Vectorized anti-complement antibody and its administration

Abstract

This invention describes compositions and methods for the delivery of fully human post-translationally modified therapeutic monoclonal antibodies, or their antigen-binding fragments, that bind to C5, to human subjects for ocular indications, particularly for the treatment of AMD. The nucleotide sequence encoding the antibody is delivered via an rAAV vector that targets ocular tissue cells for transgene expression.

Description

[Technical Field]

[0001] Sequence listing reference The contents of the electronic sequence listing (38013_0037P1_SL.xml, size: 387,877 bytes, and creation date: May 16, 2024) are incorporated in their entirety hereby by reference.

[0002] 1. Technical field This paper describes compositions and methods for delivering C5-binding therapeutic scFv. It also describes a method for administering C5-binding therapeutic scFv to human subjects diagnosed with age-related macular degeneration (AMD). [Background technology]

[0003] 2. Background technology Therapeutic mAbs have been shown to be effective in treating several diseases and conditions. However, because these drugs are only effective for a short period, they often require repeated injections over a long period, which places a considerable treatment burden on patients.

[0004] The complement system is a crucial component of the immune system that enhances the elimination of microorganisms and damaged cells, promotes inflammation, and attacks the cell membranes of pathogens. Three biochemical pathways activate the complement system: 1) the classical complement system, 2) the alternative complement pathway, and 3) the lectin pathway.

[0005] Age-related macular degeneration (AMD) causes progressive and permanent vision impairment. There are two types of AMD: atrophic and exudative. Atrophic AMD accounts for approximately 85-90% of the 196 million AMD cases worldwide. Complement system overactivation is a key driving factor for AMD. Furthermore, more than one million patients with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) may also benefit from interventions to counteract intraocular overactivation of complement.

[0006] More effective treatments are needed to reduce the treatment burden on patients with AMD. Intravitreal drug delivery is a promising mode of drug administration in patients because it delivers a large amount of drug to target tissue and eliminates the risk of systemic toxicity. Reducing or eliminating the need for regular ocular administration would reduce the burden on patients and improve therapy. [Overview of the Initiative]

[0007] 3. Outline of the Invention Therapeutic antibodies containing scFv (single-strand variable fragments), delivered via gene therapy, offer several advantages over injected or infused therapeutic antibodies, as they dissipate over time, resulting in peak and trough levels. The sustained expression of transgene product antibodies allows for more consistent levels of antibody or protein at the site of action, as opposed to repeated antibody injections, and is less risky and more convenient for patients due to the reduced need for injections. Furthermore, antibodies and other proteins expressed from transgenes undergo post-translational modification in a different manner than those directly injected, due to the different microenvironments present during and after translation. While not constrained by any particular theory, this results in antibodies with different diffusion, bioactivity, distribution, affinity, pharmacokinetic, and immunogenicity properties, making them "bio better" than directly injected antibodies. In addition, factors such as anti-C5 antibodies (see Clark et al., 2014, J.Immunol. 193:4962) can inhibit complement activation, inhibit intraocular drusen deposition, and reduce the progression of atrophic AMD. Provided herein are compositions and methods for anti-C5 gene therapy, particularly recombinant AAV gene therapy, which target the eye and are designed to generate a depot of a transgene for the expression of anti-C5 scFv as disclosed herein, thereby resulting in therapeutic or prophylactic levels of antibodies (in ocular tissue and, in embodiments, also in serum) within 20, 30, 40, 50, 60, or 90 days of administration of an rAAV composition for the treatment or reduction of the progression of atrophic AMD and associated geographic atrophy.

[0008] This invention describes compositions and methods for ocular or systemic delivery of anti-C5 scFv (e.g., a fully human glycosylated scFv form of a therapeutic mAb) to patients (human subjects) diagnosed with AMD or other conditions for which treatment with therapeutic anti-C5 scFv is indicated. In embodiments, the scFv includes a VH having the amino acid sequence of SEQ ID NO: 170 and a VL having the amino acid sequence of SEQ ID NO: 171, or a VH having the amino acid sequence of SEQ ID NO: 172 and a VL having the amino acid sequence of SEQ ID NO: 173, or a VH having the amino acid sequence of SEQ ID NO: 174 and a VL having the amino acid sequence of SEQ ID NO: 175, or a VH having the amino acid sequence of SEQ ID NO: 176 and a VL having the amino acid sequence of SEQ ID NO: 177. In embodiments, the scFv has the 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 a signal sequence.

[0009] Delivery can be advantageously achieved by administering, for example, a viral vector or other DNA expression construct encoding a therapeutic anti-C5 scFv form to a subject diagnosed with a condition suitable for treatment with therapeutic anti-C5 scFv, via gene therapy, to create a permanent depot in the eye of a patient, or in an alternative embodiment, in the liver and / or muscle, where the scFv continuously supplies HuPTM scFv, e.g., a human glycosylated transgene product, to one or more ocular tissues in which the scFv exerts its therapeutic or prophylactic effect. In embodiments, the scFv includes a VH having the amino acid sequence of SEQ ID NO: 170 and a VL having the amino acid sequence of SEQ ID NO: 171, or a VH having the amino acid sequence of SEQ ID NO: 172 and a VL having the amino acid sequence of SEQ ID NO: 173, or a VH having the amino acid sequence of SEQ ID NO: 174 and a VL having the amino acid sequence of SEQ ID NO: 175, or a VH having the amino acid sequence of SEQ ID NO: 176 and a VL having the amino acid sequence of SEQ ID NO: 177. In this embodiment, scFv has amino acid sequences 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 a signal sequence.

[0010] Provided are gene therapy vectors, particularly rAAV gene therapy vectors, which, when administered to a human subject, result in the expression of anti-C5 scFv disclosed herein, achieving maximum or steady-state concentrations in ocular tissue, e.g., aqueous humor, vitreous fluid, or serum, for example, 20, 30, 40, 50, 60, or 90 days after administration of a vector encoding an anti-C5 antibody. In embodiments, the gene therapy vector comprises an artificial genome containing a nucleotide sequence encoding anti-C5 scFv operably linked to a regulatory sequence that promotes expression in a target tissue. In embodiments, the artificial genome comprises one of the nucleotide sequences SEQ ID NOs: 185, 196, 199, 201, 203, 205, 207, or 202. In embodiments, the gene therapy vector comprises an expression cassette containing the nucleotide sequence SEQ ID NOs: 186, 195, 198, 200, 202, 204, 206, or 208, adjacent to the ITR sequence.

[0011] The recombinant vector used to deliver the transgene includes a non-replicating recombinant adeno-associated virus vector ("rAAV"). In embodiments, the AAV type has tropism to ocular tissues, including, for example, retinal cells, RPE, choroid, Bruch membrane (BrM) and its epithelial cells, choroidal capillaries and their epithelial cells, photoreceptor cells (rods and cones), and retinal ganglion cells. The AAV type may be, for example, AAV8, AAV9, AAV3B, or a subtype of AAVrh73 (or its variant). However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia virus vectors, or nonviral expression vectors referred to as "naked DNA" constructs. Transgene expression may be controlled by constitutive expression elements such as the CAG promoter, or by tissue-specific expression regulatory elements, particularly ocular, liver, and / or muscle-specific regulatory elements, for example, one or more elements from Tables 1 and 1a.

[0012] In certain embodiments, the anti-C5 scFv is an scFv derived from C5-A-mab, C5-B-mab, C5-C-mab, or C5-D-mab; see Figure 1C for structure and Table 5 for amino acid sequence, for example. In other embodiments, a recombinant AAV vector is provided containing a transgene encoding the scFv form of a BB5.1 antibody, which can be used as a surrogate for the anti-C5 binding antibody 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), rats, or mouse models for the preclinical evaluation of atrophic AMD. The amino acid sequences of the scFv forms of BB5.1 are provided in Table 5.

[0013] In other embodiments, the construct expresses scFv such as GGGGSGGGGSGGGGS (SEQ ID NO: 41), in which heavy and light chain variable domains are connected via a flexible, non-cleavable linker. In certain embodiments, the construct expresses NH2-V from the N-terminus. L -Linker-V H -COOH or NH2-V H -Linker-V L -Expresses COOH. In certain embodiments, the construct has an NH2-signal or leader sequence -V from the N-terminus. L -GGGGSGGGGSGGGGS-V H -COOH, or NH2-signal or leader sequence -V H -GGGGSGGGGSGGGGS-V L - Code COOH.

[0014] In addition, scFv expressed from transgenes in vivo is unlikely to contain antibody-related degradation products produced by recombinant techniques such as protein aggregation and protein oxidation. Aggregation is a problem associated with protein production and storage resulting from high protein concentrations, surface interactions with manufacturing equipment and containers, and purification in certain buffer systems. These conditions that promote aggregation are not present 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 stressful cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed from transgenes in vivo may also be oxidized under stressful conditions. However, humans and many other organisms have antioxidant defense systems that not only reduce oxidative stress but can also repair and / or reverse oxidation. Therefore, proteins produced in vivo are unlikely to be in an oxidized form. Both aggregation and oxidation can affect potency, pharmacokinetics (clearance), and immunogenicity.

[0015] Combination therapy involving systemic delivery of anti-C5 scFv to patients in conjunction with the administration of other available treatments is included in the methods provided herein. Additional treatments may be administered before, concurrently with, or after gene therapy. Such additional treatments may include, but are not limited to, combination therapy with therapeutic scFv.

[0016] Also provided are methods of manufacturing viral vectors, particularly AAV-based viral vectors. In certain embodiments, a method of producing recombinant AAV, the cis-expression cassette adjacent to the AAV ITR (the cis-expression cassette contains a transgene encoding a therapeutic antibody operably linked to an expression control element that controls the expression of the transgene in human cells), a trans-expression cassette lacking the AAV ITR (the trans-expression cassette drives the expression of AAV rep and capsid proteins in host cells in culture and is operably linked to an expression control element that supplies the rep and cap proteins in trans and encodes AAV rep and capsid proteins), culturing a host cell containing an artificial genome containing adenoviral helper functions sufficient to allow replication and packaging of the artificial genome by AAV capsid proteins, and recovering recombinant AAV that capsidizes the artificial genome from the cell culture. A method is provided that includes:

[0017] 3.1. Embodiments Embodiment 1. A composition comprising an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector comprises (a) a viral AAV capsid having tropism for ocular tissue cells, and (b) an artificial genome comprising an expression cassette adjacent to the AAV inverted terminal repeat (ITR), the expression cassette comprising a transgene encoding a single-chain variable fragment (scFv) that binds to C5, 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 contains the amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) the VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) the VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) the VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, The composition, wherein the transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. Embodiment 2. The composition according to Embodiment 1, wherein the viral capsid comprises a VP1 capsid protein that 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 rh10 (AAVrh10), 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 hu12 (AAV.hu12), or serotype hu26 (AAV.hu26). Embodiment 3. The composition according to claim 1 or Embodiment 2, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof. Embodiment 4. The composition according to any one of Embodiments 1 to 3, wherein the human eye tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells). Embodiment 5. The composition according to any one of Embodiments 1 to 4, wherein one or more regulatory sequences include regulatory sequences from Table 1 or Table 1a. Embodiment 6. The composition according to Embodiment 5, wherein the one or more regulatory sequences include a CAG promoter (SEQ ID NO: 44), a mutant CAG promoter (SEQ ID NO: 221, 222, or 223), a CB promoter (SEQ ID NO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 47 or 139), a mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 134, 135, or 136), a human red opsin (RedO) promoter (SEQ ID NO: 134), or a Best1 / GRK1 tandem promoter (SEQ ID NO: 143). Embodiment 7. The composition according to Embodiment 6, wherein one or more regulatory sequences include a mutant CAG promoter (SEQ ID NO: 221, 222, or 223). Embodiment 8. The composition according to 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 the human eye tissue cells. Embodiment 9. The composition according to Embodiment 8, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2. Embodiment 10. The composition according to Embodiment 9, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55). Embodiment 11. The composition according to any one of Embodiments 1 to 10, wherein the scFv bonded to C5 has the structure: signal sequence-VH-linker-VL. Embodiment 12. The composition according to any one of Embodiments 1 to 10, wherein the scFv bonded to C5 has the structure: signal sequence-VL-linker-VH. Embodiment 13. The composition according to 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 GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42). Embodiment 14. The composition according to Embodiment 13, wherein the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 42). Embodiment 15.i) The composition according to any one of Embodiments 1 to 14, wherein VH comprises the amino acid sequence of SEQ ID NO: 170 and VL comprises the amino acid sequence of SEQ ID NO: 171, or ii) VH comprises the amino acid sequence of SEQ ID NO: 172 and VL comprises the amino acid sequence of SEQ ID NO: 173, or iii) VH comprises the amino acid sequence of SEQ ID NO: 174 and VL comprises the amino acid sequence of SEQ ID NO: 175, or iv) VH comprises the amino acid sequence of SEQ ID NO: 176 and VL comprises the amino acid sequence of SEQ ID NO: 177. Embodiment 16. The composition according to any one of Embodiments 1 to 15, wherein the introduced gene encodes a polypeptide having the 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. Embodiment 17. The composition according to Embodiment 16, wherein the introduced gene encodes a polypeptide having amino acid sequence number 180. Embodiment 18. The composition according to any one of Embodiments 1 to 17, wherein the artificial genome comprises the 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. Embodiment 19. The composition according to Embodiment 18, wherein the artificial genome comprises a nucleotide sequence encoding C5-D-mab.scFv (SEQ ID NO: 207, or SEQ ID NO: 208, or SEQ ID NO: 209). Embodiment 20. A pharmaceutical composition for use in treating age-related macular degeneration (AMD) in human subjects requiring treatment, comprising an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector is A viral capsid that is tropic towards ocular tissue cells, An artificial genome comprising an expression cassette adjacent to an AAV reverse terminal repeat (ITR), wherein the expression cassette comprises a transgene encoding a single-stranded variable fragment (scFv) that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker, i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence that is 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence that is 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence that is 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence that is 90% identical thereto The transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. The pharmaceutical composition wherein the AAV vector is formulated for subretinal, intravitreous, intranasal, intrachorionic, parachoroidal, or systemic administration to a human subject. Embodiment 21. The viral capsid is 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 rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AA) The pharmaceutical composition according to Embodiment 20, comprising a VP1 capsid protein whose amino acid sequence is at least 95% identical to that of the VP1 capsid protein of serotype Vrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu12 (AAV.hu12), or serotype hu26 (AAV.hu26). Embodiment 22. The pharmaceutical composition according to claim 20 or Embodiment 21, wherein the viral capsid is AAV9, AAV8, AAV3B, or AAVrh73, or a variant thereof. Embodiment 23. The pharmaceutical composition according to any one of Embodiments 20 to 22, wherein the human eye tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells). Embodiment 24. The pharmaceutical composition according to any one of Embodiments 20 to 23, wherein the one or more regulatory sequences include regulatory sequences from Table 1 or Table 1a. Embodiment 25. The pharmaceutical composition according to Embodiment 24, wherein one or more regulatory sequences include a CAG promoter (SEQ ID NO: 44), a mutant CAG promoter (SEQ ID NO: 221, 222, or 223), a CB promoter (SEQ ID NO: 144 or 145), a human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 47 or 139), a mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 134, 135, or 136), a human red opsin (RedO) promoter (SEQ ID NO: 134), or a Best1 / GRK1 tandem promoter (SEQ ID NO: 143). Embodiment 26. The pharmaceutical composition according to Embodiment 25, wherein one or more regulatory sequences include a mutant CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223). Embodiment 27. The pharmaceutical composition according to 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 the human eye tissue cells. Embodiment 28. The pharmaceutical composition according to Embodiment 27, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55) or a signal sequence from Table 2. Embodiment 29. The pharmaceutical composition according to Embodiment 28, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55). Embodiment 30. The pharmaceutical composition according to any one of Embodiments 20 to 29, wherein the scFv bound to C5 has the structure: signal sequence-VH-linker-VL. Embodiment 31. The pharmaceutical composition according to any one of Embodiments 20 to 29, wherein the scFv bound to C5 has the structure: signal sequence-VL-linker-VH. Embodiment 32. The pharmaceutical composition according to any one of Embodiments 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). Embodiment 33. The pharmaceutical composition according to Embodiment 32, wherein the linker is GGGGSGGGGSGGGGS (Sequence ID 42). Embodiment 34. A pharmaceutical composition according to any one of Embodiments 20 to 33, wherein VH comprises the amino acid sequence of SEQ ID NO: 170 and VL comprises the amino acid sequence of SEQ ID NO: 171, or ii) VH comprises the amino acid sequence of SEQ ID NO: 172 and VL comprises the amino acid sequence of SEQ ID NO: 173, or iii) VH comprises the amino acid sequence of SEQ ID NO: 174 and VL comprises the amino acid sequence of SEQ ID NO: 175, or iv) VH comprises the amino acid sequence of SEQ ID NO: 176 and VL comprises the amino acid sequence of SEQ ID NO: 177. Embodiment 35. The pharmaceutical composition according to any one of Embodiments 20 to 34, wherein the introduced gene encodes a polypeptide having the 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. Embodiment 36. The pharmaceutical composition according to Embodiment 35, wherein the introduced gene encodes a polypeptide having amino acid sequence number 180. Embodiment 37. A pharmaceutical composition according to any one of Embodiments 20 to 36, wherein the artificial genome comprises the 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. Embodiment 38. The pharmaceutical composition according to Embodiment 37, wherein the artificial genome comprises the nucleotide sequence of SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO: 209. Embodiment 39. The pharmaceutically active composition according to any one of claims 20 to 38, wherein the scFv bound to C5 inhibits both the classical complement pathway and the alternative complement pathway. Embodiment 40. The pharmaceutical composition according to any one of claims 20 to 39, wherein the scFv bound to C5 inhibits membrane invasion complex (MAC) formation. Embodiment 41. A method for producing recombinant AAV, A cis-expression cassette adjacent to the AAV ITR, wherein the cis-expression cassette includes a transgene encoding scFv that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker, i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and the transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells, an artificial genome comprising the cis-expression cassette, A trans-expression cassette lacking an AAV ITR, wherein the trans-expression cassette drives the expression of AAV rep and AAV capsid protein in host cells in culture, and encodes the AAV rep and AAV capsid protein operably linked to an expression regulatory element that supplies the AAV rep and AAV capsid protein in trans, and the capsid has ocular cell tropism, The process involves culturing host cells containing sufficient adenovirus helper function to enable replication and packaging of the artificial genome by the AAV capsid protein, The method comprising recovering recombinant AAV that forms a capsid around the artificial genome from the cell culture. Embodiment 42. The method according to Embodiment 41, wherein the transgene encodes an scFv containing variable weight domains and variable light chain domains of C5-D-mab, C5-A-mab, C5-C-mab, and C5-B-mab, and the AAV capsid protein is AAV8, AAV9, AAV3B, or AAVrh73, or a variant thereof. Embodiment 43. The method according to Embodiment 41 or 42, wherein the ocular tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells). Embodiment 44. The method according to any one of Embodiments 41 to 43, wherein the artificial genome includes the 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. Embodiment 45. A host cell, A cis-expression cassette adjacent to the AAV ITR, wherein the cis-expression cassette includes a transgene encoding scFv that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker, i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence that is 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence that is 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence that is 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence that is 90% identical thereto The host cell comprising a plasmid comprising the cis-expression cassette, wherein the transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. Embodiment 46. The host cell according to Embodiment 45, wherein the ocular tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rod, cone, and / or retinal ganglion cells). Embodiment 47.i) The host cell according to Embodiment 45 or 46, wherein VH contains the amino acid sequence of SEQ ID NO: 170 and VL contains the amino acid sequence of SEQ ID NO: 171, or ii) VH contains the amino acid sequence of SEQ ID NO: 172 and VL contains the amino acid sequence of SEQ ID NO: 173, or iii) VH contains the amino acid sequence of SEQ ID NO: 174 and VL contains the amino acid sequence of SEQ ID NO: 175, or iv) VH contains amino acid sequence NO: 176 and VL contains the amino acid sequence of SEQ ID NO: 177. Embodiment 48. A host cell according to any one of Embodiments 45 to 47, wherein the introduced gene encodes a polypeptide having the amino acid sequence 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. Embodiment 49. A host cell according to Embodiment 48, wherein the artificial genome comprises the 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. Embodiment 50. The composition according to 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 0.5% to about 1.0% w / v hyaluronic acid. Embodiment 51. The composition according to 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 (2.5% w / v) sucrose and about 0.5% to about 1.0% w / v hyaluronic acid. Embodiment 52. The composition according to 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 of potassium chloride, about 0.2 mg / mL of potassium dihydrogen phosphate, about 5.84 mg / mL of sodium chloride, about 1.15 mg / mL of anhydrous disodium hydrogen phosphate, about 25.0 mg / mL (2.5% w / v) of sucrose, about 0.002% (0.02 mg / mL) of poloxamer 188, and about 0.7% w / v of hyaluronic acid. Embodiment 53. The composition according to 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 of potassium chloride, about 0.2 mg / mL of potassium dihydrogen phosphate, about 5.84 mg / mL of sodium chloride, about 1.15 mg / mL of anhydrous disodium hydrogen phosphate, about 40.0 mg / mL (4% w / v) of sucrose, about 0.001% (0.01 mg / mL) of poloxamer 188, and about 1% of high viscosity grade carboxymethylcellulose (CMC). [Brief explanation of the drawing]

