[go: up one dir, main page]

WO2025217230A1 - Anticorps anti-complément vectorisés et agents de complément et leur administration - Google Patents

Anticorps anti-complément vectorisés et agents de complément et leur administration

Info

Publication number
WO2025217230A1
WO2025217230A1 PCT/US2025/023760 US2025023760W WO2025217230A1 WO 2025217230 A1 WO2025217230 A1 WO 2025217230A1 US 2025023760 W US2025023760 W US 2025023760W WO 2025217230 A1 WO2025217230 A1 WO 2025217230A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
capsid protein
aav
scfv
mab
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023760
Other languages
English (en)
Inventor
Brendan N. Lilley
Jessica GUMERSON
April R. TEPE
Samantha YOST
Elad FIRNBERG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regenxbio Inc
Original Assignee
Regenxbio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regenxbio Inc filed Critical Regenxbio Inc
Publication of WO2025217230A1 publication Critical patent/WO2025217230A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • compositions and methods are described for the delivery of a fully human post- translationally modified (HuPTM) proteins, including therapeutic monoclonal antibodies (“mAbs”) that bind to C5.
  • HuPTM antigen-binding fragments of a therapeutic mAb that bind to C5 e.g., a fully human-glycosylated (HuGly) Fab of the therapeutic mAb — to a human subject diagnosed with Age-Related Macular Degeneration (AMD).
  • ALD Age-Related Macular Degeneration
  • Therapeutic mAbs have been shown to be effective in treating a number of diseases and conditions. However, because these agents are effective for only a short period of time, repeated injections for long durations are often required, thereby creating considerable treatment burden for patients.
  • 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.
  • Age-Related Macular Degeneration causes progressive and permanent vision impairment.
  • AMD Age-Related Macular Degeneration
  • 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 over-active complement in the eye.
  • G geographic atrophy
  • AMD age-related macular degeneration
  • 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.
  • Therapeutic antibodies and other proteins delivered by gene therapy have several advantages over injected or infused therapeutic antibodies that dissipate over time resulting in peak and trough levels. Sustained expression of the transgene product antibody or protein, as opposed to injecting an antibody or protein repeatedly, allows for a more consistent level of antibody or 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, antibodies and other 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.
  • compositions and methods for anti-C5 gene therapy designed to target the eye and generate a depot of transgenes for expression of anti- C5 antibodies, including, C5-D-mab scFv mAb (H-L) (amino acid sequence of SEQ ID NO: 195 or 203), C5-D-mab scFv mAb (L-H) (SEQ ID NO: 196), C5-A-mab scFv mAb (H-L) (SEQ ID NO: 197), C5-A-mab scFv mAb (L-H) (SEQ ID NO: 198), C5-C-mab scFv mAb (H-L) (SEQ ID NO: 199), C5- C-mab scFv mAb (L-H) (SEQ ID NO: 200), C5-B-mab sc
  • the administration including by ocular administration, including, in embodiments, administration to the suprachoroidal space of the eye, results in a therapeutic or prophylactic levels (in ocular tissues and, also, in embodiments, in serum) of the antibody 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.
  • the rAAV composition is administered to the suprachoroidal space by a microneedle or microinjector device.
  • compositions and methods which comprise AAV gene therapy vectors for the ocular delivery of anti-C5 scFv antibodies, including the C5-D-mab scFv (H-L) having an amino acid sequence of SEQ ID NO: 195 or 203 (with leader sequence), the C5-A-mab scFv (H-L) having an amino acid sequence of SEQ ID NO: 197, the C5-C-mab scFv (H-L) having an amino acid sequence of SEQ ID NO: 199, and the C5-B-mab scFv (H-L) having an amino acid sequence of SEQ ID NO: 201.
  • AAV gene therapy vectors for the ocular delivery of anti-C5 scFv antibodies including the C5-D-mab scFv (H-L) having an amino acid sequence of SEQ ID NO: 195 or 203 (with leader sequence), the C5-A-mab scFv (H-
  • Methods are described for delivery of the C5-D mab or C5-A mab to a patient (human subject) diagnosed with AMD or other condition indicated for treatment with the therapeutic anti-C5 mAb. Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic anti-C5 mAb, such as C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203), C5-D-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 196), C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197), C5-A-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 198), C5-C-mab Vectorized scFv mAb (H-L) (SEQ ID NO:
  • gene therapy vectors particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of an anti-C5 antibody, such as C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203) or C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197).
  • an anti-C5 antibody such as C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203) or C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197).
  • the sequence encoding the C5-D-mab scFv (H-L) or C5-A-mab scFv (H-L), optionally with a signal sequence at the N-terminus, is provided in an expression cassette where the coding sequence is operably linked to regulatory elements, such as a CAG promoter, and other regulatory elements, such as polyA signal sequences and introns, flanked by AAV ITR sequences.
  • regulatory elements such as a CAG promoter
  • other regulatory elements such as polyA signal sequences and introns, flanked by AAV ITR sequences.
  • 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.
  • Nucleotide sequences of exemplary constructs including coding sequences for the transgene products, expression cassettes and artificial genomes are provided in Table 8 herein and include SEQ ID NO: 225, 226, 227, 228, 229, 233, 234, 235, 236, 237, 238, 239, 240, 242, 276, 277, 278, and 279.
  • the construct encoding the C5-D-mab scFv (HL) or C5-A-mab scFv (HL) is packaged in a recombinant AAV (rAAV) which 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.
  • rAAV recombinant AAV
  • the AAV has an engineered AAV3B or AAV8 capsid protein which has an ocular tissue homing peptide or 7 to 9 amino acids inserted into either VR-IV or VR-VIII of the capsid protein.
  • the engineered rAAV comprises a capsid protein of AAV3B 455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249) (see Table A).
  • [001 1 ] Provided are methods of treating ocular indications, including Age-Related Macular Degeneration (AMD), by ocular, including suprachoroidal, administration to a patient suffering therefrom, a pharmaceutical composition comprising the rAAV comprising a transgene encoding the C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203) or C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197), including encoded by a polynucleotide comprising the nucleotide sequence of 219-220, or 224-242, packaged in a capsid comprising a capsid protein of AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456
  • the administration provides a therapeutically effective amount to the target ocular tissues of the subject for treatment of AMD, including to achieve a maximum or steady state 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.
  • the rAAV is administered to the suprachoroidal space by a microneedle or microinjector device.
  • the constructs express an scFv in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker, such as GGGGSGGGGSGGGGS (SEQ ID NO: 128).
  • the construct expresses, from the N-terminus, NH2-V L -linker-Vn-COOH or NH2-V n -linker-V L -COOH.
  • the construct encodes, from the N-terminus, NFF-signal or leader sequence- V L -GGGGSGGGGSGGGGS- VH-COOH or NFL-signal or leader sequence-V H - GGGGSGGGGSGGGGS-V L -COOH.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • 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 antibody 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 genes 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.
  • a recombinant adeno-associated virus (rAAV) vector comprising
  • an AAV capsid comprising viral capsid protein; wherein the viral capsid protein comprises a 7 amino acid peptide inserted into a AAV3B or AAV8 capsid protein as a parental capsid protein and is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8 590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249); and
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene comprising a nucleotide sequence encoding C5-D-mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5-A-mab.ScFv.HL (SEQ ID NO: 197), and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; wherein the rAAV vector has enhanced tropism to ocular tissue compared to an rAAV vector prepared from the parental capsid protein.
  • ITRs AAV inverted terminal repeats
  • rAAV vector of embodiment 1 wherein said ocular tissue with enhanced tropism is retina, RPE choroid or sclera tissue.
  • rAAV vector of embodiment 2 wherein the rAAV vector exhibits an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, or 25 fold greater transduction of ocular tissue than an rAAV vector comprising the parental capsid.
  • rAAV vector of embodiment 4 wherein the rAAV vector has an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or 40 fold lower transduction of liver than an rAAV vector comprising the parental capsid protein.
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243).
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV3B.455.DVTPLLS (SEQ ID NO: 244).
  • rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV3B.455.LVSVSLP (SEQ ID NO: 245).
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV8.456.RIQMGTK (SEQ ID NO: 246).
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247).
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV8.590.GDNTTFRRA (SEQ ID NO: 248).
  • the rAAV vector any one of embodiments 1 to 5, wherein the viral capsid protein is AAV8.590.GRTIRGDLA (SEQ ID NO: 249).
  • the rAAV vector any one of embodiments 1 to 14, wherein the transgene encodes a signal sequence at the N-terminus of the scFv that directs secretion and post-translational modification in said human ocular tissue cells.
  • the rAAV vector of any one of embodiments 1 to 16 wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
  • CAG promoter SEQ ID NO: 2
  • a mutated CAG promoter SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31
  • a CB promoter SEQ ID NO: 24 or 25
  • GRK1 human rhodopsin kinase
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a transgene encoding an anti-C5 scFv having an amino acid sequence of SEQ ID NO: 195, or 203 operatively linked to a regulatory sequence which promotes expression in ocular tissue and flanked by ITR sequences.
  • a pharmaceutical composition comprising the rAAV vector of any one of embodiments 1 to 24 and a pharmaceutically acceptable carrier.
  • composition according to embodiment 26 wherein said composition is formulated for suprachoroidal administration with a microneedle.
  • a method of delivering a transgene to a cell comprising contacting said cell with the rAAV vector of any one of embodiments 1-24.
  • a method of delivering a transgene to a target tissue of a subject in need thereof comprising administering to said subject the rAAV vector of any one of embodiments 1-24.
  • a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) vector comprising,
  • an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein comprises a 7 amino acid peptide inserted into a AAV3B or AAV8 capsid protein as a parental capsid protein and is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 345), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456 RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249); and
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding C5-D- mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5-A-mab.ScFv.HL (SEQ ID NO: 197), and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; wherein the rAAV vector has enhanced tropism to ocular tissue compared to an rAAV vector prepared from the parental capsid protein.
  • ITRs AAV inverted terminal repeats
  • composition of embodiment 31, wherein said ocular tissue with enhanced tropism is retina, RPE choroid or sclera tissue.
  • composition of embodiment 32 wherein the rAAV vector exhibits an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, or 25 fold greater transduction of ocular tissue than an rAAV vector comprising the parental capsid.
  • composition any one of embodiments 31 to 35, wherein the viral capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243).
  • the composition any one of embodiments 31 to 35, wherein the viral capsid protein is AAV3B.455.DVTPLLS (SEQ ID NO: 244).
  • the composition any one of embodiments 31 to 35, wherein the viral capsid protein is AAV3B.455.LVSVSLP (SEQ ID NO: 245).
  • the composition of any one of embodiments 33 to 35, wherein the parental capsid is AAV3B (SEQ ID NO: 254).
  • composition any one of embodiments 31 to 39, wherein the viral capsid protein is AAV8.456.RIQMGTK (SEQ ID NO: 246).
  • the composition any one of embodiments 31 to 39, wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247).
  • the composition any one of embodiments 31 to 39, wherein the viral capsid protein is AAV8.590.GDNTTFRRA (SEQ ID NO: 248).
  • the composition any one of embodiments 31 to 39, wherein the viral capsid protein is AAV8.590.GRTIRGDLA (SEQ ID NO: 249).
  • the composition of any one of embodiments 33 to 35, wherein the parental capsid is AAV8 (SEQ ID NO: 260).
  • the composition of any one of embodiments 31 to 46, wherein the regulatory sequence comprises a regulatory sequence from Table 1 or Table la.
  • composition of any one of embodiments 31 to 47, wherein the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a mutated CAG promoter (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31), a CB promoter (SEQ ID NO: 24 or 25), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 16-18), a human red opsin (RedO) promoter (SEQ ID NO: 14) or a Bestl/GRKl tandem promoter (SEQ ID NO: 26).
  • CAG promoter SEQ ID NO: 2
  • a mutated CAG promoter SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31
  • CB promoter SEQ ID NO: 24 or 25
  • GRK1 human rhodopsin kinase
  • CAR mouse cone
  • composition of embodiment 44, wherein the regulatory sequence is a mutated CAG promoter (SEQ ID NO: 29).
  • composition of any one of embodiments 31 to 49, wherein the artificial genome comprises the nucleotide sequence of SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 276, or SEQ ID NO: 277.
  • a pharmaceutical composition for use in treating Age-Related Macular Degeneration in a subject comprising or a method for treating Age-Related Macular Degeneration (AMD) in a human subject in need thereof comprising administering a therapeutically effective amount of recombinant adeno-associated virus (rAAV) vector comprising,
  • an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein comprises a 7 amino acid peptide inserted into a AAV3B or AAV8 capsid protein as a parental capsid protein and is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249); and
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding C5-D- mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5-A-mab.ScFv.HL (SEQ ID NO: 197), and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; wherein the rAAV vector has enhanced tropism to ocular tissue compared to an rAAV vector prepared from the parental capsid protein.
  • ITRs AAV inverted terminal repeats
  • the pharmaceutical composition or method for treating according to embodiment 51 wherein said ocular tissue with enhanced tropism is retina, RPE choroid or sclera tissue.
  • the pharmaceutical composition or method for treating according to embodiment 52 wherein the rAAV vector exhibits an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, or 25 fold greater transduction of ocular tissue than an rAAV vector comprising the parental capsid.
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV3B.455.DVTPLLS (SEQ ID NO: 244).
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV3B.455.LVSVSLP (SEQ ID NO: 245).
  • the pharmaceutical composition or method for treating according to any one of embodiments 53 to 55, wherein the parental capsid is AAV3B (SEQ ID NO: 254).
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV8.456.RIQMGTK (SEQ ID NO: 246).
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247).
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV8.590.GDNTTFRRA (SEQ ID NO: 248).
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 55, wherein the viral capsid protein is AAV8.590.GRTIRGDLA (SEQ ID NO: 249).
  • the pharmaceutical composition or method for treating according to any one of embodiments 53 to 55, wherein the parental capsid is AAV8 (SEQ ID NO: 260).
  • the regulatory sequence is a CAG promoter (SEQ ID NO: 2), a mutated CAG promoter (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31), a CB promoter (SEQ ID NO: 24 or 25), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 16-18), a human red opsin (RedO) promoter (SEQ ID NO: 14) or a Bestl/GRKl tandem promoter (SEQ ID NO: 26).
  • CAG promoter SEQ ID NO: 2
  • a mutated CAG promoter SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31
  • a CB promoter SEQ ID NO: 24 or 25
  • GRK1 human rhodopsin kinase
  • CAR mouse cone arresting
  • RedO human red ops
  • the pharmaceutical composition or method for treating according to any one of embodiments 51 to 74 which results in a therapeutic or prophylactic levels in ocular tissues of the antibody within 20 days, 30 days, 40 days, 50 days, 60 days, or 90 days of administration of the rAAV composition.
  • the pharmaceutical composition of any one of embodiments 25 to 50 or the composition for use in treatment of AMD of any one of embodiments 51 to 77 wherein the pharmaceutical composition or composition or composition for use in the treatment of AMD comprises between about 0.5% to about 1.0% w/v hyaluronic acid.
  • composition of any one of embodiments 25 to 50 or the composition for use in treatment of AMD of any one of embodiments 51 to 77 wherein the pharmaceutical composition or composition or composition for use in the treatment of AMD comprises about (2.5% w/v) sucrose and between about 0.5% to about 1.0% w/v hyaluronic acid.
  • composition of any one of embodiments 25 to 50 or the composition for use in treatment of AMD of any one of embodiments 51 to 77 wherein the pharmaceutical composition or composition or composition for use in the treatment of AMD comprises about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.7% w/v hyaluronic acid.
  • the pharmaceutical composition or composition or composition for use in the treatment of AMD comprises about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL
  • the pharmaceutical composition of any one of embodiments 25-50 or the composition for use in treatment of AMD of any one of embodiments 51-77 wherein the pharmaceutical composition or composition or composition for use in the treatment of AMD comprises about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 40.0 mg/mL (4% w/v) sucrose, about 0.001% (0.01 mg/mL) poloxamer 188, and about 1% carboxymethylcellulose (CMC) high viscosity grade.
  • CMC carboxymethylcellulose
  • a method of producing recombinant AAVs comprising:
  • an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding C5-D- mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5-A-mab.ScFv.HL (SEQ ID NO: 197), and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells;
  • trans expression cassette lacking AAV ITRs
  • 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 cell tropism
  • the AAV capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249);
  • a host cell comprising: a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding C5-D-mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5- A-mab.ScFv.HL (SEQ ID NO: 197) operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.
  • a method for treating a human subject diagnosed with age-related macular degeneration comprising delivering to an eye of the human subject a therapeutically effective amount of an anti-C5 scFv antibody produced by the human subject's ocular cells transduced with a recombinant adeno-associated virus (rAAV) vector carrying an artificial genome which comprises an expression cassette comprising a transgene encoding an anti-C5 scFv antibody, by a single dose administration of the rAAV vector via suprachoroidal (SC) injection or subretinal (SR) injection to the eye of the human subject.
  • SC suprachoroidal
  • SR subretinal
  • a pharmaceutical composition for use in treating a human subj ect diagnosed with age-related macular degeneration comprising delivering to an eye of the human subject a therapeutically effective amount of an anti-C5 scFv antibody produced by the human subject's ocular cells transduced with a recombinant adeno-associated virus (rAAV) vector carrying an artificial genome containing an expression cassette comprising a transgene encoding an anti-C5 scFv antibody, by a single dose administration of the rAAV vector via suprachoroidal (SC) injection or subretinal (SR) injection to the eye of the human subject.
  • SC suprachoroidal
  • SR subretinal
  • rAAV vector comprises an engineered AAV8 capsid protein AAV8.456.RQKNAMV (SEQ ID NO: 247).
  • a method for enhancing the survival and/or the function of the photoreceptors of an eye comprising delivering to the eye of a mammal a therapeutically effective amount of an anti- C5 scFv antibody produced by the mammal's ocular cells transduced with a recombinant adeno-associated virus (rAAV) vector carrying an expression cassette encoding the anti-C5 scFv antibody, by a single dose administration of the rAAV vector via suprachoroidal (SC) injection or subretinal (SR) injection to the mammal’s eye.
  • SC suprachoroidal
  • SR subretinal
  • the method of embodiment 92 wherein the mammal is a human, non-human primate, mouse or a pig.
  • the rAAV vector comprises an engineered AAV8 capsid protein AAV8.456.RQKNAMV (SEQ ID NO: 247).
  • the expression cassette comprises a transgene encoding C5-D-mab.ScFv.HL (SEQ ID NO: 195 or 203) or C5-A-mab.ScFv.HL (SEQ ID NO: 197).
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 225.
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 229.
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 232..
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 234.
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 236. .
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 238. .
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 240. .
  • An rAAV vector comprising (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 242.
  • a pharmaceutical composition for use in treating or a method for treating Age-Related Macular Degeneration in a subject comprising or a method for treating Age-Related Macular Degeneration (AMD) in a human subject in need thereof comprising administering a therapeutically effective amount of recombinant adeno-associated virus (rAAV) vector of any one of embodiments 97-104. .
  • the pharmaceutical composition or method for treating according to embodiment 105 wherein the rAAV vector is formulated for suprachoroidal administration to said human subject.
  • the pharmaceutical composition or method for treating according to embodiment 106 wherein the rAAV vector is formulated for suprachoroidal administration with a microneedle or microinjector.
  • the pharmaceutical composition or method for treating according to any one of embodiments 105 to 111 wherein the pharmaceutical composition comprises between about 0.5% to about 1.0% w/v hyaluronic acid. .
