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WO2024238859A1 - Vectorized c5 inhibitor agents and administration thereof - Google Patents

Vectorized c5 inhibitor agents and administration thereof Download PDF

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Publication number
WO2024238859A1
WO2024238859A1 PCT/US2024/029800 US2024029800W WO2024238859A1 WO 2024238859 A1 WO2024238859 A1 WO 2024238859A1 US 2024029800 W US2024029800 W US 2024029800W WO 2024238859 A1 WO2024238859 A1 WO 2024238859A1
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seq
serotype
aav
tick
cells
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Joseph Bruder
Wei-Hua Lee
Mi SHI
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Regenxbio Inc
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Regenxbio Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
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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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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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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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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

Definitions

  • tick C5 inhibitor protein e.g., a protein comprising the amino acid sequence of SEQ ID NO: 129, may inhibit complement activation and drusen deposit in the eye to inhibit, reduce the progression of dry AMD.
  • compositions and methods are described for the ocular or systemic delivery of a tick C5 inhibitor protein (for example, a fully human-glycosylated), to a patient (human subject) diagnosed with AMD or other condition indicated for treatment with the therapeutic tick C5 inhibitor protein.
  • Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein — to create a permanent depot in the eye, or in alternative embodiments, liver and/or muscle, of the patient that continuously supplies the human post-translationally modified (HuPTM) tick C5 inhibitor peptide to one or more ocular tissues where the peptide exerts its therapeutic or prophylactic effect.
  • Human post-translationally modified (HuPTM) tick C5 inhibitor peptide to one or more ocular tissues where the peptide exerts its therapeutic or prophylactic effect.
  • gene therapy vectors particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of tick C5 inhibitor protein to achieve a maximum or steady states concentrations in ocular tissues, such as aqueous humor, vitreous humor, or in serum for example, 20, 30, 40, 50, 60 or 90 days after administration of the vector encoding the tick C5 inhibitor protein.
  • the recombinant vector used for delivering the transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”).
  • the AAV type has a tropism for ocular tissues, including, for example, retinal cells, RPE, choroid, Bruch’s membrane (BrM) and epithelial cells thereof, choriocapillaris and epithelial cells thereof, photoreceptor cells (rods and cones) and retinal ganglion cells.
  • the AAV type may be, for example, AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof) subtype of AAV.
  • viral vectors including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs.
  • Expression of the transgene can be controlled by constitutive expression elements, such as a CAG promoter, or tissue-specific expression control elements, particularly elements that are ocular tissue, liver and/or muscle specific control elements, for example one or more elements of Tables 1 and la.
  • the HuPTM tick C5 inhibitor protein encoded by the transgene can include, but is not limited to, one of SEQ ID NOs: 129 - 130 (encoding a HuPTM tick C5 inhibitor protein without a signal sequence) or one of SEQ ID Nos: 131- 134 (encoding a HuPTM tick C5 inhibitor protein having a signal sequence).
  • proteins expressed from transgenes in vivo are not likely to contain degradation products associated with proteins produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is an issue associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and purification with certain buffer systems. These conditions, which promote aggregation, do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage, and is caused by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed from transgenes in vivo may also oxidize in a stressed condition.
  • HuPTM tick C5 inhibitor protein in ocular tissue cells of the human subject should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein to a patient (human subject) diagnosed with a disease indication for that tick C5 inhibitor protein, to create a permanent depot in the subject that continuously supplies the human post- translationally modifiedtransgene product produced by the subject’s transduced cells.
  • the cDNA construct for the HuPTM tick C5 inhibitor protein should include a signal peptide (or more than one signal peptide, e.g., a native signal sequence and a mutated IL-2 signal sequence in tandem) that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • a signal peptide or more than one signal peptide, e.g., a native signal sequence and a mutated IL-2 signal sequence in tandem
  • the HuPTM tick C5 inhibitor protein can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients.
  • the recombinant tick C5 inhibitor protein produced by human cell lines includes a heterologous signal peptide such as a mutated IL-2 signal sequence, or more than one signal peptide, e.g., a native signal sequence and a mutated IL-2 signal sequence in tandem.
  • Combination therapies involving systemic delivery of the HuPTM tick C5 inhibitor protein to the patient accompanied by administration of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Such additional treatments can include but are not limited to co-therapy with a therapeutic (recombinant) tick C5 inhibitor protein.
  • kits for producing recombinant AAV s comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic tick C5 inhibitor protein operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
  • Embodiment 1 A pharmaceutical composition for treating AMD in a human subj ect in need thereof, comprising a recombinant adeno-associated virus (AAV) vector comprising: (a) a viral capsid that has a tropism for ocular tissue cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein said AAV vector is formulated for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic administration to said human subject.
  • AAV adeno-associated virus
  • Embodiment 2 The pharmaceutical composition of embodiment 1, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu1), ser
  • Embodiment 3 The pharmaceutical composition of embodiment 1 or embodiment 2, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
  • Embodiment 4 The pharmaceutical composition of any one of embodiments 1 to 3, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photreceptor cells (rods, cones and/or retinal ganglion cells).
  • the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photreceptor cells (rods, cones and/or retinal ganglion cells).
  • Embodiment 5 The pharmaceutical composition of any one of embodiments 1 to 4, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
  • Embodiment 6 The pharmaceutical composition of embodiment 5, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
  • the transgene comprises a signal sequence.
  • Embodiment 8 The pharmaceutical composition of embodiment 7, wherein the signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
  • Embodiment 9 The pharmaceutical composition of any one of embodiments 1 to 8, wherein the tick C5 inhibitor protein comprises an amino acid sequence of SEQ ID NO: 129.
  • Embodiment 10 The pharmaceutical composition of any one of embodiments 1 to 9, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143 .
  • Embodiment 11 The pharmaceutical composition of any one of embodiments 1 to 10, wherein the artificial genome comprises construct CAG. tick. C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
  • a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising: (a) a viral capsid that has a tropism for ocular tissue cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein the transgene encodes a signal sequence at the N- terminus of said tick C5 inhibitor protein that directs secretion and post translational modification of said tick C5 inhibitor protein in human ocular tissue cells.
  • AAV adeno-associated virus
  • Embodiment 13 The pharmaceutical composition of embodiment 12, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu1), ser
  • Embodiment 14 The pharmaceutical composition of embodiment 13, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
  • Embodiment 15 The pharmaceutical composition of any one of embodiments 12 to 14, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
  • the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
  • Embodiment 16 The pharmaceutical composition of any one of embodiments 12 to 15, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
  • Embodiment 17 The pharmaceutical composition of embodiment 16, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
  • CAG promoter SEQ ID NO: 2
  • CB promoter SEQ ID NO: 17 or SEQ ID NO: 18
  • GRK1 human rhodopsin kinase
  • CAR mouse cone arresting
  • RedO human red opsin
  • SEQ ID NO: 19 Bestl/GRKl tandem promoter
  • Embodiment 18 The pharmaceutical composition of any one of embodiments 12 to 17, wherein the transgene comprises a signal sequence.
  • Embodiment 21 The composition of any one of embodiments 12 to 20, wherein the tick C5 inhibitor protein has an amino acid sequence of SEQ ID NO: 129.
  • Embodiment 22 The pharmaceutical composition of any one of embodiments 12 to 21, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) CAG promoter (SEQ ID NO: 2), b) optionally, a VH4 intron (SEQ ID NO: 36) or other intron and c) a rabbit (3-globin poly A signal (SEQ ID NO: 38); and (3) nucleic acid sequences coding for a tick C5 inhibitor protein.
  • the AAV2 inverted terminal repeats are the 5’ ITR (SEQ ID NO: 48) and the 3’ ITR (SEQ ID NO: 149).
  • the viral vectors provided herein are lentivirus-based viral vectors.
  • lentiviral vectors provided herein are derived from human lentiviruses.
  • lentiviral vectors provided herein are derived from non-human lentiviruses.
  • lentiviral vectors provided herein are packaged into a lentiviral capsid.
  • lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.
  • the viral vectors provided herein are alphavirus-based viral vectors.
  • alphavirus vectors provided herein are recombinant, replicationdefective alphaviruses.
  • alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.
  • the viral vectors provided herein are AAV based viral vectors.
  • the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for synthesis of the capsid proteins) (the rep and cap proteins may be provided by the packaging cells in trans). Multiple AAV serotypes have been identified.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV with tropism to ocular tissues, liver and/or muscle.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAVhu21, AAV.hul2, or AAV.hu26.
  • AAV based vectors provided herein are or comprise components from one or more of AAV8, AAV3B, AAV9, AAV10, AAVrh73, or AAVrhlO serotypes.
  • the capsid protein is a variant of the AAV8 capsid protein (SEQ ID NO: 114), AAV3B capsid protein (SEQ ID NO: 108), or AAVrh73 capsid protein (SEQ ID NO: 120), and the capsid protein is e. ., at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 114), AAV9 (SEQ ID NO: 115), AAV3B capsid protein (SEQ ID NO: 108), or AAVrh73 capsid protein (SEQ ID NO: 120), while retaining the biological function of the native capsid.
  • the encoded AAV capsid has the sequence of SEQ ID NO: 114 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV8, AAV3B, or AAVrh73 capsid.
  • FIG. 1 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS.
  • the AAV vector comprises an AAV8, AAV3B, or AAVrh73, capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions that are not present at that position in the native AAV capsid sequence as identified in the SUBS row of FIG. 1.
  • Amino acid sequence for AAV8, AAV9, AAV3B, or AAVrh73 capsids are provided in FIG. 1.
  • amino acid sequence of hu37 capsid can be found in international application PCT WO 2005/033321 (SEQ ID NO: 88 thereof) and the amino acid sequence for the rh8 capsid can be found in international application PCT WO 03/042397 (SEQ ID NO:97).
  • amino acid sequence for the rh64Rl sequence is found in W02006/110689 (a R697W substitution of the Rh.64 sequence, which is SEQ ID NO: 43 of WO 2006/110689).
  • AAV-based vectors comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu31, AAV.hu32, AAVAnc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAV
  • AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu31, AAVhu32, AAV.Anc80, AAV.Anc80L65, AAV7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAVHSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC
  • rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
  • AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAVrh8, AAVrhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAVrh73, AAV.Rh74, AAVRHM4-1, AAV.hu37, AAV.hu31, AAV.hu32, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAVHSC2, AAVHSC3, AAV
  • the recombinant AAV for us in compositions and methods herein is AAVS3 (including variants thereof) (see e.g., US Patent Application No. 20200079821, which is incorporated herein by reference in its entirety).
  • rAAV particles comprise the capsids of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 10,301,648, such as AAV.rh46 or AAVrh73.
  • the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety).
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B.
  • the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors).
  • the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.
  • rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
  • rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g., SEQ ID Nos: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID Nos: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID Nos: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g., SEQ ID Nos: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID Nos: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID No., SEQ ID Nos:
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No.
  • WO 2003/052051 see, e.g., SEQ ID NO: 2 of '051 publication
  • WO 2005/033321 see, e.g., SEQ ID Nos: 123 and 88 of '321 publication
  • WO 03/042397 see, e.g., SEQ ID Nos: 2, 81, 85, and 97 of '397 publication
  • WO 2006/068888 see, e.g., SEQ ID Nos: 1 and 3-6 of '888 publication
  • WO 2006/110689 see, e.g., SEQ ID Nos: 5-38 of '689 publication
  • W02009/104964 see, e.g, SEQ ID Nos: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication
  • W0 2010/127097 see, e.g, SEQ ID Nos: 5-38 of '097 publication
  • WO 2015/191508 see, e.g, SEQ ID Nos: 80-294 of '508
  • rAAV particles comprise a pseudotyped AAV capsid.
  • the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g, Duan et al, J. Virol, 75:7662-7671 (2001); Halbert et al, J. Virol, 74: 1524-1532 (2000); Zolotukhin et al. Methods 28: 158-167 (2002); and Auricchio et al. Hum. Molec. Genet. 10:3075-3081, (2001).
  • AAV8-based, AAV3B-based, and AAVrh73-based viral vectors are used in certain of the methods described herein.
  • Nucleotide sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.
  • AAV e.g., AAV8, AAV3B, AAVrh73, or AAVrhl0
  • AAV capsids including AAV8, AAV3B, AAVrh73 and AAVrhlO are provided in FIG. 1.
  • a single-stranded AAV may be used supra.
  • a self-complementary vector e.g., scAAV
  • scAAV single-stranded AAV
  • the viral vectors used in the methods described herein are adenovirus based viral vectors.
  • a recombinant adenovirus vector may be used to transfer in the transgene encoding the tick C5 inhibitor protein.
  • the recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene is inserted between the packaging signal and the 3’ ITR, with or without staffer sequences to keep the genome close to wild-type size of approximately 36 kb.
  • An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.
  • the viral vectors used in the methods described herein are lentivirus based viral vectors.
  • a recombinant lentivirus vector may be used to transfer in the transgene encoding the HuPTM tick C5 inhibitor protein.
  • Four plasmids are used to make the construct: Gag/pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (e.g., VSV-G), and Cis plasmid with the packaging elements and the tick C5 inhibitor protein.
  • the four plasmids are co-transfected into cells (e.g., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others.
  • the lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs/should be done).
  • the supernatant is fdtered (0.45 pm) and then magnesium chloride and benzonase added.
  • Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with/without column chromatography.
  • Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, andAusubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.
  • the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g., to detect or select for cells that have taken up the polynucleotide.
  • the promoter is a CAG promoter (SEQ ID NO: 2) (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety).
  • the CAG promoter (SEQ ID NO: 2) or CB7 promoter (SEQ ID NO: 1) includes other expression control elements that enhance expression of the transgene driven by the vector.
  • the other expression control elements include chicken 0-actin intron and/or rabbit P-globin polyA signal (SEQ ID NO: 38).
  • the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements.
  • the one or more promoter elements may be inverted or moved relative to one another.
  • the elements of the promoter are positioned to function cooperatively.
