding RBPJ can be fused to a constitutive KRAB domain.
  • the KRAB domain can encode a protein having the amino acid sequence LVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEK GEEPW (SEQ ID NO: 9).
  • the nucleic acid sequence capable of encoding RBPJ can be SEQ ID NO: 2.
  • the nucleic acid sequence capable of encoding RBPJ can comprise a R216H point mutation.
  • the nucleic acid sequence capable of encoding RBPJ comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequence capable of encoding RBPJ comprises up to 20 nucleotides that are different from the RBPJ gene set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the RBPJ gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are different from the RBPJ gene set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequence capable of encoding RBPJ comprises more than 20 nucleotides that are different from the RBPJ gene set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequence capable of encoding RBPJ comprises insertions relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequences encoding RBPJ comprises insertions relative to SEQ ID NO: 1 or SEQ ID NO: 2 that do not introduce a frameshift mutation.
  • an insertion in the nucleic acid sequence relative to SEQ ID NO: 1 or SEQ ID NO: 2 involves the insertion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • an insertion in the nucleic acid sequence relative to SEQ ID NO: 1 or SEQ ID NO: 2 leads to an increase in the total number of amino acid residues in the resultant PGM1 protein (e.g., an increase of 1-3, 15, 3-10, 5-10, 5-15, or 10-20 amino acid residues).
  • the nucleic acid sequence capable of encoding RBPJ comprises deletions relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequences capable of encoding RBPJ comprises deletions relative to SEQ ID NO: 1 or SEQ ID NO: 2 that do not introduce a frameshift mutation.
  • a deletion in the nucleic acid sequence relative to SEQ ID NO: 1 or SEQ ID NO: 2 involves the deletion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.). In some aspects, a deletion in the nucleic acid sequence relative to SEQ ID NO: 1 or SEQ ID NO: 2 leads to an decrease in the total number of amino acid residues in the resultant RBPJ protein (e.g., a decrease of 1-3, 1-5, 3-10, 5-10, 5-15, or 10-20 amino acid residues). In some aspects, the nucleic acid sequence capable of encoding RBPJ can be a codon- optimized sequence (e.g., codon optimized for expression in mammalian cells).
  • a codon-optimized sequence capable of encoding RBPJ comprises reduced GC content relative to a wild-type sequence that has not been codon-optimized. In some aspects, a codon-optimized sequence capable of encoding RBPJ comprises a 1-5%, 3-5%, 3-10%, 5- 10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50%, or 30-60% reduction in GC content relative to a wild-type sequence that has not been codon-optimized. In some aspects, a codon- optimized sequence encoding RBPJ comprises fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized.
  • a codon- optimized sequence encoding RBPJ comprises 1-5, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20- 40, 25-50, or 30-60 fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding RBPJ comprises fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding RBPJ comprises 1-3, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized.
  • the nucleotide sequence encoding PGM1 is SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleic acid sequence capable of encoding NFIX can be SEQ ID NO: 3.
  • the nucleic acid sequence capable of encoding NFIX comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3.
  • the nucleic acid sequence capable of encoding NFIX comprises up to 20 nucleotides that are different from the NFIX gene set forth in SEQ ID NO: 3.
  • the NFIX gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are different from the NFIX gene set forth in SEQ ID NO: 3.
  • the nucleic acid sequence capable of encoding NFIX comprises more than 20 nucleotides that are different from the NFIX gene set forth in SEQ ID NO: 3. In some aspects, the nucleic acid sequence capable of encoding NFIX comprises insertions relative to SEQ ID NO: 3. In some aspects, the nucleic acid sequences encoding NFIX comprises insertions relative to SEQ ID NO: 3 that do not introduce a frameshift mutation. In some aspects, an insertion in the nucleic acid sequence relative to SEQ ID NO: 3 involves the insertion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • an insertion in the nucleic acid sequence relative to SEQ ID NO: 3 leads to an increase in the total number of amino acid residues in the resultant NFIX protein (e.g., an increase of 1-3, 15, 3-10, 5-10, 5-15, or 10-20 amino acid residues).
  • the nucleic acid sequence encoding NFIX comprises deletions relative to SEQ ID NO: 3.
  • the nucleic acid sequences encoding NFIX comprises deletions relative to SEQ ID NO: 3 that do not introduce a frameshift mutation.
  • a deletion in the nucleic acid sequence relative to SEQ ID NO: 3 involves the deletion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • a deletion in the nucleic acid sequence relative to SEQ ID NO: 3 leads to an decrease in the total number of amino acid residues in the resultant NFIX protein (e.g., a decrease of 1-3, 1- 5, 3-10, 5-10, 5-15, or 10-20 amino acid residues).
  • the nucleic acid sequence capable of encoding NFIX can be a codon- optimized sequence (e.g., codon optimized for expression in mammalian cells).
  • a codon-optimized sequence encoding NFIX comprises reduced GC content relative to a wild-type sequence that has not been codon-optimized.
  • a codon- optimized sequence encoding NFIX comprises a 1-5%, 3-5%, 3-10%, 5-10%, 5-15%, 10- 20%, 15-30%, 20-40%, 25-50%, or 30-60% reduction in GC content relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding NFIX comprises fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding NFIX comprises 1-5, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized. In some aspects, a codon-optimized sequence encoding NFIX comprises fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon- optimized.
  • a codon-optimized sequence encoding NFIX comprises 1-3, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized.
  • the nucleotide sequence encoding NFIX is SEQ ID NO: 3.
  • the nucleic acid sequence encoding YAP can be found at NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequence capable of encoding YAP comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequence capable of encoding YAP comprises up to 20 nucleotides that are different from the YAP gene set forth in NCBI Reference Sequence: NC_000011.10.
  • the YAP gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are different from the YAP gene set forth in NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequence encoding YAP gene comprises more than 20 nucleotides that are different from the YAP gene set forth in NCBI Reference Sequence: NC_000011.10. In some aspects, the nucleic acid sequence encoding YAP comprises insertions relative to NCBI Reference Sequence: NC_000011.10. In some aspects, the nucleic acid sequences encoding PGM1 comprises insertions relative to NCBI Reference Sequence: NC_000011.10that do not introduce a frameshift mutation. In some aspects, an insertion in the nucleic acid sequence relative to NCBI Reference Sequence: NC_000011.10involves the insertion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • the nucleic acid sequence encoding YAP comprises deletions relative to NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequences encoding PGM1 comprises deletions relative to NCBI Reference Sequence: NC_000011.10that do not introduce a frameshift mutation.
  • the nucleic acid sequence encoding YAP can be a codon-optimized sequence (e.g., codon optimized for expression in mammalian cells).
  • a codon-optimized sequence encoding YAP comprises reduced GC content relative to a wild- type sequence that has not been codon-optimized. In some aspects, a codon-optimized sequence encoding YAP comprises a 1-5%, 3-5%, 3-10%, 5-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50%, or 30-60% reduction in GC content relative to a wild-type sequence that has not been codon-optimized. In some aspects, a codon-optimized sequence encoding YAP comprises fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding YAP comprises 1-5, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon- optimized. In some aspects, a codon-optimized sequence encoding YAP comprises fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding YAP comprises 1-3, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer CpG dinucleotide islands relative to a wild- type sequence that has not been codon-optimized.
