WO2024129992A1 - Activateurs entraînant l'expression de motoneurones moteurs - Google Patents

Activateurs entraînant l'expression de motoneurones moteurs Download PDF

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WO2024129992A1
WO2024129992A1 PCT/US2023/084062 US2023084062W WO2024129992A1 WO 2024129992 A1 WO2024129992 A1 WO 2024129992A1 US 2023084062 W US2023084062 W US 2023084062W WO 2024129992 A1 WO2024129992 A1 WO 2024129992A1
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seq
nucleic acid
promoter
protein
family
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Sinisa HRVATIN
Michael E. Greenberg
Mark Aurel NAGY
Eric C. Griffith
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Harvard University
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Harvard University
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • compositions related to regulatory elements such as elements directing cell type specific expression.
  • SMA Spinal muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • MNs spinal motor neurons
  • SMA resulting from loss-of-function mutations in the SMN1 gene, represents a particularly appealing candidate for gene therapy-based interventions, and an adeno-associated virus (AAV)-based treatment to restore SMN1 expression was recently reported to improve motor function in an early-stage single-site clinical trial.
  • AAV adeno-associated virus
  • the present disclosure provides methods and compositions for generating cell-type-specific AAV drivers, to generate novel AAVs capable of driving restricted gene expression within spinal cord MNs.
  • the resulting viral constructs will represent promising candidates for the basis of next-generation motor neuron disease or disorder (e.g. , SMA and ALS) gene therapeutics.
  • next-generation motor neuron disease or disorder e.g. , SMA and ALS
  • the present invention provides a nucleic acid comprising a regulatory element sequence that has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81- 83 or a fragment thereof.
  • the regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises at least one additional regulatory element sequence. In some embodiments, the nucleic acid comprises at least two, three, four, five or six additional regulatory element sequences. In some embodiments, the at least one additional regulatory element sequence has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83.
  • the at least one additional regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the at least one additional regulatory sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises two or more identical copies of a regulatory element sequence selected from the group consisting of SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83. In some embodiments, the nucleic acid comprises two, three, four, five or six identical copies of the regulatory element sequence.
  • the nucleic acid comprises at least two or more versions of a regulatory element sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, wherein the first version of the regulatory element sequence differs from the second version of the regulatory element sequence.
  • the regulatory element sequence comprises SEQ ID NO: 81.
  • the regulatory element sequence comprises SEQ ID NO: 11.
  • the regulatory element sequence comprises SEQ ID NO: 83.
  • the nucleic acid comprises three copies of SEQ ID NO: 81.
  • the nucleic acid comprises three copies of SEQ ID NO: 11.
  • the nucleic acid comprises three copies of SEQ ID NO: 83. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid further comprises a heterologous gene. In some embodiments, the regulatory element comprises SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises a sequence that is at least 500 nucleotides, at least 400 nucleotides, at least 350 nucleotides, at least 300 nucleotides, at least 250 nucleotides, at least 200 nucleotides, at least 150 nucleotides, at least 100 nucleotides, at least 50 nucleotides, or at least 25 nucleotides.
  • the regulatory element comprises one or more transcription factor binding sites.
  • the one or more transcription factor binding sites is selected from the group consisting of a binding site for Lhx3 (TTAATTAG), a binding site for Lhx4 (SEQ ID NO: 16), a binding site for Mnx1 (TTAATTAA), a binding site for Isl2 (GCACTTAA), a binding site for RREB1 (SEQ ID NO: 19), a binding site for STAT4 (SEQ ID NO: 20), a binding site for Esrrb (SEQ ID NO: 21), a binding site for Myb (AACTGCCA), and a combination thereof.
  • the heterologous gene is naturally expressed in a neuron.
  • the neuron is selected from the group consisting of a motor neuron, a sensory neuron, and an interneuron. In some embodiments, the neuron is a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron, but is not expressed in dorsal root ganglia.
  • the heterologous gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin
  • the heterologous gene is SMN1.
  • the SMN1 gene comprises the sequence set forth in SEQ ID NO: 25-28.
  • the SMN1 gene comprises the sequence set forth in SEQ ID NO: 28.
  • the heterologous gene is an inhibitory nucleic acid.
  • the inhibitory nucleic acid is a microRNA (miRNA), artificial microRNA (amiRNA), or short hairpin RNA (shRNA).
  • the inhibitory nucleic acid is a microRNA, wherein the microRNA binds to mRNA of a target gene.
  • the target gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin Mu DNA
  • the target gene is SOD1. In some embodiments, the SOD1 gene comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the target gene is C9orf72. In some embodiments, the C9orf72 gene comprises the sequence set forth in SEQ ID NO: 35, 36, or 37.
  • the promoter is a promoter from a gene expressed in motor neurons, optionally wherein the gene expressed in motor neurons is selected from the group consisting of a Dnajc22, Sycp1, Slit3, Hrasls5, Otop3, Ccnb3, Nlrp3, Hormad1, Chat, Anxa4, Tnfsf4, Myo3b, Cdh15, Nr2e1, Il17f, Apela, Gnb3, Pappa, Tmprss15, Crp, Nxpe5, Tex21, Ttc24, Ttc23l, Ahnak2, Vipr2, Gstt4, Aox3, Plac8l1, Grin3b, Adam2, Col5a1, Clca3a1, Serpinb7, Edn2, Mgarp, Atp12a, Lhx4, Pip5kl1, Slc25a48, Tfcp2l1, Clec18a, Spint2, Il22ra1, Galp, Mei1, Aox1, Prph,
  • the gene expressed in motor neurons is ChAT, Slc5a7, Isl1, Mnx1, Lhx3, or Lhx4.
  • the promoter is selected from the group consisting of beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55), a choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23), CAG promoter (pCAG) (optionally comprising SEQ ID NO: 24 or 57), minimal CMV promoter, human synapsin promoter, chicken beta actin, PGK promoter, Efla promoter, ubiquitin promoter, a TATA-box containing promoter, Slc5a7 promoter, Isl1 promoter, Mnx1 promoter, Lhx3 promoter, Lhx4 promoter, and variants thereof.
  • the promoter comprises the beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55). In some embodiments, the promoter comprises the choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23). In some embodiments, the promoter comprises the minimal CMV promoter. In some embodiments, the nucleic acid comprises SEQ ID NOs: 84, 85 or 86. In some embodiments, the nucleic acid is associated with an adeno-associated virus comprising a capsid that crosses the blood brain barrier and/or the blood spinal cord barrier.
  • the adeno-associated virus comprising a capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh.10, AAV1 R6, AAV1 R7, rAAVrh.8, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, and AAV- PHP.eB.
  • the present invention provides a vector comprising the nucleic acid of the above aspects or any other aspect of the invention delineated herein.
  • the vector is a viral vector.
  • the viral vector is a recombinant adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • the present invention provides a recombinant adeno- associated viral (rAAV) vector comprising a regulatory element sequence that has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83 or a fragment thereof.
  • the regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification.
  • the nucleic acid comprises at least one additional regulatory element sequence. In some embodiments, the nucleic acid comprises at least two, three, four, five or six additional regulatory element sequences. In some embodiments, the at least one additional regulatory element sequence has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83.
  • the at least one additional regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the at least one additional regulatory sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises two or more identical copies of a regulatory element sequence selected from the group consisting of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the nucleic acid comprises two, three, four, five or six identical copies of the regulatory element sequence.
  • the nucleic acid comprises at least two or more versions of a regulatory element sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, wherein the first version of the regulatory element sequence differs from the second version of the regulatory element sequence.
  • the regulatory element sequence comprises SEQ ID NO: 81.
  • the regulatory element sequence comprises SEQ ID NO: 11.
  • the regulatory element sequence comprises SEQ ID NO: 83.
  • the nucleic acid comprises three copies of SEQ ID NO: 81.
  • the nucleic acid comprises three copies of SEQ ID NO: 11.
  • the nucleic acid comprises three copies of SEQ ID NO: 83. In some embodiments, the nucleic acid further comprises a heterologous gene. In some embodiments, the regulatory element comprises SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the regulatory element comprises a sequence that is at least 500 nucleotides, at least 400 nucleotides, at least 350 nucleotides, at least 300 nucleotides, at least 250 nucleotides, at least 200 nucleotides, at least 150 nucleotides, at least 100 nucleotides, at least 50 nucleotides, or at least 25 nucleotides.
  • the regulatory element comprises one or more transcription factor binding sites.
  • the one or more transcription factor binding sites is selected from the group consisting of a binding site for Lhx3 (TTAATTAG), a binding site for Lhx4 (SEQ ID NO: 16), a binding site for Mnx1 (TTAATTAA), a binding site for Isl2 (GCACTTAA), a binding site for RREB1 (SEQ ID NO: 19), a binding site for STAT4 (SEQ ID NO: 20), a binding site for Esrrb (SEQ ID NO: 21), a binding site for Myb (AACTGCCA), and a combination thereof.
  • the heterologous gene is naturally expressed in a neuron.
  • the neuron is selected from the group consisting of a motor neuron, a sensory neuron, and an interneuron. In some embodiments, the neuron is a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron, but is not expressed in dorsal root ganglia.
  • the heterologous gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin
  • the heterologous gene is SMN1.
  • the SMN1 gene comprises the sequence set forth in SEQ ID NO: 28.
  • the heterologous gene is an inhibitory nucleic acid.
  • the inhibitory nucleic acid is a microRNA (miRNA), artificial microRNA (amiRNA), or short hairpin RNA (shRNA).
  • the inhibitory nucleic acid is a microRNA, wherein the microRNA binds to mRNA of a target gene.
  • the target gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin Mu DNA
  • the target gene is SOD1. In some embodiments, the SOD1 gene comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the target gene is C9orf72. In some embodiments, the C9orf72 gene comprises the sequence set forth in SEQ ID NO: 35, 36, or 37. In some embodiments, the rAAV further comprises a promoter.
  • the promoter is a promoter from a gene expressed in motor neurons, optionally wherein the gene expressed in motor neurons is selected from the group consisting of a Dnajc22, Sycp1, Slit3, Hrasls5, Otop3, Ccnb3, Nlrp3, Hormad1, Chat, Anxa4, Tnfsf4, Myo3b, Cdh15, Nr2e1, Il17f, Apela, Gnb3, Pappa, Tmprss15, Crp, Nxpe5, Tex21, Ttc24, Ttc23l, Ahnak2, Vipr2, Gstt4, Aox3, Plac8l1, Grin3b, Adam2, Col5a1, Clca3a1, Serpinb7, Edn2, Mgarp, Atp12a, Lhx4, Pip5kl1, Slc25a48, Tfcp2l1, Clec18a, Spint2, Il22ra1, Galp, Mei1, Aox1, Prph,
  • the gene expressed in motor neurons is ChAT, Slc5a7, Isl1, Mnx1, Lhx3, or Lhx4.
  • the promoter is selected from the group consisting of beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55), a choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23), CAG promoter (pCAG) (optionally comprising SEQ ID NO: 24 or 57), minimal CMV promoter, human synapsin promoter, chicken beta actin, PGK promoter, Efla promoter, ubiquitin promoter, a TATA-box containing promoter, Slc5a7 promoter, Isl1 promoter, Mnx1 promoter, Lhx3 promoter, Lhx4 promoter, and variants thereof.
  • the promoter comprises the beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55). In some embodiments, the promoter comprises the choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23). In some embodiments, the promoter comprises the minimal CMV promoter. In some embodiments, the nucleic acid comprises SEQ ID NOs: 84, 85 or 86. In some embodiments, the rAAV vector is replication-competent.
  • the present invention provides a transgenic cell comprising the nucleic acid of the above aspects or any other aspect of the invention delineated herein and/or a vector of the above aspects or any other aspect of the invention delineated herein.
  • the transgenic cell is a neuron.