  • the elements of the promoter are positioned to function independently.
  • the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter.
  • the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs.
  • the vectors provided herein comprise one or more tissue specific promoters (e.g., a retinal-specific promoter).
  • the viral vectors provided herein comprises a ocular tissue cell specific promoter, such as, human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), or a human red opsin (RedO) promoter (SEQ ID NO: 7).
  • GRK1 human rhodopsin kinase
  • CAR mouse cone arresting
  • RedO human red opsin
  • nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette. Regulatory elements, in general, have multiple functions as recognition sites for transcription initiation or regulation, coordination with cellspecific machinery to drive expression upon signaling, and to enhance expression of the downstream gene.
  • the promoter is an inducible promoter. In certain embodiments the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF- la binding site. In certain embodiments, the promoter comprises a HlF-2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schbdel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated by reference herein in its entirety.
  • the promoter comprises a binding site for a hypoxia induced transcription factor other than a HIF transcription factor.
  • the viral vectors provided herein comprise one or more IRES sites that is preferentially translated in hypoxia.
  • the hypoxia-inducible promoter is the human N-WASP promoter, see, e.g., Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 (incorporated by reference for the teaching of the N-WASP promoter) or is the hypoxia-induced promoter of human Epo, see, e.g., Tsuchiya et al., 1993, J. Biochem. 113:395-400 (incorporated by reference for the disclosure of the Epo hypoxia-inducible promoter).
  • the promoter is a drug inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof.
  • constructs containing certain ubiquitous and tissue-specific promoters include synthetic and tandem promoters. Examples and nucleotide sequences of promoters are provided in Tables 1 and la below. Table 1 also includes the nucleotide sequences of other regulatory elements useful for the expression cassettes provided herein. Table 1. Promoter and Other Regulatory Element Sequences Table la. Other regulatory and ITR sequences [0106]
  • the viral vectors provided herein comprise one or more regulatory elements other than a promoter.
  • the viral vectors provided herein comprise an enhancer.
  • the viral vectors provided herein comprise a repressor.
  • the viral vectors provided herein comprise an intron (e.g. VH4 intron (SEQ ID NO: 36), SV40 intron (SEQ ID NO: 37), or a chimeric intron ([3-globin/Ig Intron) (SEQ ID NO: 35).
  • the viral vectors may also include a Kozak sequence to promote translation of the transgene product, for example GCCACC (SEQ ID NO: 39).
  • the viral vectors provided herein comprise a polyadenylation sequence downstream of the coding region of the transgene.
  • Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure.
  • Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit -globin gene (SEQ ID NO: 38), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.
  • signal peptides may retain function and exert activity after being cleaved from the expressed protein as “post-targeting functions”.
  • the signal peptide is selected from signal peptides of the most abundant proteins secreted by the cells used for expression to avoid the post-targeting functions.
  • Exemplary sequences are the native tick C5 inhibitor signal peptide, MLVLVTLIFSFSANIAYA (SEQ ID NO: 52), which can be encoded by a nucleotide sequence of SEQ ID NO: 63, and a mutant IL2 signal peptide, MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), which can be encoded by a nucleotide sequence of SEQ ID NO: 64 or SEQ ID NO: 65 (see Table 2).
  • more than one signal sequence may be used, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in Example 2 (SEQ ID NO: 132).
  • signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the tick C5 inhibitor protein in the eye/CNS, muscle, or liver are provided in Tables 2A/2B and Table 3, below.
  • the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs.
  • UTRs are optimized for the desired level of protein expression.
  • the UTRs are optimized for the mRNA half-life of the transgene.
  • the UTRs are optimized for the stability of the mRNA of the transgene.
  • the UTRs are optimized for the secondary structure of the mRNA of the transgene.
  • the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences.
  • ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector.
  • the ITR is from an AAV, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No.
  • nucleotide sequences encoding the ITRs may, for example, comprise the nucleotide sequences of SEQ ID NO: 48 (5’-ITR) and SEQ ID NO: 50 or SEQ ID NO: 149 (3’-ITR).
  • the modified ITRs used to produce self-complementary vector e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171 -82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos.
  • nucleotide sequences encoding the modified ITRs may, for example, comprise the nucleotide sequences of SEQ ID NO: 48 (5’-ITR) and SEQ ID NO: 50 or SEQ ID NO: 149 (3’-ITR) or modified for scAAV, SEQ ID NO: 49 (m 5’ITR) and SEQ ID NO: 51 or SEQ ID NO: 150 (m 3’ ITR).
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 129 (see Table 4).
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 129 and has C5 inhibitory activity.
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 130 (see Table 4).
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 130 and has C5 inhibitory activity.
  • the tick C5 inhibitor protein may have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and/or muscle cells.
  • the signal sequence may have the amino acid sequence of the endogenous tickC5 inhibitor sequence, which is MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) (underlined in Table 4) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Table 2 that correspond to the proteins secreted by ocular tissue cell types.
  • the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 131 (having a native tick C5 inhibitor signal sequence).
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 131 and has C5 inhibitory activity.
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 132 (having a mutant IL2 signal sequence and a native C5 inhibitor signal sequence).
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 132 and has C5 inhibitory activity.
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 133 (having a mutant IL2 signal sequence).
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 133 and has C5 inhibitory activity.
  • vectors including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 134.
  • the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 134 and has C5 inhibitory activity.
  • vectors including AAV vectors, comprising a transgene encoding the tick C5 inhibitor protein, having an amino acid sequence of SEQ ID NO: 129.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the tick C5 inhibitor protein may be encoded by a nucleotide sequence comprising SEQ ID NO: 135 (see Table 5).
  • the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 135.
  • the tick C5 inhibitor protein may be encoded by a nucleotide sequence comprising SEQ ID NO: 136 (see Table 5).
  • the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 136.
  • the tick C5 inhibitor protein (having a mutant IL2 signal sequence and a native signal sequence) may be encoded by a nucleotide sequence comprising SEQ ID NO: 140 (see Table 5).
  • the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 140.
  • the tick C5 inhibitor protein (having a mutant IL2 signal sequence) may be encoded by a nucleotide sequence comprising SEQ ID NO: 143 (see Table 5).
  • the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 143.
  • Expression of tick C5 inhibitor protein may be directed by a constitutive or a tissue specific promoter.
  • the transgene contains a CAG promoter (SEQ ID NO: 2), a CB promoter or CB long promoter (SEQ ID NO: 17 or 18), or a GRK1 (SEQ ID NO: 5) promoter.
  • the promoter may be a tissue specific promoter (or regulatory sequence including promoter and enhancer elements) such as the GRK1 promoter (SEQ ID NO: 5 or 12), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
  • GRK1 promoter SEQ ID NO: 5 or 12
  • CAR mouse cone arresting
  • RedO human red opsin
  • an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence (SEQ ID NO: 36).
  • the transgenes may contain elements provided in Table 1 or la.
  • the transgenes may be packaged into AAV, including AAV8 or AAV3B.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) Control elements, which include a) an ocular-tissue specific promoter or constitutive promoter, b) optionally an intron, such as a chicken 0-actin intron or VH4 intron (SEQ ID NO: 36) and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the tick C5 inhibitor protein.
  • an expression cassette comprising a nucleotide sequence of SEQ ID NO: 138.
  • the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 138 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • an expression cassette comprising a nucleotide sequence of SEQ ID NO: 141.
  • the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 141 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • an expression cassette comprising a nucleotide sequence of SEQ ID NO: 144.
  • the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 147 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • an artificial genomes comprising the nucleotide sequence of SEQ ID NO: 139.
  • the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 139 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 142.
  • the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 142 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 145.
  • the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 145 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 148.
  • the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 148 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 114), or, alternatively, an AAV9 (SEQ ID NO: 115), AAV3B (SEQ ID NO: 108), or AAVrh73 (SEQ ID NO: 120) capsid (or a variant thereof); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inihibitor; operably linked to one or more regulatory sequences that control expression of the transgene in ocular tissue type cells, such as RPE cells, BrM cells, choriocapillaris cells, photoreceptor cells (rods and/or cones), retinal ganglion cells.
  • ITRs AAV inverted terminal repeats
  • the artificial genome is self-complementary.
  • the artificial genome is single stranded.
  • the construct or artificial genome may comprise the nucleotide sequence of SEQ ID NO: 138, 141, 144 or 147 (promoter to polyAtail).
  • the artificial genome may comprise or consist of the nucleotide sequence of SEQ ID NO: 139, 142, 145 or 148 (ITR to ITR).
  • the artificial genome may comprise of the nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 138, 141, 144 or 147 (promoter to poly A) or any one of SEQ ID NOs: 139, 142, 145 or 148 (ITR to ITR) as described herein wherein the nucleotide sequence encodes a tick C5 inhibitor protein that is i) expressed in a target cell and ii) has C5 inhibitory activity.
  • Table 4 provides the amino acid sequences of tick C5 inhibitor proteins.
  • Table 5 provides nucleotide sequences encoding the tick C5 inhibitor proteins, transgene coding sequences, and artificial genomes disclosed herein.
  • the rAAV vectors that encode and express the tick C5 inihibitor protein may be administered to treat or prevent or ameliorate symptoms of a disease or condition amenable to treatment, prevention or amelioration of symptoms with the tick C5 inihibitor protein, such as dry AMD. Also provided are methods of expressing HuPTM tick C5 inihibitor protein in human cells using the rAAV vectors and constructs encoding them.
  • the viral vectors provided herein may be manufactured using host cells.
  • the viral vectors provided herein may be manufactured using mammalian host cells, for example, A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.
  • the host cells are stably transformed with the sequences encoding the transgene and associated elements (e.g., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes e.g., the rep and cap genes of AAV).
  • the means of producing viruses in the host cells for example, the replication and capsid genes e.g., the rep and cap genes of AAV.
  • Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis.
  • Virions may be recovered, for example, by CsCb sedimentation.
  • baculovirus expression systems in insect cells may be used to produce AAV vectors.
  • AAV vectors See Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045- 1054 which is incorporated by reference herein in its entirety for manufacturing techniques.
  • in vitro assays e.g., cell culture assays
  • transgene expression from a vector described herein thus indicating, e.g., potency of the vector.
  • in vitro neutralization assays can be used to measure the activity of the transgene expressed from a vector described herein.
  • hemolysis assays and binding kinetic and affinity for C5, as described in Examples 5 and 6, respectively can be used to assess activity of transgenes expressed from a vector described herein.
  • other characteristics of the expressed product can be determined, for example determination of the glycosylation and tyrosine sulfation patterns.
  • Vector genome concentration (GC) or vector genome copies can be evaluated using digital PCR (dPCR) or ddPCRTM (BioRad Technologies, Hercules, CA, USA).
  • dPCR digital PCR
  • ddPCRTM BioRad Technologies, Hercules, CA, USA.
  • ocular tissue samples such as aqueous and/or vitreous humor samples, are obtained at several timepoints.
  • mice are sacrificed at various timepoints post injection. Ocular tissue samples are subjected to total DNA extraction and dPCR assay for vector copy numbers. Copies of vector genome (transgene) per gram of tissue may be measured in a single biopsy sample, or measured in various tissue sections at sequential timepoints will reveal spread of AAV throughout the eye.
  • Vector copy in specific cell types or tissues may indicate sustained expression of the transgene by the tissue.
  • compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • a formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered.
  • adjuvant e.g., Freund's complete and incomplete adjuvant
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM as known in the art.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight polypeptides proteins, such as serum albumin and gelatin
  • hydrophilic polymers such as
  • the pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol
  • methods for treating dry AMD (age-related AMD) or other indication that can be treated with a tick C5 inhibitor protein, in a subject in need thereof comprising the administration of recombinant AAV particles comprising an expression cassette encoding a tick C5 inhibitor protein
  • a subj ect in need thereof includes a subj ect suffering from dry AMD, or a subject pre-disposed thereto, e.g., a subject at risk of developing dry AMD, or other indication that may be treated with a tick C5 inhibitor protein.
  • Subjects to whom such gene therapy is administered can be those responsive to a tick C5 inhibitor protein.
  • the methods encompass treating patients who have been diagnosed with dry AMD, and, in certain embodiments, identified as responsive to treatment with a tick C5 inhibitor protein, or considered a good candidate for therapy with a tick C5 inhibitor protein.
  • the patients have previously been treated with a tick C5 inhibitor protein or other C5 inhibitor, such as an anti-C5 inhibitor antibody.
  • the tick C5 inhibitor protein e.g., produced in human cell culture, bioreactors, etc.
  • kits for treating dry AMD or other indication amenable to treatment with a tick C5 inhibitor protein in a human subject in need thereof comprising: administering to the eye, for example, intravitreal, subretinal, suprachoroidal, intracameral, or intranasal, or liver and/or muscle by systemic administration (including intravenous or intramuscular) of said subject a therapeutically effective amount of a recombinant nucleotide expression vector, such as an AAV vector, comprising a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells (such as retinal cells, BrM cells, choriocapillaris cells, RPE cells and/or choroid cells), so that a depot is formed that releases the tick C5 inhibitor protein.
  • a recombinant nucleotide expression vector such as an AAV vector
  • Subretinal, intravitreal, intracameral, or suprachoroidal administration should result in expression of the transgene product in one or more of the following retinal cell types: Bruch’s membrane (BrM), including epithelial cells thereof, choriocapillaris, human photoreceptor cells (cone cells, rod cells); horizontal cells; bipolar cells; amarcrine cells; retina ganglion cells (midget cell, parasol cell, bistratified cell, giant retina ganglion cell, photosensitive ganglion cell, and muller glia); and retinal pigment epithelial cells or other ocular tissue cell: cornea cells, iris cells, ciliary body cells, a schlemm’s canal cells, a trabecular meshwork cells, RPE-choroid tissue cells, or optic nerve cells.
  • Bruch’s membrane BrM
  • epithelial cells thereof choriocapillaris
  • human photoreceptor cells cone cells, rod cells
  • horizontal cells bipolar cells
  • amarcrine cells retina ganglion
  • Recombinant vectors and pharmaceutical compositions for treating diseases or disorders in a subject in need thereof are described in Section 5.1.