  • An rAAV vector comprising
  • an AAV capsid comprising viral capsid protein; wherein the viral capsid protein comprises a 7 amino acid peptide inserted into a AAV3B or AAV8 capsid protein as a parental capsid protein and is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), or AAV3B.455.LVSVSLP (SEQ ID NO: 245); and
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a therapeutic transgene, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; wherein the rAAV vector has enhanced tropism to ocular tissue compared to an rAAV vector prepared from the parental capsid protein.
  • the rAAV vector of embodiment 116, comprising the viral capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243) .
  • the rAAV vector of embodiment 116, comprising the viral capsid protein is
  • AAV3B.455.DVTPLLS (SEQ ID NO: 244).
  • the rAAV vector of embodiment 116, comprising the viral capsid protein is
  • a pharmaceutical composition comprising the rAAV vector of any one of embodiments
  • compositions 116 to 119 and a pharmaceutically acceptable carrier are formulated for subretinal, intravitreal, intranasal, intracameral, or suprachoroidal administration. .
  • the pharmaceutical composition according to embodiment 120, wherein said composition is formulated for suprachoroidal administration. .
  • the pharmaceutical composition according to embodiment 120, wherein said composition is formulated for suprachoroidal administration with a microneedle. .
  • a method of delivering a transgene to a cell comprising contacting said cell with the rAAV vector of any one of embodiments 116-119. .
  • a method of producing recombinant AAVs comprising: (a) culturing a host cell containing:
  • an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a therapeutic transgene, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells;
  • 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 cell tropism, wherein the AAV capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), or AAV3B.455.LVSVSLP (SEQ ID NO: 245);
  • a host cell comprising: a plasmid comprising 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 cell tropism, wherein the AAV capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), or AAV3B.455.LVSVSLP (SEQ ID NO: 245).
  • AAV capsid protein is AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), or AAV3B.455.LVSVSLP (SEQ ID NO: 245).
  • FIGS. 1A-1C Schematics of rAAV vector genome constructs containing an expression cassette encoding the heavy and light chains of a therapeutic mAb separated by a Furin-2A linker, operably linked to a promoter, flanked by the AAV ITRs.
  • the transgene can comprise nucleotide sequences encoding the A) full-length heavy and light chains with Fc regions, B) the heavy and light chains of the Fab portion, or C) a single chain variable fragment (scFv) connecting the heavy and light chains of the antibody with a linker.
  • FIGS. 2A-2E The amino acid sequence of a transgene construct for the Fab region of A) C5-D-mab IgGl, B) C5-A-mab IgGl, C) C5-A-mab IgG2, D) C5-C-mab, and E) C5-B-mab therapeutic antibodies to C5.
  • Glycosylation sites are boldface.
  • Glutamine glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine- O-sulfation sites (italics) are as indicated in the legend.
  • Complementarity-determining regions (CDR) are underscored. The hinge region is highlighted in grey.
  • FIG. 4 Glycans that can be attached to HuGlyFab regions of full length mAbs or the antigen-binding domains. (Adapted from Bondt et al., 2014, Mol & Cell Proteomics 13.1 : 3029-3039). [0019] FIG. 5. Clustal Multiple Sequence Alignment of constant heavy chain regions (CH2 and CH3) of IgGl (SEQ ID NO: 192), lgG2 (SEQ ID NO: 193), and lgG4 (SEQ ID NO: 194). The numbering of the amino acids is in EU-format.
  • FIGs. 6A-6B shows expression of full length C5-D-mab and C5-A-mab levels after transfection of HEK293T cells with pITR-CAG-C5-A-mab and pITR-CAG-C5-D-mab on A) nonreducing and B) reducing gels.
  • FIGS. 7A and 7B show the results of the ability of cis plasmid-expressed vectorized 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.
  • FIGS 8A-8F show that the recombinant purified forms of each C5 inhibitor suppressed classical and alternative complement pathways in hemolysis inhibition assays against A) 50% human C5, classical complement pathway conditions, testing anti-hC5 (C5-D-mab formats) and C5 inhibitor, B) 50% human C5, alternative complement pathway conditions, testing anti-hC5 (C5-D-mab formats) and C5 inhibitor C) 50% mouse C5, classical complement pathway conditions, testing anti-hC5 (C5- D-mab formats) and anti-mC5 (BB5.1 mAb formats), D) 50% mouse C5, classic complement pathway conditions, comparing anti-hC5 (C5-D-mab full-length mAb) and anti-mC5 (BB5.1 full-length mAb), E) 50% mouse C5, classic complement pathway conditions, comparing anti-hC5 (C5-D-mab Fab mAb) and anti-mC5 (BB5.1 Fab mAb), and F) 50% mouse C5, classic complement pathway conditions
  • FIGS. 9A-H measure membrane attack complex (MAC) formation in A-C) ARPE-19 cells and D-F) iPSC-derived RPE cells, G) TP level in apical and basal compartments, and H) mRNA/cDNA of AAV.
  • MAC membrane attack complex
  • FIGS. 10A-10D show the results of AAV8-encoding C5 inhibitors injected into wildtype mouse eyes via subretinal (SR) administration at 1E8 and 3E8 vg/eye.
  • AAV8.CAG.anti-hC5 (C5- D-mab) vectors were formatted as IgG (full-length), Fab or scFV vectorized antibodies and administered subretinally (SR) at each dose, and AAV8.CAG.anti-mC5 (BB5.1) vectors were administered SR at each dose, while purified recombinant anti-mC5 IgG (BB5.1) or isotype controls were delivered intraperitoneal (ip).
  • A) represents measurement of transgene product (TP) as ng/eye (RNA transcript); B) represents measurement of transgene product (TP) as pmol/eye (protein). C) represents transgene product (TP) as pmol/eye (protein) in the retina. D) represents transgene product (TP) as pmol/eye (protein) in RPE/Choroid/Sclera.
  • FIG. 11 is a diagram of full-length CAG promoter, the CAG-Del5 deletion mutant, the
  • CAG-Delm deletion mutant and the CAG-Del 3 deletion mutant are CAG-Delm deletion mutant and the CAG-Del 3 deletion mutant.
  • FIG. 12 is a bar graph showing the relative promoter strength of the CAG-Del5 deletion mutant (25%), the CAG-Delm deletion mutant (61%) and the CAG-Del 3 deletion mutant (11%) compared to the full-length CAG promoter (100%).
  • FIGS. 13A-D are bar graphs showing the expression of C5-D-mab scFv (in ng) at day 1 (A), day 2 (B), day 3 (C) and day 6 (D) after transduction of HEK293T cells with AAV8 ss.CAG.
  • C5-D.scFv open circles
  • AAV8 sc-CAG-Delm AAV8 sc-CAG-Delm.
  • C5-D.scFv closed circles at a multiplicity of infection (MOI) of 5e4, 1.6e4 or 5e5.
  • FIGS. 14A-14C show A) ONL thickness, B) total retinal thickness, C) total retina ONL, as BB5.1 antibody significantly ameliorated ONL and retinal thinning as measured by OCT post NaIO3 induction.
  • FIGS. 15A-15F show dark-adapted ERG recordings (a-wave amplitudes A, C and E; b-wave amplitudes B, D and F) and b-wave amplitudes of mice cohorts receiving i.p. antibody treatments at baseline, 3 and 7 days after NaI03 administration.
  • FIGS. 16A-16C show vector biodistribution (DNA, genome copies per pg of tissue) A) and transcript copy (# copies (DNA) per pg RNA) B) in bar graphs for various ocular tissues of animals (day 85) following treatment with 3E12 GC per eye by suprachoroidal administration of vector. Vector biodistribution (DNA) detected in peripheral tissues was also measured (C).
  • FIGS. 17A-17C show transgene product (TP) for the scFv antibody levels detected in aqueous humor (AH) following AAV8.CAG.C5-D-mab.scFv SCS dosing at collection intervals (day 15, 29, 57 and 85).
  • FIGS. 18A-18B show TP expression levels in vitreous humor (VH) collected at the end of the study (day 85) following SCS delivery of 3el2 GC/eye AAV8.CAG.C5-D-mab-scFv.
  • FIGS. 19A-19H shows AAV8-mediated TP production levels (ng/mg tissue) in ocular tissue homogenates: A) retina, B) RPE-choroid, C) sclera, and D) Sample 3 only tissues. TP results are illustrated exclusive of the an eye in the cohort that displayed no or low biodistribution; E) retina, F) RPE-choroid, G) sclera , and H) Sample 3 only tissues. TP results are illustrated inclusive of the an eye in the cohort that displayed no or low biodistribution.
  • FIGS. 20A-20C show TP levels in A) serum, B) peripheral vector genome biodistribution (liver), and C) time course following AAV8 vector delivery of CAG.C5-D-mab-scFv.
  • FIGS. 21A-21C show A) vector biodistribution (DNA, genome copies per pg of tissue) and B) transcript copy (# copies (DNA) per pg RNA) in bar graphs for various ocular tissues of animals (day 85) following treatment with 3E12 GC per eye by suprachoroidal administration of AAV3B vector.
  • FIGS. 22A-22C show transgene product (TP) for the scFv antibody levels detected in aqueous humor (AH) following AAV3B.CAG.C5-D-mab.scFv SCS dosing at collection intervals (day 15, 29, 57 and 85).
  • C Time course of the AH collection antibody levels for all eyes.
  • FIGS. 23A-23B show TP expression levels in vitreous humor (VH) collected at the end of the study (day 85) following SCS delivery of 3el2 GC/eye AAV3B.CAG.C5-D-mab-scFv.
  • FIGS. 24A-24H show AAV3B-mediated TP production levels (ng/mg tissue) in ocular tissue homogenates: A) retina, B) RPE-choroid, C) sclera, and D) Sample 3 only tissues. TP results are illustrated exclusive of the an eye in the cohort that displayed no or low biodistribution or +ATPA; E) retina, F) RPE-choroid, G) sclera, and H) Sample 3 only tissues. TP results are illustrated inclusive of the an eye in the cohort that displayed no or low biodistribution or +ATPA.
  • FIGS. 25A-25C show TP expression levels in A) serum levels, B) peripheral vector genome biodistribution (liver), and C) time course following AAV3B vector delivery of CAG.C5-D- mab-scFv.
  • FIG. 26 shows vector genome biodistribution (Vector DNA, GC per pg of tissue) were plotted for various ocular tissues of animals (retina, RPE-choroid and sclera) at end of study (day 29) post-injection of AAV 8 -anti -C 5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or AAV8-NS-scFv at a dose of lElO or 1E11 GC/eye.
  • AAV 8 -anti -C 5 scFV AAV8.CAG.C5-D-mab.ScFv.HL
  • AAV8-NS-scFv at a dose of lElO or 1E11 GC/eye.
  • FIGS. 27A-27B show AH collected at two and four weeks post-injection of (A) AAV8- anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or (B) AAV8-NS-scFv at a dose of 1E10 or 1E11 GC/eye.
  • FIGS. 28A-28B show VH collected at two and four weeks post-inj ection of (A) AAV8- anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or (B) AAV8-NS-scFv at a dose of 1E10 or 1E11 GC/eye.
  • FIGS. 29A-29B show A) AH and B) VH collected at two and four weeks post-injection of AAV8-anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL; 1E10 or 1E11 GC/eye), AAV8-NS-scFv (1E10 or 1E11 GC/eye) or AAV8-NS-IgG (full-length Non-specific mab; 3E9 or 1E10 GC/eye) showing greater TP expression levels for vectorized ScFv antibodies.
  • AAV8-anti-C5 scFV AAV8.CAG.C5-D-mab.ScFv.HL
  • AAV8-NS-scFv 1E10 or 1E11 GC/eye
  • AAV8-NS-IgG full-length Non-specific mab
  • 3E9 or 1E10 GC/eye showing greater TP expression levels for vectorized ScFv antibodies.
  • FIGS. 30A-30B show ocular tissues (retina, RPE-choroid and sclera) collected at two and four weeks post-injection of A) AAV8-anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or B) AAV8-NS-scFv at a dose of 1E10 or 1E11 GC/eye.
  • FIGS. 30A-30B show ocular tissues (retina, RPE-choroid and sclera) collected at two and four weeks post-injection of A) AAV8-anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or B) AAV8-NS-scFv at a dose of 1E10 or 1E11 GC/eye.
  • TP transgene product
  • FIGS. 32A-32B show transgene product A) expression and B) biodistribution of PEPIN3.1, PEPIN8.1, PEPIN8.2 and PEPIN8.4 in the retina at 3el2 GC/eye (solid) and 3el 1 GC/eye (brick).
  • FIGS. 34A-34B show In vitro transduction efficiency of rAAV comprising various capsids (AAV8, AAV3B, 3.1, 3.2, 8.1, 8.2, 8.3, and 8.4) in A) iRPE cells at day 21 and B) ARPE cells at 48 hours .
  • FIGS. 35A-35D show transgene product expression (scFvOl ng/mg) in A) retinal cells and B) RPE cells, and biodistribution (GC/pg DNA) in C) retinal cells and D) RPE cells for AAV3B- 3.1 variant vector administered at 3el2 GC/eye (solid) or 3el 1 GC/eye (hatched).
  • FIG. 36 shows transgene expression of AAV8 and AAV3B-3.1 in the vitreous humor at day 29.
  • FIGS. 37A-37D show NHP eyes were harvested at 29 days post-administration with 3el2 GC/eye AAV8.2 and anti-hC5-scFv01 transgene product (TP) levels were measured from A) AH, B) VH, C) retina lysates, and D) RPE-Choroid lysates from a macular punch, distal and proximal tissue strips.
  • TP transgene product
  • FIGs. 38A-38B illustrate the fold-change difference of round 3 engineered capsids with increased transduction of A) retina and B) RPE-choroid compared to parental AAV8 following an SCS dose of 3xl0 12 GC/eye to NHPs.
  • compositions and methods are described for the ocular delivery of a fully human post- translationally modified (HuPTM) therapeutic monoclonal antibody (mAb) or a HuPTM antigen- binding fragment of a therapeutic anti-C5 mAb (for example, a fully human-glycosylated Fab (HuGlyFab) or scFv of a therapeutic mAb) to a patient (human subject) diagnosed with AMD (including dry AMD and dry AMD with geographic atrophy) or other indication indicated for treatment with the therapeutic mAb. Delivery may be advantageously accomplished via gene therapy — e.
  • gene therapy vectors particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of an anti-C5 antibody, such as C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203), C5-D-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 196), C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197), C5-A-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 198), C5-C-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 199), C5-C- mab Vectorized scFv mAb (L-H) (SEQ ID NO: 200), C5-B-mab Vectorized scFv mAb (H-L) (SEQ
  • the sequence encoding the C5-D-mab scFv (H-L), optionally with a signal sequence at the N-terminus, is provided in an expression cassette where the coding sequence is operably linked to regulatory elements, such as a CAG promoter, and other regulatory elements, such as polyA signal sequences, or flanked by AAV ITR sequences.
  • regulatory elements such as a CAG promoter
  • other regulatory elements such as polyA signal sequences, or flanked by AAV ITR sequences.
  • 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.
  • Nucleotide sequences of exemplary constructs are provided in Table 8 herein and include SEQ ID NO; 205-242.
  • the construct encoding the C5-D-mab scFv (HL) or C5-A-mab scFv (HL) is packaged in a recombinant AAV (rAAV) which 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.
  • rAAV recombinant AAV
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 225.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 229.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 232.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 234.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 236.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 238.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 240.
  • the rAAV vector comprises (a) an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and (b) an artificial genome comprising a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 242.
  • compositions and methods provided herein ocularly or systemically deliver anti- 05 antibodies or antigen binding fragments thereof, particularly C5-D-mab scFv or C5-A-mab scFv, 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 an anti-C5 antibody or antigen binding fragment thereof.
  • 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
  • viral vectors for delivery of transgenes encoding the therapeutic anti-C5 antibodies or antigen-binding fragments thereof, 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 heavy and light chains of the anti- C5 antibody or antigen binding fragment that promote the expression of the antibody or antigen binding fragment in the cells, in embodiments, in the ocular tissue cells.
  • regulatory elements including constitutive promoters, such as CAG, and ocular tissue-specific regulatory elements, are provided in Table 1 and Table la and in Example 13 (including certain modified CAG promoters) herein.
  • 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 anti-C5 scFv is present at therapeutically effective levels in the serum or in ocular tissues of said human subject.
  • nucleic acids e.g., polynucleotides
  • 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 C5-D-mab and C5-A-mab heavy and light chains are provided in Table 8 (SEQ ID NOs: 205 and 206). Useful signal sequences for the expression of the scFv of the therapeutic antibodies in human cells are disclosed herein, for example in Tables 2 and 3. Exemplary recombinant expression constructs are shown in FIGS. 1A, IB and 1C
  • HuPTM mAb or HuPTM Fab 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 full-length HuPTM mAh or HuPTM Fab or other antigen binding fragment, such as an scFv, of a therapeutic mAb, to a patient (human subject) diagnosed with a disease indication for that mAb or protein, to create a permanent depot in the subject that continuously supplies the human-glycosylated, sulfated transgene product produced by the subject’s transduced cells.
  • the cDNA construct for the HuPTM mAb or HuPTM Fab or HuPTM scFv should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • 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 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.
  • Combination therapies involving delivery of the full-length HuPTM mAb or HuPTM Fab or HuPTM scFv or antigen binding fragment thereof 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 therapeutic mAb.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • 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 antibody 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 genes 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.
  • Viral vectors or other DNA expression constructs encoding an anti-C5 scFv, particularly a HuGlyFab or HuGlyscFv, or a hyperglycosylated derivative of a HuPTM mAb antigenbinding fragment 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.
  • 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.
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes the C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203) or C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197) 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), Chicken Betaactin promoter-chicken beta actin intron-rabbit beta-globin splice acceptor) with mutations (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31), cytomegalovirus (CMV) promoter, EF-1 alpha promoter (SEQ ID NO: 4), mUla (SEQ ID NO: 3), UB6 promoter, chicken beta-actin (CBA) promoter, and ocular-tissue specific promoters, such as human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 5 or 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS:
  • the expression cassette (promoter to poly A) comprises the nucleotide sequence of SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 278, or SEQ ID NO: 279.
  • nucleic acids e.g., polynucleotides
  • 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).
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) one or more control elements, b) optionally, a chicken 0-actin or other intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for C5-D-mab Vectorized scFv mAb (H-L) (having an amino acid sequence of SEQ ID NO: 195 or 203) or C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197). Exemplary constructs are shown in FIGS. 1A, IB and 1C and are provided in Table 8.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) GRK1 promoter (SEQ ID NO: 5), b) optionally, a VH4 intron (SEQ ID NO: 8) or other intron and c) a rabbit P-globin polyA signal (SEQ ID NO: 6); and (3) nucleic acid sequences coding for C5-D-mab Vectorized scFv mAb (H-L) (having an amino acid sequence of SEQ ID NO: 195 or 203).
  • the artificial genome comprises the nucleotide sequence of SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 276, or SEQ ID NO: 277.
  • 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, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAVhu21, AAV.hul2, or AAVhu26.
  • AAV based vectors provided herein are or comprise components from one or more of AAV8, AAV3B, AAV9, AAV10, AAVrh73, or AAVrhlO serotypes.
  • viral vectors in which the capsid protein is a variant VP1 protein of AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249) (see Table A).
  • the capsid protein is a variant of the AAV8 capsid protein (SEQ ID NO:260), AAV3B capsid protein (SEQ ID NO:254), or AAVrh73 capsid protein (SEQ ID NO:266), and the capsid protein is e.g., at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO:260), AAV9 (SEQ ID NO: 261), AAV3B capsid protein (SEQ ID NO:254), or AAVrh73 capsid protein (SEQ ID NO:266), while retaining the biological function of the native capsid.
  • the encoded AAV capsid has the sequence of SEQ ID NO: 260 with
  • FIG. 3 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,
  • 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, AAVrhlO, AAV.rh20, AAVrh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAVHSC1, AAV.HSC2, AAV.HSC3,
  • 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, AAVrhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAVRh74, AAVRHM4-1, AAV.hu37, AAV.Anc80, AAVAnc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVHSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAVHSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAVHS
  • 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, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAVAnc80, rAAVAnc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAVHSC4, AAV.HSC5, AAV.HSC
  • 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...
  • 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 PatNos. 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 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.
  • 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 e.g., AAV8, AAV3B, AAVrh73, or AAVrhl0
  • a transgene e.g, an HuPTM Fab or HuPTM scFv or protein.
  • the amino acid sequences of AAV capsids, including AAV8, AAV3B, AAVrh73 and AAVrhlO are provided in FIG. 3.
  • the AAV vector comprises an engineered capsid protein that comprises a 7 to 9 amino acid peptide that homes to or targets ocular tissue, or enhances transduction and/or integration of the viral genome, and is inserted into an AAV8 or AAV3B capsid protein.
  • the AAV type may be, for example, AAV3B.455.DLMLPGS (capsid protein having an amino acid sequence of SEQ ID NO: 243), AAV3B.455.DVTPLLS (capsid protein having an amino acid sequence of SEQ ID NO: 244), AAV3B.455.LVSVSLP (capsid protein having an amino acid sequence of SEQ ID NO: 245), AAV8.456.RIQMGTK (capsid protein having an amino acid sequence of capsid protein SEQ ID NO: 246), AAV8.456.RQKNAMV (capsid protein having an amino acid sequence of SEQ ID NO: 247), AAV8.590.GDNTTFRRA (capsid protein having an amino acid sequence of SEQ ID NO:
  • 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 scFv.
  • 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 stuffer sequences to keep the genome close to wildtype 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.
