WO2024091495A1 - Compositions et méthodes de traitement de la rétinite pigmentaire - Google Patents

Compositions et méthodes de traitement de la rétinite pigmentaire Download PDF

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WO2024091495A1
WO2024091495A1 PCT/US2023/035795 US2023035795W WO2024091495A1 WO 2024091495 A1 WO2024091495 A1 WO 2024091495A1 US 2023035795 W US2023035795 W US 2023035795W WO 2024091495 A1 WO2024091495 A1 WO 2024091495A1
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nucleic acid
acid sequence
sequence
seq
aav
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PCT/US2023/035795
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English (en)
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Edward Raymond Guilmette
Meihua JU
Ashley MACKEY
Devin Scott MCDOUGALD
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Biogen Ma Inc.
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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • Retinitis pigmentosa is a degenerative genetic disease that affects cells in the retina. Retinitis pigmentosa is characterized by a decrease or loss of vision. Initial symptoms of retinitis pigmentosa have been reported to include impaired vision in low light settings, e.g., at night. Further progression may involve loss of peripheral vision followed by loss of central vision and subsequently, total blindness. Mutations in rhodopsin (RHO) have been reported as having a causative role in some instances of retinal degeneration, e.g . retinitis pigmentosa. There are currently no approved treatments for retinitis pigmentosa associated with mutant RHO, and accordingly, there is a need for therapeutic modalities to treat RHO-associated retinal degeneration.
  • RHO rhodopsin
  • the present disclosure provides technologies (e.g., inhibitory nucleic acids, vectors, compositions, methods, etc.) for treating various conditions, disorders or diseases associated with rhodopsin (RHO).
  • the present disclosure provides inhibitory nucleic acids that can hybridize to a RHO transcript.
  • the present disclosure provides vectors comprising one or more inhibitor ⁇ - nucleic acids that can hybridize to a RHO transcript.
  • the present disclosure provides inhibitory nucleic acids and vectors comprising one or more inhibitory nucleic acids that when administered or delivered to a system comprising or expressing a RHO transcript can reduce the level of a RHO transcript.
  • a provided technology reduces levels of a RHO transcript and/or polypeptide in a system.
  • the present disclosure provides technologies for preventing and/or treating various conditions, disorders or diseases associated with RHO.
  • the present disclosure provides an inhibitory nucleic acid that inhibits expression of a target nucleic acid sequence .
  • the inhibitory nucleic acid is a miRN A.
  • the target nucleic acid sequence is a RHO transcript.
  • the target nucleic acid sequence is a mutant RHO transcript.
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a modified AAV genome and a capsid.
  • rAAV recombinant adeno-associated virus
  • the modified AAV genome comprises: (i) a 5 ’ inverted terminal repeat (ITR); (ii) a promoter; (iii) a synthetic intron; (iv) a miRNA sequence located within the synthetic intron, wherein the miRNA sequence comprises a guide strand sequence that targets a pathogenic nucleic acid sequence encoding a pathogenic polypeptide, and a scaffold sequence: (v) a therapeutic nucleic acid sequence encoding a therapeutic polypeptide: and (vi) a 3' inverted terminal repeat (ITR).
  • the miRNA sequence and the therapeutic nucleic acid sequence are operably linked to the promoter.
  • the pathogenic polypeptide and the therapeutic polypeptide share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity.
  • the present disclosure provides a recombinant adeno-associated vims (rAAV) vector comprising a modified AAV genome and a capsid.
  • the modified AAV genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) a therapeutic nucleic acid sequence that encodes a therapeutic polypeptide; (iv) a synthetic intron; (v) a miRNA sequence located within the synthetic intron, wherein tire miRNA sequence comprises a guide strand sequence that targets a pathogenic nucleic acid sequence encoding a pathogenic polypeptide, and a scaffold sequence; and (vi) a 3' inverted terminal repeat (ITR).
  • ITR inverted terminal repeat
  • the miRNA sequence and the therapeutic nucleic acid sequence are operably linked to the promoter.
  • the pathogenic polypeptide and the therapeutic polypeptide share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity.
  • the modified AAV genome comprises a 5 ’ ITR. In some embodiments, the modified AAV genome comprises a 3’ ITR. In some embodiments, the modified AAV genome comprises a 5’ ITR and a 3' ITR. In some embodiments, the 5' ITR comprises or is an AAV2 5' ITR. In some embodiments, tire 3’ ITR comprises or is an AAV2 3’ ITR. In some embodiments, the 5’ ITR comprises or is the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 3’ ITR comprises or is the nucleic acid sequence of SEQ ID NO: 3.
  • the modified AAV genome comprises a promoter.
  • the promoter comprises or is a human RHO promoter or portion thereof.
  • the promoter comprises or is a human rhodopsin kinase (GRK1) promoter or portion thereof.
  • the promoter comprises or is a CAG promoter or portion thereof.
  • the promoter comprises or is the nucleic acid sequence of SEQ ID NO: 4, 5, 6, or 7.
  • tire promoter comprises or is tire nucleic acid sequence of SEQ ID NO: 4.
  • tire promoter comprises or is the nucleic acid sequence of SEQ ID NO: 5.
  • the promoter comprises or is the nucleic acid sequence of SEQ ID NO: 6.
  • the promoter comprises or is the nucleic acid sequence of SEQ ID NO: 7.
  • the modified AAV genome comprises a synthetic intron.
  • the synthetic intron comprises a splice donor site.
  • the synthetic intron comprises a splice acceptor site.
  • the synthetic intron comprises a splice donor site and a splice acceptor site.
  • the splice donor site comprises or is the nucleic acid sequence of SEQ ID NO: 8.
  • the splice donor site comprises or is the nucleic acid sequence of SEQ ID NO: 9.
  • the modified AAV genome comprises a miRNA sequence.
  • the miRNA sequence comprises a guide strand sequence.
  • the miRNA sequence comprises a scaffold sequence.
  • the miRNA sequence comprises a guide strand sequence and a scaffold sequence.
  • the guide strand sequence targets a pathogenic nucleic acid sequence encoding a pathogenic polypeptide.
  • the pathogenic polypeptide comprises or is rhodopsin.
  • the pathogenic polypeptide comprises or is mutant rhodopsin.
  • the guide strand sequence comprises or is a nucleic acid sequence comprising about 10 or more (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, or 30) contiguous nucleobases of a RHO transcript. In some embodiments, the guide strand sequence comprises or is a nucleic acid sequence comprising about 10 or more (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous nucleobases of a mutant RHO transcript.
  • the guide strand sequence comprises or is a nucleic acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the guide strand sequence comprises or is tire nucleic acid sequence of SEQ ID NO: 1.
  • the scaffold sequence comprises or is amiR-E scaffold sequence, amiR-155 scaffold sequence, or an UltramiR scaffold sequence.
  • the scaffold sequence comprises or is a miR-E scaffold sequence.
  • the scaffold sequence comprises or is a miR-155 scaffold sequence.
  • the scaffold sequence comprises or is an UltramiR scaffold sequence. In some embodiments, the scaffold sequence comprises or is the nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In some embodiments, the scaffold sequence comprises or is the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the scaffold sequence comprises or is the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the scaffold sequence comprises or is the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the miRNA sequence comprises or is the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. In some embodiments, the miRNA sequence comprises or is the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the miRNA sequence comprises or is the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the miRNA sequence comprises or is the nucleic acid sequence of SEQ ID NO: 15.
  • the miRNA sequence is located within tire synthetic intron.
  • the splice donor site is upstream (5’) of the miRNA sequence.
  • the splice acceptor site is downstream (3’) of the miRNA sequence.
  • the splice donor site is upstream (5’) of the miRNA sequence and the splice acceptor site is downstream (3’) of the miRNA sequence.
  • the modified AAV genome comprises a therapeutic nucleic acid sequence encoding a therapeutic polypeptide.
  • the therapeutic nucleic acid sequence encoding a therapeutic polypeptide comprises or is resistant to targeting by a guide strand sequence.
  • the therapeutic polypeptide comprises or is wild-type (WT) rhodopsin.
  • WT wild-type
  • the therapeutic nucleic acid sequence encoding a therapeutic polypeptide comprises about 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations such that the therapeutic nucleic acid sequence is resistant to targeting by tire guide strand sequence.
  • the therapeutic nucleic acid sequence encoding a therapeutic polypeptide comprises or is resistant to targeting by a guide strand sequence that comprises or is the nucleic acid sequence of SEQ ID NO: 1.
  • the therapeutic nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO: 16.
  • the therapeutic polypeptide comprises or is rhodopsin.
  • the modified AAV genome comprises a 3’ untranslated region (UTR) element.
  • the 3' UTR element comprises a woodchuck hepatitis vims post- transcriptional regulator element (WPRE) or polyadenylation signal sequence, or combination thereof.
  • WPRE woodchuck hepatitis vims post- transcriptional regulator element
  • the WPRE comprises or is tire nucleic acid sequence of SEQ ID NO: 17.
  • the polyadenylation signal sequence comprises or is a human growth hormone (hGH) polyadenylation signal sequence, a bovine growth hormone (bGH) polyadenylation signal sequence, a PGK polyadenylation signal sequence, a simian vims 40 (SV40) polyadenylation signal sequence, a human beta globin polyadenylation signal sequence, or a synthetic polyadenylation signal sequence.
  • the polyadenylation signal sequence comprises or is an hGH polyadenylation signal sequence.
  • the polyadenylation signal sequence comprises or is a bGH polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence comprises or is a PGK polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence comprises or is an SV40 polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence comprises or is a human beta globin polyadcnylation signal sequence. In some embodiments, the polyadenylation signal sequence comprises or is a synthetic polyadenylation signal sequence.
  • the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the polyadenylation signal comprises or is the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the polyadenylation signal sequence comprises or is the nucleic acid sequence of SEQ ID NO: 23.
  • the synthetic intron is located upstream (5’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the synthetic intron is located downstream (3’) of the promoter and upstream (5’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the synthetic intron is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the synthetic intron is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide and upstream (5 ’ ) of the 3 ’ UTR element.
  • the synthetic intron is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide and upstream (5’) of the WPRE. In some embodiments, the synthetic intron is located downstream (3’) of the WPRE and upstream (5‘) of the polyadenylation signal sequence. In some embodiments, the synthetic intron is located downstream (3’) of the polyadenylation signal sequence and upstream (5’) of the 3’ ITR.
  • the miRNA sequence is located upstream (5 ’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the miRNA sequence is located downstream (3’) of the promoter and upstream (5’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the miRNA sequence is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the miRNA sequence is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide and upstream (5’) of the 3' UTR element.
  • the miRNA sequence is located downstream (3’) of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide and upstream (5’) of the WPRE. In some embodiments, the miRNA sequence is located downstream (3’) of the WPRE and upstream (5’) of the polyadenylation signal sequence. In some embodiments, the miRNA sequence is located downstream (3’) of the polyadenylation signal sequence and upstream (5’) of the 3’ ITR.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 11.
  • the modified AAV genome comprises tire nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16. SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 13.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 15.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 11.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 17. SEQ ID NO: 18, and SEQ ID NO: 13.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 15.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 11.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 15.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 7, SEQ ID NO: 16. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 11.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 13.
  • the modified AAV genome comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 15.
  • the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.
  • the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 24.
  • the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 25.
  • the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the modified AAV genome comprises or is tire nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 32.
  • the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the modified AAV genome comprises or is the nucleic acid sequence of SEQ ID NO: 40.
  • the rAAV vector comprises a capsid.
  • the capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid.