  • Such vectors should have a tropism for human ocular tissue, or liver and/or muscle cells and can include non-replicating rAAV, particularly those bearing an AAV3B, AAV8, AAAV9, AAV10, AAVrhlO, or AAVrh73 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters ocular tissue cells, e g., by introducing the recombinant vector into the eye.
  • Such vectors should further comprise one or more regulatory sequences that control expression of the transgene in human ocular tissue cells and/or human liver and muscle cells include, but are not limited to, human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7), a CAG promoter (SEQ ID NO: 2), a CB promoter or CBlong promoter (SEQ ID NO: 17 or 18) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19) (see also Tables 1 and la).
  • GRK1 promoter SEQ ID NOS: 5 or 12
  • CAR mouse cone arresting
  • RedO human red opsin
  • SEQ ID NO: 7 a CAG promoter
  • CB promoter or CBlong promoter SEQ ID NO: 17 or 18
  • the methods described herein treat, slow the progression of, reduce the severity of or prevent dry (age related) AMD in a human subject in need of the treatment.
  • the treatment, slowing progression of, reduction of severity or prevention may be assessed relative to the subject prior to treatment, a comparable untreated subject or according to the natural history of the disease.
  • method of the invention may reduce the progression of geographic atrophy, including within the fovea, slow retinal cell loss, slow the loss of central vision, increase or slow the loss of visual acuity, etc.
  • the subject may be at risk or have a predisposition to develop dry AMD based upon age, and/or risk factors such as history of smoking, obesity, cardiovascular disease or diabetes.
  • compositions and methods are described for the delivery of tick C5 inhibitor protein for treating dry AMD.
  • the HuPTM tick C5 inhibitor protein has the amino acid sequence of SEQ ID NO: 129 or SEQ ID NO: 130.
  • compositions and methods for delivery of HuPTM tick C5 inhibitor protein (amino acid sequence in Table 4). Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein to patients (human subjects) diagnosed with dry AMD to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a tick C5 inhibitor protein
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding a tick C5 inhibitor protein, or variants thereof, as detailed herein.
  • the transgene may also encode a tick C5 inhibitor protein that contains altered glycosylation sites.
  • tick C5 inhibitor protein may be SEQ ID NO: 129.
  • the viral vector has an AAV capsid with tropism for human ocular tissues and may be an AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof, for example having 90%, 95% or 99% sequence identity to the capsid sequence of AAV8, AAV9, AAV3B, or AAVrh73).
  • the transgene is operably linked by regulatory sequences that promote expression of the transgene in human ocular tissue cells (including in retinal cells, RPE, choroid, BrM, choriocapillaris, photoreceptor cells, retinal ganglion cells), for example a CAG promoter (SEQ ID NO: 2), or an ocular specific promoter, such as a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
  • a CAG promoter SEQ ID NO: 2
  • an ocular specific promoter such as a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR
  • Regulatory sequences may also include polyadenylation signal sequences.
  • the expression cassette comprising the transgene and operably linked regulatory sequences are flanked by ITR sequences, as an artificial AAV genome.
  • the flanking ITR sequences may be configured to provide a single stranded AAV (ssAAV) genome.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters one or more ocular tissue cells.
  • the recombinant AAV comprises an artificial genome of (or is produced using a cis plasmid or construct comprising) ss.CAG.tick.C5.
  • the artificial genome is self complementary. In certain embodiments, the artificial genome is single stranded.
  • Therapeutically effective doses of any of these recombinant vectors should be administered in any manner such that the recombinant vector enters ocular tissue cells (e.g., retinal cells), e.g., via subretinal, intravitreal, intracameral, or suprachoroidal injection or intranasal administration.
  • the vector is administered peripherally (for example, intravenously, intramuscularly or subcutaneously) such that the recombinant vector transduces liver and/or muscle cells, creating a depot in liver and/or muscle tissue which express the transgene product into the bloodstream, delivering the therapeutic to ocular tissues.
  • subretinal, intravitreal, intracameral, suprachoroidal administration should result in expression of the transgene product in cells of the eye, creating a depot in one or more ocular tissue cells of the patient that continuously supplies the tick C5 inhibitor protein to ocular tissues of the subject.
  • the transgene expression results in therapeutically effective levels of the tick C5 inhibitor protein in the aqueous humor, the vitreous humor, retinal tissue, the RPE, the BrM or choriocapillaris.
  • Subjects to whom such gene therapy is administered can be those responsive to anticomplement therapy.
  • the methods encompass treating patients who have been diagnosed with dry AMD, or have one or more symptoms associated therewith, and identified as responsive to treatment with a complement inhibitor, such as an anti-C3 or C5 antibody, or considered a good candidate for therapy with a tick C5 inhibitor protein.
  • the patients have previously been treated with crovalimab, eculizumab, ravulizumab, tesidolumab or other complement activation inhibitor, and have been found to be responsive to thereto.
  • the tick C5 inhibitor protein e.g., produced in cell culture, bioreactors, etc.
  • administration of the recombinant AAV comprising a construct for expressing a transgene encoding a tick C5 inhibitor protein in ocular tissues results in reduction or slowing the progression of one or more symptoms of dry AMD within 10 days, 20 days, 30 days, 40 days, 6 months, 9 months or 1 year after administration of the AAV.
  • the administration results in a slowing or reduction in the rate of the progression of geographic atrophy, including of the fovea, in the subject relative to an untreated subject or as expected in the subject based upon natural history of dry AMD, for example as measured by fundus autofluorescence (FAF).
  • FAF fundus autofluorescence
  • the administration results in an improvement or reduction in the rate of loss of visual acuity or best corrected visual acuity (BCVA), for example, as measured by a standard ETDRS chart or to improve visual function as measured by dark adaptation methodology; to improve contrast sensitivity by the Pelli-Robson test or to reduce the drusen area or accumulation of drusen.
  • BCVA best corrected visual acuity
  • the dose of therapeutic gene delivered by gene therapy is sufficient to inhibit complement activation without exacerbating choroidal neovascularization (CNV).
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic (intravenous, intramuscular or subcutaneous) administration comprise a suspension of the recombinant vector comprising the transgene encoding the tick C5 inhibitor protein in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the goal of gene therapy treatment provided herein is to slow or arrest the progression of or relieve one or more symptoms of dry AMD, such as to reduce the rate of geographic atrophy or improve visual acuity (or reduce the rate of loss of visual acuity).
  • Combinations of delivery of the tick C5 inhibitor protein, to the eye, liver and/or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • Available treatments for a subject with dry AMD that could be combined with the gene therapy provided herein include but are not limited to, elamipretide, risuteganib, photobiomodulation, brimonidine tartrate, kamuvudine, Xiflam, or doxycycline, and others and administration with the tick C5 inhibitor protein. 5.6. Monitoring of Efficacy
  • compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD.
  • the assessment may be determined in animal models or in human subjects.
  • the efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or reduction in geographic atrophy, including of the fovea, to be assessed by visual inspection.
  • BCVA best corrected visual acuity
  • compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD.
  • the assessment may be determined in animal models or in human subjects.
  • the efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or an increase in logMAR.
  • Physical changes to the eye, including changes in geographic atrophy may be measured Optical Coherence Tomography, using methods known in the art.
  • compositions and methods described herein may be assessed for efficacy using in vitro complement inhibition assays, such as membrane attack complex (“MAC”) formation, C5a generation and hemolysis.
  • Complement inhibition assays can be performed in any appropriate cell type, such as ARPE19 cells (MAC and C5a assays), iPSC-derived RPE cells (MAC and C5a assays) or sheep/rabbit erythrocytes (hemolysis assay).
  • MAC formation assays measure the deposition of MAC on the surface of RPE cells (% relative inhibition of MAC formation).
  • Hemolysis assays allow the comparison of complement inhibition among different complement inhibitors (50% complement inhibition dose (ng/ml) (CHso; AHso).
  • Animal models may be used to assess the recombinant vectors encoding the tick C5 inhibitor proteins for expression, therapeutic effect and adverse effects.
  • Animal models may include a humanized C3-/C5- rodent model or aNaIO3 induction rat or mouse model, or a CFH-/- mouse model.
  • Animals may be administered vectors described herein, for example, subretinally or suprachoroi dally, and then assessed for geographic atrophy (or change therein) by OCT, retinal pathology (damage to RPE), and other assessments of dry AMD pathology, as well as reduction in C3a or C5a, cleavage of C3 or C5 or other markers of complement activation.
  • Endpoints may include, but are not limited to, mean change in geographic atrophy in the study eye from baseline to 12, 16, 20, 24, or 28 weeks or at time of administration, if earlier, proportion of responders in the study eye at 12, 16, 20, 24, or 28 weeks, mean change in best corrected visual acuity from baseline to 12, 16, 20, 24, or 28 weeks, change from baseline in quality of life/patient reported outcome assessments, mean change in visual acuity from baseline to 12, 16, 20, 24, or 28 weeks.
  • a tick C5 inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequence of SEQ ID NO. 129).
  • the transgene also comprises nucleotide sequences that encodes the native tick C6 inhibitor signal peptide, MLVLVTLIFSFSANIAYA (SEQ ID NO: 52).
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • tissue-specific promoter such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO:
  • the artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO:
  • a Tick C5 Inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 129).
  • the transgene also comprises nucleotide sequences that encode i) a mutant IL2 signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and ii) the native tick C5 inhibitor signal peptide, e.g., MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) arranged in tandem with the mutant IL2 signal peptide at the N-terminus of the protein.
  • a mutant IL2 signal peptide e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54)
  • the native tick C5 inhibitor signal peptide e.g., MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) arranged in tandem with
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • constitutive promoters such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as an ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 141.
  • the artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 142.
  • Tick C5 Inhibitor (mutated IL2 Signal Peptide) cDNA Based Vector
  • a Tick C5 Inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 129).
  • the transgene also comprises nucleotide sequences that encode a mutated IL2 signal peptide, MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54).
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • tissue-specific promoter such as an ocular tissuespecific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 144.
  • the artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 145.
  • a Tick C5 Inhibitor (having mutations at N78Q and N102Q) cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 130).
  • the transgene also comprises nucleotide sequences that encode a native signal peptide, e.g., MLVLVTLIFSFSANIAYA (SEQ ID NO: 52).
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • tissue-specific promoter such as an ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxiainducible promoter, may be used.
  • the vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 147.
  • the artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 148.
  • Cis- plasmids were initially screened in the assay following transfection in 293 T cells and then subsequently packaged as AAV8 viral vectors (including scAAV8 vectors) for further study.
  • CP complement activation
  • CH50 sheep erythrocytes coated with rabbit antibodies
  • Such classical complement pathway-related hemolysis inhibition assay was employed using supernatant collected from HEK cells transfected with the plasmids (encoding complement inhibitors as described herein).
  • the supernatants (containing the complement inhibitor, or negative controls containing media without inhibitor or containing an antibody or antigen binding fragement expressed from the plasmid to a non-complement related target) were collected and applied to sheep erythrocytes coated with optimum levels of rabbit anti-sheep erythrocyte IgM antibodies suspended at 5 x 10 8 cells/ml in Gelatin Veronal Buffered saline (GVB++ Buffer) in the wells of an assay plate.
  • GVB++ Buffer Gelatin Veronal Buffered saline
  • AB2 plasmids (different vectorized forms IgG, Fab or ScFv) or a plasmid expressing the tick C5 inhibitor (native signal sequence) (SEQ ID NO: 138) were tested in the assay for the % of lysis of the EA as an indication of inhibition of complement activation.
  • the tick C5 inhibitor (CI5) displayed strong inhibition of complement (FIGS. 2A-2B, middle bar).
  • the native tick C5 inihibitor protein inhibits human C5-mediated hemolysis in the classical complement pathway (FIG. 2A) better than it inhibits mouse C5-mediated hemolysis (FIG. 2B).
  • C5 inhibitor protein resulting from the IL2 signal peptide-cassette expressed in HEK293 cells displayed equivalent inhibition of complement (compared to native signal pwptide), and the C5 inhibitor protein resulting from the dual mutant IL2 / native tick inhibitor signal peptide-expressed cassette in HEK293 cells suppresses complement pathway activation in hemolysis inhibition assays at varying degrees (data not shown).
  • C5 inhibitor tick C5 inhibitor
  • diamond 0 SEQ ID NO: 129
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated Human Complement C5 Protein was firstly immobilized onto SA biosensor. AB1 IgG and Ab fragments and native tick C5 inhibitor protein was applied as analyte for association and dissociation steps. Table 6. Assay conditions for human C5 assay after optimization b. Affinity and kinetics for cynomolgous Complement C5:
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated cyno C5 antigen was firstly immobilized onto SA biosensor. AB1 IgG and Ab fragments and the tick C5 inhibitor protein were applied as analyte for association and dissociation steps.
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated mouse C5 antigen was firstly immobilized onto SA biosensor. AB2 IgG and Ab fragments were applied as analyte for association and dissociation steps.
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated mouse C5 antigen was firstly immobilized onto SA biosensor. AB 1 IgG and Ab fragments and Tick C5 inhibitor protein were applied as analyte for association and dissociation steps.

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Abstract

Compositions and methods are described for the delivery of a tick C5 inhibitor protein to a human subject for treatment of an ocular indication, particularly AMD. Also provided are compositions and methods for the delivery of a tick C5 inhibitor protein to a human subject for treatment of an ocular indication, particularly AMD. The nucleotide sequence encoding the tick C5 inhibitor protein is delivered in a rAAV vector that targets ocular tissue cells for expression of the transgene.

Description

VECTORIZED C5 INHIBITOR AGENTS AND ADMINISTRATION THEREOF
REFERENCE TO SEQUENCE LISTING
[0001] The contents of the electronic sequence listing (38013_0035Pl_SL.xml; Size: 206,321 bytes; and Date of Creation: May 16, 2024) is herein incorporated by reference in its entirety.