  • a vector for use in the methods described herein is one that encodes an HuPTM mAb, such that, upon introduction of the vector into a relevant cell, a glycosylated and/or tyrosine sulfated variant of the HuPTM mAb is expressed by the cell.
  • 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 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.
  • the promoter is a constitutive promoter.
  • 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 (SEQ ID NO: 2) or CB7 promoter (SEQ ID NO: 1) or a mutated CAG promoter (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31) includes other expression control elements that enhance expression of the transgene driven by the vector.
  • the other expression control elements include chicken P-actin intron and/or rabbit P-globin polyA signal (SEQ ID NO: 6).
  • the promoter comprises a TATA box.
  • 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 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 16-18), or a human red opsin (RedO) promoter (SEQ ID NO: 14).
  • 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 HIF -2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG (SEQ ID NO: 275) motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schodel, 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 hypoxiainducible 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.
  • 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: 8), SV40 intron (SEQ ID NO: 27), or a chimeric intron (P-globin/Ig Intron) (SEQ ID NO: 7). The viral vectors may also include a Kozak sequence to promote translation of the transgene product, for example GCCACC (SEQ ID NO: 28).
  • VH4 intron SEQ ID NO: 8
  • SV40 intron SEQ ID NO: 27
  • a chimeric intron P-globin/Ig Intron
  • the viral vectors may also include a Kozak sequence to promote translation of the transgene product, for example GCCACC (SEQ ID NO: 28).
  • 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: 6), 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.
  • the vectors provided herein comprise components that modulate protein delivery.
  • 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”.
  • the signal peptides allow for the transgene product to achieve the proper packaging e.g., glycosylation) in the cell.
  • the signal peptides allow for the transgene product to achieve the proper localization in the cell.
  • the signal peptides allow for the transgene product to achieve secretion from the cell.
  • a signal sequence for protein production in a gene therapy context or in cell culture 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.
  • a human antibody signal peptide may be used to express IgGs in CHO or other cells.
  • Another approach is to identify signal peptides optimized for the particular host cells used for expression. 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.
  • the signal peptide of human albumin the most abundant protein in plasma, was found to substantially increase protein production yield in CHO cells.
  • the signal peptide 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.
  • the signal sequence is fused to both the heavy and light chain sequences.
  • one signal sequence is present in the transgene and is fused to whichever sequence (heavy or light chain) is at the N-terminus of the transgene.
  • MYRMQLLLLIALSLALVTNS SEQ ID NO: 55
  • SEQ ID NO: 56 SEQ ID NO: 56
  • signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the HuPTM mAb or Fab or scFv in the eye/CNS, muscle, or liver are provided in Tables 2, 3, and 4, respectively, 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 NOS:9 (5 ’-ITR) or 11 (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 NOS:9 (5’-ITR) or 11 (3’-ITR) or modified for scAAV, SEQ ID NO: 10 (m 5’ITR) or SEQ ID NO: 12 (m 3’ ITR).
  • transgenes encode an scFv disclosed herein.
  • the transgenes encode a full length heavy chain (including the heavy chain variable domain, the heavy chain constant domain 1 (CHI), the hinge and Fc domain) and a full length light chain (light chain variable domain and light chain constant domain) that upon expression associate to form antigen-binding antibodies with Fc domains.
  • the recombinant AAV constructs express the intact (i.e., full length) or substantially intact HuPTM mAb in a cell, cell culture, or in a subject.
  • the nucleotide sequences encoding the heavy and light chains may be codon optimized for expression in human cells and have reduced incidence of CpG dimers in the sequence to promote expression in human cells. See for example, the codon optimized sequences of C5-D-mab (SEQ ID NOs: 205, 206, 214, or 215) or C5-A-mab (SEQ ID NO: 207-209, or 216-218) of Table 8.
  • the transgenes encode a full-length form of any of the therapeutic antibodies disclosed herein, for example, the Fab fragment of which depicted in FIGS. 2A-2E herein and including, in certain embodiments, the associated Fc domain provided in Table 6.
  • the full length mAb encoded by the transgene described herein preferably have the Fc domain of the full-length therapeutic antibody or is an Fc domain of the same type of immunoglobulin as the therapeutic antibody to be expressed.
  • the Fc region is an IgG Fc region, but in other embodiments, the Fc region may be an IgA, IgD, IgE, or IgM.
  • the Fc domain is preferably of the same isotype as the therapeutic antibody to be expressed, for example, if the therapeutic antibody is an IgGl isotype, then the antibody expressed by the transgene comprises an IgGl Fc domain.
  • the antibody expressed from the transgene may have an IgGl, IgG2, IgG3 or IgG4 Fc domain.
  • the Fc region of the intact mAb has one or more effector functions that vary with the antibody isotype.
  • the effector functions can be the same as that of the wild-type or the therapeutic antibody or can be modified therefrom to add, enhance, modify, or inhibit one or more effector functions using the Fc modifications disclosed in Section 5.1.9, infra.
  • the HuPTM mAb transgene encodes a mAb comprising an Fc polypeptide comprising 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 the Fc domain polypeptides of the therapeutic antibodies described herein as set forth in Table 6 for C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab, or an exemplary Fc domain of an IgGl, IgG2 or IgG4 isotype as set forth in Table 6.
  • the HuPTM mAb comprises a Fc polypeptide of a sequence that is a variant of the Fc polypeptide sequence in Table 6 in that the sequence has been modified with one or more of the techniques described in Section 5.1.9, infra, to alter the Fc polypeptide’s effector function.
  • the transgene encodes a surrogate antibody for use in NHP or other animal models, for example the antibody BB5.1 as a C5 binding surrogate for at least C5-A-mab or C5-C-mab.
  • constructs comprising a transgene that encodes an scFv that bind to C5.
  • the transgene encodes an scFv comprising an amino acid sequence of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, or SEQ ID NO: 204.
  • the nucleotide sequence encoding the anti-C5 scFv is SEQ ID NO: 224, SEQ ID NO: 227, or SEQ ID NO: 230.
  • nucleic acids comprising nucleotide sequence encoding an anti-C5 scFv, including having an amino acid sequence of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO: 203, or SEQ ID NO:204.
  • exemplary recombinant AAV constructs such as the constructs shown in FIG. 1A, for gene therapy administration to a human subject in order to express an intact or substantially intact HuPTM mAb in the subject.
  • Gene therapy constructs are designed such that both the heavy and light chains are expressed in tandem from the vector including the Fc domain polypeptide of the heavy chain.
  • the transgene encodes a transgene with heavy and light chain Fab fragment polypeptides as shown in Table 7, yet have a heavy chain that further comprises an Fc domain polypeptide C terminal to the hinge region of the heavy chain (including an IgGl, IgG2 or IgG4 Fc domain or the C5-D-mab, C5-A-mab, C5-C-mab, C5-B- mab as in Table 6).
  • the transgene is a nucleotide sequence that encodes the following: Signal sequence-heavy chain Fab portion (including hinge region)-heavy chain Fc polypeptide-Furin-2A linker-signal sequence-light chain Fab portion.
  • 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 P- actin intron or VH4 intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the heavy chain Fab of an anti-C5 mAb (e.g.
  • an Fc polypeptide associated with the therapeutic antibody (Table 6) or of the same isotype as the native form of the therapeutic antibody, such as an IgG isotype amino acid sequence from Table 6; and the light chain of an anti-C5 mAb (e.g.
  • 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 P-actin intron or VH4 intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding an scFv in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker, such as GGGGSGGGGSGGGGS (SEQ ID NO: 128).
  • the construct expresses, from the N-terminus, NFF-signal sequence- V L -linker-V H -COOH or NFF-signal sequence- V H -linker-V L - COOH.
  • the construct encodes, from the N-terminus, NFF-signal sequence- V L -GGGGSGGGGSGGGGS-VH-COOH or NFL-signal sequence- V H - GGGGSGGGGSGGGGS-V L - COOH.
  • the linker is GGGGS (SEQ ID NO: 126), GGGGSGGGGS (SEQ ID NO: 127), GGGGSGGGGSGGGGS (SEQ ID NO: 128), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 129) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 130).
  • the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.
  • the VH is SEQ ID NO: 184 and VL is SEQ ID NO: 185, wherein VH is SEQ ID NO: 186 and VL is SEQ ID NO: 187, wherein VH is SEQ ID NO: 188 and the VL is SEQ ID NO: 189, or wherein VH is SEQ ID NO: 190 and VL is SEQ ID NO: 191.
  • Exemplary constructs are provided in FIG. 1C and Table 7 (amino acid sequences).
  • polynucleotides comprising nucleotide sequences encoding the heavy and light chain variable regions are SEQ ID NO: 205 and SEQ ID NO: 206, or SEQ ID NO: 207 and SEQ ID NO: 209, or SEQ ID NO: 208 and SEQ ID NO: 209, or SEQ ID NO: 210 and SEQ ID NO: 211, or SEQ ID NO: 212 and SEQ ID NO: 213.
  • Exemplary constructs are provided in FIG. 1C and Table 8 (nucleotide sequences).
  • the nucleotide sequences encoding the scFvs are SEQ ID NO: 224, SEQ ID NO: 227 or SEQ ID NO: 230.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 260), or, alternatively, an AAV9, AAV3B, or AAVrh73 capsid (or a variant thereof) or alternatively AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249) (see Table A); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs),
  • the rAAV vectors that encode and express the full-length therapeutic antibodies 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 therapeutic antibodies, such as dry AMD. Also provided are methods of expressing HuPTM mAbs in human cells using the rAAV vectors and constructs encoding them.
  • the transgenes express antigen binding fragments, e.g. a Fab fragment (an HuGlyFab) or a F(ab’)2, nanobody, or an scFv based upon a therapeutic antibody disclosed herein.
  • FIGS. 2A-2E and section 5.4. provide the amino acid sequence of the heavy and light chains of the Fab fragments of the therapeutic antibodies (see also Table 7, which provides the amino acid sequences of the Fab heavy and light chains of the therapeutic antibodies).
  • constructs comprising a transgene that encodes an scFv that bind to C5.
  • the transgene encodes an scFv comprising an amino acid sequence of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, or SEQ ID NO: 204
  • the nucleotide sequence encoding the anti-C5 scFv is SEQ ID NO: 224, SEQ ID NO: 227, or SEQ ID NO: 230.
  • nucleotide sequences are codon optimized for expression in human cells. See for example, the codon optimized sequences encoding C5-D-mab (SEQ ID NOs: 205 and 206) or C5-A-mab (SEQ ID Nos: 207-209) in Table 8.
  • the transgene may encode a Fab fragment using nucleotide sequences encoding the amino acid sequences provided in Table 7, but not including the portion of the hinge region on the heavy chain that forms interchain di-sulfide bonds (e.g., the portion containing the sequence CPPCPA (SEQ ID NO: 132)).
  • Heavy chain Fab domain sequences that do not contain a CPPCP (SEQ ID NO: 131) sequence of the hinge region at the C-terminus will not form intrachain disulfide bonds and, thus, will form Fab fragments with the corresponding light chain Fab domain sequences, whereas those heavy chain Fab domain sequences with a portion of the hinge region at the C-terminus containing the sequence CPPCP (SEQ ID NO: 131) will form intrachain disulfide bonds and, thus, will form Fab2 fragments.
  • the transgene may encode a scFv comprising a light chain variable domain and a heavy chain variable domain connected by a flexible linker in between (where the heavy chain variable domain may be either at the N-terminal end or the C-terminal end of the scFv), and optionally, may further comprise a Fc polypeptide (e.g., IgGl, IgG2, IgG3, or IgG4) on the C-terminal end of the heavy chain.
  • a Fc polypeptide e.g., IgGl, IgG2, IgG3, or IgG4
  • the transgene may encode F(ab’)2 fragments comprising a nucleotide sequence that encodes the light chain and the heavy chain sequence that includes at least the sequence CPPCA (SEQ ID NO: 133) of the hinge region, as depicted in FIGS. 2A-2E which depict various regions of the hinge region that may be included at the C-terminus of the heavy chain sequence.
  • the hinge region is the sequence EPKSCDKTH (SEQ ID NO: 159).
  • Pre-existing anti-hinge antibodies (AHA) may cause immunogenicity and reduce efficacy.
  • C-terminal ends with D221 or ends with a mutation T225L or with L242 can reduce binding to AHA.
  • AHA See, e.g., Brezski, 2008, J Immunol 181: 3183-92 and Kim, 2016, 8: 1536-1547.
  • the risk of AHA is lower since the hinge region of IgG2 is not as susceptible to enzymatic cleavage required to generate endogenous AHA. (See, e.g., Brezski, 2011, MAbs 3: 558-567).
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or inducible (e.g., hypoxia-inducible or rifamycin- inducible) promoter sequence or a tissue specific promoter/regulatory region, for example, one of the regulatory regions provided in Table 1 or la, and b) a sequence encoding the transgene (e.g., a HuGlyFab or scFv).
  • the sequence encoding the transgene comprises multiple ORFs separated by IRES elements.
  • the ORFs encode the heavy and light chain domains of the HuGlyFab.
  • the sequence encoding the transgene comprises multiple subunits in one ORF separated by F/F2A sequences or F/T2A sequences. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain domains of the HuGlyFab separated by an F/F2A sequence or a F/T2A sequence. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain variable domains of the HuGlyFab separated by a flexible peptide linker (as an scFv).
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or an inducible promoter sequence or a tissue specific promoter, such as one of the promoters or regulatory regions in Table 1 or la, and b) a sequence encoding the transgene (e.g., a HuGlyFab), wherein the transgene comprises a nucleotide sequence encoding a signal peptide, a light chain and a heavy chain Fab portion separated by an IRES element.
  • a constitutive or an inducible promoter sequence or a tissue specific promoter such as one of the promoters or regulatory regions in Table 1 or la
  • a sequence encoding the transgene e.g., a HuGlyFab
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence or regulatory element listed in Table 1 or la, and b) a sequence encoding the transgene comprising a signal peptide, a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence (SEQ ID NOs: 119 or 120) or a F/T2A sequence (SEQ ID NOs: 117 or 118) or a flexible peptide linker.
  • a constitutive or a hypoxia-inducible promoter sequence or regulatory element listed in Table 1 or la and b) a sequence encoding the transgene comprising a signal peptide, a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence (SEQ ID NOs: 119 or 120) or a F/T2A sequence (SEQ ID NOs: 117 or 118) or a flexible peptide link
  • the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or an inducible promoter sequence or a tissue specific promoter or regulatory region, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene e.g., a HuGlyFab), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence.
  • the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or an inducible promoter sequence or a tissue specific regulatory region, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., HuGlyFab), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal, and wherein the transgene encodes a light chain and a heavy chain sequence separated by a cleavable F/2A sequence.
  • a first ITR sequence e.g., HuGlyFab
  • the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or an inducible promoter sequence or a tissue specific regulatory region, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene e.g., VH-(linker)-VL or VL-(linker)-VH), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence.
  • the transgenes encode full length or substantially full length heavy and light chains that associate to form a full length or intact antibody.
  • substantially intact or substantially full length refers to a mAb having a heavy chain sequence that is at least 95% identical to the full-length heavy chain mAb amino acid sequence and a light chain sequence that is at least 95% identical to the full-length light chain mAb amino acid sequence.
  • the transgenes comprise nucleotide sequences that encode, for example, the light and heavy chains of the Fab fragments including the hinge region of the heavy chain and C-terminal of the heavy chain of the Fab fragment, an Fc domain peptide.
  • Table 6 provides the amino acid sequence of the Fc polypeptides for C5-D-mab, C5-A-mab, C5-C-mab, and C5-B-mab.
  • an IgGl, IgG2, or IgG4 Fc domain the sequences of which are provided in Table 6 may be utilized.
  • Fc region refers to a dimer of two "Fc polypeptides” (or “Fc domains”), each "Fc polypeptide” comprising the heavy chain constant region of an antibody excluding the first constant region immunoglobulin domain.
  • an "Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers.
  • Fc polypeptide refers to at least the last two constant region immunoglobulin domains of IgA, IgD, and IgG, or the last three constant region immunoglobulin domains of IgE and IgM and may also include part or all of the flexible hinge N-terminal to these domains.
  • Fc polypeptide comprises immunoglobulin domains Cgamma2 (Cy2, often referred to as CH2 domain) and Cgamma3 (Cy3, also referred to as CH3 domain) and may include the lower part of the hinge domain between Cgammal (Cyl, also referred to as CHI domain) and CH2 domain.
  • the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting at T223 or C226 or P230, to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, Va.).
  • Fc polypeptide comprises immunoglobulin domains Calpha2 (Ca2) and Calpha3 (Ca3) and may include the lower part of the hinge between Calphal (Cal) and Ca2.
  • the Fc polypeptide is that of the therapeutic antibody or is the
  • the Fc polypeptide corresponding to the isotype of the therapeutic antibody).
  • the Fc polypeptide is an IgG Fc polypeptide.
  • the Fc polypeptide may be from the IgGl, IgG2, or IgG4 isotype (see Table 6) or may be an IgG3 Fc domain, depending, for example, upon the desired effector activity of the therapeutic antibody.
  • the engineered heavy chain constant region (CH), which includes the Fc domain is chimeric. As such, a chimeric CH region combines CH domains derived from more than one immunoglobulin isotype and/or subtype.
  • the chimeric (or hybrid) CH region comprises part or all of an Fc region from IgG, IgA and/or IgM.
  • the chimeric CH region comprises part or all a CH2 domain derived from a human IgGl, human IgG2, or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgGl, human IgG2, or human IgG4 molecule.
  • the chimeric CH region contains a chimeric hinge region.
  • the recombinant vectors encode therapeutic antibodies comprising an engineered (mutant) Fc regions, e.g. engineered Fc regions of an IgG constant region.
  • Modifications to an antibody constant region, Fc region or Fc fragment of an IgG antibody may alter one or more effector functions such as Fc receptor binding or neonatal Fc receptor (FcRn) binding and thus half-life, CDC activity, ADCC activity, and/or ADPC activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG heavy chain constant region without the recited modification(s).
  • the antibody may be engineered to provide an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits altered binding (as compared to a reference or wild-type constant region without the recited modification(s)) to one or more Fc receptors (e.g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRIV, or FcRn receptor).
  • Fc receptors e.g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRIV, or FcRn receptor.
  • the antibody an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits a one or more altered effector functions such as CDC, ADCC, or ADCP activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s).
  • Appector function refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcyR-mediated effector functions such as ADCC and ADCP and complement-mediated effector functions such as CDC. [0123] An “effector cell” refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions.
  • Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • nonspecific cytotoxic effector (immune) cells that express Fey Rs recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • ADCP antibody dependent cell-mediated phagocytosis
  • FcyRs cytotoxic effector cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • CDC or “complement-dependent cytotoxicity” refers to the reaction wherein one or more complement protein components recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • the modifications of the Fc domain include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an IgG constant region (see FIG. 5): 233, 234, 235, 236, 237, 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,
  • the Fc region comprises an amino acid addition, deletion, or substitution of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 of the IgG.
  • 251-256, 285-290, 308-314, 385-389, and 428-436 (EU numbering of Kabat; see FIG. 5) is substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine.
  • a non-histidine residue is substituted with a histidine residue.
  • a histidine residue is substituted with a non-histidine residue.
  • Enhancement of FcRn binding by an antibody having an engineered Fc leads to preferential binding of the affinity -enhanced antibody to FcRn as compared to antibody having wild- type Fc, and thus leads to a net enhanced recycling of the FcRn-affinity-enhanced antibody, which results in further increased antibody half-life.
  • An enhanced recycling approach allows highly effective targeting and clearance of antigens, including e.g. "high titer" circulating antigens, such as C5, cytokines, or bacterial or viral antigens.
  • modified constant region, Fc region or Fc fragment of an IgG antibody with enhanced binding to FcRn in serum as compared to a wild-type Fc region are engineered modifications.
  • antibodies e.g. IgG antibodies
  • antibodies, e.g. IgG antibodies are engineered to exhibit enhanced binding (e.g.
  • FcRn in endosomes e.g., at an acidic pH, e.g., at or below pH 6.0
  • a wildtype IgG and/or reference antibody binding to FcRn at an acidic pH as well as in comparison to binding to FcRn in serum (e.g., at a neutral pH, e.g., at or above pH 7.4).
  • an engineered antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits an improved serum or resident tissue half-life, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s);
  • Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., LN/Y/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y or A), including 428L and 434A; or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., LN/Y/W or T
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P) (EU numbering; see FIG 5).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • a 428L, 2591 e.g., V2591
  • 308F e.g.,
  • the Fc region can be a mutant form such as hlgGl Fc including M252 mutations, e.g. M252Y and S254T and T256E (“YTE mutation”) exhibit enhanced affinity for human FcRn (Dall’Acqua, et al., 2002, J Immunol 169:5171-5180) and subsequent crystal structure of this mutant antibody bound to hFcRn resulting in the creation of two salt bridges (Oganesyan, et al. 2014, IBC 289(11): 7812-7824).
  • Antibodies having the YTE mutation have been administered to monkeys and humans, and have significantly improved pharmacokinetic properties (Haraya, et al., 2019, Drug Metabolism and Pharmacokinetics, 34(1):25-41).
  • modifications to one or more amino acid residues in the Fc region may reduce half-life in systemic circulation (serum), however result in improved retainment in tissues (e g. in the eye) by disabling FcRn binding (e.g. H435A, EU numbering of Kabat) (Ding et al., 2017, MAbs 9:269-284; and Kim, 1999, Eur J Immunol 29:2819).
  • FcRn binding e.g. H435A, EU numbering of Kabat
  • the Fc domain may be engineered to activate all, some, or none of the normal Fc effector functions, without affecting the Fc polypeptide’s (e.g. antibody's) desired pharmacokinetic properties.
  • Fc polypeptides having altered effector function may be desirable as they may reduce unwanted side effects, such as activation of effector cells, by the therapeutic protein.
  • Methods to alter or even ablate effector function may include mutation(s) or modification(s) to the hinge region amino acid residues of an antibody.
  • IgG Fc domain mutants comprising 234A, 237A, and 238S substitutions, according to the EU numbering system, exhibit decreased complement dependent lysis and/or cell mediated destruction.
  • Deletions and/or substitutions in the lower hinge e.g. where positions 233-236 within a hinge domain (EU numbering) are deleted or modified to glycine, have been shown in the art to significantly reduce ADCC and CDC activity.
  • the Fc domain is an aglycosylated Fc domain that has a substitution at residue 297 or 299 to alter the glycosylation site at 297 such that the Fc domain is not glycosylated.
  • Such aglycosylated Fc domains may have reduced ADCC or other effector activity.
  • Non-limiting examples of proteins comprising mutant and/or chimeric CH regions having altered effector functions, and methods of engineering and testing mutant antibodies, are described in the art, e.g. K.L. Amour, et al., Eur. J. Immunol. 1999, 29:2613-2624; Lazar et al., Proc. Natl. Acad. Sci. USA 2006, 103:4005; US Patent Application Publication No. 20070135620A1 published June 14, 2007; US Patent Application Publication No. 20080154025 Al, published June 26, 2008; US Patent Application Publication No. 20100234572 Al, published September 16, 2010; US Patent Application Publication No. 20120225058 Al, published September 6, 2012; US Patent Application Publication No.
  • the DNA encoding the C-terminal lysine (-K) or glycine-lysine (-GK) of the Fc terminus can be deleted to produce a more homogeneous antibody product in situ.
  • 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 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 CsCk 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.
  • Vero-E6 cells a cell line derived from the kidney of an African green monkey, or HeLa cells engineered to stably express the ACE2 receptor (HeLa-ACE2), can be used to assess neutralization activity of transgenes expressed from a vector described herein.
  • glycosylation and tyrosine sulfation patterns associated with the HuGlyFab can be determined, for example determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlyFab. Glycosylation patterns and methods of determining the same are discussed in Section 5.3, while tyrosine sulfation patterns and methods of determining the same are discussed in Section 5.3.
  • benefits resulting from glycosylation/sulfation of the cell-expressed HuGlyFab can be determined using assays known in the art, e.g., the methods described in Section 5.3.
  • 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.
  • 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.
  • 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.
  • 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 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 PLURON1CSTM 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
  • 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
  • the pharmaceutical composition comprises viscosity promoting agent(s).
  • Example formulations for the delivery of AAV include those in WO 2022/076549 and WO 2021/071835, each of which is hereby incorporated by reference in their entireties.
  • the reference pharmaceutical composition comprises 1% carboxymethyl cellulose high viscosity grade. In some embodiments, the reference pharmaceutical composition comprises 0.2 to 15% carboxymethyl cellulose (CMC) high viscosity grade, CMC high viscosity grade, CMC medium viscosity grade, hydroxypropyl methyl cellulose (HPMC), HPMC, hydroxyethyl cellulose (HES), CMC low viscosity grade, and/or poloxamer 407.
  • CMC carboxymethyl cellulose
  • HPMC hydroxypropyl methyl cellulose
  • HES hydroxyethyl cellulose
  • the pharmaceutical composition comprises hyaluronic acid as a viscosity promoting agent.
  • the hyaluronic acid comprises a molecular weight of from about 1 MDa to about 2 MDa.
  • the hyaluronic acid can comprise a molecular weight of, for example, about 1 MDa, about 1.5 MDa, about 1.58 MDa, or about 2.0 MDa.
  • the hyaluronic acid comprises a molecular weight of about 1.58 MDa.
  • the hyaluronic acid comprises a molecular weight of about 2.0 MDa.
  • the HA may be obtained from various sources including ThermoFisher, Lifecore Biomedical, and the like, and is pharmaceutical grade HA.
  • Hyaluronic acid is also known as Sodium hyaluronate [CAS No. 9067-32- 7, Formula: (Ci4H2oNOnNa)n], Various sources include but are not limited to the list in Table B.
  • the pharmaceutical formulation comprises from about 0.5% w/v to about 1.0% w/v hyaluronic acid. In embodiments, the pharmaceutical formulation comprises from about 0.6% w/v to about 0.9% w/v hyaluronic acid. In embodiments, the pharmaceutical formulation comprises from about 0.7% w/v to about 0.8% w/v hyaluronic acid.
  • the pharmaceutical formulation comprises about 0.5% w/v hyaluronic acid, about 0.6% w/v hyaluronic acid, about 0.7% w/v hyaluronic acid, about 0.8% w/v hyaluronic acid, about 0.9% w/v hyaluronic acid or about 1.0% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 40.0 mg/mL (4% w/v) sucrose, and about 0.001% (0.01 mg/mL) poloxamer 188.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.5% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.6% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.7% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.8% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 0.9% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 25.0 mg/mL (2.5% w/v) sucrose, about 0.002% (0.02 mg/mL) poloxamer 188 and about 1.0% w/v hyaluronic acid.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 40.0 mg/mL (4% w/v) sucrose, and about 0.001% (0.01 mg/mL) poloxamer 188.
  • the AAV is in a pre-formulated solution at: about 0.2 mg/mL potassium chloride, about 0.2 mg/mL potassium phosphate monobasic, about 5.84 mg/mL sodium chloride, about 1.15 mg/mL sodium phosphate dibasic anhydrous, about 40.0 mg/mL (4% w/v) sucrose, about 0.001% (0.01 mg/mL) poloxamer 188, and about 1% carboxymethylcellulose (CMC) high viscosity grade.
  • CMC carboxymethylcellulose
  • methods for treating dry AMD (age-related AMD) or dry AMD with geographic atrophy, or other indications that can be treated with an anti-C5 antibody in a subject in need thereof comprising the administration, including ocular administration, of recombinant AAV particles comprising an expression cassette encoding an C5-D-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 195 or 203), C5-D-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 196), C5-A-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 197), C5-A-mab Vectorized scFv mAb (L-H) (SEQ ID NO: 198), C5- C-mab Vectorized scFv mAb (H-L) (SEQ ID NO: 198), C5- C-mab Vectorized scFv mAb (H-
  • a subject in need thereof includes a subject 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 an anti-C5 antibody.
  • Subjects to whom such gene therapy is administered can be those responsive to anti-C5 antibody e.g., C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab antibody.
  • the methods encompass treating patients who have been diagnosed with dry AMD, and, in certain embodiments, identified as responsive to treatment with an anti-C5 antibody, or considered a good candidate for therapy with an anti-C5 antibody.
  • the patients have previously been treated with an anti-C5 antibody.
  • the anti-C5 antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • kits for treating dry AMD or other indication amenable to treatment with an anti-C5 antibody 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 C5-D-mab scFv (HL) or C5-A-mab scFv (HL) 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 a HuPTM form of mAb or antigen-binding fragment
  • 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
  • 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 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 16-18), a human red opsin (RedO) promoter (SEQ ID NO: 14), a CAG promoter (SEQ ID NO: 2), a mutated CAG promoter (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31), a CB promoter or CB Long promoter (SEQ ID NO: 24 or 25) or a Bestl/GRKl tandem promoter (SEQ ID NOS
  • the artificial genome comprises one of the nucleotide sequences of SEQ ID NO 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 276, or SEQ ID NO: 277.
  • the vector is self-complementary. In embodiments, the vector is single stranded.
  • 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.
  • treating a subject with the AAV vectors and pharmaceutical compositions disclosed herein can reduce the likelihood of vision threatening events.
  • Vision threatening events include a number of vision-threatening complications, such as geographic atrophy lesions, geographic atrophy lesion progression and/or growth.
  • the methods 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 HuPTM mAb or the antigen-binding fragment thereof, such as HuPTM Fab, that bind to C5 (and inhibit complement activation) derived from an anti-C5 antibody and indicated for treating dry AMD.
  • the HuPTM mAb has the amino acid sequence of C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab or an antigen binding fragment thereof.
  • the amino acid sequence of Fab fragment of these antibodies is provided in FIGS. 2A-2E (see also Table 7, which also provides amino acid sequences of certain full length and scFv constructs).
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an anti-C5 HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) 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.
  • a viral vector or other DNA expression construct encoding an anti-C5 HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) 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.
  • the artificial genome is self-complementary.
  • the construct or artificial genome may comprise or consist of the nucleotide sequence of SEQ ID NOs: 225, 229, 232, 234, 236, 238, 242, 276, or 277.
  • the artificial genome may comprise of the nucleotide sequence of any one of SEQ ID NOs: 225, 229, 232, 234, 236, 238, 242, 276, or 277.
  • 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: 225, 229, 232, 234, 236, 238, 242, 276, or 277, which encodes and expresses a C5 antibody or antigen binding fragment thereof as described herein.
  • Table 7 provides the amino acid sequences of Fab, scFv and full length heavy and light chains of the anti-C5 antibodies, and the expression products of the transgenes, including the signal sequences and linkers, such as Furin/T2a linkers.
  • Table 8 provides a nucleotide sequence encoding the Fab and full length heavy and light chains of the antibodies, transgene coding sequences, and artificial genomes disclosed herein.
  • kits for treating human subjects for dry AMD by administration of a viral vector containing a transgene encoding an anti-C5 scFv which has an amino acid sequence of SEQ ID NO: 195, 196, 197, 198, 199, 200, 201, or 202.
  • methods of treating human subjects for dry AMD by administration of a viral vector containing a transgene encoding an anti-C5 scFv which has an amino acid sequence of one of SEQ ID NOs: 195, 196, 197, 198, 199, 200, 201, 202, 203, or 204.
  • the scFv may be encoded by the nucleotide sequence of SEQ ID NO: 224, 227, or 230.
  • the scFv may include a signal peptide and, in embodiments, has an amino acid sequence of SEQ ID NO: 203.
  • the viral vector has an AAV capsid with tropism for human ocular tissues and comprises a capsid protein of AAV3B.455.DLMLPGS (SEQ ID NO: 243), AAV3B.455.DVTPLLS (SEQ ID NO: 244), AAV3B.455.LVSVSLP (SEQ ID NO: 245), AAV8.456.RIQMGTK (SEQ ID NO: 246), AAV8.456.RQKNAMV (SEQ ID NO: 247), AAV8.590.GDNTTFRRA (SEQ ID NO: 248), or AAV8.590.GRTIRGDLA (SEQ ID NO: 249) (see Table A).
  • the engineered capsids exhibit superior transgene product expression in retinal and RPE-choroid tissue, as well as aqueous and vitreous humor, following suprachoroidal delivery of an rAAV vector comprising any one of the capsid proteins of SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, or SEQ ID NO: 249, compared to a corresponding parental capsid.
  • 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 (SEQ ID NO: 2) promoter or a mutated CAG promoter (SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31), or ocular specific promoter, such as a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS:5 or 19), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 16-18), a human red opsin (RedO) promoter (SEQ ID NO: 14) or a Bestl/GRKl tandem promoter (SEQ ID NO: 26).
  • a CAG SEQ ID NO: 2
  • a mutated CAG promoter SEQ ID NO: 29, SEQ ID NO: 30 or S
  • Regulatory sequences may also include polyadenylation signal sequences and introns.
  • 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 self-complementary AAV (scAAV) genome.
  • the recombinant vectors including those as shown in FIGS. 2A-2E, 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) CAG.C5-D-mab.scFv (SEQ ID NO: 225) or CAG.C5-A-mab.scFv (SEQ ID NO: 276).
  • the recombinant AAV comprises an artificial genome of (or is produced using a cis plasmid or construct comprising) C5-D-mab.scFv (SEQ ID NO: 224 or SEQ ID NO: 227 or SEQ ID NO: 230).
  • the expression cassette (promoter to poly A) comprises SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 278, or SEQ ID NO: 279.
  • the artificial genome comprises SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 276, or SEQ ID NO: 277.
  • the artificial genome is self- complementary.
  • the transgene encoding an anti-C5 scFv is for a C5-D scFv, a C5-A scFv, a C5-C scFv, or a C5-D scFv.
  • the anti-C5 scFv has an amino acid sequence of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, or SEQ ID NO: 202.
  • transgene which is a scFv.
  • the transgene encodes an scFv with the structure: signal sequence - VH - linker - VL - polyA.
  • the transgene encodes an scFv with the structure: signal sequence - VL - linker - VH - poly A.
  • the linker is GGGGS (SEQ ID NO: 126), GGGGSGGGGS (SEQ ID NO: 127), GGGGSGGGGSGGGGS (SEQ ID NO: 128), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 129) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 130).
  • the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.
  • the VH is SEQ ID NO: 184 and VL is SEQ ID NO: 185, wherein VH is SEQ ID NO: 186 and VL is SEQ ID NO: 187, wherein VH is SEQ ID NO: 188 and the VL is SEQ ID NO: 189, or wherein VH is SEQ ID NO: 190 and VL is SEQ ID NO: 191.
  • AAV vectors comprising an AAV capsid comprising a viral capsid protein; wherein the viral capsid protein is AAV8.456.RQKNAMV (SEQ ID NO: 247) and an artificial genome comprising SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 276, or SEQ ID NO: 277, or an artificial genome that is 95%, 96%, 97%, 98%, or 99% identical thereto and encodes a functional anti-C5 scFv.
  • AAV8.456.RQKNAMV SEQ ID NO: 247
  • an artificial genome comprising SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240,
  • Therapeutically elfective 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.
  • suprachoroidal administration results in expression of the transgene product in cells of the eye, creating a depot in one or more ocular tissue cells such as RPE or photoreceptor cells of the patient that continuously supplies the anti-C5 scFv to ocular tissues of the subject.
  • the transgene expression results in therapeutically effective levels of the anti-C5 scFv in the aqueous humor, the vitreous humor, retinal tissue, the RPE, the BrM or choriocapillaris.
  • a method of suprachoroidal administration for treating a pathology of the eye comprising administering to the suprachoroidal space in the eye of a human subject in need of treatment a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product such that the therapeutic product is expressed and results in treatment of the pathology of the eye.
  • the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device.
  • the suprachoroidal drug delivery device comprises a microneedle.
  • the suprachoroidal drug delivery device is a microinjector.
  • SC suprachoroidal space
  • scleral flap technique catheters and standard hypodermic needles, as well as with microneedles.
  • a hollow-bore microneedle with a length matched to the thickness of the sclera, for example a 900 pm-long or 1100 pm-long microneedle (SCS Microinjector®, Clearside Biomedical, Inc.) inserted in the eye has been shown to deliver therapeutic agents to the posterior segment of the eye via the suprachoroidal space (Rai UDJ, et al. The suprachoroidal pathway: a new drug delivery route to the back of the eye. Drug Discov Today.
  • Recombinant AAV vector compositions such as the rAAV compositions described herein, achieve delivery of the transgene to the posterior segment of the eye, including retinal cells, RPE and photoreceptor cells, via suprachoroidal administration (Ewing, TM, et al. 2022 Retinal Physician 19:24-26).
  • a method of administering an rAAV vector composition comprises inserting a needle, e.g. a microneedle, into the suprachoroidal space of the eye without the needle penetrating through the choroid (“suprachoroidal administration or injection”).
  • rAAV vector compositions can be injected into the eye using a microneedle, typically locating the needle approximately 4-4.5 mm from the limbus, into the pars plana.
  • the microneedle is inserted into the sclera at a perpendicular angle, then the needle hub is depressed to create a sealing gasket effect, then the injection of a pharmaceutical composition comprising the rAAV vector is performed over 5-10 seconds.
  • the administration step is performed under local anesthesia (e.g. anesthesia administered before the suprachoroidal injection).
  • topical steroid or other anti-inflammatory may be applied to the eye before or after insertion of the needle.
  • Oxular Limited is developing a SCS device and delivery system (Oxulumis) that advances an illuminated cannula in the suprachoroidal space.
  • the Orbit device (Gyroscope) is a specially-designed system enabling cannulation of the suprachoroidal space with a flexible cannula (for example, the flexible cannula is inserted between the sclera and the choroid).
  • a microneedle inside the cannula is advanced into the subretinal space to enable targeted dose delivery.
  • Ab interno access to the SCS can also be achieved using micro-stents, which serve as minimally-invasive glaucoma surgery (MIGS) devices.
  • MIGS minimally-invasive glaucoma surgery
  • Examples include the CyPass® Micro-Stent (Alcon, Fort Worth, Texas, US) and i Stent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS to drain the aqueous humor without forming a filtering bleb.
  • the suprachoroidal drug delivery device is a syringe with a 1 millimeter 30 gauge needle.
  • the syringe has a larger circumference (e g., 29 gauge needle).
  • a microneedle or syringe is used to administer the rAAV compositions described herein.
  • a microneedle or syringe comprises a needle having an effective length of about 2000 microns or less.
  • a microneedle or syringe comprises a needle having an effective length between about 500 microns to about 2000 microns, or between about 800 microns to about 1200 microns.
  • a microneedle or syringe comprises a needle having an effective length of about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1000 pm, about 1100 pm, or about 1200 pm.
  • a microneedle or syringe is selected based on the viscosity of a pharmaceutical composition (e.g., liquid formulation). In some embodiments, a microneedle is selected based on the pressure resulted in the eye (e.g., in the SCS) when a pharmaceutical composition (e.g., liquid formulation) is administered.
  • a pharmaceutical composition e.g., liquid formulation
  • a pharmaceutical composition having medium or high viscosity may benefit from the use of a wider microneedle for injection.
  • the pressure in the SCS is lower when a wider microneedle is used as compared to the pressure obtained when a narrower microneedle is used.
  • a 27 gauge needle is used. In some embodiments, a 28 gauge needle is used. In some embodiments, a 29 gauge needle is used. In some embodiments, a 30 gauge needle is used. In some embodiments, a 31 gauge needle is used. In some embodiments, a gauge that is smaller than a 27 gauge needle is used. In some embodiments, a gauge that is larger than a 27 gauge needle is used. In some embodiments, a gauge that is smaller than a 28 gauge needle is used. In some embodiments, a gauge that is smaller than a 30 gauge needle is used. In some embodiments, a gauge that is higher than a 30 gauge needle is used.
  • Devices used to carry out the methods described herein comprise any one of the devices disclosed in International Publication No. WO2011139713, International Publication No.
  • 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 an anti-C5 antibody, or considered a good candidate for therapy with an anti-C5 antibody.
  • the patients have previously been treated with C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab or other complement activation inhibitor, and have been found to be responsive to thereto.
  • the anti-C5 transgene product e.g., produced in cell culture, bioreactors, etc.
  • administration of the recombinant AAV comprising a construct for expressing a transgene encoding the C5-D-mab scFv (HL) or C5-A-mab scFv (HL) 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 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 C5-D-mab scFv (HL) or C5-A-mab scFv (HL) 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.
  • Combinations of delivery of the C5-D-mab scFv (HL) or C5-A-mab scFv (HL), 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 anti-C5 antibody.
  • 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 C5-D-mab scFv (HL) or C5-A-mab scFv (HL) for expression, therapeutic effect and adverse effects.
  • Animal models may include a humanized C3-/C5- rodent model or a NaIO3 induction rat or mouse model, or a CFH-/- mouse model.