[0018] 4. Brief explanation of the drawing [Figure 1] Schematic diagram of an rAAV vector genome construct containing an expression cassette encoding the heavy and light chains of a therapeutic mAb, isolated by a Furin-2A linker, operably linked to the promoter and adjacent to the AAV ITR. The transgene may contain nucleotide sequences encoding full-length heavy and light chains having an Fc region (A), Fab region heavy and light chains (B), or a single-stranded variable fragment (scFv) (C) that connects the antibody heavy and light chains to the linker. [Figure 2A] Amino acid sequences of the transgene constructs for the Fab regions of the C5-D-mab(A), C5-A-mab IgG1(B), C5-A-mab IgG2(C), C5-C-mab(D), and C5-B-mab(E) therapeutic antibodies against C5. Glycosylation sites are shown in bold. Glutaming glycosylation sites; aspartate (N) glycosylation sites; non-consensual aspartate (N) glycosylation sites; and tyrosine-O-sulfation sites (italicized) are as shown in the legend. Complementarity-determining regions (CDRs) are underlined. Hinge regions are highlighted in gray. [Figure 2B]Amino acid sequences of the transgene constructs for the Fab regions of the C5-D-mab(A), C5-A-mab IgG1(B), C5-A-mab IgG2(C), C5-C-mab(D), and C5-B-mab(E) therapeutic antibodies against C5. Glycosylation sites are shown in bold. Glutaming glycosylation sites; aspartate (N) glycosylation sites; non-consensual aspartate (N) glycosylation sites; and tyrosine-O-sulfation sites (italicized) are as shown in the legend. Complementarity-determining regions (CDRs) are underlined. Hinge regions are highlighted in gray. [Figure 2C] Amino acid sequences of the transgene constructs for the Fab regions of the C5-D-mab(A), C5-A-mab IgG1(B), C5-A-mab IgG2(C), C5-C-mab(D), and C5-B-mab(E) therapeutic antibodies against C5. Glycosylation sites are shown in bold. Glutaming glycosylation sites; aspartate (N) glycosylation sites; non-consensual aspartate (N) glycosylation sites; and tyrosine-O-sulfation sites (italicized) are as shown in the legend. Complementarity-determining regions (CDRs) are underlined. Hinge regions are highlighted in gray. [Figure 2D] Amino acid sequences of the transgene constructs for the Fab regions of the C5-D-mab(A), C5-A-mab IgG1(B), C5-A-mab IgG2(C), C5-C-mab(D), and C5-B-mab(E) therapeutic antibodies against C5. Glycosylation sites are shown in bold. Glutaming glycosylation sites; aspartate (N) glycosylation sites; non-consensual aspartate (N) glycosylation sites; and tyrosine-O-sulfation sites (italicized) are as shown in the legend. Complementarity-determining regions (CDRs) are underlined. Hinge regions are highlighted in gray. [Figure 2E]Amino acid sequences of the transgene constructs for the Fab regions of the C5-D-mab(A), C5-A-mab IgG1(B), C5-A-mab IgG2(C), C5-C-mab(D), and C5-B-mab(E) therapeutic antibodies against C5. Glycosylation sites are shown in bold. Glutaming glycosylation sites; aspartate (N) glycosylation sites; non-consensual aspartate (N) glycosylation sites; and tyrosine-O-sulfation sites (italicized) are as shown in the legend. Complementarity-determining regions (CDRs) are underlined. Hinge regions are highlighted in gray. [Figure 3-1] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-2] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-3] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-4]Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-5] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-6] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 3-7] Clusteral multiple sequence alignments of various capsids exhibiting ocular tissue tropism. Amino acid substitutions (shown in bold in the lower row) can be performed on the AAV8 capsid by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray = hypervariable regions. The amino acid sequences of AAV capsids are assigned sequence ID numbers as shown in Figure 3. [Figure 4]The results show the ability of cis-plasmid-expressed vectorized antibodies to suppress complement in HEK293 cells in hemolysis inhibition assays using (A) 1.5% normal human serum or (B) 20% normal mouse serum. A) Human C5 inhibitor, IgG=C5-D-mab full-length mAb, Fab=C5-D-mab Fab, scFv=C5-D-mab scFv mAb compared to recombinant C5 inhibitor (C5I) protein, isotype, and vehicle control. B) Mouse C5 inhibitor, IgG=BB5.1 full-length mAb, Fab=BB5.1 Fab, scFv=BB5.1 scFv mAb compared to recombinant C5 inhibitor (C5I) protein, isotype, and vehicle control. [Figure 5] The recombinant purified forms of each C5 inhibitor were tested as follows: A) 50% human C5, classical complement pathway conditions, anti-hC5 (C5-D-mab format) and C5 inhibitor; B) 50% human C5, alternative complement pathway conditions, anti-hC5 (C5-D-mab format) and C5 inhibitor; C) 50% mouse C5, classical complement pathway conditions, anti-hC5 (C5-D-mab format) and anti-mC5 (BB5.1 mAb format); D) 50% mouse C5, classical complement pathway conditions, anti-hC5 (C5-D-mab full-length mAb) and anti-mC5 (BB5.1 full-length mAb) were compared; E) 50% mouse C5, classical complement pathway conditions, anti-hC5 (C5-D-mab Fab mAb) and anti-mC5 (BB5.1 Fab mAb) In a hemolysis inhibition assay comparing mAbs, and comparing F) 50% mouse C5, classical complement pathway conditions, anti-hC5 (C5-D-mab scFv mAb), and anti-mC5 (BB5.1 scFv mAb), we demonstrate that both classical and alternative complement pathways were suppressed. [Figure 6A] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6B] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6C]We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6D] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6E] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6F] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6G] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 6H] We measure the formation of membrane invasion complexes (MACs) in ARPE-19 cells (Figure 9A-C) or iPSC-derived RPE cells (Figure 9D-H). [Figure 7A] The results of C5 inhibitors encoding AAV8, administered subretinally (SR) to the eyes of wild-type mice using 1E8 and 3E8 vg / eye, are shown. The AAV8.CAG.anti-hC5(C5-D-mab) vector was formatted as IgG (full-length), Fab, or scFV vectorized antibody and administered subretinally (SR) at each dose. The AAV8.CAG.anti-mC5(BB5.1) vector was administered SR at each dose, and purified recombinant anti-mC5 IgG(BB5.1) or isotype control was delivered intraperitoneally (ip). Transgene product (TP) measurements are expressed as ng / eye (RNA transcript). [Figure 7B]The results of C5 inhibitors encoding AAV8, administered subretinally (SR) to the eyes of wild-type mice at doses of 1E8 and 3E8 vg / eye, are shown. The AAV8.CAG.anti-hC5(C5-D-mab) vector was formatted as IgG (full-length), Fab, or scFV vectorized antibody and administered subretinally (SR) at each dose. The AAV8.CAG.anti-mC5(BB5.1) vector was administered SR at each dose, and purified recombinant anti-mC5 IgG(BB5.1) or isotype control was delivered intraperitoneally (ip). Transgene product (TP) measurements are expressed as mol / eye (protein). [Figure 7C] The results of C5 inhibitors encoding AAV8, administered subretinally (SR) to the eyes of wild-type mice using 1E8 and 3E8 vg / eye, are shown. The AAV8.CAG.anti-hC5(C5-D-mab) vector was formatted as IgG (full-length), Fab, or scFV vectorized antibody and administered subretinally (SR) at each dose. The AAV8.CAG.anti-mC5(BB5.1) vector was administered SR at each dose, and purified recombinant anti-mC5 IgG(BB5.1) or isotype control was delivered intraperitoneally (ip). Transgene product (TP) in the retina is expressed as pmol / eye (protein). [Figure 7D] The results of C5 inhibitors encoding AAV8, administered subretinally (SR) to the eyes of wild-type mice using 1E8 and 3E8 vg / eye, are shown. The AAV8.CAG.anti-hC5(C5-D-mab) vector was formatted as IgG (full-length), Fab, or scFV vectorized antibody and administered subretinally (SR) at each dose. The AAV8.CAG.anti-mC5(BB5.1) vector was administered SR at each dose, and purified recombinant anti-mC5 IgG(BB5.1) or isotype control was delivered intraperitoneally (ip). Transgene product (TP) in the RPE / choroid / sclera is represented as pmol / eye (protein). [Figure 8] This figure shows the full-length CAG promoter, the CAG-Del5 deletion mutant, the CAG-Delm deletion mutant, and the CAG-Del3 deletion mutant. [Figure 9]This bar graph shows the relative promoter strength of CAG-Del5 deletion mutants (25%), CAG-Delm deletion mutants (61%), and CAG-Del3 deletion mutants (11%) compared to the full-length CAG promoter (100%). [Figure 10] This bar graph shows the expression of C5-D-mab scFv(ng) on ​​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 (white circles) or AAV8 sc-CAG-Delm. C5-D.scFv (black circles) in multiple infection modalities (MOI) of 5e4, 1.6e4, or 5e5. [Figure 11] The BB5.1 antibody significantly improved ONL and retinal thinning, as measured by OCT after NaIO3 induction. [Figure 12] Dark-adapted ERG recordings (a-wave amplitudes A, C, and E; b-wave amplitudes B, D, and F) and b-wave amplitudes of mouse cohorts treated with ip antibody at baseline, 3, and 7 days after NaIO3 administration. [Figure 13] After treating each eye with 3E12 GC by intrachoroidal administration of the vector, the in vivo distribution of the vector (DNA, genome copies per 1 μg of tissue) (A) and transcriptional copies (copy number (DNA) per 1 μg of RNA) (B) were plotted as bar graphs for various eye tissues of the animals (day 85). The in vivo distribution of the vector (DNA) detected in peripheral tissues was also measured (C). [Figure 14] This shows the transgene product (TP) for scFv antibody levels detected in aqueous humor (AH) after administration of AAV8.CAG.C5-D-mab.scFv SCS at collection intervals (days 15, 29, 57, and 85). A: Dataset of antibody levels, including eyes, in cohorts that showed no or low vector biodistribution. B: Dataset excluding eyes at 29 days that showed no or low vector biodistribution. C: Time course of AH collected antibody levels for all eyes. [Figure 15]TP expression levels in vitreous fluid (VH) collected at the end of the study (day 85) after SCS delivery of AAV8.CAG.C5-D-mab-scFv to 3e12 GC / ocular. A is the dataset of TP levels including ocular cells in cohorts that showed no or low vector biodistribution. B is the dataset excluding ocular cells that showed no or low vector biodistribution. [Figure 16] Ocular tissue homogenates: TP results for AAV8-mediated TP production levels (ng / mg tissue) in the retina (A), RPE-choroid (B), sclera (C), and tissue from sample 3 only (D) are shown, excluding eyes in cohorts that showed no or low biodistribution. TP results for the retina (E), RPE-choroid (F), sclera (G), and tissue from sample 3 only (H) are shown, including eyes in cohorts that showed no or low biodistribution. [Figure 17] Serum levels (A), peripheral vector genome distribution (liver) (B), and TP levels over time (C) after AAV8 vector delivery of CAG.C5-D-mab-scFv. [Figure 18] After treatment with 3E12 GC per eye by intrachoroidal administration of the AAV3B vector, the in vivo distribution of the vector (DNA, genome copies per μg of tissue) (A) and transcriptional copies (copy number (DNA) per μg of RNA) (B) were plotted as bar graphs for various eye tissues of the animals (day 85). The in vivo distribution of the vector (DNA) detected in peripheral tissues was also measured (C). [Figure 19]This shows the transgene product (TP) for scFv antibody levels detected in aqueous humor (AH) after administration of AAV3B.CAG.C5-D-mab.scFv SCS at collection intervals (days 15, 29, 57, and 85). A is the antibody level dataset including all data points (including those that did not show vector biodistribution or showed low vector biodistribution). B is the dataset excluding eyes at day 29 that did not show biodistribution, showed low biodistribution, or showed high ATPA. C is the time course of AH collected antibody levels for all eyes. [Figure 20] TP expression levels in vitreous fluid (VH) collected at the end of the study (day 85) after SCS delivery of AAV3B.CAG.C5-D-mab-scFv to 3e12 GC / eyes. A is the dataset of TP levels including eyes that showed no biodistribution, low biodistribution, or high ATPA. B is the dataset excluding eyes that showed no vector biodistribution, low vector biodistribution, or +ATPA. [Figure 21] Ocular tissue homogenates: TP results for AAV3B-mediated TP production levels (ng / mg tissue) in the retina (A), RPE-choroid (B), sclera (C), and tissue from sample 3 only (D) are shown, excluding eyes in cohorts that showed no biodistribution, a low biodistribution, or +ATPA. TP results for the retina (E), RPE-choroid (F), sclera (G), and tissue from sample 3 only (H) are shown, including eyes in cohorts that showed no biodistribution, a low biodistribution, or +ATPA. [Figure 22] Serum level (A), peripheral vector genome distribution (liver) (B), and TP expression levels over time (C) after delivery of CAG.C5-D-mab-scFv via AAV3B vector. [Figure 23]The in vivo distribution of the vector genome (vector DNA, GC per 1 μg of tissue) was plotted for various ocular tissues (retina, RPE-choroid, and sclera) of animals at the end of the study (day 29) after 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. [Figure 24] AH was collected 2 and 4 weeks after injection of (A) AAV8-a anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL) or (B) AAV8-NS-scFv at a dose of 1E10 or 1E11 GC / eye. [Figure 25] VH collected 2 and 4 weeks after 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. [Figure 26] AH(A) and VH(B) collected 2 and 4 weeks after injection of AAV8-anti-C5 scFV (AAV8.CAG.C5-D-mab.ScFv.HL; 1E10 or 1E11 GC / eye), AAV8-NS-scFv (1E10 or 1E11 GC / eye), or AAV8-NS-IgG (full-length nonspecific mab; 3E9 or 1E10 GC / eye), which show higher TP expression levels for vectorized ScFv antibodies. [Figure 27] Ocular tissue (retina, RPE-choroid, and sclera) collected 2 and 4 weeks after 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 / ocular. [Figure 28] Schematic diagram of human anti-C5 inhibitors in different formats [Figure 29A]AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29B] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29C] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29D] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29E]AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29F] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29G] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29H] AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 29I]AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) rescues sodium iodate-induced functional and structural damage in a mouse model of geographic atrophy (GA) compared to a negative (non-specific mab) control (AAV.anti-NS-scFv01), as measured by ERG (A, B), OCT imaging, and measurement of the outer retinal layer (C-F), as well as H&E staining of the central eye section and measurement of ONL thickness (G-I). [Figure 30] Anti-C5-D-mab.scFv (anti-hC5-scFv01) levels were measured from aqueous humor (A) by punctures at days 15, 29, 57, and 85 of NHP administered with AAV8.CAG.C5-D-mab.scFv, from vitreous fluid (B) at 3 months, and from macular, distal, and proximal ocular tissue (D) collected according to scheme (C). The in vivo distribution of AAV was measured from ocular tissue shown in (C) using ddPCR. Two of the four eyes were collected for histological analysis. [Figure 31] Vitreous fluid from the eyes of three miniature pigs (Miniature Pig 1 (MP1), MP2, MP3) administered SCS with 3e12 GC / eye was tested for anti-C5-D-mab.scFv (anti-hC5-scFv01) protein levels and inhibition of classical pathway hemolysis. Anti-C5-D-mab.scFv expressed in vivo was as active as purified protein diluted with naive VH. [Modes for carrying out the invention]