1. INTRODUCTION
[0002] Compositions and methods are described for the delivery of tick C5 inhibitor protein to a human subject diagnosed with Age-Related Macular Degeneration (AMD).
2. BACKGROUND OF THE INVENTION
[0003] The complement system is a critical element of the immune system that enhances the clearing of microbes and damaged cells, promotes inflammation, and attacks a pathogen’s cell membrane. Three biochemical pathways activate the complement system, 1) the classical complement system, 2) the alternative complement pathway and 3) the lectin pathway.
[0004] Age-Related Macular Degeneration (AMD) causes progressive and permanent vision impairment. There are two forms of AMD, dry AMD and wet AMD. Dry AMD accounts for about 85- 90% of the 196 million global AMD cases. Overactivation of the complement system is an important driver of AMD. Over one million patients who also present with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) may also benefit from intervention to counteract overactive complement in the eye.
[0005] There is a need for more effective treatments that reduce the treatment burden on patients suffering from AMD. Intravitreal medications have become a promising mode of drug administration in patients as they provide high volume of drug to the target tissues, eliminating the risk of systemic toxicity. Reducing or eliminating the need for periodic ocular administration would reduce patient burden and improve therapy.
3. SUMMARY OF THE INVENTION
[0006] Proteins delivered by gene therapy have several advantages over injected or infused therapeutic proteins that dissipate over time resulting in peak and trough levels. Sustained expression of the transgene product protein, as opposed to injecting a protein repeatedly, allows for a more consistent level of protein to be present at the site of action, and is less risky and more convenient for patients, since fewer injections need to be made. Furthermore, proteins expressed from transgenes are post-translationally modified in a different manner than those that are directly injected because of the different microenvironment present during and after translation. Without being bound by any particular theory, this results in proteins that have different diffusion, bioactivity, distribution, affinity, pharmacokinetic, and immunogenicity characteristics, such that the proteins delivered to the site of action are “biobetters” in comparison with directly injected proteins. In addition, factors such as tick C5 inhibitor protein, e.g., a protein comprising the amino acid sequence of SEQ ID NO: 129, may inhibit complement activation and drusen deposit in the eye to inhibit, reduce the progression of dry AMD. Accordingly, provided herein are compositions and methods for tick C5 inhibitor protein therapy, including recombinant AAV gene therapy, designed to target the eye and generate a depot of transgenes for expression of tick C5 inhibitor protein that result in a therapeutic or prophylactic levels (in ocular tissues and, also, in embodiments in serum) of the tick C5 inhibitor protein within 20 days, 30 days, 40 days, 50 days, 60 days, or 90 days of administration of the rAAV composition for treatment or reduction in the progression of dry AMD and the geographic atrophy associated therewith.
[0007] Compositions and methods are described for the ocular or systemic delivery of a tick C5 inhibitor protein (for example, a fully human-glycosylated), to a patient (human subject) diagnosed with AMD or other condition indicated for treatment with the therapeutic tick C5 inhibitor protein. Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein — to create a permanent depot in the eye, or in alternative embodiments, liver and/or muscle, of the patient that continuously supplies the human post-translationally modified (HuPTM) tick C5 inhibitor peptide to one or more ocular tissues where the peptide exerts its therapeutic or prophylactic effect.
[0008] Provided are gene therapy vectors, particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of tick C5 inhibitor protein to achieve a maximum or steady states concentrations in ocular tissues, such as aqueous humor, vitreous humor, or in serum for example, 20, 30, 40, 50, 60 or 90 days after administration of the vector encoding the tick C5 inhibitor protein.
[0009] The recombinant vector used for delivering the transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”). In embodiments, the AAV type has a tropism for ocular tissues, including, for example, retinal cells, RPE, choroid, Bruch’s membrane (BrM) and epithelial cells thereof, choriocapillaris and epithelial cells thereof, photoreceptor cells (rods and cones) and retinal ganglion cells. The AAV type may be, for example, AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof) subtype of AAV. However, other viral vectors may be used, including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive expression elements, such as a CAG promoter, or tissue-specific expression control elements, particularly elements that are ocular tissue, liver and/or muscle specific control elements, for example one or more elements of Tables 1 and la.
[0010] In certain embodiments, the HuPTM tick C5 inhibitor protein encoded by the transgene can include, but is not limited to, one of SEQ ID NOs: 129 - 130 (encoding a HuPTM tick C5 inhibitor protein without a signal sequence) or one of SEQ ID Nos: 131- 134 (encoding a HuPTM tick C5 inhibitor protein having a signal sequence).
[001 1] In addition, proteins expressed from transgenes in vivo are not likely to contain degradation products associated with proteins produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is an issue associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and purification with certain buffer systems. These conditions, which promote aggregation, do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage, and is caused by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed from transgenes in vivo may also oxidize in a stressed condition. However, humans, and many other organisms, are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but sometimes also repairs and/or reverses the oxidation. Thus, proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, pharmacokinetics (clearance), and immunogenicity.
[0012] The production of HuPTM tick C5 inhibitor protein in ocular tissue cells of the human subject should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein to a patient (human subject) diagnosed with a disease indication for that tick C5 inhibitor protein, to create a permanent depot in the subject that continuously supplies the human post- translationally modifiedtransgene product produced by the subject’s transduced cells. The cDNA construct for the HuPTM tick C5 inhibitor protein should include a signal peptide (or more than one signal peptide, e.g., a native signal sequence and a mutated IL-2 signal sequence in tandem) that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
[0013] As an alternative, or an additional treatment to gene therapy, the HuPTM tick C5 inhibitor protein can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients. The recombinant tick C5 inhibitor protein produced by human cell lines includes a heterologous signal peptide such as a mutated IL-2 signal sequence, or more than one signal peptide, e.g., a native signal sequence and a mutated IL-2 signal sequence in tandem.
[0014] Combination therapies involving systemic delivery of the HuPTM tick C5 inhibitor protein to the patient accompanied by administration of other available treatments are encompassed by the methods provided herein. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Such additional treatments can include but are not limited to co-therapy with a therapeutic (recombinant) tick C5 inhibitor protein.
[0015] Also provided are methods of manufacturing the viral vectors, particularly the AAV based viral vectors. In specific embodiments, provided are methods of producing recombinant AAV s comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic tick C5 inhibitor protein operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture. 3.1. EMBODIMENTS
[0016] Embodiment 1. A pharmaceutical composition for treating AMD in a human subj ect in need thereof, comprising a recombinant adeno-associated virus (AAV) vector comprising: (a) a viral capsid that has a tropism for ocular tissue cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein said AAV vector is formulated for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic administration to said human subject.
[0017] Embodiment 2. The pharmaceutical composition of embodiment 1, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).
[0018] Embodiment 3. The pharmaceutical composition of embodiment 1 or embodiment 2, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
[0019] Embodiment 4. The pharmaceutical composition of any one of embodiments 1 to 3, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photreceptor cells (rods, cones and/or retinal ganglion cells).
[0020] Embodiment 5. The pharmaceutical composition of any one of embodiments 1 to 4, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
[0021] Embodiment 6. The pharmaceutical composition of embodiment 5, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19). [0022] Embodiment 7. The pharmaceutical composition of any one of embodiments 1 to 6, wherein the transgene comprises a signal sequence.
[0023] Embodiment 8. The pharmaceutical composition of embodiment 7, wherein the signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
[0024] Embodiment 9. The pharmaceutical composition of any one of embodiments 1 to 8, wherein the tick C5 inhibitor protein comprises an amino acid sequence of SEQ ID NO: 129.
[0025] Embodiment 10. The pharmaceutical composition of any one of embodiments 1 to 9, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143 .
[0026] Embodiment 11. The pharmaceutical composition of any one of embodiments 1 to 10, wherein the artificial genome comprises construct CAG. tick. C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
[0027] Embodiment 12. A pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising: (a) a viral capsid that has a tropism for ocular tissue cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein the transgene encodes a signal sequence at the N- terminus of said tick C5 inhibitor protein that directs secretion and post translational modification of said tick C5 inhibitor protein in human ocular tissue cells.
[0028] Embodiment 13. The pharmaceutical composition of embodiment 12, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu!2 (AAV.hul2), or serotype hu26 (AAV.hu26).
[0029] Embodiment 14. The pharmaceutical composition of embodiment 13, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
[0030] Embodiment 15. The pharmaceutical composition of any one of embodiments 12 to 14, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
[0031 ] Embodiment 16. The pharmaceutical composition of any one of embodiments 12 to 15, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
[0032] Embodiment 17. The pharmaceutical composition of embodiment 16, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
[0033] Embodiment 18. The pharmaceutical composition of any one of embodiments 12 to 17, wherein the transgene comprises a signal sequence.
[0034] Embodiment 19. The pharmaceutical composition of any one of embodiments 12 to 18, wherein said signal sequence is MLVLVTLIFSFSANIAYA (SEQ ID NO: 52), MYRMQLLSCIALILALVTNS (SEQ ID NO: 53) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), or a combination thereof or a signal sequence from Table 2.
[0035] Embodiment 20. The composition of embodiment 19, wherein said signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
[0036] Embodiment 21. The composition of any one of embodiments 12 to 20, wherein the tick C5 inhibitor protein has an amino acid sequence of SEQ ID NO: 129. [0037] Embodiment 22. The pharmaceutical composition of any one of embodiments 12 to 21, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143.
[0038] Embodiment 23. The composition of any one of embodiments 12 to 22, wherein the artificial genome is single stranded.
[0039] Embodiment 24. The composition of any one of embodiments 12 to 23 wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
[0040] Embodiment 25. A nucleic acid encoding an expression cassette, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 138, SEQ ID NO: 141 or SEQ ID NO: 144.
[0041 ] Embodiment 26. A nucleic acid encoding an artificial genome, wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142 or SEQ ID NO: 145).
[0042] Embodiment 27. A plasmid comprising the nucleic acid of embodiment 25.
[0043] Embodiment 28. A method of treating AMD in a human subject in need thereof, comprising subretinally, intravitreally, intranasally, intracamerally, suprachoroi dally, or systemically administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV vector comprising (a) a viral capsid that has a tropism for ocular tissue cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells.
[0044] Embodiment 29. The method of embodiment 28, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAVhu51), serotype hu21 (AAV.hu21), serotype hu!2 (AAV.hul2), or serotype hu26 (AAV.hu26).
[0045] Embodiment 30. The method of embodiment 29, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
[0046] Embodiment 3 E The method of any one of embodiments 28 to 30, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
[0047] Embodiment 32. The method of any one of embodiments 28 to 31, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
[0048] Embodiment 33. The method of embodiment 32, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
[0049] Embodiment 34. The method of any one of embodiments 28 to 33, wherein the transgene comprises a signal sequence.
[0050] Embodiment 35. The method of embodiment 34, wherein the signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
[0051 ] Embodiment 36. The method of any one of embodiments 28 to 35, wherein the tick C5 inhibitor protein comprises an amino acid sequence of SEQ ID NO: 129.
[0052] Embodiment 37. The method of any one of embodiments 28 to 36, wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
[0053] Embodiment 38. The method of any one of embodiments 28 to 37, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 135, 137, 140 or 143. [0054] Embodiment 39. The method of any one of embodiments 28 to 38, wherein the therapeutically effective amount is determined to be sufficient to improve best corrected visual acuity (BCVA) by >= 2 ETDRS lines or increase in logMAR; to decrease the mean rate of change in geographic atrophy as measured by fundus autofluorescence (FAF); to improve visual function as measured by dark adaptation methodology; to improve contrast sensitivity by the Pelli-Robson test, or reduce the drusen area within 10 weeks, 20 weeks, 6 months or 1 year of administration.
[0055] Embodiment 40. A method of producing recombinant AAVs comprising: (a) culturing a host cell containing: (i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has ocular tissue tropism; (ii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
[0056] Embodiment 41. The method of embodiment 40, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
[0057] Embodiment 42. A host cell comprising: a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.
[0058] Embodiment 43. The host cell of embodiment 42, wherein the transgene encodes a tick C5 inhibitor protein (SEQ ID NO: 129). 4. BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1. Clustal Multiple Sequence Alignment of various capsids with ocular tissue tropism. Amino acid substitutions (shown in bold in the bottom rows) can be made to AAV8 capsids by “recruiting” amino acid residues from the corresponding position of other aligned AAV capsids. Sequence shown in gray = hypervariable regions. The amino acid sequences of the AAV capsids are assigned sequence ID numbers as indicated in FIG. 1.
[0060] FIGS. 2A and 2B show the results of the ability of cis plasmid-expressed C5 tick inhibitor protein compared to vectorized C5 antibodies in HEK293 cells to suppress complement in a hemolysis inhibition assay with (A) 1.5% normal human serum or (B) 20% normal mouse serum. The same anti-C5 inhibitors, IgG = AB1 full-length mAb, Fab = AB1 Fab, scFv = AB1 scFv mAb, and C5I=recombinant tick C5 inhibitor protein and isotype and vehicle controls were tested against human (A) or mouse (B) serum containing human or mouse C5, respectively..
[0061] FIGS. 3A-3B show that the recombinant purified forms of C5 tick inhibitor and anti- C5 antibodies suppressed classical and alternative complement pathways in hemolysis inhibition assays against A) 50% human C5, classical complement pathway conditions, testing anti-hC5 (AB1 formats) and tick C5 inhibitor protein, B) 50% human C5, alternative complement pathway conditions, testing anti-hC5 (AB 1 formats) and tick C5 inhibitor protein.