  • Animals may be administered vectors described herein, for example, subretinally or suprachoroidally, 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 C5-D-mab Fab cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of C5-D-mab (amino acid sequences being SEQ ID NOs. 168 and 169, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain is the nucleotide sequence of SEQ ID NOs. 205 and 206, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:55).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 4, particularly, SEQ ID NO: 118 or 120) to create a bicistronic vector.
  • 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: 26), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • 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: 26)
  • an inducible promoter such as a hypoxia-inducible promoter
  • a C5-D-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the heavy and light chain sequences of C5-D-mab scFv and a linker sequence (amino acid sequences for VH-linker-VL and VL-linker-VH being SEQ ID NOs. 195, 196, and 203).
  • the nucleotide sequence coding for the scFv portion of the heavy and light chain is the nucleotide sequence of SEQ ID NO. 224, respectively.
  • the transgene comprises a nucleotide sequence encoding a linker sequence (amino acid sequence being SEQ ID NOs: 126, 127, 128, 129, and 130).
  • the transgene also comprises nucleotide sequences that encodes a interleukin 2 signal peptide (SEQ ID NO: 56) (amino acid sequence being MYRMQLLLLIALSLALVTNS (SEQ ID NO:55)).
  • 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 a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 26), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the artificial genome from the 5’ ITR to the 3’ ITR is shown in SEQ ID NO: 225 or 232.
  • An C5-A-mab Fab IgGl cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of C5-A-mab (amino acid sequences being SEQ ID NOs. 170 and 172, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain is the nucleotide sequence of SEQ ID NOs. 207 and 209, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:55).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 4, particularly, SEQ ID NO: 118 or 120) to create a bicistronic vector.
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • constitutive promoters such as mUla, EFla, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissuespecific promoter, particularly GRK1 promoter (SEQ ID NO:5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 26), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the sequence of the vector from the 5’ ITR to the 3’ ITR is shown in SEQ ID NO: 221.
  • An C5-A-mab Fab cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of C5-A-mab (amino acid sequences being SEQ ID NOs. 171 and 172, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain is the nucleotide sequence of SEQ ID NOs. 208 and 209, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:55).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 4, particularly, SEQ ID NO: 118 or 120) to create a bicistronic vector.
  • the vector additionally includes the constitutive promoter CAG (SEQ ID NO: 2).
  • constitutive promoters such as mUla, EFla, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissuespecific promoter, particularly GRK1 promoter (SEQ ID NO:5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 26), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the sequence of the vector from the 5’ ITR to the 3’ ITR is shown in SEQ ID NO: 222.
  • a C5-A-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding the heavy and light chain sequences of C5-A-mab scFv and a linker sequence (amino acid sequences for Vn-linker-VL and Vt-linker-Vn being SEQ ID NOs. 197 and 198, respectively). Additionally, the transgene comprises a nucleotide sequence encoding a linker sequence (amino acid sequence being SEQ ID NOs: 126, 127, 128, 129, and 130). The transgene also comprises nucleotide sequences that encodes a interleukin 2 signal peptide (SEQ ID NO: 56) (amino acid sequence being MYRMQLLLLIALSLALVTNS (SEQ ID NO:55)).
  • 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 a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO: 5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 26), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.
  • the artificial genome from the 5’ ITR to the 3’ ITR is shown in SEQ ID NO: 276 and 277.
  • EXAMPLE 4 C5-C-mab Fab cDNA-Based Vector
  • a C5-C-mab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of C5-C-mab (amino acid sequences may be SEQ ID NOs. 173 and 174, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 210 and 211, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:55).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 4, particularly, SEQ ID NO: 118 or 120) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 2), mUla, EFla, 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: 26), or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 2), mUla, EFla, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:5)
  • a C5-B-mab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of C5-B-mab (amino acid sequences being SEQ ID NOs. 175 and 176, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 212 and 213, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:55).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 4, particularly, SEQ ID NO: 118 or 120) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 2), mUla, EFla, a CB or CB long promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:5), or a BEST1/GRK1 tandem promoter (SEQ ID NO: 26), or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 2), mUla, EFla, a CB or CB long promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:5), or a BEST1/GRK1 tandem promoter (SEQ ID
  • Cis plasmids pITR-CAG-C5-A-mab.IgGl (SEQ ID NO: 221) or pITR-CAG-C5-D- mab (SEQ ID NO: 219) were transfected into HEK293 cells.
  • Supernatant and pellet samples were collected and run on a non-reducing gel (FIG. 6A), with a band representing full length antibodies observed in the supernatant of the cell culture, or run on a reducing gel (FIG. 6B), indicating heavy and light chains of the antibodies seen in both cell pellet and supernatant.
  • ANaIO3 induced model of dry AMD in rodents will be used to assess the anti-C3, anti-C5 antibodies and CFHL-1 protein AAV constructs.
  • AAV8 constructs AAV8.CAG.C5-D-mab.Fab (SEQ ID NO: 219), AAV8.CAG.C5-D-mab.full (SEQ ID NO:NO:220) , AAV8.C5-A-mab.Fab.IgGl (SEQ ID NO: 221), AAV8.C5-A-mab.Fab.IgG2 (SEQ ID NO: 222), and AAV8.C5-A-mab.full (SEQ ID NO: 223) will be administered to humanized C3-/C5- mice at a dose of 1E7, 1E8 or 1E9 subretinally or suprachoroi dally.
  • NaIO3 will be administered to the mouse to induce geographic atrophy.
  • the eyes of the mice will be assessed by fundus and for visual function deficits and then will be sacrificed and eyes assessed for inhibition of RPE damage and photoreceptor loss and for transgene, C3 and C5 levels.
  • 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 plasmids (encoding complement inhibitors as described herein) transfected into HEK293T cells.
  • the supernatants (containing the complement inhibitor, or negative controls containing media without inhibitor or containing a vectorized antibody 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
  • 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.
  • C5 inhibitors expressed in HEK293 cells suppress complement pathway activation in hemolysis inhibition assays at varying degrees.
  • the scFv formats displayed strong inhibition of complement (FIGS. 7A-B).
  • Recombinant purified forms of each C5 inhibitor displayed potent inhibition of complement activation in classical and alternative hemolysis assays (FIGS 8A-F).
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated Human Complement C5 Protein was firstly immobilized onto SA biosensor. C5-D-mab IgG and Ab fragments and Coversin was applied as analyte for association and dissociation steps. Table 9. Assay conditions for human C5 assay after optimization b. Affinity and kinetics for cynomolgus Complement C5:
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated cyno C5 antigen was firstly immobilized onto SA biosensor. C5-D-mab IgG and Ab fragments and a recombinant 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. BB5.1 IgG and Ab fragments were applied as analyte for association and dissociation steps. Table 11. Assay conditions for mouse C5 assay after optimization (surrogate anti-mouse C5 mAb)
  • the assay was performed at 30°C and at 1000 rpm. Biotinylated mouse C5 antigen was firstly immobilized onto SA biosensor. C5-D-mab IgG and Ab fragments and Coversin were applied as analyte for association and dissociation steps.
  • C5-D-mab All three formats of anti-human C5 inhibitor (C5-D-mab) demonstrated KD values for human and cyno C5 in the low to high picomolar range, whereas the C5 inhibitor bound less strongly with a low nanomolar affinity constant.
  • MAC Membrane attack complex
  • C5 inhibitors prevented C5 cleavage and reduced membrane attack complex (MAC) formation (FIGS. 9A-C, ARPE19; FIGS. 9D-9H, iPSC-derived RPE).
  • iPSC-derived RPE transduced with AAV.anti-hC5 scFV (C5-D-mab scFv) at increasing MOIs demonstrate a dose-dependent increase in transgene product (TP) level in apical and basal compartments (FIG. 9G).
  • the TP level (FIG. 9G) was consistent with mRNA/cDNA of AAV measured by ddPCR (FIG. 9H).
  • AAV8-encoding C5 inhibitors were injected into wild-type mouse eyes via subretinal (SR) administration at 1E8 and 3E8 vg/eye.
  • Anti-C5 scFV anti-hC5: C5-D-mab or anti-mC5: BB5.1
  • IgG and AAV.anti-hC5.Fab SR delivery showed similar TP level in the retina and RPE / Choroid / Sclera, AAV.anti-hC5.scFV had higher level of TP in the RPE/Choroid/Sclera suggesting better penetration of the anti-hC5.scFv to the outer ocular layers (FIG. IOC and FIG. 10D and Table 14).
  • One-time subretinal delivery of AAV yielded high levels of TP in the mouse eye.
  • CAG deletion mutants Del5, DelM and Del3 operably linked to a C5-D-mab scFv were made using standard molecular biology techniques. See FIG. 11.
  • CAG-Delm has about 60% of the promoter strength as full-length CAG. See FIG. 12.
  • the objective of this study is to evaluate the ocular tropism of up to two different adeno- associated virus (AAV) pools/libraries following suprachoroidal administration to female cynomolgus monkeys. See Table 15. Following dosing on Day 1, animals will be observed for 3 to 12 weeks for biodistribution sample collection to investigate the transduction protocol.
  • AAV adeno- associated virus
  • GC Genome copies a: Dose levels are based on a dose volume of 100 pl/eye b: Animals in Groups 1 and 2 will be designated as terminal sacrifice animals (based on survival). Procedures
  • Animals will be dosed lOOpl/eye via suprachoroidal injection on Day 1 of the dosing phase. The right eye will be dosed first. All post-dose collection times will be based on the time of the dosing of the left eye.
  • Ophthalmic examinations will be performed at least twice pre-dose. During the dosing phase, ophthalmic examinations will be performed on days 3, 8, 15, 17, 29, 31 , 42, 57, 59, and 85.
  • animals will be anesthetized with ketamine. Animals will be examined with a slit-lamp biomicroscope and indirect ophthalmoscope. The adnexa and anterior portion of both eyes will be examined using a slit-lamp biomicroscope. The ocular fundus of both eyes will be examined (where visible) using an indirect ophthalmoscope. Prior to examination with the indirect ophthalmoscope, pupils will be dilated with a mydriatic agent (e.g., 1% tropicamide).
  • a mydriatic agent e.g., 1% tropicamide
  • Intraocular pressure measurements will be performed in conjunction with ophthalmic examinations (OEs). On days of OEs, intraocular pressure measurements (lOPs) will be conducted on eyes that have been previously dilated. Intraocular pressure measurements will be performed at least twice pre-dose. During the dosing phase, ophthalmic examinations will be performed on days 3, 8, 15, 17, 29, 31, 42, 57, 59, and 85.
  • animals will be anesthetized with ketamine.
  • the IOP measurements will be done using an applanation tonometer.
  • a topical anesthetic e.g., 0.5% proparacaine
  • Spectral domain optical coherence tomography OCT or sdOCT will be performed at least once pre-dose. During the dosing phase, OCT will be performed once during weeks 2, 4, 8, and 12.
  • Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and maintained on sevoflurane. Pupils will be dilated with a mydriatic agent. OCT will be performed and the data will be evaluated. Imaging will be done in a manner to obtain axial views of the retinal surface in the posterior fundus. The instruments will be set to perform standard retinal scans (macular volume scans and/or line scans and/or circle scans). Additional methods or scans may be used. A 55 degree lens may be used, if necessary.
  • Ocular photography will be performed at least once pre-dose. During the dosing phase, ocular photography will be performed once during weeks 2, 4, 8, and 12.
  • Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and dexmedetomidine. Pupils will be dilated with a mydriatic agent. Photographs will be taken with a wide angle lens and a digital fundus camera. Color photographs will be taken of each eye to include stereoscopic photographs of the posterior pole and nonstereoscopic photographs of two midperipheral fields (temporal and nasal); additional images will also be taken superior temporal, if possible.
  • Fundus Autofluorescent Imagining will be performed at least once pre-dose. During the dosing phase, fundus autofluorescent imaging will be performed once during weeks 2, 4, 8, and 12.
  • Anti-AAV 2 and AAV8 neutralizing antibody analysis will be performed within 5 days of animal transfer pre-dose. During the dosing phase, anti-AAV 2 and AAV8 neutralizing antibody analysis will be performed prior to dosing on day 1 and on each day of scheduled sacrifice.
  • 2.4mL of blood will be taken from the femoral vein. An alternate site may be used if necessary, and the site of blood collection will be documented. Blood samples will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of collection, and serum will be harvested into two approximately equal aliquots. Following harvesting, samples will be placed on dry ice until stored in a freezer. Anti-AAV9 Total Antibody (TAB) Analysis
  • Anti-AAV9 total antibody analysis will be performed within 5 days of animal transfer pre-dose.
  • 2.4mL of blood will be taken from the femoral vein. An alternate site may be used if necessary, and the site of blood collection will be documented. Blood samples will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of collection, and serum will be harvested into two approximately equal aliquots. Following harvesting, samples will be placed on dry ice until stored in a freezer.
  • PBMCs Peripheral blood mononuclear cells
  • ELISPOT ELISPOT at least once pre-dose.
  • PBMCs will be isolated on days 15, 29, 57, and 85. Briefly, a 3mL blood sample will be collected from the femoral vein. An alternate site may be used if necessary.
  • Whole blood will be collected during the dosing phase on days 3, 8, 15, 29, 57, and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary.
  • Serum Blood samples for serum collection will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of collection, and serum will be harvested into 3 approximately equal aliquots. Following harvesting, whole blood and serum samples will be placed on dry ice until stored in a freezer.
  • Aqueous humor will be collected once during pre-dose. Aqueous humor will be collected once on days 15, 22, 29, 57 and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary.
  • Aqueous humor samples from each eye will be placed into separate tubes with Watson barcoded labels, snap frozen in liquid nitrogen, and placed on dry ice until stored in a freezer. Aqueous humor samples will be analyzed for transgene product. Blood Collection for Clinical Chemistry
  • Blood will be collected at least twice during pre-dose. Blood will be collected during the dosing phase on days 8, 15, 29, 57, and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary. 1 mL will be collected for hematology, 1.8 mL will be collected for coagulation and 1 mL will be collected for clinical chemistry. Tests are provided in Table 16.
  • All other ocular tissues will be collected as single samples (one sample/tube). The tissues will be rinsed with saline and blotted dry, as appropriate. Following collection, samples will be placed in separate tubes and flash-frozen with liquid nitrogen and stored on dry ice (unless immediately stored in a freezer).
  • Ocular fluids and tissues will be collected using ultra-clean procedures, according to Labcorp SOPs, in order to minimize the risk for potential contamination.
  • any work surfaces and non-disposable tools used will be cleaned with DNA Away Surface Decontaminant (Thermo Scientific, Catalog No. 7010 or equivalent) and RNAse decontamination solution (Invitrogen RNaseZap or equivalent) between animals.
  • a NalCh induced model of dry AMD in rodents was used to assess the anti-mouse C5 (mC5) antibody BB5.1.
  • Recombinant anti- mC5 antibody BB5.1 (group 1) and an irrelevant isotype matched (mouse IgGl) antibody (group 2) were administered to C57BL/6J mice at a dose of 1 mg intraperitoneally (i.p.) daily.
  • Table 19 Mice were evaluated on Day 0 for baseline electroretinography (ERG)/body weights (BW)/OCT, and then each administered a test antibody daily starting at Day 1 to Day 9.
  • H&E hematoxylin & eosin
  • RPE retinal pigment epithelium
  • 1HC retinal pigment epithelium
  • n 8 whole globes/group were snap frozen
  • n 8 whole globes were fixed in 4% paraformaldehyde (PFA).
  • ERGs were performed on both eyes using the Diagnosys ERG systems. Animals were dark adapted for a period of at least 12 hours prior to ERG. Under dark adaptation, eyes were dilated using a cocktail of tropicamide HC1 1% and phenylephrine HC1 2.5%. Before ERGs were recorded, pupil dilatation was checked to ensure full dilation. Animals were positioned on the ERG machine, then proparacaine HC1 0.5% and GenTeal were applied to the eyes, followed by the electrode contact and reference leads. Animals were placed on a warm water blanket to control body temperature and contact lens leads were placed on the eyes. A reference subcutaneous lead was placed in the head and a ground lead was placed near the tail of the animal. Light-adapted ERG with 30 cd/m2 background light immediately followed the dark-adapted series. There was a 5 minute light adaptation period before the light-adapted signals were collected. Background light was used constantly for all light- adapted measurements.
  • Step 1-9 0.001-10 cd*s/m2 scotopic ERG, each step advancing half log units in light intensity. Both ERG traces and oscillatory potentials were collected.
  • Step 10 Dark-adapted 150 cd/m2 scotopic c-wave measurement.
  • Step 1-3 1-10 cd*s/m2 photopic ERG, each step advancing half log units in light intensity. Both ERG traces and oscillatory potentials will be collected.
  • Step 4-7 3.0 c cd*s/m2 photopic ERG flicker ERG at 10-40 Hz, each step advancing 10 Hz.
  • the a-wave amplitude of Group 2 increased to -87 pV at 1.00 cd*s/m2.
  • the Day 10 a-wave amplitude in both groups remained generally within the 30-40 pV range at all light intensities.
  • Rods are a type of photoreceptor cell in the retina that are sensitive to light levels and provide vision in low light. Rods are concentrated in the outer areas of the retina and also provide peripheral vision.
  • group 1 the retinal lesions were multifocal and mild, while in group 2, there were moderate to severe and diffuse. Furthermore, there was severe retinal photoreceptor disruption and diffuse retinal thinning in group 2 eyes, not observed in group 1.
  • Eyes of group 1 were characterized in 3 out of 4 of animals with mild multifocal areas of RPE proliferation with migration into the retina, mild photoreceptor layer thinning, and retinal outer nuclear layer disorganization. Eyes of group 2 all had moderate to severe and diffuse areas of RPE proliferation with moderate to severe photoreceptor layer disruption, outer nuclear layer disorganization, and diffuse retinal thinning.
  • the observed lesions in group 1 were multifocal and mild, and in group 2, the lesions were diffuse and moderate to severe. Additionally, there was more severe retinal photoreceptor destruction and diffuse retinal thinning in group 2 eyes that was not observed in group 1 eyes.
  • mice will be evaluated for ERG/body weights (BW)/0CT analogously to the methods in Example 16A, and then each administered a test AAV vector at a dose of 3E13 vg/kg IV (Table 20). On Day 28, mice will again be evaluated for OCT/Fundus/ERG and NalOs administered to each mouse intravenously on Day 29 to induce an inflammatory event in the eye.
  • mice will be assessed (OCT imaging, ERG, etc.) and then sacrificed, and the eyes will be further assessed for inhibition of RPE damage and photoreceptor loss and for vector genome (DNA and RNA) and transgene product (TP) levels.
  • Table 21 Study Design [0261 ] Two eyes administered AAV8, two eyes administered AAV3B, and one untreated eye were fixed and processed for histopathological analysis, with tissues from the remaining eyes collected frozen for DNA/RNA biodistribution and transgene product analysis. These tissues included: vitreous humor, retina, RPE/choroid, sclera, trabecular meshwork, iris/ciliary body, cornea, lens, and optic nerve.
  • the posterior segment was dissected by cutting the posterior eye cup into quadrants and collecting a distal and a proximal strip from each quadrant. Each strip was then separated into retina, RPE/choroid, and sclera for analysis.
  • sample 3 and 4 The distal samples and the proximal samples from the temporal quadrants were each pooled (sample 3 and 4, respectively) for transgene product analysis (by ELISA), in addition to sample 5, which contained the macula.
  • Nasal samples were taken for biodistribution, and both DNA and RNA were extracted from each sample.
  • Peripheral tissues, including liver, heart, kidney, and spleen, from each animal were also collected frozen for biodistribution. Serum was also collected throughout the study to assess transgene product levels and anti -transgene product antibodies (ATPA).
  • ATPA transgene product antibodies
  • AAV8 anti-C5-ScFv Vector results Vector genome copy levels were assessed by ddPCR using primer/probes against polyA; for retina, RPE/choroid, and sclera, each data point represents a single sample from a single tissue sample from a single animal; for the other ocular tissues, each data point represents the singular extraction from that tissue.
  • Vector genome copy levels ranged from ⁇ le2-le7 GC/pg DNA (FIG. 16A). In the posterior segment, vector genome copies were highest in the sclera, followed by RPE/choroid, followed by retina, as is typical of delivery of AAV to the SCS. Vector DNA was also detected in a number of other ocular tissues, which did not always result in detectable RNA expression.
  • RNA expression in retina and RPE/choroid were similar (FIG. 16B). Biodistribution in the left eye of animal 1 was determined to be profoundly lower than expected. Vector genome biodistribution was also evaluated in peripheral tissues, with vector genomes detected in the spleen of all 3 AAV8-dosed animals, in the liver of 2 of the 3 animals, and in the kidney of 1 of the 3 animals (FIG. 16C).
  • anti-C5-scFv was also detected in the retina, RPE/choroid, and sclera of animals 2 and 3, with the greatest transgene product levels detected in RPE/choroid, achieving up to 6ng anti-C5-scFv/mg tissue (FIGS. 19A-H).