[0019] 5. Modes for Carrying Out the Invention This invention describes compositions and methods for the systemic delivery of fully human post-translationally modified (HuP™) therapeutic anti-C5 mAb scFv to patients (human subjects) diagnosed with AMD (including atrophic AMD) or other indications suitable for treatment with therapeutic scFv. Delivery can be advantageously achieved by administering, for example, a viral vector or other DNA expression construct encoding therapeutic scFv to a patient (human subject) diagnosed with a condition suitable for treatment with therapeutic mAb, via gene therapy, to create a permanent depot in the patient's tissue or organ, particularly the eye, but in embodiments, the liver or muscle, to continuously supply the HuP™ scFv, e.g., a human glycosylated transgene product, to the target eye tissue where its anti-C5 scFv exerts its therapeutic effect.

[0020] In certain embodiments, HuPTM scFv is a HuPTMscFv that binds to the scFv form of C5, particularly 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 of the antibody, as well as its full length and Fab form).

[0021] The compositions and methods provided herein are for the delivery of anti-C5 scFv, particularly the scFv form of C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab antibodies, from a depot of viral genomes to the eye, or systemically, at any level in serum, for example, in the eye (including retinal tissue) of a subject, or intra-liver / muscle, intraocular tissue (e.g., in vitreous fluid or aqueous humor or retinal tissue, RPE, BrM, and / or choroid), or in which the treatment or alleviation of symptoms of AMD or other indications that can be treated with anti-C5 antibodies is therapeutic or prophylactic. In embodiments, a viral vector for delivery of a transgene encoding therapeutic anti-C5 scFv to cells in a human subject, including one or more ocular tissue cells, and in embodiments, a regulatory element operably linked to a nucleotide sequence encoding anti-C5 scFv that promotes scFv expression in ocular tissue cells is identified herein. Constitutive promoters such as CAG, and such regulatory elements, including ocular tissue-specific regulatory elements, are provided herein in Tables 1 and 1a, and in Example 13 (including a specific modified CAG promoter). Thus, such viral vectors may be delivered to human subjects in appropriate doses such that anti-C5 scFv is present in the serum or ocular tissue of the human subject at a therapeutically effective level at least 20, 30, 40, 50, or 60 days after administration. In embodiments, the therapeutically effective level of anti-C5 scFv is determined for reduction of geographic atrophy (or slowing the progression of geographic atrophy compared to untreated individuals, based on either disease control or natural history), reduction of drusen deposition, or reduction of other metrics of atrophic AMD, in order to improve best corrected visual acuity (BCVA) by two or more ETDRS lines (in human clinical trials, animal models, etc.).

[0022] In the embodiment, scFv includes a VH having the amino acid sequence of SEQ ID NO: 170 and a VL having the amino acid sequence of SEQ ID NO: 171, or a VH having the amino acid sequence of SEQ ID NO: 172 and a VL having the amino acid sequence of SEQ ID NO: 173, or a VH having the amino acid sequence of SEQ ID NO: 174 and a VL having the amino acid sequence of SEQ ID NO: 175, or a VH having the amino acid sequence of SEQ ID NO: 176 and a VL having the amino acid sequence of SEQ ID NO: 177. In the embodiment, scFv has the 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 a signal sequence.

[0023] The recombinant vectors used to deliver the transgene include non-replicating recombinant adeno-associated virus vectors ("rAAV"). rAAVs are particularly attractive vectors for several reasons, as they can be modified to preferentially target specific organs of choice, and hundreds of capsid serotypes exist to be selected to obtain desired tissue specificity and / or to avoid neutralization by existing patient antibodies against certain AAVs. The AAV type used herein preferentially targets the eye, i.e., is tropic against retinal cells. Such rAAVs include, but are not limited to, AAV-based vectors containing a capsid component from one or more of the following serotypes: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrh10, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAV.hu21, AAV.hu12, or AAV.hu26. In certain embodiments, the AAV-based vectors provided herein contain a capsid from one or more of the following serotypes: AAV3B, AAV8, AAV9, AAVrh10, AAV10, or AAVrh73.

[0024] However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia virus vectors, or nonviral expression vectors referred to as "naked DNA" constructs. Transgene expression can be controlled by constitutive or tissue-specific expression regulatory elements.

[0025] In some embodiments, the construct expresses scFv, such as GGGGSGGGGSGGGGS (SEQ ID NO: 53), in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker. Exemplary non-cleavable linkers are found in Table 4. In certain embodiments, the construct expresses NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH from the N-terminus to the C-terminus. In other embodiments, the construct expresses NH2 signal or localization sequence-VL-linker-VH-COOH or NH2-signal or localization sequence-VH-linker-VL-COOH from the N-terminus to the C-terminus.

[0026] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon optimization technique known to those skilled in the art (see, for example, the overview in Quax et al., 2015, Mol Cell 59:149-161), and may also be optimized to reduce CpG dimers. Codon-optimized sequences for the heavy and light chains of C5-D-mab and C5-A-mab are provided in Table 6 (SEQ ID NOs. 26-30 and 49), and these sequences may be incorporated into a transgene to encode the anti-C5 scFv described herein. Useful signal sequences for scFv expression and localization in human cells are disclosed herein, for example, in Tables 2 and 3. An exemplary recombinant expression construct is shown in Figure 1C.

[0027] The production of HuPTM scFv should result in a "bio-better" molecule for treating diseases achieved through gene therapy, for example, by administering a viral vector or another DNA expression construct encoding HuPTM scFv derived from a therapeutic mAb to a patient (human subject) diagnosed with the disease indication for that mAb, thereby creating a permanent depot in the subject that continuously supplies human glycosylated sulfated transgene products produced by the subject's transduced cells. The cDNA construct of HuPTM scFv should contain a signal peptide to ensure proper co-translation and post-translational processing (glycosylation and protein sulfated protein) by transduced human cells.

[0028] Pharmaceutical compositions suitable for administration to human subjects include a suspension of a recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and an optional excipient. Such a formulation buffer may contain one or more of the following: polysaccharides, surfactants, polymers, or oils.

[0029] As an alternative or additional treatment to gene therapy, HuPTM scFv can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients. Examples of human cell lines that may be used for such recombinant glycoprotein production include, but are not limited to, human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6, or RPE (see, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6):1110-1122 (for an overview of human cell lines or HuPTM scFv products that may be used for recombinant production of HuPTM mAb, the entire article is incorporated by reference)). To ensure complete glycosylation, particularly sialylation, and tyrosine-sulfation, the cell lines used for production may be enhanced by manipulating the host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes, which are involved in tyrosine-O-sulfation in human cells.

[0030] It is not essential that all molecules produced by either gene therapy or protein therapy approaches be completely glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfatedness to demonstrate efficacy. The goal of the gene therapy treatment of this invention is to slow or halt disease progression.

[0031] Combination therapy involving the delivery of HuPTM scFv to patients in conjunction with the administration of other available treatments is included in the method of the present invention. Additional treatments may be administered before, concurrently with, or after gene therapy. Such additional treatments may include, but are not limited to, combination therapy with therapeutic scFv.

[0032] Methods for producing viral vectors, particularly AAV-based viral vectors, are also provided. In certain embodiments, a method for producing recombinant AAV is provided, comprising culturing host cells containing an artificial genome containing an artificial genome with sufficient adenovirus helper function to enable replication and packaging of the artificial genome by the AAV capsid protein; and recovering recombinant AAV from the cell culture that capsids the artificial genome.

[0033] 5.1 Construct Viral vectors encoding anti-C5 scFv, including HuPTM anti-C5 scFv, or other DNA expression constructs are provided herein. The viral vectors and other DNA expression constructs provided herein include any preferred method for delivering the transgene to target cells. Means of transgene delivery include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetically modified mRNA, unmodified mRNA, small molecules, biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector that targets ocular tissue cells, or a vector that has tropism toward ocular tissue cells.

[0034] In some embodiments, the Disclosure provides a nucleic acid for use, comprising a nucleotide sequence encoding HuPTM scFv derived from an anti-C5 antibody disclosed herein, as a transgene described herein, operably linked to a ubiquitous promoter, an eye tissue-specific promoter, or an inducible promoter, the promoter being selected for the expression of the transgene in a target tissue. The promoters may include, for example, the CB7 / CAG promoter (SEQ ID NO: 44) and associated upstream regulatory sequences, the CAG promoter (CMS initial enhancer, chicken beta-actin promoter-chicken beta-actin intron-rabbit beta-globin splice acceptor) (SEQ ID NO: 45), the mutated chicken beta-actin promoter-chicken beta-actin intron-rabbit beta-globin splice acceptor) (SEQ ID NO: 221, 222, or 223), the cytomegalovirus (CMV) promoter, the EF-1 alpha promoter (SEQ ID NO: 47), mU1a (SEQ ID NO: 46), the UB6 promoter, the chicken beta-actin (CBA) promoter, and eye tissue-specific promoters such as the human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 48 or 137), the mouse cone arrestin (CAR) promoter (SEQ ID NOs: 134-136), or the human red opsin (RedO) promoter (SEQ ID NO: 132). See Tables 1 and 1a for a list of useful promoters. See also Example 13 for promoters.

[0035] In certain embodiments, recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides) are provided herein. The nucleic acids may include DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of a promoter sequence, a sequence of the gene of interest (a transgene, e.g., a nucleotide sequence encoding HuPTM scFv), an untranslated region, and a stop sequence. In certain embodiments, the viral vector provided herein comprises a promoter operably ligated to the gene of interest.

[0036] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon-optimized, for example, through any codon optimization technique known to those skilled in the art (see, for example, the overview in Quax et al., 2015, Mol Cell 59:149-161).

[0037] In certain embodiments, the constructs described herein include the following components: (1) an AAV2 reverse terminal repeat adjacent to the expression cassette, (2) one or more regulatory elements, b) optionally chicken beta-actin or other introns, and c) a rabbit beta-globin polyA signal, and (3) a nucleic acid sequence encoding scFv. Exemplary constructs are shown in Figures 1A, 1B, and 1C.

[0038] In certain embodiments, the construct described herein comprises the following components: (1) an AAV2 reverse terminal repeat adjacent to the expression cassette, (2) a GRK1 promoter (SEQ ID NO: 48), (b) optionally a VH4 intron (SEQ ID NO: 51) or another intron, and (c) a rabbit beta-globin polyA signal (SEQ ID NO: 49), and (3) a nucleic acid sequence encoding scFv.

[0039] 5.1.1 mRNA vectors In certain embodiments, as an alternative to a DNA vector, the vector provided herein is a modified mRNA encoding the gene of interest (e.g., a transgene encoding HuPTM scFv). The synthesis of modified and unmodified mRNAs for the delivery of transgenes to retinal pigment epithelial cells is described, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672 (which is incorporated herein by reference in its entirety). In certain embodiments, modified mRNA encoding HuPTMmAb or HuPTM scFv is provided herein.

[0040] 5.1.2 Viral vectors Viral vectors include adenoviruses, adeno-associated viruses (AAVs, e.g., AAV8, AAV9, AAVrh10, AAV10), lentiviruses, helper-dependent adenoviruses, herpes simplex viruses, poxviruses, Sendai virus (hemagglutinin virus of Japan (HVJ)), alphaviruses, vaccinia viruses, and retroviral vectors. Retroviral vectors include mouse leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors. Alphaviral vectors include Semryki forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In certain embodiments, the viral vector is a hybrid vector, e.g., an AAV vector placed in a "non-responsive" adenovirus vector. In certain embodiments, viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus are provided herein. In certain embodiments, the second virus is vesicular stomatitis virus (VSV). In more specific embodiments, the envelope protein is the VSV-G protein.

[0041] In certain embodiments, the viral vector provided herein is an HIV-based viral vector. In certain embodiments, the HIV-based vector provided herein comprises at least two polynucleotides, wherein the gag and pol genes are derived from the HIV genome and the env gene is derived from another virus.

[0042] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus-based vectors provided herein are modified so that they do not contain one or more initial (IE) genes, thereby making them non-cytotoxic.