  • the nucleotide sequence encoding YAP is NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequence encoding OCT-4 can be Gene ID: 5460.
  • the nucleic acid sequence capable of encoding OCT-4 comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to Gene ID: 5460. In some aspects, the nucleic acid sequence capable of encoding OCT-4 comprises up to 20 nucleotides that are different from the OCT-4 gene set forth in Gene ID: 5460. In some aspects, the OCT-4 gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are different from the OCT-4 gene set forth in Gene ID: 5460.
  • the nucleic acid sequence encoding OCT-4 gene comprises more than 20 nucleotides that are different from the OCT-4 gene set forth in Gene ID: 5460.
  • the nucleic acid sequence capable of encoding OCT-4 comprises insertions relative to Gene ID: 5460.
  • the nucleic acid sequences encoding OCT-4 comprises insertions relative to Gene ID: 5460 that do not introduce a frameshift mutation.
  • an insertion in the nucleic acid sequence relative to Gene ID: 5460 involves the insertion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • an insertion in the nucleic acid sequence relative to Gene ID: 5460 leads to an increase in the total number of amino acid residues in the resultant OCT-4 protein (e.g., an increase of 1-3, 15, 3-10, 5-10, 5-15, or 10-20 amino acid residues).
  • the nucleic acid sequence capable of encoding OCT-4 comprises deletions relative to Gene ID: 5460.
  • the nucleic acid sequences encoding PGM1 comprises deletions relative to Gene ID: 5460 that do not introduce a frameshift mutation.
  • a deletion in the nucleic acid sequence relative to Gene ID: 5460 involves the deletion of multiples of 3 nucleotides (e.g., 3, 6, 9, 12, 15, 18, etc.).
  • a deletion in the nucleic acid sequence relative to Gene ID: 5460 leads to an decrease in the total number of amino acid residues in the resultant OCT-4 protein (e.g., a decrease of 1-3, 1-5, 3-10, 5-10, 5-15, or 10-20 amino acid residues).
  • the nucleic acid sequence capable of encoding OCT-4 can be a codon-optimized sequence (e.g., codon optimized for expression in mammalian cells).
  • a codon-optimized sequence encoding OCT-4 comprises reduced GC content relative to a wild-type sequence that has not been codon-optimized.
  • a codon- optimized sequence encoding OCT-4 comprises a 1-5%, 3-5%, 3-10%, 5-10%, 5-15%, 10- 20%, 15-30%, 20-40%, 25-50%, or 30-60% reduction in GC content relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding OCT-4 comprises fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized.
  • a codon-optimized sequence encoding OCT-4 comprises 1-5, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer guanine and/or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized. In some aspects, a codon-optimized sequence encoding OCT-4 comprises fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon- optimized.
  • a codon-optimized sequence encoding OCT-4 comprises 1-3, 3- 5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized.
  • the nucleotide sequence encoding OCT-4 is Gene ID: 5460. Promoters. In the constructs disclosed herein, the promoter can be a constitutive promoter.
  • the promoter can be a constitutive promoter, for example a CAG promoter, a chicken beta-actin (CBA) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [e.g., Boshart et al., Cell, 41:521-530 (1985)], a CMV enhanced chicken ⁇ -actin promoter (CB), a SV40 promoter, a dihydrofolate reductase promoter, a (3-actin promoter, a phosphoglycerol kinase (PGK) promoter, or an EFla promoter [Invitrogen].
  • CB CMV enhanced chicken ⁇ -actin promoter
  • PGK phosphoglycerol kinase
  • a promoter can be a CAG promoter. In some aspects, a promoter can be an enhanced chicken ⁇ -actin promoter. In some aspects, a promoter can be a U6 promoter. In some aspects, the promoter can be a CB6 promoter. In some aspects, the promoter can be a JeT promoter. In some aspects, a promoter can be a CB promoter.
  • the CBA promoter has the sequence of TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGG GGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGG CGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA AAGTTTCCTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAG CGCGCGGCGGGCG (SEQ ID NO: 10).
  • the vector can further comprise conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the vector or infected with the virus produced by the disclosure.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • the recombinant AAV vector comprises a posttranscriptional response element.
  • posttranscriptional response element refers to a nucleic acid sequence that, when transcribed, adopts a tertiary structure that enhances expression of a gene.
  • posttranscriptional regulatory elements include, but are not limited to, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MPMV), and the 5' untranslated region of the human heat shock protein 70 (Hsp705'UTR).
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • RTE mouse RNA transport element
  • CTE constitutive transport element of the simian retrovirus type 1
  • MMV Mason-Pfizer monkey virus
  • Hsp705'UTR the recombinant AAV vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).
  • a polyadenylation sequence can be inserted following the transgene sequences and optionally before a 3' AAV ITR sequence.
  • a recombinant AAV construct useful in the disclosure can also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • the polyA signal sequence can be a simian virus 40 (SV40) poly A sequence having the nucleotide sequence of ATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTG AAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT AACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGG TTTTTTAAAGCGG (SEQ ID NO: 11).
  • SV40 simian virus 40
  • the expression cassette can be flanked by adeno-associated virus inverted terminal repe ats. Recombinant AAVs.
  • the disclosed polynucleotides can be inserted into a vector.
  • the vector can be an expression vector.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).
  • “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector.
  • the invention is intended to include other vectors which serve equivalent functions.
  • the vectors are used to deliver the disclosed nucleic acid sequences to cells to ultimately result in expression of the proteins encoded by the nucleic acid sequences. These proteins can then be used in the methods disclosed herein.
  • disclosed are vectors that comprise one or more of the polynucleotides disclosed herein.
  • the vector can be a viral vector.
  • the vector can be an adeno-associated viral vector.
  • the vector can be a viral vector.
  • the viral vector can be an adeno-associated viral vector (AAV).
  • AAV adeno-associated viral vector
  • the AAV can be AAV9.
  • the vector can be a non-viral vector, such as a DNA based vector.
  • the isolated nucleic acids disclosed herein can be recombinant adeno-associated viruses (rAAVs) vectors.
  • rAAV vectors described herein can be composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector that can be packaged into a capsid protein and delivered to a selected target cell.
  • the transgene can be a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule or other gene product, of interest.
  • the nucleic acid coding sequence can be operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue.
  • Adeno-associated virus AAV is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs).
  • ITRs nucleotide inverted terminal repeat
  • the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994).
  • AAV-1 is provided in GenBank Accession No. NC_002077
  • the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829
  • the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829
  • the AAV-5 genome is provided in GenBank Accession No. AF085716
  • the complete genome of AAV-6 is provided in GenBank Accession No.
  • AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al.
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy.
  • AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible.
  • the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal.
