Vectorized Anti-complement antibodies and administration thereof

Abstract

Compositions and methods are described for the delivery of a fully human post- translationally modified therapeutic monoclonal antibody, or an antigen binding fragment thereof, that binds to C5 to a human subject for treatment of an ocular indication, particularly AMD. The nucleotide sequence encoding the antibody is delivered in a rAAV vector that targets ocular tissue cells for expression of the transgene.

Description

VECTORIZED ANTI-COMPLEMENT ANTIBODIES AND ADMINISTRATION THEREOFREFERENCE TO SEQUENCE LISTING[1] The contents of the electronic sequence listing (38013_0037Pl_SL.xml; Size: 387,877 bytes; and Date of Creation: May 16, 2024) is herein incorporated by reference in its entirety.1. INTRODUCTION[2] Compositions and methods are described for the delivery of therapeutic scFvs, that bind to C5. Also described are methods of administering therapeutic scFvs, that bind to C5 — to a human subject diagnosed with Age-Related Macular Degeneration (AMD).2. BACKGROUND OF THE INVENTION[3] 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.[4] 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.[5] Age-Related Macular Degeneration (AMD) causes progressive and permanent vision impairment. There are two forms of AMD, dry AMD and wet AMD. Dry AMD accounts for about 85-90% of the 196 million global AMD cases. Overactivation of the complement system is an important driver of AMD. Over one million patients who also present with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) may also benefit from intervention to counteract over-active complement in the eye.[6] There is a need for more effective treatments that reduce the treatment burden on patients suffering from AMD. Intravitreal medications have become a promising mode of drug administration in patients as they provide high volume of drug to the target tissues, eliminating the risk of systemic toxicity. Reducing or eliminating the need for periodic ocular administration would reduce patient burden and improve therapy.3. SUMMARY OF THE INVENTION[7] Therapeutic antibodies, including scFvs (single-chain variable fragments), 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, as opposed to injecting an antibody 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. Without being bound by any particular theory, this results in antibodies that have different diffusion, bioactivity, distribution, affinity, pharmacokinetic, and immunogenicity characteristics, such that the antibodies delivered to the site of action are “biobetters” in comparison with directly injected antibodies. In addition, factors such as anti-C5 antibodies (see Clark et al., 2014, J. Immunol. 193:4962) may inhibit complement activation and drusen deposit in the eye to inhibit, reduce the progression of dry AMD. Accordingly, provided herein are compositions and methods for anti-C5 gene therapy, particularly recombinant AAV gene therapy, designed to target the eye and generate a depot of transgenes for expression of anti-C5 scFvs as disclosed herein, that result 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.[8] Compositions and methods are described for the ocular or systemic deliveryof an anti-C5 scFv (for example, a fully human-glycosylated scFv form of a therapeutic mAb), to a patient (human subject) diagnosed with AMD or other condition indicated for treatment with the therapeutic anti-C5 scFv. In embodiments, the scFv comprises a VH with an amino acid sequence of SEQ ID NO: 170 and a VL with an amino acid sequence of SEQ ID NO: 171, or a VH with an amino acid sequence of SEQ ID NO: 172 and a VL with an amino acid sequence of SEQ ID NO: 173, or a VH with an amino acid sequence of SEQ ID NO: 174 and a VL with an amino acid sequence of SEQ ID NO: 175, or a VH with an amino acid sequence of SEQ ID NO: 176 and a VL with an amino acid sequence of SEQ ID NO: 177. In embodiments, the scFv has an amino acid sequence SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 190, which may or may not include the signal sequence.[9] Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic anti- 05 scFv form to a subject diagnosed with a condition indicated for treatment with the therapeutic anti-C5 scFv — to create a permanent depot in the eye, or in alternative embodiments, liver and / or muscle, of the patient that continuously supplies the HuPTM scFv, e.g., a human-glycosylated transgene product, to one or more ocular tissues where the scFv exerts its therapeutic or prophylactic effect. In embodiments, the scFv comprise a VH with an amino acid sequence of SEQ ID NO: 170 and a VL with an amino acid sequence of SEQ ID NO: 171, or a VH with an amino acid sequence of SEQ ID NO: 172 and a VL with an amino acid sequence of SEQ ID NO: 173, or a VH with an amino acid sequence of SEQ ID NO: 174 and a VL with an amino acid sequence of SEQ ID NO: 175, or a VH with an amino acid sequence of SEQ ID NO: 176 and a VL with an amino acid sequence of SEQ ID NO: 177. In embodiments, the scFv has an amino acid sequence SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 190, which may or may not include the signal sequence.

[0010] Provided are gene therapy vectors, particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of an anti-C5 scFv as disclosed herein, to achieve a maximum or steady states concentrations in ocular tissues, such as aqueous humor, vitreous humor, or in serum for example, 20, 30, 40, 50, 60 or 90 days after administration of the vector encoding the anti-C5 antibody. In embodiments, the gene therapy vector comprises an artificial genome comprising a nucleotide sequence encoding the anti-C5 scFv operably linked to regulatory sequences that promote expression in the target tissue. In embodiments, the artificial genome comprises the nucleotide sequence of one of SEQ ID NO: 185, 196, 199, 201, 203, 205, 207, or 202. In embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence of SEQ ID NO: 186, 195, 198, 200, 202, 204, 206, or 208, flanked by ITR sequences.

[0011] The recombinant vector used for delivering the transgene includes nonreplicating recombinant adeno-associated virus vectors (“rAAV”). In embodiments, the AAV type has a tropism for ocular tissues, including, for example, retinal cells, RPE, choroid, Bruch’s membrane (BrM) and epithelial cells thereof, choriocapillaris and epithelial cells thereof, photoreceptor cells (rods and cones) and retinal ganglion cells. The AAV type may be, for example, AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof) subtype of AAV. However, other viral vectors may be used, including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive expression elements, such as a CAG promoter, or tissue-specific expression control elements, particularly elements that are ocular tissue, liver and / or muscle specific control elements, for example one or more elements of Tables 1 and la.

[0012] In certain embodiments, the antiC5 scFv is an scFv derived from C5-A- mab, C5-B-mab, C5-C-mab, C5-D-mab, see, for example, FIG. 1C for structure and Table 5 for amino acid sequences. In other embodiments, provided are recombinant AAV vectors comprising a transgene encoding an scFv form of an BB5.1 antibody which may be used as a surrogate for anti C5-binding antibodies C5-A-mab, C5-B-mab, C5-C-mab, or C5-D-mab, in non-human animal models, such as non-human primates (cynomolgus monkeys) rat or mouse models, for pre-clinical assessment for Dry AMD. Amino acid sequences of scFv forms of BB5.1 are provided in Table 5.

[0013] . In other embodiments, 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: 41). In certain embodiments, the construct expresses, from the N-terminus, NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In certain embodiments, the construct encodes, from the N-terminus, NFb-signal or leader sequence- VL-GGGGSGGGGSGGGGS-VH-COOH or NFb-signal or leader sequence- VH- GGGGSGGGGSGGGGS-VL-COOH.

[0014] In addition, scFvs expressed from transgenes in vivo are not likely to contain degradation products associated with antibodies produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is an issue associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and purification with certain buffer systems. These conditions, which promote aggregation, do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage, and is caused by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed from transgenes in vivo may also oxidize in a stressed condition. However, humans, and many other organisms, are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but sometimes also repairs and / or reverses the oxidation. Thus, proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, pharmacokinetics (clearance), and immunogenicity.

[0015] Combination therapies involving systemic delivery of the anti-C5 scFv to the patient accompanied by administration of other available treatments are encompassed by the methods provided herein. The additional treatments may be administered before,concurrently or subsequent to the gene therapy treatment. Such additional treatments can include but are not limited to co-therapy with the therapeutic scFv.

[0016] Also provided are methods of manufacturing the viral vectors, particularly the AAV based viral vectors. In specific embodiments, provided are methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a 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 functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.3.1. EMBODIMENTS

[0017] Embodiment 1. A composition comprising an adeno-associated virus (AAV) vector comprising:(a) a viral AAV capsid that has a tropism for ocular tissue cells; and(b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a single chain variable fragment (scFv) that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprisesan amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.

[0018] Embodiment 2. The composition of embodiment 1, wherein the viral capsid comprises a VP1 capsid protein which is at least 95% identical to the amino acid sequence of the VP1 capsid protein of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAVhu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).

[0019] Embodiment 3. The composition of claim 1 or embodiment 2, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof.

[0020] Embodiment 4. The composition of any one of embodiments 1 to 3, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

[0021] Embodiment 5. The composition of any one of embodiments 1 to 4, wherein the one or more regulatory sequences comprises a regulatory sequence from Table 1 or Table la.

[0022] Embodiment 6. The composition of embodiment 5, wherein the one or more regulatory sequences comprises a CAG promoter (SEQ ID NO: 44), a mutated CAGpromoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), a CB promoter (SEQ ID NO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 47 or 139), a mouse cone arresting (CAR) promoter (SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136), a human red opsin (RedO) promoter (SEQ ID NO: 134) or a Bestl / GRKl tandem promoter (SEQ ID NO: 143).

[0023] Embodiment 7. The composition of embodiment 6, wherein the one or more regulatory sequences comprises a mutated CAG promoter (SEQ ID NO: 221, 222 or 223).

[0024] Embodiment 8. The composition of any one of embodiments 1 to 7, 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.

[0025] Embodiment 9. The composition of embodiment 8, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.

[0026] Embodiment 10. The composition of embodiment 9, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).

[0027] Embodiment 11. The composition of any one of embodiments 1 to 10, wherein the scFv that binds to C5 has the structure: signal sequence - VH - linker - VL.

[0028] Embodiment 12. The composition of any one of embodiments 1 to 10, wherein the scFv that binds to C5 has the structure: signal sequence - VL - linker - VH.

[0029] Embodiment 13. The composition of any one of embodiments 1 to 12, wherein the linker is GGGGS (SEQ ID NO: 38), GGGGSGGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 42).

[0030] Embodiment 14. The composition of embodiment 13, wherein the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 42).

[0031] Embodiment 15. The composition of any one of embodiments 1 to 14, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprisesan amino acid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence of SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

[0032] Embodiment 16. The composition of any one of embodiments 1 to 15, wherein the transgene encodes a polypeptide having an amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188.

[0033] Embodiment 17. The composition of embodiment 16, wherein the transgene encodes a polypeptide having an amino acid sequence SEQ ID NO: 180.

[0034] Embodiment 18. The composition of any one of embodiment 1 to 17, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

[0035] Embodiment 19. The composition of embodiment 18, wherein the artificial genome comprises the nucleotide sequence encoding C5-D-mab.scFv (SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209).

[0036] Embodiment 20. A pharmaceutical composition for use in treating Age- Related Macular Degeneration (AMD) in a human subject in need thereof, comprising an adeno-associated virus (AAV) vector comprising: a viral capsid that has a tropism for ocular tissue cells; andan artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a single chain variable fragment (scFv) that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, 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 said AAV vector is formulated for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic administration to said human subject.

[0037] Embodiment 21. The pharmaceutical composition of embodiments 20, wherein the viral capsid comprises a VP1 capsid protein which is at least 95% identical to the amino acid sequence of the VP1 capsid protein of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).

[0038] Embodiment 22. The pharmaceutical composition of claim 20 or embodiments 21, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof.

[0039] Embodiment 23. The pharmaceutical composition of any one of embodiments 20 to 22, wherein the human ocular tissue cells are retinal cells, RPE -choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

[0040] Embodiment 24. The pharmaceutical composition of any one of embodiments 20 to 23, wherein the one or more regulatory sequences comprises a regulatory sequence from Table 1 or Table la.

[0041] Embodiment 25. The pharmaceutical composition of embodiment 24, wherein the one or more regulatory sequences comprises a CAG promoter (SEQ ID NO: 44), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), a CB promoter (SEQ ID NO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 47 or 139), a mouse cone arresting (CAR) promoter (SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136), a human red opsin (RedO) promoter (SEQ ID NO: 134) or a Bestl / GRKl tandem promoter (SEQ ID NO: 143).

[0042] Embodiment 26. The pharmaceutical composition of embodiment 25, wherein the one or more regulatory sequences comprises a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223).

[0043] Embodiment 27. The pharmaceutical composition of any one of embodiments 20 to 26, 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.

[0044] Embodiment 28. The pharmaceutical composition of embodiment 27, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.

[0045] Embodiment 29. The pharmaceutical composition of embodiment 28, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).

[0046] Embodiment 30. The pharmaceutical composition of any one of embodiments 20 to 29, wherein the scFv that binds to C5 has the structure: signal sequence- VH - linker - VL.

[0047] Embodiment 31. The pharmaceutical composition of any one of embodiments 20 to 29, wherein the scFv that binds to C5 has the structure: signal sequence- VL - linker - VH.

[0048] Embodiment 32. The pharmaceutical composition of any one of embodiments 20 to 31, wherein the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGS GGGGS GGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) orGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0049] Embodiment 33. The pharmaceutical composition of embodiment 32, wherein the linker is GGGGS GGGGS GGGGS (SEQ ID NO: 42).

[0050] Embodiment 34. The pharmaceutical composition of any one of embodiments 20 to 33, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprises an amino acid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence of SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

[0051] Embodiment 35. The pharmaceutical composition of any one of embodiments 20 to 34, wherein the transgene encodes a polypeptide having an amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188.

[0052] Embodiment 36. The pharmaceutical composition of embodiments 35, wherein the transgene encodes a polypeptide having an amino acid sequence SEQ ID NO: 180.

[0053] Embodiment 37. The pharmaceutical composition of any one of embodiments 20 to 36, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

[0054] Embodiment 38. The pharmaceutical composition of embodiment 37, wherein the artificial genome comprises the nucleotide sequence of SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209.

[0055] Embodiment 39. The pharmaceutical composition of any one of claims 20 to 38, wherein the scFv that binds to C5 inhibits both classical and alternative complement pathways.

[0056] Embodiment 40. The pharmaceutical composition of any one of claims 20 to 39, wherein the scFv that binds to C5 inhibits membrane attack complex (MAC) formation.

[0057] Embodiment 41. A method of producing recombinant AAVs comprising: culturing a host cell containing: an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding an scFv that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variablelight domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells; 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; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.

[0058] Embodiment 42. The method of embodiment 41, wherein the transgene encodes an scFv that comprises a variable heavy domain and a variable light chain domain of, C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab, wherein the AAV capsid protein is an AAV8, AAV9, AAV3B, or AAVrh73, or variant thereof.

[0059] Embodiment 43. The method of embodiment 41 or embodiment 42, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglioncells).

[0060] Embodiment 44. The method of any one of embodiments ms 41 to 43, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

[0061] Embodiment 45. A host cell comprising: a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding an scFv that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.

[0062] Embodiment 46. The host cell of embodiment 45, wherein the ocular tissuecells retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

[0063] Embodiment 47. The host cells of embodiment 45 or embodiment 46, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprises an amino acid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

[0064] Embodiment 48. The host cell of any one of embodiments 45 to 47, wherein the transgene encodes a polypeptide having an amino acid sequence of of SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189.

[0065] Embodiment 49. The host cell of embodiment 48, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

[0066] Embodiment 50. The composition of any one of embodiments 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition comprises between about 0.5% to about 1.0% w / v hyaluronic acid.

[0067] Embodiment 51. The composition of any one of embodiments 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, whereinthe composition or pharmaceutical composition comprises about (2.5% w / v) sucrose and between about 0.5% to about 1.0% w / v hyaluronic acid.

[0068] Embodiment 52. The composition of any one of embodiments 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition 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.

[0069] Embodiment 53. The composition of any one of embodiments 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition 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.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] 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 full-length heavy and light chains with Fc regions (A), the heavy and light chains of the Fab portion (B), or a single chain variable fragment (scFv) connecting the heavy and light chains of the antibody with a linker (C).

[0071] FIGS. 2A-2E The amino acid sequence of a transgene construct for the Fab region of C5-D-mab (A), C5-A-mab IgGl (B), C5-A-mab IgG2 (C) C5-C-mab (D), and C5-B-mab (E) 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.

[0072] FIG. 3. Clustal Multiple Sequence Alignment of various capsids with ocular tissue tropism. Amino acid substitutions (shown in bold in the bottom rows) can be made to AAV8 capsids by “recruiting” amino acid residues from the corresponding position of other aligned AAV capsids. Sequence shown in gray = hypervariable regions. The amino acid sequences of the AAV capsids are assigned sequence ID numbers as indicated in FIG. 3

[0073] FIGS. 4A and 4B 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. A) human C5 inhibitors, IgG = C5-D-mab full-length mAb, Fab = C5-D-mab Fab, scFv = C5-D-mab scFv mAb, compared to a recombinant C5 inhibitor (C5I) protein and isotype and vehicle controls. B) mouse C5 inhibitors, IgG = BB5.1 full-length mAb, Fab = BB5.1 Fab, scFv = BB5.1 scFv mAb, compared to a recombinant C5 inhibitor (C5I) protein and isotype and vehicle controls.

[0074] FIGS. 5A-5F 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, comparing anti-hC5 (C5-D-mab scFv mAb) and anti- mC5 (BB5.1 scFv mAb).

[0075] FIGS. 6A-6H measure membrane attack complex (MAC) formation in ARPE-19 cells (FIGS. 9A-C) or iPSC-derived RPE cells (FIGS. 9D-H).