5. DETAILED DESCRIPTION OF THE INVENTION
[0062] Compositions and methods are described for the systemic delivery of a human post- translationally modified (HuPTM) version of a tick C5 inhibitor protein (SEQ ID NO: 129) to a patient (human subject) diagnosed with AMD (including dry AMD). Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding tick C5 inhibitor protein to a patient (human subject) diagnosed with a condition indicated for treatment — to create a permanent depot in a tissue or organ of the patient, particularly the eye, but, in embodiments, liver or muscle, that continuously supplies the tick C5 inhibitor protein, e.g., a human-glycosylated transgene product, into ocular tissues of the subject to where the thereof exerts its therapeutic effect. [0063] The compositions and methods provided herein ocularly or systemically deliver the tick C5 inhibitor protein, from a depot of viral genomes, for example, in the subject’s eye (including retinal tissue), or liver/muscle, at a level either in the ocular tissue (e.g., in the vitreous or aqueous humor or retinal tissue, RPE, BrM and/or choroid), or in the serum that is therapeutically or prophylactically effective to treat or ameliorate the symptoms of AMD or other indication that may be treated with tick C5 inhibitor protein. Identified herein are viral vectors for delivery of transgenes encoding the therapeutic tick C5 inhibitor protein, to cells in the human subject, including, in embodiments, one or more ocular tissue cells, and regulatory elements operably linked to the nucleotide sequence encoding the tick C5 inhibitor protein, in embodiments, in the ocular tissue cells. Such regulatory elements, including constitutive promoters, such as CAG, and ocular tissue-specific regulatory elements, are provided in Table 1 and Table la. Accordingly, such viral vectors may be delivered to the human subject at appropriate dosages, such that at least 20, 30, 40, 50 or 60 days after administration, the tick C5 inhibitor protein is present at therapeutically effective levels in the serum or in ocular tissues of said human subject. In embodiments, the therapeutically effective level of the tick C5 inhibitor protein is determined (in human trials, animal models, etc.) to improve best corrected visual acuity (BCVA) by >= 2 ETDRS lines, reduction in geographic atrophy (or slow the progression of geographic atrophy relative to untreated individual either based upon controls or natural history of the disease), reduction in drusen deposits or other metric of dry AMD.
[0064] Provided is the amino acid sequence for tick C5 inhibitor protein (without a signal sequence) (SEQ ID NO: 129), encoded by nucleotide sequence of SEQ ID NO: 135. Aso provided is the amino acid sequence for the tick C5 inhibitor protein having the native tick inhibitor signal sequence (SEQ ID NO: 131), the amino acid sequence for the tick C5 inhibitor protein having two signal sequences, a native tick inhibitor signal sequence and a mutated IL2 signal sequence (SEQ ID NO: 132), and the tick C5 inhibitor protein having the mutated IL2 signal sequence (SEQ ID NO: 133), which are encoded by nucleotide sequences of SEQ ID NO: 137, 140, and 143, respectively.
[0065] The recombinant vector used for delivering the transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”). rAAVs are particularly attractive vectors for a number of reasons -they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to obtain the desired tissue specificity, and/or to avoid neutralization by pre-existing patient antibodies to some AAVs. The AAV types for use here in preferentially target the eye, i.e., have a tropism for retinal cells. Such rAAVs include but are not limited to AAV based vectors comprising capsid components from one or more of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAV. hu21, AAV.hu 12, or AAV.hu26. In certain embodiments, AAV based vectors provided herein comprise capsids from one or more of AAV3B, AAV8, AAV9, AAVrhlO, AAV 10, or AAVrh73 serotypes.
[0066] However, other viral vectors may be used, including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements.
[0067] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161) and may also be optimized to reduce CpG dimers. Codon optimized sequences of the tick C5 inhibitor protein are provided in Table 5 (SEQ ID NOs: 135 and 136). Useful signal sequences for the expression of the tick C5 inhibitor proteins are disclosed herein, for example, the native tick inhibitor signal sequence (SEQ ID NO: 52), a mutated IL2 signal sequence (SEQ ID NO: 54), and the additional signal sequences shown in Tables 2A/2B and 3.
[0068] The production of HuPTM protein should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein to a patient (human subject), to create a permanent depot in the subject that continuously supplies the human-post-translationally modified transgene product produced by the subject’s transduced cells. The cDNA construct for the tick C5 inhibitor protein should include a signal peptide that ensures proper co- and post-translational processing by the transduced human cells.
[0069] Pharmaceutical compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
[0070] As an alternative, or an additional treatment to gene therapy, the tick C5 inhibitor protein can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients. Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to human embryonic kidney 293 cells (HEK293), fibro sarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6, or RPE to name a few (e.g., see Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6): 1110-1122, which is incorporated by reference in its entirety for a review of the human cell lines that could be used for the recombinant production of the HuPTM tick C5 inhibitor protein. To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and/or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation in human cells.
[0071] It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy. The goal of gene therapy treatment of the invention is to slow or arrest the progression of disease.
[0072] Combination therapies involving delivery of the tick C5 inhibitor protein to the patient accompanied by administration of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Such additional treatments can include but are not limited to co-therapy with the tick C5 inhibitor protein.
[0073] Also provided are methods of manufacturing the viral vectors, particularly the AAV based viral vectors. In specific embodiments, provided are methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a tick C5 inhibitor protein operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
5.1 CONSTRUCTS
[0074] Viral vectors or other DNA expression constructs encoding tick C5 inhibitor protein are provided herein. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to a target cell. The means of delivery of a transgene include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g, gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector targeted ocular tissue cells or a vector that has a tropism for ocular tissue cells.
[0075] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes a tick C5 inhibitor protein, as a transgene described herein, operatively linked to an ubiquitous promoter, an ocular tissue-specific promoter, or an inducible promoter, wherein the promoter is selected for expression in tissue targeted for expression of the transgene. Promoters may, for example, be a CB7/CAG promoter (SEQ ID NO: 1) and associated upstream regulatory sequences, CAG promoter (CMS early enhancer, Chicken Beta-actin promoter-chicken beta actin intron-rabbit beta-globin splice acceptor) (SEQ ID NO: 2), cytomegalovirus (CMV) promoter, mUla (SEQ ID NO: 3), EF-1 alpha promoter (SEQ ID NO: 4), UB6 promoter (SEQ ID NO: 6), chicken beta-actin (CBA) promoter, and ocular-tissue specific promoters, such as human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), or a human red opsin (RedO) promoter (SEQ ID NO: 7). See Tables 1 and la for a list of useful promoters.
[0076] In certain embodiments, provided herein are recombinant vectors that comprise one or more nucleic acids (e.g., polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., the nucleotide sequences encoding tick C5 inhibitor protein), untranslated regions, and termination sequences. In certain embodiments, viral vectors provided herein comprise a promoter operably linked to the gene of interest.
[0077] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149- 161).
[0078] In an embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) one or more control elements, a) a CAG promoter (SEQ ID NO: 2), b) optionally, a chicken 0-actin intron (or other intron and c) a rabbit 0-globin poly A signal (SEQ ID NO: 38); and (3) nucleic acid sequences coding for the tick C5 inhibitor protein.
[0079] In an embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) CAG promoter (SEQ ID NO: 2), b) optionally, a VH4 intron (SEQ ID NO: 36) or other intron and c) a rabbit (3-globin poly A signal (SEQ ID NO: 38); and (3) nucleic acid sequences coding for a tick C5 inhibitor protein. In an embodiment, the AAV2 inverted terminal repeats are the 5’ ITR (SEQ ID NO: 48) and the 3’ ITR (SEQ ID NO: 149).
5.1.1 mRNA Vectors
[0080] In certain embodiments, as an alternative to DNA vectors, the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, a tick C5 inhibitor protein). The synthesis of modified and unmodified mRNA for delivery of a transgene to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9): 5661-5672, which is incorporated by reference herein in its entirety. In certain embodiments, provided herein is a modified mRNA encoding for a HuPTM tick C5 inhibitor protein.
5.1.2 Viral Vectors
[0081 ] Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8, AAV9, AAVrhlO, AAV10), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors. Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HlV)-based vectors. Alphavirus vectors include semliki forest virus (SFV) and sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are altered such that they are replication-deficient in humans. In certain embodiments, the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitus virus (VSV). In more specific embodiments, the envelope protein is VSV- G protein.
[0082] In certain embodiments, the viral vectors provided herein are HIV based viral vectors. In certain embodiments, HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
[0083] In certain embodiments, the viral vectors provided herein are herpes simplex virusbased viral vectors. In certain embodiments, herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.
[0084] In certain embodiments, the viral vectors provided herein are MLV based viral vectors. In certain embodiments, MLV-based vectors provided herein comprise up to 8 kb of heterologous DNA in place of the viral genes.
[0085] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, lentiviral vectors provided herein are packaged into a lentiviral capsid. In certain embodiments, lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site. [0086] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, alphavirus vectors provided herein are recombinant, replicationdefective alphaviruses. In certain embodiments, alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.
[0087] In certain embodiments, the viral vectors provided herein are AAV based viral vectors. In certain embodiments, the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for synthesis of the capsid proteins) (the rep and cap proteins may be provided by the packaging cells in trans). Multiple AAV serotypes have been identified. In certain embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In preferred embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV with tropism to ocular tissues, liver and/or muscle. In certain embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAVhu21, AAV.hul2, or AAV.hu26. In certain embodiments, AAV based vectors provided herein are or comprise components from one or more of AAV8, AAV3B, AAV9, AAV10, AAVrh73, or AAVrhlO serotypes. Provided are viral vectors in which the capsid protein is a variant of the AAV8 capsid protein (SEQ ID NO: 114), AAV3B capsid protein (SEQ ID NO: 108), or AAVrh73 capsid protein (SEQ ID NO: 120), and the capsid protein is e. ., at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 114), AAV9 (SEQ ID NO: 115), AAV3B capsid protein (SEQ ID NO: 108), or AAVrh73 capsid protein (SEQ ID NO: 120), while retaining the biological function of the native capsid. In certain embodiments, the encoded AAV capsid has the sequence of SEQ ID NO: 114 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV8, AAV3B, or AAVrh73 capsid. FIG. 1 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS. Accordingly, in specific embodiments, the AAV vector comprises an AAV8, AAV3B, or AAVrh73, capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions that are not present at that position in the native AAV capsid sequence as identified in the SUBS row of FIG. 1. Amino acid sequence for AAV8, AAV9, AAV3B, or AAVrh73 capsids are provided in FIG. 1.
[0088] The amino acid sequence of hu37 capsid can be found in international application PCT WO 2005/033321 (SEQ ID NO: 88 thereof) and the amino acid sequence for the rh8 capsid can be found in international application PCT WO 03/042397 (SEQ ID NO:97). The amino acid sequence for the rh64Rl sequence is found in W02006/110689 (a R697W substitution of the Rh.64 sequence, which is SEQ ID NO: 43 of WO 2006/110689).
[0089] In some embodiments, AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu31, AAV.hu32, AAVAnc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAVHSC9, AAV.HSC10, AAV.HSC11, AAVHSC12, AAV.HSC13, AAV.HSC14, AAVHSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof. In some embodiments, AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu31, AAVhu32, AAV.Anc80, AAV.Anc80L65, AAV7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAVHSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSCl l, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAVHSC16 or other rAAV particles, or combinations of two or more thereof serotypes. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAVrh8, AAVrhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAVrh73, AAV.Rh74, AAVRHM4-1, AAV.hu37, AAV.hu31, AAV.hu32, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAVHSC2, AAVHSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAVHSC7, AAV.HSC8, AAV.HSC9, AAV.HSC1O, AAVHSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAVHSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.
[0090] In particular embodiments, the recombinant AAV for us in compositions and methods herein is AAVS3 (including variants thereof) (see e.g., US Patent Application No. 20200079821, which is incorporated herein by reference in its entirety). In particular embodiments, rAAV particles comprise the capsids of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In particular embodiments, the AAV for use in compositions and methods herein is any AAV disclosed in US 10,301,648, such as AAV.rh46 or AAVrh73. In some embodiments, the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety). In particular embodiments, the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B. In particular embodiments, the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors). In particular embodiments, the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335.
[00 1 ] In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
[0092] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g., SEQ ID Nos: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID Nos: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID Nos: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g., SEQ ID Nos: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID Nos: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID Nos: 5- 38 of '097 publication), and WO 2015/191508 (see, e.g., SEQ ID Nos: 80-294 of '508 publication), and U.S. Appl. Publ. No. 2015/0023924 (see, e.g., SEQ ID Nos: 1, 5-10 of '924 publication), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g., SEQ ID Nos: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID Nos: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID Nos: 1 and 3-6 of '888 publication), WO 2006/110689 (see, e.g., SEQ ID Nos: 5-38 of '689 publication) W02009/104964 (see, e.g, SEQ ID Nos: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication), W0 2010/127097 (see, e.g, SEQ ID Nos: 5-38 of '097 publication), and WO 2015/191508 (see, e.g, SEQ ID Nos: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g, SEQ ID Nos: 1, 5-10 of '924 publication).
[0093] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g, Duan et al, J. Virol, 75:7662-7671 (2001); Halbert et al, J. Virol, 74: 1524-1532 (2000); Zolotukhin et al. Methods 28: 158-167 (2002); and Auricchio et al. Hum. Molec. Genet. 10:3075-3081, (2001).
[0094] AAV8-based, AAV3B-based, and AAVrh73-based viral vectors are used in certain of the methods described herein. Nucleotide sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV (e.g., AAV8, AAV3B, AAVrh73, or AAVrhl0)-based viral vectors encoding a transgene (e.g., HuPTM tick C5 inhibitor protein). The amino acid sequences of AAV capsids, including AAV8, AAV3B, AAVrh73 and AAVrhlO are provided in FIG. 1.
[0095] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248- 1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).
[0096] In certain embodiments, the viral vectors used in the methods described herein are adenovirus based viral vectors. A recombinant adenovirus vector may be used to transfer in the transgene encoding the tick C5 inhibitor protein. The recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3’ ITR, with or without staffer sequences to keep the genome close to wild-type size of approximately 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.
[0097] In certain embodiments, the viral vectors used in the methods described herein are lentivirus based viral vectors. A recombinant lentivirus vector may be used to transfer in the transgene encoding the HuPTM tick C5 inhibitor protein. Four plasmids are used to make the construct: Gag/pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (e.g., VSV-G), and Cis plasmid with the packaging elements and the tick C5 inhibitor protein.