  • the transgenic cell is selected from the group consisting of a motor neuron, a sensory neuron, and an interneuron.
  • the transgenic cell is a motor neuron.
  • the transgenic cell is murine, human, or non-human primate.
  • the present invention provides a composition comprising the nucleic acid of the above aspects or any other aspect of the invention delineated herein, the vector of the above aspects or any other aspect of the invention delineated herein, the rAAV vector of the above aspects or any other aspect of the invention delineated herein, or the transgenic cell of the above aspects or any other aspect of the invention delineated herein; and a pharmaceutically acceptable excipient.
  • the present invention provides a method for selectively expressing a heterologous gene within a population of neural cells in vivo or in vitro, the method comprising providing the composition of the above aspects or any other aspect of the invention delineated herein in a sufficient dosage and for a sufficient time to a sample or a subject comprising the population of neural cells thereby selectively expressing the gene within the population of neural cells.
  • the present invention provides a method for selectively expressing a heterologous gene within a population of neural cells in vivo or in vitro, the method comprising providing a composition comprising a nucleic acid of the above aspects or any other aspect of the invention delineated herein and a pharmaceutically acceptable excipient in a therapeutically effective dosage to a sample or a subject comprising the population of neural cells thereby selectively expressing the gene within the population of neural cells.
  • the composition is a lipid formulation.
  • the lipid formulation comprises one or more cationic lipids, non-cationic lipids, and/or PEG-modified lipids, or a combination thereof.
  • the pharmaceutical composition comprises a lipid nanoparticle.
  • the providing comprises administering to a living subject.
  • the living subject is a human, non-human primate, or a mouse.
  • the administering to a living subject is through injection.
  • the injection comprises intracerebroventricular (ICV) or intravenous (IV) injection, optionally into the cisterna magna (ICM).
  • the present invention provides a method for the treatment of a motor neuron disease or disorder in a subject in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) comprising a regulatory element sequence that has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83 or a fragment thereof to the subject, wherein one or more symptoms associated with the motor neuron disease or disorder are inhibited or prevented.
  • the regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification.
  • the nucleic acid comprises at least one additional regulatory element sequence. In some embodiments, the nucleic acid comprises at least two, three, four, five or six additional regulatory element sequences. In some embodiments, the at least one additional regulatory element sequence has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83.
  • the at least one additional regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the at least one additional regulatory sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises two or more identical copies of a regulatory element sequence selected from the group consisting of SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83. In some embodiments, the nucleic acid comprises two, three, four, five or six identical copies of the regulatory element sequence.
  • the nucleic acid comprises at least two or more versions of a regulatory element sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, wherein the first version of the regulatory element sequence differs from the second version of the regulatory element sequence.
  • the regulatory element sequence comprises SEQ ID NO: 81.
  • the regulatory element sequence comprises SEQ ID NO: 11.
  • the regulatory element sequence comprises SEQ ID NO: 83.
  • the nucleic acid comprises three copies of SEQ ID NO: 81.
  • the nucleic acid comprises three copies of SEQ ID NO: 11.
  • the nucleic acid comprises three copies of SEQ ID NO: 83. In some embodiments, the nucleic acid further comprises a heterologous gene. In some embodiments, the regulatory element comprises SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the regulatory element comprises a sequence that is at least 500 nucleotides, at least 400 nucleotides, at least 350 nucleotides, at least 300 nucleotides, at least 250 nucleotides, at least 200 nucleotides, at least 150 nucleotides, at least 100 nucleotides, at least 50 nucleotides, or at least 25 nucleotides.
  • the regulatory element comprises one or more transcription factor binding sites.
  • the one or more transcription factor binding sites is selected from the group consisting of a binding site for Lhx3 (TTAATTAG), a binding site for Lhx4 (SEQ ID NO: 16), a binding site for Mnx1 (TTAATTAA), a binding site for Isl2 (GCACTTAA), a binding site for RREB1 (SEQ ID NO: 19), a binding site for STAT4 (SEQ ID NO: 20), a binding site for Esrrb (SEQ ID NO: 21), a binding site for Myb (AACTGCCA), and a combination thereof.
  • the rAAV further comprises a promoter.
  • the promoter is a promoter from a gene expressed in motor neurons, optionally wherein the gene expressed in motor neurons is selected from the group consisting of a Dnajc22, Sycp1, Slit3, Hrasls5, Otop3, Ccnb3, Nlrp3, Hormad1, Chat, Anxa4, Tnfsf4, Myo3b, Cdh15, Nr2e1, Il17f, Apela, Gnb3, Pappa, Tmprss15, Crp, Nxpe5, Tex21, Ttc24, Ttc23l, Ahnak2, Vipr2, Gstt4, Aox3, Plac8l1, Grin3b, Adam2, Col5a1, Clca3a1, Serpinb7, Edn2, Mgarp, Atp12a, Lhx4, Pip5kl1, Slc25a48, Tfcp2l1, Clec18a, Spint2, Il22ra1, Galp, Mei1, Aox1, Prph,
  • the gene expressed in motor neurons is ChAT, Slc5a7, Isl1, Mnx1, Lhx3, or Lhx4.
  • the promoter is selected from the group consisting of beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55), a choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23), CAG promoter (pCAG) (optionally comprising SEQ ID NO: 24 or 57), minimal CMV promoter, human synapsin promoter, chicken beta actin, PGK promoter, Efla promoter, ubiquitin promoter, a TATA-box containing promoter, Slc5a7 promoter, Isl1 promoter, Mnx1 promoter, Lhx3 promoter, Lhx4 promoter, and variants thereof.
  • the promoter comprises the beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55). In some embodiments, the promoter comprises the choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23). In some embodiments, the promoter comprises the minimal CMV promoter. In some embodiments, the nucleic acid comprises SEQ ID NOs: 84, 85 or 86. In some embodiments, the heterologous gene is naturally expressed in a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron. In some embodiments, the heterologous gene is selectively expressed in a motor neuron, but is not expressed in dorsal root ganglia.
  • the heterologous gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin
  • the heterologous gene is SMN1.
  • the SMN1 gene comprises the sequence set forth in SEQ ID NO: 28.
  • the heterologous gene is an inhibitory nucleic acid.
  • the inhibitory nucleic acid is a microRNA (miRNA), artificial microRNA (amiRNA), or short hairpin RNA (shRNA).
  • the inhibitory nucleic acid is a microRNA, wherein the microRNA binds to mRNA of a target gene.
  • the target gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin Mu DNA
  • the target gene is SOD1. In some embodiments, the SOD1 gene comprises the sequence set forth SEQ ID NO: 33. In some embodiments, the target gene is C9orf72. In some embodiments, the C9orf72 gene comprises the sequence set forth SEQ ID NO: 35, 36, or 37. In some embodiments, the target gene is silenced.
  • the motor neuron disease or disorder is Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA) (e.g., non-5q SMA, Spinal muscular atrophy with congenital bone fractures, Autosomal dominant spinal muscular atrophy, and lower extremity-predominant 2 (SMALED2)), Distal hereditary motor neuropathy (dHMN) (e.g., autosomal dominant, autosomal recessive, type 1, 2, 4, 5, 6, 7, 7b), Triosephosphate isomerase deficiency (TIP), Hereditary spastic paraplegia (HSP) (also known as familial spastic paraparesis (FSP))(e.g., autosomal dominant or autosomal recessive or X-linked), amyotrophic lateral sclerosis (ALS), ALS with frontotemporal dementia (FTD), Adrenomyeloneuropathy (AMN), Allgrove syndrome (also known as triple A (3A) syndrome), Friedreich at
  • the one or more symptoms associated with the motor neuron disease or disorder are muscle weakness and decreased muscle tone, limited mobility, breathing problems, problems eating and swallowing, delayed gross motor skills, congenital hemolytic anemia, progressive neuromuscular dysfunction, spontaneous tongue movements, behavioral/cognitive symptoms, cerebellar degeneration, or scoliosis.
  • the present invention provides a method of silencing the expression of a target gene in a motor neuron, the method comprising contacting a recombinant adeno-associated virus (rAAV) comprising a regulatory element sequence that has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83 or a fragment thereof to the motor neuron.
  • rAAV recombinant adeno-associated virus
  • the regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises at least one additional regulatory element sequence. In some embodiments, the nucleic acid comprises at least two, three, four, five or six additional regulatory element sequences. In some embodiments, the at least one additional regulatory element sequence has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83.
  • the at least one additional regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the at least one additional regulatory sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification. In some embodiments, the nucleic acid comprises two or more identical copies of a regulatory element sequence selected from the group consisting of SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83. In some embodiments, the nucleic acid comprises two, three, four, five or six identical copies of the regulatory element sequence.
  • the nucleic acid comprises at least two or more versions of a regulatory element sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, wherein the first version of the regulatory element sequence differs from the second version of the regulatory element sequence.
  • the regulatory element sequence comprises SEQ ID NO: 81.
  • the regulatory element sequence comprises SEQ ID NO: 11.
  • the regulatory element sequence comprises SEQ ID NO: 83.
  • the nucleic acid comprises three copies of SEQ ID NO: 81.
  • the nucleic acid comprises three copies of SEQ ID NO: 11.
  • the nucleic acid comprises three copies of SEQ ID NO: 83. In some embodiments, the nucleic acid further comprising a heterologous gene.
  • the regulatory element comprises SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In some embodiments, the regulatory element comprises a sequence that is at least 500 nucleotides, at least 400 nucleotides, at least 350 nucleotides, at least 300 nucleotides, at least 250 nucleotides, at least 200 nucleotides, at least 150 nucleotides, at least 100 nucleotides, at least 50 nucleotides, or at least 25 nucleotides.
  • the regulatory element comprises one or more transcription factor binding sites.
  • the one or more transcription factor binding sites is selected from the group consisting of a binding site for Lhx3 (TTAATTAG), a binding site for Lhx4 (SEQ ID NO: 16), a binding site for Mnx1 (TTAATTAA), a binding site for Isl2 (GCACTTAA), a binding site for RREB1 (SEQ ID NO: 19), a binding site for STAT4 (SEQ ID NO: 20), a binding site for Esrrb (SEQ ID NO: 21), a binding site for Myb (AACTGCCA), and a combination thereof.
  • the rAAV further comprises a promoter.
  • the promoter is a promoter from a gene expressed in motor neurons, optionally wherein the gene expressed in motor neurons is selected from the group consisting of a Dnajc22, Sycp1, Slit3, Hrasls5, Otop3, Ccnb3, Nlrp3, Hormad1, Chat, Anxa4, Tnfsf4, Myo3b, Cdh15, Nr2e1, Il17f, Apela, Gnb3, Pappa, Tmprss15, Crp, Nxpe5, Tex21, Ttc24, Ttc23l, Ahnak2, Vipr2, Gstt4, Aox3, Plac8l1, Grin3b, Adam2, Col5a1, Clca3a1, Serpinb7, Edn2, Mgarp, Atp12a, Lhx4, Pip5kl1, Slc25a48, Tfcp2l1, Clec18a, Spint2, Il22ra1, Galp, Mei1, Aox1, Prph,
  • the gene expressed in motor neurons is ChAT, Slc5a7, Isl1, Mnx1, Lhx3, or Lhx4.
  • the promoter is selected from the group consisting of beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55), a choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23), CAG promoter (pCAG) (optionally comprising SEQ ID NO: 24 or 57), minimal CMV promoter, human synapsin promoter, chicken beta actin, PGK promoter, Efla promoter, ubiquitin promoter, a TATA-box containing promoter, Slc5a7 promoter, Isl1 promoter, Mnx1 promoter, Lhx3 promoter, Lhx4 promoter, and variants thereof.