  • the rep and cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56.degree. C. to 65.degree. C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized.
  • AAV-infected cells are not resistant to superinfection.
  • Multiple studies have demonstrated long-term (>1.5 years) recombinant AAV- mediated protein expression in muscle. See, Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also, Chao et al., Mol Ther, 2: 619-623 (2000) and Chao et al., Mol Ther, 4: 217-222 (2001).
  • AAV is a standard abbreviation for adeno-associated virus.
  • Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus.
  • General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol.1, pp.169-228, and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York).
  • AAV vector refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs).
  • ITRs AAV terminal repeat sequences
  • AAV virion or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”. Thus, production of AAV vector particle necessarily includes production of AAV vector; as such a vector is contained within an AAV vector particle.
  • a heterologous polynucleotide i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell
  • Recombinant AAV genomes of the invention comprise nucleic acid molecule of the invention and one or more AAV ITRs flanking a nucleic acid molecule.
  • AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAVrh.74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV- 13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • DNA plasmids of the invention comprise rAAV genomes of the invention.
  • the DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El-deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles.
  • helper virus of AAV e.g., adenovirus, El-deleted adenovirus or herpesvirus
  • rAAV particles in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.
  • Methods of generating a packaging cell comprise creating a cell line that stably expresses all the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6.
  • the packaging cell line is then infected with a helper virus such as adenovirus.
  • a helper virus such as adenovirus.
  • packaging cells that produce infectious rAAV.
  • packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line).
  • packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).
  • Recombinant AAV (i.e., infectious encapsidated rAAV particles) of the invention comprise a rAAV genome.
  • the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes.
  • rAAV examples of rAAV that may be constructed to comprise the nucleic acid molecules of the invention are set out in International Patent Application No. PCT/US2012/047999 (WO 2013/016352) incorporated by reference herein in its entirety.
  • the rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No.6,566,118 and WO 98/09657.
  • compositions comprising rAAV of the present invention.
  • Compositions of the invention comprise rAAV and a pharmaceutically acceptable carrier.
  • the compositions may also comprise other ingredients such as diluents and adjuvants.
  • Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers and surfactants such as pluronics.
  • Titers of rAAV to be administered in methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art.
  • Titers of rAAV may range from about 1.times.10.sup.6, about 1.times.10.sup.7, about 1.times.10.sup.8, about 1.times.10.sup.9, about 1.times.10.sup.10, about 1.times.10.sup.11, about 1.times.10.sup.12, about 1.times.10.sup.13to about 1.times.10.sup.14 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg).
  • an isolated nucleic acid as described herein comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof and a second region comprising a transgene encoding PGM1.
  • the isolated nucleic acid e.g., the recombinant AAV vector
  • the transgene can also comprise a region encoding, for example, a protein and/or an expression control sequence (e.g., a poly-A tail).
  • vectors comprising a single, cis-acting wild-type ITR.
  • the ITR can be a 5’ ITR. In some aspects, the ITR can be a 3' ITR. ITR sequences are about 145 bp in length. In some aspects, the entire sequences encoding the ITR(s) can be used in the molecule, although some degree of minor modification of these sequences is permissible. In some aspects, an ITR can be mutated at its terminal resolution site (TR), which inhibits replication at the vector terminus where the TR has been mutated and results in the formation of a self-complementary AAV.
  • TR terminal resolution site
  • a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements can be flanked by the 5’ AAV ITR sequence and a 3' hairpin-forming RNA sequence can be used.
  • AAV ITR sequences can be obtained from any known AAV, including presently identified mammalian AAV types.
  • an ITR sequence can be an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, and/or AAVrh10 ITR sequence.
  • vector is an adeno-associated viral vector of a serotype 2 (AAV2), a serotype 6 (AAV6), or a serotype AAV-DJ.
  • serotype AAV2 can be AAV2.7m8.
  • the AAV serotype can have a capsid that is at least 95% identical to any of the AAV serotype capsids.
  • the AAV capsid can have the amino acid sequence: MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLG PFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTS FGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQ PARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADG VGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFG YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKT IANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNF
  • the vectors can comprise one or more of the polynucleotides disclosed herein.
  • the vectors disclosed herein can comprise the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 1 encoding the human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain and the expression cassette further comprising a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 3 encoding the human nuclear factor 1 X-type (hNFIX) protein.
  • the vectors can comprise one or more of the polynucleotides disclosed herein.
  • the vectors disclosed herein can comprise the expression cassette comprises a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 2 encoding the human recombination signal binding protein (hRBPJ) protein comprising a R216H point mutation and the expression cassette further comprising a transcriptional regulatory region comprising a promoter operatively linked to a nucleotide sequence as set forth in SEQ ID NO: 3 encoding the human nuclear factor 1 X-type (hNFIX) protein.
  • hRBPJ human recombination signal binding protein
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human recombination signal binding protein (hRBPJ) protein fused to a constitutive KRAB domain, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • hRBPJ human recombination signal binding protein
  • the nucleic acid sequence encoding hRBPJ having a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 1.
  • the expression cassettes can further comprise: a nucleic acid sequence capable of encoding the human nuclear factor 1 X-type (hNFIX) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human recombination signal binding protein (hRBPJ) protein operably linked to one or more regulatory elements; and a polyadenylation tail signal, wherein the nucleic acid sequence capable of encoding hRBPJ has a nucleic acid sequence of SEQ ID NO: 2.
  • the expression cassettes can further comprise: a nucleic acid sequence capable of encoding the human nuclear factor 1 X-type (hNFIX) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human recombination signal binding protein (hRBPJ) protein operably linked to one or more regulatory elements; and a polyadenylation tail signal, wherein the nucleic acid sequence encoding hRBPJ having a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 2.
  • the expression cassettes can further comprise: a nucleic acid sequence capable of encoding the human nuclear factor 1 X-type (hNFIX) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • the nucleic acid sequence is capable of encoding hRBPJ comprising a nucleic acid sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or a sequence reverse complementary thereto. In some aspects, the nucleic acid sequence is capable of encoding hNFIX comprising or consisting of the nucleic acid sequence of SEQ ID NO: 3 or a sequence reverse complementary thereto.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human nuclear factor 1 X-type (hNFIX) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • the nucleic acid sequence is capable of encoding hNFIX has a nucleic acid sequence of SEQ ID NO: 3.
  • the nucleic acid sequence capable of encoding hNFIX comprises a nucleic acid sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 3 or a sequence reverse complementary thereto.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence capable of encoding human Yes1 associated transcriptional regulator (hYAP) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • hYAP human Yes1 associated transcriptional regulator
  • the nucleic acid sequence capable of encoding hYAP having a sequence at least 90% identical to the sequence set forth in NCBI Reference Sequence: NC_000011.10.
  • the nucleic acid sequence encoding hYAP comprises a nucleic acid sequence having at least 85% identity to the nucleotide sequence of NCBI Reference Sequence: NC_000011.10 or a sequence reverse complementary thereto.