  • Transgene product (TP) levels correlated with vector genome levels in retina and RPE/choroid, but did not in sclera.
  • Minimal anti-C5-scFv was detected in the serum of animal 3 at any timepoint; serum aC5-scFv increased over time in both animals 1 and 2, decreasing by day 85, which correlated well with the degree of liver biodistribution in those animals (FIGS. 20A-20C).
  • Ophthalmic examination found that AAV8.CAG.aC5-scFv was well-tolerated in this study, with a single eye from a single animal presenting with mild (0.5+) vitreous cell on D29. No treatment-related changes in intraocular pressure were noted, and OCT assessments also found no treatment-related changes in ocular structure throughout the study. Histopathology analysis found slight inflammation in the sclera of 1/2 eyes that was determined to be related to the test article. All other histopathological findings were considered spontaneous, and no findings were considered adverse as there was no evidence of retinal degeneration nor clinical findings impacting ocular function.
  • AAV3B-anti-C5-scFv Vector results Vector genome copy levels ranged from ⁇ le2-le6 GC/pg DNA (based on ddPCR using primer/probes against poly A; for retina, RPE/choroid, and sclera, each data point represents a single sample from a single strip from a single animal; for the other ocular tissues, each data point represents the singular extraction from that tissue) (FIGS. 21A-21C). In the posterior segment, vector genome copies were highest in the sclera, followed by RPE/choroid, followed by retina, as is typical of delivery of AAV to the SCS.
  • Vector genome levels in the posterior segment of P0102-OS were suggestive of poor transduction; transgene product data are presented +/- the datapoints from this eye.
  • Vector DNA was also detected in a number of other ocular tissues. RNA expression was highest in retina and RPE/choroid, where transcript copies were similar, followed by iris/ciliary body and trabecular meshwork. Vector genomes were detected in the spleen of all 3 AAV3B-dosed animals, and biodistribution in heart, kidney, and liver was considered undetectable, as no datapoints gave signal >5-fold above background.
  • Histopathology analysis established the presence of choroidal infdtrates in P0101-OD and P0103-OD, as well as inflammation in the sclera of P0101-OD, which was considered test article related. All other histopathological findings were considered spontaneous, and no findings were considered adverse as there was no evidence of retinal degeneration nor clinical findings impacting ocular function.
  • TP (ScFv levels) were measured via ELISA using antigen-coated plates (coated with C5) and detecting TP with Protein-L HRP subset of values were confirmed by bioanalytical measurement by mass spectrometry demonstrating “free” and “total” TP levels are similar.
  • TP expression is variable, but mostly dose-dependent for both scFvs (FIGS. 27A-27B). Two animals (non-specific scFV group) had detectable ATPA in serum, but still demonstrated increase in TP from DI 5 to D29. No ATPA was detected in anti-C5 treated animals.
  • TP expression was measured via ELISA in Vitreous humor (VH) using antigen-coated plates (C5) and detecting TP with Protein-L HRP (FIGS. 28A-28B). A subset of values were confirmed as before with mass spectrometry demonstrating “free” and “total” TP levels similar.
  • TP expression is variable, but dose-dependent for both scFvs. Two animals (non-specific scFV group) had detectable ATPA in serum, but had TP expression within range of other samples, and no ATPA was detected in the anti-C5 treated animals.
  • Vectorized scFv expression data was compared to a non-specific vectorized IgG (full-length) antibody.
  • TP expression was also measured various ocular tissues.
  • the four punches of tissues after vector biodistribution were analyzed and the remaining posterior tissues from these eyes were separated into three tissue layers and homogenized for TP expression.
  • TP was measured using same ELISA method as for AH and VH.
  • Subretinal delivery of AAV-anti-C5-scFv vectors results in high anti-C5 scFv (TP) expression in retina, RPE-choroid, and sclera (FIGS. 30A- 30B).
  • This example relates to the evaluation of biodistribution and expression of an AAV transgene product from different formulations of AAV vector carrying the transgene (e.g., AAVScFvAntibody) in animals (e.g., minipigs, such as Yucatan minipig) after a single suprachoroidal injection.
  • AAV vector carrying the transgene e.g., AAVScFvAntibody
  • animals e.g., minipigs, such as Yucatan minipig
  • three (3) pigs receive each test article formulation via bilateral suprachoroidal injection using a 29-gauge needle approximately 1110 pm in length (performed once).
  • Timepoints indicated as ‘days’ will occur during the day indicated [0274] Test articles 3-5 were provided as follows:
  • Test article 3 AAV8.CAG.C5-D-mab.scFv.HL provided as a pre-formulated solution at a concentration of 3xl0 13 GC/mL (Frozen (-80°C)) in the following: 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 40.0 mg/mL (4% w/v) sucrose, and 0.001% (0.01 mg/mL) poloxamer 188 (Formulation 1).
  • Test article 4 AAV8.CAG.C5-D-mab.scFv.HL provided as a pre-formulated solution at a concentration of 3xl0 13 GC/mL (Frozen (-80°C)) in the following: 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 25.0 mg/mL (2.5% w/v) sucrose, 0.002% (0.02 mg/mL) poloxamer 188 and 0.7% hyaluronic acid (Formulation 2 ⁇ HA).
  • Test article 5 AAV8.CAG.C5-D-mab scFv.HL provided as a pre-formulated solution at a concentration of 3xl0 12 GC/mL (Frozen (-80°C)) in the following: 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 40.0 mg/mL (4% w/v) sucrose, and 0.001% (0.01 mg/mL) poloxamer 188 (Formulation 1). Table 23:
  • Suprachoroidal Dosing (Day 1): Animals will be fasted the night prior to dosing. Approximately fifteen ( 15) minutes prior to anesthesia, 1.0% tropicamide HC1 will be applied topically to the ocular surface to induce mydriasis and atropine (0.05 mg/kg) IM or glycopyrrolate (0.01 mg/kg) IM will be administered to reduce risk of aspiration while under sedation. Animals will receive a single dose of buprenorphine (0.01-0.05 mg/kg) IM as an analgesic, and animals will be anesthetized per IACUC. The area around both eyes, including the eyelid, will be cleaned and sterilized, including the ocular surface.
  • Test article will be administered by suprachoroidal injection over 5-10 seconds using a Sponsor provided 29-gauge needle approximately 1100 pm in length, delivered to the supra-temporal quadrant 4-mm from the limbus between 10 and 11 o’clock in the right eye and between 1 and 2 o’clock in the left eye. Following the injection, the needle will be kept in the eye for approximately 5 seconds before being withdrawn. Upon withdrawal of the needle, a cotton-tipped applicator will be placed over the injection site for approximately 10 seconds. Atopical drop of antibiotic ophthalmic solution will be applied to the ocular surface.
  • OCT optical coherence Tomography
  • OCT Optical Coherence Tomography
  • Color Fundus Imaging Blood Collections (for Serum, Plasma, & Whole Blood), and Tissue Collections will take place as per the experimental design.
  • Affinity assays will be performed on anti-hC5-scFv01 demonstrate the species-specific affinity for human C5, mouse C5 and cynomolgus macaque C5.
  • Bio-layer interferometry is an optical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern.
  • BLI will be used to examine the affinity of anti-hC5-scFv01 to various antigens. Briefly, the biosensor tip will be contacted to biotinylated antigen. Next, the biosensor tip will be contacted to anti-hC5-scFv01. Association of the ligand to the antigen will be measured.
  • Octet machine (Sartorious, Model: RED384), as supported by BLI technology was used to measure KD values between ligands (C5 proteins) and analytes (Anti-hC5-scFv01).
  • C5 proteins C5 proteins
  • Anti-hC5-scFv01 Recombinant C5 proteins from all species were produced using CHO cell expression platforms.
  • C5 proteins, as ligands for the assay were biotinylated and loaded onto streptavidin sensors. The association and dissociation rates of C5:scFv01 complex were measured by Octet, and KD values were calculated.
  • Anti-hC5-scFv01 showed a strong binding affinity to all recombinant C5 proteins with KD values of -200 pM to human C5 protein, of 92 pM to cynomolgus C5 protein, and of -15 nM to mouse C5 protein.
  • Anti-hC5-scFv01 shows a strong binding affinity in vitro towards the target protein, human C5.
  • anti-hC5-scFv01 binds to C5 proteins from other species, albeit with slightly higher affinity to cynomolgus C5, and substantially lower affinity to mouse C5, demonstrating the suitability of anti-hC5-scFv01 for use as a C5 inhibitor in studies of mouse and non-human primate animal models.
  • CP complement activation
  • CH50 sheep erythrocytes coated with rabbit antibodies
  • Such classical complement pathway-related hemolysis inhibition assay will be 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 fragment expressed from the plasmid to a non-complement related target) will be 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
  • Example 19B Inhibition of Complement activation PKPD human iRPE cells
  • MAC Membrane attack complex
  • Cells will be fixed in 4% paraformaldehyde for 20 min, at RT and immunostained with antibodies or cell stain against C5b-9 (Invitrogen, MA5-28502), ZO-1 (Invitrogen, REF 40220), Phalloidin (AF-568 Phalloidin: Invitrogen A 12380) and DAPI.
  • Example 20 In Vivo Mouse IV and/or Transgenic PD study
  • a NaIO3 induced model of dry AMD in rodents will be used to assess the modified AAV anti-hC5-scFv01 (also called C5-D-mab scFv (HL) (SEQ ID NO: 203)).
  • Recombinant modified AAV anti-hC5-scFv01 vectors (groups 2-4 and 6) will be administered to C57BL/6J mice at a according to Table 3. Mice will be evaluated on Day 0 for baseline electroretinography (ERG)/body weights (BW)/OCT, and then each administered a test antibody at Day 0.
  • ERP electroretinography
  • BW body weights
  • NaIO3 will be administered to each mouse intravenously to induce an inflammatory event in the eye. Mice will again be evaluated for ERG/OCT on Day 32 and on Day 36, respectively, then sacrificed on Day 36 for tissue collection.
  • the induced inflammation from injection of NaIO3 results in degeneration of the retinal pigment epithelium (RPE) layer of the eye causing RPE loss, and also the thinning of the photoreceptor layer, the outer nuclear layer (ONL) containing mostly photoreceptors. Thickness of the ONL will be visualized by optical coherence tomography (OCT) or immunohistochemical staining of the tissue.
  • RPE retinal pigment epithelium
  • ONL the outer nuclear layer
  • Antibody product levels will be detected in the eye as assessed following i.p. injection of recombinant modified AAV anti-hC5-scFv01 vectors.
  • ERGs will be performed on both eyes using the Diagnosys ERG systems. Animals will be dark adapted for a period of at least 12 hours prior to ERG. Under dark adaptation, eyes will be dilated using a cocktail of tropicamide HC1 1% and phenylephrine HC1 2.5%. Before ERGs were recorded, pupil dilatation will be checked to ensure full dilation. Animals will be positioned on the ERG machine, then proparacaine HC1 0.5% and GenTeal will be applied to the eyes, followed by the electrode contact and reference leads. Animals will be placed on a warm water blanket to control body temperature and contact lens leads will be placed on the eyes.
  • a reference subcutaneous lead will be placed in the head and a ground lead was placed near the tail of the animal.
  • Light-adapted ERG with 30 cd/m2 background light will immediately follow the dark-adapted series. There will be a 5 minute light adaptation period before the light-adapted signals will be collected. Background light will be used constantly for all light-adapted measurements.
  • Step 1-9 0.001-10 cd*s/m2 scotopic ERG, each step advancing half log units in light intensity. Both ERG traces and oscillatory potentials will be collected.
  • Step 10 Dark-adapted 150 cd/m2 scotopic c-wave measurement.
  • Step 1-3 1-10 cd*s/m2 photopic ERG, each step advancing half log units in light intensity. Both ERG traces and oscillatory potentials will be collected.
  • Step 4-7 3.0 c cd*s/m2 photopic ERG flicker ERG at 10-40 Hz, each step advancing 10 Hz.
  • NHP will be administered AAV vectored anti-hC5-scFv01 (groups 2 and 3) or vehicle control according to Table 4.
  • Biofluids Blood (MOV), AH, VH, 2) Tissue collections for biodistribution (BioD) (Collected and analyzed): Eye (lens , iris- ciliary body, cornea, RPE, Choroid, Retina, Sclera [15 samples per eye]), Optic nerve, Ovary/teste, liver, heart, 3) BioD Collections collected and stored: Adrenal Gland, Brain (3 samples), Cerebellum, DRG (2 pairs from 4 regions), Kidney, Lung, Lymph nodes (2), spleen, 4) VCN (biodistribution), 5) mRNA Transgene, 6) TP, 7) C5 and other complement factors, and 8) VH for activity assay.
  • BioD Biodistribution
  • NHP will be administered AAV vectored anti-hC5-scFV (group 2) or vehicle control according to Table 30.
  • BioD collections Eye (lens , iris-ciliary body, cornea, RPE, Choroid, Retina, Sclera [15 samples per eye]), Adrenal gland, brain (3 samples), Cerebellum, DRG (2 pairs from 4 regions), Heart, Kidney, Liver, Lung, Lymph nodes (4), Muscle (quad), Optic nerve, Ovary/teste, Spinal cord, spleen (remaining tissues saved for histopath), 2) VCN (biodistribution), 3) mRNA Transgene, 4) TP, 5) C5 and other complement factors, and 6) VH for activity assay.
  • NHP will be administered AAV vectored anti-hC5-scFV (groups 2 and 3) or vehicle control according to Table 6 Table 29:
  • AAV capsid sequences modified by peptide insertions with (AAV8.590 library hits) or without additional substitutions (AAV8.456 library hits) were further evaluated in in vivo for biodistribution in test animals using next generation sequencing (NGS) and quantitative PCR.
  • NGS next generation sequencing
  • formalin fixed eyes may be, e.g. sectioned at 40pm thickness on a vibrating blade microtome (VT1000S, Leica) and the floating sections probed with antibodies against transgene (or viewed for fluorescence if fluorescent transgene) to look at the cellular distribution of the delivered vectors.
  • VT1000S vibrating blade microtome
  • NHP Library Down-selection Summary As described hereinabove, several high diversity peptide insertion libraries (up to 10 8 variants per library) were designed and produced, each encoding AAV cap under the control of the CMV promoter with randomized 7-mer insertions at VR- IV or VR-VIII of AAV8. The libraries were delivered intraocularly to non-human primates (NHP, cynomolgus macaques). Leveraging a directed evolution platform NAVIGATE (Novel AAV Vector Intelligent Guided Adaptation Through Evolution), 2-4k novel variants overall were identified and enriched in retina and RPE-choroid from each high diversity library, which were then re-packaged and administered to NHP via SCS delivery.
  • NAVIGATE Novel AAV Vector Intelligent Guided Adaptation Through Evolution
  • capsid variants (12 AAV8.VR-IV and 10 AAV8.VR- VIII) were chosen for final library down-selection.
  • Each variant (as well as parental control AAV8) was produced individually with genomes encoding a barcoded CAG.tdTomato cassette, pooled, and delivered to NHP, again by SCS delivery, at a dose of 3el2 GC/eye.
  • TdTomato genome (cDNA) and transcripts (mRNA) were detected by digital PCR and plotted against a reference gene.
  • Results show that relative abundance adjusted for input (RAAFI) (DNA and mRNA) was increased in retina and RPE-choroid (RPE-C) for several AAV8.456 or AAV8.590 vectors in the pool compared to parental AAV8 or wtAAV3B vector (FIGS. 38A-B).
  • RPE-C RPE-choroid
  • FIGs. 38A-38B also illustrate the enrichment of SCS-delivered vectors having engineered capsids packaging a fluorescent protein, showing the majority of the tested capsids resulted in greater than 10X abundance over parental AAV8 capsid.
  • the data from the present NHP study for AAV8.456 and AAV8.590 mRNA distribution in ocular tissues shows that RNA expression of transgene from these variant capsids is greatly enriched and transduction of retina and RPE-choroid tissue over scleral tissue is better than wildtype AAV8 upon SCS administration which may be desirable for certain transgenes.
  • AAV8 variants AAV8.1, AAV8.2, AAV8.4 (also named PEPIN8.1, PEPIN8.2 and PEPIN8.4) were administered as individual vectors carrying the anti-hC5- scFvOl transgene in an NHP study similar to above.
  • AAV8.VR-IV/AAV8.VR-VIII insertion variants were up to >30-fold and up to >50-fold improved, respectively, when compared to the parental serotypes.
  • Most AAV8-based insertion variants also expressed >20-fold more transgene mRNA in RPE-choroid compared to AAV8, with the top variant producing in excess of 200-fold more mRNA than AAV8. Numerous variants with advantageous off- target transduction profiles were also identified.
  • TP Protein
  • A AH
  • VH B
  • Retina lysates C
  • RPE- Choroid lysates D
  • AAV3B.455 capsids from a library of AAV3B variants was performed.
  • TdTomato genome (cDNA) and transcripts (mRNA) were detected by digital PCR and plotted against a reference gene. Results show that relative abundance adjusted for input (RAAFI) (DNA and mRNA) was increased in retina and RPE-choroid for several AAV3B.455 vectors in the pool compared to parental AAV3B or wtAAV8 vector. Three capsids/vector with high abundance are shown in Table 31.
  • a variant capsid library and a subset of single vectors was assessed via SCS delivery in Yucatan minipigs.
  • pooled capsids (vectors) were administered to one cohort (2 animals, 2 eyes collected each). Following 4 weeks, animals were sacrificed and eyes and several peripheral tissues were collected (Day 15 and Day 29).
  • scFv genome cDNA
  • transcripts mRNA
  • Retina and RPE tissues as well as aqueous humor (AH) and vitreous humor (VH) were tested for transgene product and DNAbiodistribution (BD) at Day 29 (TP measured as ng/mL protein, DNA genome copies (GC)/pg) (FIGS. 31A-D).
  • AAV8 variants from the libraries outperform AAV8 following single vector SCS delivery of scFvOl transgene in minipigs in a dose-dependent manner.
  • AAV8 variants achieve TP concentrations up to 344ng/mg in retina, consistent with biodistribution. (FIG. 32A-32B).
  • AAV8.3 achieves >153X AAV8 expression levels if transgene product in AAV8 in AH.
  • Results show that transgene product (TP) in aqueous humor for the AAV8.456.RIQMGTK (8.1), AAV8.456.RQKNAMV (8.2), AAV8.590.GDNTTFRRA (8.3), and AAV8.590.GRTIRGDLA (8.4) vectors were increased relative to wildtype AAV8 vector.
  • AAV8.456.RIQMGTK (8.1) and AAV8.456.RQKNAMV (8.2) vectors exhibited about 50-fold and 150-fold change, respectively, in TP concentration relative to wtAAV8 vector at the high dose at Day 15.
  • scFv genome cDNA
  • transcripts mRNA
  • Retina and RPE tissues as well as aqueous humor (AH) and vitreous humor (VH) were tested for transgene product and DNA biodistribution (BD) at Day 29 (TP measured as ng/mL protein, DNA genome copies (GC)/pg) (FIGs. 35A-35D)
  • Novel AAV3B variants from the library screen outperformed wild-type AAV8 (wtAAV8) following single vector SCS delivery of scFvOl transgene in minipigs in a dose-dependent manner.
  • the AAV3B-3.1 (AAV3B.455.DLMLPGS) variant achieved significantly higher transgene product (TP) concentrations (+++) in retina, consistent with biodistribution and dosedependent, compared to wtAAV8 (FIGs. 35A and 35C).
  • AAV3B-3.1 also achieved significantly higher transgene product (TP) concentrations, calculated to 28-fold higher than wtAAV8 at its maximum concentration, in RPE-choroid (RPE-C). TP production in RPE-C appears consistent with biodistribution and is dose-dependent. (FIGs. 35B and 35D.)
  • AAV3B-3.1 also achieved higher expression levels of transgene product in VH (+++,
  • results also showed that transgene product (TP) in aqueous humor for the single AAV3B.455 vector (capsid AAV3B.455.DLMLPGS / AAV3B-3.1) was increased (++) compared to parental capsid or an AAV8 wildtype capsid.
  • the AAV3B.455 capsid exhibited over 50-fold change (up to 93 fold at the maximum concentration) in TP concentration in AH compared to wtAAV8 vector at the high dose.
  • mice commonly used as a model for human geographic atrophy (GA) or dry age-related macular degeneration (AMD)
  • AAV8- anti-hC5-scFv01 vector was assessed by AAV8.2-anti-hC5-scFv01 vector, each administered by tail vein (intravenous) injection in methods analogous to Examples 14A, 14B and 20.
  • the vectors carrying anti- C5 transgene were compared to an AAV8.2.null vector (control), a vector carrying a transgene that does not encode for any protein.
  • mice Male C57BL/6J mice were dosed via tail vein with AAV at 3E13 vg/kg body weight (dose volume 5 mL/kg).
  • SI sodium iodate
  • mice were euthanized and eyes collected for either measurement of transgene product levels via ELISA, analysis of AAV biodistribution via ddPCR, or histology following fixation in Davidson’s fixative.
  • Study groups were masked, and measurements of photoreceptor layer and total retina thickness were taken from Optical Coherence Tomography (OCT) scans or H&E images using FIJFImageJ.
  • OCT Optical Coherence Tomography
  • ERG electroretinogram
  • H&E Hematoxylin/Eosin
  • mice in each group were treated prophylactically with a single intravenous (IV) injection via the tail vein with either vehicle or one of four doses of AAV8.2.CAG.anti-hC5 on Day 0.
  • AAV8.2.CAG.anti-hC5-scFv01 encodes a human-specific singlechain fragment variable antibody (scFv) with high affinity for human C5 and lower affinity for mouse C5.
  • the transgene cassette is packaged in an AAV8-derived variant capsid.
  • Retinal degeneration was induced on Day 28 by intravenous administration of NaIO3 in Groups 1-5 only, and Group 6 served as a control. Disease progression was assessed in all groups over the next 7 days.
  • Electroretinography (ERG) was performed on all animals on Day 24 and Day 31, four days before and three days after sodium iodate administration. OCT was performed on Day 35 prior to termination. Blood was collected for isolation of serum at baseline prior to dosing, and on Days 7, 21, and 35.
  • EMG Electroretinography
  • OCT was performed on Day 35 prior to termination.
  • Blood was collected for isolation of serum at baseline prior to dosing, and on Days 7, 21, and 35.
  • the right eye (OD) from each animal and biopsies from liver, heart, kidney, brain, lung, and spleen from each animal were collected, fixed, and processed to H&E sections for microscopic evaluation and image analysis (eyes only).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Ophthalmology & Optometry (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des compositions et des procédés pour l'administration d'un AAV qui comprend un transgène codant pour un scFv qui se lie à C5 chez un sujet humain pour le traitement d'une indication oculaire, en particulier la DMLA. La séquence nucléotidique codant l'anticorps est administrée dans un vecteur VAAr qui cible des cellules de tissu oculaire pour l'expression du transgène.
PCT/US2025/023760 2024-04-08 2025-04-08 Anticorps anti-complément vectorisés et agents de complément et leur administration Pending WO2025217230A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202463631448P 2024-04-08 2024-04-08
US63/631,448 2024-04-08
US202463642642P 2024-05-03 2024-05-03
US63/642,642 2024-05-03
US202463703110P 2024-10-03 2024-10-03
US63/703,110 2024-10-03