[0076] FIGS. 7A-7D show the results of AAV8-encoding C5 inhibitors injected into wild-type 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.

[0077] FIG. 8 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.

[0078] FIG. 9 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%).

[0079] FIGS. 10A-10D 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) or AAV8 sc-CAG-Delm. C5-D.scFv (closed circles) at a multiplicity of infection (MOI) of 5e4, 1.6e4 or 5e5.

[0080] FIGS. 11A-11C: BB5.1 antibody significantly ameliorated ONL and retinal thinning as measured by OCT post NaIO3 induction.

[0081] FIGS. 12A-12F: 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 NaIO3 administration.

[0082] FIGS. 13A-13C Vector biodistribution (DNA, genome copies per pg of tissue)(A) and transcript copy (# copies (DNA) per pg RNA)(B) were plotted 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).

[0083] FIGS. 14A-14C: Transgene product (TP) is illustrated 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). A: Data set of antibody levels including eyes in the cohort that displayed no or low vector biodistribution; B: Data set excluding eyes after day 29 that displayed no or low biodistribution; C: Time course of the AH collection antibody levels for all eyes.

[0084] FIGS. 15A-15B: 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. A: Data set of TP levels including an eye in the cohort that displayed no or low biodistribution; B: Data set excluding the eyes that displayed no or low vector biodistribution.

[0085] FIGS. 16A-16H: AAV8-mediated TP production levels (ng / mg tissue) in ocular tissue homogenates: retina (A), RPE-choroid (B), sclera (C), and Sample 3 only tissues(D) TP results are illustrated exclusive of the an eye in the cohort that displayed no or low biodistribution; retina (E), RPE-choroid (F), sclera (G), and Sample 3 only tissues (H) TP results are illustrated inclusive of the an eye in the cohort that displayed no or low biodistribution.

[0086] FIGS. 17A-17C: TP levels showing serum levels (A), peripheral vector genome biodistribution (liver)(B), and time course (C) following AAV8 vector delivery of CAG.C5-D-mab-scFv.

[0087] FIGS. 18A-18C Vector biodistribution (DNA, genome copies per pg of tissue)(A) and transcript copy (# copies (DNA) per pg RNA) (B) were plotted in bar graphs for various ocular tissues of animals (day 85) following treatment with 3E12 GC per eyeby suprachoroidal administration of AAV3B vector. Vector biodistribution (DNA) detected in peripheral tissues was also measured (C).

[0088] FIGS. 19A-19C: Transgene product (TP) is illustrated 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). A: Data set of antibody levels including all data points (including any that displayed no or low vector biodistribution); B: Data set excluding eyes after day 29 that displayed no or low biodistribution or high ATPA; C: Time course of the AH collection antibody levels for all eyes.

[0089] FIGS. 20A-20B: 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. A: Data set of TP levels including eyes that displayed no or low biodistribution or high ATPA; B: Data set excluding the eyes that displayed no or low vector biodistribution or +ATPA.

[0090] FIGS. 21A-21H: AAV3B-mediated TP production levels (ng / mg tissue) in ocular tissue homogenates: retina (A), RPE-choroid (B), sclera (C), and Sample 3 only tissues (D) TP results are illustrated exclusive of the an eye in the cohort that displayed no or low biodistribution or +ATPA; retina (E), RPE-choroid (F), sclera (G), and Sample 3 only tissues (H) TP results are illustrated inclusive of the an eye in the cohort that displayed no or low biodistribution or +ATPA.

[0091] FIGS. 22A-22C: TP expression levels showing serum levels (A), peripheral vector genome biodistribution (liver) (B), and time course (C) following AAV3B vector delivery of CAG.C5-D-mab-scFv.

[0092] FIG. 23: 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 AAV8-anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or AAV8-NS-scFv at a dose of 1E10 or 1E11 GC / eye.

[0093] FIGS. 24A-24B: 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 lElO or lEll GC / eye.

[0094] FIGS. 25A-25B: VH 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 lElO or lEll GC / eye.

[0095] FIGS. 26A-26B: AH (A) and VH (B) collected at two and four weeks postinjection 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 Nonspecific mab; 3E9 or 1E10 GC / eye) showing greater TP expression levels for vectorized ScFv antibodies.

[0096] FIGS. 27A-27B: 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.

[0097] FIG. 28 Schematic of human anti-C5 inhibitors of different formats

[0098] FIGS. 29A-29I Sodium Iodate-induced functional and structural damage in a mouse model of Geographic Atrophy (GA) is rescued by AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) compared to a negative (non-specific mab) control (AAV.anti-NS- scFvOl) as measured by ERG (A, B), OCT imaging and measurement of outer retinal layers (C-F) and H&E staining of central eye sections and measurement of ONL thickness (G-I).

[0099] FIGS. 30A-30E Anti-C5-D-mab.scFv (anti-hC5-scFv01) levels were measured from aqueous humor taps in NHPs administered the AAV8.CAG.C5-D- mab.scFv on days 15, 29, 57, and 85 (A), and at 3 months from vitreous humor (B) and from macule, distal, and promixal ocular tissues (D) collected according to the scheme (C). AAV bio-distribution was measured from ocular tissues indicated in (C) using ddPCR. Two of four eyes were collected for histological analysis.

[0100] FIG. 31 Vitreous humor from the eyes of 3 minipigs (minipig 1 (MP1),MP2, MP3) dosed SCS with 3el2 GC / eye was tested for anti-C5-D-mab.scFv (anti-hC5- scFvOl) protein levels and for inhibition of classical pathway hemolysis. In vivo expressed anti-C5-D-mab.scFv was as active as purified protein diluted in naive VH.5. DETAILED DESCRIPTION OF THE INVENTION

[0101] Compositions and methods are described for the systemic delivery of a fully human post-translationally modified (HuPTM) therapeutic anti-C5 mAb scFv to a patient (human subject) diagnosed with AMD (including dry AMD) or other indication indicated for treatment with the therapeutic scFv. Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic scFv to a patient (human subject) diagnosed with a condition indicated for treatment with the therapeutic mAb — to create a permanent depot in a tissue or organ of the patient, particularly the eye, but, in embodiments, liver or muscle, that continuously supplies the HuPTM scFv, e.g., a human-glycosylated transgene product, into ocular tissues of the subject to where the anti-C5 scFv there of exerts its therapeutic effect.

[0102] In certain embodiments, the HuPTM scFv is a HuPTMscFv that binds C5, particularly. scFv forms of C5-D-mab, C5-A-mab, C5-C-mab or C5-B-mab (see Table 5 for the VH, VL and scFv amino acid sequences and also full length and Fab forms of the antibodies)).

[0103] The compositions and methods provided herein ocularly or systemically deliver anti-C5 scFvs, particularly, scFv forms of C5-D-mab, C5-A-mab, C5-C-mab or C5- B-mab antibodies 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. Identified herein are viral vectors for delivery of transgenes encoding the therapeutic anti-C5 scFvs, 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 anti-C5 scFv thatpromote the expression of the scFv in the cells, in embodiments, in the ocular tissue cells. Such regulatory elements, including constitutive promoters, such as CAG, and ocular tissue-specific regulatory elements, are provided in Table 1 and Table la and in Example 13 (including certain modified CAG promoters) herein. Accordingly, such viral vectors may be delivered to the human subject at appropriate dosages, such that at least 20, 30, 40, 50 or 60 days after administration, the anti-C5 scFv is present at therapeutically effective levels in the serum or in ocular tissues of said human subject. In embodiments, the therapeutically effective level of the anti-C5 scFv thereof, is determined (in human trials, animal models, etc.) to improve best corrected visual acuity (BCVA) by >= 2 ETDRS lines, reduction in geographic atrophy (or slow the progression of geographic atrophy relative to untreated individual either based upon controls or natural history of the disease), reduction in drusen deposits or other metric of dry AMD.

[0104] In embodiments, the scFv comprises a VH with an amino acid sequence of SEQ ID NO: 170 and a VL with an amino acid sequence of SEQ ID NO: 171, or a VH with an amino acid sequence of SEQ ID NO: 172 and a VL with an amino acid sequence of SEQ ID NO: 173, or a VH with an amino acid sequence of SEQ ID NO: 174 and a VL with an amino acid sequence of SEQ ID NO: 175, or a VH with an amino acid sequence of SEQ ID NO: 176 and a VL with an amino acid sequence of SEQ ID NO: 177. In embodiments, the scFv has an amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 190, which may or may not include the signal sequence.

[0105] The recombinant vector used for delivering the transgene includes nonreplicating recombinant adeno-associated virus vectors (“rAAV”). rAAVs are particularly attractive vectors for a number of reasons -they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to obtain the desired tissue specificity, and / or to avoid neutralization by pre-existing patient antibodies to some AAVs. The AAV types for use here in preferentially target the eye, i.e., have a tropism for retinal cells. Such rAAVs include but are not limited to AAV basedvectors comprising capsid components from one or more of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAVhu51, AAVhu21, AAVhul2, or AAV.hu26. In certain embodiments, AAV based vectors provided herein comprise capsids from one or more of AAV3B, AAV8, AAV9, AAVrhlO, AAV10, or AAVrh73 serotypes.

[0106] However, other viral vectors may be used, including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements.

[0107] In some embodiments, the constructs express a scFv in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker, such as GGGGSGGGGSGGGGS (SEQ ID NO: 53). Exemplary non-cleavable linkers are found in Table 4. In certain embodiments, the construct expresses, from the N-terminus to C- terminus, NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In other embodiments, the construct expresses, from the N-terminus to C-terminus, NH2-signal or localization sequence- VL-linker-VH-COOH or NH2- signal or localization sequence- VH- linker-VL-COOH.

[0108] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codonoptimization 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 6 (SEQ ID NOs: 26 to 30 and 49) and these sequences may be incorporated into a transgene to encode the anti-C5 scFvs described herein. Useful signal sequences for the expression and localization of the scFvs in human cells are disclosed herein, for example in Tables 2 and 3. Exemplary recombinant expression constructs are shown in FIG. 1C.

[0109] The production of HuPTM scFv should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viralvector or other DNA expression construct encoding a HuPTM scFv derived from a therapeutic mAbto a patient (human subject) diagnosed with a disease indication for that mAb, 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 scFv should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.

[0110] Pharmaceutical compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.[I l l] As an alternative, or an additional treatment to gene therapy, the HuPTM scFv can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients. Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to human embryonic kidney 293 cells (HEK293), fibro sarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6, or RPE to name a few e.g., see Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6): 1110-1122, which is incorporated by reference in its entirety for a review of the human cell lines that could be used for the recombinant production of the HuPTM mAb or HuPTM scFv product. To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6- sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O- sulfation in human cells.

[0112] It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation)and sulfation to demonstrate efficacy. The goal of gene therapy treatment of the invention is to slow or arrest the progression of disease.

[0113] Combination therapies involving delivery of the HuPTM scFv 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 scFv.

[0114] Also provided are methods of manufacturing the viral vectors, particularly the AAV based viral vectors. In specific embodiments, provided are methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a 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 functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.5.1 CONSTRUCTS

[0115] Viral vectors or other DNA expression constructs encoding an anti-C5 scFv, including a HuPTM anti-C5 scFv, are provided herein. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to a target cell. The means of delivery of a transgene include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targetedvector, e.g., a vector targeted ocular tissue cells or a vector that has a tropism for ocular tissue cells.

[0116] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes a HuPTM scFv derived from an anti-C5 antibody disclosed hereinor 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: 44) 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: 45), Chicken Beta-actin promoter-chicken beta actin intron-rabbit beta-globin splice acceptor) with mutations (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), cytomegalovirus (CMV) promoter, EF-1 alpha promoter (SEQ ID NO: 47), mUla (SEQ ID NO: 46), UB6 promoter, chicken beta-actin (CBA) promoter, and ocular-tissue specific promoters, such as human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 48 or 137), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), or a human red opsin (RedO) promoter (SEQ ID NO: 132). See Tables 1 and la for a list of useful promoters. See also Example 13 for promoters.

[0117] In certain embodiments, provided herein are recombinant vectors that comprise one or more nucleic acids (e.g., polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., the nucleotide sequence encoding the HuPTM scFv ), untranslated regions, and termination sequences. In certain embodiments, viral vectors provided herein comprise a promoter operably linked to the gene of interest.

[0118] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).

[0119] In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) one or more control elements, b) optionally, a chicken [Cacti n or other intron and c) a rabbit 0-globin poly A signal; and (3) nucleic acid sequences coding for the scFv, , . Exemplary constructs are shown in FIGS. 1A, IB and 1C.

[0120] In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) GRK1 promoter (SEQ ID NO: 48), b) optionally, a VH4 intron (SEQ ID NO: 51) or other intron and c) a rabbit P-globin polyA signal (SEQ ID NO: 49); and (3) nucleic acid sequences coding for a scFv .5.1.1 mRNA Vectors

[0121] In certain embodiments, as an alternative to DNA vectors, the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, encoding a HuPTM scFv ). The synthesis of modified and unmodified mRNA for delivery of a transgene to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is incorporated by reference herein in its entirety. In certain embodiments, provided herein is a modified mRNA encoding for a HuPTMmAb or HuPTM scFv.5.1.2 Viral vectors

[0122] Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8, AAV9, AAVrhlO, AAV10), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors. Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HlV)-based vectors. Alphavirus vectors include semliki forest virus (SFV) and sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors providedherein are altered such that they are replication-deficient in humans. In certain embodiments, the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitus virus (VSV). In more specific embodiments, the envelope protein is VSV-G protein.

[0123] In certain embodiments, the viral vectors provided herein are HIV based viral vectors. In certain embodiments, HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.

[0124] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.

[0125] In certain embodiments, the viral vectors provided herein are MLV based viral vectors. In certain embodiments, MLV-based vectors provided herein comprise up to 8 kb of heterologous DNA in place of the viral genes.

[0126] In certain embodiments, the viral vectors provided herein are lentivirus- based viral vectors. In certain embodiments, lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, lentiviral vectors provided herein are packaged into a lentiviral capsid. In certain embodiments, lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.

[0127] In certain embodiments, the viral vectors provided herein are alphavirusbased viral vectors. In certain embodiments, alphavirus vectors provided herein are recombinant, replication-defective alphaviruses. In certain embodiments, alphavirusreplicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.

[0128] In certain embodiments, the viral vectors provided herein are AAV based viral vectors. In certain embodiments, the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of the capsid proteins) (the rep and cap proteins may be provided by the packaging cells in trans). Multiple AAV serotypes have been identified. In certain embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In preferred embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV with tropism to ocular tissues, liver and / or muscle. In certain embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAV.hu21, AAV.hul2, or AAV.hu26. In certain embodiments, AAV based vectors provided herein are or comprise components from one or more of AAV8, AAV3B, AAV9, AAV10, AAVrh73, or AAVrhlO serotypes. Provided are viral vectors in which the capsid protein is a variant of the AAV8 capsid protein (SEQ ID NO: 116), AAV3B capsid protein (SEQ ID NO: 110), or AAVrh73 capsid protein (SEQ ID NO: 112), 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: 116), AAV9 (SEQ ID NO: 117), AAV3B capsid protein (SEQ ID NO: 110), or AAVrh73 capsid protein (SEQ ID NO: 122), while retaining the biological function of the native capsid. In certain embodiments, the encoded AAV capsid has the sequence of SEQ ID NO: 116 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV8, AAV3B, or AAVrh73 capsid. FIG. 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. Accordingly, in specific embodiments, the AAV vector comprises anAAV8, AAV3B, or AAVrh73, capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions that are not present at that position in the native AAV capsid sequence as identified in the SUBS row of FIG. 3. Amino acid sequence for AAV8, AAV9, AAV3B, or AAVrh73 capsids are provided in FIG. 3.

[0129] The amino acid sequence of hu37 capsid can be found in international application PCT WO 2005 / 033321 (SEQ ID NO: 88 thereof) and the amino acid sequence for the rh8 capsid can be found in international application PCT WO 03 / 042397 (SEQ ID NO:97). The amino acid sequence for the rh64Rl sequence is found in W02006 / 110689 (a R697W substitution of the Rh.64 sequence, which is SEQ ID NO: 43 of WO 2006 / 110689).

[0130] In some embodiments, AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAVPHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAVHSC14, AAV.HSC15, or AAVHSC16 or other rAAV particles, or combinations of two or more thereof. In some embodiments, AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAVrh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAVAnc80L65, AAV7m8, AAV.PHP.B, AAVPHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVHSC1, AAV.HSC2, AAV.HSC3, AAVHSC4, AAVHSC5, AAV.HSC6, AAVHSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAVHSC11, AAV.HSC12, AAVHSC13, AAV.HSC14, AAVHSC15, or AAV.HSC16 or other rAAV particles, orcombinations of two or more thereof serotypes. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and / or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAVHSC1, AAVHSC2, AAV.HSC3, AAVHSC4, AAV.HSC5, AAV.HSC6, AAVHSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAVHSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.