  • the expression level of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide from a rAAV vector comprising a modified AAV genome comprising a synthetic intron is increased relative to an expression level of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide from a rAAV vector comprising a modified AAV genome not comprising a synthetic intron.
  • the expression level of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide from tire rAAV vector is increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. 55, 60, 65, 70, 75, 80, 85, 90.
  • the expression level of the therapeutic nucleic acid sequence encoding a therapeutic polypeptide from the rAAV vector is increased by about 5 to 110, 6 to 100, 7 to 90, 8 to 80, 9 to 70, 10 to 60 fold relative to the expression level of the nucleic acid sequence from a rAAV vector comprising a modified AAV genome not comprising a synthetic intron.
  • the present disclosure provides a pharmaceutical composition comprising any of the rAAV vectors described herein.
  • Various pharmaceutically acceptable carriers are available in the art.
  • the phannaceutical composition comprises a pharmaceutically acceptable carrier.
  • the present disclosure describes usefill technologies for assessing rAAV vectors and compositions thereof. Certain useful technologies are described in the Examples.
  • provided technologies can be utilized for various purposes. For example, in some embodiments, provided technologies are useful for preventing and/or treating various diseases, disorders, or conditions associated with rhodopsin (RHO).
  • RHO rhodopsin
  • the present disclosure provides a method of treating a subject with a disease, disorder, or condition. In some embodiments, the method of treating a subject with a disease, disorder, or condition comprises administering or delivering a therapeutically effective amount of any of the rAAV vectors described herein. In some embodiments, the method of treating a subject with disease, disorder, or condition comprises administering or delivering a therapeutically effective amount of a pharmaceutical composition comprising any of the rAAV vectors described herein.
  • rAAV vectors or compositions thereof are administered or delivered by intraocular injection.
  • rAAV vectors or compositions thereof are administered or delivered by intravitreal injection.
  • rAAV vectors or compositions thereof are administered or delivered by subretinal injection.
  • the disease, disorder, or condition is a rhodopsin (RHO) associated disease, disorder, or condition.
  • RHO rhodopsin
  • the disease, disorder, or condition is retinal degeneration.
  • the disease, disorder, or condition is retinitis pigmentosa (RP).
  • FIG. 1 Schematic of an exemplary cargo, in part.
  • a rod-specific promoter, huRHOP805 is positioned upstream of a synthetic intron containing a RHO-targcting miRNA.
  • the synthetic intron comprises RHO-254 miRNA (amiR-254) within a scaffold (e.g., miRE) and further flanked by a synthetic splice donor (SD) and a splice acceptor (SA).
  • SA synthetic splice donor
  • SA splice acceptor
  • Other exemplary cargos may comprise various components.
  • Figure 2A Figure 2A.
  • FIG. 1 Shown are micrographs of expression of RHO-P23H-HA (top) and wild-type RHO or GFP (bottom) in HEK293T cells expressing RHO-P23H-HA following transfection with plasmids comprising control cargoes (e g., p029 (SEQ ID NO: 24). p201 (SEQ ID NO: 29)) or miRNA-containing cargos (e.g.. p 181 (SEQ ID NO: 25), p 185 (SEQ ID NO: 26), p!93 (SEQ ID NO: 28), p228 (SEQ ID NO: 40)).
  • Control cargos comprise either GFP (p029) or RHO cDNA (p201) whereas miRNA-containing cargos comprise RHO cDNA and RHO-254 miRNA.
  • Cargo identifiers are listed above the column of corresponding micrographs.
  • FIG. 2B Shown are average counts of RHO-P23H-HA + HEK293T cells following transfection with plasmids comprising control cargos (e.g., p029 (SEQ ID NO: 24), p201 (SEQ ID NO: 29)) or miRNA-containing cargos (e.g..
  • Control cargos comprise either GFP (p029) or RHO cDNA (p201), whereas miRNA-containing cargos comprise RHO cDNA and RHO-254 miRNA.
  • the X axis denotes the cargo, while the Y axis represents the number of RHO-P23H-HA + cells. Average results for five or six technical replicates (as represented by outlined circles) are shown. Error bars represent standard error of the mean (SEM).
  • FIG. 2C Shown are average counts of RHO+ HEK293T cells following transfection with control cargo (e g., p201 (SEQ ID NO: 29)) or miRNA-containing cargos (e.g., p!81 (SEQ ID NO: 25), p 185 (SEQ ID NO: 26).
  • control cargo e g., p201 (SEQ ID NO: 29)
  • miRNA-containing cargos e.g., p!81 (SEQ ID NO: 25), p 185 (SEQ ID NO: 26).
  • Control cargo comprises RHO cDNA (p201), whereas miRNA-containing cargos comprise RHO cDNA and RHO-254 miRNA.
  • the X axis denotes the cargo, while the Y axis represents the number of RHO+ cells. Average results for five or six technical replicates (as represented by outlined circles) are shown. Error bars represent standard error of the mean (SEM).
  • Figure 3 Exemplary results of a splicing assay.
  • Control cargo plasmids comprising no miRNA-containing synthetic intron
  • candidate cargo plasmids comprising a miRNA-containing synthetic intron
  • Control cargo plasmids comprising no miRNA-containing synthetic intron
  • candidate cargo plasmids comprising a miRNA-containing synthetic intron
  • the location of the miRNA-containing synthetic intron (e.g., 5’ to the resRHO cDNA, 3’ to the resRHO cDNA and 5’ to the WPRE, 3’ to both the resRHO cDNA and the WPRE) within the candidate cargo is specified by the label above each gel image.
  • Predicted sizes (in base pairs, bp) of unspliced and spliced products are denoted above each gel image. Lanes are identified by the letters A, B, or C, which correspond to the samples listed in the table below the gel image.
  • FIG. 4A Schematic of constructs used for a luciferase expression assay.
  • Each construct comprises a huRHOP805 promoter operably linked to a downstream renilla luciferase (RLuc) gene followed by a polyadenylation (polyA) sequence and a HSV-TK promoter operably linked to a downstream firefly luciferase (FFLuc) gene followed by a polyadenylation (polyA) sequence.
  • the construct further comprises an intron between the huRHOP805 promoter and RLuc gene . The type of intron is varied.
  • Figure 4B Shown are averaged results from a luciferase expression assay using plasmids comprising the constructs depicted in Fig. 4A transfected into HEK293T cells alongside CRISPR activation (CRISPRa) components to stimulate tire rhodopsin (RHO) promoter.
  • Constructs comprising a synthetic intron comprising a RHO-254 miRNA displayed higher ratios of Renilla luciferase (RLuc) expression versus firefly luciferase (FFLuc) expression as compared to no intron or the other, conventional introns tested.
  • the X axis denotes the type of intron included in the tested construct, while the Y axis represents the ratio of renilla luciferase (RLuc) expression to firefly luciferase (FFLuc) expression as measured by luminescence.
  • RLuc renilla luciferase
  • FFLuc firefly luciferase
  • Fold change in RLuc:FFLuc for the tested intron compared to no intron is denoted above the corresponding bar. Average results for six technical replicates (as represented by outlined circles) are shown. Error bars represent standard error of the mean.
  • FIG. 5 A Schematic of constructs for a luciferase-based RHO knockdown assay.
  • Plasmid 1 comprises a cargo comprising a CAG promoter operably linked to a cargo comprising a RHO-targeting miRNA.
  • Plasmid 2 comprises a renilla luciferase (RLuc)-human RHO cDNA fusion sequence operably linked to a SV40 promoter and a firefly luciferase (FFLuc) sequence linked to a TK promoter.
  • RLuc renilla luciferase
  • FFLuc firefly luciferase
  • Figure 5B Shown are averaged results from a luciferase-based RHO knockdown assay using the plasmids comprising the cargos depicted in Fig. 5A transfected into HEK293T cells. Cells were assayed using the Promega Dual-Glo Luciferase Assay System at 48 hours post-transfection.
  • the X axis denotes the cargo contained in plasmid 1 (e.g., p332 (SEQ ID NO: 46), p333 (SEQ ID NO: 47), p334 (SEQ ID NO: 48), p335 (SEQ ID NO: 49), p336 (SEQ ID NO: 50), or a control plasmid comprising eGFP), while the Y axis represents the normalized ratio of renilla luciferase (RLuc) expression to firefly luciferase (FFLuc) expression as measured by luminescence. Average results for five technical replicates (as represented by outlined circles) are shown. Error bars represent standard error of the mean.
  • FIG. 6 Shown are average counts of HA + HEK293T cells following transduction with AAV 8 vectors comprising miRNA-containing cargos (e.g., pOGTX244 (SEQ ID NO: 43), pOGTX245 (SEQ ID NO: 44)).
  • AAV 8 vectors comprising miRNA-containing cargos (e.g., pOGTX244 (SEQ ID NO: 43), pOGTX245 (SEQ ID NO: 44)).
  • AAVR universal AAV receptor
  • Cells were previously transfected with CRISPR activation (CRISPRa) components to stimulate expression of the rhodopsin (RHO) promoter. Cells were assayed at 48 hours post-transduction using immunohistochemistry against the HA tag.
  • CRISPRa CRISPR activation
  • RHO rhodopsin
  • the X axis denotes the cargo and multiplicity of infection (MOI), while the Y axis represents the number of HA + cells. Average results for four technical replicates (as represented by outlined circles) are shown. Error bars represent standard error of the mean.
  • FIG. 7A Shown is a schematic of an exemplary cargo (eGFP) to investigate RHO promoter expression in the NHP retina.
  • eGFP exemplary cargo
  • Figure 7B Shown are images of confocal scanning laser ophthalmoscopy (cSLO) of samples from cynomolgus macaques that received subretinal injections of AAV5 or AAV8 vectors encoding eGFP driven by 7 the human RHO promoter.
  • cSLO confocal scanning laser ophthalmoscopy
  • Figure 7C Shown are images of retinal cross sections from cynomolgus macaques that received subretinal injections of AAV5 or AAV8 vectors encoding eGFP driven by the human RHO promoter.
  • Figure 8 Shown are graphs of scotopic a- and b-wave amplitudes calculated across vector groups and dose levels in NHPs that received AAV5 or AAV8 vectors encoding a tagged version of the cargo for pOGTX228. Shaded bars reflect different luminance levels tested pre-dose or post-dose. Data represented as mean ⁇ SD.
  • Figure 9 Shown are graphs of scotopic a- and b-wave amplitudes calculated across vector groups and dose levels in NHPs that received AAV5 or AAV8 vectors encoding a tagged version of the cargo for pOGTX181. Shaded bars reflect different luminance levels tested pre-dose or post-dose. Data represented as mean ⁇ SD. *p ⁇ 0.05.
  • Figure 10 Shown are graphs of photopic a-wave amplitudes calculated across vector groups and dose levels in NHPs that received AAV5 or AAV8 vectors encoding a tagged version of tire cargo for pOGTX228 or pOGTX181. Shaded bars reflect different luminance levels tested pre-dose or post-dose. Data represented as mean ⁇ SD at one luminance level.
  • Figure 11 Shown are histological images of retina from NHP injected with high doses of AAV5 vectors containing tagged pOGTX228 (pOGTX245) cargo.
  • Figure 12 Shown arc histological images of retina from NHP injected with low doses of AAV5 vectors containing tagged pOGTX228 (pOGTX245) cargo.
  • Figure 13 A Schematic of an exemplary construct containing amiRNA targeting RHO mRNA.
  • Figure 13B Shown are graphs of in vitro cargo characterization in a P23H mutant silencing assay.
  • FIG. 13C Shown are histological images of retina from wild-type mice injected with the AAV vector comprising the exemplary construct shown in FIG. 13 A.