[0043] In certain embodiments, the viral vector provided herein is an MLV-based viral vector. In certain embodiments, the MLV-based vector provided herein contains up to 8 kb of heterologous DNA instead of viral genes.

[0044] In certain embodiments, the viral vector provided herein is a lentivirus-based viral vector. In certain embodiments, the lentivirus vector provided herein is derived from a human lentivirus. In certain embodiments, the lentivirus vector provided herein is derived from a non-human lentivirus. In certain embodiments, the lentivirus vector provided herein is packaged in a lentivirus capsid. In certain embodiments, the lentivirus vector provided herein includes one or more of the following elements: a long terminal repeat, a primer binding site, a polypurine tube, an att site, and a capsid-forming site.

[0045] In certain embodiments, the viral vector provided herein is an alphavirus-based viral vector. In certain embodiments, the alphavirus vector provided herein is a recombinant replication-deficient alphavirus. In certain embodiments, the alphavirus replicon within the alphavirus vector provided herein targets a specific cell type by displaying a functional heterologous ligand on its virion surface.

[0046] In certain embodiments, the viral vector provided herein is an AAV-based viral vector. In certain embodiments, the AAV-based vector provided herein does not encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for the synthesis of the capsid protein) (the rep and cap proteins may be provided by packaging cells in trans). Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vector provided herein comprises components from one or more serotypes of AAV. In preferred embodiments, the AAV-based vector provided herein comprises components from one or more serotypes of AAV having tropism to eye tissue, liver, and / or muscle. In certain embodiments, the AAV-based vectors provided herein include a capsid component from one or more of the following: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrh10, AAVrh20, AAVrh39, AAVhu.37, AAVrh73, AAVrh74, AAV.hu51, AAV.hu21, AAV.hu12, or AAV.hu26. In certain embodiments, the AAV-based vectors provided herein include a component from one or more of the following: AAV8, AAV3B, AAV9, AAV10, AAVrh73, or AAVrh10 serotypes. The present invention provides a viral vector in which the capsid protein is a variant of 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 at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical in amino acid sequence to, for example, 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 natural capsid.In certain embodiments, the encoded AAV capsid has the sequence of Sequence 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, retaining the biological function of the AAV8, AAV3B, or AAVrh73 capsid. Figure 3 provides comparative alignments of the amino acid sequences of capsid proteins of different AAV serotypes with potential amino acids that may be substituted at specific positions in the aligned sequences, based on a comparison of row-labeled subs. Therefore, in certain embodiments, the AAV vector includes an AAV8, AAV3B, or AAVrh73 capsid variant having 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 the positions of the native AAV capsid sequence identified in the SUBS row of Figure 3. The amino acid sequences of the AAV8, AAV9, AAV3B, or AAVrh73 capsids are provided in Figure 3.

[0047] The amino acid sequence of the hu37 capsid can be found in international application PCT WO2005 / 033321 (sequence number 88), and the amino acid sequence of the rh8 capsid can be found in international application PCT WO03 / 042397 (sequence number 97). The amino acid sequence of the rh64R1 sequence can be found in WO2006 / 110689 (R697W substitution of the Rh.64 sequence, which is sequence number 43 of WO2006 / 110689).

[0048] In some embodiments, the AAV-based vector comprises components from one or more serotypes of AAV. In some embodiments, the AAV-based vectors provided herein include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, Includes capsid components from one or more of the following: AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or combinations of two or more thereof.In some embodiments, the AAV-based vectors provided herein are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B It includes components from one or more of the following rAAV particles, or combinations of two or more serotypes: AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles.In some embodiments, the rAAV particles include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, A AV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m 8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HS The VP1, VP2 and / or VP3 sequences of AAV capsid serotypes selected from C3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifications, or pseudotypes, are at least 80% identical, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., contain capsid proteins that are up to 100% identical.

[0049] In certain embodiments, the recombinant AAV for use in the compositions and methods herein is AAVS3 (including its variants) (see, for example, U.S. Patent Application No. 20200079821, which is incorporated herein in its entirety by reference). In certain embodiments, the rAAV particles include a capsid of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418, which is incorporated herein in its entirety by reference. In certain embodiments, the AAV for use in the compositions and methods herein is any AAV disclosed in U.S. 10,301,648, such as AAV.rh46 or AAV.rh73. In some embodiments, the recombinant AAV for use in the compositions and methods herein is Anc80 or Anc80L65 (see, for example, Zinn et al., 2015, Cell Rep. 12(6):1056-1068, which is incorporated herein by reference in its entirety). In certain embodiments, the AAV for use in the compositions and methods herein is any AAV disclosed in US9,585,971, e.g., AAV-PHP.B. In certain embodiments, the AAV for use in the compositions and methods herein is an AAV2 / Rec2 or AAV2 / Rec3 vector having a hybrid capsid sequence derived from AAV8 and serotype cy5, rh20, or rh39 (see, for example, Issa et al., 2013, PLoS One 8(4):e60361, which is incorporated herein by reference).In certain embodiments, the AAV for use in the compositions and methods herein is the AAV disclosed in any of the following (each of which is incorporated herein by reference in whole): US7,282,199, US7,906,111, US8,524,446, US8,999,678, US8,628,966, US8,927,514, US8,734,809, US9,284,357, US9,409,953, US9,169,299, US9,193,956, US9,458,517, US9,587,282, US2015 / 0374803, US2015 / 0126588, US2017 / 0067908, US2013 / 0224836, US2016 / 0215024, US2017 / 0051257, PCT / US2015 / 034799, and PCT / EP2015 / 053335. In some embodiments, rAAV particles are used in the following patents and patent applications (each of which is incorporated herein by reference in its entirety): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803 and 2015 / 012658. The AAV capsid has at least 80% identical capsid proteins to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following international patent applications: PCT / US2015 / 034799, PCT / EP2015 / 053335, 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 capsid proteins.

[0050] In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313 (each of which is incorporated herein by reference in its entirety) (e.g., AAV.Rh74 and RHM4-1). In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2014 / 172669 (each of which is incorporated herein by reference in its entirety), such as AAV rh.74. In some embodiments, the rAAV particles include the AAV2 / 5 capsid 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 herein by reference in its entirety). In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2017 / 070491, which is incorporated entirely herein by reference, e.g., AAV2tYF. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent Nos. 8,628,966, U.S. 8,927,514, U.S. 9,923,120 and WO2016 / 049230 (each of which is incorporated entirely by reference) (e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16).

[0051] In some embodiments, rAAV particles are represented by International Patent Publication No. WO2003 / 052051 (see, for example, SEQ ID NO. 2 in Publication '051), WO2005 / 033321 (see, for example, SEQ ID NOs. 123 and 88 in Publication '321), WO03 / 042397 (see, for example, SEQ ID NOs. 2, 81, 85, and 97 in Publication '397), WO2006 / 068888 (see, for example, SEQ ID NOs. 1 and 3-6 in Publication '888), WO2006 / 110689 (see, for example, SEQ ID NOs. 5-38 in Publication '689), WO20 The capsid protein is disclosed in 09 / 104964 (see, for example, SEQ ID NOs. 1-5, 7, 9, 20, 22, 24 and 31 of '964), WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38 of '097), and WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294 of '508), as well as in U.S. Patent Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10 of '924) (the contents of each of these are incorporated herein by reference in their entirety).In some embodiments, rAAV particles are represented by International Patent Publication No. WO2003 / 052051 (see, for example, SEQ ID NO: 2 in Publication '051'), WO2005 / 033321 (see, for example, SEQ ID NOs: 123 and 88 in Publication '321'), WO03 / 042397 (see, for example, SEQ ID NOs: 2, 81, 85 and 97 in Publication '397'), WO2006 / 068888 (see, for example, SEQ ID NOs: 1 and 3-6 in Publication '888'), WO2006 / 110689 (see, for example, SEQ ID NOs: 5-38 in Publication '689'), WO2009 / 104964 (see, for example, SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 in Publication 964). The AAV capsid has at least 80% identical capsid proteins to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38 in '097), WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294 in '508), and U.S. Patent Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10 in '924), 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 capsid proteins.

[0052] In additional embodiments, the rAAV particles include a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsid is an rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsid. Methods for producing and using pseudotyped rAAV particles are known in the art (see, for example, 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)).

[0053] AAV8-based, AAV3B-based, and AAVrh73-based viral vectors are used in specific methods described herein. Nucleotide sequences of AAV-based viral vectors, as well as methods for preparing recombinant AAVs and AAV capsids, are described, for example, in U.S. Patents 7,282,199B2, 7,790,449B2, 8,318,480B2, 8,962,332B2, and International Patent Application PCT / EP2014 / 076466 (each of which is incorporated herein in whole by reference). In one embodiment, AAV (e.g., AAV8, AAV3B, AAVrh73, or AAVrh10)-based viral vectors encoding a transgene (e.g., HuPTM Fab or HuPTM scFv, or a protein) are provided herein. The amino acid sequences of AAV capsids, including AAV8, AAV3B, AAVrh73, and AAVrh10, are provided in Figure 3.

[0054] In certain embodiments, single-stranded AAVs (ssAAVs) may be used as described above. In certain embodiments, self-complementary vectors, such as scAAVs, may be used (see, for example, 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. Patents No. 6,596,535; No. 7,125,717; and No. 7,456,683 (each of which is incorporated herein by reference in its entirety)).

[0055] In certain embodiments, the viral vector used in the method described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors can be used to introduce into a transgene encoding HuPTM scFv o. Recombinant adenoviruses can be first-generation vectors with or without an E1 deletion, or with an expression cassette inserted into either deletion region. Recombinant adenoviruses can be second-generation vectors containing complete or partial deletions of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus terminal inverted repeat and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3'ITR and may or may not contain a stuffer sequence to maintain the genome at approximately 36 kb, near the wild-type size. An exemplary protocol for producing adenovirus vectors can be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27 (the entire protocol is incorporated herein by reference).

[0056] In certain embodiments, the viral vector used in the method described herein is a lentivirus-based viral vector. Recombinant lentiviral vectors can be used to transfer HuPTM mAb antigen-binding fragments within a transgene. Four plasmids are used to construct the construct: a Gag / pol sequence containing the plasmid, a Rev sequence containing the plasmid, an envelope protein (e.g., VSV-G) containing the plasmid, and a cis-plasmid having a packaging element and a C5 antigen-binding scFv gene.

[0057] For lentiviral vector production, four plasmids are co-transfected into cells (e.g., HEK293-based cells), thereby allowing the use of polyethyleneimine or calcium phosphate as transfection agents, among other things. The lentivirus is then collected in the supernatant (cell collection is unnecessary / should not be performed as the lentivirus needs to budding from the cells to be active). The supernatant is filtered (0.45 μm), and then magnesium chloride and benzonase are added. Further downstream processes can vary considerably, with the use of TFF and column chromatography being the most GMP-compatible. Others use ultracentrifugation with or without column chromatography. Exemplary protocols for lentiviral vector production can 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 are incorporated herein by reference in their entirety).

[0058] In certain embodiments, the vector for use in the method described herein is a vector encoding HuPTM scFv such that, upon introduction of the vector into the relevant cells, a glycosylated and / or tyrosine-sulfated variant of HuPTM mAb is expressed by the cells.

[0059] 5.1.3 Gene expression promoters and modifiers In certain embodiments, the vectors provided herein include components that regulate gene delivery or gene expression (e.g., “expression regulatory elements”). In certain embodiments, the vectors provided herein include components that regulate gene expression. In certain embodiments, the vectors provided herein include components that affect binding to or targeting of cells. In certain embodiments, the vectors provided herein include components that affect the localization of polynucleotides (e.g., transgenes) within cells after uptake. In certain embodiments, the vectors provided herein include components that can be used, for example, as detectable or selectable markers for detecting or selecting cells that have taken up polynucleotides.

[0060] In certain embodiments, the viral vectors provided herein include one or more promoters that control the expression of the transgene. These promoters (and other transcription-controlling regulatory elements such as enhancers) may be constitutive (promoting ubiquitous expression) or may be specifically or selectively expressed in the eye. In certain embodiments, the promoters are constitutive promoters.

[0061] In certain embodiments, the promoter is a CAG promoter (SEQ ID NO: 74) (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663, which is incorporated herein by reference in its entirety). In some embodiments, the CAG (SEQ ID NO: 45) or CB7 promoter (SEQ ID NO: 44), or a mutant (deletion) CAG promoter (SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223) includes other expression regulatory elements that enhance the expression of the vector-driven transgene. In certain embodiments, the other expression regulatory elements include chicken β-actin introns and / or rabbit β-globin polyA signaling (SEQ ID NO: 49). In certain embodiments, the promoter includes a TATA box. In certain embodiments, the promoter includes one or more elements. In certain embodiments, one or more promoter elements may be oriented in opposite directions or moved relative to each other. In certain embodiments, the elements of the promoter are arranged to function cooperatively. In certain embodiments, the elements of the promoter are arranged to function independently. In certain embodiments, the viral vector provided herein comprises one or more promoters selected from the group consisting of the human CMV earliest gene promoter, the SV40 early promoter, the Roussarcoma virus (RS) long-terminal repeat, and the rat insulin promoter. In certain embodiments, the vector provided herein comprises one or more long-terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTR.

[0062] In certain embodiments, the vectors provided herein include one or more tissue-specific promoters (e.g., retina-specific promoters). In certain embodiments, the viral vectors provided herein include ocular tissue cell-specific promoters such as human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 48 or 137), mouse cone arrestin (CAR) promoter (SEQ ID NO: 134-136), and human red opsin (RedO) promoter (SEQ ID NO: 132).

[0063] The invention provides nucleic acid regulatory elements that are chimeric with respect to the tandem sequence of elements within an expression cassette. Generally, regulatory elements have multiple functions, such as transcription initiation or regulation, coordination with cell-specific mechanisms to drive expression during signal transduction, and recognition sites to enhance the expression of downstream genes.

[0064] In certain embodiments, the promoter is an inductive promoter. In certain embodiments, the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter includes a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter includes a HIF-1α binding site. In certain embodiments, the promoter includes a HIF-2α binding site. In certain embodiments, the HIF binding site includes an RCGTG (SEQ ID NO: 232) motif. For further details regarding the location and sequence of the HIF binding site, see, for example, Schodel, et al., Blood, 2011, 117(23):e207-e217 (which is incorporated herein by reference in its entirety). In certain embodiments, the promoter includes binding sites for hypoxia-inducible transcription factors other than HIF transcription factors. In certain embodiments, the viral vector provided herein includes one or more IRES sites that are preferentially translated under hypoxic conditions. For teachings regarding hypoxia-inducible gene expression and the factors involved, see, for example, Kenneth and Rocha, Biochem J., 2008, 414:19-29 (which is incorporated herein by reference in its entirety). In certain embodiments, the hypoxia-inducible promoter is either the human N-WASP promoter (see, for example, Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 (which is incorporated by reference for teachings on the N-WASP promoter)) or the human Epo hypoxia-inducible promoter (see, for example, Tsuchiya et al., 1993, J. Biochem. 113:395-400 (which is incorporated by reference for disclosures on the Epo hypoxia-inducible promoter)). In other embodiments, the promoter is a drug-inducible promoter, such as a promoter induced by administration of rapamycin or an analogue thereof.See, for example, PCT Publications WO94 / 18317, WO96 / 20951, WO96 / 41865, WO99 / 10508, WO99 / 10510, WO99 / 36553, and WO99 / 41258, as well as US7,067,526 (these in their entirety are incorporated herein by reference for the disclosure of drug-inducible promoters).

[0065] This specification provides constructs containing certain ubiquitous and tissue-specific promoters. Such promoters include synthetic promoters and tandem promoters. Examples of promoters and their nucleotide sequences are provided in Tables 1 and 1a below. Table 1 also includes nucleotide sequences of other regulatory elements useful for expression cassettes provided herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0066] In certain embodiments, the viral vector provided herein includes one or more regulatory elements other than a promoter. In certain embodiments, the viral vector provided herein includes an enhancer. In certain embodiments, the viral vector provided herein includes a repressor. In certain embodiments, the viral vector provided herein includes an intron (e.g., VH4 intron (SEQ ID NO: 51), SV40 intron (SEQ ID NO: 145), or chimeric intron (β-globin / Ig intron) (SEQ ID NO: 50). The viral vector may also include a Kozak sequence, e.g., GCCACC (SEQ ID NO: 15), to facilitate the translation of the transgene product.