Publications (1)

Publication Number Publication Date
WO2025217230A1 true WO2025217230A1 (fr) 2025-10-16

Family

ID=95651360

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/023760 Pending WO2025217230A1 (fr) 2024-04-08 2025-04-08 Anticorps anti-complément vectorisés et agents de complément et leur administration

Country Status (1)

Country Link
WO (1) WO2025217230A1 (fr)

Citations (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018317A1 (fr) 1993-02-12 1994-08-18 The Board Of Trustees Of The Leland Stanford Junior University Transcription regulee de genes cibles et d'autres evenements biologiques
WO1996020951A1 (fr) 1994-12-29 1996-07-11 Massachusetts Institute Of Technology Proteines chimeres de liaison d'adn
WO1996041865A1 (fr) 1995-06-07 1996-12-27 Ariad Gene Therapeutics, Inc. Regulation d'evenements biologiques fondee sur la rapamycine
WO1999010510A2 (fr) 1997-08-26 1999-03-04 Ariad Gene Therapeutics, Inc. Proteines de fusion a domaine de dimerisation, de trimerisation ou de tetramerisation, et a domaine additionnel d'activation de transcription heterologue, d'inhibition de transcription, de liaison d'adn ou de liaison de ligand
WO1999010508A1 (fr) 1997-08-27 1999-03-04 Ariad Gene Therapeutics, Inc. Activateurs transcriptionnels chimeres, compositions et applications afferentes
WO1999036553A2 (fr) 1998-01-15 1999-07-22 Ariad Gene Therapeutics, Inc. Regulation de phenomenes biologiques au moyen de proteines chimeres multimeres
WO1999041258A1 (fr) 1998-02-13 1999-08-19 President And Fellows Of Harvard College Agents de dimerisation, production et utilisation
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
WO2003052051A2 (fr) 2001-12-17 2003-06-26 The Trustees Of The University Of Pennsylvania Sequences du serotype 8 du virus associe a l'adenovirus (aav), vecteurs les contenant et utilisations correspondantes
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
US7067526B1 (en) 1999-08-24 2006-06-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
WO2006068888A1 (fr) 2004-12-22 2006-06-29 Raytheon Company Systeme et technique d'etalonnage de reseaux de radars
WO2006110689A2 (fr) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Procede d'augmentation de la fonction d'un vecteur aav
US20070135620A1 (en) 2004-11-12 2007-06-14 Xencor, Inc. Fc variants with altered binding to FcRn
US20080154025A1 (en) 2003-03-03 2008-06-26 Xencor, Inc. Fc Variants with Increased Affinity for FcyRIIc
US7456683B2 (en) 2005-06-09 2008-11-25 Panasonic Corporation Amplitude error compensating device and quadrature skew error compensating device
WO2009104964A1 (fr) 2008-02-19 2009-08-27 Amsterdam Molecular Therapeutics B.V. Optimisation de l'expression de protéines rep et cap parvovirales dans des cellules d'insectes
US20100234572A1 (en) 2004-11-12 2010-09-16 Xencor, Inc. Fc Variants with altered binding to FcRn
WO2010127097A1 (fr) 2009-04-30 2010-11-04 The Trustees Of The University Of Pennsylvania Compositions pour cibler des cellules des voies respiratoires conductrices comprenant des constructions de virus adéno-associé
WO2011139713A2 (fr) 2010-04-26 2011-11-10 Emory University Procédés et dispositifs d'administration de médicament à un tissu oculaire à l'aide d'une micro-aiguille
US20120225058A1 (en) 2004-10-21 2012-09-06 Xencor, Inc. Novel immunoglobulin insertions, deletions, and substitutions
WO2012145601A2 (fr) * 2011-04-22 2012-10-26 The Regents Of The University Of California Virions de virus adéno-associé avec capside variant et procédés d'utilisation de ceux-ci
US20130224836A1 (en) 2010-10-27 2013-08-29 Jichi Medical University Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells
US8628966B2 (en) 2010-04-30 2014-01-14 City Of Hope CD34-derived recombinant adeno-associated vectors for stem cell transduction and systemic therapeutic gene transfer
WO2014036009A1 (fr) 2012-08-27 2014-03-06 Clearside Biomedical, Inc. Appareil et procédés d'administration de médicaments à l'aide de micro-aiguilles
WO2014074823A1 (fr) 2012-11-08 2014-05-15 Clearside Biomedical, Inc. Procédés et dispositifs pour le traitement de maladies oculaires chez des sujets humains
US8734809B2 (en) 2009-05-28 2014-05-27 University Of Massachusetts AAV's and uses thereof
WO2014172669A1 (fr) 2013-04-20 2014-10-23 Research Institute At Nationwide Children's Hospital Administration de virus adéno-associé recombinant de constructions polynucléotidiques u7snarn ciblant l'exon 2
WO2014179698A2 (fr) 2013-05-03 2014-11-06 Clearside Biomedical, Inc. Appareil et procédés pour une injection oculaire
US8927514B2 (en) 2010-04-30 2015-01-06 City Of Hope Recombinant adeno-associated vectors for targeted treatment
US20150023924A1 (en) 2013-07-22 2015-01-22 The Children's Hospital Of Philadelphia Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues
US20150126588A1 (en) 2012-05-09 2015-05-07 Oregon Health & Science University Adeno associated virus plasmids and vectors
WO2015126694A1 (fr) 2014-02-12 2015-08-27 Ethicon Endo-Surgery, Inc. Procédé et appareil pour administration suprochoroïdienne d'un agent thérapeutique
US9169299B2 (en) 2011-08-24 2015-10-27 The Board Of Trustees Of The Leleand Stanford Junior University AAV capsid proteins for nucleic acid transfer
US20150337053A1 (en) 2009-11-30 2015-11-26 Janssen Biotech, Inc. Antibody Fc Mutants with Ablated Effector Functions
WO2015191508A1 (fr) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Capsides chimériques
US20150374803A1 (en) 2013-03-13 2015-12-31 The Children's Hospital Of Philadelphia Adeno-associated virus vectors and methods of use thereof
WO2016040635A1 (fr) 2014-09-11 2016-03-17 Ethicon Endo-Surgery, Inc. Appareil motorisé d'injection suprachoroïdale d'un agent thérapeutique
WO2016044404A2 (fr) 2014-09-18 2016-03-24 Ethicon Endo-Surgery, Inc. Dispositif d'administration d'un agent thérapeutique
WO2016049230A1 (fr) 2014-09-24 2016-03-31 City Of Hope Variants de vecteur de virus adénoassocié pour une édition de haute efficacité du génome et procédés correspondants
US9359437B2 (en) 2013-02-01 2016-06-07 Regeneron Pharmaceuticals, Inc. Antibodies comprising chimeric constant domains
US20160215024A1 (en) 2013-10-11 2016-07-28 Massachusetts Eye & Ear Infirmary Methods of Predicting Ancestral Virus Sequences and Uses Thereof
US9409953B2 (en) 2011-02-10 2016-08-09 The University Of North Carolina At Chapel Hill Viral vectors with modified transduction profiles and methods of making and using the same
WO2016161010A2 (fr) 2015-03-30 2016-10-06 Regeneron Pharmaceuticals, Inc. Régions constantes de chaînes lourdes présentant une liaison réduite aux récepteurs fc gamma
US9585971B2 (en) 2013-09-13 2017-03-07 California Institute Of Technology Recombinant AAV capsid protein
US20170067908A1 (en) 2014-04-25 2017-03-09 Oregon Health & Science University Methods of viral neutralizing antibody epitope mapping
WO2017070491A1 (fr) 2015-10-23 2017-04-27 Applied Genetic Technologies Corporation Formulations ophtalmiques
WO2017156227A1 (fr) 2016-03-09 2017-09-14 Janssen Biotech, Inc. Appareil pour l'administration sous-rétinienne d'un agent thérapeutique par l'intermédiaire d'une aiguille courbe
WO2017218613A2 (fr) 2016-06-17 2017-12-21 Janssen Biotech, Inc. Appareil de guidage pour entrée tangentielle dans un espace suprachoroïdal
US9923120B2 (en) 2015-09-26 2018-03-20 Nichia Corporation Semiconductor light emitting element and method of producing the same
US10053517B2 (en) 2011-09-26 2018-08-21 Jn Biosciences Llc Hybrid constant regions
WO2019053466A1 (fr) 2017-09-15 2019-03-21 Oxular Limited Compositions médicamenteuses ophtalmiques
WO2019202603A1 (fr) 2018-04-19 2019-10-24 Everads Therapy Ltd Dispositif d'injection d'une substance dans une couche intermédiaire d'un tissu ou d'un organe corporel
US20200079821A1 (en) 2015-05-11 2020-03-12 Ucl Business Ltd Capsid
WO2021071835A1 (fr) 2019-10-07 2021-04-15 Regenxbio Inc. Composition pharmaceutique de vecteur de virus adéno-associé et méthodes
WO2021188803A1 (fr) 2020-03-19 2021-09-23 Clearside Biomedical, Inc. Compositions et méthodes pour traiter des troubles oculaires
US11273072B2 (en) 2017-01-13 2022-03-15 Gyroscope Therapeutics Limited Suprachoroidal injection device
WO2022076549A1 (fr) 2020-10-07 2022-04-14 Regenxbio Inc. Formulations pour administration suprachoroïdienne, telles que formulations à viscosité élevée
WO2022076711A2 (fr) * 2020-10-07 2022-04-14 Regenxbio Inc. Virus adéno-associés pour administration oculaire de thérapie génique
WO2023215806A2 (fr) * 2022-05-03 2023-11-09 Regenxbio Inc. Anticorps anti-complément vectorisés et agents de complément et leur administration