  • Figure 14 Shown are mean fold changes of mouse RHO (mRHO) expression relative to mouse GAPDH (mGAPDH) in wild-type C57BL6/J mice at 8 weeks post-subretinal injection of 1.5 x 10 9 vg/eye of an AAV8 vector comprising a pOGTX347 cargo (SEQ ID NO: 52) (AAV8-RHO.3) or vehicle (PBS + 0.001% pluronic F-68) alone.
  • SEQ ID NO: 52 pOGTX347 cargo
  • PBS + 0.001% pluronic F-68 vehicle alone.
  • Each circle represents data for a single whole mouse retina. Error bars represent standard deviation.
  • Figure 15 Shown are exemplary micrographs of retinal sections from non-human primates that received a subretinal injection of 5 x 10 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44).
  • Top left image displays HA epitope tag (which is attached to vector-borne RHO); bottom left image displays DAPI; bottom right image displays RHO; top right image displays merged view of the HA epitope tag, DAPI, and RHO.
  • Figure 16 Shown are exemplary micrographs of retinal sections from non-human primates that received a subretinal injection of 1 x 10 10 vg/eye of an AAV5 vector comprising a pOGTX244 cargo (SEQ ID NO: 43).
  • Top left image displays DAPI
  • top right image displays HA epitope tag (which is attached to vector-borne RHO); bottom right image displays RHO; bottom left image displays merged view of the HA epitope tag, DAPI, and RHO.
  • Figure 17 Shown are exemplary micrographs of retinal sections from non-human primates that received a subretinal injection of 5 x 10 10 vg/eye of an AAV5 vector comprising a pOGTX347 cargo (SEQ ID NO: 52).
  • Top left image displays HA epitope tag (which is attached to vector-borne RHO); top left right displays DAPE bottom right image displays RHO: top right image displays merged view of the HA epitope tag, DAPI, and RHO.
  • Figure 18 Shown are mean fold changes of amiR-254 expression relative to endogenous GAPDH in non-human primates (Maccica fasciciilciris) at 8 weeks post-subretinal injection of 5 x 1 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44) (AAV-RH0.1; animals 3001 and 3601), 1 x I 10 vg/eye of an AAV5 vector comprising apOGTX244 cargo (SEQ ID NO: 43) (AAV-RHO.2; animals 2001 and 2501), 5 x I 10 vg/eye of an AAV5 vector comprising a pOGTX347 cargo (SEQ ID NO: 52) (AAV -RHO.3; animals 4001 and 4501), orvehicle (PBS + 0.001% pluronic F-68) alone (animal 1001).
  • Each circle represents data for A of a single subretinal bleb (4 s
  • Figure 19 Shown are mean fold changes of endogenous RHO (mfRHO) expression relative to endogenous GAPDH (mfGAPDH) in non-human primates (Macaca fascicularis) at 8 weeks post- subretinal injection of 5 x 1 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44) (AAV-RHO.
  • Figure 20 Shown is exemplar ⁇ - uveitis scoring of non-human primate before and following subretinal injection of 5 x 1 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44) (animals NHP 3001 and NHP 3601), 1 x I 10 vg/eye of an AAV5 vector comprising a pOGTX244 cargo (SEQ ID NO: 43) (AAV-RHO.2; animals NHP 2001 and NHP 2501), 5 x I 10 vg/eye of an AAV5 vector comprising a pOGTX347 cargo (SEQ ID NO: 52) (AAV-RHO.3; animals NHP 4001 and NHP 4501), or vehicle (PBS + 0.001% pluronic F-68) alone (animal NHP 1001).
  • Figure 21 A Shown are graphs of mean scotopic b-wave amplitudes at a luminance intensity of 0.01 cd*s/m 2 (left graph) or 3.0 cd*s/m 2 (right graph) in non-human primates pre-dose (left bar for each condition) or at the end of study (8 weeks following injection; right bar for each condition).
  • Animals received a subretinal injection of 5 x I 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44) (AAV-RHO.
  • Figure 21B Shown is a graph of mean scotopic b-wave amplitude at a luminance intensity of 10.0 cd*s/m 2 in non-human primates pre-dose (left bar for each condition) or at the end of study (8 weeks following injection; right bar for each condition). Animals received a subretinal injection of 5 x I 10 vg/eye of an AAV5 vector comprising a pOGTX245 cargo (SEQ ID NO: 44) (AAV-RHO.l).
  • Figure 22 Shown are graphs of mean scotopic a-wave amplitudes at a luminance intensity of 3.0 cd*s/m 2 (left graph) or 10.0 cd*s/m 2 (right graph) in non-human primates pre-dose (left bar for each condition) or at the end of study (8 weeks following injection; right bar for each condition).
  • Each circle represents data for one eye. Error bars represent standard deviation.
  • Figure 23 Shown are graphs of mean photopic b-wave implicit time (left graph) and a-wave implicit time (right graph) at a luminance intensity of 3.0 cd*s/m 2 in non -human primates pre-dose (left bar for each condition) or at the end of study (8 weeks following injection; right bar for each condition).
  • Each circle represents data for one eye. Error bars represent standard deviation.
  • the term “a” or “an” may be understood to mean '‘at least one”; (ii) the term ‘'or” may be understood to mean “and/or”: (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps: (iv) the term “another” may be understood to mean at least an additional/second one or more: and (v) where ranges are provided, endpoints are included.
  • Adeno-associated virus As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in w hole or in part, of family Parvoviridae and genus Dependoparvovirus. AAV is a small, replication-defective, non-enveloped virus.
  • AAV may include, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV serotype rhlO, AAV serotype rh74, AAV from the HSC 1-17 series, AAV from the CBr, CLv or CLg series, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any variant of any of the foregoing.
  • AAV may also include engineered or chimeric versions of a wild-type AAV that include one or more insertions, deletions and/or substitutions within the Cap polypeptide(s) that affect one or more properties of the wildtype AAV serotype, including without limitation tropism and evasion of neutralizing antibodies (e.g., AAV- DJ, AAV-PHP.B, AAV-PHP.N, AAV.CAP-B1 to AAV.CAP-B25 and variants thereof).
  • Wild-type AAV is replication deficient and requires co-infection of cells by a helper vims (e.g., adenovirus, herpes, or vaccinia vims) or supplementation of helper viral genes in order to replicate.
  • helper vims e.g., adenovirus, herpes, or vaccinia vims
  • Administration refers to tire administration of a composition to a subject. Administration may be by any appropriate route.
  • administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, subretinal, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, vitreal, or any combination thereof.
  • administration may be subretinal.
  • a preferred method of administration will reduce or prevent an immune response from a subject receiving treatment.
  • agent may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof.
  • an agent can be or comprise a cell or organism, or a fraction, extract, or component thereof.
  • an agent is agent is or comprises a natural product in that it is found in and/or is obtained from nature.
  • an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
  • potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
  • agents that may be utilized in accordance with the present disclosure include small molecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs, miRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, small molecules, etc.
  • an agent is or comprises a polymer.
  • an agent is not a polymer and/or is substantially free of any polymer.
  • an agent contains at least one polymeric moiety.
  • an agent lacks or is substantially free of any polymeric moiety.
  • nucleic acid basepairing refers to oligonucleotide hybridization related by base-pairing rules.
  • sequence “C- A-G-T” is complementary to the sequence “G-T-C-A.”
  • Complementarity can be partial or total. Tirus, any degree of partial complementarity is intended to be included within tire scope of the term “complementary” provided that the partial complementarity permits oligonucleotide hybridization. Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
  • expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Identity refers to the overall relatedness between polymeric molecules, e.g.. between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between tire two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Representative algorithms and computer programs useful in determining the percent identity between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be detennined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • MicroRNA As used herein, the term “microRNA” or “miRNA” refers to a small, non-coding RNA molecule that can function in transcriptional and/or post-transcriptional regulation of target gene expression. The terms encompass a mature miRNA sequence or a precursor miRNA sequence, including a primary transcript (pri -miRNA) and a stem-loop precursor (pre-miRNA). The biogenesis of a naturally occurring miRNA initiates in the nucleus by RNA polymerase II transcription, generating a primary transcript (pri -miRNA).
  • the primary transcript is cleaved by Drosha ribonuclease III enzyme to produce an approximately 70 nt stem-loop precursor miRNA (pre-miRNA).
  • pre-miRNA is then actively exported to tire cytoplasm where it is cleaved by Dicer ribonuclease to form the mature miRNA, which includes an “antisense strand” or “guide strand” (that includes a region that is substantially complementary to a target sequence) and a “sense strand” or “passenger strand” (that includes a region that is substantially complementary to a region of the antisense strand).
  • a guide strand may be perfectly complementary to a target region of a target RNA or may have less than perfect complementarity to a target region of a target RNA.
  • the guide strand of this miRNA is incorporated into an RNA-induced silencing complex (RISC) that recognizes target mRNAs through base pairing with the miRNA, and commonly results in translational inhibition or destabilization of the target mRNA.
  • RISC RNA-induced silencing complex
  • target mRNA recognition occurs through imperfect base pairing with the mRNA.
  • an miRNA is synthetic or engineered, and target mRNA recognition occurs through perfect base pairing with the mRNA.
  • the target mRNA contains a sequence complementary to a “seed” sequence of the miRNA, which usually corresponds to nucleotides 2-8 of the miRNA.
  • a seed sequence of the miRNA usually corresponds to nucleotides 2-8 of the miRNA.
  • Information concerning miRNAs and associated pri -miRNA and pre-miRNA sequences is available in miRNA databases such as miRBase (Griffiths-Jones et al. 2008 Nucl Acids Res 36, (Database Issue: D154-D158) and the NCBI human genome database.
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodicstcr linkage.
  • ‘'nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester scaffold.
  • a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the scaffold, are considered within the scope of the present disclosure.
  • a nucleic acid has one or more phosphorothioate and/or 5‘-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methvlcytidmc.
  • a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2 ’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid can comprise or consist of one or more inhibitory nucleic acids (e.g., small RNA molecules).
  • an inhibitory nucleic acid comprises or consists of an RNA molecule (e.g., a small RNA molecule) that inhibits gene expression (e.g., via mRNA degradation) or inhibits translation (e.g., decreases the level of gene expression or translation of a transcript as compared to a relevant control).
  • an inhibitory nucleic acid comprises or consists of one or more siRNA, miRNA, shRNA, gRNA, or any combination thereof. In some embodiments, an inhibitory nucleic acid can be single stranded or double stranded.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually: ocularly: transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • a pharmaceutical composition is formulated for subretinal administration,
  • Polypeptide As used herein, the term “polypeptide”, in its broadest sense, refers to a sequential chain of amino acids linked together via peptide bonds. In some embodiments, a polypeptide is a protein. In some embodiments, a polypeptide is or comprises a fragment or portion, variant, derivative, or analog of a protein. Unless otherwise specified, peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.
  • Recombinant adeno-associated viral (rAA V) particle A “recombinant adeno-associated viral
  • rAAV particle refers to an infectious, replication-defective viral particle comprising an AAV protein shell encapsulating at least one payload that is flanked on both sides by inverted terminal repeats (ITRs) in a vector.
  • ITRs inverted terminal repeats
  • An rAAV particle can be produced in suitable host cells described herein (e.g., HEK293 cells, CHO-K cells, HeLa cells, or a variant thereof).
  • host cells are transfected with one or more vectors encoding: at least one payload flanked by an ITR on either side of the at least one payload, at least one Rep polypeptide, at least one Cap polypeptide, and at least one helper polypeptide, such that the host cells are capable of producing Rep, Cap and helper polypeptides necessary for packaging of rAAV particles.