[0131] In particular embodiments, the recombinant AAV for us in compositions and methods herein is AAVS3 (including variants thereof) (see e.g., US Patent Application No. 20200079821, which is incorporated herein by reference in its entirety). In particular embodiments, rAAV particles comprise the capsids of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In particular embodiments, the AAV for use in compositions and methods herein is any AAV disclosed in US 10,301,648, such as AAVrh46 or AAV.rh73. In some embodiments, the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety). In particular embodiments, the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV- PHP.B. In particular embodiments, the AAV for use in compositions and methods herein is an AAV2 / Rec2 or AAV2 / Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors). In particular embodiments, the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199;US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015 / 0374803; US 2015 / 0126588; US 2017 / 0067908; US 2013 / 0224836; US 2016 / 0215024; US 2017 / 0051257; PCT / US2015 / 034799; and PCT7EP2015 / 053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos.PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0132] In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015 / 013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014 / 172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2 / 5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017 / 070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[0133] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of 051 publication), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of '321 publication), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006 / 110689, (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38 of '097 publication), and WO 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924 publication), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of '321 publication), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication), W02010 / 127097 (see, e.g., SEQ ID NOs: 5-38 of '097 publication), and WO 2015 / 191508 (see, e g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924 publication).

[0134] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin et al., Methods 28: 158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[0135] AAV8-based, AAV3B-based, and AAVrh73-based viral vectors are used in certain of the methods described herein. Nucleotide sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV (e.g., AAV8, AAV3B, AAVrh73, or AAVrhlO)-based viral vectors encoding a transgene (e. ., 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.

[0136] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0137] In certain embodiments, the viral vectors used in the methods described herein are adenovirus based viral vectors. A recombinant adenovirus vector may be used to transfer in the transgene encoding the HuPTM scFv o. 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 : S 18-S27, which is incorporated by reference herein in its entirety.

[0138] In certain embodiments, the viral vectors used in the methods described herein are lentivirus based viral vectors. A recombinant lentivirus vector may be used to transfer in the transgene encoding the HuPTM mAb antigen binding fragment. Four plasmids are used to make the construct: Gag / pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (e.g., VSV-G), and Cis plasmid with the packaging elements and the C5 antigen-binding scFv gene.

[0139] For lentiviral vector production, the four plasmids are co-transfected into cells (e.g., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others. The lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs / should be done). The supernatant is filtered (0.45 pm) and then magnesium chloride and benzonase added. Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with / without column chromatography. Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, and Ausubel et al., 2012, “Production of CGMP- Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.

[0140] In a specific embodiment, a vector for use in the methods described herein is one that encodes an HuPTM scFv, 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.5.1.3 Promoters and Modifiers of Gene Expression

[0141] In certain embodiments, the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, thevectors 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.

[0142] In certain embodiments, the viral vectors provided herein comprise one or more promoters that control expression of the transgene. These promoters (and other regulatory elements that control transcription, such as enhancers) may be constitutive (promote ubiquitous expression) or may specifically or selectively express in the eye. In certain embodiments, the promoter is a constitutive promoter.

[0143] In certain embodiments, the promoter is a CAG promoter (SEQ ID NO: 74) (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety). In some embodiments, the CAG (SEQ ID NO: 45) or CB7 promoter (SEQ ID NO: 44) or a mutated (deleted) CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), includes other expression control elements that enhance expression of the transgene driven by the vector. In certain embodiments, the other expression control elements include chicken [3-actin intron and / or rabbit 0-globin polyA signal (SEQ ID NO: 49). In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another. In certain embodiments, the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1 , and HIV-2 LTRs.

[0144] In certain embodiments, the vectors provided herein comprise one or more tissue specific promoters (e.g., a retinal-specific promoter). In particular embodiments, theviral vectors provided herein comprises a ocular tissue cell specific promoter, such as, human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 48 or 137), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), or a human red opsin (RedO) promoter (SEQ ID NO: 132).

[0145] Provided are nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette. Regulatory elements, in general, have multiple functions as recognition sites for transcription initiation or regulation, coordination with cell-specific machinery to drive expression upon signaling, and to enhance expression of the downstream gene.

[0146] In certain embodiments, the promoter is an inducible promoter. In certain embodiments the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF-la binding site. In certain embodiments, the promoter comprises a HIF-2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG (SEQ ID NO: 232) motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schbdel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated by reference herein in its entirety. In certain embodiments, the promoter comprises a binding site for a hypoxia induced transcription factor other than a HIF transcription factor. In certain embodiments, the viral vectors provided herein comprise one or more IRES sites that is preferentially translated in hypoxia. For teachings regarding hypoxia-inducible gene expression and the factors involved therein, see, e.g., Kenneth and Rocha, Biochem J., 2008, 414: 19-29, which is incorporated by reference herein in its entirety. In specific embodiments, the hypoxia-inducible promoter is the human N-WASP promoter, see, e.g., Salvi, 2017, Biochemistry and Biophysics Reports 9: 13-21 (incorporated by reference for the teaching of the N-WASP promoter) or is the hypoxia- induced promoter of human Epo, see, e.g., Tsuchiya et al., 1993, J. Biochem. 113:395-400 (incorporated by reference for the disclosure of the Epo hypoxia-inducible promoter). In other embodiments, the promoter is a drug inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof. See, e.g., the disclosureof rapamycin inducible promoters in PCT publications WO94 / 18317, WO 96 / 20951, WO 96 / 41865, WO 99 / 10508, WO 99 / 10510, WO 99 / 36553, and WO 99 / 41258, and US 7,067,526, which are hereby incorporated by reference in their entireties for the disclosure of drug inducible promoters.

[0147] Provided herein are constructs containing certain ubiquitous and tissuespecific promoters. Such promoters include synthetic and tandem promoters. Examples and nucleotide sequences of promoters are provided in Tables 1 and la below. Table 1 also includes the nucleotide sequences of other regulatory elements useful for the expression cassettes provided herein.Table 1. Promoter and Other Regulatory Element SequencesTable la. Other regulatory sequences

[0148] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron (e.g. VH4 intron (SEQ ID NO: 51), SV40 intron (SEQ ID NO: 145), ora chimeric intron (P-globin / Ig Intron) (SEQ ID NO:50). The viral vectors may also include a Kozak sequence to promote translation of the transgene product, for example GCCACC (SEQ ID NO: 15).

[0149] In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence downstream of the coding region of the transgene. Any poly A 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 0-globin gene (SEQ ID NO: 49), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.5.1.4 Signal Peptides

[0150] In certain embodiments, the vectors provided herein comprise components that modulate protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. Signal peptides (also referred to as “signal sequences”) may also be referred to herein as “leader sequences” or “leader peptides”. In certain embodiments, the signal peptides allow for the transgene product to achieve the proper packaging (e.g., glycosylation) in the cell. In certain embodiments, the signal peptides allow for the transgene product to achieve the proper localization in the cell. In certain embodiments, the signal peptides allow for the transgene product to achieve secretion from the cell.

[0151] 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. For example, 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. For example,the signal peptide of human albumin, the most abundant protein in plasma, was found to substantially increase protein production yield in CHO cells. However, certain signal peptides may retain function and exert activity after being cleaved from the expressed protein as “post-targeting functions”. Thus, in specific embodiments, the signal peptide is selected from signal peptides of the most abundant proteins secreted by the cells used for expression to avoid the post-targeting functions. In a certain embodiment, the signal sequence is fused to both the heavy and light chain sequences. In another embodiment, 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. An exemplary sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56) which can be encoded by a nucleotide sequence of SEQ ID NO: 61 (see Table 2, FIGS 2A-2G). Alternatively, signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the HuPTM scFv in the eye / CNS, muscle, or liver are provided in Tables 2, 3, and 4, respectively, below.Table 2. Signal peptides for expression in eye / CNSTable 3. Signal peptides for expression in liver cells.5.1.5 Linkers for scFv Constructs

[0152] Flexible peptide linker. In some embodiments, a single construct can be engineered to encode both the heavy and light chains (e.g. the heavy and light chain variable domains) separated by a flexible peptide linker such as those encoding a scFv. A flexible peptide linker can be composed of flexible residues like glycine and serine so that the adjacent heavy chain and light chain domains are free to move relative to one another. The construct may be arranged such that the heavy chain variable domain is at the N- terminus of the scFv, followed by the linker and then the light chain variable domain. Alternatively, the construct may be arranged such that the light chain variable domain is at the N-terminus of the scFv, followed by the linker and then the heavy chain variable domain. That is, the components may be arranged as NH2-VL-linker-Vn-C00H or NH2- Vn-linker-VL-COOH.

[0153] In certain embodiments, an expression cassette described herein is contained within a viral vector with a restraint on the size of the polynucleotide(s) therein. In certain embodiments, the expression cassette is contained within an AAV virus-based vector. Due to the size restraints of certain vectors, the vector may or may not accommodate the coding sequences for the full heavy and light chains of the therapeutic antibody but may accommodate the coding sequences of the heavy and light chains of antigen binding fragments, such as the heavy and light chains of a Fab or F(ab’)2 fragment or an scFv. In particular, the AAV vectors described herein may accommodate a transgeneof approximately 4.7 kilobases. Substitution of smaller expression elements would permit the expression of larger protein products, such as full-length therapeutic antibodies.

[0154] Commonly used flexible linkers have sequences consisting primarily of stretches of four Gly and one Ser residue (“GS” linker), an example of the most widely used flexible linker having the sequence of (Gly-Gly-Gly-Gly-Ser)n (GGGGS or G4S; SEQ ID NO: 39). By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Examples include, but are not limited to (Gly-Gly- Gly-Gly-Ser)2 (SEQ ID NO: 40), (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 41), (Gly-Gly- Gly-Gly-Ser)4 (SEQ ID NO: 42), and (Gly-Gly-Gly-Gly-Ser)5 (SEQ ID NO: 43). Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins (Chen, X. et al, Adv Drug Deliv Rev . 2013 Oct 15; 65(10): 1357-1369). See, e.g., Table 4Table 4. Linker Sequences5.1.6 Untranslated regions

[0155] In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and / or 5’ UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half-life of the transgene. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the transgene. In certainembodiments, the UTRs are optimized for the secondary structure of the mRNA of the transgene.5.1.7 Inverted terminal repeats

[0156] In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITR is from an AAV, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety). In preferred embodiments, nucleotide sequences encoding the ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS:81 (5’-ITR) or 82 (3’-ITR). In certain embodiments, the modified ITRs used to produce self-complementary vector, e.g., scAAV, may be used (see, e. ., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety). In preferred embodiments, nucleotide sequences encoding the modified ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS: 52 (5’-ITR) or 54 (3’-ITR) or modified for scAAV, SEQ ID NO: 53 (m 5’ITR) or SEQ ID NO: 55 (m 3’ ITR).5.1.8 Transgenes

[0157] The transgenes encode a HuPTM scFv based upon a anti-C5 therapeutic antibody as disclosed herein.5.1.8.1 Constructs for Expression of scFvs

[0158] In specific embodiments for expressing an scFv form of a mAb in ocular tissue cell types, 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) optionallyan intron, such as a chicken [Lactin 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: 41). In certain embodiments, the construct expresses, from the N-terminus, NFL-signal sequence-VL-linker-VH-COOH or NFL-signal sequence- VH-linker-V,-COOH. In certain embodiments, the construct encodes, from the N-terminus, NFL-signal sequence-VL-GGGGSGGGGSGGGGS-VH-COOH or NFL-signal sequence- VH- GGGGSGGGGSGGGGS-VL-COOH. In certain embodiments, the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43). In certain embodiments, the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56) or a signal sequence from Table 2. In certain embodiments, the VH is SEQ ID NO: 170 and VL is SEQ ID NO: 171, wherein VH is SEQ ID NO: 172 and VL is SEQ ID NO: 173, wherein VH is SEQ ID NO: 174 and the VL is SEQ ID NO: 175, wherein VH is SEQ ID NO: 176 and VL is SEQ ID NO: 177. Exemplary constructs are provided in FIG. 1C and Tables 7 (amino acid sequences) and 8 (nucleotide sequences).

[0159] In specific embodiments, provided are AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 116), or, alternatively, an AAV9, AAV3B, or AAVrh73 capsid (or a variant thereof); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-C5 scFv mAb; operably linked to one or more regulatory sequences that control expression of the transgene in ocular tissue type cells, such as RPE cells, BrM cells, choriocapillaris cells, photoreceptor cells (rods and / or cones), retinal ganglion cells.

[0160] In embodiments, the transgenes express scFv based upon an anti-C5 therapeutic antibody as disclosed herein. Section 5.4. and Table 5 provide the amino acid sequence of the heavy and light chains variable domains of scFv and the scFvs themselves.Certain of these nucleotide sequences are codon optimized for expression in human cells. See for example, the codon optimized sequences encoding C5-D-mab (SEQ ID NOs: 16, 17, 27, 28) or C5-A-mab (SEQ ID Nos: 18-20, and 29-31) in Table 6.Transgenes

[0161] The HuPTM scFv encoded by the transgene can include, but is not limited to, a scFv form of an antibody that binds to C5, including but not limited to C5-A-mab.scFv, C5-B-mab.scFv, C5-C-mab.scFv or C5-D-mab.scFv. The amino acid sequences of the VH and VL and the scFvs are provided in Table 5, infra.C5-A-scFv constructsAA sequences

[0162] In embodiments, the transgene encodes an scFv that binds to C5 comprising a variable heavy domain (VH) of SEQ ID NO: 172, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a variable light domain (VL) of SEQ ID NO: 173 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5 and FIG. 2B) In embodiments, the scFv that binds to C5 comprises a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 172 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B). In specific embodiments, the scFv that binds to C5 comprises a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 173 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B).

[0163] In embodiments, the VH and VL domains are connected by a flexible linker (e.g., one of SEQ ID NOs: 39-43). In embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 56).

[0164] In embodiments, the transgene has a signal or leader sequence at the N- terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence comprises the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56). In embodiments, the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Table 2. Alternatively, the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.

[0165] In embodiments, the scFv that binds to C5 has the structure: signal sequence- VH - linker - VL. In embodiments the scFv that binds to C5 comprises a VH domain having an amino acid sequence of SEQ ID NO: 172, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VL domain having an amino acid sequence of SEQ ID NO: 173, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0166] In embodiments, the scFv that binds to C5 has the structure: signal sequence- VL - linker - VH. In embodiments the scFv that binds to C5 comprises a VL domain having an amino acid sequence of SEQ ID NO: 173, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VH domain having an amino acid sequence of SEQ ID NO: 172 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0167] In embodiments, the scFvs have an amino acid sequence of SEQ ID NO: 180 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or SEQ ID NO: 264, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table5) and bind to C5. The nucleotide sequences encoding the scFvs may be codon optimized for expression in human cells.

[0168] In embodiments, the transgene encodes an scFv comprising a VH domain comprising the three CDRs that are underlined in the C5-A-mab heavy chain sequence of FIG. 2B and a VL domain having the three CDRs that are underlined in the C5-A- mab light chain sequence of FIG. 2B. In embodiments, the VH domain and the VL domain have alternate framework regions (i.e., framework regions not found or only partially found in the sequences of FIG. 2B, which are known in the art, including, but not limited to, human framework regions.Transgene sequences

[0169] In certain embodiments, anti-C5 scFv transgene comprises the nucleotide sequence of the first 369 nucleotides of SEQ ID NO: 18 or 19 (encoding the C5-A- scFv variable heavy domain), or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto which encodes an scFv which binds C5, and the first 324 nucleotides of SEQ ID NO: 20 (encoding the C5-A-scFv variable light domain) or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto and encodes and scFv which binds C5, as set forth in Table 6. In embodiments, the nucleotide sequences encoding the variable light domain and variable heavy domain are separated by a nucleotide sequence encoding a flexible linker, for example, one having an amino acid sequence of one of SEQ ID NOs: 39-43. In embodiments, the nucleotide sequence encoding the scFv (including, in embodiments, the signal sequence at the N-terminus) is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells. In embodiments, the one or more regulatory elements is a constitutive promoter. In embodiments, the one or more regulatory elements is a tissue specific promoter. In embodiments, the transgene is operably linked to a CAG promoter (SEQ ID NO: 45), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), and / or a CB promoter or CB long promoter (SEQ ID NO: 142 or 143). In embodiments, the promoter is a tissue specific promoter (or regulatorysequence including promoter and enhancer elements) such as the GRK 1 promoter (SEQ ID NO: 48 or 137), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), a human red opsin (RedO) promoter (SEQ ID NO: 132) or a Bestl / GRKl tandem promoter (SEQ ID NO: 144). In embodiments, an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence (SEQ ID NO: 70). The transgenes may further comprise a polyadenylation signal after the 3’ end of the coding sequence. The transgenes may contain elements provided in Table 1 or la. Exemplary transgenes encoding scFv forms of C5-A-mab, which are provided in Table 6, include C5-A-mab.scFv coding sequences (SEQ ID NO: 230 or 231). Exemplary expression cassette coding sequences include SEQ ID NO: 235 and 236. Exemplary artificial genome coding sequences include SEQ ID NO: 233, or 234. The artificial genome comprising the transgene may be packaged into an AAV capsid, particularly AAV8 or AAV3B.

[0170] In certain embodiments, provided is a construct encoding an artificial genome in which the transgene is operably linked to regulatory sequences and a poly A tail (for example, the expression cassettes described above) and wherein the expression cassette is flanked by ITR sequences. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single stranded. The artificial genome may comprise or consist of the nucleotide sequence of SEQ ID NO: 233 or 234. The artificial genome may comprise 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 NO: 233 or 234, which encodes and expresses an anti-C5 scFv as described herein.