Patent Citations (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018317A1 (fr) 1993-02-12 1994-08-18 The Board Of Trustees Of The Leland Stanford Junior University Transcription regulee de genes cibles et d'autres evenements biologiques
WO1996020951A1 (fr) 1994-12-29 1996-07-11 Massachusetts Institute Of Technology Proteines chimeres de liaison d'adn
WO1996041865A1 (fr) 1995-06-07 1996-12-27 Ariad Gene Therapeutics, Inc. Regulation d'evenements biologiques fondee sur la rapamycine
WO1999010510A2 (fr) 1997-08-26 1999-03-04 Ariad Gene Therapeutics, Inc. Proteines de fusion a domaine de dimerisation, de trimerisation ou de tetramerisation, et a domaine additionnel d'activation de transcription heterologue, d'inhibition de transcription, de liaison d'adn ou de liaison de ligand
WO1999010508A1 (fr) 1997-08-27 1999-03-04 Ariad Gene Therapeutics, Inc. Activateurs transcriptionnels chimeres, compositions et applications afferentes
WO1999036553A2 (fr) 1998-01-15 1999-07-22 Ariad Gene Therapeutics, Inc. Regulation de phenomenes biologiques au moyen de proteines chimeres multimeres
WO1999041258A1 (fr) 1998-02-13 1999-08-19 President And Fellows Of Harvard College Agents de dimerisation, production et utilisation
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
US7125717B2 (en) 1999-08-09 2006-10-24 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US7067526B1 (en) 1999-08-24 2006-06-27 Ariad Gene Therapeutics, Inc. 28-epirapalogs
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
US8524446B2 (en) 2001-11-13 2013-09-03 The Trustees Of The University Of Pennsylvania Method for detecting adeno-associated virus
WO2003052051A2 (fr) 2001-12-17 2003-06-26 The Trustees Of The University Of Pennsylvania Sequences du serotype 8 du virus associe a l'adenovirus (aav), vecteurs les contenant et utilisations correspondantes
US8962332B2 (en) 2001-12-17 2015-02-24 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US7790449B2 (en) 2001-12-17 2010-09-07 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing the same, and uses therefor
US8318480B2 (en) 2001-12-17 2012-11-27 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US20080154025A1 (en) 2003-03-03 2008-06-26 Xencor, Inc. Fc Variants with Increased Affinity for FcyRIIc
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
US20120225058A1 (en) 2004-10-21 2012-09-06 Xencor, Inc. Novel immunoglobulin insertions, deletions, and substitutions
US20070135620A1 (en) 2004-11-12 2007-06-14 Xencor, Inc. Fc variants with altered binding to FcRn
US20100234572A1 (en) 2004-11-12 2010-09-16 Xencor, Inc. Fc Variants with altered binding to FcRn
WO2006068888A1 (fr) 2004-12-22 2006-06-29 Raytheon Company Systeme et technique d'etalonnage de reseaux de radars
US8999678B2 (en) 2005-04-07 2015-04-07 The Trustees Of The University Of Pennsylvania Method of increasing the function of an AAV vector
US10301648B2 (en) 2005-04-07 2019-05-28 The Trustees Of The University Of Pennsylvania Method of increasing the function of an AAV vector
WO2006110689A2 (fr) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Procede d'augmentation de la fonction d'un vecteur aav
US7456683B2 (en) 2005-06-09 2008-11-25 Panasonic Corporation Amplitude error compensating device and quadrature skew error compensating device
WO2009104964A1 (fr) 2008-02-19 2009-08-27 Amsterdam Molecular Therapeutics B.V. Optimisation de l'expression de protéines rep et cap parvovirales dans des cellules d'insectes
WO2010127097A1 (fr) 2009-04-30 2010-11-04 The Trustees Of The University Of Pennsylvania Compositions pour cibler des cellules des voies respiratoires conductrices comprenant des constructions de virus adéno-associé
US9284357B2 (en) 2009-05-28 2016-03-15 University Of Massachusetts AAV's and uses thereof
US8734809B2 (en) 2009-05-28 2014-05-27 University Of Massachusetts AAV's and uses thereof
US20150337053A1 (en) 2009-11-30 2015-11-26 Janssen Biotech, Inc. Antibody Fc Mutants with Ablated Effector Functions
WO2011139713A2 (fr) 2010-04-26 2011-11-10 Emory University Procédés et dispositifs d'administration de médicament à un tissu oculaire à l'aide d'une micro-aiguille
US8927514B2 (en) 2010-04-30 2015-01-06 City Of Hope Recombinant adeno-associated vectors for targeted treatment
US8628966B2 (en) 2010-04-30 2014-01-14 City Of Hope CD34-derived recombinant adeno-associated vectors for stem cell transduction and systemic therapeutic gene transfer
US20130224836A1 (en) 2010-10-27 2013-08-29 Jichi Medical University Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells
US9409953B2 (en) 2011-02-10 2016-08-09 The University Of North Carolina At Chapel Hill Viral vectors with modified transduction profiles and methods of making and using the same
WO2012145601A2 (fr) * 2011-04-22 2012-10-26 The Regents Of The University Of California Virions de virus adéno-associé avec capside variant et procédés d'utilisation de ceux-ci
US9458517B2 (en) 2011-04-22 2016-10-04 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9193956B2 (en) 2011-04-22 2015-11-24 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9587282B2 (en) 2011-04-22 2017-03-07 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9169299B2 (en) 2011-08-24 2015-10-27 The Board Of Trustees Of The Leleand Stanford Junior University AAV capsid proteins for nucleic acid transfer
US10053517B2 (en) 2011-09-26 2018-08-21 Jn Biosciences Llc Hybrid constant regions
US20150126588A1 (en) 2012-05-09 2015-05-07 Oregon Health & Science University Adeno associated virus plasmids and vectors
WO2014036009A1 (fr) 2012-08-27 2014-03-06 Clearside Biomedical, Inc. Appareil et procédés d'administration de médicaments à l'aide de micro-aiguilles
WO2014074823A1 (fr) 2012-11-08 2014-05-15 Clearside Biomedical, Inc. Procédés et dispositifs pour le traitement de maladies oculaires chez des sujets humains
US9359437B2 (en) 2013-02-01 2016-06-07 Regeneron Pharmaceuticals, Inc. Antibodies comprising chimeric constant domains
US20150374803A1 (en) 2013-03-13 2015-12-31 The Children's Hospital Of Philadelphia Adeno-associated virus vectors and methods of use thereof
WO2014172669A1 (fr) 2013-04-20 2014-10-23 Research Institute At Nationwide Children's Hospital Administration de virus adéno-associé recombinant de constructions polynucléotidiques u7snarn ciblant l'exon 2
WO2014179698A2 (fr) 2013-05-03 2014-11-06 Clearside Biomedical, Inc. Appareil et procédés pour une injection oculaire
US20150023924A1 (en) 2013-07-22 2015-01-22 The Children's Hospital Of Philadelphia Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues
WO2015013313A2 (fr) 2013-07-22 2015-01-29 The Children's Hospital Of Philadelphia Compositions et variants de virus adéno-associés, et méthodes et utilisations pour un transfert de gènes dans des cellules, des organes et des tissus
US9840719B2 (en) 2013-07-22 2017-12-12 The Children's Hospital Of Philadelphia Variant AAV and compositions, methods and uses for gene transfer to cells, organs and tissues
US9585971B2 (en) 2013-09-13 2017-03-07 California Institute Of Technology Recombinant AAV capsid protein
US20160215024A1 (en) 2013-10-11 2016-07-28 Massachusetts Eye & Ear Infirmary Methods of Predicting Ancestral Virus Sequences and Uses Thereof
US20170051257A1 (en) 2013-10-11 2017-02-23 Massachusetts Eye And Ear Infirmary Methods of predicting ancestral virus sequences and uses thereof
WO2015126694A1 (fr) 2014-02-12 2015-08-27 Ethicon Endo-Surgery, Inc. Procédé et appareil pour administration suprochoroïdienne d'un agent thérapeutique
US20170067908A1 (en) 2014-04-25 2017-03-09 Oregon Health & Science University Methods of viral neutralizing antibody epitope mapping
WO2015191508A1 (fr) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Capsides chimériques
WO2016040635A1 (fr) 2014-09-11 2016-03-17 Ethicon Endo-Surgery, Inc. Appareil motorisé d'injection suprachoroïdale d'un agent thérapeutique
WO2016044404A2 (fr) 2014-09-18 2016-03-24 Ethicon Endo-Surgery, Inc. Dispositif d'administration d'un agent thérapeutique
WO2016049230A1 (fr) 2014-09-24 2016-03-31 City Of Hope Variants de vecteur de virus adénoassocié pour une édition de haute efficacité du génome et procédés correspondants
WO2016161010A2 (fr) 2015-03-30 2016-10-06 Regeneron Pharmaceuticals, Inc. Régions constantes de chaînes lourdes présentant une liaison réduite aux récepteurs fc gamma
US20200079821A1 (en) 2015-05-11 2020-03-12 Ucl Business Ltd Capsid
US9923120B2 (en) 2015-09-26 2018-03-20 Nichia Corporation Semiconductor light emitting element and method of producing the same
WO2017070491A1 (fr) 2015-10-23 2017-04-27 Applied Genetic Technologies Corporation Formulations ophtalmiques
WO2017156227A1 (fr) 2016-03-09 2017-09-14 Janssen Biotech, Inc. Appareil pour l'administration sous-rétinienne d'un agent thérapeutique par l'intermédiaire d'une aiguille courbe
WO2017218613A2 (fr) 2016-06-17 2017-12-21 Janssen Biotech, Inc. Appareil de guidage pour entrée tangentielle dans un espace suprachoroïdal
US11273072B2 (en) 2017-01-13 2022-03-15 Gyroscope Therapeutics Limited Suprachoroidal injection device
WO2019053466A1 (fr) 2017-09-15 2019-03-21 Oxular Limited Compositions médicamenteuses ophtalmiques
WO2019202603A1 (fr) 2018-04-19 2019-10-24 Everads Therapy Ltd Dispositif d'injection d'une substance dans une couche intermédiaire d'un tissu ou d'un organe corporel
WO2021071835A1 (fr) 2019-10-07 2021-04-15 Regenxbio Inc. Composition pharmaceutique de vecteur de virus adéno-associé et méthodes
WO2021188803A1 (fr) 2020-03-19 2021-09-23 Clearside Biomedical, Inc. Compositions et méthodes pour traiter des troubles oculaires
WO2022076549A1 (fr) 2020-10-07 2022-04-14 Regenxbio Inc. Formulations pour administration suprachoroïdienne, telles que formulations à viscosité élevée
WO2022076711A2 (fr) * 2020-10-07 2022-04-14 Regenxbio Inc. Virus adéno-associés pour administration oculaire de thérapie génique
WO2023215806A2 (fr) * 2022-05-03 2023-11-09 Regenxbio Inc. Anticorps anti-complément vectorisés et agents de complément et leur administration

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
ALBA ET AL.: "Gutless adenovirus: last generation adenovirus for gene therapy", GENE THERAPY, vol. 12, 2005, pages S18 - S27, XP037774015, DOI: 10.1038/sj.gt.3302612
APONTE-UBILLUS ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 102, 2018, pages 1045 - 1054
AURICCHIO ET AL., HUM. MOLEC. GENET., vol. 10, 2001, pages 3075 - 3081
BONDT ET AL., MOL & CELL PROTEOMICS, vol. 13, no. 1, 2014, pages 3029 - 3039
BREMER ET AL., MABS, vol. 7, 2015, pages 672 - 680
BREZSKI, J IMMUNOL, vol. 181, 2008, pages 3183 - 92
BREZSKI, MABS, vol. 3, 2011, pages 558 - 567
CHIANG B ET AL.: "The suprachoroidal space as a route of administration to the posterior segment of the eye.", ADV DRUG DELIV REV., vol. 126, 2018, pages 58 - 66, XP093102157, DOI: 10.1016/j.addr.2018.03.001
CLARK ET AL., J. IMMUNOL., vol. 193, 2014, pages 4962
DALL'ACQUA ET AL., J IMMUNOL, vol. 169, 2002, pages 5171 - 5180
DENIZ DALKARA ET AL: "In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous", SCIENCE TRANSLATIONAL MEDICINE, vol. 5, no. 189, 12 June 2013 (2013-06-12), United States, pages 1 - 11, XP055533378, Retrieved from the Internet <URL:http://stm.sciencemag.org/content/scitransmed/5/189/189ra76.full.pdf> DOI: 10.1126/scitranslmed.3005708 *
DINCULESCU ET AL., HUM GENE THER, vol. 16, 2005, pages 649 - 663
DING ET AL., MABS, vol. 9, 2017, pages 269 - 284
DUAN ET AL., J. VIROL., vol. 75, 2001, pages 7662 - 7671
EWING, TM ET AL., RETINAL PHYSICIAN, vol. 19, 2022, pages 24 - 26
GEORGIADIS ET AL., GENE THERAPY, vol. 23, 2016, pages 857 - 862
GEORGIADIS ET AL., GENE THERAPY, vol. 25, 2018, pages 450
HALBERT ET AL., J. VIROL., vol. 74, 2000, pages 1524 - 1532
HARAYA ET AL., DRUG METABOLISM AND PHARMACOKINETICS, vol. 34, no. 1, 2019, pages 25 - 41
HU ET AL., BIOTECHNOL. PROG., vol. 33, 2017, pages 786 - 794
ISSA ET AL., PLOS ONE, vol. 8, no. 4, 2013, pages e60361
K.L. AMOUR ET AL., EUR. J. IMMUNOL., vol. 29, 1999, pages 2613 - 2624
KENNETHROCHA, BIOCHEM J., vol. 414, 2008, pages 19 - 29
KIM, EUR J IMMUNOL, vol. 29, 1999, pages 2819
LAZAR ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 4005
MCCARTY ET AL., GENE THERAPY, vol. 8, no. 16, 2001, pages 1248 - 1254
MOISSEIEV E ET AL.: "The suprachoroidal space: from potential space to a space with potential.", CLIN OPHTHALMOL., vol. 10, 2016, pages 173 - 178, XP055803016, DOI: 10.2147/OPTH.S89784
OGANESYAN ET AL., JBC, vol. 289, no. 11, 2014, pages 7812 - 7824
POWELLRIVERA-SOTO, DISCOV. MED., vol. 19, no. 102, 2015, pages 49 - 57
PUZZO ET AL., SCI. TRANSL. MED, vol. 29, no. 9, 2017, pages 418
QUAX ET AL., MOL CELL, vol. 59, 2015, pages 149 - 161
RAI UDJ ET AL.: "The suprachoroidal pathway: a new drug delivery route to the back of the eye.", DRUG DISCOV TODAY., vol. 20, no. 4, 2015, pages 491 - 495, XP055629994, DOI: 10.1016/j.drudis.2014.10.010
SALVI, BIOCHEMISTRY AND BIOPHYSICS REPORTS, vol. 9, 2017, pages 13 - 21
SCHODEL ET AL., BLOOD, vol. 117, no. 23, 2011, pages e207 - e217
TSUCHIYA ET AL., J. BIOCHEM., vol. 113, 1993, pages 395 - 400
WESTHAUS ADRIAN ET AL: "AAV capsid bioengineering in primary human retina models", SCIENTIFIC REPORTS, vol. 13, no. 1, 11 December 2023 (2023-12-11), US, XP093266292, ISSN: 2045-2322, Retrieved from the Internet <URL:https://www.nature.com/articles/s41598-023-49112-2> DOI: 10.1038/s41598-023-49112-2 *
WESTHAUS ADRIAN: "Supplemental Information: AAV capsid bioengineering in primary human retina models", SCIENTIFIC REPORTS, 11 December 2023 (2023-12-11), XP093266303 *
WU, HUMAN GENE THERAPY, vol. 18, no. 2, 2007, pages 171 - 82
YAN ET AL., J. VIROL, vol. 79, no. 1, 2005, pages 364 - 379
ZINN ET AL., CELL REP., vol. 12, no. 6, 2015, pages 1056 - 1068
ZOLOTUKHIN ET AL., METHODS, vol. 28, 2002, pages 158 - 167

Similar Documents

Publication Publication Date Title
JP7685833B2 (ja) 翻訳後修飾された完全ヒト抗ＶＥＧＦ Ｆａｂによる眼疾患の治療
JP2025000642A (ja) 翻訳後修飾された完全ヒト抗ＶＥＧＦ Ｆａｂを用いる眼疾患の治療
JP7410522B2 (ja) 抗ｆａｍ１９ａ５抗体のアデノ関連ウイルス（ａａｖ）伝達
CN113966236A (zh) 眼睛病状的基因疗法
US20230295243A1 (en) Composition and method for treating eye diseases
JP2023540094A (ja) Ｋｈ９０２（コンベルセプト）の送達のためのアデノ随伴ウイルスおよびその使用
WO2025113202A1 (fr) Vecteur d&#39;administration de gène pour administrer une protéine de fusion de récepteur du vegf humain, et son utilisation
WO2022094106A1 (fr) ANTICORPS ANTI-TNF-α VECTORISÉS POUR INDICATIONS OCULAIRES
EP4518972A2 (fr) Anticorps anti-complément vectorisés et agents de complément et leur administration
EP4213890A1 (fr) Lanadelumab vectorisé et administration de celui-ci
WO2025217230A1 (fr) Anticorps anti-complément vectorisés et agents de complément et leur administration
US20250288697A1 (en) Vectorized anti-tnf-alpha inhibitors for ocular indications
US20220389120A1 (en) Multispecific antagonists
EP4237453A1 (fr) Antagonistes de tnf-alpha vectorisés pour des indications oculaires
WO2024238867A1 (fr) Anticorps anti-complément vectorisés et leur administration
JPWO2022018516A5 (fr)
US20250099622A1 (en) Compositions and methods for expressing therapeutics
WO2024238859A1 (fr) Agents inhibiteurs de c5 vectorisés et leur administration
WO2024238853A1 (fr) Virus adéno-associés pour administration oculaire de thérapie génique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25723514

Country of ref document: EP

Kind code of ref document: A1