  • rAAV particles described herein may be used for subsequent gene delivery.
  • Subject refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes.
  • a subject is or comprises a cell or a tissue.
  • Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans).
  • a patient is a human.
  • a patient is suffering from or susceptible to one or more diseases, disorders, or conditions.
  • a patient displays one or more symptoms of a disease, disorder, or condition.
  • a patient has been diagnosed with one or more diseases, disorders, or conditions.
  • the disease, disorder, or condition is retinal degeneration.
  • the disease, disorder, or condition is retinitis pigmentosa (e.g., retinitis pigmentosa- 11 (RP11)).
  • the term ‘‘substantially” refers to tire qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • an individual who is “susceptible to” a disease, disorder and/or condition is one who has a higher risk of developing the disease, disorder and/or condition than does a member of the general public.
  • an individual who is susceptible to a disease, disorder and/or condition is predisposed to have that disease, disorder and/or condition.
  • an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder and/or condition.
  • an individual who is susceptible to a disease, disorder and/or condition may exhibit symptoms of the disease, disorder and/or condition.
  • an individual who is susceptible to a disease, disorder and/or condition may not exhibit symptoms of the disease, disorder and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, tire target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of tire disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • treatment refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • expression vectors are referred to herein as "expression vectors.”
  • the term “vector” refers to an agent capable of transporting a nucleic acid, wherein the agent comprises the nucleic acid.
  • a vector comprises or is an agent capable of transporting a nucleic acid.
  • Wild-type As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).
  • inhibitory nucleic acids, vectors, or rAAV vectors comprise one or more miRNAs that inhibit expression of a pathogenic polypeptide.
  • provided technologies e.g., inhibitory nucleic acids, rAAV vectors, compositions thereof, or methods thereof
  • provided technologies e.g., rAAV vectors or compositions thereof
  • provided technologies increase expression of a therapeutic polypeptide.
  • provided rAAV vectors comprise various sequence elements, which, among other things, provide improved expression (e.g.. one or both of greater levels and increased stability) of an inhibitory nucleic acid and/or a therapeutic nucleic acid sequence.
  • provided rAAV vectors comprise a synthetic intron comprising a miRNA sequence.
  • a rAAV vector comprises a 5’ ITR, a promoter, a synthetic intron, a miRNA sequence located within the synthetic intron, a therapeutic nucleic acid sequence encoding a therapeutic polypeptide, and/or a 3’ ITR as described herein.
  • an inhibitory nucleic acid is a miRNA.
  • a miRNA is miR-254.
  • a pathogenic polypeptide is mutant rhodopsin (RHO).
  • a therapeutic polypeptide is wild-type rhodopsin (RHO).
  • RHO refers to a gene or a gene product thereof (e.g., a nucleic acid (e g., DNA or RNA), a transcript (e.g., a RHO mRNA), or a protein encoded thereby (e.g.. a RHO polypeptide)) from a species, which may be known as rhodopsin, RHO, opsin-2, OPN2, or other terms as known to those of skill in the art.
  • a gene or a gene product thereof e.g., a nucleic acid (e g., DNA or RNA), a transcript (e.g., a RHO mRNA), or a protein encoded thereby (e.g.. a RHO polypeptide)) from a species, which may be known as rhodopsin, RHO, opsin-2, OPN2, or other terms as known to those of skill in the art.
  • a species which may be known as rhodopsin, R
  • the RHO gene is reported to encode a RHO protein, which is chiefly expressed in retinal tissue in photoreceptor cells (e.g., rod photoreceptor cells) and primarily localizes to the outer segment cilia of the cells.
  • RHO protein has been reported to be a transmembrane protein with a total of seven transmembrane domains (TMDs), an intradiscal (e.g., interior of the rod photoreceptor discs) N-terminus, and a cytoplasmic C-terminus. Further, RHO reportedly acts a light-sensitive receptor protein involved in visual phototransduction, particularly in low-light conditions.
  • RHO Reported mutations in RHO include a multitude of variants across various locations in the gene and may result in various structural (e.g., misfolding, changes in post- translational modifications, instability) or functional (e.g., altered activation) changes as compared to wild- type RHO.
  • cells containing mutant RHO may exhibit various defects in, e.g., trafficking, activation, etc.
  • a mutation in RHO is in an exon.
  • a mutation in RHO is a missense mutation. In some embodiments, a mutation in RHO is a nonsense mutation. In some embodiments, a mutation in RHO is a loss-of-function mutation. In some embodiments, a mutation in RHO is a recessive mutation. In some embodiments, a mutation in RHO is a dominant mutation.
  • a mutation in RHO is L328P, T342M, Q344R/P/ter, V345L/M, A346P, P347A/R/Q/L/S/T, ter349/Q/E, N15S, T17M, V20G, Q28H ⁇ , G51R/V, P53R, T58R/M, V87D, G89D, G106R/W, C110F/R/S/Y, E113K, L125R, W161R, A164E/V, C167R/W, P171Q/L/S, Y178N/D/C, E181K, G182S/V, C185R, C187G/Y, G188R/E, D190N/G/Y, H211R/P, C222R, P267R/L, S270R, K296N/E/M, P12R, R21C, Q28H, L40R, L46R, L47R
  • M163T A169P, P170H/R, S176F, P180A/S, Q184P, S186P/W.
  • Y191C T193M, M207R/K, V210F, I214N, P215L/T. M216R/L/K, R252P, T289P, S297R.
  • a mutation in RHO is P23H.
  • a disease, disorder, or condition is associated with RHO if the presence, level, activity, or form of RHO or products (e.g., one or both of transcripts and encoded proteins.) thereof correlates with incidence of or susceptibility to the disease, disorder, or condition (e.g., across a relevant population).
  • a disease, disorder, or condition associated with RHO may be treated or prevented by providing wild-type RHO or products thereof and/or reducing levels of mutant RHO or product thereof.
  • a disease, disorder, or condition is an inherited disease, disorder, or condition.
  • a disease, disorder, or condition is autosomal dominant.
  • a disease, disorder, or condition is a retinopathy.
  • a disease, disorder, or condition is RHO-associated retinopathy.
  • a disease, disorder, or condition is retinal degeneration.
  • a disease, disorder, or condition is retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • a disease, disorder, or condition is autosomal recessive retinitis pigmentosa (arRP). In some embodiments, a disease, disorder, or condition is autosomal dominant retinitis pigmentosa (adRP). In some embodiments, a disease, disorder, or condition is retinitis pigroentosa-4 (RP4).
  • arRP autosomal recessive retinitis pigmentosa
  • a disease, disorder, or condition is autosomal dominant retinitis pigmentosa (adRP).
  • a disease, disorder, or condition is retinitis pigroentosa-4 (RP4).
  • inhibitor ⁇ - nucleic acids can comprise a nucleic acid sequence targeting pathogenic polypeptide transcripts (e.g., pathogenic polypeptide mRNA) as provided in tire present disclosure.
  • pathogenic polypeptide transcripts e.g., pathogenic polypeptide mRNA
  • the present disclosure provides inhibitory nucleic acids that inhibit the expression of genes that cause or are implicated in retinitis pigmentosa pathogenesis.
  • the present disclosure provides inhibitory nucleic acids that target nucleic acids produced from genes that cause or are implicated in retinitis pigmentosa pathogenesis.
  • inhibitory nucleic acids comprise RNA molecules that inhibit gene expression by hybridizing to target nucleic acids produced by a gene of interest, e.g., RNA interference, CRISPR, etc.
  • inhibitory nucleic acids of the present disclosure include, but are not limited to, siRNA, shRNA, miRNA. gRNA, or any combination thereof.
  • inhibitory nucleic acids are single stranded or double stranded.
  • inhibitory nucleic acids of the present disclosure comprise miRNAs.
  • inhibitory nucleic acids of the present disclosure comprise artificial miRNAs (amiRNAs).
  • miRNAs of tire present disclosure comprise a guide strand sequence that targets a target nucleic acid of interest.
  • inhibitory nucleic acids of the present disclosure are flanked by and/or operably linked to structural and/or regulatory nucleic acid sequences, for example those described herein.
  • the present disclosure provides inhibitor ⁇ ' nucleic acids that inhibit RHO expression.
  • mutant variants of RHO such as those common in retinitis pigmentosa and described herein, are targeted by inhibitor ⁇ ' nucleic acids of the present disclosure.
  • the present disclosure provides inhibitory nucleic acids between 19 and 30 bases in length. In some embodiments, provided inhibitory nucleic acids are between 15 and 20, between 20 and 25, or between 25 and 30 bases in length. In some embodiments, the present disclosure provides inhibitory nucleic acids that are at least 30, at least 29, at least 28, at least 27, at least 26, at least 25, at least 24, at least 23, at least 22, at least 21, at least 20, at least 19, at least 18, at least 17, at least 16, or at least 15 bases in length. In some embodiments, the present disclosure provides inhibitor ⁇ - nucleic acids that are at most 30, at most 29, at most 28. at most 27, at most 26, at most 25, at most 24, at most 23, at most 22. at most 21. at most 20, at most 19, at most 18, at most 17, at most 16, or at most 15 bases in length. In some embodiments, inhibitor ⁇ ' nucleic acids can be single stranded or double stranded.
  • inhibitory nucleic acids of the present disclosure comprise or consist of one or more inhibitor ⁇ - nucleic acid sequences that arc complementary to one or more target nucleic acids (e.g., guide sequences).
  • inhibitory nucleic acids comprise or consist of one or more inhibitory nucleic acid sequences that are complementary to at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%.
  • inhibitory nucleic acids comprise or consist of one or more inhibitory nucleic acid sequences that are complementary to at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%. at most 91%. at most 90%, at most 89%, at most 88%, at most 87%, at most 86%, at most 85%, at most 84%, at most 83%, at most 82%, at most 81%, at most 80%, at most 79%, at most
  • the present disclosure provides inhibitory nucleic acids that comprise or consist of one or more inhibitory nucleic acid sequences that are complementary to at least 35, at least 34, at least 33, at least 32, at least 31, at least 30, at least 29, at least 28, at least 27, at least 26, at least 25, at least 24, at least 23, at least 22, at least 21, at least 20, at least 19, at least 18, at least 17, at least 16, at least 15, at least 14, at least 13, at least 12, at least 11. at least 10, at least 9, at least 8. at least 7. at least 6, or at least 5 bases in a target nucleic acid sequence.
  • the present disclosure provides inhibitory nucleic acids that comprise or consist of one or more inhibitory nucleic acid sequences that are complementary to at most 35, at most 34, at most 33, at most 32, at most 31, at most 30, at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7. at most 6, or at most 5 bases in a target nucleic acid sequence.
  • inhibitory nucleic acids of the present disclosure can contain contiguous and/or non-contiguous base mismatches within regions that are substantially complementarity to a target nucleic acid.
  • inhibitory nucleic acids comprise one or more base mismatches within regions that arc substantially complementary to a target nucleic acid.
  • inhibitory nucleic acids comprise at least 5, at least 4, at least 3, or at least 2 base mismatches that are contiguous within regions that are substantially complementarity to a target nucleic acid.
  • inhibitory nucleic acids comprise at most 5, at most 4, at most 3, or at most 2 base mismatches that are contiguous within regions that are substantially complementarity to a target nucleic acid.
  • the present disclosure provides inhibitory nucleic acids that comprise at least 10. at least 9, at least 8, at least 7. at least 6, at least 5, at least 4, at least 3. or at least 2 base mismatches that are noncontiguous within regions that are substantially complementarity to a target nucleic acid sequence.