[0171] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucleotides of SEQ ID NO: 30. In certain embodiments, theanti-C5 scFv transgene encodes a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29. In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucleotides of SEQ ID NO: 30 and a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29. In specific embodiments, the anti-C5 scFv comprises a variable heavy domain comprising a nucleotide sequence of the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B). In specific embodiments, the anti-C5 scFv comprises a variable light domain comprising a nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 30 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B).C5-B-scFv constructsA A sequences

[0172] In embodiments, the transgene encodes an scFv that binds to C5 comprising a variable heavy domain (VH) of SEQ ID NO: 176, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a variable light domain (VL) of SEQ ID NO: 258 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5 and FIG. 2E). In embodiments, the scFv that binds to C5 comprises a heavy chain variabledomain comprising an amino acid sequence of SEQ ID NO: 177 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2E). In specific embodiments, the scFv that binds to C5 comprises a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 177 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2E).

[0173] In embodiments, the VH and VL domains are connected by a flexible linker (e.g., one of SEQ ID NOs: 39-43). In embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 41).

[0174] In embodiments, the transgene has a signal or leader sequence at the N- terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence comprises the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56). In embodiments, the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Table 2. Alternatively, the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.

[0175] In embodiments, the scFv that binds to C5 has the structure: signal sequence - VH - linker - VL. In embodiments the scFv that binds to C5 comprises a VH domain having an amino acid sequence of SEQ ID NO: 176, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VL domain having an amino acid sequence of SEQ ID NO: 177, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0176] In embodiments, the scFv that binds to C5 has the structure: signal sequence - VL - linker - VH. In embodiments the scFv that binds to C5 comprises a VL domain having an amino acid sequence of SEQ ID NO: 177, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VH domain having an amino acid sequence of SEQ ID NO: 176 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0177] In embodiments, the scFvs have an amino acid sequence of SEQ ID NO: 187 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or SEQ ID NO: 271, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5) and bind to C5. The nucleotide sequences encoding the scFvs may be codon optimized for expression in human cells.

[0178] In embodiments, the transgene encodes an scFv comprising a VH domain comprising the three CDRs that are underlined in the C5-B-mab heavy chain sequence of FIG. 2E and a VL domain having the three CDRs that are underlined in the C5-B- mab light chain sequence of FIG. 2E. In embodiments, the VH domain and the VL domain have alternate framework regions (i.e., framework regions not found or only partially found in the sequences of FIG. 2E, which are known in the art, including, but not limited to, human framework regions.Transgene sequences

[0179] In certain embodiments, anti-C5 scFv transgene comprises the nucleotide sequence of the first 348 nucelotides of SEQ ID NO: 23 (encoding the C5-B-scFv variable heavy domain), or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto which encodes an scFv which binds C5, and the first 324 nucleotides of SEQ ID NO: 34 (encoding the C5-B-scFv variable light domain) or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical thereto and encodes and scFv which binds C5, as set forth in Table 6. In embodiments, the nucleotide sequences encoding the variable light domain and variable heavy domain are separated by a nucleotide sequence encoding a flexible linker, for example, one having an amino acid sequence of one of SEQ ID NOs: 39-43. In embodiments, the nucleotide sequence encoding the scFv (including, in embodiments, the signal sequence at the N-terminus) is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells. In embodiments, the one or more regulatory elements is a constitutive promoter. In embodiments, the one or more regulatory elements is a tissue specific promoter. In embodiments, the transgene is operably linked to a CAG promoter (SEQ ID NO: 45), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), and / or a CB promoter or CB long promoter (SEQ ID NO: 142 or 143). In embodiments, the promoter is a tissue specific promoter (or regulatory sequence including promoter and enhancer elements) such as the GRK1 promoter (SEQ ID NO: 48 or 137), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 214-216), a human red opsin (RedO) promoter (SEQ ID NO: 132) or a Bestl / GRKl tandem promoter (SEQ ID NO: 144). In embodiments, an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence (SEQ ID NO: 51). The transgenes may further comprise a polyadenylation signal after the 3’ end of the coding sequence. The transgenes may contain elements provided in Table 1 or la. Exemplary transgenes encoding scFv forms of C5-B-mab, which are provided in Table 6, include C5-B- mab.scFv coding sequences (SEQ ID NO: 226 or 227). The artificial genome comprising the transgene may be packaged into an AAV capsid, particularly AAV8 or AAV3B.

[0180] In certain embodiments, provided is a construct encoding an artificial genome in which the transgene is operably linked to regulatory sequences and a poly A tail (for example, the expression cassettes described above) and wherein the expression cassette is flanked by ITR sequences. In some embodiments, the artificialgenome is self-complementary. In some embodiments, the artificial genome is single stranded.

[0181] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucleotides of SEQ ID NO: 24. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 348 nucelotides of SEQ ID NO: 23. In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucleotides of SEQ ID NO: 34 and a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 348 nucelotides of SEQ ID NO: 23. In specific embodiments, the anti-C5 scFv comprises a variable heavy domain comprising a nucleotide sequence of the first 348 nucelotides of SEQ ID NO: 23 with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more nucleotide substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2E). In specific embodiments, the anti-C5 scFv comprises a variable light domain comprising a nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2E).C5-C-scFv constructsAA sequences

[0182] In embodiments, the transgene encodes an scFv that binds to C5 comprising a variable heavy domain (VH) of SEQ ID NO: 174, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a variable light domain (VL) of SEQ ID NO: 175 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5 and FIG. 2D) In embodiments, the scFv that binds to C5 comprises a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 174 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D). In specific embodiments, the scFv that binds to C5 comprises a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 175 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D).

[0183] In embodiments, the VH and VL domains are connected by a flexible linker (e.g., one of SEQ ID Nos: 39-43). In embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 41).

[0184] In embodiments, the transgene has a signal or leader sequence at the N- terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence comprises the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56). In embodiments, the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Table 2. Alternatively, the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.

[0185] In embodiments, the scFv that binds to C5 has the structure: signal sequence - VH - linker - VL. In embodiments the scFv that binds to C5 comprises a VH domain having an amino acid sequence of SEQ ID NO: 174, or a sequence at least about 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VL domain having an amino acid sequence of SEQ ID NO: 175, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0186] In embodiments, the scFv that binds to C5 has the structure: signal sequence - VL - linker - VH. In embodiments the scFv that binds to C5 comprises a VL domain having an amino acid sequence of SEQ ID NO: 175, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VH domain having an amino acid sequence of SEQ ID NO: 174 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0187] In embodiments, the scFvs have an amino acid sequence of SEQ ID NO: 265 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto or SEQ ID NO: 266, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5) and bind to C5. The nucleotide sequences encoding the scFvs may be codon optimized for expression in human cells.

[0188] In embodiments, the transgene encodes an scFv comprising a VH domain comprising the three CDRs that are underlined in the C5-C-mab heavy chain sequence of FIG. 2D and a VL domain having the three CDRs that are underlined in the C5-C- mab light chain sequence of FIG. 2D. In embodiments, the VH domain and the VL domain have alternate framework regions (i.e., framework regions not found or only partially found in the sequences of FIG. 2D, which are known in the art, including, but not limited to, human framework regions.Transgene sequences

[0189] In certain embodiments, anti-C5 scFv transgene comprises the nucleotide sequence of the first 366 nucleotides of SEQ ID NO: 21 (encoding the C5-C-scFv variable heavy domain), or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto which encodes an scFv which binds C5, and the first 324 nucleotides of SEQ ID NO: 22 (encoding the C5-C-scFv variable light domain) or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto and encodes and scFv which binds C5, as set forth in Table 6. In embodiments, the nucleotide sequences encoding the variable light domain and variable heavy domain are separated by a nucleotide sequence encoding a flexible linker, for example, one having an amino acid sequence of one of SEQ ID NOs: 39-43. In embodiments, the nucleotide sequence encoding the scFv (including, in embodiments, the signal sequence at the N-terminus) is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells. In embodiments, the one or more regulatory elements is a constitutive promoter. In embodiments, the one or more regulatory elements is a tissue specific promoter. In embodiments, the transgene is operably linked to a CAG promoter (SEQ ID NO: 45), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), and / or a CB promoter or CB long promoter (SEQ ID NO: 142 or 143). In embodiments, the promoter is a tissue specific promoter (or regulatory sequence including promoter and enhancer elements) such as the GRK1 promoter (SEQ ID NO: 48 or 137), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-146), a human red opsin (RedO) promoter (SEQ ID NO: 212) or a Bestl / GRKl tandem promoter (SEQ ID NO: 114). In embodiments, an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence (SEQ ID NO: 51). The transgenes may further comprise a polyadenylation signal after the 3’ end of the coding sequence. The transgenes may contain elements provided in Table 1 or la. Exemplary transgenes encoding scFv forms of C5-C-mab, which are provided in Table 6, include C5-C- mab.scFv coding sequences (SEQ ID NO: 228 or 229). The artificial genomecomprising the transgene may be packaged into an AAV capsid, particularly AAV8 or AAV3B.

[0190] In certain embodiments, provided is a construct encoding an artificial genome in which the transgene is operably linked to regulatory sequences and a poly A tail (for example, the expression cassettes described above) and wherein the expression cassette is flanked by ITR sequences. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single stranded.

[0191] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucelotides of SEQ ID NO: 22. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 366 nucleotides of SEQ ID NO: 21. In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 324 nucelotides of SEQ ID NO: 22 and a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 366 nucleotides of SEQ ID NO: 21. In specific embodiments, the anti-C5 scFv comprises a variable heavy domain comprising a nucleotide sequence of the first 366 nucleotides of SEQ ID NO: 31 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D). In specific embodiments, the anti-C5 scFv comprises a variable light domain comprising a nucleotide sequence of the first 324 nucelotides of SEQ ID NO: 22 with 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D).C5-D-scFv constructsAA sequences

[0192] In embodiments, the transgene encodes an scFv that binds to C5 comprising a variable heavy domain (VH) of SEQ ID NO: 170, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a variable light domain (VL) of SEQ ID NO: 171 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5 and FIG. 2A). In embodiments, the scFv that binds to C5 comprises a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 170 with 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A). In specific embodiments, the scFv that binds to C5 comprises a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 171 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A).

[0193] In embodiments, the VH and VL domains are connected by a flexible linker (e.g., one of SEQ ID NOs: 839-43). In embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 41).

[0194] In embodiments, the transgene has a signal or leader sequence at the N- terminus appropriate for expression and secretion in human cells, in particular, human ocular tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence comprises the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56). In embodiments, the signal sequence may have an amino acidsequence selected from any one of the signal sequences set forth in Table 2. Alternatively, the signal sequence may be appropriate for expression in muscle or liver cells, such as those listed in Table 3 infra.

[0195] In embodiments, the scFv that binds to C5 has the structure: signal sequence- VH - linker - VL. In embodiments the scFv that binds to C5 comprises a VH domain having an amino acid sequence of SEQ ID NO: 170, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VL domain having an amino acid sequence of SEQ ID NO: 171, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0196] In embodiments, the scFv that binds to C5 has the structure: signal sequence- VL - linker - VH. In embodiments the scFv that binds to C5 comprises a VL domain having an amino acid sequence of SEQ ID NO: 171, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, at the N- terminus, a flexible linker (see Table 4, for example having an amino acid sequence of one of SEQ ID NOs: 39-43), and a VH domain having an amino acid sequence of SEQ ID NO: 170 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0197] In embodiments, the scFvs have an amino acid sequence of SEQ ID NO: 261 or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto or SEQ ID NO: 179, or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto (see Table 5) and bind to C5. The nucleotide sequences encoding the scFvs may be codon optimized for expression in human cells.

[0198] In embodiments, the transgene encodes an scFv comprising a VH domain comprising the three CDRs that are underlined in the C5-D-mab heavy chain sequence of FIG. 2A and a VL domain having the three CDRs that are underlined in the C5-D-mab light chain sequence of FIG. 2A. In embodiments, the VH domain and the VL domain have alternate framework regions (i.e., framework regions not found or only partially found in the sequences of FIG. 2A, which are known in the art, including, but not limited to, human framework regions.Transgene sequences

[0199] In certain embodiments, anti-C5 scFv transgene comprises the nucleotide sequence of the first 369 or 372 nucleotides of SEQ ID NO: 16 (encoding the C5-D- scFv variable heavy domain), or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto which encodes an scFv which binds C5, and the first 333 nucleotides of SEQ ID NO: 17 (encoding the C5-D-scFv variable light domain) or a sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto and encodes and scFv which binds C5, as set forth in Table 6. In embodiments, the nucleotide sequences encoding the variable light domain and variable heavy domain are separated by a nucleotide sequence encoding a flexible linker, for example, one having an amino acid sequence of one of SEQ ID NOs: 39-43. In embodiments, the nucleotide sequence encoding the scFv (including, in embodiments, the signal sequence at the N-terminus) is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells. In embodiments, the one or more regulatory elements is a constitutive promoter. In embodiments, the one or more regulatory elements is a tissue specific promoter. In embodiments, the transgene is operably linked to a CAG promoter (SEQ ID NO: 45), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), and / or a CB promoter or CB long promoter (SEQ ID NO: 142 or 143). In embodiments, the promoter is a tissue specific promoter (or regulatory sequence including promoter and enhancer elements) such as the GRK1 promoter (SEQ ID NO: 48 or 137), (a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), a human red opsin (RedO) promoter (SEQ ID NO: 212) or a Bestl / GRKl tandem promoter (SEQ ID NO: 144). In embodiments, an intron sequence is positioned between the promoter and the coding sequence, for example a VH4 intron sequence(SEQ ID NO: 52). The transgenes may further comprise a polyadenylation signal after the 3’ end of the coding sequence. The transgenes may contain elements provided in Table 1 or la. Exemplary transgenes encoding scFv forms of C5-D-mab, which are provided in Table 6, include C5-D-mab.scFv coding sequences (SEQ ID NO: 184, 194, or 197). Exemplary expression cassette coding sequences include SEQ ID NO: 186, 195, 198, 200, 202, 204, 206, and 208. Exemplary artificial genome coding sequences include SEQ ID NO: 185, 196, 199, 201, 203, 205, 207, or 209. The artificial genome comprising the transgene may be packaged into an AAV capsid, particularly AAV8 or AAV3B.

[0200] In certain embodiments, provided is a construct encoding an artificial genome in which the transgene is operably linked to regulatory sequences and a poly A tail (for example, the expression cassettes described above) and wherein the expression cassette is flanked by ITR sequences. In some embodiments, the artificial genome is self- complementary. In some embodiments, the artificial genome is single stranded. The artificial genome may comprise or consist of the nucleotide sequence of SEQ ID NO: 185, 196, 199, 201, 203, 205, 207, or 209. The artificial genome may comprise 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 NO: 185, 196, 199, 201, 203, 205, 207, or 209, which encodes and expresses an anti-C5 scFv as described herein.

[0201] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 333 nucleotides of SEQ ID NO: 17. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 369 or 372 nucleotides of SEQ ID NO: 16. In certain embodiments, the anti-C5 scFv transgene encodes a variable light domaincomprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 333 nucleotides of SEQ ID NO: 17 and a variable heavy domain comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the first 369 or 372 nucleotides of SEQ ID NO: 16. In specific embodiments, the anti-C5 scFv comprises a variable heavy domain comprising a nucleotide sequence of the first 369 or 372 nucleotides of SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A). In specific embodiments, the anti-C5 scFv comprises a variable light domain comprising a nucleotide sequence of the first 333 nucleotides of SEQ ID NO: 17 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A).

[0202] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or inducible (e.g., hypoxiainducible 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 scFv). 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). In certain embodiments, 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 HuGlyscFv), wherein the transgene comprises anucleotide sequence encoding a signal peptide, a variable light chain and a heavy chain separated by a linker.

[0203] In certain embodiments, 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 HuGlyscFv), 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.

[0204] In certain embodiments, 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., HuGlyscFv), 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 variable domain and a heavy chain variable domain separated by a flexible linker sequence.

[0205] In certain embodiments, 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.5.1.10 Manufacture and testing of vectors

[0206] The viral vectors provided herein may be manufactured using host cells. The viral vectors provided herein may be manufactured using mammalian host cells, forexample, A549, WEHI, 10T1 / 2, BHK, MDCK, C0S1, 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.

[0207] The host cells are stably transformed with the sequences encoding the transgene and associated elements (e.g., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes e.g., the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCb sedimentation.

[0208] Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054 which is incorporated by reference herein in its entirety for manufacturing techniques.

[0209] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. In addition, in vitro neutralization assays can be used to measure the activity of the transgene expressed from a vector described herein. For example, 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. In addition, other characteristics of the expressed product can be determined, for example determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlyscFv. In addition, benefits resulting from glycosylation / sulfation of the cell-expressed HuGlyscFv can be determined using assays known in the art, e.g., the methods described in Section 5.3.

[0210] Vector genome concentration (GC) or vector genome copies can be evaluated using digital PCR (dPCR) or ddPCR™ (BioRad Technologies, Hercules, CA, USA). In one example, ocular tissue samples, such as aqueous and / or vitreous humor samples, are obtained at several timepoints. In another example, several mice are sacrificed at various timepoints post injection. Ocular tissue samples are subjected to total DNA extraction and dPCR assay for vector copy numbers. Copies of vector genome (transgene) per gram of tissue may be measured in a single biopsy sample, or measured in various tissue sections at sequential timepoints will reveal spread of AAV throughout the eye. Total DNA from collected ocular fluid or tissue is extracted with the DNeasy Blood & Tissue Kit and the DNA concentration measured using a Nanodrop spectrophotometer. To determine the vector copy numbers in each tissue sample, digital PCR is performed with Naica Crystal Digital PCR system (Stilla technologies). Two color multiplexing system is applied to simultaneously measure the transgene AAV and an endogenous control gene. In brief, the transgene probe can be labelled with FAM (6-carboxyfluorescein) dye while the endogenous control probe can be labelled with VIC fluorescent dye. The copy number of delivered vector in a specific tissue section per diploid cell is calculated as: (vector copy number) / (endogenous control)*2. Vector copy in specific cell types or tissues, such as cornea, iris, ciliary body, schlemm’s canal cells, trabecular meshwork, retinal cells, RPE cells, RPE-choroid tissue, or optic nerve cells, over time may indicate sustained expression of the transgene by the tissue.5.1.11 Compositions

[0211] Pharmaceutical compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil. In some embodiments, the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ as known in the art. The pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0212] In some embodiments, 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.