[0067] In certain embodiments, the viral vectors provided herein include a polyadenylation sequence downstream of the coding region of the transgene. Any polyA site that signals the termination of transcription and directs the synthesis of a polyA tail is suitable for use in the AAV vectors of this disclosure. Exemplary polyA signals are derived from, but are not limited to, the late SV40 gene, the rabbit β-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, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

[0068] 5.1.4 Signal Peptides In certain embodiments, the vector provided herein includes components that regulate protein delivery. In certain embodiments, the viral vector provided herein includes one or more signal peptides. The signal peptide (also referred to as the “signal sequence”) may also be referred to herein as the “leader sequence” or “leader peptide”. In certain embodiments, the signal peptide enables the transgene product to achieve appropriate packaging (e.g., glycosylation) within the cell. In certain embodiments, the signal peptide enables the transgene product to achieve appropriate localization within the cell. In certain embodiments, the signal peptide enables the transgene product to achieve secretion from the cell.

[0069] There are two common approaches to selecting signal sequences for protein production in gene therapy contexts or cell cultures. One approach is to use a signal peptide from a protein homologous to the protein being expressed. For example, a human antibody signal peptide may be used to express IgG in CHO or other cells. Another approach is to identify a signal peptide optimized for the specific host cell used for expression. Signal peptides can be exchanged between different proteins, or even between proteins of different organisms, but typically the signal sequence of the most abundant secreted protein of that cell type is used for protein expression. For example, the signal peptide of human albumin, the most abundant protein in plasma, has been found to substantially increase the rate of protein production in CHO cells. However, certain signal peptides may retain their function and exert activity after being cleaved from the expressed protein as a "post-targeted function." Therefore, in certain embodiments, the signal peptide is selected from the signal peptides of the most abundant protein secreted by the cell used for expression to avoid post-targeted function. In certain embodiments, the signal sequence is fused to both the heavy chain and light chain sequences. In another embodiment, a single signal sequence resides within the transgene and is fused to either a sequence (heavy or light chain) at the N-terminus of the transgene. An exemplary sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), which can be encoded by the nucleotide sequence of SEQ ID NO: 61 (see Table 2, Figures 2A-2G). Alternatively, signal sequences suitable for expression and capable of inducing selective or directional expression of HuPTM scFv in the eye / CNS, muscle, or liver are provided in Tables 2, 3, and 4 below, respectively. [Table 3] [Table 4-1] [Table 4-2]

[0070] 5.1.5 Linker for scFv Construct Flexible peptide linker. In some embodiments, a single construct can be engineered to encode both a heavy chain and a light chain (e.g., heavy and light chain variable domains) separated by a flexible peptide linker such as one encoding an scFv. The flexible peptide linker can be composed of flexible residues such as glycine and serine, allowing the adjacent heavy and light chain domains to move freely relative to each other. The construct can 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 can 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 can be arranged as NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L -COOH.

[0071] In certain embodiments, the expression cassettes described herein are contained within a viral vector and are restricted by 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 limitations of certain vectors, the vector may or may not be able to accommodate the coding sequences for the full heavy and light chains of a therapeutic antibody, but may be able to accommodate the coding sequences for the heavy and light chains of an antigen-binding fragment such as a Fab or F(ab’)2 fragment or the heavy and light chains of an scFv. In particular, the AAV vectors described herein may be able to accommodate a transgene of approximately 4.7 kilobases. Substitution of smaller expression elements enables the expression of larger protein products such as full-length therapeutic antibodies.

[0072] Commonly used flexible linkers have a sequence ("GS" linker) consisting mainly of an extension of four Gly residues and one Ser residue, with the most widely used example being the sequence (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 proper separation of functional domains or to maintain necessary interdomain 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). In addition to 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, for example, Table 4. [Table 5]

[0073] 5.1.6 Untranslated Areas In certain embodiments, the viral vector provided herein includes one or more untranslated regions (UTRs), e.g., 3' and / or 5' UTRs. In certain embodiments, the UTRs are optimized with respect to a desired protein expression level. In certain embodiments, the UTRs are optimized with respect to the half-life of the transgene mRNA. In certain embodiments, the UTRs are optimized with respect to the stability of the transgene mRNA. In certain embodiments, the UTRs are optimized with respect to the secondary structure of the transgene mRNA.

[0074] 5.1.7 Reverse terminal repetition In certain embodiments, the viral vectors provided herein include one or more reverse-terminal repeat (ITR) sequences. The ITR sequences may be used to package recombinant gene expression cassettes into virions of the viral vector. In certain embodiments, the ITRs are derived from AAVs, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent Nos. 7,282,199B2, 7,790,449B2, 8,318,480B2, 8,962,332B2 and International Patent Application No. PCT / EP2014 / 076466 (each of which is incorporated herein in whole by reference)). In preferred embodiments, the nucleotide sequence encoding the ITR may include, for example, the nucleotide sequence of SEQ ID NO: 81(5'-ITR) or 82(3'-ITR). In certain embodiments, self-complementary vectors, such as modified ITRs used to produce scAAV, may be used (see, for example, 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. Patents 6,596,535; 7,125,717; and 7,456,683 (each of which is incorporated herein by reference in whole)). In preferred embodiments, the nucleotide sequence encoding the modified ITR may include, for example, the nucleotide sequence of SEQ ID NO: 52 (5'-ITR) or 54 (3'-ITR), or may be modified for scAAV, SEQ ID NO: 53 (m 5'ITR), or SEQ ID NO: 55 (m 3'ITR).

[0075] 5.1.8 Transgenes The transgene encodes HuPTM scFv based on the anti-C5 therapeutic antibody disclosed herein.

[0076] 5.1.8.1 Construct for scFv expression In certain embodiments for expressing the scFv morphology of an mAb in ocular tissue cell type, the construct described herein comprises the following components: (1) an AAV2 reverse terminal repeat adjacent to the expression cassette, (2) a) an ocular tissue-specific promoter or constitutive promoter, b) optionally an intron, e.g., a chicken β-actin intron or a VH4 intron, and c) a regulatory element comprising a rabbit β-globin polyA signal, and (3) a nucleic acid sequence encoding scFv, in which 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 comprises an NH2-signal sequence-V from the N-terminus. L -Linker-V H -COOH or NH2-signal sequence-V H -Linker-V L -Expresses COOH. In certain embodiments, the construct has an NH2-signal sequence -V from the N-terminus. L -GGGGSGGGGSGGGGS-V H -COOH or NH2-signal sequence-V H -GGGGSGGGGSGGGGS-V L It encodes -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, VH is SEQ ID NO: 170 and VL is SEQ ID NO: 171, VH is SEQ ID NO: 172 and VL is SEQ ID NO: 173, VH is SEQ ID NO: 174 and VL is SEQ ID NO: 175, VH is SEQ ID NO: 176 and VL is SEQ ID NO: 177. Exemplary constructs are provided in Figure 1C and in Tables 7 (amino acid sequences) and 8 (nucleotide sequences).

[0077] In certain embodiments, an AAV vector is provided comprising an AAV8 capsid (SEQ ID NO: 116), or alternatively, a viral capsid that is at least 95% identical in amino acid sequence to an AAV9, AAV3B, or AAVrh73 capsid (or a variant thereof), and an artificial genome comprising an expression cassette adjacent to an AAV reverse terminal repeat (ITR) (the expression cassette contains a transgene encoding an anti-C5 scFv mAb operably ligated to one or more regulatory sequences that control the expression of the transgene in ocular tissue cells such as RPE cells, BrM cells, choroidal capillary cells, photoreceptor cells (rod and / or cone), and retinal ganglion cells).

[0078] In the embodiment, the transgene expresses scFv based on an anti-C5 therapeutic antibody disclosed herein. Section 5.4 and Table 5 provide the amino acid sequences of scFv and the heavy and light chain variable domains of scFv itself. Certain nucleotide sequences are codons optimized for expression in human cells. For example, see the codon-optimized sequences encoding C5-D-mab (sequences 16, 17, 27, 28) or C5-A-mab (sequences 18-20 and 29-31) in Table 6.

[0079] Transgene The HuPTM scFv encoded by the transgene may include, but are not limited to, scFv forms of antibodies that bind to C5, including C5-A-mab.scFv, C5-B-mab.scFv, C5-C-mab.scFv, or C5-D-mab.scFv. The amino acid sequences of VH and VL, as well as the scFvs, are provided in Table 5 below.

[0080] C5-A-scFv construct AA array In this embodiment, the transgene encodes a C5-binding scFv containing the variable weight domain (VH) of SEQ ID NO: 172, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the variable light domain (VL) of SEQ ID NO: 173, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5 and Figure 2B). In one embodiment, the scFv that binds to C5 includes a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 172, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2B). In a specific embodiment, the scFv that binds to C5 includes a light chain variable domain containing the amino acid sequence of SEQ ID NO: 173, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2B).

[0081] In the embodiment, the VH and VL domains are connected by a flexible linker (e.g., one of sequence numbers 39-43). In the embodiment, the linker is GGGGSGGGGSGGGGS (sequence number 56).

[0082] In embodiments, the transgene has a signal or leader sequence at its N-terminus suitable for expression and secretion in human cells, particularly human eye tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence includes 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 shown in Table 2. Alternatively, the signal sequence may be suitable for expression in muscle or hepatocytes, such as those listed in Table 3 below.

[0083] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VH-linker-VL. In the embodiment, the scFv that binds to C5 includes a VH domain having the amino acid sequence of SEQ ID NO: 172, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VL domain having the amino acid sequence of SEQ ID NO: 173, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0084] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VL-linker-VH. In the embodiment, the scFv that binds to C5 includes a VL domain having the amino acid sequence of SEQ ID NO: 173, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VH domain having the amino acid sequence of SEQ ID NO: 172, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0085] In embodiments, scFv has the amino acid sequence of SEQ ID NO: 180, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or the sequence of SEQ ID NO: 264, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5), and binds to C5. The nucleotide sequence encoding scFv can be codon-optimized for expression in human cells.

[0086] In the embodiment, the transgene encodes scFv, which includes a VH domain containing three CDRs underlined in the C5-A-mab heavy chain sequence in Figure 2B, and a VL domain having three CDRs underlined in the C5-A-mab light chain sequence in Figure 2B. In the embodiment, the VH domain and the VL domain are alternative framework regions (i.e., framework regions not found or found only partially in the sequences of Figure 2B, which include, but are not limited to, human framework regions known in the art).

[0087] Transgene sequence In certain embodiments, the anti-C5 scFv transgene includes, as shown in Table 6, the nucleotide sequence of the first 369 nucleotides of SEQ ID NO: 18 or 19 (encoding the C5-A-scFv variable heavy domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 20 (encoding the C5-A-scFv variable light domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In embodiments, the nucleotide sequences encoding the variable light domain and the variable heavy domain are separated by a plastic linker, for example, a nucleotide sequence having one of the amino acid sequences from SEQ ID NOs: 39-43. In the embodiments, the nucleotide sequence encoding scFv (in the embodiments, including a signal sequence at the N-terminus) is operably ligated to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. In the embodiments, one or more regulatory elements are constitutive promoters. In the embodiments, one or more regulatory elements are tissue-specific promoters. In the embodiments, the transgene is operably ligated to a CAG promoter (SEQ ID NO: 45), a mutant 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 the embodiment, the promoter is a tissue-specific promoter (or a regulatory sequence including a promoter and an enhancer element) such as the GRK1 promoter (SEQ ID NO: 48 or 137), the mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 134-136), the human red opsin (RedO) promoter (SEQ ID NO: 132), or the Best1 / GRK1 tandem promoter (SEQ ID NO: 144). In the embodiment, the intron sequence is located between the promoter and the coding sequence, for example, the VH4 intron sequence (SEQ ID NO: 70).The transgene may further include a polyadenylation signal after the 3' end of the coding sequence. The transgene may contain elements provided in Table 1 or 1a. An exemplary transgene encoding the scFv form of C5-A-mab provided in Table 6 is the C5-A-mab.scFv coding sequence (SEQ ID NO: 230 or 231). Exemplary expression cassette coding sequences include SEQ ID NOs: 235 and 236. Exemplary artificial genome coding sequences include SEQ ID NOs: 233 or 234. The artificial genome containing the transgene may be packaged in an AAV capsid, particularly AAV8 or AAV3B.

[0088] In certain embodiments, a construct is provided that encodes an artificial genome in which the transgene is operably ligated to a regulatory sequence and a poly-A tail (e.g., the expression cassette described above), and the expression cassette is adjacent to an ITR sequence. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single-stranded. The artificial genome may include or consist of the nucleotide sequence of SEQ ID NO: 233 or 234. The artificial genome may include a nucleotide sequence that is 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 either SEQ ID NO: 233 or 234 which encodes and expresses anti-C5 scFv as described herein.

[0089] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 30. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain containing 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 shown in the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29. In a particular embodiment, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 30, and a variable heavy domain containing 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 shown in the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29. In certain embodiments, anti-C5 scFv includes a variable heavy domain containing the nucleotide sequence of the first 369 nucleotides of SEQ ID NO: 28 or SEQ ID NO: 29, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2B). In certain embodiments, anti-C5 scFv includes a variable light domain containing the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 30, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2B).

[0090] C5-B-scFv construct AA array In this embodiment, the transgene encodes a C5-binding scFv containing the variable weight domain (VH) of SEQ ID NO: 176, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the variable light domain (VL) of SEQ ID NO: 258, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5 and Figure 2E). In one embodiment, the scFv that binds to C5 includes a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 177, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2E). In a specific embodiment, the scFv that binds to C5 includes a light chain variable domain containing the amino acid sequence of SEQ ID NO: 177, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2E).

[0091] In the embodiment, the VH and VL domains are connected by a flexible linker (e.g., one of sequence numbers 39-43). In the embodiment, the linker is GGGGSGGGGSGGGGS (sequence number 41).

[0092] In embodiments, the transgene has a signal or leader sequence at its N-terminus suitable for expression and secretion in human cells, particularly human eye tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence includes 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 shown in Table 2. Alternatively, the signal sequence may be suitable for expression in muscle or hepatocytes, such as those listed in Table 3 below.

[0093] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VH-linker-VL. In the embodiment, the scFv that binds to C5 includes a VH domain having the amino acid sequence of SEQ ID NO: 176, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VL domain having the amino acid sequence of SEQ ID NO: 177, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0094] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VL-linker-VH. In the embodiment, the scFv that binds to C5 includes a VL domain having the amino acid sequence of SEQ ID NO: 177, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VH domain having the amino acid sequence of SEQ ID NO: 176, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0095] In embodiments, scFv has the amino acid sequence of SEQ ID NO: 187, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or the sequence of SEQ ID NO: 271, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5), and binds to C5. The nucleotide sequence encoding scFv can be codon-optimized for expression in human cells.

[0096] In the embodiment, the transgene encodes scFv, which includes a VH domain containing three CDRs underlined in the C5-B-mab heavy chain sequence in Figure 2E, and a VL domain having three CDRs underlined in the C5-B-mab light chain sequence in Figure 2E. In the embodiment, the VH domain and the VL domain are alternative framework regions (i.e., framework regions not found or found only partially in the sequence in Figure 2E, which include, but are not limited to, human framework regions known in the art).

[0097] Transgene sequence In certain embodiments, the anti-C5 scFv transgene includes, as shown in Table 6, the nucleotide sequence of the first 348 nucleotides of SEQ ID NO: 23 (encoding the C5-B-scFv variable weight domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 34 (encoding the C5-B-scFv variable light domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In embodiments, the nucleotide sequences encoding the variable light domain and the variable weight domain are separated by a flexible linker, for example, a nucleotide sequence having one of the amino acid sequences from SEQ ID NOs: 39-43. In the embodiments, the nucleotide sequence encoding scFv (in the embodiments, including a signal sequence at the N-terminus) is operably ligated to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. In the embodiments, one or more regulatory elements are constitutive promoters. In the embodiments, one or more regulatory elements are tissue-specific promoters. In the embodiments, the transgene is operably ligated to a CAG promoter (SEQ ID NO: 45), a mutant 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 the embodiment, the promoter is a tissue-specific promoter (or a regulatory sequence including a promoter and an enhancer element) such as the GRK1 promoter (SEQ ID NO: 48 or 137), the mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 214-216), the human red opsin (RedO) promoter (SEQ ID NO: 132), or the Best1 / GRK1 tandem promoter (SEQ ID NO: 144). In the embodiment, the intron sequence is located between the promoter and the coding sequence, for example, the VH4 intron sequence (SEQ ID NO: 51).The transgene may further contain a polyadenylation signal after the 3' end of the coding sequence. The transgene may contain elements provided in Table 1 or 1a. An exemplary transgene encoding the scFv form of C5-B-mab provided in Table 6 is the C5-B-mab.scFv coding sequence (sequence number 226 or 227). The artificial genome containing the transgene may be packaged in an AAV capsid, particularly AAV8 or AAV3B.