[0098] For lentiviral vector production, the four plasmids are co-transfected into cells (e.g., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others. The lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs/should be done). The supernatant is fdtered (0.45 pm) and then magnesium chloride and benzonase added. Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with/without column chromatography. Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, andAusubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.
5.1.3 Promoters and Modifiers of Gene Expression
[0099] In certain embodiments, the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g., to detect or select for cells that have taken up the polynucleotide.
[0100] In certain embodiments, the viral vectors provided herein comprise one or more promoters that control expression of the transgene. These promoters (and other regulatory elements that control transcription, such as enhancers) may be constitutive (promote ubiquitous expression) or may specifically or selectively express in the eye. In certain embodiments, the promoter is a constitutive promoter.
[0101 ] In certain embodiments, the promoter is a CAG promoter (SEQ ID NO: 2) (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety). In some embodiments, the CAG promoter (SEQ ID NO: 2) or CB7 promoter (SEQ ID NO: 1), includes other expression control elements that enhance expression of the transgene driven by the vector. In certain embodiments, the other expression control elements include chicken 0-actin intron and/or rabbit P-globin polyA signal (SEQ ID NO: 38). In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another. In certain embodiments, the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs.
[0102] In certain embodiments, the vectors provided herein comprise one or more tissue specific promoters (e.g., a retinal-specific promoter). In particular embodiments, the viral vectors provided herein comprises a ocular tissue cell specific promoter, such as, human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), or a human red opsin (RedO) promoter (SEQ ID NO: 7).
[0103] Provided are nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette. Regulatory elements, in general, have multiple functions as recognition sites for transcription initiation or regulation, coordination with cellspecific machinery to drive expression upon signaling, and to enhance expression of the downstream gene.
[0104] In certain embodiments, the promoter is an inducible promoter. In certain embodiments the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF- la binding site. In certain embodiments, the promoter comprises a HlF-2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schbdel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated by reference herein in its entirety. In certain embodiments, the promoter comprises a binding site for a hypoxia induced transcription factor other than a HIF transcription factor. In certain embodiments, the viral vectors provided herein comprise one or more IRES sites that is preferentially translated in hypoxia. For teachings regarding hypoxia-inducible gene expression and the factors involved therein, see, e.g., Kenneth and Rocha, Biochem J., 2008, 414: 19-29, which is incorporated by reference herein in its entirety. In specific embodiments, the hypoxia-inducible promoter is the human N-WASP promoter, see, e.g., Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 (incorporated by reference for the teaching of the N-WASP promoter) or is the hypoxia-induced promoter of human Epo, see, e.g., Tsuchiya et al., 1993, J. Biochem. 113:395-400 (incorporated by reference for the disclosure of the Epo hypoxia-inducible promoter). In other embodiments, the promoter is a drug inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof. See, e.g., the disclosure of rapamycin inducible promoters in PCT publications WO94/18317, WO 96/20951, WO 96/41865, WO 99/10508, WO 99/10510, WO 99/36553, and WO 99/41258, and US 7,067,526, which are hereby incorporated by reference in their entireties for the disclosure of drug inducible promoters.
[0105] Provided herein are constructs containing certain ubiquitous and tissue-specific promoters. Such promoters include synthetic and tandem promoters. Examples and nucleotide sequences of promoters are provided in Tables 1 and la below. Table 1 also includes the nucleotide sequences of other regulatory elements useful for the expression cassettes provided herein. Table 1. Promoter and Other Regulatory Element Sequences
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Table la. Other regulatory and ITR sequences
Figure imgf000038_0001
Figure imgf000039_0001
[0106] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron (e.g. VH4 intron (SEQ ID NO: 36), SV40 intron (SEQ ID NO: 37), or a chimeric intron ([3-globin/Ig Intron) (SEQ ID NO: 35). The viral vectors may also include a Kozak sequence to promote translation of the transgene product, for example GCCACC (SEQ ID NO: 39).
[0107] In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence downstream of the coding region of the transgene. Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure. Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit -globin gene (SEQ ID NO: 38), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.
5.1.4 Signal Peptides
[0108] In certain embodiments, the vectors provided herein comprise components that modulate protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. Signal peptides (also referred to as “signal sequences”) may also be referred to herein as “leader sequences” or “leader peptides”. In certain embodiments, the signal peptides allow for the transgene product to achieve the proper packaging (e.g., glycosylation) in the cell. In certain embodiments, the signal peptides allow for the transgene product to achieve the proper localization in the cell. In certain embodiments, the signal peptides allow for the transgene product to achieve secretion from the cell.
[0109] There are two general approaches to select a signal sequence for protein production in a gene therapy context or in cell culture. One approach is to use a signal peptide from proteins homologous to the protein being expressed. Another approach is to identify signal peptides optimized for the particular host cells used for expression. In some embodiments, the signal peptide is a heterologous signal peptide, thus is not native or homologous to the protein being expressed. Signal peptides may be interchanged between different proteins or even between proteins of different organisms, but usually the signal sequences of the most abundant secreted proteins of that cell type are used for protein expression. For example, the signal peptide of human albumin, the most abundant protein in plasma, was found to substantially increase protein production yield in CHO cells. However, certain signal peptides may retain function and exert activity after being cleaved from the expressed protein as “post-targeting functions”. Thus, in specific embodiments, the signal peptide is selected from signal peptides of the most abundant proteins secreted by the cells used for expression to avoid the post-targeting functions. Exemplary sequences are the native tick C5 inhibitor signal peptide, MLVLVTLIFSFSANIAYA (SEQ ID NO: 52), which can be encoded by a nucleotide sequence of SEQ ID NO: 63, and a mutant IL2 signal peptide, MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), which can be encoded by a nucleotide sequence of SEQ ID NO: 64 or SEQ ID NO: 65 (see Table 2). In some embodiments, more than one signal sequence may be used, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in Example 2 (SEQ ID NO: 132). Alternatively, signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the tick C5 inhibitor protein in the eye/CNS, muscle, or liver are provided in Tables 2A/2B and Table 3, below.
Table 2A. Signal peptides for expression in eye/CNS
Figure imgf000041_0001
Table 2B - coding sequences for signal peptides
Figure imgf000041_0002
Figure imgf000042_0001
Table 3. Signal peptides for expression in liver and muscle cells.
Figure imgf000042_0002
Figure imgf000043_0001
5.1.5 Untranslated regions
[0110] In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half-life of the transgene. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the transgene. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA of the transgene.
5.1.6 Inverted terminal repeats
[01 11] In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITR is from an AAV, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety). In preferred embodiments, nucleotide sequences encoding the ITRs may, for example, comprise the nucleotide sequences of SEQ ID NO: 48 (5’-ITR) and SEQ ID NO: 50 or SEQ ID NO: 149 (3’-ITR). In certain embodiments, the modified ITRs used to produce self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171 -82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety). In preferred embodiments, nucleotide sequences encoding the modified ITRs may, for example, comprise the nucleotide sequences of SEQ ID NO: 48 (5’-ITR) and SEQ ID NO: 50 or SEQ ID NO: 149 (3’-ITR) or modified for scAAV, SEQ ID NO: 49 (m 5’ITR) and SEQ ID NO: 51 or SEQ ID NO: 150 (m 3’ ITR).
5.1.7 Transgenes
[01 12] In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 129 (see Table 4). In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 129 and has C5 inhibitory activity. In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 130 (see Table 4). In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 130 and has C5 inhibitory activity.
[01 13] The tick C5 inhibitor protein may have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and/or muscle cells. The signal sequence may have the amino acid sequence of the endogenous tickC5 inhibitor sequence, which is MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) (underlined in Table 4) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54). Alternatively, the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Table 2 that correspond to the proteins secreted by ocular tissue cell types. Alternatively, the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.
[0114] In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 131 (having a native tick C5 inhibitor signal sequence). In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 131 and has C5 inhibitory activity.
[01 15] In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 132 (having a mutant IL2 signal sequence and a native C5 inhibitor signal sequence). In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 132 and has C5 inhibitory activity.
[01 16] In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 133 (having a mutant IL2 signal sequence). In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 133 and has C5 inhibitory activity.
[0117] In certain embodiments, provided are vectors, including AAV vectors comprising a transgene encoding the tick C5 inhibitor protein having an amino acid sequence of SEQ ID NO: 134. In certain embodiments, the tick C5 inhibitor protein transgene encodes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 134 and has C5 inhibitory activity.
[0118] Alternatively, provided are vectors, including AAV vectors, comprising a transgene encoding the tick C5 inhibitor protein, having an amino acid sequence of SEQ ID NO: 129. The nucleotide sequences may be codon optimized for expression in human cells. The tick C5 inhibitor protein may be encoded by a nucleotide sequence comprising SEQ ID NO: 135 (see Table 5). In certain embodiments, the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 135. The tick C5 inhibitor protein may be encoded by a nucleotide sequence comprising SEQ ID NO: 136 (see Table 5). In certain embodiments, the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 136.
[01 19] The tick C5 inhibitor protein (having a mutant IL2 signal sequence and a native signal sequence) may be encoded by a nucleotide sequence comprising SEQ ID NO: 140 (see Table 5). In certain embodiments, the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 140.
[0120] The tick C5 inhibitor protein (having a mutant IL2 signal sequence) may be encoded by a nucleotide sequence comprising SEQ ID NO: 143 (see Table 5). In certain embodiments, the tick C5 inhibitor protein has C5 inhibitory activity is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 143.
[0121 ] Expression of tick C5 inhibitor protein may be directed by a constitutive or a tissue specific promoter. In certain embodiments, the transgene contains a CAG promoter (SEQ ID NO: 2), a CB promoter or CB long promoter (SEQ ID NO: 17 or 18), or a GRK1 (SEQ ID NO: 5) promoter. Alternatively, the promoter may be a tissue specific promoter (or regulatory sequence including promoter and enhancer elements) such as the GRK1 promoter (SEQ ID NO: 5 or 12), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19). In embodiments, an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence (SEQ ID NO: 36). The transgenes may contain elements provided in Table 1 or la. The transgenes may be packaged into AAV, including AAV8 or AAV3B.
[0122] In specific embodiments for expressing a tick C5 inhibitor protein, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) Control elements, which include a) an ocular-tissue specific promoter or constitutive promoter, b) optionally an intron, such as a chicken 0-actin intron or VH4 intron (SEQ ID NO: 36) and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the tick C5 inhibitor protein.
[0123] In specific embodiments, provided is an expression cassette comprising a nucleotide sequence of SEQ ID NO: 138. In certain embodiments, the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 138 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided is an expression cassette comprising a nucleotide sequence of SEQ ID NO: 141. In certain embodiments, the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 141 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided is an expression cassette comprising a nucleotide sequence of SEQ ID NO: 144. In certain embodiments, the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 144 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided is an expression cassette comprising a nucleotide sequence of SEQ ID NO: 147. In certain embodiments, the expression cassette is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 147 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
[0124] In specific embodiments, provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 139. In certain embodiments, the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 139 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 142. In certain embodiments, the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 142 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 145. In certain embodiments, the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 145 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity. In specific embodiments, provided are artificial genomes comprising the nucleotide sequence of SEQ ID NO: 148. In certain embodiments, the artificial genome is encoded by a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 148 and encodes a C5 tick inhibitor or analog thereof having C5 inhibitory activity.
[0125] In specific embodiments, provided are AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 114), or, alternatively, an AAV9 (SEQ ID NO: 115), AAV3B (SEQ ID NO: 108), or AAVrh73 (SEQ ID NO: 120) capsid (or a variant thereof); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inihibitor; operably linked to one or more regulatory sequences that control expression of the transgene in ocular tissue type cells, such as RPE cells, BrM cells, choriocapillaris cells, photoreceptor cells (rods and/or cones), retinal ganglion cells.
[0126] In certain embodiments, provided is a construct encoding or an artificial genome in which the transgene operably linked to regulatory sequences is flanked by ITR sequences. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single stranded. The construct or artificial genome may comprise the nucleotide sequence of SEQ ID NO: 138, 141, 144 or 147 (promoter to polyAtail). The artificial genome may comprise or consist of the nucleotide sequence of SEQ ID NO: 139, 142, 145 or 148 (ITR to ITR). The artificial genome may comprise of the nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 138, 141, 144 or 147 (promoter to poly A) or any one of SEQ ID NOs: 139, 142, 145 or 148 (ITR to ITR) as described herein wherein the nucleotide sequence encodes a tick C5 inhibitor protein that is i) expressed in a target cell and ii) has C5 inhibitory activity. [0127] Table 4 provides the amino acid sequences of tick C5 inhibitor proteins. Table 5 provides nucleotide sequences encoding the tick C5 inhibitor proteins, transgene coding sequences, and artificial genomes disclosed herein.
Table 4. Amino Acid Sequences of Tick C5 Inhibitors
Figure imgf000049_0001
Table 5. Nucleotide Sequences of Expressed Polypeptides, Proteins, and Genomes
Figure imgf000049_0002
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
[0128] The rAAV vectors that encode and express the tick C5 inihibitor protein may be administered to treat or prevent or ameliorate symptoms of a disease or condition amenable to treatment, prevention or amelioration of symptoms with the tick C5 inihibitor protein, such as dry AMD. Also provided are methods of expressing HuPTM tick C5 inihibitor protein in human cells using the rAAV vectors and constructs encoding them.
5.1.8 Manufacture and testing of vectors
[0129] The viral vectors provided herein may be manufactured using host cells. The viral vectors provided herein may be manufactured using mammalian host cells, for example, A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells. The viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.
[0130] The host cells are stably transformed with the sequences encoding the transgene and associated elements (e.g., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes e.g., the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCb sedimentation.
[0131] Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045- 1054 which is incorporated by reference herein in its entirety for manufacturing techniques.