[0213] In some embodiments, 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 methylcellulose (HPMC), HPMC, hydroxyethyl cellulose (HES), CMC low viscosity grade, and / or poloxamer 407.

[0214] In some embodiments, the pharmaceutical composition comprises hyaluronic acid as a viscosity promoting agent. In some embodiments, 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. In some embodiments, the hyaluronic acid comprises a molecular weight of about 1.58 MDa. In some embodiments, the hyaluronic acid comprises a molecular weight of about 2.0 MDa. In embodiments the HAmay 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 A.Table A: Several available sources for HA

[0215] In embodiments, 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.

[0216] In embodiments, 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.

[0217] In embodiments, 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.

[0218] In embodiments, 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.

[0219] In embodiments, 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.

[0220] In embodiments, 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, about25.0 mg / mL (2.5% w / v) sucrose, about 0.002% (0.02 mg / mL) poloxamer 188 and about 0.7% w / v hyaluronic acid.

[0221] In embodiments, 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.

[0222] In embodiments, 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.

[0223] In embodiments, 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.

[0224] In embodiments, 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.

[0225] In embodiments, 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.

[0226] In embodiments, the pharmaceutical formulation is in a sucrose containing formulation, such as those found in WO2021071835A1, which is hereby incorporated byreference in its entireity. In embodiments, the AAV is the pharmaceutical composition comprising: (a) a recombinant adeno-associated virus (AAV), (b) potassium chloride, (c) potassium phosphate monobasic, (d) sodium chloride, (e) sodium phosphate dibasic anhydrous, (f) sucrose, and (e) poloxamer 188, polysorbate 20, or polysorbate 80. In embodiments, the AAV is the pharmaceutical composition comprising (a) potassium chloride at a concentration of 0.2 g / L, (b) potassium phosphate monobasic at a concentration of 0.2 g / L, (c) sodium chloride at a concentration of 5.84 g / L, (d) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (e) sucrose at a concentration of 4% weight / volume (40 g / L), and (f) poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of 0.001% weight / volume (0.01 g / L).

[0227] In embodiments, the pharmaceutical formulation is in a formulation such as those found in WO2022076591A1, which is hereby incorporated by reference in its entireity. In embodiments, the pharmaceutical composition comprises an ionic strength of at most about 200 mM and at least about 3% aggregated recombinant AAV prior to suprachoroidal administration.

[0228] In embodiments, the pharmaceutical formulation is in a high viscosity formulation such as those found in WO2022076549A1, which is hereby incorporated by reference in its entireity. In embodiments, the pharmaceutical composition has viscosity of between about 25 cP to about 3 x 106 cP as measured at a shear rate of at most about 1 s- 1 and comprises at least one of sucrose, 4% sucrose, 6% sucrose, 10% sucrose, 2% carboxymethyl cellulose sodium salt, 1% carboxymethyl cellulose sodium salt, carboxymethyl cellulose (CMC), 0.5% CMC, 1% CMC, 2% CMC, 4% CMC, hyaluronic acid, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose sodium salt, and hydroxypropyl methylcellulose.

[0229] In embodiments, the pharmaceutical formulation is in a gel formulation such as those found in WO2022076595A1, which is hereby incorporated by reference in its entireity. In embodiments, the pharmaceutical composition has a viscosity and / or higher elastic modulus that increases with increasing temperature and optionally the pharmaceutical composition contains poloxamer 407 and poloxamer 188.5.2 Methods of Treating dry AMD

[0230] In another aspect, methods for treating dry AMD (age-related AMD) or other indication that can be treated with an anti-C5 antibody in a subject in need thereof (a composition for use in the treatment of dry AMD or other indication that can be treated with an anti-C5 antibody) comprising the administration of recombinant AAV particles comprising an expression cassette encoding an anti-C5 scFv as described herein, are provided. 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, or C5-B-mab. In particular embodiments, the methods encompass treating patients who have been diagnosed with dry AMD, and, in certain embodiments, identified as responsive to treatment with an anti-C5 antibody, or considered a good candidate for therapy with an anti-C5 antibody. In specific embodiments, the patients have previously been treated with an anti-C5 antibody. To determine responsiveness, the anti-C5 antibody scFv transgene product, (e.g., produced in human cell culture, bioreactors, etc.) may be administered directly to the subject.

[0231] In specific embodiments, provided are methods of 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 an scFv derived from C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab, 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, chori ocapil laris cells, RPE cells and / or choroid cells), so that a depot is formed that releases the scFv. Subretinal, intravitreal, intracameral, or suprachoroidal administration should result in expression of the transgene product in one or more of the following retinal celltypes: 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 schl emm’s canal cells, a trabecular meshwork cells, RPE-choroid tissue cells, or optic nerve cells.

[0232] Recombinant vectors and pharmaceutical compositions for treating diseases or disorders in a subject in need thereof are described in Section 5.1. Such vectors should have a tropism for human ocular tissue, or liver and / or muscle cells and can include nonreplicating rAAV, particularly those bearing an AAV3B, AAV8, AAAV9, AAV10, AAVrhlO, orAAVrh73 capsid. The recombinant vectors can be administered in any manner such that the recombinant vector enters ocular tissue cells, e.g., by introducing the recombinant vector into the eye. Such vectors comprise recombinant genomes encoding the anti-Cf scFv and 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:48 or 137), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), a human red opsin (RedO) promoter (SEQ ID NO: 132), a CAG promoter (SEQ ID NO: 45), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), a CB promoter or CBlong promoter (SEQ ID NO: 142 or 143) or a Bestl / GRKl tandem promoter (SEQ ID NO: 144) (see also Tables 1 and la).

[0233] The methods described herein treat, slow the progression of, reduce the severity of or prevent dry (age related) AMD in a human subject in need of the treatment. The treatment, slowing progression of, reduction of severity or prevention may be assessed relative to the subject prior to treatment, a comparable untreated subject or according to the natural history of the disease. In embodiments, 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 progressionand / 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.

[0234] 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.Gene Therapy Methods

[0235] Provided are methods of treating human subjects for dry AMD by administration of a viral vector containing a transgene encoding an anti-C5 antibody, or antigen binding fragment thereof, or CFHL-1 protein. The antibody may be C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab, and is, e.g., a full length or substantially full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof. The viral vector has an AAV capsid with tropism for human ocular tissues and may be an AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof, for example having 90%, 95% or 99% sequence identity to the capsid sequence of AAV8, AAV9, AAV3B, or AAVrh73). The transgene is operably linked by regulatory sequences that promote expression of the transgene in human ocular tissue cells (including in retinal cells, RPE, choroid, BrM, choriocapillaris, photoreceptor cells, retinal ganglion cells), for example a CAG (SEQ ID NO: 45) promoter or a mutated CAG promoter (SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID NO: 224), or ocular specific promoter, such as a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS:48 or 137), a mouse cone arresting (CAR) promoter (SEQ ID NOS: 134-136), a human red opsin (RedO) promoter (SEQ ID NO: 132) or a Bestl / GRKl tandem promoter (SEQ ID NO: 144). Regulatory sequences may also include polyadenylation signal sequences. The expression cassette comprising the transgene and operably linked regulatory sequences are flanked by ITR sequences, as an artificial AAV genome. The flanking ITR sequences may be configured to provide a self-complementary AAV (scAAV) genome. The recombinant vectors, including those as shown in FIGS. 2A- 2G, can be administered in any manner such that the recombinant vector enters one or more ocular tissue cells. In particular embodiments, the recombinant AAV comprises an artificialgenome of (or is produced using a cis plasmid or construct comprising) CAG C5-D- mab.scFv (SEQ ID NO: 186). In particular embodiments, 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: 184 or SEQ ID NO: 185 or SEQ ID NO: 186 or SEQ ID NO: 194 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 197 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209). In certain embodiments, the artificial genome is self complementary. In other embodiments, the recombinant AAV comprises a construct comprising a transgene encoding a surrogate anti-C5 antibody, or antigen binding fragment thereof, including BB5.1, for use in animal model, such as non-human primate, testing of anti-C5 antibodies. Constructs encoding a BB5.1 antibody include CAG.BB5.1 (SEQ ID NO: 48).

[0236] Provided also are method of administering recombinant AAV vectors comprising a transgene which is a scFv. In some embodiments, the transgene encodes an scFv with the structure: signal sequence - VH - linker - VL - poly A. In some embodiments, the transgene encodes an scFv with the structure: signal sequence - VL - linker - VH - poly A. In some embodiments, the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) orGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43). In some embodiments, the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56) or a signal sequence from Table 2. In some embodiments, the VH is SEQ ID NO: 170 and VL is SEQ ID NO: 171 wherein VH is SEQ ID NO: 172 and VL is SEQ ID NO: 173, wherein VH is SEQ ID NO: 174 and the VL is SEQ ID NO: 175, wherein VH is SEQ ID NO: 176 and VL is SEQ ID NO: 177.

[0237] Therapeutically effective doses of any of these recombinant vectors should be administered in any manner such that the recombinant vector enters ocular tissue cells (e.g., retinal cells), e.g., via subretinal, intravitreal, intracameral, or suprachoroidalinjection or intranasal administration. Alternatively, the vector is administered peripherally (for example, intravenously, intramuscularly or subcutaneously) such that the recombinant vector transduces liver and / or muscle cells, creating a depot in liver and / or muscle tissue which express the transgene product into the bloodstream, delivering the therapeutic to ocular tissues. Alternatively, subretinal, intravitreal, intracameral, suprachoroidal administration should result in expression of the transgene product in cells of the eye, creating a depot in one or more ocular tissue cells of the patient that continuously supplies the anti-C5 HuPTM mAb, or antigen binding fragment of the anti-C5 mAb to ocular tissues of the subject. The transgene expression results in therapeutically effective levels of the anti-C5 antibody or antigen-binding fragment thereof in the aqueous humor, the vitreous humor, retinal tissue, the RPE, the BrM or choriocapillaris.

[0238] Subjects to whom such gene therapy is administered can be those responsive to anti-complement therapy. In certain embodiments, the methods encompass treating patients who have been diagnosed with dry AMD, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-C5 antibody, or considered a good candidate for therapy with an anti-C5 antibody or CFHL-1 protein. In specific embodiments, 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. To determine responsiveness, the anti-C5 transgene product (e.g., produced in cell culture, bioreactors, etc.) may be administered directly to the subject.

[0239] In embodiments, administration of the recombinant AAV comprising a construct for expressing a transgene encoding an anti-C5 antibody or antigen-binding fragment thereof, in ocular tissues results in reduction or slowing the progression of one or more symptoms of dry AMD within 10 days, 20 days, 30 days, 40 days, 6 months, 9 months or 1 year after administration of the AAV. In embodiments, the administration results in a slowing or reduction in the rate of the progression of geographic atrophy, including of the fovea, in the subject relative to an untreated subject or as expected in the subject based upon natural history of dry AMD, for example as measured by fundus autofluorescence (FAF). In embodiments, the administration results in an improvement or reduction in therate of loss of visual acuity or best corrected visual acuity (BCVA), for example, as measured by a standard ETDRS chart or to improve visual function as measured by dark adaptation methodology; to improve contrast sensitivity by the Pelli-Robson test or to reduce the drusen area or accumulation of drusen. In other embodiments, the dose of therapeutic gene delivered by gene therapy is sufficient to inhibit complement activation without exacerbating choroidal neovascularization (CNV).

[0240] However, in all cases because the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective. The concentration of the transgene product can be measured in patient blood serum samples.

[0241] Pharmaceutical compositions suitable for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic (intravenous, intramuscular or subcutaneous) administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-C5 antibody, or antigen-binding fragment thereof, 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.

[0242] In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and / or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and / or sulfated. The goal of gene therapy treatment provided herein is to slow or arrest the progression of or relieve one or more symptoms of dry AMD, such as to reduce the rate of geographic atrophy or improve visual acuity (or reduce the rate of loss of visual acuity).

[0243] Combinations of delivery of the anti-C5 HuPTM mAb or antigen-binding fragment thereof, 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 withthe 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.5.3.N-GLYCOSYLATION, TYROSINE SULFATION, AND O-GLYCOSYLATION

[0244] The amino acid sequence (primary sequence) of HuPTM scFvs disclosed herein each comprises at least one site at which N-glycosylation or tyrosine sulfation takes place for glycosylation and / or sulfation positions within the amino acid sequences of the Fab fragments of the therapeutic antibodies).5.3.1. N-GlycosylationReverse Glycosylation Sites

[0245] The canonical N-glycosylation sequence is known in the art to be Asn-X- Ser(or Thr), wherein X can be any amino acid except Pro. However, it recently has been demonstrated that asparagine (Asn) residues of human antibodies can be glycosylated in the context of a reverse consensus motif, Ser(or Thr)-X-Asn, wherein X can be any amino acid except Pro. See Valliere-Douglass et al., 2009, J. Biol. Chem. 284:32493-32506; and Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022. As disclosed herein, certain HuGlyFabs and HuPTM scFvs disclosed herein comprise such reverse consensus sequences.Non-Consensus Glycosylation Sites

[0246] In addition to reverse N-glycosylation sites, it recently has been demonstrated that glutamine (Gin) residues of human antibodies can be glycosylated in the context of a non-consensus motif, Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022. Surprisingly, certain of the HuGlyFab fragments disclosed herein comprise such non-consensus sequences. In addition, O-glycosylation comprises the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated. The possibility of O-glycosylation confers another advantage to the therapeutic antibodies provided herein, as compared to, e.g., antigen-binding fragmentsproduced in E. coli, again because the E. coli naturally does not contain machinery equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli has been demonstrated only when the bacteria is modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.)N-Glycosylation of HuPTM scFvs

[0247] Unlike small molecule drugs, biologies usually comprise a mixture of many variants with different modifications or forms that could have a different potency, pharmacokinetics, and / or safety profile. It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy. The goal of gene therapy treatment provided herein can be, for example, to slow or arrest the progression of a disease or abnormal condition or to reduce the severity of one or more symptoms associated with the disease or abnormal condition.

[0248] When a HuPTM scFv is expressed in a human cell, the N-glycosylation sites of the antigen-binding fragment can be glycosylated with various different glycans. N- glycans of antigen-binding fragments have been characterized in the art. For example, Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 :3029-3039 (incorporated by reference herein in its entirety for its disclosure of Fab -associated N-glycans) characterizes glycans associated with Fabs, and demonstrates that Fab and Fc portions of antibodies comprise distinct glycosylation patterns, with Fab glycans being high in galactosylation, sialylation, and bisection (e.g., with bisecting GlcNAc) but low in fucosylation with respect to Fc glycans. Like Bondt, Huang et al., 2006, Anal. Biochem. 349: 197-207 (incorporated by reference herein in its entirety for it disclosure of Fab-associated N-glycans) found that most glycans of Fabs are sialylated. However, in the Fab of the antibody examined by Huang (which was produced in a murine cell background), the identified sialic residues were N-Glycolylneuraminic acid (“Neu5Gc” or “NeuGc”) (which is not natural to humans) instead of N-acetylneuraminic acid (“Neu5Ac,” the predominant human sialic acid). In addition, Song et al., 2014, Anal. Chem. 86:5661-5666 (incorporated by reference hereinin its entirety for its disclosure of Fab-associated N-glycans) describes a library of N- glycans associated with commercially available antibodies.

[0249] Importantly, when the HuPTM scFv is expressed in human cells, the need for in vitro production in prokaryotic host cells (e.g., E. coli) or eukaryotic host cells e.g., CHO cells or NSO cells) is circumvented. Instead, as a result of the methods described herein, N-glycosylation sites of the HuPTM scFv are advantageously decorated with glycans relevant to and beneficial to treatment of humans. Such an advantage is unattainable when CHO cells, NSO cells, or E. coli are utilized in antibody / antigen-binding fragment production, because e.g., CHO cells (1) do not express 2,6 sialyltransferase and thus cannot add 2,6 sialic acid during N-glycosylation; (2) can add Neu5Gc as sialic acid instead of Neu5Ac; and (3) can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis; and because (4) E. coli does not naturally contain components needed for N-glycosylation.

[0250] Assays for determining the glycosylation pattern of antibodies, including antigen-binding fragments are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows release of the attached glycans. N-acetyl groups are lost during this treatment and have to be reconstituted by re- N-acetylation. Glycans may also be released using enzymes such as glycosidases or endoglycosidases, such as PNGase F and Endo H, which cleave cleanly and with fewer side reactions than hydrazines. The free glycans can be purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2-amino benzamide. The labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(1 ):70-90. The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units. Structural information can be gathered by collecting individual peaks andsubsequently performing MS / MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified. Specific peaks of low or high molecular weight can be analyzed by MALDI-MS / MS and the result used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer, e.g., glycan, consisting of a certain number of repeat units and fragments, e.g., sugar residues, thereof. The chromatogram thus allows measurement of the polymer, e.g., glycan, length distribution. The elution time is an indication for polymer length, while fluorescence intensity correlates with molar abundance for the respective polymer, e.g., glycan. Other methods for assessing glycans associated with antigen-binding fragments include those described by Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, Huang et al., 2006, Anal. Biochem. 349: 197-207, and / or Song et al., 2014, Anal. Chem. 86:5661-5666.