  • AAV vectors comprising an expression cassette comprising: a nucleic acid sequence encoding human octamer-binding transcription factor 4 (hOCT-4) protein, operably linked to one or more regulatory elements; and a polyadenylation tail signal.
  • the nucleic acid sequence encoding hOCT- 4 has a nucleic acid sequence of Gene ID: 5460.
  • the nucleic acid sequence encoding hOCT-4 comprises a nucleic acid sequence having at least 85% identity to the nucleotide sequence of Gene ID: 5460or a sequence reverse complementary thereto.
  • the one or more regulatory elements can be a chicken-beta-actin (CBA) promoter, EF1alpha promoter, or CAG promoter; and a WPRE sequence.
  • recombinant AAV vectors can comprise a polyadenylation tail signal.
  • polyadenylation tail signal can be a simian virus 40 (SV40) polyadenylation signal.
  • the recombinant AAV vectors disclosed herein can further comprise a Kozak sequence.
  • the recombinant AAV vector can be a serotype 2 (AAV2), a serotype 6 (AAV6), or a serotype AAV-DJ.
  • the recombinant AAV vector can have a capsid that is at least 95% identical to SEQ ID NO: 12.
  • the nucleic acid sequence capable of encoding hRBPJ can be flanked by inverted terminal repeat (ITR) nucleotide sequences.
  • the nucleic acid sequence encoding hNFIX can be flanked by inverted terminal repeat (ITR) nucleotide sequences.
  • the nucleic acid sequence capable of encoding hYAP can be flanked by inverted terminal repeat (ITR) nucleotide sequences.
  • the nucleic acid sequence capable of encoding hOCT-4 can be flanked by inverted terminal repeat (ITR) nucleotide sequences.
  • the ITRs can comprise a 5’ ITR having a nucleotide sequence of SEQ ID NO: 13 and a 3’ ITR having a nucleotide sequence of SEQ ID NO: 18, or the reverse complement thereof.
  • the nucleic acid sequence capable of encoding hRBPJ can be flanked by inverted terminal repeat (ITR) nucleotide sequences.
  • the recombinant AAV vectors disclosed herein can further comprise a selectable marker.
  • components to be cultured in the host cell to package a rAAV vector in an AAV capsid can be provided to the host cell in trans.
  • any one or more of the required components e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • such a stable host cell can contain the required component(s) under the control of an inducible promoter.
  • the required component(s) can be under the control of a constitutive promoter.
  • a selected stable host cell can contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
  • the recombinant AAV vector, rep sequences, cap sequences, and helper functions useful for producing the rAAV described herein can be delivered to the packaging host cell using any appropriate genetic element (vector).
  • the selected genetic element can be delivered by any suitable method, including those described herein.
  • the methods used to construct any of compositions disclosed herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K.
  • recombinant AAVs can be produced using the triple transfection method (described in detail in U.S. Pat. No.6,001,650).
  • the recombinant AAVs can be produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector.
  • An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation.
  • the AAV helper function vector can support efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes).
  • vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No.6,001,650 and pRep6cap6 vector, described in U.S. Pat. No.6,156,303, the entirety of both incorporated by reference herein.
  • the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”).
  • the accessory functions include those functions required for AAV replication, including, without limitation, those moieties 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 any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. Table 1.
  • This marker product can be used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Marker genes can include, but are not limited to the E. coli lacZ gene, which encodes ß-galactosidase, and the gene encoding the green fluorescent protein.
  • the marker may be a selectable marker.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes.
  • the first category is based on a cell’s metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media.
  • Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • dominant selection refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet.1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell.
  • the three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.
  • METHODS Disclosed herein are methods for the converting Muller glial cells into retinal neurons in a subject in need thereof.
  • the methods can comprise administering to the subject, one or more of the polynucleotides disclosed herein, one or more of the vectors disclosed herein or any of the pharmaceutical compositions disclosed herein.
  • the retinal neurons can be retinal bipolar cells or amacrine cells.
  • the methods can comprise administering to the subject, one or more of the polynucleotides disclosed herein, one or more of the vectors disclosed herein or any of the pharmaceutical compositions disclosed herein.
  • the retinal neurons can be retinal bipolar cells or amacrine cells.
  • the degenerative retinal disease can be macular degeneration, retinitis pigmentosa, glaucoma, Leber’s congenital amaurosis, Stargardt disease, or optic neuropathy.
  • the methods can comprise administering to the subject any of the pharmaceutical compositions disclosed herein.
  • the retinal neurons can be retinal bipolar cells or amacrine cells.
  • the methods can comprise administering to the subject, one or more of the polynucleotides disclosed herein, one or more of the vectors disclosed herein or any of the pharmaceutical compositions disclosed herein.
  • the degenerative retinal disease can be macular degeneration, retinitis pigmentosa, glaucoma, Leber’s congenital amaurosis, Stargardt disease, or optic neuropathy.
  • the methods can comprise the steps of: (i) providing a cell comprising any of the polynucleotides disclosed herein, AAV cap proteins, AAV rep proteins and, optionally, viral proteins upon which AAV is dependent for replication, (ii) maintaining the cell under conditions adequate for assembly of the AAV; and (iii) purifying the adeno-associated viral vector produced by the cell.
  • the method comprising administering to the subject a therapeutically effective amount of any of the recombinant AAV vectors herein or any of the pharmaceutical compositions disclosed herein.
  • the retinal neurons can be retinal bipolar cells or amacrine cells.
  • the methods can comprise administering to the subject a therapeutically effective amount of any of the recombinant AAV vectors disclosed herein or any of the pharmaceutical compositions disclosed herein, wherein the degenerative retinal disease is macular degeneration, retinitis pigmentosa, glaucoma, Leber’s congenital amaurosis, Stargardt disease, or optic neuropathy.
  • the administering can be intravitreal.
  • the methods can comprise inhibiting Notch signaling in a Muller glial cell in the subject.
  • the Notch signaling can be Notch 1 and/or Notch 2 signaling.
  • the Notch signaling can be inhibited by directly inhibiting Rbpj.
  • the inhibiting of Rbpj can be performed with an Rbpj inhibitor.
  • the Rbpj inhibitor can be a nucleic acid, a small molecule, a peptide, or an antibody.
  • the Rbpj inhibitor can be RIN1 or CB103.
  • the methods can further comprise overexpressing Yap and/or Oct4 in the Muller glial cell in the subject.
  • the methods can comprise inhibiting Notch signaling in a Muller glial cell in the subject.
  • the Notch signaling can be Notch 1 and/or Notch 2 signaling.
  • the Notch signaling can be inhibited by directly inhibiting Rbpj.
  • the Notch signaling can be inhibited by directly inhibiting Rbpj.
  • the inhibiting of Rbpj can be performed with an Rbpj inhibitor.
  • the Rbpj inhibitor can be a nucleic acid, a small molecule, a peptide, or an antibody.
  • the Rbpj inhibitor can be RIN1 or CB103.
  • the methods can further comprise overexpressing Yap and/or Oct4 in the Muller glial cell in the subject.