[0098] In certain embodiments, a construct is provided that encodes an artificial genome in which the transgene is operably linked to a regulatory sequence and a poly-A tail (e.g., the expression cassette described above), and the expression cassette is adjacent to an ITR sequence. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single-stranded.

[0099] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 24. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain containing 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 shown in the first 348 nucleotides of SEQ ID NO: 23. In a particular embodiment, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 34, and a variable heavy domain containing 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 shown in the first 348 nucleotides of SEQ ID NO: 23. In certain embodiments, anti-C5 scFv includes a variable heavy domain containing the nucleotide sequence of the first 348 nucleotides of SEQ ID NO: 23, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2E). In certain embodiments, anti-C5 scFv includes a variable light domain containing the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 24, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2E).

[0100] C5-C-scFv construct AA array In this embodiment, the transgene encodes a C5-binding scFv containing the variable weight domain (VH) of SEQ ID NO: 174, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the variable light domain (VL) of SEQ ID NO: 175, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5 and Figure 2D). In one embodiment, the scFv that binds to C5 includes a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 174, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2D). In a specific embodiment, the scFv that binds to C5 includes a light chain variable domain containing the amino acid sequence of SEQ ID NO: 175, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2D).

[0101] In the embodiment, the VH and VL domains are connected by a flexible linker (e.g., one of sequence numbers 39-43). In the embodiment, the linker is GGGGSGGGGSGGGGS (sequence number 41).

[0102] In embodiments, the transgene has a signal or leader sequence at its N-terminus suitable for expression and secretion in human cells, particularly human eye tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence includes 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 shown in Table 2. Alternatively, the signal sequence may be suitable for expression in muscle or hepatocytes, such as those listed in Table 3 below.

[0103] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VH-linker-VL. In the embodiment, the scFv that binds to C5 includes a VH domain having the amino acid sequence of SEQ ID NO: 174, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VL domain having the amino acid sequence of SEQ ID NO: 175, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0104] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VL-linker-VH. In the embodiment, the scFv that binds to C5 includes a VL domain having the amino acid sequence of SEQ ID NO: 175, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VH domain having the amino acid sequence of SEQ ID NO: 174, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0105] In embodiments, scFv has the amino acid sequence of SEQ ID NO: 265, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or the sequence of SEQ ID NO: 266, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5), and binds to C5. The nucleotide sequence encoding scFv can be codon-optimized for expression in human cells.

[0106] In the embodiment, the transgene encodes scFv, which includes a VH domain containing three CDRs underlined in the C5-C-mab heavy chain sequence in Figure 2D, and a VL domain having three CDRs underlined in the C5-C-mab light chain sequence in Figure 2D. In the embodiment, the VH domain and the VL domain are alternative framework regions (i.e., framework regions not found or found only partially in the sequence in Figure 2D, which include, but are not limited to, human framework regions known in the art).

[0107] Transgene sequence In certain embodiments, the anti-C5 scFv transgene includes, as shown in Table 6, the nucleotide sequence of the first 366 nucleotides of SEQ ID NO: 21 (encoding the C5-C-scFv variable weight domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 22 (encoding the C5-C-scFv variable light domain) encoding the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In embodiments, the nucleotide sequences encoding the variable light domain and the variable weight domain are separated by a flexible linker, for example, a nucleotide sequence having one of the amino acid sequences from SEQ ID NOs: 39-43. In the embodiments, the nucleotide sequence encoding scFv (in the embodiments, including a signal sequence at the N-terminus) is operably ligated to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. In the embodiments, one or more regulatory elements are constitutive promoters. In the embodiments, one or more regulatory elements are tissue-specific promoters. In the embodiments, the transgene is operably ligated to a CAG promoter (SEQ ID NO: 45), a mutant 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 the embodiment, the promoter is a tissue-specific promoter (or a regulatory sequence including a promoter and an enhancer element) such as the GRK1 promoter (SEQ ID NO: 48 or 137), the mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 134-146), the human red opsin (RedO) promoter (SEQ ID NO: 212), or the Best1 / GRK1 tandem promoter (SEQ ID NO: 114). In the embodiment, the intron sequence is located between the promoter and the coding sequence, for example, the VH4 intron sequence (SEQ ID NO: 51).The transgene may further contain a polyadenylation signal after the 3' end of the coding sequence. The transgene may contain elements provided in Table 1 or 1a. An exemplary transgene encoding the scFv form of C5-C-mab provided in Table 6 is the C5-C-mab.scFv coding sequence (sequence number 228 or 229). The artificial genome containing the transgene may be packaged in an AAV capsid, particularly AAV8 or AAV3B.

[0108] In certain embodiments, a construct is provided that encodes an artificial genome in which the transgene is operably linked to a regulatory sequence and a poly-A tail (e.g., the expression cassette described above), and the expression cassette is adjacent to an ITR sequence. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single-stranded.

[0109] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 22. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain containing 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 shown in the first 366 nucleotides of SEQ ID NO: 21. In a particular embodiment, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 324 nucleotides of SEQ ID NO: 22, and a variable heavy domain containing 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 shown in the first 366 nucleotides of SEQ ID NO: 21. In certain embodiments, anti-C5 scFv includes a variable heavy domain containing the nucleotide sequence of the first 366 nucleotides of SEQ ID NO: 31, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotide substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2D). In certain embodiments, anti-C5 scFv includes a variable light domain containing the nucleotide sequence of the first 324 nucleotides of SEQ ID NO: 22, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions, or deletions, where the substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2D).

[0110] C5-D-scFv construct AA array In this embodiment, the transgene encodes a C5-binding scFv containing the variable weight domain (VH) of SEQ ID NO: 170, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the variable light domain (VL) of SEQ ID NO: 171, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5 and Figure 2A). In one embodiment, the scFv that binds to C5 includes a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 170, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2A). In a specific embodiment, the scFv that binds to C5 includes a light chain variable domain containing the amino acid sequence of SEQ ID NO: 171, which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or more amino acid substitutions, insertions, or deletions, and these substitutions, insertions, or deletions are made, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2A).

[0111] In the embodiment, the VH and VL domains are connected by a flexible linker (e.g., one of sequence numbers 839-43). In the embodiment, the linker is GGGGSGGGGSGGGGS (sequence number 41).

[0112] In embodiments, the transgene has a signal or leader sequence at its N-terminus suitable for expression and secretion in human cells, particularly human eye tissue cells (e.g., retinal cells) or liver and / or muscle cells. In embodiments, the signal sequence includes 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 shown in Table 2. Alternatively, the signal sequence may be suitable for expression in muscle or hepatocytes, such as those listed in Table 3 below.

[0113] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VH-linker-VL. In the embodiment, the scFv that binds to C5 includes a VH domain having the amino acid sequence of SEQ ID NO: 170, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VL domain having the amino acid sequence of SEQ ID NO: 171, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0114] In the embodiment, the scFv that binds to C5 has the structure: signal sequence-VL-linker-VH. In the embodiment, the scFv that binds to C5 includes a VL domain having the amino acid sequence of SEQ ID NO: 171, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, a flexible linker at the N-terminus (see Table 4, e.g., having one of the amino acid sequences from SEQ ID NOs: 39-43), and a VH domain having the amino acid sequence of SEQ ID NO: 170, or a sequence identical thereto by at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0115] In embodiments, scFv has the amino acid sequence of SEQ ID NO: 261, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or the sequence of SEQ ID NO: 179, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto (see Table 5), and binds to C5. The nucleotide sequence encoding scFv can be codon-optimized for expression in human cells.

[0116] In the embodiment, the transgene encodes scFv, which includes a VH domain containing three CDRs underlined in the C5-D-mab heavy chain sequence in Figure 2A, and a VL domain having three CDRs underlined in the C5-D-mab light chain sequence in Figure 2A. In the embodiment, the VH domain and the VL domain are alternative framework regions (i.e., framework regions not found or found only partially in the sequence in Figure 2A, which include, but are not limited to, human framework regions known in the art).

[0117] Transgene sequence In certain embodiments, the anti-C5 scFv transgene includes, as shown in Table 6, the nucleotide sequence of the first 369 or 372 nucleotides of SEQ ID NO: 16 (encoding the C5-D-scFv variable weight domain), which encodes the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the nucleotide sequence of the first 333 nucleotides of SEQ ID NO: 17 (encoding the C5-D-scFv variable light domain), which encodes the C5-binding scFv, or a sequence that is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In embodiments, the nucleotide sequences encoding the variable light domain and the variable weight domain are separated by a flexible linker, for example, a nucleotide sequence having one of the amino acid sequences from SEQ ID NOs: 39-43. In the embodiments, the nucleotide sequence encoding scFv (in the embodiments, including a signal sequence at the N-terminus) is operably ligated to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. In the embodiments, one or more regulatory elements are constitutive promoters. In the embodiments, one or more regulatory elements are tissue-specific promoters. In the embodiments, the transgene is operably ligated to a CAG promoter (SEQ ID NO: 45), a mutant 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 the embodiment, the promoter is a tissue-specific promoter (or a regulatory sequence including a promoter and an enhancer element) such as the GRK1 promoter (SEQ ID NO: 48 or 137), the mouse pyramidal arrestin (CAR) promoter (SEQ ID NO: 134-136), the human red opsin (RedO) promoter (SEQ ID NO: 212), or the Best1 / GRK1 tandem promoter (SEQ ID NO: 144). In the embodiment, the intron sequence is located between the promoter and the coding sequence, for example, the VH4 intron sequence (SEQ ID NO: 52).The transgene may further include a polyadenylation signal after the 3' end of the coding sequence. The transgene may contain elements provided in Table 1 or 1a. An exemplary transgene encoding the scFv form of C5-D-mab provided in Table 6 is the C5-D-mab.scFv coding sequence (sequence numbers 184, 194, or 197). Exemplary expression cassette coding sequences include sequence numbers 186, 195, 198, 200, 202, 204, 206, and 208. Exemplary artificial genome coding sequences include sequence numbers 185, 196, 199, 201, 203, 205, 207, or 209. The artificial genome containing the transgene may be packaged in an AAV capsid, particularly AAV8 or AAV3B.

[0118] In certain embodiments, a construct is provided that encodes an artificial genome in which the transgene is operably linked to a regulatory sequence and a poly-A tail (e.g., the expression cassette described above), and the expression cassette is adjacent to an ITR sequence. In some embodiments, the artificial genome is self-complementary. In some embodiments, the artificial genome is single-stranded. The artificial genome may include, or consist of, the nucleotide sequences of SEQ ID NOs: 185, 196, 199, 201, 203, 205, 207, or 209. The artificial genome may contain nucleotide sequences that are 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 about 97%, at least about 98%, or at least about 99% identical to any one of sequence numbers 185, 196, 199, 201, 203, 205, 207, or 209 that encode and express anti-C5 scFv as described herein.

[0119] In certain embodiments, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 333 nucleotides of SEQ ID NO: 17. In certain embodiments, the anti-C5 scFv transgene encodes a variable heavy domain containing 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 shown in the first 369 or 372 nucleotides of SEQ ID NO: 16. In a particular embodiment, the anti-C5 scFv transgene encodes a variable light domain containing 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 shown in the first 333 nucleotides of SEQ ID NO: 17, and a variable heavy domain containing 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 shown in the first 369 or 372 nucleotides of SEQ ID NO: 16. In certain embodiments, anti-C5 scFv includes a variable heavy domain containing the nucleotide sequence of the first 369 or 372 nucleotides of SEQ ID NO: 16, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotide substitutions, insertions, or deletions, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2A). In certain embodiments, anti-C5 scFv includes a variable light domain containing the nucleotide sequence of the first 333 nucleotides of SEQ ID NO: 17, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more amino acid substitutions, insertions, or deletions, for example, in the framework region (e.g., the region outside the CDR, which is underlined in Figure 2A).

[0120] In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a constitutive or inducible (e.g., hypoxia-inducible or rifamycin-inducible) promoter sequence, or a tissue-specific promoter / regulatory region, e.g., one of the regulatory regions provided in Table 1 or Table 1a, and b) a sequence encoding a transgene (e.g., scFv). In certain embodiments, the sequence containing the transgene encodes the heavy and light chain variable domains of HuGlyFab, separated by a flexible peptide linker (as scFv). In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a constitutive or inducible promoter sequence, or a tissue-specific promoter (e.g., one of the promoters or regulatory regions in Table 1 or 1a), and b) a sequence encoding a transgene (e.g., HuGlyscFv) (the transgene comprises a signal peptide separated by a linker, a variable light chain, and a nucleotide sequence encoding the heavy chain).

[0121] In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or 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 a transgene (e.g., HuGlyscFv), i) a second UTR sequence, j) a fourth linker sequence, k) a poly(A) sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0122] In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or inducible promoter sequence or 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 a transgene (e.g., HuGlyscFv), i) a second UTR sequence, j) a fourth linker sequence, k) a poly(A) sequence, l) a fifth linker sequence, and m) a second ITR sequence (the transgene includes a signal, and the transgene encodes a light chain variable domain and a heavy chain variable domain separated by the flexible linker sequence).

[0123] In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or 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 a 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, l) a fifth linker sequence, and m) a second ITR sequence.

[0124] 5.1.10 Manufacturing and testing of vectors The viral vectors provided herein may be produced using host cells. The viral vectors provided herein may be produced using mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided herein may be produced using host cells from humans, monkeys, mice, rats, rabbits, or hamsters.

[0125] Host cells are stably transformed using the transgene and associated elements (e.g., vector genome), as well as means for producing the virus in the host cell, such as sequences encoding replication and capsid genes (e.g., the rep and cap genes of AAV). For a method of producing a recombinant AAV vector with the AAV8 capsid, see Section IV of the Embodiments for Carrying Out the Invention of U.S. Patent No. 7,282,199B2 (which is incorporated herein by reference in its entirety). The genomic copy titer of the vector can be determined, for example, by TAQMAN® analysis. Virons can be recovered, for example, by CsCl2 precipitation.

[0126] Alternatively, AAV vectors may be produced using a baculovirus expression system in insect cells. For an overview, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054 (the entire description of the manufacturing technique is incorporated herein by reference).

[0127] In vitro assays, such as cell culture assays, can be used to measure transgene expression from the vectors described herein and thus, for example, to demonstrate the potency of the vectors. In addition, in vitro neutralization assays can be used to measure the activity of transgenes expressed from the vectors described herein. For example, the neutralizing activity of transgenes expressed from the vectors described herein can be evaluated using Vero-E6 cells, a cell line derived from the kidney of African green monkeys, or HeLa cells engineered to stably express the ACE2 receptor (HeLa-ACE2). In addition, other characteristics of the expressed product, such as the glycosylation and tyrosine sulfate patterns associated with HuGlyscFv, can be determined. Furthermore, the benefits arising from the glycosylation / sulfation of cell-expressed HuGlyscFv can be determined using assays known in the art, such as those described in Section 5.3.