[0251] Homogeneity or heterogeneity of the glycan patterns associated with antibodies (including antigen-binding fragments), as it relates to both glycan length or size and numbers glycans present across glycosylation sites, can be assessed using methods known in the art, e.g., methods that measure glycan length or size and hydrodynamic radius. HPLC, such as size exclusion, normal phase, reversed phase, and anion exchange HPLC, as well as capillary electrophoresis, allows the measurement of the hydrodynamic radius. Higher numbers of glycosylation sites in a protein lead to higher variation in hydrodynamic radius compared to a carrier with less glycosylation sites. However, when single glycan chains are analyzed, they may be more homogenous due to the more controlled length. Glycan length can be measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis. In addition, homogeneity can also mean that certain glycosylation site usage patterns change to a broader / narrower range. These factors can be measured by Glycopeptide LC-MS / MS.

[0252] In certain embodiments, the HuPTM scFvs also do not contain detectable NeuGc and / or a-Gal. By “detectable NeuGc” or “detectable a-Gal” or “does not contain or does not have NeuGc or a-Gal” means herein that the HuPTM scFv does not contain NeuGc or a-Gal moieties detectable by standard assay methods known in the art. Forexample, NeuGc may be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of A-Acetyl-and A-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.” J. Chromatogr., B: Biomed. 377, 111-119, which is hereby incorporated by reference for the method of detecting NeuGc. Alternatively, NeuGc may be detected by mass spectrometry. The a-Gal may be detected using an ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring a-Gal epitope expression on cells by a monoclonal anti-Gal antibody.” Transplantation. 65(8): 1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profding of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.” Landes Bioscience. 5(5):699- 710. See also the references cited in Platts-Mills et al., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal” Immunol Allergy Clin North Am. 35(2): 247-260.Benefits of N-Glycosylation

[0253] N-glycosylation confers numerous benefits on the HuPTM scFv described herein. Such benefits are unattainable by production of antigen-binding fragments in E. coli, because E. colt does not naturally possess components needed for N-glycosylation. Further, some benefits are unattainable through antibody production in, e.g, CHO cells (or murine cells such as NSO cells), because CHO cells lack components needed for addition of certain glycans (e.g, 2,6 sialic acid and bisecting GlcNAc) and because either CHO or murine cell lines add N-N-Glycolylneuraminic acid (“Neu5Gc” or “NeuGc”) which is not natural to humans (and potentially immunogenic), instead of N-Acetylneuraminic acid (“Neu5Ac”) the predominant human sialic acid. See, e.g., Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6): 1110-1122; Huang et al., 2006, Anal. Biochem. 349: 197-207 (NeuGc is the predominant sialic acid in murine cell lines such as SP2 / 0 and NSO); and Song et al., 2014, Anal. Chem. 86:5661-5666, each of which is incorporated by reference herein in its entirety). Moreover, CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat. Biotech. 28:1153-1156. The human glycosylation pattern of the HuPTM scFv described herein should reduce immunogenicity of the transgene product and improve efficacy.

[0254] While non-canonical glycosylation sites usually result in low level glycosylation (e.g., 1-5%) of the antibody population, the functional benefits may be significant (See, e.g., van de Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441). For example, Fab glycosylation may affect the stability, half-life, and binding characteristics of an antibody. To determine the effects of Fab glycosylation on the affinity of the antibody for its target, any technique known to one of skill in the art may be used, for example, enzyme linked immunosorbent assay (ELISA), or surface plasmon resonance (SPR). To determine the effects of Fab glycosylation on the half-life of the antibody, any technique known to one of skill in the art may be used, for example, by measurement of the levels of radioactivity in the blood or organs in a subject to whom a radiolabelled antibody has been administered. To determine the effects of Fab glycosylation on the stability, for example, levels of aggregation or protein unfolding, of the antibody, any technique known to one of skill in the art may be used, for example, differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), e.g., size exclusion high performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurement.

[0255] The presence of sialic acid on HuPTM scFv used in the methods described herein can impact clearance rate of the HuPTM scFv. Accordingly, sialic acid patterns of a HuPTM scFv can be used to generate a therapeutic having an optimized clearance rate. Methods of assessing antigen-binding fragment clearance rate are known in the art. See, e.g., Huang et al., 2006, Anal. Biochem. 349: 197-207.

[0256] In another specific embodiment, a benefit conferred by N-glycosylation is reduced aggregation. Occupied N-glycosylation sites can mask aggregation prone amino acid residues, resulting in decreased aggregation. Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuPTM scFv that is less prone to aggregation when expressed, e.g., expressed in human cells. Methods of assessing aggregation of antibodies are knownin the art. See, e.g., Courtois et al., 2016, mAbs 8:99-112 which is incorporated by reference herein in its entirety.

[0257] In another specific embodiment, a benefit conferred by N-glycosylation is reduced immunogenicity. Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuPTM scFv that is less prone to immunogenicity when expressed, e.g., expressed in human ocular tissue cells, human CNS cells, human liver cells or human muscle cells.

[0258] In another specific embodiment, a benefit conferred by N-glycosylation is protein stability. N-glycosylation of proteins is well-known to confer stability on them, and methods of assessing protein stability resulting from N-glycosylation are known in the art. See, e.g., Sola and Griebenow, 2009, 1 Pharm Sci., 98(4): 1223-1245.

[0259] In another specific embodiment, a benefit conferred by N-glycosylation is altered binding affinity. It is known in the art that the presence of N-glycosylation sites in the variable domains of an antibody can increase the affinity of the antibody for its antigen. See, e.g., Bovenkamp et al., 2016, J. Immunol. 196:1435-1441. Assays for measuring antibody binding affinity are known in the art. See, e.g., Wright et al., 1991, EMBO J. 10:2717-2723; and Leibiger et al., 1999, Biochem. J. 338:529-538.5.3.2 Tyrosine Sulfation

[0260] Tyrosine sulfation occurs at tyrosine (Y) residues with glutamate (E) or aspartate (D) within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid, but not a basic amino acid, e.g., arginine (R), lysine (K), or histidine (H) that abolishes sulfation. The HuPTM scFvs described herein comprise tyrosine sulfation sites.

[0261] Importantly, tyrosine-sulfated antigen-binding fragments cannot be produced in E. coli, which naturally does not possess the enzymes required for tyrosinesulfation. Further, CHO cells are deficient for tyrosine sulfation-they are not secretory cells and have a limited capacity for post-translational tyrosine-sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537. Advantageously, the methodsprovided herein call for expression HuPTM scFvs in human cells that are secretory and have capacity for tyrosine sulfation.

[0262] Tyrosine sulfation is advantageous for several reasons. For example, tyrosine-sulfation of the antigen-binding fragment of therapeutic antibodies against targets has been shown to dramatically increase avidity for antigen and activity. See, e.g., Loos et al., 2015, PNAS 112: 12675-12680, and Choe et al., 2003, Cell 114: 161-170. Assays for detection tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.Amino Acid and Nucleotide Sequences

[0263] In certain embodiments, the anti-C5 antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six C5-A-mab CDRs which are underlined in the heavy and light chain variable domain sequences of FIG. 2B and 2C which ar Table 5 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. Table 6 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.Table 5. Amino Acid Sequences of Heavy and Light Chains and ProteinTable 6. Nucleotide Sequences of Heavy and Light Chains, Expressed Polypeptides, Proteins, and Genomes5.6. Monitoring of Efficacy

[0264] The compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD. The assessment may be determined in animal models or in human subjects. The efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or reduction in geographic atrophy, including of the fovea, to be assessed by visual inspection.

[0265] The compositions and methods described herein may be assessed for efficacy using any method for assessing efficacy in treating, preventing, or ameliorating dry AMD. The assessment may be determined in animal models or in human subjects. The efficacy on visual deficits may be measured by best corrected visual acuity (BCVA), for example, assessing the increase in numbers of letters or lines and where efficacy may be assessed as an increase in greater than or equal to 2 ETDRS lines or an increase in logMAR. Physical changes to the eye, including changes in geographic atrophy may be measured Optical Coherence Tomography, using methods known in the art.

[0266] The compositions and methods described herein may be assessed for efficacy using in vitro complement inhibition assays, such as membrane attack complex (“MAC”) formation, C5a generation and hemolysis. Complement inhibition assays can be performed in any appropriate cell type, such as ARPE19 cells (MAC and C5a assays), iPSC-derived RPE cells (MAC and C5a assays) or sheep / rabbit erythrocytes (hemolysis assay). MAC formation assays measure the deposition of MAC on the surface of RPE cells (% relative inhibition of MAC formation). C5a generation assays measure the ability of theC5 antibody to prevent C5 cleavage (less C5 cleavage = less C5a). Hemolysis assays allow the comparison of complement inhibition among different complement inhibitors (50% complement inhibition dose (ng / ml) (CH50; AH50).

[0267] Animal models may be used to assess the recombinant vectors encoding the anti-C5 antibodies for expression, therapeutic effect and adverse effects. Animal models may include a humanized C5- rodent model (Example 9) or a NaIO3 induction rat or mouse model (also see Example 9). 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 C5a, cleavage of C5 or other markers of complement activation.

[0268] Endpoints may include, but are not limited to, mean change in geographic atrophy in the study eye from baseline to 12, 16, 20, 24, or 28 weeks or at time of administration, if earlier, proportion of responders in the study eye at 12, 16, 20, 24, or 28 weeks, mean change in best corrected visual acuity from baseline to 12, 16, 20, 24, or 28 weeks, change from baseline in quality of life / patient reported outcome assessments, mean change in visual acuity from baseline to 12, 16, 20, 24, or 28 weeks.6 EXAMPLES6.1 EXAMPLE 1: C5-D-mab scFv cDNA-Based Vector

[0269] A C5-D-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of the C5-D-mab (amino acid sequences being SEQ ID NOs. 170 and 171, respectively) connected by nucleotide sequence encoding a flexible linker (for example, GGGGSGGGGSGGGGS (SEQ ID NO: 41) or Table 4. The scFv may have the structure VH-Linker-VL or VL-Linker-VH and have the amino acid sequence of SEQ ID NO: 178 or 179, respectively. The scFv encoded by the transgene may further comprise at its N-terminus a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:56) and have an amino acid sequence of SEQ ID NO: 188 (signal sequence- VH-linker-VL) or SEQ ID NO: 190 (signal sequence- VL-linker-VH) The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 45) operatively linked to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:58), or a BEST1 / GRK1 tandem promoter (SEQ ID NO: 144), or an inducible promoter, such as a hypoxia-inducible promoter, may be used. The artificial genome from the 5’ ITR to the 3’ ITR has a nucleotide sequence of SEQ ID NO: 212 or 214. A schematic of various anti-C5 inhibitors is depicted in FIG. 28. The nucleotide coding sequence for the C5-D-mab scFv (H-L) cassette including the CAG promoter, signal sequence, scFv transgene, and polyA sequence has a nucleotide sequence of SEQ ID No: 186.6.2 EXAMPLE 2: C5-A-mab scFv cDNA-Based Vector

[0270] A C5-A-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of the C5-A-mab (amino acid sequences being SEQ ID NOs. 172, and 173, respectively) connected by nucleotide sequence encoding a flexible linker (for example, GGGGSGGGGSGGGGS (SEQ ID NO: 41) or Table 4. The scFv may have the structure VH-Linker-VL or VL-Linker-VH and have the amino acid sequence of SEQ ID NO: 180 or 181, respectively. The scFv encoded by the transgene may further comprise at its N-terminus a signal peptide , e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:56) and have an amino acid sequence of SEQ ID NO: 189 (signal sequence-VH- linker-VL) or SEQ ID NO: 190 (signal sequence- VL-linker-VH) ,. The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 45) operatively linked to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:48), or a BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or an inducible promoter, such as a hypoxia-inducible promoter, may be used. Artificial genomes include or comprise the nucleotide sequences of SEQ ID NOs: 233-236.6.3 EXAMPLE 3: C5-C-mab Fab cDNA-Based Vector

[0271] A C5-C-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of the C5-C-mab (amino acid sequences being SEQ ID NOs. 174 and 175, respectively) connected by nucleotide sequence encoding a flexible linker (for example, GGGGSGGGGSGGGGS (SEQ ID NO: 41) or Table 4. The scFv may have the structure VH-Linker-VL or VL-Linker-VH and have the amino acid sequence of SEQ ID NO: 265 and 266, respectively. The scFv encoded by the transgene may further comprise at its N-terminus a signal peptide , e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:56) and have an amino acid sequence of SEQ ID NO: 189 (signal sequence-VH- linker-VL) or SEQ ID NO: 190 (signal sequence- VL-linker-VH) ,. The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 45) operatively linked to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:48), or a BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.6.4 EXAMPLE 4: C5-B-mab scFv cDNA-Based Vector

[0272] A C5-B-mab scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of the C5-B-mab (amino acid sequences being SEQ ID NOs. 176 and 177, respectively) connected by nucleotide sequence encoding a flexible linker (for example, GGGGSGGGGSGGGGS (SEQ ID NO: 41 ) or Table 4. The scFv may have the structure VH-Linker-VL or VL -Linker- VH and have the amino acid sequence of SEQ ID NO: 187 and 188, respectively. The scFv encoded by the transgene may further comprise at its N-terminus a signal peptide , e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:56) and have an amino acid sequence of SEQ ID NO: 188 (signal sequence-VH- linker-VL) or SEQ ID NO: 189 (signal sequence- VL-linker-VH) ,. The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 45) operatively linked to thenucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:48), or a BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.6.5 EXAMPLE S: BB5.1 scFv cDNA-Based Vector

[0273] A BB5.1 scFv cDNA-based vector was constructed comprising a transgene comprising nucleotide sequences encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of the C5-D-mab (amino acid sequences being SEQ ID NOs. 191 and 192, respectively) connected by nucleotide sequence encoding a flexible linker (for example, GGGGSGGGGSGGGGS (SEQ ID NO: 41) or Table 4. The scFv may have the structure VH-Linker-VL or VL-Linker-VH and have the amino acid sequence of SEQ ID NO: 25 or 26, respectively. The scFv encoded by the transgene may further comprise at its N-terminus a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:56) and have an amino acid sequence of SEQ ID NO: 194 (signal sequence- VH-linker-VL) or SEQ ID NO: 195 (signal sequence- VL-linker-VH) ,. The vector additionally includes the constitutive promoter CAG (SEQ ID NO: 45) operatively linked to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, such as mUla, EFla, CB7, a CB or CB long promoter, a tissue-specific promoter, such as a ocular tissue-specific promoter, particularly GRK1 promoter (SEQ ID NO:48), or a BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or an inducible promoter, such as a hypoxia-inducible promoter, may be used.6.7 EXAMPLE 7: NaIO3 Induction Mouse Model

[0274] A NaI03 induced model of dry AMD in rodents (inducing RPE damage) will be used to assess the anti-C5 antibodies AAV constructs. AAV8 constructs, AAV8.CAG.C5-D-mab.Fab (SEQ ID NO: 33), AAV8.CAG.C5-D-mab.full (SEQ ID NO:NO:34) , AAV8.C5-A-mab.Fab.IgGl (SEQ ID NO: 35), AAV8.C5-A-mab.Fab.IgG2 (SEQ ID NO: 36), AAV8.C5-A-mab.full (SEQ ID NO: 37), AAV8.CAG.BB5 1 (SEQ IDNO: 38), will be administered to humanized C5- mice at a dose of 1E7, 1E8 or 1E9 subretinally or suprachoroidally. 28 days later, NaI03 will be administered to the mouse to induce geographic atrophy. One week later, 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, and C5 levels.6.8 EXAMPLE 8: Hemolysis Assay

[0275] All C5 inhibitor expression cassettes utilized in this study were constructed with a CAG promoter and rabbit beta-globin polyA. All transgenes were codon-optimized and CpG depleted. Cis-plasmids were initially screened in the assay following transfection in 293T cells and then subsequently packaged as AAV8 viral vectors (including scAAV8 vectors) for further study.

[0276] The classical pathway of complement activation (CP) is initiated primarily by immune complexes. The standard assay for the overall functional activity of this pathway is the CH50. This assay uses sheep erythrocytes coated with rabbit antibodies (referred to as EA) to activate the complement system.

[0277] 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 108cells / ml in Gelatin Veronal Buffered saline (GVB++ Buffer) in the wells of an assay plate. Percent hemolysis was compared to a positive test hemolytic solution containing normal human serum that has been titrated up to 50% hemolysis. Percent hemolysis was calculated as such: % hemolysis = (test sample hemolysis (OD405)-background hemolysis (OD405)) / (maximal hemolysis (OD405)- background hemolysis (OD405)) X 100.