  • the promoter comprises the beta globin promoter (pBG) (optionally comprising SEQ ID NO: 55). In some embodiments, the promoter comprises the choline acetyltransferase promoter (pChAT) (optionally comprising SEQ ID NO: 23). In some embodiments, the promoter comprises the minimal CMV promoter. In some embodiments, the nucleic acid comprises SEQ ID NOs: 84, 85 or 86. In some embodiments, the heterologous gene is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is a microRNA (miRNA), artificial microRNA (amiRNA), or short hairpin RNA (shRNA).
  • miRNA microRNA
  • amiRNA artificial microRNA
  • shRNA short hairpin RNA
  • the inhibitory nucleic acid is a microRNA, wherein the microRNA binds to mRNA of a target gene.
  • the target gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation
  • SSN1 motor neuro
  • the target gene is SOD1. In some embodiments, the SOD1 gene comprises the sequence set forth SEQ ID NO: 33. In some embodiments, the target gene is C9orf72. In some embodiments, the C9orf72 gene comprises the sequence set forth SEQ ID NO: 35, 36, or 37.
  • the neuron is from a subject. In some embodiments, the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject has been diagnosed or is suspected of having a motor neuron disease or disorder.
  • the motor neuron disease or disorder is Spinal- bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA) (e.g., non-5q SMA, Spinal muscular atrophy with congenital bone fractures, Autosomal dominant spinal muscular atrophy, and lower extremity-predominant 2 (SMALED2)), Distal hereditary motor neuropathy (dHMN) (e.g., autosomal dominant, autosomal recessive, type 1, 2, 4, 5, 6, 7, 7b), Triosephosphate isomerase deficiency (TIP), Hereditary spastic paraplegia (HSP) (also known as familial spastic paraparesis (FSP))(e.g., autosomal dominant or autosomal recessive or X-linked), amyotrophic lateral sclerosis (ALS), ALS with frontotemporal dementia (FTD), Adrenomyeloneuropathy (AMN), Allgrove syndrome (also known as triple A (3A) syndrome), Friedreich at
  • the promoter is pBG (optionally comprising SEQ ID NO: 55).
  • the nucleic acid further comprises a pBG intron (optionally comprising SEQ ID NO: 56), optionally wherein there is a linker between the pBG and pBG intron of zero to 500 nucleotides.
  • the promoter is pBG (optionally comprising SEQ ID NO: 55).
  • the rAAV vector further comprises a pBG intron (optionally comprising SEQ ID NO: 56), optionally wherein there is a linker between the pBG and pBG intron of zero to 500 nucleotides.
  • the promoter is pBG (optionally comprising SEQ ID NO: 55). In some embodiments, the rAAV further comprises a pBG intron (optionally comprising SEQ ID NO: 56), optionally wherein there is a linker between the pBG and pBG intron of zero to 500 nucleotides. In some embodiments, the promoter is pBG (optionally comprising SEQ ID NO: 55). In some embodiments, the rAAV further comprises a pBG intron (optionally comprising SEQ ID NO: 56), optionally wherein there is a linker between the pBG and pBG intron of zero to 500 nucleotides.
  • FIG.1A depicts expression of GFP in the spinal cord under the control of the Enh98 enhancer and beta globin promoter (pBG).
  • FIG.1B depicts expression of GFP in the spinal cord under the control of only the beta globin promoter (pBG).
  • FIG.2 depicts a graph quantifying the expression of GFP in the spinal cord under the control of the Enh57 and Enh98 enhancer compared to no enhancer and a saline control. Expression was compared across dorsal cells, the ventral horn, and dorsal root ganglion (DRG).
  • FIG.3 depicts expression of GFP in the spinal cord under the control of CAG promoter. 3E+13 gc/ml.
  • FIGs.9A-9G are related to motor neuron cis-regulatory element identification.
  • FIG.9A depicts the experimental design.
  • FIG.9B depicts an immunohistochemistry example of Chat-Sun1 cross labeling motor neuron nuclear envelope.
  • FIG.9C depicts an example of IP-specific and nonspecific cis-regulatory element ATAC-seq data.
  • FIG.9D depicts a genome-wide fixed-line-plot of ATAC-seq signal for all spinal cord peaks.
  • FIG.9E depicts summary plots showing average ATAC- seq signal intensity (left) and conservation (right) across spinal cord peaks.
  • FIG.9F depicts an MA plot of Enh MN-enrichment as a function of mean ATAC signal for each peak.
  • FIG.9G depicts a subselection of putative MN-selective Enhs by conservation.
  • FIGs.10A-10E are related to preliminary enhancer screening by confocal microscopy.
  • FIG. 10A depicts a volcano plot (top) and plot of conservation (bottom) demonstrating candidate element selection thresholds.
  • FIG.10B depicts a table of selected elements.
  • FIG.10C depicts vector maps of screen AAV genomes.
  • FIG.10D depicts representative images from screen for all constructs evaluated by confocal microscopy.
  • FIG.10E depicts quantification of native GFP signal intensity in ventral and dorsal horns for all constructs evaluated.
  • FIGs.11A-11G are related to immunohistochemistry quantification of hit specificity.
  • FIG. 11A depicts representative images for all conditions assayed by IHC.
  • FIG.11B depicts percentage of GFP positivity quantification for NeuN+ Chat+ and NeuN+ Chat- neurons of spinal cord.
  • FIG.11C depicts mean GFP signal intensity quantification for NeuN+ Chat+ and NeuN+ Chat- neurons of spinal cord.
  • FIG.11D depicts relative GFP signal intensity of Enh98 compared to CAG in NeuN+ Chat+ and NeuN+ Chat- neurons of spinal cord.
  • FIG.11E depicts representative images for off-target GFP expression in DRG.
  • FIG.11F depicts percentage of GFP positivity quantification for neurons of the DRG.
  • FIG.11G depicts mean GFP signal intensity quantification for neurons of the DRG.
  • FIGs.12A-12F are related to the identification of core functional components of Enh98.
  • FIG.12A depicts a scatter plot of TF motif significance as a function of enrichment for expression of that TF in motor neurons (left) and associated position-weight matrix (PWM) representation for significantly enriched motifs (denoted in green, right).
  • PWM position-weight matrix
  • FIG.12B depicts a genomic map of TFBS position and truncated Enh98 construct design.
  • FIG.12C depicts a percentage of GFP positivity quantification for NeuN+ Chat+ and Neun+ Chat- neurons of spinal cord.
  • FIG.12D depicts a mean GFP signal intensity quantification for NeuN+ Chat+ and Neun+ Chat- neurons of spinal cord.
  • FIG. 12E depicts distributions of GFP intensity of Enh98-pBG and Enh98-pCHAT promoter in the ventral horn of spinal cord and DRG.
  • FIG.12F depicts distributions of GFP intensity for all truncated constructs compared to CAG in the DRG.
  • FIG.13A depicts heat map showing gene expression of specific markers in various cell types.
  • FIG.13B depicts a volcano plot of the fold change of gene expression of the markers shown in FIG. 13A.
  • FIG.13C depicts IP-specific and nonspecific Enh Fragment distribution.
  • FIG.13D depicts ATAC-seq principal component analysis (PCA)
  • FIG.13E depicts ATAC-seq correlation.
  • FIG.14A depicts percent positive GFP cells comparing NeuN+/Chat-, NeuN+/Chat+ interneurons, NeuN+/Chat+ visceral motor neurons, and NeuN+/Chat+ skeletal motor neurons when different Enhancers were used.
  • Enhancers Enh57, Enh98, and Enh119.
  • Controls Saline, ⁇ Enh, and CAG promoter.
  • FIG.14B depicts mean GFP intensity in cells from FIG.14A.
  • FIG.15A is a schematic of transcription factor binding site (TFBS) positions and human Enh98 in native genomic context.
  • FIG.15B is a schematic of synthetic constructs tested in AAV: 1) triplicate concatemers of the core sequences from each of mouse and human Enh98 (3mCORE, 3hCORE) and a similar triplicate concatemer of a synthetic 129 bp sequence comprising just the mCORE TFBS, with relative order and orientation preserved and all native inter-motif sequences replaced by 15 bp linkers (3mTFBS).
  • FIG.15C are representative spinal images for all conditions assayed by immunohistochemistry for GFP.
  • FIG.15D depicts a bar graph of the percentage of GFP- positive MNs (NeuN+ Chat+, dark bars) and other spinal neurons (NeuN+ Chat-, light bars) for each construct.
  • FIG.15E depicts a bar graph of the mean GFP signal intensity in MNs (NeuN+ Chat+, dark bars) and other spinal neurons (NeuN+ Chat-, light bars) for each construct.
  • FIG.15F are representative images for off-target GFP expression in DRG.
  • FIG.15G depicts a bar graph of the percentage of GFP-positive neurons in the DRG.
  • FIG.15H depicts a bar graph of the mean GFP signal intensity of neurons in the DRG.
  • FIG.16A are representative images of neurons transduced in vitro with the indicated GRE- AAVs.
  • FIG.16B depicts a bar graph of the quantification of percentage of GFP-positive nuclei per well.
  • FIG.16C depicts a bar graph of the quantification of mean intensity of GFP per well in each condition. Each dot represents one well; error bars represent standard deviation.
  • DETAILED DESCRIPTION The present disclosure provides compositions and methods for cell-type specific expression of a heterologous gene. Also described herein are compositions and methods for expression of a heterologous gene comprising one or more regulatory elements which, when operably linked to a heterologous gene, can facilitate the expression of the heterologous in one or more target cell types or tissues.
  • the one or more regulatory elements disclosed herein drive expression of a heterologous gene in a cell or in vivo, in vitro, and/or ex vivo.
  • the present disclosure also provides a viral vector comprising a heterologous gene operably linked to a regulatory element, which induces expression of the heterologous gene in a cell-type specific manner.
  • the regulatory element is SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the heterologous gene is survival of motor neuron 1 (SMN1).
  • the viral vector is a recombinant adeno-associated vector (rAAV).
  • a recombinant AAV viral particle comprises the rAAV comprising the heterologous gene operably linked to the regulatory element.
  • the heterologous gene is expressed in a neuron.
  • the heterologous gene is expressed preferentially in a motor neuron and little to no expression in a non-motor neuron cell, for example, a dorsal root ganglia cell.
  • the present disclosure provides for a method of treating a subject having a motor neuron disease or disorder, comprising administering a recombinant adeno-associated virus (rAAV) which comprises a heterologous gene operably linked to a regulatory element, wherein one or more symptoms associated with the motor neuron disease or disorder are inhibited or prevented.
  • rAAV recombinant adeno-associated virus
  • the heterologous gene is preferentially expressed in motor neuron and little to no expression in a non-motor neuron cell, for example, a dorsal root ganglia cell.
  • the regulatory element is SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, or a variant or fragment thereof.
  • the heterologous gene is survival of motor neuron 1 (SMN1).
  • SSN1 motor neuron 1
  • Definitions In order that the present invention may be more readily understood, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.
  • ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.
  • the term “about” or “approximately” means within 5%, or more preferably within 1%, of a given value or range.
  • the term “encode” or “encoding” refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” may therefore be used in some embodiments herein to capture potential lack of completeness inherent in many biological and chemical phenomena. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
  • regulatory elements refers to elements that can function to modulate gene expression selectivity in a cell type of interest at a DNA and/or RNA level. Regulatory elements can function to modulate gene expression at the transcriptional phase, post-transcriptional phase, or at the translational phase of gene expression. Regulatory elements include, but are not limited to, promoter, enhancer, intronic, or other non-coding sequences. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and/or transcriptional termination. In some cases, regulatory elements can recruit transcriptional factors to a coding region that increase gene expression selectivity in a cell type of interest.
  • regulatory elements can increase the rate at which RNA transcripts are produced, increase the stability of RNA produced, and/or increase the rate of protein synthesis from RNA transcripts.