  • the methods can further comprise administering a Yap agonist to the subject or contacting the Muller glial cell with a Yap agonist.
  • the Yap agonist can be a large tumor suppressor (LATS) kinase inhibitor or a Yap-Tead complex activator.
  • LATS large tumor suppressor
  • the large tumor suppressor (LATS) kinase inhibitor can be GA-017, TRULI (Lats-IN-1) or TDI 011536.
  • the Yap-Tead complex activator can be TT-10 (TAZ-K).
  • the methods can further comprise overexpressing of Yap in the Muller glial cell by viral-mediated overexpression of dominant-active Yap in the Muller glial cell.
  • overexpressing Yap in the Muller glial cell of the subject can be carried out by administering a vector comprising a nucleic acid sequences capable of encoding a dominant-active Yap.
  • the methods can further comprise overexpressing Oct4 in the Muller glial cell by viral-mediated overexpression of Oct4 in the Muller glial cell.
  • overexpressing Oct4 in the Muller glial cell of the subject can be carried out by administering a vector comprising a nucleic acid sequences capable of encoding Oct4.
  • the viral-mediated overexpression can be AAV-mediated overexpression.
  • methods can increase or enhance neurogenesis in the Muller glial cell in the subject.
  • the subject has a retinal injury or dystrophy.
  • the retinal injury or dystrophy can be macular degeneration, retinitis pigmintosa, Leber’s congenital amarosis, stargat disease, glycoma, or optic neuropathy.
  • the subject does not have a retinal injury or dystrophy.
  • the methods can further comprise inhibiting Nfia/b/x in the Muller glial cell in the subject.
  • the Nfia/b/x inhibitors can be an Nfia/b/x antagonist.
  • the step of inhibiting Nfia/b/x in the Muller glial cell can be performed using CRISPR.
  • the methods can further comprise overexpressing Atoh7 and/or Neurog2 and/or Foxn4 and/or Insm1 in the Muller glial cell.
  • overexpressing Atoh7 and/or Neurog2 and/or Foxn4 and/or Insm1 in the Muller glial cell can lead or result in an increase in neurogenesis.
  • the methods of overexpressing Atoh7 and/or Neurog2 and/or Foxn4 and/or Insm1 in the Muller glial cell can accomplished by exogenous delivery.
  • exogenous delivery can be genetic (e.g., nucleic acid or peptide based).
  • the rAAV encoding any of the protein disclosed herein can be administered by any method known in the art.
  • the rAAV can delivered by intravitreal administration.
  • methods for delivering a transgene to heart tissue in a subject can comprise co-administering of an effective amount of a rAAV by two different administration routes, e.g., by intracardiac administration and by intravenous administration. Co-administration of the rAAV can be performed at approximately the same time, or different times.
  • the dose of rAAV to be administered in methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art.
  • Titers of each rAAV administered may range from about 1x10 6 , about 1x10 7 , about 1x10 8 , about 1x10 9 , about 1x10 10 , about 1x10 11 , about 1x10 12 , about 1x10 13 , about 1x10 14 , or to about 1x10 15 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., 1x10 7 vg, 1x10 8 vg, 1x10 9 vg, 1x10 10 vg, 1x10 11 vg, 1x10 12 vg, 1x10 13 vg, 1x10 14 vg, 1x10 15 , respectively).
  • vg viral genomes
  • Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of bodyweight (i.e., 1x10 10 vg/kg, 1x10 11 vg/kg, 1x10 12 vg/kg, 1x10 13 vg/kg, 1x10 14 vg/kg, 1x10 15 vg/kg respectively).
  • bodyweight i.e., 1x10 10 vg/kg, 1x10 11 vg/kg, 1x10 12 vg/kg, 1x10 13 vg/kg, 1x10 14 vg/kg, 1x10 15 vg/kg respectively.
  • Methods for titering AAV are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).
  • the composition e.g., a pharmaceutical composition
  • compositions comprising a recombinant AAV comprising at least one modified genetic regulatory sequence or element can further comprise a pharmaceutically acceptable carrier.
  • suitable carriers can be selected for the indication for which the rAAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • buffering solutions e.g., phosphate buffered saline
  • suitable carriers include but are not limited to sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • compositions disclosed herein can also include, in addition to the rAAV and carrier(s), other pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • the rAAV can be administered in a pharmaceutical composition comprising phosphate buffered saline (PBS), pH 7.3 and 0.001% of a pharmaceutically acceptable non-ionic surfactant, such as, for example, pluronic F-68 (PF68), or other appropriate pharmaceutically acceptable buffers or excipients.
  • PBS phosphate buffered saline
  • PF68 pluronic F-68
  • the formulation may be frozen until ready for use and then thawed and administered.
  • the compositions disclosed herein can comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes).
  • a composition can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.
  • rAAVs can be administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., injection into the liver, skeletal muscle, heart), oral, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration.
  • the route of administration can be by direct injection.
  • the route of administration can be by intravenous delivery. Routes of administration can be combined, if desired.
  • the dose of rAAV virions required to achieve a particular “therapeutic effect,” e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), the units of dose in genome copies per heart volume, will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.
  • a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.
  • An effective amount of a rAAV is an amount sufficient to target infect an animal, target a desired tissue.
  • the effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue.
  • an effective amount of the rAAV can be in the range from about 1 ml to about 100 ml of solution containing from about 10 6 to 10 16 genome copies (e.g., from 1 x 10 6 to 1 x 10 16 , inclusive).
  • the therapeutically effective dose is between 6X10 13 gc/kg to 6X10 14 gc/kg, including 7X10 13 gc/kg, 8X10 13 gc/kg, 9X10 13 gc/kg, 1X10 14 gc/kg, 2X10 14 gc/kg, 3X10 14 gc/kg, 4X10 14 gc/kg, or 5X10 14 gc/kg (or alternatively, genome copies per heart volume or other measurement appropriate for intracardiac delivery).
  • a dosage between about 10 11 to 10 12 per kg or appropriate measurement rAAV genome copies can be appropriate.
  • a dosage of between about 10 11 to 10 13 per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of between about 10 11 to 10 14 per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of between about 10 11 to 10 15 per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of about 1 x 10 14 vector genome (vg) copies per kg or appropriate measurement can be appropriate. In some aspects, the dosage can vary or be reduced when specifically targeting one or more organs. In some aspects, a dosage between about 10 7 to 10 8 rAAV genome copies per kg or appropriate measurement can be appropriate.
  • a dosage of between about 10 8 to 10 9 rAAV genome copies per kg or appropriate measurement can be appropriate. In some aspects, a dosage of between about 10 9 to 10 10 rAAV genome copies per kg or appropriate measurement can be appropriate. In some aspects, a dosage of between about 10 10 to 10 11 rAAV genome copies per kg or other appropriate measurement can be appropriate.