[0278] The neat supernatant collected from HEK293 cells transfected with vectorized C5-D-mab or BB5.1 plasmids (different vectorized forms IgG, Fab or ScFv) or a recombinant protein C5 inhibitor were tested in the assay for the % of lysis of the EA as an indication of inhibition of complement activation (FIGS. 4A-7B). Purified proteins of C5 inhibitors were tested at series concentrations (nM) to determine the minimum concentration necessary to lyse 50% of the cells (1 CH50 Unit) (FIGS 5A-5F). Activation of the classical pathway requires calcium and magnesium ions. The initial reactions include binding of Cl and activation of C2 and C4 to form a C3 convertase. This enzyme cleaves C3 which promotes cleavage of C5 and activation of the membrane attack pathway (proteins C5, C6, C7, C8 and C9). These five components assemble in the membrane of the sheep erythrocyte and lyse the cell. The release of hemoglobin is subsequently quantitated to measure the total complement activity present in the sample.

[0279] 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. 4A-B). Recombinant purified forms of each C5 inhibitor displayed potent inhibition of complement activation in classical and alternative hemolysis assays (FIGS 5A-F).6.9 EXAMPLE 9: Binding Kinetics and Affinity of Recombinant purified forms of each C5 inhibitor

[0280] The generated recombinant purified proteins of each C5 inhibitor (expressed in HEK293 cells as above) were compared by their IC50 values in both the classical and alternative complement pathways. The binding kinetics and affinity of each C5 inhibitor to human, cynomolgus macaque, and mouse C5 were measured with the OctetRED384 system, as follows. a. Affinity and kinetics for human Complement C5 :

[0281] 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 7. Assay conditions for human C5 assay after optimizationb. Affinity and kinetics for cynomolgous Complement C5:

[0282] 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.Table 8. Assay conditions for cyno C5 assay after optimizationc. Affinity and kinetics for mouse Complement C5:

[0283] 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 9. Assay conditions for mouse C5 assay after optimization (surrogate anti-mouse C5 mAb)

[0284] 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.Table 10. Assay conditions for mouse C5 assay after optimization (cross-species C5 binders)

[0285] 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. Anti -Mouse (BB5.1) and Antihuman (C5-D-mab) C5 inhibitors with the same vectorized antibody format demonstrated comparable affinity to mouse C5. See Table 11.Table 116.10 EXAMPLE 10: Evaluation of AAV expressed C5 inhibitors in iPSC- derived RPE

[0286] Membrane attack complex (MAC) formation was measured. Briefly,ARPE19 cells or 4-5 weeks old fully matured, polarized iPSC-RPE (iRPE cells, Cellular Dynamics International Catalog, #: R1102) monolayers were used in the studies. ARPE19 or iRPE cells were treated for 24 h with purified recombinant C5 inhibitors in the culture media followed by 48 h with a second treatment of purified recombinant C5 inhibitors in the culture media along with 5% CC-HS (NHS, Complement technology), or 5% CI-HS (heat-inactivated CC-HS). Cells were fixed in 4% paraformaldehyde for 20 min, at RT andimmunostained with antibodies or cell stain against C5b-9 (Invitrogen, MA5-28502), ZO- 1 (Invitrogen, REF 40220), Phalloidin (AF-568 Phalloidin: Invitrogen A12380) and DAPI.

[0287] C5 inhibitors prevented C5 cleavage and reduced membrane attack complex (MAC) formation (FIGS. 6A-C, ARPE19; FIGS. 6D-6H, 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. 6G). The TP level (FIG. 6G) was consistent with mRNA / cDNA of AAV measured by ddPCR (FIG. 6H).6.11 EXAMPLE 11: Evaluation of AAV expressed C5 inhibitors in vivo

[0288] 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) delivered via AAV administered subretinally demonstrated a >10-fold higher TP level (FIG. 7A = ng / eye; FIG. 7B = pmol / eye) than IgG (full-length antibody) and Fab forms and similar levels to purified anti-mC5 IgG delivered intraperitoneal (IP) once per day (data not shown). While AAV. anti -h5. 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. 7C and FIG. 7D and Table 12). Onetime subretinal delivery of AAV yielded high levels of TP in the mouse eye.

[0289] The scFv antibody fragment transgene products demonstrated improved expression and distribution to outer ocular layers following subretinal delivery in mice.Table 126.12 EXAMPLE 12: CAG Promoter Studies

[0290] CAG deletion mutants Del5, DelM and Del3 operably linked to a C5-D- mab scFv were made using standard molecular biology techniques. See FIG. 8. CAG- Delm has about 60% of the promoter strength as full-length CAG. See FIG. 9.

[0291] Either full length CAG or CAGdelm promoters controlling expression of a C5-D-mab scFv were packaged in an AAV8 capsid. The resultant AAV8 particles were used to transduce HEK293T cells at an MOI of 5e4, 1.5e5 or 5e5 and C5-D-mab scFv expression was measured (in ng) on days 1, 2, 3 and 6 post-transduction. See FIG. 10A- 10D. Even though the CAG del promoter has about 60% activity of full-length CAG, when combined with scFV formatted mAh in a condensed scAAV vector the expression levels are increased compared to ssAAV.6.13 EXAMPLE 13: Investigational Study of AAV Test Articles Following Suprachoroidal Administration in NHP

[0292] 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 13. Following dosing on Day 1, animals will be observed for 3 to 12 weeks for biodistribution sample collection to investigate the transduction protocol.Table 13 - Group Assignment and Dose LevelsGC: 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

[0293] Animals will be dosed 100jj.l / 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.Opthalmic Examinations

[0294] Opthalmic 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.

[0295] Briefly, 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).Intraocular Pressure Measurements

[0296] Intraocular pressure measurements (lOPs) 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.

[0297] Briefly, animals will be anesthetized with ketamine. The IOP measurements will be done using an applanation tonometer. Atopical anesthetic (e.g., 0.5% proparacaine) will be applied before IOP measurements.Spectral Domain Optical Coherence Tomography

[0298] 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.

[0299] 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 - Fundus

[0300] 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.

[0301] 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 Imaging

[0302] 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.

[0303] Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and dexmedetomidine or ketamine and maintained on sevoflurane, as applicable. Pupils will be dilated with a mydriatic agent. Images will be taken with a Heidelberg SPECTRALIS® instrument. Fundus autofluorescence images, to include posterior pole and nonstereoscopic photographs of two midperipheral fields(temporal and nasal, if possible); additional images will also be taken superior temporal, if possible.Anti-AAV2 and AAV8 Neutralizing Antibody (NAM) Analysis

[0304] 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.

[0305] 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

[0306] Anti-AAV9 total antibody analysis will be performed within 5 days of animal transfer pre-dose.

[0307] 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.Peripheral Blood Mononuclear Cell Isolation for ELISPOT

[0308] Peripheral blood mononuclear cells (PBMCs) will be isolated for ELISPOT at least once pre-dose. During the dosing phase, 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 Collection

[0309] 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.

[0310] Whole Blood: Blood samples for whole blood collection will be maintained on wet ice or chilled cryo-racks following collection. Whole blood will be harvested and transferred into 3 approximately equal aliquots.

[0311] 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 Collection

[0312] 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.

[0313] 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

[0314] 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 L 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 14.Table 14 - Blood TestsOcular Fluid and Frozen Ocular Tissue Collection for Biodistribution and Transgene Product Analysis

[0315] The following ocular fluid / tissues from the left eye from 2 of 3 animals in Groups 1 and 2, and both eyes from 1 of 3 animals in Groups 1 and 2. Each eye (as applicable) will be enucleated. Immediately following enucleation, a sample of aqueous humor will be collected and divided into two approximately equal aliquots. Vitreous humor and the ocular tissues will then be collected. Vitreous humor will be collected and divided into two approximately equal aliquots. See Table 15.

[0316] The anterior segment of the eye will be removed and the eyes will be divided into four approximately equal quadrants (superior temporal [to include the area of the dose site], superior nasal, inferior temporal, and inferior nasal). Two approximatelyequal strips (located distal, and proximal to the optic disc) from each quadrant will be collected.

[0317] 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).

[0318] Ocular fluids and tissues will be collected using ultra-clean procedures, according to Labcorp SOPs, in order to minimize the risk for potential contamination. In addition, 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.Table 15 - Ocular Tissue CollectionFrozen Systemic Tissue Collection for Biodistribution and Transgene Product Analysis

[0319] The following tissues (see Table 16) will be collected and frozen for analysis of biodistribution and transgene product analysis.Table 16 - Tissue Collection6.14 EXAMPLES 14A, 14B, and 14C: NaIO3Induction Mouse Models

[0320] A. ANalCh induced model of dry AMD in rodents (sodium iodate-induced retinal degeneration model) 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 17. 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. On Day 3, NalOi was administered to each mouse intravenously to induce an inflammatory event in the eye. Mice were again evaluated for ERG / OCT on Day 6 and on Day 10, then sacrificed on Day 10 for tissue collection. Without being bound by any one theory, the induced inflammation from injection of NalOi 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 can be visualized by optical coherence tomography (OCT) or immunohistochemical staining of the tissue.

[0321] n = 8 eyes / group were processed for hematoxylin & eosin (H&E) histopathology, n = 8 eyes / group were processed for retinal pigment epithelium (RPE) / choroid flatmount immunohistochemistry (IHC), n = 8 whole globes / group were snap frozen, and n = 8 whole globes were fixed in 4% paraformaldehyde (PFA).Table 17: NaIO3 Study Design A

[0322] Antibody product levels were detected in the eye as assessed following i.p. injection of recombinant anti-C5 BB5.1 antibody in Example 13 (FIGS. 7A-7B). In this NalCh experiment, intraperitoneal injections of recombinant anti-C5 antibody (BB5.1) were shown to have a protective effect with respect to degeneration of the ONL in eyes of the treated mice. Figures 11A-11C. BB5.1 antibody significantly ameliorated ONL and retinal thinning as measured by OCT three days and seven days post NalCh induction (day 6 and day 10, respectively).

[0323] 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 HC12.5%. 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 adaptationperiod before the light-adapted signals were collected. Background light was used constantly for all light-adapted measurements.

[0324] Dark-adapted ERG:

[0325] 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.

[0326] Step 10: Dark-adapted 150 cd / m2 scotopic c-wave measurement.

[0327] Light-adapted ERG:

[0328] 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.

[0329] Step 4-7: 3.0 c cd*s / m2 photopic ERG flicker ERG at 10-40 Hz, each step advancing 10 Hz.

[0330] Following ERG and OCT procedures, animals received atipamezole (0.1-1 mg / kg SQ) to reverse the xylazine effects and were allowed to recover normally from the procedures.

[0331] During the scotopic a-wave measurements, the a-wave amplitudes of both groups at all timepoints was -20-50 pV from -3.00 cd*s / m2 to -1.52 cd*s / m2 (Figure 3). At -1.52 cd*s / m2 s, the a-wave amplitude of both groups at baseline increased to -220-240 pV at 1.00 cd*s / m2; the a-wave amplitude of Group 1 on Day 6 began increasing to a similar amplitude after -1.00 cd*s / m2. At -1.00 cd*s / m2 on Day 6, 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.

[0332] The Dark-adapted ERG recordings made at 3 and 7 days after NalCh administration compared to baseline recordings showed that IP delivery of BB5.1 significantly improved a- and b-wave amplitudes 3 days following NalOi administration, indicative of improved rod function (FIGS. 15A-15F).

[0333] H&E histopathology was performed on n = 8 eyes / group. In general, in group 1, the retinal lesions were multifocal and mild, while in group 2, there were moderateto 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.

[0334] B. A NaIO.3 induced model of dry AMD was then performed to assess AAV vector-expressed anti-C5 antibodies. AAV8.CAG.anti-hC5-D-mab.scFv.HL and AAV8.CAG.anti-mC5-BB5.1.IgG will be administered to C57BL / 6J mice at a dose of 3E13 intravenously (IV). Briefly, mice will be evaluated for ERG / body weights (BW) / OCT analogously to the methods in Example 16A, and then each administered a test AAV vector at a dose of 3E13 vg / kg IV (Table 18). On Day 28, mice will again be evaluated for OCT / Fundus / ERG and NalO.i administered to each mouse intravenously on Day 29 to induce an inflammatory event in the eye.Table 18: NaKh Study Design B

[0335] Several days following administration of NalCh (-Day 32), the eyes of the 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.

[0336] NalOs Study Design C

[0337] Briefly, male C57B1 / 6J mice were injected IV with AAV8 vectors encoding anti-C5-D-mab.scFv (anti-hC5-scFv01), anti-mC5-IgG01 (mouse full-length mAb targeting mC5, positive control) and anti-NS-scFvOl (non-specific scFv, negative control) at a dose of 3el3 GC / kg body weight. Vector was injected at Day 1, and NalOs was injected at Day 29. Serum was collected at Day 0, Day 29 and Day 35 to assess protein expression levels and hemolytic function using sensitized sheep erythrocytes. Eyes were collected at necropsy and anti-C5-D-mab.scFv levels in whole eye lysates and in serum were measuredby ELISA. The eyes designated for histological analysis were collected in Davidson’s fixative and processed to paraffin blocks. Measurements of ONL thickness on H&E stained slides and photoreceptor layers from OCT images were taken in Imaged.

[0338] The Dark-adapted ERG recordings after NalOs administration compared to baseline recordings showed that delivery of anti-C5 scFv via AAV 29 days prior to injury significantly improved a- and b-wave amplitudes following NalO? administration, indicative of improved rod function (FIGs. 29A-29B). OCT imaging, measurement of outer retinal layers and ONL thickness after NaI0.3 administration showed AAV8.CAG.C5- D-mab.scFv (AAV.anti-hC5-scFv01) was comparable to an anti-mC5-IgG in reducing degeneration of photoreceptor cells and thus structural integrity of the retinal outer nuclear layer as compared to a non-specific antibody, anti-NS-scFvOl (FIGS. 29C-29I).6.15 EXAMPLE 15: Non-human primate (NHP) SCS administration of anti-C5 antibody-expressing vectors

[0339] Two cohorts of three female cynomolgus macaques each were dosed suprachoroi dally in the superior temporal quadrant using a custom MedOne transscleral needle with either 1) AAV8.CAG.C5-D-mab.scFv.HL (n=5 eyes dosed at 3el2 GC / eye in lOOuL; both eyes of two animals were dosed, while the right eye of the third animal was dosed and the left eye of this animal was not dosed and was used as a negative control), or 2) AAV3B.CAG. C5-D-mab.scFv.HL (n=5 eyes dosed at 3el2 GC / eye in lOOuL, n=l eye dosed at ~2el2 GC / eye in 70uL). (Table 19.) Animals dosed with AAV8 were anti-AAV8 neutralizing antibody (Nab) and total antibody (Tab) negative, and animals dosed with AAV3B were anti-AAV3B Tab negative. Ophthalmic exams, fundus photography, optical coherence tomography, and fundus autofluorescence imaging were performed and intraocular pressure measurements and aqueous humor taps were also collected at regular intervals, and animals were euthanized on study day 85.Table 19: Study Design

[0340] 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. 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 alsocollected frozen for biodistribution. Serum was also collected throughout the study to assess transgene product levels and anti-transgene product antibodies (ATPA).

[0341] AAV8 anti-C5-ScFv Vector results: Vector genome copy levels were assessed by ddPCR using primer / probes against poly A; 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 / ug DNA (FIG. 13A). 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. 13B). 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. 13C).

[0342] With the exception of an animal which established low vector transduction based on biodistribution data, anti-C5-scFv was detected in aqueous humor from all treated eyes throughout the course of the study (FIGS. 14A and 14B). The expression was largely stable, averaging 0.68ug anti-C5-scFv in AH at D29, with the exception of the right eye of animal 1, which rose throughout the course of the study (FIG. 14C). Excluding animal 1 left eye (low vector levels), anti-C5-scFv levels in the day 85 vitreous humor (VH) averaged 1.2 ug / mL (FIG. 15A and 15B). 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 (FIG. 16). 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 17A-17C).

[0343] 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.

[0344] AAV3B-anti-C5-scFv Vector results: Vector genome copy levels ranged from ~le2-le6 GC / ug DNA (based on ddPCR using primer / probes against polyA; 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 / cili ary 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.

[0345] With the exception of P0102-OS (established low vector transduction based on biodistribution data), anti-C5-scFv was detected in aqueous humor from all treated eyes through study day 29 (FIGS. 19A-19B), averaging 1.5 ug aC5-scFv / mL AH. After D29, anti-C5-scFv levels continued to rise in 3 / 5 eyes, while levels in the AH of both eyes from P0101 dropped to undetectable levels, correlating with the development of ATPA (FIG. 19C). Excluding the one eye with poor transduction and the other from the animal that developed ATPA, anti-C5-scFv levels in the D85 vitreous humor averaged 16.5 ug / mL(FIGS 20A-20B). Excluding the one eye with poor transduction and the other from the animal that developed ATPA, aC5-scFv was detected in the retina, RPE / choroid, and sclera of the remaining treated eyes, with the greatest transgene product levels detected in RPE / choroid, achieving up to 225ng aC5-scFv / mg tissue (FIGS 21A-21H). Transgene product levels correlated with vector genome levels in retina and RPE / choroid but did not in sclera. Minimal to no anti-C5-scFv was detected in the serum all three animals at any timepoint; low levels of detected anti-C5-scFv in the serum at D85 correlated well with the degree of liver biodistribution in those animals (FIGS 22A-22C). One of three animals (P0101) developed ATPA, first detected at D57, that correlated with a reduction in detectable anti-C5-scFv levels in AH, VH, and ocular tissues at D85.