  • the present disclosure provides inhibitory nucleic acids that comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2 base mismatches that are noncontiguous within regions that are substantially complementarity to a target nucleic acid sequence
  • the present disclosure provides inhibitory nucleic acids that comprise or consist of inhibitory nucleic acid sequences that are substantially complementary to a target nucleic acid sequence.
  • a target nucleic acid sequence is a RHO nucleic acid sequence.
  • inhibitory nucleic acid sequences comprise or consist of miRNA, siRNA, shRNA, gRNA, or any combination thereof.
  • inhibitory nucleic acid sequences of the present disclosure comprise or consist of one or more miRNA.
  • miRNA of the present disclosure comprise guide strand sequences that are substantially complementary to one or more target nucleic acid sequences.
  • a target nucleic acid sequence comprises a wild-type RHO nucleic acid sequence, or mutant or variant RHO nucleic acid sequence.
  • targeted RHO nucleic acid sequences include RHO mRNA sequences.
  • targeted RHO mRNA sequences comprise sequences from human RHO mRNA.
  • inhibitory nucleic acid sequences of the present disclosure are at least 99%, at least 98%, at least 97%. at least 96%, at least 95%, at least 94%, at least 93%. at least 92%, at least 91%, at least 90%, at least 89%.
  • inhibitory nucleic acid sequences of the present disclosure are at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 89%, at most 88%, at most 87%, at most 86%, at most 85%, at most 84%, at most 83%, at most 82%, at most 81%, at most 80%, at most 79%, at most 78%, at most 77%, at most 76%, at most 75%, at most 74%, at most 73%, at most 72%, at most 71%, at most 70%, at most 69%, at most 68%, at most 67%, at most 66%, at most 65%, at most 64%, at most 63%, at most 62%, at most 61%, at most 60%, at most 59%, at most 58%, at most 57%, at most 56%, at most 55%, at most 54%.
  • inhibitory nucleic acid sequences of the present disclosure comprise or consist of one or more of SEQ ID NOs: 1, 11, 13, or 15.
  • inhibitory nucleic acids of the present disclosure inhibit expression of genes that cause or are implicated in one or more diseases, disorders, or conditions (e.g., retinal degeneration, retinitis pigmentosa, RP4).
  • inhibitory nucleic acids inhibit gene expression by hybridizing to target nucleic acids produced by a gene of interest, e.g., by RNA interference, CRISPR, etc.
  • a cell or tissue treated with inhibitory nucleic acids of the present disclosure exhibits a reduction in expression of a target nucleic acid of least 10%, at least 20%.
  • a cell or tissue treated with inhibitory nucleic acids of the present disclosure exhibits a reduction in expression of a target nucleic acid of most 20%, at most 30%, at most 40%, at most 50%, at most 60% at most 70%, at most 80%, or at most 90% compared to expression of a target nucleic acid in a cell or tissue not treated with inhibitory nucleic acids of the present disclosure.
  • the present disclosure recognizes that guide strand to passenger strand ratio provided by an inhibitory nucleic acid (e.g., miRNA) plays a role in effective targeting of a target nucleic acid.
  • inhibitory nucleic acids provide a guide strand to passenger strand ratio of at least 2 or at least 3 when administered to a subject.
  • inhibitory' nucleic acids provide a guide strand to passenger strand ratio greater than 2.
  • the present disclosure recognizes that guide strand production level plays a role in effective targeting of a target nucleic acid.
  • Guide strand production level may be defined as percent of the sequencing reads that match a guide strand of a miRNA (e.g., artificial miRNA) relative to total number of sequencing reads matching all mature endogenous miRNAs in a sample. This is a proxy for the number of a-miR guide strand molecules relative to the number of endogenous miRNA molecules, expressed as a percentage.
  • inhibitory nucleic acids provide a guide strand production level of at least 0.01 %, at least 0.1 %, at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%.
  • inhibitory nucleic acids provide a guide strand production level of at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, or at most 35%.
  • guide strand potency which may be defined as the percent decrease of a target gene (e.g., RHO) expression levels
  • guide strand accuracy of certain inhibitory nucleic acids is recognized by the present disclosure to play a role in effective targeting of a target nucleic acid.
  • Guide strand accuracy may be defined as tire fraction of a-miR guide strands that match a designed sequence with maximum one nucleotide mismatch, and further, have the exact length of the designed sequence or are longer.
  • inhibitory nucleic acids of the present disclosure provide guide strand accuracy of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • inhibitory nucleic acids of the present disclosure provide guide strand accuracy of at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%. at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 99%. In some embodiments, inhibitory nucleic acids of the present disclosure provide guide strand accuracy greater than 80%.
  • the present disclosure provides inhibitory nucleic acids that comprise or consist of one or more miRNAs that inhibit the expression of genes that cause or are implicated in retinitis pigmentosa pathogenesis.
  • miRNAs of the present disclosure comprise scaffold sequences of wild-type miRNAs.
  • wild type miRNA scaffold sequences include, but are not limited to, miR-155, miR-30a, mIR-122, miR-150, miR-21, miR-20a, miR- 16-1, and combinations thereof. It is contemplated that any wild-type miRNA scaffold known by those skilled in the art to facilitate inhibition of a target nucleic acid can be utilized in accordance with the present disclosure.
  • miRNAs of the present disclosure comprise modified and/or engineered miRNA scaffolds.
  • modified and engineered miRNA scaffolds include miR-E, miR-3G, miR-16-2, ultramiR, engineered variants of miR-155, or any combination thereof.
  • miRNA scaffolds discussed herein comprise one of SEQ ID NOs: 10, 12, or 14.
  • inhibitory nucleic acids of the present disclosure are modified to include one or more chemically modified nucleotides to obtain one or more desirable qualities (e.g., enhanced silencing of a target gene, enhanced stability, or combinations thereof).
  • chemically modified nucleotides of the present disclosure include, but are not limited to, 2'-deoxy nucleotides, 2’-0Me nucleotides, thioate linked nucleotides, 2 ’-fluorouridine, 2'-fluorocytidine, N3-methyluridine, 5- bromouridine. 5 -iodouridine, 2,6-diaminopurine, and combinations thereof.
  • a vector can comprise an inhibitory nucleic acid as provided in the present disclosure.
  • a vector is a bacterial artificial chromosome (BAC), a cosmid, a phagemid, a plasmid, or a viral vector.
  • a vector is a recombinant vector.
  • a vector is a recombinant BAC. a recombinant cosmid, a recombinant phagemid. a recombinant plasmid, or a recombinant viral vector.
  • suitable carrier e.g., liposomes, cell-penetrating peptides, antibodies, etc.
  • a vector is a viral vector.
  • a viral vector is a recombinant viral vector.
  • Recombinant viral vectors have become widely used for inserting nucleic acid sequences (e.g., a gene or an inhibitory nucleic acid) into mammalian cells (e.g., human cells).
  • nucleic acid sequences e.g., a gene or an inhibitory nucleic acid
  • mammalian cells e.g., human cells.
  • Many forms of viral vectors can be used to deliver a payload (e.g., a payload described herein, e.g., an isolated nucleic acid as described herein) to a cell, tissue, or organism.
  • Non-limiting examples of recombinant viral vectors include, but are not limited to, adeno- associated virus (AAV), retrovirus (e.g., Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, murine mammary tumor virus, or Rous sarcoma virus), adenovirus, SV40-type virus, polyomavirus, Epstein-Barr vims, papilloma vims, herpes vims, vaccinia vims, baculovims, or polio vims.
  • a recombinant viral vector comprises or is a retroviral vector.
  • Retrovimses are enveloped vimses that belong to viral family Retroviridae. Protocols for production of replication-deficient retrovimses are known in the art (See, e.g., Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry. E. J.. Methods in Molecular Biology, Vol. 7, Humana Press. Inc., Cliffion, N.J. (1991), each of which is hereby incorporated by reference in its entirety )- A number of retroviral systems are known in the art (See, e.g., U.S. Pat Nos. 5,994,136, 6,165,782, and 6,428,953, each of which is hereby incorporated by reference in its entirety 7 ).
  • a retrovirus comprises or is a lentivims of Retroviridae family.
  • a lentivirus comprises or is human immunodeficiency viruses (e.g., HIV-1 or HIV-2), simian immunodeficiency vims (S1V), feline immunodeficiency vims (FIV), equine infections anemia (EIA), or visna vims.
  • a recombinant viral vector comprises or is an adenovims vector.
  • An adenovims vector may be from any origin, subgroup, subtype, serotype, or mixture thereof.
  • an adenovims can be of subgroup A (e.g., serotypes 12, 18, or 31).
  • subgroup B e.g.. serotypes 3, 7, 11, 14,
  • subgroup C e.g., serotypes 1, 2, 5, or 6
  • subgroup D e.g., serotypes 8, 9, 10, 13, 15,
  • Adenoviral serotypes 1 through 51 are available from tire American Type Culture Collection (ATCC, Manassas, VA, USA).
  • Non-group C adenovimses can be used to prepare replication-deficient adenoviral vectors.
  • Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U.S. Pat. Nos. 5,801,030, 5,837,511, and 5,849,561, and International Patent Applications WO 97/12986 and WO 98/53087, each of which is hereby incorporated by reference in its entirety.
  • Further examples of adenoviral vectors can be found in U.S. Publication Nos. 20150093831, 20140248305, 20120283318, 20100008889, 20090175897 and 20090088398, each of which is hereby incorporated by reference in its entirety.
  • a recombinant viral vector comprises or is an alphavims.
  • alphavimses include, but are not limited to, Sindbis vims, Aura vims, Babanki vims, Barmah Forest vims, Bebam vims, Cabassou vims, Chikungunya vims, Eastern equine encephalitis vims, Everglades vims, Fort Morgan vims, Getah vims, Highlands J vims, Kyzylagach vims, Mayaro vims, Me Tri vims. Middelburg vims, Mosso das Pedras vims, Mucambo vims, Ndumu vims.
  • O'nyong-nyong vims Pixuna vims, Rio Negro vims, Ross River vims, Salmon pancreas disease vims, Semliki Forest vims, Southern elephant seal vims, Tonate vims, Trocara vims, Una vims, Venezuelan equine encephalitis vims, Western equine encephalitis vims, and Whataroa vims.
  • a genome of such viruses encodes nonstmctural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in host cell cytoplasm.
  • a recombinant viral vector comprises or is an AAV vector.
  • AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6): 1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3): 141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699- 708 (2012), each of which is hereby incorporated by reference in its entirety).
  • Methods for generating and using AAV vectors are described, for example, in U.S. Pat. Nos. 5,139.941 and 4,797,368, each of which is hereby incorporated by reference in its entirety.
  • AAV vectors for use in methods, compositions, and systems described herein may be of any AAV serotype.
  • AAV serotypes generally have different tropisms to infect different tissues.
  • an AAV serotype is selected based on a tropism.
  • AAV serotypes have been characterized including, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5.
  • an AAV vector is derived from an AAV genome sequence or a variant thereof as described in US Patent Nos. 7,906,111; 6,759,237; 7,105,345; 7,186,552; 9,163,260; 9,567,607; 4,797,368; 5,139,941; 5,252,479; 6,261,834; 7,718.424; 8,507.267; 8,846.389; 6,984.517; 7,479.554; 6,156.303; 8,906,675: 7,198,951; 10,041.090: 9,790,472: 10.308.958; 10,526,617; 7.282,199; 7.790.449; 8,962,332; 9,587,250; 10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01: 8,574,583: 8,906,387: 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,9
  • an AAV vector comprises or is a single -stranded (ss) or self- complementary (sc) AAV vector.