  • a potential side-effect for administering an AAV to a subject can be an immune response in the subject to the AAV, including inflammation, and, and may depend on the route of administration, and in particularly, when the administration of an AAV is systemic. In some aspects, a subject can be immunosuppressed prior to administration of one or more rAAVs as described herein.
  • immunosuppressed or “immunosuppression” refers to a decrease in the activation or efficacy of an immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., multiple, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, methotrexate, and any combination thereof.
  • agents including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, methotrexate, and any combination thereof.
  • methods disclosed herein can further comprise the step of inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered a rAAV (e.g., an rAAV or pharmaceutical composition as disclosed herein).
  • a subject can be immunosuppressed (e.g., immunosuppression is induced in the subject) between about 30 days and about 0 days (e.g., any time between 30 days until administration of the rAAV, inclusive) prior to administration of the rAAV to the subject.
  • the subject can be pretreated with immune suppression agent (e.g., rituximab, sirolimus, and/or prednisone) for at least 7 days.
  • immune suppression agent e.g., rituximab, sirolimus, and/or prednisone
  • a subject can be immunosuppressed (e.g., administered one or more immunosuppressants) for between 1 day and 1 year after administration of the rAAV or pharmaceutical composition.
  • rAAV compositions can be formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., —10 13 GC/ml or more).
  • Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions are well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
  • these formulations can contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and can be conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of active compound in each therapeutically-useful composition can be prepared in such a way that a suitable dosage can be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations can be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens can be desirable.
  • rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions as disclosed herein either subcutaneously, intrapancreatically, intranasally, intracardiacally, parenterally, intravenously, intramuscularly, or orally, intraperitoneally, by inhalation, intravitreally.
  • the administration modalities as described in U.S. Pat. Nos.5,543,158; 5,641,515 and 5,399,363 can be used to deliver rAAVs.
  • a preferred mode of administration can be intravenous delivery.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form can be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and
  • isotonic agents for example, sugars or sodium chloride can be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art.
  • one dosage can be dissolved in 1 ml of isotonic NaC1 solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • the rAAV is formulated in phosphate buffered saline (PBS) at pH 7.3, including 0.001% of a pharmaceutically acceptable non-ionic surfactant, such as, for example, PF68.
  • PBS phosphate buffered saline
  • PF68 phosphate buffered saline
  • Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
  • Sterile injectable solutions can be prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the rAAV compositions disclosed herein can be also be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which can be formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium
  • compositions can be easily administered in a variety of dosage forms such as injectable solutions, drug- release capsules, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the rAAV vector delivered transgenes can be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations can be used for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
  • the formation and use of liposomes is generally known to those of skill in the art.
  • liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No.5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos.5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals.
  • Liposomes can be formed from phospholipids that can be dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Angstroms, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • nanocapsule formulations of the rAAV can be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 p.m.) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • the methods can include administering one or more additional therapeutic agents to a subject who has been administered an rAAV or pharmaceutical composition as described herein.
  • rAAV rAAV
  • administration of the rAAV described herein to a human subject suffering from a retinal injury, disease or dystrophy will within 1 week, 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks or 1 year after the administration will result in reduction in one or more biomarkers or hallmarks of the disease or injury.
  • “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that can be undetectable. As used herein the terms development or progression refer to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.
  • the subject can be a human, a mouse, a rat, a pig, a dog, a cat, or a non-human primate.
  • a subject has or is suspected of having a retinal disease or injury.
  • the rAAVs disclosed herein can be administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
  • Pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., to the heart), oral, inhalation (including intranasal and intratracheal delivery), intravenous, intramuscular, subcutaneous, intradermal, intravitreally, and other parental routes of administration.
  • the dose of rAAV virions required to achieve a particular “therapeutic effect,” e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), can vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.
  • a rAAV virion dose range to treat a patient having a particular retinal disease or injury based on the aforementioned factors, as well as other factors that are well known in the art.
  • an effective amount of an rAAV is an amount sufficient to target infect a subject or target a desired tissue.
  • an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model.
  • the effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue.
  • an effective amount of the rAAV can be in the range of from about 1 ml to about 100 ml of solution containing from about 10 9 to 10 16 genome copies.
  • the effective amount of rAAV can be 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 genome copies per kg.
  • the effective amount of rAAV can be 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some cases, a dosage between about 6X10 09 to 6X10 14 rAAV genome copies can be appropriate.
  • rAAV compositions can be formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., —10 13 GC/ml or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc.
  • kits comprising any of the agents described herein.
  • any of the agents disclosed herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications.
  • a kit can include one or more containers housing the components of the disclosure and instructions for use.
  • kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents.
  • the agents in a kit can be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • Kits for research purposes can contain the components in appropriate concentrations or quantities for running various experiments.
  • kits for producing a rAAV can comprise a container housing an isolated nucleic acid encoding any of the proteins disclosed herein or a portion thereof.
  • the kits can further comprise instructions for producing the rAAV.
  • the kit further comprises at least one container housing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene.
  • the kits can comprise a container housing a recombinant AAV as described supra.
  • the kits can further comprises a container housing a pharmaceutically acceptable carrier.
  • a kit can comprise one container housing a rAAV and a second container housing a buffer suitable for injection of the rAAV into a subject.
  • the container can be a syringe.
  • the kits can be designed to facilitate use of the methods described herein by researchers and can take many forms.
  • compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • some of the compositions can be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure.
  • Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions can be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions can be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.
  • the kits disclosed herein can also contain any one or more of the components described herein in one or more containers.
  • the kits can include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kits can include a container housing agents described herein.
  • the agents can be in the form of a liquid, gel or solid (powder).
  • the agents can be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it can be housed in a vial or other container for storage. A second container can have other agents prepared sterilely.
  • the kits can include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • the kits can have one or more or all of the components required to administer the agents to an animal, such as a syringe, topical application devices, or iv needle tubing and bag, particularly in the case of the kits for producing specific somatic animal models.
  • the method disclosed herein can involve transfecting cells with total cellular DNAs isolated from the tissues that potentially harbor proviral AAV genomes at very low abundance and supplementing with helper virus function (e.g., adenovirus) to trigger and/or boost AAV rep and cap gene transcription in the transfected cell.
  • helper virus function e.g., adenovirus
  • RNA from the transfected cells can provide a template for RT-PCR amplification of cDNA and the detection of novel AAVs.
  • the cells can also be infected with a helper virus, such as an Adenovirus or a Herpes Virus.
  • a helper virus such as an Adenovirus or a Herpes Virus.
  • the helper functions can be provided by an adenovirus.
  • the adenovirus can be a wild-type adenovirus, and can be of human or non-human origin, for example, non-human primate (NHP) origin.
  • adenoviruses known to infect non- human animals e.g., chimpanzees, mouse
  • can also be employed in the methods of the disclosure See, e.g., U.S. Pat. No.6,083,716).
  • recombinant viruses or non-viral vectors e.g., plasmids, episomes, etc.
  • recombinant viruses are known in the art and may be prepared according to published techniques. See, e.g., U.S. Pat. No.5,871,982 and U.S. Pat. No.6,251,677, which describe a hybrid Ad/AAV virus.