[0346] Ophthalmic exams and ocular imaging found that AAV3B.CAG.anti-C5-D- mab.scFv was initially well-tolerated up to D57 / week 8, when the retinas in both eyes of P0101 and P0103 were noted to present with a mottled appearance. Both eyes of P0101 presented with perivascular sheathing on D57 that lessened in intensity by D85 as well as minimal or slight vitreous cell on D57 / D85, correlating with the development of ATPA. No treatment-related changes in intraocular pressure were noted. Histopathology analysis established the presence of choroidal infiltrates 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.

[0347] Suprachoroidal delivery of an AAV vector encoding a complement inhibitor optimized for ocular expression resulted in localized and durable expression of bioactive protein, thus representing a minimally invasive approach to treat dry AMD that would reduce the treatment burden and deliver therapeutic molecules directly into the site of AMD pathogenesis.

[0348] In vivo expression in NHPs

[0349] Briefly, cynomolgus macaques were dosed with AAV vectors expressinganti-C5-D-mab.scFv at 3el2 GC / eye intwo different studies. In a three-month study, AAV8 vector was dosed and AH taps were collected at days 15, 29, 57 and 85. Ocular tissues from two eyes were collected, dissected according to the scheme in FIG. 29C and separated into retina, RPE-Choroid and sclera. Anti-C5-D-mab.scFv (anti-hC5-scFv01) levels were measured by ELISA from temporal tissues while ddPCR to measure AAV vector genomes was done in nasal tissues. Two eyes were collected in Davidson’s fixative and processed into paraffin blocks for histological analysis. Anti-C5-D-mab.scFv levels were detected in aqueous and vitreous humor of NHPs at days 15, 29, 57 and 85 (FIGS. 29A-29B). In particular, anti-C5-D-mab.scFv was largely detected in distal and proximal areas of RPE- C tissue (FIG. 29D). Lastly, genome copies (DNA) were more abundant in RPE-C and sclera tissues compared to retina after SCS delivery (FIG. 29E). The vector was well tolerated throughout as demonstrated with SD-OCT, fundus autofluorescence imaging and histological analysis (data not shown).6.16 EXAMPLE 16: NHP Subretinal administration

[0350] Four cynomolgus macaques each were dosed subretinally (SR) with AAV8.CAG.C5-D-mab.scFv.HL or vehicle (n=l eye dosed with vehicle; n-3 eyes dosed at lelO GC / eye; n=4 eyes dosed at lei 1 GC / eye). One animal developed a cataract due to injection procedure (not TA related) and that animal was replaced. Several animals were also dosed with an AAV8 vector expressing a non-specific (NS) scFv using the same scFv framework.

[0351] Four punches of posterior ocular tissues were taken and the single punch with highest biodistribution (BD) is plotted (assumed dose site). Vector DNA was measured in ocular tissues 4 weeks-post SR injection. Similar results for vector transduction were seen in anti-C5 versus non-specific scFv. (FIG. 23.) Poor transduction of sclera tissue was anticipated and confirmed.

[0352] Aqueous humor (AH) was collected at day 15 and day 29 (two weeks and four weeks post-dose, respectively). TP (ScFv levels) were measured via ELISA using antigen-coated plates (coated with C5) and detecting TP with Protein-L HRP subset ofvalues were confirmed by bioanalytical measurement by mass spectrometry demonstrating “free” and “total” TP levels are similar. TP expression is variable, but mostly dosedependent for both scFvs. (FIGS. 27A-27B) Two animals (non-specific scFV group) had detectable ATPA in serum, but still demonstrated increase in TP from D 15 to D29. No ATPA was detected in anti-C5 treated animals.

[0353] TP expression was measured via ELISA in Vitreous humor (VH) using antigen-coated plates (C5) and detecting TP with Protein-L HRP (FIGS. 25A-25B). 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. Note that the IgG data resulted from a different study and used different assays to measure the non-specific (non-C5) TP. However, the study plan was nearly identical and performed at the same research laboratory. (FIGS. 26A-26B) Vectorized scFvs were more highly expressed in AH than the vectorized IgG format in this model. IgG was poorly tolerated at doses higher than 1E10, therefore a limited amount of data to compare expression at equivalent doses except the more highly expressed scFv 1E10 dose.

[0354] TP expression was also measured various ocular tissues. The four punches of tissues after vector biodistribution (DNA & RNA) 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 and RPE-choroid (FIGS. 27A-27B).

[0355] Expression and tolerability in NHP following injections either subretinally (one month study duration) or suprachoroidally (3 -month study duration, Example 17, infra) were tested. Tolerability assessed with in vivo imaging, and also for histopathology, determined that anti-C5 scFv -AAV vector was well tolerated and yielded high expressionof bioactive C5 inhibitor in ocular fluids and tissues (including RPE / choroid) with minimal levels of C5 inhibitor in serum. In vivo expressed C5 inhibitors present in NHP vitreous humor (from subretinally injected eyes) was also examined and found to be bioactive.6.17 EXAMPLE 17: One-Month Evaluation of Expression of Formulated AAV Vectors Administered Suprachoroidally in Yucatan Minipigs

[0356] 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., AAV.ScFvAntibody) in animals (e.g., minipigs, such as Yucatan minipig) after a single suprachoroidal injection. In brief, three (3) pigs receive each test article formulation via bilateral suprachoroidal injection using a 29-gauge needle approximately 1110 pm in length (performed once).Table 20: Experimental Design#- OU = Oculus Uterque (both eyes)*- allowable time windows for timed collations are as follows: < 10 minutes, no window; 11-59 minutes, ± 1 minute; 1-6 hours, ± 5 minutes; 7-24 hours, ± 10 minutes; 24 hours or greater (Example: 48 or 72 hour), ± 15 minutes. Timepoints indicated as ‘days’ will occur during the day indicated

[0357] Test articles 3-5 were provided as follows:

[0358] Test article 3: AAV8.CAG.C5-D-mab.scFv.EIL provided as a preformulated solution at a concentration of 3xl013GC / 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).

[0359] Test article 4: AAV8.CAG.C5-D-mab.scFv.HL provided as a preformulated solution at a concentration of 3xl013GC / 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).

[0360] Test article 5: AAV8.CAG.C5-D-mab.scFv.HL provided as a preformulated solution at a concentration of 3xl012GC / 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 21:

[0361] 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. Animals will then be positioned beneath a surgical microscope with the first eye upwards, and a surgical drape and eyelid speculum will be placed. The 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 cottontipped applicator will be placed over the injection site for approximately 10 seconds. A topical drop of antibiotic ophthalmic solution will be applied to the ocular surface. Following dosing, animals will undergo post-dose OCT (to examine injection site as well as nasal -temporal area near injection site) and fundus imaging. Animals will be weighed prior to the start of dosing, weekly, and at the time of euthanasia. Ocular Examinations (OEs), Optical Coherence Tomography (OCT), Color Fundus Imaging, Blood Collections(for Serum, Plasma, & Whole Blood), and Tissue Collections will take place as per the experimental design.Bioactivity of Transgene Product:

[0362] Briefly, aqueous humor taps were collected pre-dose and at 15 and 29 days post-dose from Yucatan minipig eyes dosed SCS with AAV8.CAG.C5-D-mab.scFv (AAV8-anti-hC5-scFv01) vector at 3el2 GC / eye. Vitreous humor and ocular tissues were collected at necropsy and expression of anti-C5-D-mab.scFv was measured with ELISA. Vitreous humor from animals with appropriate levels of expression were used for testing of bioactivity in hemolysis assays. Minipigs dosed with AAV. Anti-C5-D-mab.scFv (anti- hC5-scFv01) strongly inhibited complement pathway activation (classical pathway hemolysis) compared to controls (FIG. 31).6.18 EXAMPLE 18: Transduction and bioactivity evaluation of scFV-AAV in iPSC-derived RPE cells

[0363] Retinal pigmented epithelial cells (RPEs) function as a barrier and homeostasis regulator of the retina and may be disrupted in response to inflammation. Briefly, human induced pluripotent stem cell (iPSC) derived RPEs were cultured and maintained in porous membrane inserts for up to 60 days, allowing the cells to develop characteristic transepithelial electric resistance (TER) and observe changes induced by the expression of C5 inhibitors by the cells.

[0364] Once RPE culture conditions and readout measurements were established (see Example 12, infra), RPEs were transduced with AAV s expressing anti -human C5 scFv. iPSC-derived RPE cultured in inserts has potential as a platform in human-specific ocular gene therapy development for therapeutic targets such as C5 inhibition, among other targets.

[0365] iPSC-derived RPE cells transduced with AAV vectors expressing C5 inhibitors were evaluated for inhibition of membrane attack complex (MAC) using immunohi stochemi stry.EQUIVALENTS

[0366] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0367] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.

Claims

What is claimed is:1 . A composition comprising an adeno-associated virus (AAV) vector comprising:(a) a viral AAV capsid that has a tropism for ocular tissue cells; and(b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a single chain variable fragment (scFv) that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.

2. The composition of claim 1, wherein the viral capsid comprises a VP1 capsid protein which is at least 95% identical to the amino acid sequence of the VP1 capsid protein of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73(AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).

3. The composition of claim 1 or claim 2, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof.

4. The composition of any one of claims 1 to 3, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

5. The composition of any one of claims 1 to 4, wherein the one or more regulatory sequences comprises a regulatory sequence from Table 1 or Table la.

6. The composition of claim 5, wherein the one or more regulatory sequences comprises a CAG promoter (SEQ ID NO: 44), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), a CB promoter (SEQ ID NO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 47 or 139), a mouse cone arresting (CAR) promoter (SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136), a human red opsin (RedO) promoter (SEQ ID NO: 134) or a Bestl / GRKl tandem promoter (SEQ ID NO: 143).

7. The composition of claim 6, wherein the one or more regulatory sequences comprises a mutated CAG promoter (SEQ ID NO: 221, 222 or 223).

8. The composition of any one of claims 1 to 7, 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.

9. The composition of claim 8, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.

10. The composition of claim 9, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).

11. The composition of any one of claims 1 to 10, wherein the scFv that binds to C5 has the structure: signal sequence - VH - linker - VL.

12. The composition of any one of claims 1 to 10, wherein the scFv that binds to C5 has the structure: signal sequence - VL - linker - VH.

13. The composition of any one of claims 1 to 12, wherein the linker is GGGGS (SEQ ID NO: 38), GGGGSGGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42).

14. The composition of claim 13, wherein the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 42).

15. The composition of any one of claims 1 to 14, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprises an amino acid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence of SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

16. The composition of any one of claims 1 to 15, wherein the transgene encodes a polypeptide having an amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188.

17. The composition of claim 16, wherein the transgene encodes a polypeptide having an amino acid sequence SEQ ID NO: 180.

18. The composition of any one of claims 1 to 17, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQID NO: 231 or SEQ ID NO: 233or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

19. The composition of claim 18, wherein the artificial genome comprises the nucleotide sequence encoding C5-D-mab.scFv (SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209).

20. A pharmaceutical composition for use in treating Age-Related Macular Degeneration (AMD) in a human subject in need thereof, comprising an adeno- associated virus (AAV) vector comprising:(a) a viral capsid that has a tropism for ocular tissue cells; and(b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a single chain variable fragment (scFv) that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 ora sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, 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 said AAV vector is formulated for subretinal, intravitreal, intranasal, intracameral, suprachoroidal, or systemic administration to said human subject.

21. The pharmaceutical composition of claim 20, wherein the viral capsid comprises a VP1 capsid protein which is at least 95% identical to the amino acid sequence of the VP1 capsid protein of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or serotype hu26 (AAV.hu26).

22. The pharmaceutical composition of claim 20 or claim 21, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof.

23. The pharmaceutical composition of any one of claims 20 to 22, wherein the human ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

24. The pharmaceutical composition of any one of claims 20 to 23, wherein the one or more regulatory sequences comprises a regulatory sequence from Table 1 or Table la.

25. The pharmaceutical composition of claim 24, wherein the one or more regulatory sequences comprises a CAG promoter (SEQ ID NO: 44), a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223), a CB promoter (SEQ IDNO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NOS: 47 or 139), a mouse cone arresting (CAR) promoter (SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136), a human red opsin (RedO) promoter (SEQ ID NO: 134) or a Bestl / GRKl tandem promoter (SEQ ID NO: 143).

26. The pharmaceutical composition of claim 25, wherein the one or more regulatory sequences comprises a mutated CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222 or SEQ ID NO: 223).

27. The pharmaceutical composition of any one of claims 20 to 26, 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.

28. The pharmaceutical composition of claim 27, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2.

29. The pharmaceutical composition of claim 28, wherein said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).

30. The pharmaceutical composition of any one of claims 20 to 29, wherein the scFv that binds to C5 has the structure: signal sequence - VH - linker - VL.

31. The pharmaceutical composition of any one of claims 20 to 29, wherein the scFv that binds to C5 has the structure: signal sequence - VL - linker - VH.

32. The pharmaceutical composition of any one of claims 20 to 31, wherein the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43).

33. The pharmaceutical composition of claim 32, wherein the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 42).

34. The pharmaceutical composition of any one of claims 20 to 33, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprises an aminoacid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence of SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

35. The pharmaceutical composition of any one of claims 20 to 34, wherein the transgene encodes a polypeptide having an amino acid sequence of SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188.

36. The pharmaceutical composition of claim 35, wherein the transgene encodes a polypeptide having an amino acid sequence SEQ ID NO: 180.

37. The pharmaceutical composition of any one of claims 20 to 36, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

38. The pharmaceutical composition of claim 37, wherein the artificial genome comprises the nucleotide sequence of SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209.

39. The pharmaceutical composition of any one of claims 20 to 38, wherein the scFv that binds to C5 inhibits both classical and alternative complement pathways.

40. The pharmaceutical composition of any one of claims 20 to 39, wherein the scFv that binds to C5 inhibits membrane attack complex (MAC) formation.

41. A method of producing recombinant AAVs comprising:(a) culturing a host cell containing:(i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding an scFv that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by a polypeptide linker, wherein i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells;(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture andsupply the AAV rep and the AAV capsid protein in trans, wherein the capsid has ocular tissue cell tropism;(iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and(b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.

42. The method of claim 41, wherein the transgene encodes an scFv that comprises a variable heavy domain and a variable light chain domain of, C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab, wherein the AAV capsid protein is an AAV8, AAV9, AAV3B, or AAVrh73, or variant thereof.

43. The method of claim 41 or claim 42, wherein the ocular tissue cells are retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

44. The method of any one of claims 41 to 43, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ ID NO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ ID NO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ ID NO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ ID NO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ ID NO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

45. A host cell comprising: a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding an scFv that binds to C5, wherein the scFv that binds to C5 comprises a variable heavy domain (VH) and a variable light domain (VL) covalently linked by apolypeptide linker, i) the VH comprises an amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) wherein the VH comprises an amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto and the VL comprises an amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) wherein the VH comprises an amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto and VL comprises an amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and wherein the transgene is operably linked to one or more regulatory sequences that promote expression of the transgene in human ocular tissue cells.

46. The host cell of claim 45, wherein the ocular tissue cells retinal cells, RPE-choroid tissue cells, BrM epithelial cells, choriocapillaris epithelial cells, or photoreceptor cells (rods, cones and / or retinal ganglion cells).

47. The host cells of claim 45 or claim 46, wherein i) VH comprises an amino acid sequence of SEQ ID NO: 170 and VL comprises an amino acid sequence of SEQ ID NO: 171, or ii) VH comprises an amino acid sequence of SEQ ID NO: 172 and VL comprises an amino acid sequence of SEQ ID NO: 173, or iii) VH comprises an amino acid sequence of SEQ ID NO: 174 and VL comprises an amino acid sequence of SEQ ID NO: 175, or iv) VH comprises an amino acid sequence SEQ ID NO: 176 and VL comprises an amino acid sequence of SEQ ID NO: 177.

48. The host cell of any one of claims 45 to 47, wherein the transgene encodes a polypeptide having an amino acid sequence of of SEQ ID NO: 180, SEQ ID NO:181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189.

49. The host cell of claim 48, wherein the artificial genome comprises a nucleotide sequence of SEQ ID NO: 186 or SEQ ID NO: 187 or SEQ ID NO: 188 or SEQ ID NO: 195 or SEQ ID NO: 196 or SEQ ID NO: 198 or SEQ ID NO: 199 or SEQ IDNO: 200 or SEQ ID NO: 201 or SEQ ID NO: 202 or SEQ ID NO: 203 or SEQ IDNO: 204 or SEQ ID NO: 205 or SEQ ID NO: 206 or SEQ ID NO: 207 or SEQ IDNO: 208 or SEQ ID NO: 209 or SEQ ID NO: 210 or SEQ ID NO: 211 or SEQ IDNO: 224 or SEQ ID NO: 225 or SEQ ID NO: 226 or SEQ ID NO: 227 or SEQ IDNO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230 or SEQ ID NO: 231 or SEQ IDNO: 233 or SEQ ID NO: 234 or SEQ ID NO: 235 or SEQ ID NO: 236.

50. The composition of any one of claims 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition comprises between about 0.5% to about 1.0% w / v hyaluronic acid.

51. The composition of any one of claims 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition comprises about (2.5% w / v) sucrose and between about 0.5% to about 1.0% w / v hyaluronic acid.

52. The composition of any one of claims 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition 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.

53. The composition of any one of claims 1 to 19, or the pharmaceutical composition for use according to any one of claims 20 to 40, wherein the composition or pharmaceutical composition comprises about 0.2 mg / mL potassium chloride, about0.2 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.

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