  • an AAV vector comprises an expression construct and one or more regions comprising ITR sequences (e.g., wild-type ITR sequences or engineered or modified ITR sequences) flanking an expression construct.
  • an expression construct comprises an enhancer, a promoter, a nucleic acid sequence encoding a product of interest (e.g.. a polypeptide), or a 3’ UTR element (e.g.. a WPRE. a polyadenylation signal), or a combination thereof.
  • an AAV vector comprises or is a recombinant AAV (rAAV) vector.
  • AAVs Adeno-Associated Viruses
  • AAVs adcno-associatcd viruses
  • present disclosure provides recombinant AAV (rAAV) vectors.
  • present disclosure provides rAAV vectors comprising (i) a modified AAV genome and (ii) a capsid.
  • AAV is reportedly a small, non-enveloped virus that packages a single-stranded, linear DNA genome, approximately 4.7-5 kb long.
  • a member of the family Parvoviridae, AAV was discovered in 1965 as a contaminant of adenovirus isolates.
  • AAV has not been associated with any human or animal disease, even though most humans (>70%) are seropositive for one or more serotypes (Calcedo et al. (2011); Calcedo et al. (2009)). Both positive and negative DNA strands are packaged equally well, and infection can be initiated with particles containing either strand.
  • Tire viral genome comprises three open reading frames (ORFs), rep (replication), cap (capsid), and aap (assembly-activating protein), which together code for eight proteins (Rep78, Rep68, Rep52, Rep40, VP1. VP2. VP3, and AAP) expressed from three promoters (p5, pl9. and p40).
  • the mature capsid consists of the amino acid sequence of only one ORF (cap) and the packaged DNA.
  • rAAVs recombinant AAVs
  • the present disclosure recognizes that the coding regions of AAV are flanked by inverted terminal repeats (ITRs) that are typically 145 bases long in wild-type AAVs and have a complex T-shaped structure. These repeats are the origins for DNA replication and serve as the primary packaging signal (McLaughlin et al., 1988; Hauswirth et al., 1977).
  • ITRs are the only cis-active sequences required for making rAAVs and the only AAV-encoded sequences present in AAV vectors (McLaughlin et al. (1988); Samulski et al. (1989)).
  • AAV ITRs have enhancer activity in the presence of Rep protein, they have minimal promoter or enhancer activity in the absence of Rep protein.
  • transgenes cloned into an AAV vector must be engineered with an appropriate enhancer, promoter, polyadenylation signal, and/or splice sites to ensure correct gene expression.
  • ITRs of the present disclosure can include ITRs from any AAV serotype.
  • ITRs of the present disclosure can include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6. AAV7, AAV8, AAV9, AAV 10. AAV 11, AAV 12, or any combination thereof.
  • ITRs of the present disclosure may comprise engineered or modified ITRs using methods known in the art.
  • ITRs of the present disclosure may comprise one or more sequence modifications (e.g., deletions, substitutions) as compared to a wild-type ITR sequence.
  • ITRs of an AAV vector can be derived from any AAV serotype (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV- HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1 to AAV.CAP-B25, or variants or hybrids thereof).
  • AAV serotype e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74, AAV- HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, A
  • ITRs are derived from one or more other serotypes, e.g., as described in US Patent Nos. 7,906,111: 6,759,237; 7,105,345; 7,186.552; 9,163.260; 9,567.607; 4,797.368; 5,139.941; 5,252.479; 6,261.834; 7,718.424; 8,507.267; 8,846.389; 6,984,517; 7,479,554; 6,156,303; 8,906,675: 7,198,951; 10,041,090; 9,790,472: 10,308,958; 10,526,617; 7,282,199; 7,790,449; 8,962,332; 9,587,250; 10,590,435; 10,265,417; 10,485,883; 7,588,772; 8,067,01; 8,574,583; 8,906,387; 8,734,809; 9,284,357; 10,035,825; 8,628,966; 8,927
  • ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (See, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and described in Kessler et al.. PNAS. 1996 Nov 26;93(24): 14082-7; Machida. Methods in Molecular MedicineTM. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 ⁇ Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus; and U.S. Pat. Nos. 5,139,941 and 5,962,313; each of which is hereby incorporated by reference in its entirety).
  • the present disclosure encompasses the recognition that more than 110 distinct primate AAV capsid sequences have been reportedly isolated. Each of those AAV capsids that have unique serological profiles has been named as a particular AAV serotype.
  • the present disclosure further appreciates that at least 12 primate serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12) have been described.
  • a capsid from any serotype can be used.
  • a modified or engineered capsid including, but not limited to those described herein, can be used in accordance with the present disclosure.
  • a capsid from an AAV5 serotype is used.
  • AAV8 and AAV9 have been found to transduce striated muscle with efficiencies at least as high. AAV8 and AAV9 are considered to have the highest level of hepatocyte transduction.
  • rAAV6 and rAAV9 transduce much of the entire airway epithelium, while rAAV5 transduction is limited to lung alveolar cells.
  • rAAV serotypes 1, 4, 5, 7, and 8 have been found to be efficient transducers of neurons in various regions of the brain.
  • rAAVl and rAAV5 have also been reported to transduce ependymal and glial cells.
  • rAAV serotypes 1. 4, 5, 7. 8, and 9 efficiently transduce retinal pigment epithelium, while rAAV5, rAAV7, and rAAV8 transduce photoreceptors as well.
  • rAAVl, rAAV8, and rAAV9 have shown the highest reported transduction in pancreas tissue, primarily in acinar cells.
  • the kidney appears to be a relatively difficult organ to transduce, although proximal tubule cells have been transduced by rAAV2 at low levels, as have glomeruli by rAAV9. Additionally, rAAVl has been shown to transduce adipose tissue, albeit with the aid of a nonionic surfactant.
  • the present disclosure additionally encompasses the recognition that it may be advantageous to modify wild type AAV capsids, or engineer AAV capsids, to achieve modified tissue tropism and/or immune system evasion.
  • One method of achieving these advantages is to produce vector in the presence of cap genes for multiple serotypes. Depending on the ratio of capsid proteins from each serotype, the resulting “mosaic”’ virions can exhibit a combined tropism for cell type or, in some cases, can acquire tropism not exhibited by either serotype individually. Some studies have involved attaching exogenous molecules to the capsid.
  • One example utilizes a bi-specific antibody obtained by fusing Fc regions of two different antibodies: an anti-capsid antibody and an anti-cell marker antibody, thereby conferring rAAV2 tropism to transduction-resistant megakaryocyte cell lines.
  • Another example adopted the approach of biotinylating the capsid and subsequently binding it to a streptavidin conjugate carry ing epidermal growth factor or fibroblast growth factor. This approach was shown to produce at least a ten-fold increase in the transduction of cells that highly express the epidermal growth factor or fibroblast growth factor receptor, respectively.
  • Tire present disclosure also appreciates that as an alternative to attaching molecules to the capsid surface, it may be advantageous to engineer a modification directly into the cap gene.
  • green fluorescent protein (238 amino acids) can be inserted into AAV2 VP1 and VP2.
  • the transduction efficiencies of the VP 1 -GFP and VP2-GFP vectors were 3 and 5 orders of magnitude lower, respectively, than the efficiency of wild-type capsid, the transduction in HeLa cells did occur, suggesting a tolerance for inserted sequences in capsid proteins.
  • cap genes for tissue targeting a number of researchers have inserted peptide sequences on the basis of known ligand-rcccptor interactions, or have selected for peptides in phage-display libraries. Another strategy has been to insert random sequences of amino acids, followed by in vitro selection of the best performing capsids. Instead of introducing target-specific peptides, some experiments modified the capsids generically, pending subsequent modification toward targets of choice. For example, a binding site for the Fc portion of antibodies was inserted into tire capsid, followed by binding of different antibodies specific for receptors of various cell lines.
  • Another such modification is to insert a biotin-binding site into the capsid, thereby facilitating metabolic biotinylation and allowing flexible targeting with any avidin- conjugated ligands.
  • rAAV of tire present disclosure can be produced and isolated according to any appropriate method, e.g., methods described in Clement and Grieger, 2016, Grieger et al., 2016, and Martin et al., 2013, the contents of each which are incorporated herein by reference in their entirety.
  • the methods typically involve culturing a host cell which contains a nucleic acid sequence (e.g., a cap gene) encoding an AAV capsid protein or fragment thereof; a functional rep gene; a nucleic acid sequence or vector comprising AAV ITRs (e.g., an AAV 5’ ITR and an AAV 3’ ITR) and a nucleic acid sequence encoding a product of interest (e.g., an inhibitory nucleic acid sequence (e.g., a miRNA) and/or a polypeptide (e.g., a wild-type polypeptide)); and sufficient helper functions to pennit packaging of tire recombinant AAV vector into the AAV capsid proteins.
  • a nucleic acid sequence e.g., a cap gene
  • AAV capsid protein or fragment thereof e.g., an AAV 5’ ITR and an AAV 3’ ITR
  • a nucleic acid sequence encoding a product of interest e
  • the components to be cultured in the host cell to package an isolated nucleic acid sequence or vector in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the required components e.g., isolated nucleic acid sequence or vector, rep sequences, cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell will contain the required component or components under the control of an inducible promoter.
  • the required component or components may be under tire control of a constitutive promoter. Examples of suitable promoters are provided herein.
  • a selected stable host cell may contain a selected component or components under the control of a constitutive promoter and other selected component or components under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters.
  • Still other stable host cells are known in the art or may be generated by one of skill in the art.
  • Tire modified AAV genome, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (e.g., a vector).
  • the selected genetic element may be delivered by any suitable method (e.g., transfection), including those described herein.
  • the methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • rAAVs may be produced using the triple transfection method (e.g., as described in detail in U.S. Pat. No. 6,001,650, the contents of which relating to the triple transfection method are incorporated herein by reference).
  • the rAAVs are produced by transfecting a host cell with a suitable vector (comprising a nucleic acid sequence encoding a product of interest, e.g., a polypeptide) to be packaged into rAAV particles, an AAV rep/cap vector, and a helper function vector.
  • An AAV rep/cap vector encodes rep and cap sequences, which function in trans for productive AAV replication and encapsidation.
  • the AAV rep/cap vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes).
  • vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein.
  • the helper function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., ‘'helper functions”).
  • helper functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
  • Viral-based helper functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
  • a rAAV vector comprises a modified AAV genome as described herein.
  • a rAAV vector comprises a modified AAV genome as described herein and a capsid.
  • a rAAV vector comprises a modified AAV genome comprising an inhibitory nucleic acid (e.g., a miRNA) as described herein and a capsid.
  • a rAAV vector comprises a modified AAV genome comprising a therapeutic nucleic acid sequence encoding a therapeutic polypeptide as described herein and a capsid.
  • a rAAV vector comprises a modified AAV genome comprising an inhibitory nucleic acid sequence (e.g., a miRNA) and a therapeutic nucleic acid sequence encoding a therapeutic polypeptide; and a capsid.
  • a rAAV vector comprises a modified AAV genome comprising (i) 5 ’ ITR, (ii) a promoter, (iii) a synthetic intron, (iv) an inhibitor ⁇ ' nucleic acid sequence located within the synthetic intron, (v) a therapeutic nucleic acid sequence encoding a therapeutic polypeptide, and (vi) a 3 ' ITR; and a capsid.
  • a rAAV vector comprises a modified AAV genome comprising (i) 5’ ITR.