[0132] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. In addition, in vitro neutralization assays can be used to measure the activity of the transgene expressed from a vector described herein. For example, hemolysis assays and binding kinetic and affinity for C5, as described in Examples 5 and 6, respectively, can be used to assess activity of transgenes expressed from a vector described herein. In addition, other characteristics of the expressed product can be determined, for example determination of the glycosylation and tyrosine sulfation patterns.
[0133] Vector genome concentration (GC) or vector genome copies can be evaluated using digital PCR (dPCR) or ddPCR™ (BioRad Technologies, Hercules, CA, USA). In one example, ocular tissue samples, such as aqueous and/or vitreous humor samples, are obtained at several timepoints. In another example, several mice are sacrificed at various timepoints post injection. Ocular tissue samples are subjected to total DNA extraction and dPCR assay for vector copy numbers. Copies of vector genome (transgene) per gram of tissue may be measured in a single biopsy sample, or measured in various tissue sections at sequential timepoints will reveal spread of AAV throughout the eye. Total DNA from collected ocular fluid or tissue is extracted with the DNeasy Blood & Tissue Kit and the DNA concentration measured using a Nanodrop spectrophotometer. To determine the vector copy numbers in each tissue sample, digital PCR is performed with Naica Crystal Digital PCR system (Stilla technologies). Two color multiplexing system is applied to simultaneously measure the transgene AAV and an endogenous control gene. In brief, the transgene probe can be labelled with FAM (6- carboxyfluorescein) dye while the endogenous control probe can be labelled with VIC fluorescent dye. The copy number of delivered vector in a specific tissue section per diploid cell is calculated as: (vector copy number)/(endogenous control)*2. Vector copy in specific cell types or tissues, such as cornea, iris, ciliary body, schlemm’s canal cells, trabecular meshwork, retinal cells, RPE cells, RPE-choroid tissue, or optic nerve cells, over time may indicate sustained expression of the transgene by the tissue.
5.1.9 Compositions
[0134] Pharmaceutical compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil. In some embodiments, the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ as known in the art. The pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
5.2 Methods of Treating dry AMD
[0135] In another aspect, methods for treating dry AMD (age-related AMD) or other indication that can be treated with a tick C5 inhibitor protein, in a subject in need thereof (a composition for use in the treatment of dry AMD or other indication that can be treated with a tick C5 inhibitor protein) comprising the administration of recombinant AAV particles comprising an expression cassette encoding a tick C5 inhibitor protein, are provided. A subj ect in need thereof includes a subj ect suffering from dry AMD, or a subject pre-disposed thereto, e.g., a subject at risk of developing dry AMD, or other indication that may be treated with a tick C5 inhibitor protein. Subjects to whom such gene therapy is administered can be those responsive to a tick C5 inhibitor protein. In particular embodiments, the methods encompass treating patients who have been diagnosed with dry AMD, and, in certain embodiments, identified as responsive to treatment with a tick C5 inhibitor protein, or considered a good candidate for therapy with a tick C5 inhibitor protein. In specific embodiments, the patients have previously been treated with a tick C5 inhibitor protein or other C5 inhibitor, such as an anti-C5 inhibitor antibody. To determine responsiveness, the tick C5 inhibitor protein (e.g., produced in human cell culture, bioreactors, etc.) may be administered directly to the subject.
[0136] In specific embodiments, provided are methods of treating dry AMD or other indication amenable to treatment with a tick C5 inhibitor protein in a human subject in need thereof comprising: administering to the eye, for example, intravitreal, subretinal, suprachoroidal, intracameral, or intranasal, or liver and/or muscle by systemic administration (including intravenous or intramuscular) of said subject a therapeutically effective amount of a recombinant nucleotide expression vector, such as an AAV vector, comprising a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells (such as retinal cells, BrM cells, choriocapillaris cells, RPE cells and/or choroid cells), so that a depot is formed that releases the tick C5 inhibitor protein. Subretinal, intravitreal, intracameral, or suprachoroidal administration should result in expression of the transgene product in one or more of the following retinal cell types: Bruch’s membrane (BrM), including epithelial cells thereof, choriocapillaris, human photoreceptor cells (cone cells, rod cells); horizontal cells; bipolar cells; amarcrine cells; retina ganglion cells (midget cell, parasol cell, bistratified cell, giant retina ganglion cell, photosensitive ganglion cell, and muller glia); and retinal pigment epithelial cells or other ocular tissue cell: cornea cells, iris cells, ciliary body cells, a schlemm’s canal cells, a trabecular meshwork cells, RPE-choroid tissue cells, or optic nerve cells.
[0137] Recombinant vectors and pharmaceutical compositions for treating diseases or disorders in a subject in need thereof are described in Section 5.1. Such vectors should have a tropism for human ocular tissue, or liver and/or muscle cells and can include non-replicating rAAV, particularly those bearing an AAV3B, AAV8, AAAV9, AAV10, AAVrhlO, or AAVrh73 capsid. The recombinant vectors can be administered in any manner such that the recombinant vector enters ocular tissue cells, e g., by introducing the recombinant vector into the eye. Such vectors should further comprise one or more regulatory sequences that control expression of the transgene in human ocular tissue cells and/or human liver and muscle cells include, but are not limited to, human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7), a CAG promoter (SEQ ID NO: 2), a CB promoter or CBlong promoter (SEQ ID NO: 17 or 18) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19) (see also Tables 1 and la).
[0138 j The methods described herein treat, slow the progression of, reduce the severity of or prevent dry (age related) AMD in a human subject in need of the treatment. The treatment, slowing progression of, reduction of severity or prevention may be assessed relative to the subject prior to treatment, a comparable untreated subject or according to the natural history of the disease. In particular, method of the invention may reduce the progression of geographic atrophy, including within the fovea, slow retinal cell loss, slow the loss of central vision, increase or slow the loss of visual acuity, etc.
[0139] The subject may be at risk or have a predisposition to develop dry AMD based upon age, and/or risk factors such as history of smoking, obesity, cardiovascular disease or diabetes.
5.3 Tick C5 Inhibitor Constructs and Formulations for Dry AMD
[0140] Compositions and methods are described for the delivery of tick C5 inhibitor protein for treating dry AMD. In certain embodiments, the HuPTM tick C5 inhibitor protein has the amino acid sequence of SEQ ID NO: 129 or SEQ ID NO: 130. In other embodiments, provided are compositions and methods for delivery of HuPTM tick C5 inhibitor protein (amino acid sequence in Table 4). Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a tick C5 inhibitor protein to patients (human subjects) diagnosed with dry AMD to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
Transgenes
[0141] Provided are recombinant vectors containing a transgene encoding a tick C5 inhibitor protein, that can be administered to deliver the tick C5 inhibitor protein in a patient. The transgene is a nucleic acid comprising the nucleotide sequences encoding a tick C5 inhibitor protein, or variants thereof, as detailed herein. The transgene may also encode a tick C5 inhibitor protein that contains altered glycosylation sites.
Gene Therapy Methods
[0142] Provided are methods of treating human subjects for dry AMD by administration of a viral vector containing a transgene encoding a tick C5 inhibitor protein. The tick C5 inhibitor protein may be SEQ ID NO: 129. The viral vector has an AAV capsid with tropism for human ocular tissues and may be an AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof, for example having 90%, 95% or 99% sequence identity to the capsid sequence of AAV8, AAV9, AAV3B, or AAVrh73). The transgene is operably linked by regulatory sequences that promote expression of the transgene in human ocular tissue cells (including in retinal cells, RPE, choroid, BrM, choriocapillaris, photoreceptor cells, retinal ganglion cells), for example a CAG promoter (SEQ ID NO: 2), or an ocular specific promoter, such as a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 12), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 9-11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19). Regulatory sequences may also include polyadenylation signal sequences. The expression cassette comprising the transgene and operably linked regulatory sequences are flanked by ITR sequences, as an artificial AAV genome. The flanking ITR sequences may be configured to provide a single stranded AAV (ssAAV) genome. The recombinant vectors can be administered in any manner such that the recombinant vector enters one or more ocular tissue cells. In particular embodiments, the recombinant AAV comprises an artificial genome of (or is produced using a cis plasmid or construct comprising) ss.CAG.tick.C5. inhibitor (SEQ ID NO: 139), ss.CAG.tick.C5.inhibitor (SEQ ID NO: 142), ss.CAG.tick.C5.inhibitor (SEQ ID NO: 145) or ss.CAG.tick.C5. inhibitor (SEQ ID NO: 148). In certain embodiments, the artificial genome is self complementary. In certain embodiments, the artificial genome is single stranded.
[0143] Therapeutically effective doses of any of these recombinant vectors should be administered in any manner such that the recombinant vector enters ocular tissue cells (e.g., retinal cells), e.g., via subretinal, intravitreal, intracameral, or suprachoroidal injection or intranasal administration. Alternatively, the vector is administered peripherally (for example, intravenously, intramuscularly or subcutaneously) such that the recombinant vector transduces liver and/or muscle cells, creating a depot in liver and/or muscle tissue which express the transgene product into the bloodstream, delivering the therapeutic to ocular tissues. Alternatively, subretinal, intravitreal, intracameral, suprachoroidal administration should result in expression of the transgene product in cells of the eye, creating a depot in one or more ocular tissue cells of the patient that continuously supplies the tick C5 inhibitor protein to ocular tissues of the subject. The transgene expression results in therapeutically effective levels of the tick C5 inhibitor protein in the aqueous humor, the vitreous humor, retinal tissue, the RPE, the BrM or choriocapillaris.
[0144] Subjects to whom such gene therapy is administered can be those responsive to anticomplement therapy. In certain embodiments, the methods encompass treating patients who have been diagnosed with dry AMD, or have one or more symptoms associated therewith, and identified as responsive to treatment with a complement inhibitor, such as an anti-C3 or C5 antibody, or considered a good candidate for therapy with a tick C5 inhibitor protein. In specific embodiments, the patients have previously been treated with crovalimab, eculizumab, ravulizumab, tesidolumab or other complement activation inhibitor, and have been found to be responsive to thereto. To determine responsiveness, the tick C5 inhibitor protein (e.g., produced in cell culture, bioreactors, etc.) may be administered directly to the subject.
[0145] In embodiments, administration of the recombinant AAV comprising a construct for expressing a transgene encoding a tick C5 inhibitor protein in ocular tissues results in reduction or slowing the progression of one or more symptoms of dry AMD within 10 days, 20 days, 30 days, 40 days, 6 months, 9 months or 1 year after administration of the AAV. In embodiments, the administration results in a slowing or reduction in the rate of the progression of geographic atrophy, including of the fovea, in the subject relative to an untreated subject or as expected in the subject based upon natural history of dry AMD, for example as measured by fundus autofluorescence (FAF). In embodiments, the administration results in an improvement or reduction in the rate of loss of visual acuity or best corrected visual acuity (BCVA), for example, as measured by a standard ETDRS chart or to improve visual function as measured by dark adaptation methodology; to improve contrast sensitivity by the Pelli-Robson test or to reduce the drusen area or accumulation of drusen. In other embodiments, the dose of therapeutic gene delivered by gene therapy is sufficient to inhibit complement activation without exacerbating choroidal neovascularization (CNV).
[0146] However, in all cases because the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective. The concentration of the transgene product can be measured in patient blood serum samples.
[0147] Pharmaceutical compositions suitable for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic (intravenous, intramuscular or subcutaneous) administration comprise a suspension of the recombinant vector comprising the transgene encoding the tick C5 inhibitor protein in a formulation buffer comprising a physiologically compatible aqueous buffer. The formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
[0148] The goal of gene therapy treatment provided herein is to slow or arrest the progression of or relieve one or more symptoms of dry AMD, such as to reduce the rate of geographic atrophy or improve visual acuity (or reduce the rate of loss of visual acuity).
[0149] Combinations of delivery of the tick C5 inhibitor protein, to the eye, liver and/or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein. The additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment. Available treatments for a subject with dry AMD that could be combined with the gene therapy provided herein include but are not limited to, elamipretide, risuteganib, photobiomodulation, brimonidine tartrate, kamuvudine, Xiflam, or doxycycline, and others and administration with the tick C5 inhibitor protein. 5.6. Monitoring of Efficacy
[0150] The compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD. The assessment may be determined in animal models or in human subjects. The efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or reduction in geographic atrophy, including of the fovea, to be assessed by visual inspection.
[0151 ] The compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD. The assessment may be determined in animal models or in human subjects. The efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or an increase in logMAR. Physical changes to the eye, including changes in geographic atrophy may be measured Optical Coherence Tomography, using methods known in the art.
[0152] The compositions and methods described herein may be assessed for efficacy using in vitro complement inhibition assays, such as membrane attack complex (“MAC”) formation, C5a generation and hemolysis. Complement inhibition assays can be performed in any appropriate cell type, such as ARPE19 cells (MAC and C5a assays), iPSC-derived RPE cells (MAC and C5a assays) or sheep/rabbit erythrocytes (hemolysis assay). MAC formation assays measure the deposition of MAC on the surface of RPE cells (% relative inhibition of MAC formation). C5a generation assays measure the ability of the C5 antibody to prevent C5 cleavage (less C5 cleavage = less C5a). Hemolysis assays allow the comparison of complement inhibition among different complement inhibitors (50% complement inhibition dose (ng/ml) (CHso; AHso).
[0153] Animal models may be used to assess the recombinant vectors encoding the tick C5 inhibitor proteins for expression, therapeutic effect and adverse effects. Animal models may include a humanized C3-/C5- rodent model or aNaIO3 induction rat or mouse model, or a CFH-/- mouse model. Animals may be administered vectors described herein, for example, subretinally or suprachoroi dally, and then assessed for geographic atrophy (or change therein) by OCT, retinal pathology (damage to RPE), and other assessments of dry AMD pathology, as well as reduction in C3a or C5a, cleavage of C3 or C5 or other markers of complement activation.