[0128] Vector genome concentration (GC) or vector genome copy can be evaluated using digital PCR (dPCR) or ddPCR® (BioRad Technologies, Hercules, CA, USA). In one example, ocular tissue samples, such as aqueous humor and / or vitreous humor samples, are obtained at several time points. In another example, several mice are sacrificed at various time points after injection. Ocular tissue samples are subjected to total DNA extraction and dPCR assays for vector copy number. The number of vector genome (transgene) copies per gram of tissue may be measured in a single biopsy sample or in various tissue sections at consecutive time points to reveal the extent of AAV throughout the eye. Total DNA from the collected ocular fluid or tissue is extracted using the DNeasy Blood & Tissue Kit and DNA concentration measured using a Nanodrop spectrophotometer. Digital PCR is performed using a Naica Crystal Digital PCR system (Stilla Technologies) to determine the vector copy number for each tissue sample. A two-color multiplexing system is applied to simultaneously measure the transgene AAV and endogenous control gene. In short, the transgene probe can be labeled with FAM (6-carboxyfluorescein) dye, while the endogenous control probe can be labeled with VIC fluorescent dye. The number of delivered vector copies per diploid cell in a particular tissue section is calculated as follows: (number of vector copies) / (endogenous control) × 2. Over time, vector copies in specific cell types or tissues such as cornea, iris, ciliary body, Schlemm's canal cells, trabecular meshwork, retinal cells, RPE cells, RPE choroidal tissue, or optic nerve cells may indicate sustained expression of the transgene by the tissue.

[0129] 5.1.11 Composition Pharmaceutical compositions suitable for administration to human subjects include a suspension of a recombinant vector in a formulation buffer, comprising a physiologically compatible aqueous buffer, a surfactant, and an optional excipient. Such a formulation buffer may contain one or more of the following: polysaccharides, surfactants, polymers, or oils. 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 federal or state regulatory authority for use in animals, more specifically in humans, or listed in the United States Pharmacopeia or other commonly recognized pharmacopoeias. The term “carrier” refers to a diluent, adjuvant (e.g., Freund’s complete and incomplete adjuvants), excipient, or vehicle administered with the drug. Such pharmaceutical carriers may be sterile liquids such as water and oil, including petroleum, animal, plant, or synthetic origins, including, for example, peanut oil, soybean oil, mineral oil, and sesame oil. Water is a common carrier when the pharmaceutical composition is administered intravenously. Physiological saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly as injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.Further examples of pharmaceutically acceptable carriers, excipients, and stabilizers include buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides; proteins, e.g., serum albumin and gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®, which are known in the art. The pharmaceutical compositions of the present invention may also include, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0130] In some embodiments, the pharmaceutical composition includes viscosity enhancers(s). Exemplary formulations for the delivery of AAV include WO2022 / 076549 and WO2021 / 071835, each of which is incorporated herein by reference in whole.

[0131] In some embodiments, the reference pharmaceutical composition comprises 1% high-viscosity grade carboxymethylcellulose. In some embodiments, the reference pharmaceutical composition comprises 0.2-15% high-viscosity grade carboxymethylcellulose (CMC), high-viscosity grade CMC, medium-viscosity grade CMC, hydroxypropyl methylcellulose (HPMC), HPMC, hydroxyethylcellulose (HES), low-viscosity grade CMC, and / or poloxamer 407.

[0132] In some embodiments, the pharmaceutical composition contains hyaluronic acid as a viscosity enhancer. In some embodiments, the hyaluronic acid has a molecular weight of about 1 MDa to about 2 MDa. The hyaluronic acid can include, for example, molecular weights of about 1 MDa, about 1.5 MDa, about 1.58 MDa, or about 2.0 MDa. In some embodiments, the hyaluronic acid has a molecular weight of about 1.58 MDa. In some embodiments, the hyaluronic acid has a molecular weight of about 2.0 MDa. In embodiments, HA may be obtained from various sources including ThermoFisher, Lifecore Biomedical, etc., and is pharmaceutical grade HA. Hyaluronic acid is also known as sodium hyaluronate [CAS No. 9067-32-7, chemical formula: (C 14 H 20 NO 11 Na)n]. Various sources include, but are not limited to, the list in Table A. [Table 6]

[0133] In the embodiment, the pharmaceutical formulation contains approximately 0.5% w / v to approximately 1.0% w / v of hyaluronic acid. In the embodiment, the pharmaceutical formulation contains approximately 0.6% w / v to approximately 0.9% w / v of hyaluronic acid. In the embodiment, the pharmaceutical formulation contains approximately 0.7% w / v to approximately 0.8% w / v of hyaluronic acid.

[0134] In this embodiment, the pharmaceutical formulation contains approximately 0.5% w / v hyaluronic acid, approximately 0.6% w / v hyaluronic acid, approximately 0.7% w / v hyaluronic acid, approximately 0.8% w / v hyaluronic acid, approximately 0.9% w / v hyaluronic acid, or approximately 1.0% w / v hyaluronic acid.

[0135] In this embodiment, AAV is in a pre-formulated solution of about 0.2 mg / mL potassium chloride, about 0.2 mg / mL potassium dihydrogen phosphate, about 5.84 mg / mL sodium chloride, about 1.15 mg / mL anhydrous disodium hydrogen phosphate, about 40.0 mg / mL (4% w / v) sucrose, and about 0.001% (0.01 mg / mL) poloxamer 188.

[0136] In the embodiment, AAV is in a pre-formulated solution of about 0.2 mg / mL potassium chloride, about 0.2 mg / mL potassium dihydrogen phosphate, about 5.84 mg / mL sodium chloride, about 1.15 mg / mL anhydrous disodium hydrogen phosphate, 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.

[0137] In the embodiment, AAV is in a pre-formulated solution of about 0.2 mg / mL potassium chloride, about 0.2 mg / mL potassium dihydrogen phosphate, about 5.84 mg / mL sodium chloride, about 1.15 mg / mL anhydrous disodium hydrogen phosphate, 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.

[0138] In this embodiment, AAV is in a pre-formulated solution of approximately 0.2 mg / mL potassium chloride, approximately 0.2 mg / mL potassium dihydrogen phosphate, approximately 5.84 mg / mL sodium chloride, approximately 1.15 mg / mL anhydrous disodium hydrogen phosphate, approximately 25.0 mg / mL (2.5% w / v) sucrose, approximately 0.002% (0.02 mg / mL) poloxamer 188, and approximately 0.7% w / v hyaluronic acid.

[0139] In this embodiment, AAV is in a pre-formulated solution of approximately 0.2 mg / mL potassium chloride, approximately 0.2 mg / mL potassium dihydrogen phosphate, approximately 5.84 mg / mL sodium chloride, approximately 1.15 mg / mL anhydrous disodium hydrogen phosphate, approximately 25.0 mg / mL (2.5% w / v) sucrose, approximately 0.002% (0.02 mg / mL) poloxamer 188, and approximately 0.8% w / v hyaluronic acid.

[0140] In this embodiment, AAV is in a pre-formulated solution of approximately 0.2 mg / mL potassium chloride, approximately 0.2 mg / mL potassium dihydrogen phosphate, approximately 5.84 mg / mL sodium chloride, approximately 1.15 mg / mL anhydrous disodium hydrogen phosphate, approximately 25.0 mg / mL (2.5% w / v) sucrose, approximately 0.002% (0.02 mg / mL) poloxamer 188, and approximately 0.9% w / v hyaluronic acid.

[0141] In this embodiment, AAV is in a pre-formulated solution of approximately 0.2 mg / mL potassium chloride, approximately 0.2 mg / mL potassium dihydrogen phosphate, approximately 5.84 mg / mL sodium chloride, approximately 1.15 mg / mL anhydrous disodium hydrogen phosphate, approximately 25.0 mg / mL (2.5% w / v) sucrose, approximately 0.002% (0.02 mg / mL) poloxamer 188, and approximately 1.0% w / v hyaluronic acid.

[0142] In this embodiment, AAV is in a pre-formulated solution of about 0.2 mg / mL potassium chloride, about 0.2 mg / mL potassium dihydrogen phosphate, about 5.84 mg / mL sodium chloride, about 1.15 mg / mL anhydrous disodium hydrogen phosphate, about 40.0 mg / mL (4% w / v) sucrose, and about 0.001% (0.01 mg / mL) poloxamer 188.

[0143] In the embodiment, AAV is in a pre-formulated solution of about 0.2 mg / mL potassium chloride, about 0.2 mg / mL potassium dihydrogen phosphate, about 5.84 mg / mL sodium chloride, about 1.15 mg / mL anhydrous disodium hydrogen phosphate, about 40.0 mg / mL (4% w / v) sucrose, about 0.001% (0.01 mg / mL) poloxamer 188, and about 1% high viscosity grade carboxymethylcellulose (CMC).

[0144] In embodiments, the pharmaceutical formulation is in a sucrose-containing formulation such as that found in WO2021071835A1 (which is incorporated herein in whole by reference). In embodiments, AAV is a pharmaceutical composition comprising (a) recombinant adeno-associated virus (AAV), (b) potassium chloride, (c) potassium dihydrogen phosphate, (d) sodium chloride, (e) disodium hydrogen phosphate anhydrous, (f) sucrose, and (e) poloxamer 188, polysorbate 20, or polysorbate 80. In this embodiment, AAV is a pharmaceutical composition comprising (a) potassium chloride at a concentration of 0.2 g / L, (b) potassium dihydrogen phosphate at a concentration of 0.2 g / L, (c) sodium chloride at a concentration of 5.84 g / L, (d) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L, (e) sucrose (40 g / L) at a concentration of 4 wt / vol%, and (f) poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of 0.001 wt / vol% (0.01 g / L).

[0145] In the embodiments, the pharmaceutical formulation is in a formulation such as that found in WO2022076591A1 (which is incorporated herein in its entirety by reference). In the embodiments, the pharmaceutical composition contains an ionic strength of at most about 200 mM and at least about 3% aggregated recombinant AAV before choroidal administration.

[0146] In embodiments, the pharmaceutical formulation is in a high-viscosity formulation such as that found in WO2022076549A1 (which is incorporated herein in whole by reference). In embodiments, the pharmaceutical composition has a viscosity of about 25 cP to about 3 × 10⁶ cP when measured at a maximum shear rate of about 1 s⁻¹ and comprises at least one of sucrose, 4% sucrose, 6% sucrose, 10% sucrose, 2% sodium carboxymethylcellulose salt, 1% sodium carboxymethylcellulose salt, carboxymethylcellulose (CMC), 0.5% CMC, 1% CMC, 2% CMC, 4% CMC, hyaluronic acid, polyvinyl alcohol, hydroxyethylcellulose, sodium carboxymethylcellulose salt, and hydroxypropylmethylcellulose.

[0147] In the embodiments, the pharmaceutical formulation is in a gel formulation such as that found in WO2022076595A1 (which is incorporated herein in its entirety by reference). In the embodiments, the pharmaceutical composition has a viscosity that increases with increasing temperature and / or a higher modulus of elasticity, and optionally, the pharmaceutical composition contains poloxamer 407 and poloxamer 188.

[0148] 5.2 Treatment methods for atrophic AMD In another embodiment, the present invention provides a method for treating in subjects requiring treatment of atrophic AMD (age-related AMD) or other indications treatable with an anti-C5 antibody (a composition for use in the treatment of atrophic AMD or other indications treatable with an anti-C5 antibody), the method comprising administering recombinant AAV particles containing an expression cassette encoding the anti-C5 scFv described herein. Subjects requiring treatment include subjects with atrophic AMD or those predisposed to it, e.g., subjects at risk of developing atrophic AMD or other indications treatable with an anti-C5 antibody. Subjects to which such gene therapy is administered may be responsive to an anti-C5 antibody, e.g., C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab. In certain embodiments, the method encompasses treating patients diagnosed with atrophic AMD who, in certain embodiments, are identified as responsive to treatment with an anti-C5 antibody or are considered good candidates for therapy with an anti-C5 antibody. In certain embodiments, the patient has been previously treated with an anti-C5 antibody. To determine the response, the anti-C5 antibody scFv transgene product (e.g., produced in human cell cultures, bioreactors, etc.) can be directly administered to the target.

[0149] In certain embodiments, there are methods for treating subjects requiring treatment for atrophic AMD or other indications suitable for treatment with an anti-C5 antibody, comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector, such as an AAV vector containing a transgene encoding 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 the expression of the transgene in human ocular tissue cells (e.g., retinal cells, BrM cells, choroidal capillary cells, RPE cells, and / or choroidal cells) such that a depot releasing scFv is formed, to the eye of the subject, for example, intravitreously, subretinally, superchoroidally, anteriorly chamber, or intranasally, or systemically (including intravenously or intramuscularly), to the liver and / or muscle. Subretinal, intravitreous, anterior chamber, or choroidal administration should result in expression of the transgene product in one or more of the following retinal cell types: Bruch's membrane (BrM) (including its epithelial cells), choroidal capillaries, human photoreceptor cells (cone cells, rod cells); horizontal cells; bipolar cells; amarcline cells; retinal ganglion cells (midget cells, parasol cells, bilaminated cells, giant retinal ganglion cells, photosensitive ganglion cells, and Müller glia); and retinal pigment epithelial cells or other ocular tissue cells: corneal cells, iris cells, ciliary cells, Schlemm's canal cells, trabecular meshwork cells, RPE choroidal tissue cells, or optic nerve cells.

[0150] Recombinant vectors and pharmaceutical compositions for treating diseases or disorders in subjects requiring such treatment are described in Section 5.1. Such vectors should be tropic to human eye tissue, or liver and / or muscle cells, and may include those carrying non-replicating rAAVs, particularly AAV3B, AAV8, AAAV9, AAV10, AAVrh10, or AAVrh73 capsids. Recombinant vectors may be administered in any manner, for example, by introducing the recombinant vector into the eye, so that the recombinant vector enters the ocular tissue cells. Such vectors should contain a recombinant genome encoding anti-Cf scFv and further contain one or more regulatory sequences that control the expression of the transgene in human eye tissue cells and / or human liver and muscle cells, including, but not limited to, human rhodopsin kinase (GRK1) promoters (SEQ ID NO: 48 or 137), mouse pyramidal arrestin (CAR) promoters (SEQ ID NOs: 134-136), human red opsin (RedO) promoter (SEQ ID NO: 132), CAG promoter (SEQ ID NO: 45), mutant CAG promoters (SEQ ID NOs: 221, 222, or 223), CB promoters or CB long promoters (SEQ ID NOs: 142 or 143), or Best1 / GRK1 tandem promoters (SEQ ID NO: 144) (see also Tables 1 and 1a).

[0151] The methods described herein treat, slow the progression of, reduce the severity of, or prevent atrophic (age-related) AMD in human subjects requiring treatment. Treatment, slowing of progression, reduction of severity, or prevention may be evaluated by the subject before treatment, an equivalent untreated subject, or 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 several vision-threatening complications such as geographic atrophy lesions, progression of geographic atrophy lesions, and / or growth. The methods of the present invention may reduce the progression of geographic atrophy, including in the fovea, slow the loss of retinal cells, slow the loss of central visual field, and increase or slow the loss of visual acuity, etc.

[0152] The subjects are at risk of developing atrophic AMD, or may be predisposed to it, based on age and / or risk factors such as smoking history, obesity, cardiovascular disease, or diabetes.

[0153] Methods of gene therapy A method is provided for treating human subjects with atrophic AMD by administering a viral vector containing an anti-C5 antibody or its antigen-binding fragment, or a transgene encoding the CFHL-1 protein. The antibody may be C5-D-mab, C5-A-mab, C5-C-mab, or C5-B-mab, and may be, for example, a full-length or substantially full-length antibody or its Fab fragment, or another antigen-binding fragment. The viral vector may have an AAV capsid tropized to human eye tissue and may be AAV8, AAV9, AAV3B, or AAVrh73 (or a variant thereof, e.g., having 90%, 95%, or 99% sequence identity to the capsid sequence of AAV8, AAV9, AAV3B, or AAVrh73). The transgene is operably linked by a regulatory sequence that promotes the expression of the transgene in human eye tissue cells (including retinal cells, RPE, choroid, BrM, choroidal capillaries, photoreceptor cells, and retinal ganglion cells), such as the CAG (SEQ ID NO: 45) promoter or a mutant CAG promoter (SEQ ID NO: 222, 223, or 224), or an eye-specific promoter, such as the human rhodopsin kinase (GRK1) promoter (SEQ ID NO: 48 or 137), the mouse cone arrestin (CAR) promoter (SEQ ID NO: 134-136), the human red opsin (RedO) promoter (SEQ ID NO: 132), or the Best1 / GRK1 tandem promoter (SEQ ID NO: 144). The regulatory sequence may also include a polyadenylation signal sequence. The expression cassette, containing the transgene and the operably linked regulatory sequence, is adjacent to an ITR sequence as an artificial AAV genome. The adjacent ITR sequence may be configured to provide a self-complementary AAV (scAAV) genome. Recombinant vectors, including those shown in Figures 2A–2G, can be administered in any manner so that the recombinant vector enters one or more eye tissues. In certain embodiments, recombinant AAV contains (or is produced using) the artificial genome of CAG.C5-D-mab.scFv (SEQ ID NO: 186).In certain embodiments, recombinant AAV includes (or is produced using a cis-plasmid or construct containing) the artificial genome of C5-D-mab.scFv (SEQ ID NO: 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, or 209). In certain embodiments, the artificial genome is self-complementary. In other embodiments, recombinant AAV includes a construct containing a surrogate anti-C5 antibody containing BB5.1, or a transgene encoding its antigen-binding fragment, for use in animal models such as non-human primates to test for anti-C5 antibodies. An example of a construct encoding a BB5.1 antibody is CAG.BB5.1 (SEQ ID NO: 48).