  • a rAAV vector comprises a modified AAV genome comprising (i) 5’ ITR, (ii) a promoter, (iii) a synthetic intron, (iv) a miRNA sequence located within the synthetic intron (wherein the miRNA sequence comprises a guide strand sequence that targets a pathogenic nucleic acid sequence encoding a pathogenic polypeptide, and a scaffold sequence), (v) a therapeutic nucleic acid sequence encoding a therapeutic polypeptide, and (vi) a 3’ ITR; and a capsid.
  • a miRNA sequence is miR-254.
  • a pathogenic polypeptide is mutant RHO.
  • a therapeutic polypeptide is wild-type RHO.
  • an AAV serotype may have or comprise a mutation in an AAV2 sequence (e.g., as described in Wu et al., J Virol. 2000 Sep; 74(18); 8635-47, which is hereby incorporated by reference in its entirety).
  • Other AAVs are described in, e.g., Sharma et al., Brain Res Bull. 2010 Feb 15; 81(2-3): 273, which is hereby incorporated by reference in its entirety.
  • an AAV comprises or is a naturally occurring AAV.
  • an AAV is a modified AAV or a variant of a naturally occurring AAV.
  • an AAV may be generated by directed evolution, e.g., by DNA shuffling, peptide insertion, or random mutagenesis, in order to introduce modifications into the AAV sequence to improve one or more properties for gene therapy. In some embodiments, such modifications avoid or lessen an immune response or recognition by neutralizing antibodies and/or allow for more efficient and/or targeted transduction (See, e g., Asuri et al., Molecular Therapy 20.2 (2012): 329-338, which is hereby incorporated by reference in its entirety).
  • a modified AAV is modified to include a specific tropism.
  • an AAV may be a dual or triple AAV composition, e.g., for the delivery of large payloads (e.g., payloads of greater than approximately 5kb) and/or to address safety concerns associated with administration of single AAV particles.
  • a dual AAV composition may include two separate AAV particles, each including a fragment of a foil sequence of a large payload of interest, and when recombined, the fragments form the foil sequence of the large payload of interest or a functional portion thereof.
  • a triple AAV composition may include three separate AAV particles, each including a fragment of a sequence of a large payload of interest, and when recombined, tire fragments form the foil sequence of foe large payload of interest or a functional portion thereof.
  • AAV e.g., dual or triple AAV compositions
  • fragments of a payload of interest recombine and generate a single mRNA transcript of foe entire payload of interest.
  • fragmented payloads include nonoverlapping sequences.
  • fragmented payloads include one or more specified overlapping sequences.
  • multiple AAVs for dual or triple transfection may be the same type of AAV (e.g.. same serotype and/or same construct).
  • multiple AAVs for dual or triple transfection may be different types of AAV (e.g., different serotype and/or different construct).
  • a rAAV vector comprises an inhibitory nucleic acid and/or a therapeutic nucleic acid sequence encoding a therapeutic polypeptide as described herein.
  • a rAAV vector comprises a modified AAV genome encapsidated by a viral capsid.
  • a viral capsid comprises 60 capsid protein subunits.
  • a viral capsid comprises VP1, VP2, and VP3.
  • VP1, VP2, and VP3 subunits are present in a capsid at a ratio of about 1: 1: 10, respectively.
  • a rAAV may comprise or be based on a serotype selected from any following serotypes or variants thereof including, but not limited to, AAV9.68, AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/liu.4O, AAV 12, AAV127.2/hu.41, AAVI27.5/hu.42, AAV128.1/hu.43, AAVI28.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAVI45.5/hu.54, AAVI45.6/hu.55, AAV16.12/hu.l 1, AAV16.3, AAV16.8/hu.
  • An AAV serotype may be from any number of species.
  • an AAV may be or comprise an avian AAV (AAAV), e.g., as described in U.S. Patent No. 9,238,800, which is hereby incorporated by reference in its entirety.
  • An AAV serotype may be or comprise a bovine AAV (BAAV), e.g., as described in U.S. Patent Nos. 9,193,769 or 7,427,396, each of which is hereby incorporated by reference in its entirety.
  • An AAV may be or comprise a caprine AAV. e.g., as described in U.S. Patent No. 7427396, which is hereby incorporated by reference in its entirety.
  • An AAV serotype may also be a variant or hybrid of any of the foregoing.
  • a rAAV may be or comprise a serotype generated from an AAV2 capsid library.
  • a rAAV may be or comprise a serotype generated from an AAV5 capsid library.
  • a rAAV comprises a capsid including modified capsid proteins (e.g., capsid proteins comprising a modified VP3 region). Methods of producing modified capsid proteins are known in the art (See, e.g., US20130310443, which is hereby incorporated by reference in its entirety).
  • a rAAV comprises a modified capsid protein comprising at least one non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid (e.g., a surface exposed tyrosine) in a wild-type capsid protein.
  • a rAAV comprises a modified capsid protein comprising a non-tyrosine amino acid (e.g., a phenylalanine) at a position that corresponds to a surface- exposed tyrosine amino acid in a wild-type capsid protein, a non-threonine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in a wild-type capsid protein, a nonlysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface-exposed lysine amino acid in a wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in a wild-type capsid protein, or a combination thereof.
  • a rAAV comprises a capsid that
  • properties and/or activities of provided inhibitory nucleic acids, rAAV vectors, and compositions thereof can be characterized and/or assessed using various technologies available to those skilled in the art, e.g., biochemical assays, cell-based assays, animal models, or clinical trials. Certain useful technologies are described in the Examples. Those skilled in the art reading the present disclosure will readily appreciate that other technologies, e.g., in vitro models (e.g., cell lines) for various diseases, disorders, or conditions, animal models for various diseases, disorders, or conditions, etc. may be designed and/or utilized to assess provided technologies (e.g., inhibitory nucleic acids, rAAV vectors, compositions, or methods.) in accordance with the present disclosure.
  • technologies e.g., inhibitory nucleic acids, rAAV vectors, compositions, or methods.
  • compositions of the present disclosure may be administered in any form, including tablet, powder, or liquid, formulated into a pharmaceutically acceptable carrier or excipient, depending on the condition of the patient.
  • non-active ingredients well known in the art such as binders, fdlers, coatings, preservatives, coloring agents, flavoring agents and other additives may optionally be formulated with one or more administered agents, or left out completely if there is a risk of negative side effects to the patient such as increased the risk of intestinal inflammation or interference with the absorption of particular compounds.
  • the present disclosure provides pharmaceutical compositions comprising aprovided composition, e.g., an inhibitory nucleic acid or rAAV vector.
  • a provided composition e.g., an inhibitory nucleic acid or rAAV vector.
  • an inhibitory nucleic acid or rAAV vector are provided as pharmaceutical compositions.
  • a pharmaceutical composition is suitable for administration or delivery of an inhibitory nucleic acid or rAAV vector, to an area or portion of a body affected by a disease, disorder, or condition.
  • a pharmaceutical composition comprises a therapeutically effective amount of an inhibitory nucleic acid or rAAV vector provided herein.
  • a phannaceutical composition comprises a therapeutically effective amount of an inhibitory nucleic acid or rAAV vector, and a phannaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier is a buffer.
  • a pharmaceutical composition is formulated for intravenous injection, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration, or otic administration.
  • a pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop, or an ear drop.
  • a pharmaceutical composition is formulated for subretinal administration.
  • Various technologies may be utilized to administer or deliver provided an inhibitory nucleic acids, rAAV vectors, or compositions thereof.
  • provided inhibitory nucleic acids, rAAV vectors, or compositions thereof are delivered to the eye.
  • provided inhibitory nucleic acids, rAAV vectors, or compositions thereof are delivered to the subretinal space.
  • provided inhibitory nucleic acids, rAAV vectors, or compositions thereof are delivered to a subject by subretinal administration.
  • subretinal administration is by injection, by, e.g., a syringe.
  • an injection is a bolus injection.
  • a rAAV vector, or composition thereof is administered to a subject at a dose of at least 10 8 , at least 10 9 , at least IO 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 1 ’, at least IO 16 , at least 10 17 , at least 10 18 , at least 10 19 , at least IO 20 , at least 10 21 , at least IO 22 , at least 10 23 , at least IO 24 , at least IO 25 , or at least 10 26 genome copies per subject.
  • a rAAV vector, or composition thereof is administered to a subject at a dose of at most 10 8 , at most 10 9 , at most IO 10 , at most 10 11 , at most 10 12 , at most 10 13 , at most 10 14 , at most 10 15 , at most 10 16 . at most 10 17 , at most IO 18 , at most 10 19 , at most IO 20 , at most 10 21 , at most IO 22 , at most 10 23 , at most 10 24 , at most 10 25 , or at most 10 26 genome copies per subject.
  • a rAAV vector, or composition thereof is administered to a subject at a dose of at least 10 8 , at least 10 9 , at least IO 10 , at least 10 11 , at least IO 12 , at least 10 13 , or at least IO 14 genome copies per eye.
  • a rAAV vector, or composition thereof is administered to a subject at a dose of at most 10 8 , at most 10 9 , at most IO 10 , at most 10 11 , at most 10 12 . at most 10 13 . or at most 10 14 genome copies per eye.
  • a rAAV vector, or composition thereof is administered to a subject at a dose within a range of about 10 8 to about 10 26 , IO 10 to about 10 24 , or 10 12 to about 10 22 genome copies per subject. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose within a range of about 10 8 to about 10 14 genome copies, about 10 9 to about 10 13 genome copies, or about 10 10 to about 10 12 genome copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 10 9 genome copies per eye.
  • a rAAV vector, or composition thereof is administered to a subject at a dose of about 5 x 10 9 genomes copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 10 10 genome copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 5 x 10 10 genomes copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 10 11 genome copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 5 x 10 11 genome copies per eye. In some embodiments, a rAAV vector, or composition thereof is administered to a subject at a dose of about 10 12 genome copies per eye.
  • nucleic acid sequences may be used in the provided technologies of the present disclosure. Exemplary sequences are provided below.
  • Example 1 Design of cargo comprising miR-254 and resRHO.
  • a miRNA targeting RHO mRNA was designed.
  • the artificial miRNA (amiRNA) miR-254 (SEQ ID NO: 1) was selected for silencing of the human and mouse RHO gene.
  • a miR-254-resistant RHO nucleic acid sequence (resRHO) (SEQ ID NO: 17) was developed by introducing mutations within the miR- 254-targeted region of the RHO cDNA.
  • miR-254 was embedded in various miRNA scaffolds (e.g., miR-30, miR-155, miR-E, UltramiR).
  • the miRNA-containing scaffold was further placed in a synthetic intron comprising a splice donor (SD) site upstream (5’) of the miRNA-containing scaffold and a splice acceptor (SA) site downstream (3’) of the miRNA-containing scaffold.
  • Constructs further comprised a promoter upstream (5‘) of the miRNA-containing synthetic intron.
  • SD splice donor
  • SA splice acceptor
  • Cargo constructs comprising miR-254 in a miRNA scaffold, optionally in a synthetic intron comprising SD and SA sites, and resRHO were designed and created.
  • Each cargo construct further comprised a WPRE (e.g., SEQ ID NO: 16) downstream (3’) of the resRHO nucleic acid sequence and a polyadenylation (polyA) signal (e.g., hGH polyA (SEQ ID NO: 17)) downstream (3’) of tire WPRE.
  • WPRE e.g., SEQ ID NO: 16
  • polyA polyadenylation
  • the miRNA-containing synthetic intron described above was located in one of the following locations: (i) downstream (3’) of the promoter and upstream (5‘) of the resRHO nucleic acid sequence; (ii) downstream (3 ? ) of the resRHO nucleic acid sequence and upstream (5') of the WPRE; or (iii) downstream (3’) of the WPRE and upstream (5’) of the polyA signal.