[0154] Endpoints may include, but are not limited to, mean change in geographic atrophy in the study eye from baseline to 12, 16, 20, 24, or 28 weeks or at time of administration, if earlier, proportion of responders in the study eye at 12, 16, 20, 24, or 28 weeks, mean change in best corrected visual acuity from baseline to 12, 16, 20, 24, or 28 weeks, change from baseline in quality of life/patient reported outcome assessments, mean change in visual acuity from baseline to 12, 16, 20, 24, or 28 weeks.
6 EXAMPLES
6.1 EXAMPLE 1: Tick C5 Inhibitor cDNA Based Vector
[0155] A tick C5 inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequence of SEQ ID NO. 129). The transgene also comprises nucleotide sequences that encodes the native tick C6 inhibitor signal peptide, MLVLVTLIFSFSANIAYA (SEQ ID NO: 52). The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2). Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used. The vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO:
138. The artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO:
139.
6.2 EXAMPLE 2: Tick C5 Inhibitor (Dual mutant IL2 and Native Tick C5 Inhibitor Signal Peptides) cDNA Based Vector
[0156] A Tick C5 Inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 129). The transgene also comprises nucleotide sequences that encode i) a mutant IL2 signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and ii) the native tick C5 inhibitor signal peptide, e.g., MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) arranged in tandem with the mutant IL2 signal peptide at the N-terminus of the protein. The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2). Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as an ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used. The vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 141. The artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 142.
6.3 EXAMPLE 3: Tick C5 Inhibitor (mutated IL2 Signal Peptide) cDNA Based Vector [0157] A Tick C5 Inhibitor cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 129). The transgene also comprises nucleotide sequences that encode a mutated IL2 signal peptide, MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54). The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2). Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as an ocular tissuespecific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxia-inducible promoter, may be used. The vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 144. The artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 145.
6.4 EXAMPLE 4: Mutated Tick C5 Inhibitor (Native Signal Peptide) cDNA Based Vector
[0158] A Tick C5 Inhibitor (having mutations at N78Q and N102Q) cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the tick C5 inhibitor protein (amino acid sequences of SEQ ID NO. 130). The transgene also comprises nucleotide sequences that encode a native signal peptide, e.g., MLVLVTLIFSFSANIAYA (SEQ ID NO: 52). The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2). Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as an ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 19), or an inducible promoter, such as a hypoxiainducible promoter, may be used. The vector from the CAG promoter to the poly A tail has a nucleotide sequence of SEQ ID NO: 147. The artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 148.
6.5 EXAMPLE 5: Hemolysis Assay
[0159] All C5 inhibitor expression cassettes utilized in this study were constructed with a CAG promoter and rabbit beta-globin polyA. All transgenes were codon-optimized and CpG depleted. Cis- plasmids were initially screened in the assay following transfection in 293 T cells and then subsequently packaged as AAV8 viral vectors (including scAAV8 vectors) for further study.
[0160] The classical pathway of complement activation (CP) is initiated primarily by immune complexes. The standard assay for the overall functional activity of this pathway is the CH50. This assay uses sheep erythrocytes coated with rabbit antibodies (referred to as EA) to activate the complement system.
[0161 ] Such classical complement pathway-related hemolysis inhibition assay was employed using supernatant collected from HEK cells transfected with the plasmids (encoding complement inhibitors as described herein). The supernatants (containing the complement inhibitor, or negative controls containing media without inhibitor or containing an antibody or antigen binding fragement expressed from the plasmid to a non-complement related target) were collected and applied to sheep erythrocytes coated with optimum levels of rabbit anti-sheep erythrocyte IgM antibodies suspended at 5 x 108 cells/ml in Gelatin Veronal Buffered saline (GVB++ Buffer) in the wells of an assay plate. Percent hemolysis was compared to a positive test hemolytic solution containing normal human serum that has been titrated up to 50% hemolysis. Percent hemolysis was calculated as such: % hemolysis = (test sample hemolysis (OD405)-background hemolysis (OD405))/(maximal hemolysis (OD405)- background hemolysis (OD405)) X 100.
[0162] The neat supernatant collected from HEK293 cells transfected with vectorized AB 1 or
AB2 plasmids (different vectorized forms IgG, Fab or ScFv) or a plasmid expressing the tick C5 inhibitor (native signal sequence) (SEQ ID NO: 138) were tested in the assay for the % of lysis of the EA as an indication of inhibition of complement activation. The tick C5 inhibitor (CI5) displayed strong inhibition of complement (FIGS. 2A-2B, middle bar). The native tick C5 inihibitor protein inhibits human C5-mediated hemolysis in the classical complement pathway (FIG. 2A) better than it inhibits mouse C5-mediated hemolysis (FIG. 2B). C5 inhibitor protein resulting from the IL2 signal peptide-cassette expressed in HEK293 cells displayed equivalent inhibition of complement (compared to native signal pwptide), and the C5 inhibitor protein resulting from the dual mutant IL2 / native tick inhibitor signal peptide-expressed cassette in HEK293 cells suppresses complement pathway activation in hemolysis inhibition assays at varying degrees (data not shown).
[0163] Purified proteins of C5 inhibitors were tested at series concentrations (nM) to determine the minimum concentration necessary to lyse 50% of the cells (1 CH50 Unit) (FIGS. 3A-3B). Activation of the classical pathway requires calcium and magnesium ions. The initial reactions include binding of Cl and activation of C2 and C4 to form a C3 convertase. This enzyme cleaves C3 which promotes cleavage of C5 and activation of the membrane attack pathway (proteins C5, C6, C7, C8 and C9). These five components assemble in the membrane of the sheep erythrocyte and lyse the cell. The release of hemoglobin is subsequently quantitated to measure the total complement activity present in the sample.
[0164] Recombinant purified forms of each C5 inhibitor, including tick C5 inhibitor (“C5 inhibitor,” diamond 0) (SEQ ID NO: 129), displayed potent inhibition of complement activation in both the classical and alternative hemolysis pathway assays (FIGS. 3A-B).
6.6 EXAMPLE 6: Binding Kinetics and Affinity of Recombinant purified forms of each C5 inhibitor
[0165] The generated recombinant purified proteins of each C5 inhibitor (expressed in HEK293 cells as above) were compared by their IC50 values in both the classical and alternative complement pathways. The binding kinetics and affinity of each C5 inhibitor to human, cynomolgous macaque, and mouse C5 were measured with the OctetRED384 system, as follows. a. Affinity and kinetics for human Complement C5:
[0166] The assay was performed at 30°C and at 1000 rpm. Biotinylated Human Complement C5 Protein was firstly immobilized onto SA biosensor. AB1 IgG and Ab fragments and native tick C5 inhibitor protein was applied as analyte for association and dissociation steps. Table 6. Assay conditions for human C5 assay after optimization
Figure imgf000071_0001
b. Affinity and kinetics for cynomolgous Complement C5:
[0167] The assay was performed at 30°C and at 1000 rpm. Biotinylated cyno C5 antigen was firstly immobilized onto SA biosensor. AB1 IgG and Ab fragments and the tick C5 inhibitor protein were applied as analyte for association and dissociation steps.
Table 7. Assay conditions for cyno C5 assay after optimization
Figure imgf000071_0002
c. Affinity and kinetics for mouse Complement C5:
[0168] The assay was performed at 30°C and at 1000 rpm. Biotinylated mouse C5 antigen was firstly immobilized onto SA biosensor. AB2 IgG and Ab fragments were applied as analyte for association and dissociation steps.
Table 8. Assay conditions for mouse C5 assay after optimization (surrogate anti-mouse C5 mAb)
Figure imgf000072_0001
[0169] The assay was performed at 30°C and at 1000 rpm. Biotinylated mouse C5 antigen was firstly immobilized onto SA biosensor. AB 1 IgG and Ab fragments and Tick C5 inhibitor protein were applied as analyte for association and dissociation steps.
Table 9. Assay conditions for mouse C5 assay after optimization (cross-species C5 binders)
Figure imgf000072_0002
[0170] All three formats of anti -human C5 inhibitor (AB 1) demonstrated KD values for human and cyno C5 in the low to high picomolar range, whereas the tick C5 inhibitor protein bound less strongly with a low nanomolar affinity constant. Anti-Mouse (AB2) and Anti-human (AB1) C5 inhibitors with the same vectorized antibody format demonstrated comparable affinity to mouse C5. See Table 10
Table 10
Figure imgf000073_0001
EQU IVALENTS
[0171 ] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
[0172] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.

Claims

What is claimed is:
1. A pharmaceutical composition for treating AMD in a human subj ect in need thereof, comprising a recombinant adeno-associated virus (AAV) vector comprising:
(a) a viral capsid that has a tropism for ocular tissue cells; and
(b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein said AAV vector is formulated for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic administration to said human subject.
2. The pharmaceutical composition of claim 1, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).
3. The pharmaceutical composition of claim 1 or claim 2, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
4. The pharmaceutical composition of any one of claims 1 to 3, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photreceptor cells (rods, cones and/or retinal ganglion cells).
5. The pharmaceutical composition of any one of claims 1 to 4, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
6. The pharmaceutical composition of claim 5, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
7. The pharmaceutical composition of any one of claims 1 to 6, wherein the transgene comprises a signal sequence.
8. The pharmaceutical composition of claim 7, wherein the signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), MLVLVTL1FSFSANIAYA (SEQ ID NO: 52) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
9. The pharmaceutical composition of any one of claims 1 to 8, wherein the tick C5 inhibitor protein comprises an amino acid sequence of SEQ ID NO: 129.
10. The pharmaceutical composition of any one of claims 1 to 9, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143.
11. The pharmaceutical composition of any one of claims 1 to 10, wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
12. A pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising: a) a viral capsid that has a tropism for ocular tissue cells; and b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells; wherein the transgene encodes a signal sequence at the N-terminus of said tick C5 inhibitor protein that directs secretion and post translational modification of said tick C5 inhibitor protein in human ocular tissue cells.
13. The pharmaceutical composition of claim 12, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu!2 (AAV.hul2), or serotype hu26 (AAV.hu26).
14. The pharmaceutical composition of claim 13, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
15. The pharmaceutical composition of any one of claims 12 to 14, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
16. The pharmaceutical composition of any one of claims 12 to 15, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
17. The pharmaceutical composition of claim 16, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
18. The pharmaceutical composition of any one of claims 12 to 17, wherein the transgene comprises a signal sequence.
19. The pharmaceutical composition of any one of claims 12 to 18, wherein said signal sequence is MLVLVTLIFSFSANIAYA (SEQ ID NO: 52), MYRMQLLSCIALILALVTNS (SEQ ID NO: 53) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), or a combination thereof or a signal sequence from Table 2.
20. The composition of claim 19, wherein said signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
21. The composition of any one of claims 12 to 20, wherein the tick C5 inhibitor protein has an amino acid sequence of SEQ ID NO: 129.
22. The pharmaceutical composition of any one of claims 12 to 21, wherein the transgene comprises the nucleotide sequence of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, or SEQ ID NO: 143.
23. The composition of any one of claims 12 to 22, wherein the artificial genome is single stranded.
24. The composition of any one of claims 12 to 23 wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
25. A nucleic acid encoding an expression cassette, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 138, SEQ ID NO: 141 or SEQ ID NO: 144.
26. A nucleic acid encoding an artificial genome, wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142 or SEQ ID NO: 145).
27. A plasmid comprising the nucleic acid of claim 25.
28. A method of treating AMD in a human subject in need thereof, comprising subretinally, intravitreally, intranasally, intracamerally, suprachoroidally, or systemically administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV vector comprising
(a) a viral capsid that has a tropism for ocular tissue cells; and
(b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that control expression of the transgene in human ocular tissue cells.
29. The method of claim 28, wherein the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).
30. The method of claim 29, wherein the AAV capsid is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof.
31. The method of any one of claims 28 to 30, wherein the ocular tissue cells are retinal cells, RPE- choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
32. The method of any one of claims 28 to 31, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
33. The method of claim 32, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a CB promoter (SEQ ID NO: 17 or SEQ ID NO: 18), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 5 or SEQ ID NO: 12), a mouse cone arresting (CAR) promoter (SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), a human red opsin (RedO) promoter (SEQ ID NO: 7) or a Bestl/GRKl tandem promoter (SEQ ID NO: 19).
34. The method of any one of claims 28 to 33, wherein the transgene comprises a signal sequence.
35. The method of claim 34, wherein the signal sequence comprises MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54), MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) or MYRMQLLLLIALSLALVTNS (SEQ ID NO: 54) and MLVLVTLIFSFSANIAYA (SEQ ID NO: 52) in tandem.
36. The method of any one of claims 28 to 35, wherein the tick C5 inhibitor protein comprises an amino acid sequence of SEQ ID NO: 129.
37. The method of any one of claims 28 to 36, wherein the artificial genome comprises construct CAG.tick.C5. inhibitor (SEQ ID NO: 139, SEQ ID NO: 142, or SEQ ID NO: 145).
38. The method of any one of claims 28 to 37, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 135, 137, 140 or 143.
39. The method of any one of claims 28 to 38, wherein the therapeutically effective amount is determined to be sufficient to improve best corrected visual acuity (BCVA) by >= 2 ETDRS lines or increase in logMAR; to decrease the mean rate of change in geographic atrophy as measured by fundus autofluorescence (FAF); to improve visual function as measured by dark adaptation methodology; to improve contrast sensitivity by the Pelli-Robson test, or reduce the drusen area within 10 weeks, 20 weeks, 6 months or 1 year of administration.
40. A method of producing recombinant AAVs comprising:
(a) culturing a host cell containing:
(i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a tick C5 inhibitor protein, operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells;
(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has ocular tissue tropism;
(iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and
(b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
41. The method of claim 40, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and/or retinal ganglion cells).
42. A host cell comprising: a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a tick C5 inhibitor protein, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.
43. The host cell of claim 42, wherein the transgene encodes a tick C5 inhibitor protein (SEQ ID NO: 129).
PCT/US2024/029800 2023-05-16 2024-05-16 Vectorized c5 inhibitor agents and administration thereof Pending WO2024238859A1 (en)

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