  • Regulatory elements are nucleic acid sequences or genetic elements which are capable of influencing (e.g., increasing) expression of a gene (e.g., a reporter gene such as EGFP or luciferase; a transgene; or a therapeutic gene) in one or more cell types or tissues.
  • a regulatory element can be a transgene, an intron, a promoter, an enhancer, UTR, an inverted terminal repeat (ITR) sequence, a long terminal repeat sequence (LTR), stability element, posttranslational response element, or a polyA sequence, or a combination thereof.
  • the regulatory element is derived from a human sequence (e.g., SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83).
  • the regulatory element is a variant of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, for example, containing a substitute mutation.
  • the regulatory element includes a fragment or fragments of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, which serves to modulate gene expression.
  • the regulatory element sequences used to induce cell-type specific expression accordingly to methods and compositions disclosed herein include SEQ ID NOs: 1-14, 60- 75, 78, 79, or 81-83.
  • the nucleic acid can comprise one or more regulatory element sequences.
  • the nucleic acid comprises one regulatory element sequence.
  • the nucleic acid comprises at least one additional regulatory element sequence, for example, two, three, four, five, six, or more regulatory element sequences.
  • the at least one additional regulatory element sequence has at least 85% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83. In one embodiment, the at least one additional regulatory element sequence has at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. In one embodiment, the at least one additional regulatory sequence comprises at least one modification relative to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, optionally a substitute modification.
  • the nucleic acid sequence comprises two or more identical copies, for example, three, four, five or six copies, of a regulatory element selected from the group consisting of SEQ ID NOs: 1-14, 60-75, 78, 79, and 81-83.
  • the nucleic acid comprises at least two or more versions of a regulatory element sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, wherein the first version of the regulatory element sequence differs from the second version of the regulatory element sequence.
  • the nucleic acid may include a first version of SEQ ID NO: 1 having 95% identity to SEQ ID NO: 1, and a second version of SEQ ID NO: 1 having 100% identity to SEQ ID NO: 1. Further by way of example, the nucleic acid may have a third and fourth versions of SEQ ID NO: 1, having 90% and 98% identity to SEQ ID NO: 1.
  • “enhancers” or “enhancer elements” induce expression of a gene, e.g., heterologous gene. In some embodiments, enhancers can induce expression of a heterologous gene in a cell-type specific manner.
  • cell-type specific or “cell-type specific induced expression” refer to expression being induced in certain cell types and not all cell types.
  • cell-type specific expression is induced in a specific cell type, e.g., neuron cell, but not other cell types, e.g., a non-neural cell.
  • the cell-type specific expression is induced in a specific cell type, e.g., motor neuron, and little to no expression in other cell types, e.g., dorsal cells.
  • Cell-type specific induced expression does not eliminate the possibility that expression can occur in other cell-types at a low level.
  • cell-type specific induced expression results in expression of a heterologous gene in a specific cell-type at a higher level when compared to a control cell-type.
  • the specific enhancers described herein sometimes are referred to with the prefix “Enh”, or alternatively may be referred to as cis-regulatory elements (“CREs”) or gene regulatory elements (“GREs”). These terms and prefixes as used herein are interchangeable.
  • the present disclosure provides for cell-type specific regulatory elements that induce expression of a heterologous gene in a specific cell-type.
  • the regulatory element is SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83, a variant thereof or a fragment thereof.
  • the regulatory element has at least about 80% identity with the entire sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. Accordingly, in one embodiment, the regulatory element has at least about 90% identity with the entire sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83. Accordingly, in one embodiment, the regulatory element has at least about 95% identity with the entire sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the entire sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises the sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element consists of the sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 500 nucleotides, at least about 450 nucleotides, at least about 400 nucleotides, at least about 350 nucleotides, at least about 300 nucleotides, at least about 250 nucleotides, at least about 200 nucleotides, at least about 150 nucleotides, at least about 100 nucleotides, at least about 50 nucleotides, or at least 25 nucleotides of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises about 25 nucleotides to about 500 nucleotides, about 25 nucleotides to about 400 nucleotides, about 25 nucleotides to about 300 nucleotides, about 25 nucleotides to about 200 nucleotides, about 25 nucleotides to about 100 nucleotides, about 25 nucleotides to about 50 nucleotides, about 50 nucleotides to about 500 nucleotides, about 50 nucleotides to about 400 nucleotides, about 50 nucleotides to about 300 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 100 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 200 nucleotides, about 200 nucleotides, about 200
  • the regulatory element comprises no more than 500 nucleotides, no more than 400 nucleotides, no more than 300 nucleotides, no more than 200 nucleotides, no more than 100 nucleotides, no more than 50 nucleotides, or no more than 25 nucleotides of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 500 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 500 nucleotide sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 400 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 400 nucleotide sequence of SEQ ID NOs: 1-14, 60- 75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 300 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 300 nucleotide sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 200 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 200 nucleotide sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 100 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 100 nucleotide sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the regulatory element comprises at least about 50 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 50 nucleotide sequence of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83.
  • the present disclosure provides for cell-type specific regulatory elements that induce expression of a heterologous gene in a specific cell-type.
  • the regulatory element is SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element has at least about 80% identity with the entire sequence of SEQ ID NOs: 7-14 or 60-65. Accordingly, in one embodiment, the regulatory element has at least about 90% identity with the entire sequence of SEQ ID NOs: 7-14 or 60-65. Accordingly, in one embodiment, the regulatory element has at least about 95% identity with the entire sequence of SEQ ID NOs: 7-14 or 60-65. Accordingly, in one embodiment, the regulatory element has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the entire sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises the sequence of SEQ ID NOs: 7-14 or 60-65. In yet another embodiment the regulatory element consists of the sequence of SEQ ID NOs: 7-14 or 60-65. In one embodiment, the regulatory element comprises at least about 500 nucleotides, at least about 450 nucleotides, at least about 400 nucleotides, at least about 350 nucleotides, at least about 300 nucleotides, at least about 250 nucleotides, at least about 200 nucleotides, at least about 150 nucleotides, at least about 100 nucleotides, at least about 50 nucleotides, or at least 25 nucleotides of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises about 25 nucleotides to about 500 nucleotides, about 25 nucleotides to about 400 nucleotides, about 25 nucleotides to about 300 nucleotides, about 25 nucleotides to about 200 nucleotides, about 25 nucleotides to about 100 nucleotides, about 25 nucleotides to about 50 nucleotides, about 50 nucleotides to about 500 nucleotides, about 50 nucleotides to about 400 nucleotides, about 50 nucleotides to about 300 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 100 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 200 nucleotides, about 200 nucleotides, about 200
  • the regulatory element comprises no more than 500 nucleotides, no more than 400 nucleotides, no more than 300 nucleotides, no more than 200 nucleotides, no more than 100 nucleotides, no more than 50 nucleotides, or no more than 25 nucleotides of SEQ ID NOs: 7-14 or 60-65. In some embodiments, the regulatory element comprises at least about 500 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 500 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises at least about 400 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 400 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises at least about 300 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 300 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises at least about 200 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 200 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises at least about 100 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 100 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element comprises at least about 50 nucleotides and has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the equivalent 50 nucleotide sequence of SEQ ID NOs: 7-14 or 60-65.
  • the regulatory element of SEQ ID NOs: 1-14, 60-75, 78, 79, or 81-83 comprise sequences that are transcription factor binding sites.
  • the transcription factor binding sites are, but not limited to, LIM Homeobox 3 (Lhx3) (TTAATTAG), LIM Homeobox 4 (Lhx4) (TAATTAATTAAGT (SEQ ID NO: 16)), Motor Neuron and Pancreas Homeobox 1 (Mnx1) (TTAATTAA), Insulin gene enhancer protein ISL-2 (Isl2) (GCACTTAA), Ras Responsive Element Binding Protein 1 (RREB1) (GCACTGGGGATGGGGGTGGG (SEQ ID NO: 19)), Signal Transducer And Activator Of Transcription 4 (STAT4) (TTTCCGGGAATGGC (SEQ ID NO: 20), Estrogen Related Receptor Beta (Esrrb) (TGGCCAAGGGCA (SEQ ID NO: 21)), and Myb (AACTGCCA).
  • LIM Homeobox 3 Lhx3
  • LIM Homeobox 4 LIM Homeobox 4
  • TATTAAGT SEQ ID NO: 16
  • Mnx1 TTA
  • the enhancer contains transcription factor binding sites LIM Homeobox 3 (Lhx3), LIM Homeobox 4 (Lhx4), Motor Neuron and Pancreas Homeobox 1 (Mnx1), Insulin gene enhancer protein ISL-2 (Isl2), Ras Responsive Element Binding Protein 1 (RREB1), Signal Transducer And Activator Of Transcription 4 (STAT4), and Estrogen Related Receptor Beta (Esrrb), or a combination thereof.
  • the transcription factor binding sites originate from human, mouse, or both.
  • the transcription factor binding site for Lhx3 has 90% identity with the entire sequence of TTAATTAG.
  • the transcription factor binding site for Lhx3 has at least about 95% identity with the entire sequence of TTAATTAG. In a further embodiment, the transcription factor binding site for Lhx3 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of TTAATTAG. In another embodiment, the transcription factor binding site for Lhx3 comprises the sequence of TTAATTAG. In yet another embodiment, the transcription factor binding site for Lhx3 consists of the sequence of TTAATTAG. In some embodiments, the transcription factor binding site for Lhx4 has 90% identity with the entire sequence of SEQ ID NO: 16.
  • the transcription factor binding site for Lhx4 has at least about 95% identity with the entire sequence of SEQ ID NO: 16. In a further embodiment, the transcription factor binding site for Lhx4 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 16. In another embodiment, the transcription factor binding site for Lhx4 comprises the sequence of SEQ ID NO: 16. In yet another embodiment the transcription factor binding site for Lhx4 consists of the sequence of SEQ ID NO: 16. In some embodiments, the transcription factor binding site for Mnx1 has 90% identity with the entire sequence of TTAATTAA.
  • the transcription factor binding site for Mnx1 has at least about 95% identity with the entire sequence of TTAATTAA. In a further embodiment, the transcription factor binding site for Mnx1 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of TTAATTAA. In another embodiment, the transcription factor binding site for Mnx1 comprises the sequence of TTAATTAA. In yet another embodiment. the transcription factor binding site for Mnx1 consists of the sequence of TTAATTAA. In some embodiments, the transcription factor binding site for Isl2 has 90% identity with the entire sequence of GCACTTAA.
  • the transcription factor binding site for Isl2 has at least about 95% identity with the entire sequence of GCACTTAA. In a further embodiment, the transcription factor binding site for Isl2 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of GCACTTAA. In another embodiment, the transcription factor binding site for Isl2 comprises the sequence of GCACTTAA. In yet another embodiment, the transcription factor binding site for Isl2 consists of the sequence of GCACTTAA. In some embodiments, the transcription factor binding site for RREB1 has 90% identity with the entire sequence of SEQ ID NO: 19.
  • the transcription factor binding site for RREB1 has at least about 95% identity with the entire sequence of SEQ ID NO: 19. In a further embodiment, the transcription factor binding site for RREB1 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 19. In another embodiment, the transcription factor binding site for RREB1 comprises the sequence of SEQ ID NO: 19. In yet another embodiment, the transcription factor binding site for RREB1 consists of the sequence of SEQ ID NO: 19. In some embodiments, the transcription factor binding site for STAT4 has 90% identity with the entire sequence of SEQ ID NO: 20.
  • the transcription factor binding site for STAT4 has at least about 95% identity with the entire sequence of SEQ ID NO: 20. In a further embodiment, the transcription factor binding site for STAT4 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 20. In another embodiment, the transcription factor binding site for STAT4 comprises the sequence of SEQ ID NO: 20. In yet another embodiment, the transcription factor binding site for STAT4 consists of the sequence of SEQ ID NO: 20. In some embodiments, the transcription factor binding site for Esrrb has 90% identity with the entire sequence of SEQ ID NO: 21.