  • Some cargo designs included two miRNA-containing synthetic introns placed in two of the above listed locations (e.g., (i) and (ii)).
  • HEK293T cells expressing RHO-P23H-HA were transfected with plasmid vectors comprising cargoes as described above.
  • Control plasmid vectors comprising a GFP gene (p029) or resRHO only with no miRNA (p201) were also transfected.
  • HEK293T cells were transfected with vectors comprising candidate cargoes as described above or with a control cargo containing no miRNA-containing synthetic intron (as a control).
  • CRISPRa components were also introduced into the same cells in order to provide activation of the huRHOP-805 promoter.
  • RNA was collected and used to perform RT-PCR.
  • the RNA was used to generate cDNA and a region corresponding to the miRNA-containing synthetic intron was amplified using primers specific to flanking (5‘ and 3’) sequences. Tire resulting amplified products were examined using gel electrophoresis as known in the art.
  • cargo constructs comprised a first expression cassette comprising, from 5’ to 3’, a huRHOP-805 promoter, an intron, a renilla luciferase (RLuc) nucleic acid sequence, and a polyA signal and a second expression cassette comprising, from 5’ to 3’.
  • a HSV-TK promoter a firefly luciferase nucleic acid sequence (FFLuc), and a polyA signal.
  • HEK293T cells were transfected with pairs of plasmids as depicted in Fig. 5A.
  • One plasmid comprised a CAG promoter driving expression of a cargo comprising a RHO-targeting miRNA (e.g., miR- 254) and RHO cDNA (or, as a control, eGFP), while the other plasmid comprised a renilla luciferase (RLuc)-human RHO cDNA fusion sequence operably linked to a SV40 promoter and a firefly luciferase (FFLuc) sequence operably linked to a TK promoter.
  • RLuc renilla luciferase
  • FFLuc firefly luciferase
  • RLuc-hRHO fusion construct As evaluated by normalized RLuc:FFLuc signal, and as seen in Fig. 5B, robust knockdown (c.g., -85-90%) of the RLuc-hRHO fusion construct was observed when co-cxprcsscd with miRNA- containing cargos. The control eGFP cargo did not lead to any notable knockdown of the RLuc-hRHO fusion construct.
  • provided technologies e.g., miRNAs, cargos, modified AAV genomes
  • target transcripts and/or products thereof e.g., polypeptides
  • Example 6 Cargo expression from an AAV8 vector.
  • AAV8 vectors encoding the cargos for pOGTX244 (SEQ ID NO: 43) or pOGTX245 (SEQ ID NO: 44) were evaluated in HEK293T cells that overexpressed the universal AAV receptor (AAVR) to enhance vector uptake. These cells were also previously transfected with CRISPR activation components to stimulate expression of the RHO promoter. Cells were transduced with AAV8 vectors at different multiplicity of infection (MOI) ranging from le4 to le6. Cells were assayed at 48 hours post-transduction for expression analysis by immunohistochemistry against the HA tag. IHC reveals robust expression of the 5’ synthetic intron design (pOGTX244) in comparison to the 3’ cargo design (pOGTX245), thus recapitulating the improved expression performance seen previously based on cargo plasmid transfection (FIG. 6).
  • MOI multiplicity of infection
  • Cynomolgus macaques received subretinal injections of AAV5 or AAV8 vectors encoding eGFP driven by the human RHO promoter (FIG. 7A).
  • the fovea (cone- enriched) was devoid of eGFP expression validating the rod-specificity of the promoter.
  • Example 8 Effect of AAV-RHO candidates upon retinal function in NHP retina.
  • Electroretinograms were performed prior to AAV5 or AAV8 vector administration and 8 weeks post-injection (before termination) in NHP retina. Scotopic a- and b-wave amplitudes were calculated across AAV5 and AAV8 vector groups and dose levels. Administration of AAV5 or AAV8 vectors encoding a tagged version of pOGTX228 cargo showed no significant differences between time points. These data demonstrated a trending (but non-significant) reduction in a- and b-wave amplitudes with the AAV8 vector (high dose) at the 8 week time point (FIG. 8). However, there were observing changes in retinal function with vector candidates encoding a tagged version of pOGTX181 cargo.
  • Example 8 Structural effects of AAV-RHO gene transfer in the NHP retina.
  • FIG. 13A A miRNA targeting RHO mRNA was designed (FIG. 13A). Cargo characterization was performed in vitro in a P23H mutant silencing assay as described in Example 2 (FIG. 13B). Subretinal injection of the AAV candidate in wild-type mouse retinal showed that vector-expressed RHO (HA signal) localized correctly to the photoreceptor outer segments (OS: FIG. 13C).
  • An AAV8 vector comprising a pOGTX347 cargo (AAV8-RHO.3) was designed and constructed.
  • the pOGTX347 cargo (SEQ ID NO: 52) comprised a pOGTX254 cargo (SEQ ID NO: 45) (which comprises a miRNA targeting RHO mRNA) with an additional sequence encoding a HA epitope tag fused to the C-tenninus of the encoded RHO protein.
  • Wild-type C57BL/6 mice were subretinally injected with 1.5 x 10 9 vg/eye of AAV8-RHO.3 in one eye and vehicle (PBS + 0.001% pluronic F-68) alone in the other eye.
  • mice were sacrificed and retinas collected. Levels of expression of mouse RHO and mouse GAPDH in collected retinas were quantified by qPCR.
  • the AAV8 vector comprising a pOGTX347 cargo was able to provide knockdown of endogenous RHO expression in vivo.
  • an AAV vector comprising a miRNA targeting RHO mRNA as provided herein can provide knockdown of endogenous RHO expression in vivo.
  • an AAV vector comprising a miRNA targeting RHO mRNA as provided herein can reduce level of endogenous RHO expression in vivo by at least about 50%, 55%, 60%, 65%, 70%, or 75% as compared to a reference condition.
  • a reference condition is absence of the AAV vector comprising a miRNA targeting RHO mRNA.
  • a reference condition is vehicle alone.
  • AAV5 vectors comprising various cargoes comprising a miRNA targeting RHO mRNA (miR- 254) and a miR-254-resistant RHO nucleic acid sequence (resRHO) were designed and constructed.
  • AAV- RHO.1 comprised a pOGTX245 cargo
  • AAV-RHO.2 comprised a pOGTX244 cargo
  • AAV-RHO.3 comprised a pOGTX347 cargo.
  • the pOGTX245 cargo (SEQ ID NO: 44) comprised a pOGTX228 cargo (SEQ ID NO: 40) with an additional sequence encoding a HA epitope tag fused to the C-terminus of the encoded RHO protein.
  • the pOGTX244 cargo (SEQ ID NO: 43) comprised a pOGTX181 cargo (SEQ ID NO: 25) with an additional sequence encoding a HA epitope tag fused to the C-terminus of the encoded RHO protein.
  • Hie pOGTX347 cargo (SEQ ID NO: 52) comprised a pOGTX254 cargo (SEQ ID NO: 45) with an additional sequence encoding a HA epitope tag fused to the C-terminus of the encoded RHO protein.
  • Non-human primates (NHP), Macaca fascicularis, were subretinally injected with 5 x I0 lu vg/eye of AAV-RHO.1, 1 x 10 10 vg/eye of AAV-RHO.2, 5 x 10 10 vg/eye of AAV-RHO.3, or vehicle (PBS + 0.001% pluronic F-68) alone.
  • a total of 7 animals were injected, with 2 animals utilized for each AAV and 1 animal utilized for vehicle alone. Both eyes of each animal were injected.
  • Scotopic and photopic electroretinograms (ERG) were conducted for both eyes for each animal prior to dosing and at 8 weeks post-injection, prior to sacrifice. Animals were sacrificed at 8 weeks post-injection and retinas collected. Levels of expression of miR-254, M. fascicularis RHO, and M. fascicularis GAPDH in collected retinas were quantified by qPCR.
  • Retinal sections were examined using immunofluorescence using methods available in the art. Hie retinal sections were stained to detect expression of tire HA epitope tag fused to the C-terminus of the resRHO protein, total RHO protein, and cellular nuclei (DAPI). Exemplary micrographs of retinal sections from NHP treated with AAV-RHO. 1, AAV-RHO.2, and AAV-RHO.3 are shown in Figure 15, Figure 16, and Figure 17, respectively. As shown in the aforementioned figures, injection with an AAV vector provided herein can provide robust expression of resRHO.
  • an AAV vector provided herein can provide a high level of expression of a mRNA targeting RHO, e.g., miR-254.
  • Knockdown of expression of endogenous M. fascicularis RHO mRNA was confirmed by quantification of expression within subretinal blebs. For each animal, expression oiM. fascicularis RHO and M. fascicularis GAPDH was quantified for two samples, wherein each sample comprised a half of a subretinal bleb. As shown in Figure 19, significant knockdown of M. fascicularis RHO expression was detected in animals treated with either AAV-RHO.2 (74% knockdown) or AAV-RHO.3 (56% knockdown). Meanwhile, knockdown ofM. fascicularis RHO expression was not observed in animals treated with either AAV-RHO.1 or vehicle alone.
  • an AAV vector comprising a miRNA targeting RHO mRNA as provided herein can provide knockdown of endogenous RHO expression in vivo.
  • an AAV vector comprising a miRNA targeting RHO mRNA as provided herein can reduce level of endogenous RHO expression in vivo by at least about 50%, 55%, 60%, 65%, 70%, or 75% as compared to a reference condition.
  • a reference condition is absence of the AAV vector comprising a miRNA targeting RHO mRNA.
  • a reference condition is vehicle alone.
  • an AAV vector provided herein can be injected into an eye with minimal or no resulting inflammation of the eye.

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Abstract

Entre autres, la présente invention concerne des compositions, par exemple, des vecteurs de virus adéno-associé recombinant (rAAV), comprenant, par exemple, un acide nucléique inhibiteur (par exemple, un miARN) et/ou une séquence d'acide nucléique thérapeutique codant pour un polypeptide thérapeutique. Dans certains modes de réalisation, la présente invention concerne des procédés de diminution de l'expression d'un polypeptide pathogène (par exemple, un polypeptide RHO mutant) et l'augmentation de l'expression d'un polypeptide thérapeutique (par exemple, un polypeptide RHO de type sauvage), par exemple, chez un sujet. Dans certains modes de réalisation, la présente invention concerne des méthodes de traitement de la dégénérescence rétinienne chez un sujet. Dans certains modes de réalisation, la présente invention concerne des méthodes de traitement de la rétinite pigmentaire (RP) chez un sujet.
PCT/US2023/035795 2022-10-24 2023-10-24 Compositions et méthodes de traitement de la rétinite pigmentaire WO2024091495A1 (fr)

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WO2021108755A2 (fr) * 2019-11-28 2021-06-03 Regenxbio Inc. Constructions de thérapie génique de la microdystrophine et leurs utilisations

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Publication number Priority date Publication date Assignee Title
WO2021108755A2 (fr) * 2019-11-28 2021-06-03 Regenxbio Inc. Constructions de thérapie génique de la microdystrophine et leurs utilisations

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Title
DING DAN, CHEN KAIYUAN, CHEN YUEDAN, LI HONG, XIE KABIN: "Engineering Introns to Express RNA Guides for Cas9- and Cpf1-Mediated Multiplex Genome Editing", MOLECULAR PLANT, vol. 11, no. 4, 1 April 2018 (2018-04-01), pages 542 - 552, XP055863659, ISSN: 1674-2052, DOI: 10.1016/j.molp.2018.02.005 *

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