  • a variety of adenovirus strains are available from the American Type Culture Collection, Manassas, Va., or available by request from a variety of commercial and institutional sources.
  • sequences of many such strains are available from a variety of databases including, e.g., PubMed and GenBank.
  • Cells can also be transfected with a vector (e.g., helper vector) which provides helper functions to the AAV.
  • the vector providing helper functions can provide adenovirus functions, including, e.g., Ela, E lb, E2a, E4ORF6.
  • the sequences of adenovirus gene providing these functions can be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types known in the art.
  • the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging.
  • an isolated capsid gene can be used to construct and package recombinant AAV vectors, using methods well known in the art, to determine functional characteristics associated with the novel capsid protein encoded by the gene.
  • isolated capsid genes can be used to construct and package recombinant AAV (rAAV) vectors comprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase, etc.).
  • the rAAV vector can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues of the animal.
  • an animal e.g., mouse
  • Other methods for characterizing isolated capsid genes are disclosed herein and still others are well known in the art.
  • the kits disclosed can have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag.
  • the kits can be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
  • kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.
  • the kits can also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
  • the instructions included within the kit can involve methods for detecting a latent AAV in a cell.
  • kits of the disclosure can include, instructions, a negative and/or positive control, containers, diluents and buffers for the sample, sample preparation tubes and a printed or electronic table of reference AAV sequence for sequence comparisons.
  • Example 1 Intravitreal Injection of Adeno-Associated Virus Cloning, production and intravitreal injection of Adeno-Associated Virus.
  • the Addgene #50473 construct comprising a GFAP promoter was used for overexpression of the mCherry control insert, full-length Insm1, and full-length Atoh7 and Neurog2.
  • the EF1a minipromoter was used to drive expression of Cre.
  • the EGFP sequence was replaced by the mCherry sequence.
  • the P2A and T2A ribosomal self-cleaving peptides are used to simultaneously express the transcription factor(s) 5’ to the mCherry reporter as a single polypeptide, which is then cleaved to generate the transcription factor and mCherry. Coding sequences of different transcription factors were synthesized by GeneWiz. AAV constructs were packaged into AAV2.7m8 by Boston Children’s Hospital Viral Core. Two months old GLASTCreERT2; Sun1-GFP mice first subject to 4-OHT i.p induction (4 once-daily intraperitoneal injections of 4-OHT at 1mg/dose in corn oil) to selectively label Muller glia cells with Sun1-GFP.
  • retinas were were intravitreally injected with GFAP AAV constructs using a microsyringe with a 33G blunt-ended needle.
  • the following constructs were tested: GFAP-mCherry, GFAP-Insm1- P2A-mCherry, GFAP-Atoh7-T2A-Ascl1-P2A-mCherry, and EF1a-DIO-Cre-T2A-mCherry.
  • Retinas were collected 21 days later for analysis. Fixation, sectioning, immunohistochemistry and imaging. Collection and immunohistochemical analysis of retinas was performed (Hoang et al.2020).
  • Retinas were dissected in 1x PBS and incubated in 30% sucrose overnight at 4°C. Retinas were then embedded in OCT (VWR, #95057-838), cryosectioned at 16 ⁇ m thickness, and stored at -20°C.
  • Sections were dried for 30 mins in a 37°C incubator and washed 3x5 min with 0.1% TritonX-100 in PBS (PBST) and incubated in 10% Horse Serum (ThermoFisher, #26050070), 0.4% TritonX-100 in 1x PBS (blocking buffer) for 2 h at room temperature (RT). Sections were then incubated with primary antibodies in the blocking buffer overnight at 4°C.
  • PBST TritonX-100 in PBS
  • RT room temperature
  • FIG.1A shows the schematic of generation of Muller glia-specific loss of function mutant of Rbpj, and analysis of gene expression in Rbpj-deficient glia.
  • FIG.1B shows ScRNA-Seq analysis revealing that Rbpj-deficient Muller glia show reduced expression of genes specific to resting glia and induction of genes specific to neurogenic retinal progenitors.
  • FIG.1C shows a UMAP plot of cells following analysis of retina in whole control and Rbpj-deficient retina.
  • FIG.1D shows a UMAP plot of expression of the neurogenic bHLH factors Ascl1 and Neurog2 in control and Rbpj-deficient Muller glia.
  • FIG.2A shows that a limited number of Sun1-GFP-positive cells are labeled with the amacrine/retinal ganglion cell marker HuC/D four weeks following tamoxifen induction (inset shows high magnification image).
  • FIG.2B shows the quantification of the relative fraction of Sun1-GFP-positive cells that express either HuC/D or the bipolar cell marker Otx2 in uninjured and NMDA-treated retina.
  • FIG.2C shows representative images Rpbj-deficient retinas stained with Otx2, HuC/D and the amacrine cell-specific marker glycine.
  • FIG.3A is a schematic showing injection and analysis of AAV overexpression constructs.
  • FIG.3B shows representative immunohistochemical images showing lack of co- expression of neuronal markers in Gfap-mCherry control viruses, but induction of both bipolar and amacrine cell-specific markers following Muller glia-specific overexpression of Insm1 and Atoh7/Neurog2.
  • FIG.4 shows the results of reprogramming mammalian Muller glia for retinal repair.
  • FIG.5 shows integrated regulatory network analysis (IReNA).
  • IReNA integrated regulatory network analysis
  • FIG.6 shows that the loss of Rbpj leads to injury-independent mouse Muller glia reprogramming.
  • Example 2 Efficient Induction of Proliferation and Neurogenesis in Mammalian Muller Glia By Combined Suppression of Notch Signaling and Overexpression of Oct4 and Activated Yap
  • the common Notch transcriptional mediator Rbpj was selectively deleted in adult mouse Muller glia while simultaneously tracing the fate of Muller glia-derived cells.
  • Rbpj Loss of function of Rbpj results in global inhibition of Notch signaling, and induces a limited level of injury-induced glia-to-neuron conversion when disrupted in CNS astrocytes.
  • w Rbpj was selectively deleted in Muller glia using GlastCreER T2 ;Rbpj lox/lox ;Sun1-GFP mice (FIG.7A).
  • mice express a tamoxifen- inducible Cre recombinase under the control of regulatory elements of the Muller glia- specific Glast (Slc1a3) gene, which in turn induces both Cre-dependent removal of exon 4 of Rbpj and generates a functional null mutation, and also induces expression of Sun1-GFP, which permanently labels the nuclear membrane of the Muller glia and Muller glia-derived cells.
  • Cre recombination was induced in both GlastCreER T2 ;Rbpj lox/lox ;Sun1-GFP and GlastCreER T2 ;Sun1-GFP controls with five daily intraperitoneal injections of tamoxifen beginning at postnatal day (P) 21, and collected retinas at multiple timepoints ranging from 3 weeks to 6 months post tamoxifen injection (FIG.7B), and performed immunostaining for cell type-specific markers. From 6 weeks onwards, a progressive increase in the number of Muller glia-derived neurons, as measured by coexpression of GFP and Otx2, was observed.