  • the transcription factor binding site for Esrrb has at least about 95% identity with the entire sequence of SEQ ID NO: 21. In a further embodiment, the transcription factor binding site for Esrrb has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 21. In another embodiment, the transcription factor binding site for Esrrb comprises the sequence of SEQ ID NO: 21. In yet another embodiment, the transcription factor binding site for Esrrb consists of the sequence of SEQ ID NO: 21. In some embodiments, the transcription factor binding site for Myb has 90% identity with the entire sequence of AACTGCCA.
  • the transcription factor binding site for Myb has at least about 95% identity with the entire sequence of AACTGCCA. In a further embodiment, the transcription factor binding site for Myb has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of AACTGCCA. In another embodiment, the transcription factor binding site for Myb comprises the sequence of AACTGCCA. In yet another embodiment, the transcription factor binding site for Myb consists of the sequence of AACTGCCA. Promoters A “promoter” as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene.
  • Promoters are generally located 5’ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Promoters, as described herein, are promoters of genes expressed in motor neurons.
  • Motor neuron enriched genes include, but are not limited to, Dnajc22, Sycp1, Slit3, Hrasls5, Otop3, Ccnb3, Nlrp3, Hormad1, Chat, Anxa4, Tnfsf4, Myo3b, Cdh15, Nr2e1, Il17f, Apela, Gnb3, Pappa, Tmprss15, Crp, Nxpe5, Tex21, Ttc24, Ttc23l, Ahnak2, Vipr2, Gstt4, Aox3, Plac8l1, Grin3b, Adam2, Col5a1, Clca3a1, Serpinb7, Edn2, Mgarp, Atp12a, Lhx4, Pip5kl1, Slc25a48, Tfcp2l1, Clec18a, Spint2, Il22ra1, Galp, Mei1, Aox1, Prph, Slc25a54, Cdhr1, Tgm6, Ppm1j, Esrp1, Gem,
  • Promoters include, but not limited to, beta globin promoter (pBG) (for example, comprising SEQ ID NO: 55) and choline acetyltransferase promoter (pChAT) (for example, comprising SEQ ID NO: 23), CAG promoter (pCAG) (for example, comprising SEQ ID NO: 24 or 57), minimal CMV promoter, human synapsin promoter, chicken beta actin, PGK promoter, Efla promoter, ubiquitin promoter, TATA-box containing promoters, or fragments thereof.
  • pBG beta globin promoter
  • pChAT choline acetyltransferase promoter
  • CAG promoter pCAG
  • minimal CMV promoter human synapsin promoter
  • human synapsin promoter chicken beta actin
  • PGK promoter PGK promoter
  • Efla promoter ubiquitin promoter
  • TATA-box containing promoters or fragment
  • the promoter is of genes expressed selectively in motor neurons (e.g., Chat, Slc5a7, Isl1, Mnx1, Lhx3, Lhx4, and other genes listed above).
  • the promoter is a beta globin promoter (pBG).
  • the pBG promoter comprises the pBG promoter alone (for example, comprising SEQ ID NO: 55).
  • the pBG promoter is attached to a pBG intron (for example, SEQ ID NO: 56).
  • the pBG promoter and the pBG intron are connected by Xn, where “X” can be nucleotides C, G, T, or A, and “n” can be zero nucleotides up to and including 500 nucleotides.
  • the nucleic acid sequence, vector or virus comprises pBG-X (0-500) -pBG intron (SEQ ID NO: 22).
  • the term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions.
  • the term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites.
  • the term further refers to a coding sequence for a desired expression product of a polynucleotide sequence such as a polypeptide, peptide, protein or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA).
  • the sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.
  • heterologous gene refers gene provided to the target cell by an exogenous source, such as a viral vector, e.g., rAAV.
  • the gene encodes a polypeptide or a nucleic acid molecule, such as microRNA (miRNA), artificial microRNA (amiRNA), and short hairpin RNA (shRNA).
  • miRNA microRNA
  • amiRNA artificial microRNA
  • shRNA short hairpin RNA
  • the heterologous gene is selected from the group consisting of survival of motor neuron 1 (SMN1), AR (androgen receptor), BICD2 (BICD Cargo Adaptor 2), TRIP4 (Thyroid Hormone Receptor Interactor 4), HSPB1 (Heat Shock Protein Family B (Small) Member 1), HSPB8 (Heat Shock Protein Family B (Small) Member 8), HSPB3 (Heat Shock Protein Family B (Small) Member 3), FBXO38 (F-Box Protein 38), REEP1 (Receptor Accessory Protein 1), BSCL2 (BSCL2 Lipid Droplet Biogenesis Associated, Seipin), GARS1 (Glycyl-TRNA Synthetase 1), SLC5A7 (Solute Carrier Family 5 Member 7), TRPV4 (Transient Receptor Potential Cation Channel Subfamily V Member 4), ATP7A (ATPase Copper Transporting Alpha), IGHMBP2 (Immunoglobulin
  • the heterologous gene is SMN1. In some embodiments, the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 25. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 25. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 25. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 25. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 25.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 26. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 26. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 26. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 26. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 26.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 27. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 27. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 27. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 27. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 27.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 28. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 28. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 28. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 28. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 28.
  • the heterologous gene encodes a transcriptional regulator (e.g., represses expression of a gene or enhances expression of a target gene).
  • the transcription regulator is an engineered zinc finger polypeptide, Transcription activator-like effector nucleases (TALEN), or Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) or dCas9 (nuclease deficient Cas9), rtTA ( reverse tetracycline-controlled transactivator), tetracycline transactivator (tTA), ribozymes, RNA-editing proteins, other DNA editing enzymes (e.g., DNA base editing proteins, prime editing proteins, CRISPR family proteins, etc.).
  • TALEN Transcription activator-like effector nucleases
  • Cas9 CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12
  • dCas9 nuclease deficient Cas9
  • the transcriptional regulator regulates expression of one or more target genes.
  • the one or more target gene is SMN1, AR, BICD2, TRIP4, HSPB1, HSPB8, HSPB3, FBXO38, REEP1, BSCL2, GARS1, SLC5A7, TRPV4, ATP7A, IGHMBP2, DCTN1, DYNC1H1, PLEKHG5, SIGMAR1, DNAJB2, SMAX3, TPI1, ATL1, SPAST, NIPA1, KIAA1096, KIF5A, RTN2, Hsp60, SPG37, SPG41, SLC33A1, REEP2, CPT1C, UBAP1, ALDH18A1, SPG11, CYP7B1, SPG7, ZFYVE26, SPG20, SPG21(ACP33), GJC2, SPG24, DDHD1, KIF1A, AP5Z1, FARS2, L1CAM, PLP1, ALS2, WDR7, TBK1, ABCD1,
  • the heterologous gene encodes a microRNA.
  • the microRNA inhibits expression of one or more target genes.
  • the target gene is SMN1, AR, BICD2, TRIP4, HSPB1, HSPB8, HSPB3, FBXO38, REEP1, BSCL2, GARS1, SLC5A7, TRPV4, ATP7A, IGHMBP2, DCTN1, DYNC1H1, PLEKHG5, SIGMAR1, DNAJB2, SMAX3, TPI1, ATL1, SPAST, NIPA1, KIAA1096, KIF5A, RTN2, Hsp60, SPG37, SPG41, SLC33A1, REEP2, CPT1C, UBAP1, ALDH18A1, SPG11, CYP7B1, SPG7, ZFYVE26, SPG20, SPG21(ACP33), GJC2, SPG24, DDHD1, KIF1A, AP5Z1, FARS2, L1CAM, PLP1,
  • the target gene is SOD1.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 33. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 33. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 33.
  • the heterologous gene comprises the sequence of SEQ ID NO: 33. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 33.
  • the target gene is C9orf72.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 35. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 35. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 35.
  • the heterologous gene comprises the sequence of SEQ ID NO: 35. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 35.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 36. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 36. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 36. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 36. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 36.
  • the heterologous gene has 90% identity with the entire sequence of SEQ ID NO: 37. Accordingly, in one embodiment, the heterologous gene has at least about 95% identity with the entire sequence of SEQ ID NO: 37. Accordingly, in one embodiment, the heterologous gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 37. In another embodiment, the heterologous gene comprises the sequence of SEQ ID NO: 37. In yet another embodiment the heterologous gene consists of the sequence of SEQ ID NO: 37. Exemplary survival of motor neuron 1 (SMN1) nucleic acid sequence
  • SSN1 motor neuron 1 amino acid sequences
  • SOD1 superoxide dismutase 1 nucleotide sequence
  • SOD1 superoxide dismutase 1 amino acid sequence
  • C9orf72 nucleotide sequence Exemplary survival of motor neuron 1 amino acid sequence
  • Exemplary C9orf72 amino acid sequence Viral Vector Viral vector is widely used to refer to a nucleic acid molecule that includes virus-derived nucleic acid elements that facilitate transfer and expression of non-native nucleic acid molecules within a cell.
  • adeno-associated viral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from AAV.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, and so on.
  • hybrid vector refers to a vector including structural and/or functional genetic elements from more than one virus type.
  • adenovirus vector refers to those constructs containing adenovirus sequences sufficient to (a) support packaging of an expression construct and (b) to express a coding sequence that has been cloned therein in a sense or antisense orientation.
  • a recombinant Adenovirus vector includes a genetically engineered form of an adenovirus.
  • adenovirus a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • AAV vector in the context of the present invention includes without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the E1 region encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut-off.
  • the products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • MLP major late promoter
  • the MLP is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.
  • TPL 5'-tripartite leader
  • adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication- defective adenovirus vector for use in some embodiments, since Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector is replication defective and will not have an adenovirus E1 region.
  • the position of insertion of the construct within the adenovirus sequences is not critical.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of a deleted E3 region in E3 replacement vectors or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adeno- Associated Virus is a parvovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Various serotypes have been isolated, of which AAV -2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter.
  • the AAV DNA is 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, pl9, and p40, according to their map position.
  • scAAV refers to a self-complementary AAV.
  • pAAV refers to a plasmid adeno- associated virus.
  • rAAV refers to a recombinant adeno-associated virus.
  • Other viral vectors may also be employed. For example, vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells. Retrovirus.
  • Retroviruses are a common tool for gene delivery.
  • “Retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus.”
  • the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
  • Illustrative retroviruses suitable for use in some embodiments include: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • Harvey murine sarcoma virus HaMuSV
  • murine mammary tumor virus MoMTV
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RSV Rous Sarcoma Virus
  • lentivirus refers to a group (or
  • Illustrative lentiviruses include: HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV based vector backbones i.e., HIV cis- acting sequence elements
  • a safety enhancement for the use of some vectors can be provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
  • heterologous promoters which can be used for this purpose include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system.
  • the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed.
  • the heterologous promoter can be inducible, such that transcription of all or part of the viral genome will occur only when the induction factors are present.
  • Induction factors include one or more chemical compounds or the physiological conditions such as temperature or pH, in which the host cells are cultured.
  • viral vectors include a TAR element.
  • TAR refers to the "trans-activation response" genetic element located in the R region of lentiviral LTRs. This element interacts with the lentiviral trans-activator (tat) genetic element to enhance viral replication.
  • the "R region” refers to the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly(A) tract.
  • the R region is also defined as being flanked by the U3 and U5 regions.
  • the R region plays a role during reverse transcription in permitting the transfer of nascent DNA from one end of the genome to the other.
  • expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • a variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid. Examples include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al, 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res.26(21):4818-4827, 1998); and the like (Liu et al., 1995, Genes Dev., 9: 1766).
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • HPRE hepatitis B virus
  • vectors include a posttranscriptional regulatory element such as a WPRE or HPRE.
  • vectors lack or do not include a posttranscriptional regulatory element such as a WPRE or HPRE. Elements directing the efficient termination and polyadenylation of a heterologous nucleic acid transcript can increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.
  • vectors include a polyadenylation sequence 3' of a polynucleotide encoding a molecule (e.g., protein) to be expressed.
  • the term "poly(A) site” or "poly(A) sequence” denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
  • Particular embodiments may utilize BGHpA or SV40pA.
  • a preferred embodiment of an expression construct includes a terminator element. These elements can serve to enhance transcript levels and to minimize read through from the construct into other plasmid sequences.
  • a viral vector further includes one or more insulator elements.
  • Insulator elements may contribute to protecting viral vector-expressed sequences, e.g., effector elements or expressible elements, from integration site effects, which may be mediated by as- acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et al, PNAS., USA, 99: 16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001).
  • transfer sequences i.e., position effect; see, e.g., Burgess-Beusse et al, PNAS., USA, 99: 16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001.
  • viral transfer vectors include one or more insulator elements at the 3' LTR and upon integration of the provirus into the host genome, the provirus includes the one or more insulators at both the 5' LTR and 3' LTR, by virtue of duplicating the 3' LTR.
  • Suitable insulators for use in particular embodiments include the chicken b-globin insulator (see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer blocking insulators (Liu et al., Nature Biotechnology, 33: 198, 2015). Beyond the foregoing description, a wide range of suitable expression vector types will be known to a person of ordinary skill in the art.
  • suitable expression vectors include any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cell, such as pUC or Bluescript plasmid series.
  • vectors e.g., AAV
  • BBB blood-brain barrier
  • BSCB blood-spinal cord barrier
  • vectors are modified to include capsids that cross the BBB or BSCB.
  • AAV with viral capsids that cross the blood brain barrier include AAV9 (Gombash et al., Front Mol Neurosci.2014; 7:81), AAVrh.10 (Yang, et al., Mol Ther.2014; 22(7): 1299-1309), AAV1 R6, AAV1 R7 (Albright et al., Mol Ther.
  • AAV comprises AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (including types AAV3A and AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), and AAV type 11 (AAV11) and any other AAV now known or later discovered.
  • AAV comprises AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (including types AAV3A and AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), and AAV type 11 (AAV11) and any other AAV now known or later discovered.
  • AAV comprises AAV type
  • the AAV genome comprises AAV1 (GenBank Accession No. NC_002077, AF063497) Adeno-associated NC_002077, AAV2 (GenBank Accession No. NC_001401), AAV3 (GenBank Accession No. NC_001729), AAV3B (GenBank Accession No. NC_001863), AAV4 (GenBank Accession No. NC_001829), AAV5 (GenBank Accession No. Y18065, AF085716), or AAV6 (GenBank Accession No. NC_001862).
  • the AAV comprises a capsid protein VP1 gene of Hu.48 (GenBank Accession No. AY530611), Hu 43 (GenBank Accession No.
  • AY530606 Hu 44 (GenBank Accession No. AY530607), Hu 46 (GenBank Accession No. AY530609), Hu.19 (GenBank Accession No. AY530584), Hu.20 (GenBank Accession No. AY530586), Hu 23 (GenBank Accession No. AY530589), Hu22 (GenBank Accession No. AY530588), Hu24 (GenBank Accession No. AY530590), Hu21 (GenBank Accession No. AY530587), Hu27 (GenBank Accession No. AY530592), Hu28 (GenBank Accession No. AY530593), Hu29 (GenBank Accession No. AY530594), Hu63 (GenBank Accession No.
  • AY530624 Hu64 (GenBank Accession No. AY530625), Hu13 (GenBank Accession No. AY530578), Hu56 (GenBank Accession No. AY530618), Hu57 (GenBank Accession No. AY530619), Hu49 (GenBank Accession No. AY530612), Hu58 (GenBank Accession No. AY530620), Hu34 (GenBank Accession No. AY530598), Hu35 (GenBank Accession No. AY53059), Hu45 (GenBank Accession No. AY530608), Hu47 (GenBank Accession No. AY530610), Hu51 (GenBank Accession No. AY530613), Hu52 (GenBank Accession No. AY53061), Hu T41 (GenBank Accession No.
  • Hu S17 (GenBank Accession No. AY695376), Hu T88 (GenBank Accession No. AY695375), Hu T71 (GenBank Accession No. AY695374), Hu T70 (GenBank Accession No. AY695373), Hu T40 (GenBank Accession No. AY695372), Hu T32 (GenBank Accession No. AY695371), Hu T17 (GenBank Accession No. AY695370), Hu LG15 (GenBank Accession No. AY695377), Hu9 (GenBank Accession No. AY530629), Hu10 (GenBank Accession No. AY530576), Hu11 (GenBank Accession No. AY530577), Hu53 (GenBank Accession No.
  • AY530615 Hu55 (GenBank Accession No. AY530617), Hu54 (GenBank Accession No. AY530616), Hu7 (GenBank Accession No. AY530628), Hu18 (GenBank Accession No. AY530583), Hu15 (GenBank Accession No. AY530580), Hu16 (GenBank Accession No. AY530581), Hu25 (GenBank Accession No. AY530591), Hu60 (GenBank Accession No. AY530622) Hu3 (GenBank Accession No. AY530595), Hu1 (GenBank Accession No. AY530575), Hu4 (GenBank Accession No. AY530602), Hu2 (GenBank Accession No.
  • AY530585 Hu61 (GenBank Accession No. AY530623), Rh62 (GenBank Accession No. AY530573), Rh48 (GenBank Accession No. AY530561), Rh54 (GenBank Accession No. AY530567), Rh55 (GenBank Accession No. AY530568), Rh35 (GenBank Accession No. AY243000), Rh38 (GenBank Accession No. AY530558), Hu66 (GenBank Accession No. AY530626), Hu42 (GenBank Accession No. AY530605), Hu67 (GenBank Accession No. AY530627), Hu40 (GenBank Accession No. AY530603), Hu41 (GenBank Accession No.
  • AY530604 Hu37 (GenBank Accession No. AY530600), Rh40 (GenBank Accession No. AY530559), Hu17 (GenBank Accession No. AY530582), Hu6 (GenBank Accession No. AY530621), Rh25 (GenBank Accession No. AY530557), Pi2 (GenBank Accession No. AY530554), Pi1 (GenBank Accession No. AY530553), Pi3 (GenBank Accession No. AY530555), Rh57 (GenBank Accession No. AY530569), Rh50 (GenBank Accession No. AY530563), Rh49 (GenBank Accession No. AY530562), Hu39 (GenBank Accession No.
  • AY530601 Rh58 (GenBank Accession No. AY530570), Rh61 (GenBank Accession No. AY530572), Rh52 (GenBank Accession No. AY530565), Rh53 (GenBank Accession No. AY530566), Rh51 (GenBank Accession No. AY530564), Rh64 (GenBank Accession No. AY530574), Rh43 (GenBank Accession No. AY530560), Rh1 (GenBank Accession No. AY530556), Hu14 (GenBank Accession No. AY530579), Hu31 (GenBank Accession No. AY530596), or Hu32 (GenBank Accession No. AY530597).
  • AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the BBB following intravenous injection. It transduces large sections of the central nervous system (CNS), thus permitting minimally invasive treatments (Naso et al., BioDrugs. 2017; 31 (4): 317), for example, as described in relation to clinical trials for the treatment of superior mesenteric artery (SMA) syndrome by AveXis (AVXS-101, NCT03505099) and the treatment of CLN3 gene-Related Neuronal Ceroid-Lipofuscinosis (NCT03770572).
  • SMA superior mesenteric artery
  • AveXis AVXS-101, NCT03505099
  • CLN3 gene-Related Neuronal Ceroid-Lipofuscinosis NCT03770572
  • AAVrh.10 was originally isolated from rhesus macaques and shows low seropositivity in humans when compared with other common serotypes used for gene delivery applications (Selot et al., Front Pharmacol.2017; 8: 441) and has been evaluated in clinical trials LYS-SAF302, LYSOGENE, and NCT03612869.
  • AAV1 R6 and AAV1 R7 two variants isolated from a library of chimeric AAV vectors (AAV1 capsid domains swapped into AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing significantly reduced hepatic and vascular endothelial transduction.
  • rAAVrh.8 also isolated from rhesus macaques, shows a global transduction of glial and neuronal cell types in regions of clinical importance following peripheral administration and also displays reduced peripheral tissue tropism compared to other vectors.
  • AAV-BR1 is an AAV2 variant displaying the NRGTEWD (SEQ ID NO: 42) epitope that was isolated during in vivo screening of a random AAV display peptide library. It shows high specificity accompanied by high transgene expression in the brain with minimal off-target affinity (including for the liver) (Korbelin et al., EMBO Mol Med.2016; 8(6): 609).
  • AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 43), transduces neurons in the enteric nervous system, and strongly transduces peripheral sensory afferents entering the spinal cord and brain stem.
  • AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 44). It transfers genes throughout the CNS with higher efficiency than AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.
  • AAV-PPS an AAV2 variant crated by insertion of the DSPAHPS (SEQ ID NO: 45) epitope into the capsid of AAV2, shows a dramatically improved brain tropism relative to AAV2.
  • Formulations Artificial expression constructs and vectors of the present disclosure (referred to herein as physiologically active components) can be formulated with a carrier that is suitable for administration to a cell, tissue slice, animal (e.g., mouse, non-human primate), or human.
  • Physiologically active components within compositions described herein can be prepared in neutral forms, as freebases, or as pharmacologically acceptable salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are 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.
  • 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, trimethyl
  • Carriers of physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, and the like.
  • the use of such carriers for physiologically active components is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active components, it can be used with compositions as described herein.
  • pharmaceutically-acceptable carriers refer to carriers that do not produce an allergic or similar untoward reaction when administered to a human, and in some embodiments, when administered intravenously (e.g., at the retro-orbital plexus).
  • compositions can be formulated for intravenous, intraocular, intravitreal, parenteral, subcutaneous, intracerebro-ventricular, intramuscular, intracerebroventricular, intravenous injection into the cisterna magna (ICM), intrathecal, intraspinal, oral, intraperitoneal, oral or nasal inhalation, or by direct injection in or application to one or more cells, tissues, or organs.
  • Compositions may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and/or nanoparticles.
  • lipid nanoparticle refers to a vesicle formed by one or more lipid components.
  • Lipid nanoparticles are typically used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API).
  • lipid nanoparticle compositions for such delivery are composed of synthetic ionizable or cationic lipids, phospholipids (especially compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids.
  • the sum composition of lipids typically dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cell uptake properties.
  • the “liposome” refers to lipid molecules assembled in a spherical configuration encapsulating an interior aqueous volume that is segregated from an aqueous exterior. Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery are typically composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.
  • ionizable lipid refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.
  • Ionizable lipids are also referred to as cationic lipids herein.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.
  • conjugated lipid refers to a lipid molecule conjugated with a non- lipid molecule, such as a PEG, polyoxazoline, polyamide, or polymer (e g., cationic polymer).
  • excipient refers to pharmacologically inactive ingredients that are included in a formulation with the API, e.g., ceDNA and/or lipid nanoparticles to bulk up and/or stabilize the formulation when producing a dosage form.
  • General categories of excipients include, for example, bulking agents, fillers, diluents, antiadherents, binders, coatings, disintegrants, flavours, colors, lubricants, glidants, sorbents, preservatives, sweeteners, and products used for facilitating drug absorption or solubility or for other pharmacokinetic considerations.
  • the formation and use of liposomes is generally known to those of skill in the art.