  • Muller glia-derived amacrine cells A progressive increase in generation of Muller glia-derived amacrine cells, as measured by coexpression of GFP and HuC/D, was also observed (FIG.7C-D).
  • ⁇ 15% of GFP+ cells expressed Otx2, while ⁇ 3.5% expressed HuC/D (FIG.7D).
  • Muller glia-derived neurons expressed some cone bipolar-specific markers such as Scgn by 4 months post-injection, but did not express rod bipolar markers such as Prka.
  • rod bipolar markers such as Prka.
  • No GFP+ cells expressing rod photoreceptor-specific markers such as Nrl or retinal ganglion such as Brn3b were observed, and GFP+ cells were confined to the inner nuclear layer (INL).
  • NMDA N-methyl-d-aspartate
  • Muller glia showed levels of Muller glia-derived bipolar cells that were not significantly different from those seen in age- matched Rbpj-deficient Muller glia (FIG.9D). Numbers of Muller glia-derived amacrine cells, however, were significantly lower in Notch1/2 double mutants relative to Rbpj mutants (FIG.9D). This demonstrates that Notch1/2 loss of function effectively phenocopies Rbpj loss of function.
  • GFP-positive Muller glia were isolated at P33 and analyzed using antibodies to RBPJ, with the activating histone modification H3K4me3 used as a positive control and IgG used as a negative control (FIG.11A).
  • RBPJ directly bound to cis-regulatory sites associated with Notch pathway genes, including Hes1, Hes5, and Hey2 (FIG.11B), as well as transcription factors that promote gliogenesis such as Tcf7l2.
  • Rbpj also directly bound to regulatory sites associated with some genes associated with cell cycle inhibition, including Btg2, but did not directly regulate Ascl1, Neurog2, or Hes6.
  • Notch pathway genes such as Hes1, Hes5, and Hey2 remain expressed in Nfia/b/x- deficient Muller glia, and that Nfia/b/x do not directly regulate Notch pathway genes. Furthermore, expression of Nfia/b/x is largely unaffected in Rbpj-deficient Muller glia (FIG.10), and Rbpj does not directly regulate expression of Nfia/b/x (FIG.11A). This raises the possibility that Notch signaling and NFI factors may act in parallel to inhibit neurogenic competence in adult Muller glia.
  • GFAP miniproter- based AAV constructs show ectopic neuronal expression when used to express transcription factors
  • GFAP-Oct4-mCherry constructs show generally Muller glia-specific expression.
  • scRNA-Seq was performed on FACS- isolated GFP+ cells from GlastCreER T2 ;Rbpj lox/lox ;Sun1-GFP infected with either AAV- GFAP-Oct4-mCherry or AAV- GFAP-mCherry control virus (FIG.13D). Consistent with the results of immunostaining, it was observed that Oct4 overexpression substantially increases the relative fraction of Muller glia-derived bipolar cells (FIG.13E). The relative fraction of resting Muller glia was significantly reduced, and expression of neurogenic bHLH genes was upregulated (Fig.7F, 7G).
  • mice Mice were raised and housed in a climate-controlled pathogen-free facility on a 14/10 h light/dark cycle. Mice used in this study were GlastCreER T2 ;Sun1- GFP, which were generated by crossing the GlastCreER T2 and Sun1-GFP lines (de Melo et al., 2012; Mo et al., 2015).
  • GlastCreER T2 ;Rbpj lox/lox ;Sun1-GFP mice were generated by crossing GlastCreER T2 ;Sun1GFP with conditional Rbpj lox/lox mice (Jackson Laboratories).
  • GlastCreER T2 ;Notch1 lox/lox ;Notch2 lox/lox ;Sun1-GFP mice were obtained by crossing GlastCreER T2 ;Notch1 lox/lox ;Sun1-GFP and GlastCreER T2 ;Notch2 lox/lox ;Sun1-GFP mice.
  • mice at ⁇ 3 weeks of age were intraperitoneally injected with tamoxifen (Sigma-Aldrich, #H6278- 50mg) in corn oil (Sigma-Aldrich, #C8267-500ML) at 1.5 mg/dose for five consecutive days.
  • tamoxifen Sigma-Aldrich, #H6278- 50mg
  • corn oil Sigma-Aldrich, #C8267-500ML
  • Cloning and Production of Adeno-Associated Virus The Addgene #50473 construct which contains a GFAP promoter was used in this study.
  • the EGFP sequence was replaced by the mCherry sequence.
  • the T2A ribosomal self-cleaving peptides are used to simultaneously express the transcription factor(s) 5’ to the mCherry reporter as a single polypeptide, which is then cleaved to generate the transcription factor and mCherry. Coding sequences of different transcription factors were synthesized by GeneWiz. AAV constructs were packaged into AAV2.7m8 by Boston Children’s Hospital Viral Core. GFAP AAV Delivery.
  • TAM Trigger Activated Factor AAV transduction
  • 5 daily doses of TAM 1.5mg/dose in corn oil
  • TAM Trigger Activated Factor AAV transduction
  • Titre and injection volume for each construct are listed below: Immunohistochemistry and imaging. Collection and immunohistochemical analysis of retinas was performed as described previously (Hoang et al., 2020). Briefly, mouse eye globes were fixed in 4% paraformaldehyde for 4 h at 4°C. Retinas were then embedded in OCT (VWR, #95057-838), cryosectioned at 16 ⁇ m thickness, and stored at -20°C.
  • Sections were dried for 30 min in a 37°C incubator and washed 3 x 5 min with 0.1% TritonX-100 in PBS (PBST) and incubated in 10% Horse Serum (ThermoFisher, #26050070), 0.4% TritonX- 100 in 1x PBS (blocking buffer) for 2 h at room temperature (RT). Sections were then incubated with primary antibodies in the blocking buffer overnight at 4°C.
  • PBST TritonX-100 in PBS
  • RT room temperature
  • Sections were then counterstained with DAPI in PBST, washed 4 ⁇ 5 min in PBST and mounted with ProLong Gold Antifade Mountant (Invitrogen, #P36935) under coverslips, air-dried, and stored at 4°C. Fluorescent images were captured using a Zeiss LSM 700 confocal microscope. Cell quantification and statistical analysis. Retinal cell dissociation. The retinas were dissected in fresh ice-cold PBS and retinal cells were dissociated using an optimized protocol (Fadl et al., 2020). Each sample contain a minimum of 4 retinas from 4 animals of both sex. Dissociated cells were resuspended in ice-cold HBAG Buffer.
  • scRNA-seq Single-cell RNA-sequencing

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Abstract

L'invention concerne des compositions et des méthodes utiles dans la conversion de la glie rétinienne en photorécepteurs ou en cellules ganglionnaires de la rétine.
PCT/US2023/071472 2022-08-01 2023-08-01 Compositions et méthodes de modification de cellules gliales de müller WO2024030930A2 (fr)

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