  • Liposomes have been developed with improved serum stability and circulation half-times (see, for instance, U.S. Pat. No.5,741 ,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (see, for instance U.S. Pat. Nos.5,567,434; 5,552, 157; 5,565,213; 5,738,868; and 5,795,587). The disclosure also provides for pharmaceutically acceptable nanocapsule formulations of the physiologically active components.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12): 1113-1128, 1998; Quintanar-Guerrero et al, Pharm Res.15(7): 1056- 1062, 1998; Quintanar-Guerrero et al., J. Microencapsul.15(1 ): 107-119, 1998; Douglas et al, Crit Rev Ther Drug Carrier Syst 3(3):233- 261 , 1987).
  • ultrafine particles can be designed using polymers able to be degraded in vivo.
  • Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be easily made, as described in Couvreur et al., J Pharm Sci 69(2): 199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst.5(1)1-20, 1988; zur Muhlen et al., EurJ Pharm Biopharm, 45(2): 149-155, 1998; Zambau x et al., J Control Release 50(1-3):31- 40, 1998; and U.S. Pat. No.5,145,684.
  • Injectable compositions can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No.5,466,468).
  • the form is sterile and fluid to the extent that it can be delivered by syringe. In some embodiments, it is stable under the conditions of manufacture and storage, and optionally contains one or more preservative compounds 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
  • suitable mixtures thereof e.g., vegetable oils.
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • the preparation will include an isotonic agent(s), for example, sugar(s) or sodium chloride.
  • Prolonged absorption of the injectable compositions can be accomplished by including in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.
  • Injectable compositions can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. As indicated, under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
  • Sterile compositions can be prepared by incorporating the physiologically active component in an appropriate amount of a solvent with other optional ingredients (e.g., as enumerated above), followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized physiologically active components into a sterile vehicle that contains the basic dispersion medium and the required other ingredients (e.g., from those enumerated above).
  • preferred methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the physiologically active components plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions may be in liquid form, for example, as solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non- aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives
  • compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Tablets may be coated by methods well-known in the art.
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • Inhalable compositions can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • Compositions can also include microchip devices (U.S. Pat. No.5,797,898), ophthalmic formulations (Bourlais et al, Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Pat. No.5,770,219 and U.S. Pat. No.5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899). Supplementary active ingredients can also be incorporated into the compositions.
  • compositions can include at least 0.1 % of the physiologically active components or more, although the percentage of the physiologically active components may, of course, be varied and may conveniently be between 1 or 2% and 70% or 80% or more or 0.5-99% of the weight or volume of the total composition.
  • the amount of physiologically active components in each physiologically-useful composition may be prepared in such a way that a suitable dosage will 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 will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of compositions and dosages may be desirable.
  • compositions for administration to humans, should meet sterility, pyrogenicity, and the general safety and purity standards as required by United States Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.
  • FDA United States Food and Drug Administration
  • the present disclosure includes cells including an artificial expression construct described herein.
  • a cell that has been transformed with an artificial expression construct can be used for many purposes, including in neuroanatomical studies, assessments of functioning and/or non-functioning proteins, and drug screens that assess the regulatory properties of enhancers.
  • WO 91/13150 describes a variety of cell lines, including neuronal cell lines, and methods of producing them.
  • WO 97/39117 describes a neuronal cell line and methods of producing such cell lines.
  • the neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.
  • a "neural cell” refers to a cell or cells located within the central nervous system, and includes neurons and glia, and cells derived from neurons and glia, including neoplastic and tumor cells derived from neurons or glia.
  • a "cell derived from a neural cell” refers to a cell which is derived from or originates or is differentiated from a neural cell.
  • neuronal describes something that is of, related to, or includes, neuronal cells. Neuronal cells are defined by the presence of an axon and dendrites.
  • neuronal-specific refers to something that is found, or an activity that occurs, in neuronal cells or cells derived from neuronal cells, but is not found in or occur in, or is not found substantially in or occur substantially in, non-neuronal cells or cells not derived from neuronal cells, for example glial cells such as astrocytes or oligodendrocytes.
  • Methods to differentiate stem cells into neuronal cells include replacing a stem cell culture media with a media including basic fibroblast growth factor (bFGF) heparin, an N2 supplement (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin and poly ornithine.
  • bFGF basic fibroblast growth factor
  • N2 supplement e.g., transferrin, insulin, progesterone, putrescine, and selenite
  • laminin e.g., transferrin, insulin, progesterone, putrescine, and selenite
  • laminin e.g., laminin
  • poly ornithine e.g., transferrin, insulin, progesterone, putrescine, and selenite
  • Neurol.217:407-16 describes a procedure to produce GABAergic neurons. This procedure includes exposing stem cells to all-trans-RA for three days. After subsequent culture in serum-free neuronal induction medium including IMeurobasai medium supplemented with B27, bFGF and EGF, 95% GABA neurons develop.
  • serum-free neuronal induction medium including IMeurobasai medium supplemented with B27, bFGF and EGF.
  • U.S. Publication No.2012/0329714 describes use of prolactin to increase neural stem cell numbers while U.S. Publication No.2012/0308530 describes a culture surface with amino groups that promotes neuronal differentiation into neurons, astrocytes and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by a variety of extracellular factors.
  • BDNF brain derived growth factor
  • bFGF fibroblast growth factor
  • NT-3 Neurotrophin-3
  • NT-4 Neurotrophin-4
  • CNTF ciliary neurotrophic factor
  • BMP-2 U.S. Pat. Nos.5,948,428 and 6,001 ,654
  • isobutyl 3- methylxanthine leukemia inhibitory growth factor (LIF; U.S.
  • Patent No.6,103,530 somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No.6,395,546); tetanus toxin; and transforming growth factor-a and TGF-b (U.S. Pat. Nos.5,851 ,832 and 5,753,506).
  • Transgenic animals are described below. Cell lines may also be derived from such transgenic animals.
  • transgenic Animals Another aspect of the disclosure includes transgenic animals, the genome of which contains an artificial expression construct including regulatory elements (e.g., SEQ ID NOs: 7-14 or 60-65) operatively linked to a heterologous gene.
  • the genome of a transgenic animal includes the Enh98-pBG-GFP, Enh57-pBG-GFP, Enh98-pChat-GFP, or Enh57- pChat –GFP.
  • a transgenic animal when a non-integrating vector is utilized, includes an artificial expression construct including regulatory elements (e.g., SEQ ID NO: 7-14 or 60-65) and/or Enh98- pBG-GFP, Enh57-pBG-GFP, Enh98-pChat-GFP, or Enh57- pChat –GFP within one or more of its cells.
  • regulatory elements e.g., SEQ ID NO: 7-14 or 60-65
  • Transgenic animals may be of any nonhuman species, but preferably include nonhuman primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens, and rodents such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.
  • construction of a transgenic animal results in an organism that has an engineered construct present in all cells in the same genomic integration site.
  • cell lines derived from such transgenic animals will be consistent in as much as the engineered construct will be in the same genomic integration site in all cells and hence will suffer the same position effect variegation.
  • introducing genes into cell lines or primary cell cultures can give rise to heterologous expression of the construct.
  • the artificial expression constructs of this disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art.
  • the artificial expression construct is introduced into cultured murine embryonic stem cells.
  • Transformed ES cells are then injected into a blastocyst from a host mother and the host embryo re-implanted into the mother. This results in a chimeric mouse whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo.
  • mice from which the cultured ES cells used for transgenesis are derived are chosen to have a different coat color from the host mouse into whose embryos the transformed cells are to be injected. Chimeric mice will then have a variegated coat color. As long as the germ-line tissue is derived, at least in part, from the genetically modified cells, then the chimeric mice be crossed with an appropriate strain to produce offspring that will carry the transgene.
  • sonophoresis e.g., ultrasound, as described in U.S.
  • a composition including a physiologically active component described herein is administered to a subject that has a motor neuron disease or disorder.
  • the term “motor neuron disease or disorder” refers to a disease or disorder involving the abnormal function of motor neurons resulting from abnormal protein expression, e.g., loss-of-function SMN1 protein.
  • the disease or disorder is Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA) (e.g., non-5q SMA, Spinal muscular atrophy with congenital bone fractures, Autosomal dominant spinal muscular atrophy, and lower extremity-predominant 2 (SMALED2)), Distal hereditary motor neuropathy (dHMN) (e.g., autosomal dominant, autosomal recessive, type 1, 2, 4, 5, 6, 7, 7b), Triosephosphate isomerase deficiency (TIP), Hereditary spastic paraplegia (HSP) (also known as familial spastic paraparesis (FSP))(e.g., autosomal dominant or autosomal recessive or X-linked), amyo
  • SBMA Spinal-
  • symptoms associated with the motor neuron disease or disorder are muscle weakness and decreased muscle tone, limited mobility, breathing problems, problems eating and swallowing, delayed gross motor skills, congenital hemolytic anemia, progressive neuromuscular dysfunction, spontaneous tongue movements, behavioral/cognitive symptoms, cerebellar degeneration, or scoliosis.
  • the disclosure includes the use of the artificial expression constructs described herein to modulate expression of a heterologous gene which is either partially or wholly encoded in a location downstream to that enhancer in an engineered sequence.
  • dosages for any one subject depends upon many factors, including the subject's size, surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the compounds of the disclosure will vary, but, in some embodiments, a dose could be from 10 5 to 10 100 copies of an artificial expression construct of the disclosure. In some embodiments, a patient receiving intravenous, intraspinal, retro-orbital, or intrathecal administration can be infused with from 10 6 to 10 22 copies of the artificial expression construct.
  • An "effective amount" is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes.
  • Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay.
  • constructs disclosed herein can be utilized to treat spinal muscular atrophy (SMA).
  • the methods reduce or prevent muscle weakness, or symptoms thereof in a patient in need thereof.
  • the methods provided may reduce or prevent one or more symptoms associated with SMA, e.g., muscle weakness and decreased muscle tone, limited mobility, breathing problems, problems eating and swallowing, delayed gross motor skills, spontaneous tongue movements, or scoliosis.
  • the amount of expression constructs and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings.
  • the administration of effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide an effect in the subject.
  • a single administration such as for example, a single injection of sufficient numbers of infectious particles to provide an effect in the subject.
  • the number of infectious particles administered to a mammal may be 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or even higher, infectious particles/ml given either as a single dose or divided into two or more administrations as may be required to achieve an intended effect.
  • compositions disclosed herein either by pipette, retro-orbital injection, subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricular (ICV), intravenous injection into the cisterna magna (ICM), intracerebro- ventricularly, intramuscularly, intrathecally, intraspinally, orally, intraperitoneally, by oral or nasal inhalation, or by direct application or injection to one or more cells, tissues, or organs.
  • ICV intracerebroventricular
  • ICM intravenous injection into the cisterna magna
  • intracerebro- ventricularly intramuscularly
  • intrathecally intraspinally
  • intraperitoneally by oral or nasal inhalation, or by direct application or injection to one or more cells, tissues, or organs.
  • Kits Kits and commercial packages contain an artificial expression construct described herein.
  • the expression construct can be isolated.
  • the components of an expression product can be isolated from each other.
  • the expression product can be within a vector, within a viral vector, within a cell, within a tissue slice or sample, and/or within a transgenic animal.
  • kits may further include one or more reagents, restriction enzymes, peptides, therapeutics, pharmaceutical compounds, or means for delivery of the compositions such as syringes, injectables, and the like.
  • Embodiments of a kit or commercial package will also contain instructions regarding use of the included components, for example, in basic research, electrophysiological research, neuroanatomical research, and/or the research and/or treatment of a disorder, disease or condition.
  • the constructs were packaged into adeno-associated virus AAV PhP-eB.
  • the AAVs were injected into the cerebral ventricle of ChAT-Cre; Sun1-GFP B6/C57 newborn mice in which nuclei of Chat+ spinal cord motor-neurons are labeled enabling isolation by FACS.
  • rAAV-GRE constructs were injected into the lateral ventricle of newborn mice at a titer of 3 x 10 13 genome copies/mL (2-4 ⁇ L). Two weeks following transduction, animals were sacrificed, the spinal cord and dorsal root ganglia (DRG) dissected. Mice were sacrificed and perfused with 4% PFA followed by PBS. The brain was dissected out of the skull and post-fixed with 4% PFA for 1-3 days at 4°C. The brain was mounted on the vibratome (LeicaTM VT1000S) and coronally sectioned into 100 ⁇ m slices. Sections containing VI were arrayed on glass slides and mounted using DAPI Fluoromount-G (Southern BiotechTM).
  • Sections containing V1 were imaged on a LeicaTM SPE confocal microscope using an ACS APO 10x/0.30 CS objective. Tiled VI cortical areas of -1.2 mm by -0.5 mm were imaged at a single optical section to avoid counting the same cell across multiple optical sections. Channels were imaged sequentially to avoid any optical crosstalk.
  • Spinal cord motor neuron nuclei were isolated by FACS. RNA-sequencing of spinal cord motor neurons, spinal cord non-motor neurons and DRG cells were used to measure the expression of enhancer-driven AAV vectors across these tissues. Immunostaining and/or fluorescent in situ hybridization was used to identify the cell types in which the GFP expression was observed.
  • RNA-seq Summary RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) (Buenrostro et al., 2015) were used to generate a quantitative, genome-wide dataset of chromatin accessibility in lower motor neurons of the spinal cord in adult mouse.
  • GREs gene regulatory elements
  • IHC immunohistochemistry
  • cis-regulatory element identification and selection To identify motor neuron specific enhancers (Enh), also termed as cis-regulatory elements (CREs) or gene regulatory elements (GREs), spinal motor neuron nuclei were tagged and immunopurified using the Chat-Cre; Sun1-sfGFP-6xMyc mouse line cross (Chat-Sun1, Mo et al., 2015), which stably marks the nuclear envelope of Chat-expressing cells in animals of age E12.5 or older (Rossi et al.2011; Patel et al.2021).
  • CREs motor neuron specific enhancers
  • GREs gene regulatory elements
  • this population comprises skeletal motor neurons (target) and the off-target visceral motor neuron and cholinergic interneuron populations (Sathyamurthy et al., 2018).
  • the composition of the immunolabeled population (Chatpos) by two complementary approaches was investigated. Confocal microscopy of immunohistochemically labeled Chat and GFP confirmed restriction of GFP in Sun1-Chat animals to skeletal motor neurons, identified by their distinctive large somata, positive ChAT co-staining, and anatomic localization in the ventral horn (FIG.9B), as opposed to pericanalicular (interneuron) or in the lateral horn (visceral motor neuron).
  • RNA-seq of Chatpos and putatively motor neuron-depleted flow through (Chatneg) nuclei was performed to identify differentially expressed genes across these two populations.
  • Expression of the cholinergic marker genes Slc5a8 and Chat was enriched in the Chatpos population relative to Chatneg, while excitatory (Slc17a8, Slc17a6) and inhibitory (Gad1, Slc6a5) interneuron, oligodendrocyte (Mbp, Mobp), astrocyte (Gfap, Aqp4), microglia (Cx3cr1, Tmem2), and endothelial (Cldn5) marker genes (Sathyamurthy et al.2018; Alkaslasi et al.2021; Rhee et al.2016; Patel et al.2021) showed no such enrichment, confirming successful purification of Chat-expressing populations relative to the other major cell types of the spinal cord (FIG.9C).
  • quality control metrics including nucleosomal ATAC-seq fragment size distribution, high irreproducible discovery rate (IDR), and appropriately higher correlation among than across conditions (FIG.13C - Fragment distribution, FIG.13D - ATAC-seq PCA, FIG.13E ATAC-seq correlation)(Landt et al.2012).
  • Enhs were amplified from wild-type mouse genomic DNA and incorporated into a GFP reporter AAV2 vector backbone as described previously: 5’-ITR-ENH-pBG-GFP-barcode- WPRE-polyA-ITR-3’ (Hrvatin et al., 2019) (FIG.10C - vector map, administration route).
  • a nuclear localization sequence (NLS) was incorporated into all AAV vector constructs to increase GFP overlap with ChAT and facilitate signal quantification.
  • the Enh98 and Enh119 constructs drove reporter expression in 97.0% and 91.1% in the on-target NeuN+ ChAT+ skeletal motor neuron population of the ventral horn, with off- target rates of 15.6 and 3.9% in NeuN+ ChAT- neurons (FIG.11A - representative images, FIG.11B - motor neuron fraction).
  • On-target signal intensity in the Enh98 and Enh119 conditions was significantly greater than off-target populations (.05 and .02), and greater than on-target saline or ⁇ Enh (.03 and .09) as well.
  • FIG.11C - motor neuron intensity Enhs / Motor neuron intensity CAG In the previous analysis, image window parameters were selected to emphasize intensity differences across the Enh constructs, which led to truncation of CAG signal. To compare the elements against CAG directly, alternate parameters that captured the full dynamic range of CAG signal intensity were used to evaluate the CAG, Enh98, and ⁇ ENH conditions (FIG.11C - CAG windowed images).
  • Enh98 demonstrated low off-target expression in the DRG (4.5%) comparable to the non-functional Enh57 construct (5.2%), but Enh119 failed to attain this same level of specificity with a positivity rate of 37.0%, suggesting potentially distinct mechanisms of transcriptional regulation between Enh98 and Enh119. Both constructs demonstrate significantly reduced off target DRG expression relative to CAG. Mechanistic investigation of Enh98 and identification of functional TF binding motifs Having confirmed Enh98 as the more motor neuron-selective of the hits from the screen, the key regions conferring this feature to the mouse Enh98 (mEnh98) sequence needed to be identified.
  • TF binding motifs in the JASPAR mouse database present within the full 696 bp sequence of mEnh98 were identified, and an adjusted p-value threshold of .05 was used to determine confidence in motif matching.
  • motifs whose associated TFs had non- zero and significant enrichment of expression in the purified Chatpos population (q ⁇ .05) relative to Chatneg were identified, yielding the JASPAR motifs MA0704.1 (Lhx4 and Mnx1), MA0914.1 (Isl2), MA0141.1/2 (Esrrb), MA0100.2 (Myb), and MA0518.1 (Stat4) (FIG.12A).
  • TFs all have been demonstrated to either be motor neuron defining during differentiation, or markers of motor neuron subtypes. However, none of them are solely expressed in motor neurons, and the combination of some of these factors play long understood roles in inhibitory interneuron development as well. All TF binding sites identified this way lay within a core 280-bp region of mEnh98, leading to the hypothesis that this core region was sufficient and necessary for motor neuron-selective Enh98 activity. To test this hypothesis, nine truncated or internally deleted mEnh98 vector constructs were generated: A, B, C, D, E, F, 2KO, and 5KO (FIG.12B and Table 2). Of particular note, the F construct corresponds to the core region.
  • the 2KO construct lacks only the two binding sites of TFs most associated with motor neuron identity (Isl2 and Mnx1).
  • the full length mEnh98 construct drove GFP expression in 80-90% of ChAT+ neurons (FIG. 12C).
  • Enh98 has about a 9.5-fold greater expression in the ChAT+ neurons than in ChAT- neurons (p ⁇ 2.2e-16).
  • p ⁇ 2.2e-16 We see a loss of expression in ChAT+ neurons and therefore a reduction in specificity for ChAT+ vs. ChAT- neurons for the D, 2KO, and 5KO vector constructs.
  • the core-containing constructs (A, C, E, F) roughly preserved expression strength of full-length Enh98: 9.6-fold (p ⁇ 2.2e-16) and 25-fold (p ⁇ 2.2e-16) greater expression in ChAT+ neurons than in ChAT- neurons, respectively.
  • the truncated and mutated constructs retain a similar background-like level of expression to Enh98 (FIG.12E).
  • the CAG construct had a 470-fold greater expression in the DRG (FIG.12F).
  • Enh98 truncating or knocking out key sequences in Enh98 did not amplify expression in non-target tissues such as the DRG suggests that the primary mechanism of how Enh98 achieves motor neuron-specific expression is by selectively amplifying the expression in the motor neurons.
  • the shorter Enh98 core region reproduced the desirable features of the full-length Enh98, tandem repeats of the central core sequence, or the key TFBS, were tested compared to the expression profile of Enh98.
  • mEnh98 mouse Enh98
  • hEnh98 analogous human Enh98
  • hEnh98 772 bp
  • synthetic constructs were cloned and packaged into AAV: triplicate concatemers of the core TFBS-containing sequences from each of mouse and human Enh98 (3mCORE (SEQ ID NO: 84), 3hCORE (SEQ ID NO: 85)) and a similar triplicate concatemer of a synthetic 129 bp sequence (SEQ ID NO: 83) comprising just the mCORE TF motifs, with relative order and orientation preserved and all native inter-motif sequences replaced by 15 bp linkers (3mTFBS (SEQ ID NO: 86), FIG.15B).
  • the 3mCORE sequence drove significantly higher expression in both on- and off-target populations of the spinal cord than mEnh98, suggesting that tandem enhancers are conceptually valid approach to amplification of expression levels.
  • this increased expression came at the expense of specificity.
  • the 3TFBS construct failed to attain the fraction of on-target expression observed with the full length mEnh98, but strength mean expression was comparable to mEnh98.
  • the relationship between whole and 3x CORE sequences was similar for the human sequences as to the mouse – reduced specificity, with increased mean expression intensity per cell in both on- and off-target populations (FIGs.15C-15E).
  • Enh98- and Enh119-based constructs exhibited no increase in GFP expression compared to the enhancerless negative control AAV (FIGs.16A-16C).
  • the reason for the lack of GRE-driven expression in this human MN culture system remains unclear.
  • the extremely high viral MOI needed to obtain even modest GFP expression using the CAG promoter suggests that the specific iPSC-derived MN differentiation protocol employed might have resulted in cells that were too immature for effective targeting using cell-type-restricted GREs.
  • Enh98- and Enh119-derived synthetic vectors were further evaluated using established in vivo mouse paradigm. Possibly, Enh98 and Enh119 activity in human MNs will require the use of an alternative cultures system.
  • in vivo data using these GRE-AAVs in a primate model will provide important validation of the conserved function of these cell-type-restricted MN-targeting GREs.

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Abstract

La technologie décrite dans la présente invention concerne un élément régulateur de gène, par exemple, un activateur, des vecteurs le contenant, des vecteurs adéno-associés le contenant et des cellules contenant lesdits vecteurs. Dans un autre aspect, l'invention concerne des procédés de traitement d'une maladie ou d'un trouble des motoneurones consistant à administrer lesdits vecteurs, par exemple, des vecteurs VAA. Dans un autre aspect, l'invention concerne des compositions d'acide nucléique contenant l'élément régulateur de gène selon l'invention.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008073303A2 (fr) * 2006-12-07 2008-06-19 Switchgear Genomics Éléments de régulation transcriptionnelle de voies biologiques, outils, et procédés
US20190160091A1 (en) * 2016-05-18 2019-05-30 Voyager Therapeutics, Inc. Compositions and methods for treating huntington's disease
WO2023288086A2 (fr) * 2021-07-16 2023-01-19 President And Fellows Of Harvard College Activateurs dirigeant l'expression de neurones moteurs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008073303A2 (fr) * 2006-12-07 2008-06-19 Switchgear Genomics Éléments de régulation transcriptionnelle de voies biologiques, outils, et procédés
US20190160091A1 (en) * 2016-05-18 2019-05-30 Voyager Therapeutics, Inc. Compositions and methods for treating huntington's disease
WO2023288086A2 (fr) * 2021-07-16 2023-01-19 President And Fellows Of Harvard College Activateurs dirigeant l'expression de neurones moteurs

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