[0154] A method is also provided for administering a recombinant AAV vector containing a transgene that is scFv. In some embodiments, the transgene encodes an scFv having the structure:signal sequence-VH-linker-VL-polyA. In some embodiments, the transgene encodes an scFv having the structure:signal sequence-VL-linker-VH-polyA. In some embodiments, the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), or GGGGSGGGGSGGGGSGGGGSGGGGGS (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, VH is sequence number 170, VL is sequence number 171, VH is sequence number 172, VL is sequence number 173, VH is sequence number 174, VL is sequence number 175, VH is sequence number 176, and VL is sequence number 177.

[0155] The therapeutically effective dose of any of these recombinant vectors should be administered in any manner that allows the recombinant vector to enter ocular tissue cells (e.g., retinal cells), for example, by subretinal, intravitreous, anterior chamber, or suprachoroidal injection or intranasal administration. Alternatively, the vector may be administered peripherally (e.g., intravenously, intramuscularly, or subcutaneously) so that the recombinant vector transduces liver and / or muscle cells, creating a depot in the liver and / or muscle tissue that expresses the transgene product in the bloodstream and delivers the therapeutic agent to the ocular tissue. Alternatively, subretinal, intravitreous, anterior chamber, or suprachoroidal administration should result in the expression of the transgene product in ocular cells, creating a depot in one or more ocular tissue cells of the patient that continuously supplies anti-C5 HuPTM mAb, or an antigen-binding fragment of anti-C5 mAb, to the target ocular tissue. Transgene expression results in therapeutically effective levels of anti-C5 antibodies or their antigen-binding fragments in aqueous humor, vitreous fluid, retinal tissue, RPE, BrM, or choroidal capillaries.

[0156] The subjects to whom such gene therapy is administered may be those that respond to anti-complement therapy. In certain embodiments, the method encompasses treating patients who have been diagnosed with atrophic AMD or have one or more associated symptoms and who have been identified as responsive to treatment with an anti-C5 antibody or who are considered good candidates for therapy with an anti-C5 antibody or CFHL-1 protein. In certain embodiments, the patients have previously been treated with C5-D-mab, C5-A-mab, C5-C-mab, C5-B-mab, or other complement activation inhibitors and are known to respond to them. To determine responsiveness, an anti-C5 transgene product (e.g., produced in cell cultures, bioreactors, etc.) can be directly administered to the subject.

[0157] In embodiments, administration of recombinant AAV comprising a construct for expressing a transgene encoding an anti-C5 antibody or its antigen-binding fragment in ocular tissue results in a reduction or delay in the progression of one or more symptoms of atrophic AMD within 10, 20, 30, 40 days, 6 months, 9 months, or 1 year after AAV administration. In embodiments, administration results in a delay or reduction in the rate of progression of geographic atrophy, including the fovea, as measured, for example, by fundus autofluorescence (FAF), in subjects compared to untreated subjects or in subjects based on the natural history of atrophic AMD. In embodiments, administration results in, for example, an improvement or reduction in the rate of visual acuity loss or best-corrected visual acuity (BCVA) as measured by a standard ETDRS chart, or an improvement in visual function as measured by a dark adaptation method, or an improvement in contrast sensitivity as measured by the Pelli-Robson test, or a reduction in drusen area or drusen accumulation. In other embodiments, the dose of the therapeutic gene delivered by gene therapy is sufficient to inhibit complement activation without exacerbating choroidal neovascularization (CNV).

[0158] However, in all cases, since the transgene product is continuously produced, maintaining a low concentration may be effective. Nevertheless, since the transgene product is continuously produced, maintaining a low concentration may be effective. The concentration of the transgene product can be measured in the patient's serum sample.

[0159] A pharmaceutical composition suitable for subretinal, intravitreous, intranasal, intrachorionic, superchoroidal, or systemic (intravenous, intramuscular, or subcutaneous) administration comprises a suspension of a recombinant vector containing an anti-C5 antibody or a transgene encoding its antigen-binding fragment in a formulation buffer comprising a physiologically compatible aqueous buffer. The formulation buffer may contain one or more of the following: polysaccharides, surfactants, polymers, or oils.

[0160] In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and contains at least 0.5%, 1%, or 2% glycosylation and / or sulfated material, and may contain at least 5%, 10%, or even 50% or 100% glycosylation and / or sulfated material. The goal of the gene therapy treatments provided herein is to slow, halt, or alleviate the progression of one or more symptoms of atrophic AMD, such as reducing the rate of geographic atrophy or improving (or reducing the rate of vision loss).

[0161] Combinations of delivery of anti-C5 HuP™ mAb or its antigen-binding fragment to the eye, liver, and / or muscle, accompanied by delivery of other available therapies, are included in the methods provided herein. Additional therapies may be administered before, concurrently with, or after gene therapy. Available therapies for subjects with atrophic AMD that can be combined with the gene therapy provided herein include, but are not limited to, ellamipretide, listiganib, photobiomodulatory agents, brimonidine tartrate, cambudine, Xiflam, or doxycycline, and others, as well as administration with anti-C5 antibodies.

[0162] 5.3. N-glycosylation, tyrosine sulfated, and O-glycosylation Each of the amino acid sequences (primary sequences) of HuPTM scFv disclosed herein includes at least one site in the amino acid sequence of the Fab fragment of the therapeutic antibody where N-glycosylation or tyrosine sulfated occurs.

[0163] 5.3.1. N-glycosylation Reverse glycosylation site In the art, the canonical N-glycosylation sequence is known to be Asn-X-Ser(or Thr), where X can be any amino acid except Pro. However, it has recently been demonstrated that asparagine (Asn) residues in human antibodies can be glycosylated in the context of the reverse consensus motif Ser(or Thr)-X-Asn, where 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 HuGlyFab and HuPTM scFv disclosed herein include such reverse consensus sequences.

[0164] Non-consensus glycosylated sites In addition to reverse N-glycosylation sites, it has recently been demonstrated that glutamine (Gln) residues in human antibodies can be glycosylated in the context of the non-consensus motif Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012-16022. Surprisingly, certain HuGlyFab fragments disclosed herein contain such non-consensus sequences. In addition, O-glycosylation involves the enzymatic addition of N-acetylgalactosamine to serine or threonine residues. 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 compared to, for example, antigen-binding fragments produced in E. coli, also because E. coli does not naturally contain a mechanism equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli has only been demonstrated when the bacteria are modified to contain a specific O-glycosylation mechanism. See, for example, Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.)

[0165] N-glycosylation of HuPTM scFv Unlike small molecule drugs, biological agents typically contain a mixture of many variants with different modifications or forms that may have different potency, pharmacokinetics, and / or safety profiles. It is not essential that all molecules produced by either gene therapy or protein therapy approaches be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfatedness to demonstrate efficacy. The goals of gene therapy treatments provided herein may, for example, be to slow or halt the progression of a disease or abnormal condition, or to reduce the severity of one or more symptoms associated with a disease or abnormal condition.

[0166] When HuPTM scFv is expressed in human cells, the N-glycosylation site of the antigen-binding fragment can be glycosylated with various different glycans. The N-glycans of the antigen-binding fragment have been characterized in the art. For example, Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039 (for its disclosure of Fab-related N-glycans, the entire work is incorporated herein by reference) characterizes Fab-related glycans and demonstrates that the Fab and Fc portions of the antibody contain different glycosylation patterns, with Fab glycans showing high galactosylation, sialylation, and bifurcation (e.g., with bifurcation GlcNAc), while Fc glycans show low fucosylation. Bondt, Huang et al., 2006, Anal. Biochem. 349:197-207 (disclosure of Fab-related N-glycans, the entire document is incorporated herein by reference) found that most glycans in Fab are sialylated. However, in Fab of antibodies examined by Huang (produced in a mouse cell background), the identified sialic acid residue was N-glycolylneuraminic acid ("Neu5Gc" or "NeuGc") (not native to humans) instead of N-acetylneuraminic acid ("Neu5Ac", dominant human sialic acid). In addition, Song et al., 2014, Anal. Chem. 86:5661-5666 (disclosure of Fab-related N-glycans, the entire document is incorporated herein by reference) describe a library of N-glycans associated with commercially available antibodies.

[0167] Importantly, when 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 NS0 cells) is avoided. Instead, as a result of the method described herein, the N-glycosylation site of HuPTM scFv is advantageously decorated with glycans that are therapeutically relevant and beneficial to humans. Such advantages are not achievable when CHO cells, NS0 cells, or E. coli are used for antibody / antigen binding fragment production, for example, because (1) CHO cells do not express 2,6-sialyltransferase and therefore cannot add 2,6-sialic acid during N-glycosylation, (2) Neu5Gc can be added as sialic acid instead of Neu5Ac, (3) they can also produce immunogenic glycans and α-Gal antigens that react with anti-α-Gal antibodies present in most individuals and can cause anaphylaxis at high concentrations, and (4) E. coli does not naturally contain the components necessary for N-glycosylation.

[0168] Assays for determining the glycosylation pattern of antibodies containing antigen-binding fragments are known in the art. For example, glycans can be analyzed using hydrazine degradation. First, polysaccharides are released from their associated proteins by incubation with hydrazine (the Ludger Liberate Hydrazine Degradation Glycan Release Kit (Oxfordshire, UK) can be used). Nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, allowing for the release of the attached glycan. The N-acetyl group is lost during this procedure and needs 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 are cleaved more cleanly and have fewer side effects than hydrazine. The free glycan can be purified on a carbon column and subsequently labeled at the reducing end with fluorophor 2-aminobenzamide. 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 shows the length and number of repeating units of the polysaccharide. Structural information can be collected by collecting individual peaks and subsequently performing MS / MS analysis. This allows for the confirmation of the monosaccharide composition and sequence of the repeating units, and additionally, the homogeneity of the polysaccharide composition can be identified. Specific peaks of low or high molecular weight molecules can be analyzed by MALDI-MS / MS, and the results are used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer consisting of a certain number of repeating units and their fragments, e.g., sugar residues, e.g., glycans. Therefore, the chromatogram allows for the measurement of the length distribution of polymers, e.g., glycans. Elution time is an indicator of polymer length, while fluorescence intensity correlates with the molar abundance of each polymer, e.g., glycan.Other methods for evaluating glycans associated with antigen-binding fragments include those described in 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.

[0169] The uniformity or heterogeneity of the glycan pattern associated with antibodies (including antigen-binding fragments) is related to both the length or size of the glycans and the number of glycans present at the glycosylation sites, and can therefore be evaluated using methods known in the art, such as methods for measuring the length or size and hydrodynamic radius of glycans. HPLC, including size exclusion, normal phase, reverse phase, and anion exchange HPLC, as well as capillary electrophoresis, allows for the measurement of hydrodynamic radius. The greater the number of glycosylation sites in a protein, the greater the variability in hydrodynamic radius compared to carriers with fewer glycosylation sites. However, when single glycan chains are analyzed, they may be more homogeneous due to their more controlled length. Glycan length can be measured by hydrazine degradation, SDS-PAGE, and capillary gel electrophoresis. In addition, homogeneity may also mean that the usage pattern of certain glycosylation sites varies over a wider / narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0170] In certain embodiments, HuPTM scFv also does not contain detectable NeuGc and / or α-Gal. “Detectable NeuGc” or “Detectable α-Gal” or “Does not contain or have NeuGc or α-Gal” means, as used herein, that HuPTM scFv does not contain a NeuGc or α-Gal moiety that is detectable by standard assay methods known in the art. For example, NeuGc may be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of N-Acetyl-and N-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 (a method for detecting NeuGc is incorporated herein by reference). Alternatively, NeuGc may be detected by mass spectrometry. α-Gal can be detected using ELISA (see, for example, Galili et al., 1998, “A sensitive assay for measuring α-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-profiling 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 listed in Platts-Mills et al., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal,” Immunol Allergy Clin North Am. 35(2):247-260.

[0171] Advantages of N-glycosylation N-glycosylation confers numerous benefits to HuPTM scFv as described herein. Since E. coli does not naturally possess the components necessary for N-glycosylation, such benefits cannot be achieved by the production of antigen-binding fragments in E. coli. Furthermore, since CHO cells lack the components necessary for the addition of certain glycans (e.g., 2,6-sialic acid and bisected GlcNAc), and because either CHO or mouse cell lines add NN-glycolylneuraminic acid ("Neu5Gc" or "NeuGc"), which is not naturally occurring in humans (and potentially immunogenic), instead of the dominant human sialic acid, N-acetylneuraminic acid ("Neu5Ac"), some benefits cannot be achieved, for example, through antibody production in CHO cells (or mouse cells such as NS0 cells). See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6):1110-1122, Huang et al., 2006, Anal. Biochem. 349:197-207 (NeuGc is a dominant sialic acid in mouse cell lines such as SP2 / 0 and NS0), and Song et al., 2014, Anal. Chem. 86:5661-5666 (each of which is incorporated herein in whole by reference). Furthermore, CHO cells can also produce immunogenic glycans and α-Gal antigens that react with anti-α-Gal antibodies present in most individuals, which can cause anaphylaxis at high concentrations. See, for example, Bosques, 2010, Nat. Biotech. 28:1153-1156. The human glycosylation patterns of HuPTM scFv described herein should reduce the immunogenicity of the transgene product and improve its efficacy.

[0172] Non-canonical glycosylation sites typically result in low levels of glycosylation of the antibody population (e.g., 1–5%), but the functional benefits can be significant (see, e.g., van de Bovenkamp et al., 2016, J.Immunol. 196:1435–1441). For example, Fab glycosylation can affect antibody stability, half-life, and binding properties. Any technique known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), can be used to determine the effect of Fab glycosylation on antibody affinity to a target. Any technique known to those skilled in the art, such as measuring radioactivity levels in the blood or organs of a subject to which a radiolabeled antibody has been administered, can be used to determine the effect of Fab glycosylation on antibody half-life. To determine the effect of Fab glycosylation on antibody stability, for example, aggregation level or protein elution level, any technique known to those skilled in the art may be used, such as differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), such as size exclusion high-performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidiometry.

[0173] The presence of sialic acid on HuPTM scFv used in the methods described herein may affect the clearance rate of HuPTM scFv. Therefore, the sialic acid pattern of HuPTM scFv can be used to generate therapeutic agents with optimized clearance rates. Methods for evaluating the clearance rate of antigen-binding fragments are known in the art. See, for example, Huang et al., 2006, Anal. Biochem. 349:197-207.

[0174] In another specific embodiment, the benefit conferred by N-glycosylation is reduced aggregation. Occupied N-glycosylation sites can mask amino acid residues prone to aggregation, resulting in a decrease in aggregation. Such N-glycosylation sites can be natural to the antigen-binding fragment used herein or can be engineered into the antigen-binding fragment used herein, resulting in a HuPTM scFv that is less prone to aggregation when expressed, e.g., in human cells. Methods for assessing antibody aggregation are known in the art. See, for example, Courtois et al., 2016, mAbs 8:99-112 (incorporated herein by reference in its entirety).

[0175] In another specific embodiment, the benefit conferred by N-glycosylation is reduced immunogenicity. Such N-glycosylation sites can be natural to the antigen-binding fragment used herein or can be engineered into the antigen-binding fragment used herein, resulting in a HuPTM scFv that is less immunogenic when expressed, e.g., in human eye tissue cells, human CNS cells, human liver cells, or human muscle cells.

[0176] In another specific embodiment, the benefit conferred by N-glycosylation is protein stability. Protein N-glycosylation is well known to confer stability to them, and methods for assessing protein stability resulting from N-glycosylation are known in the art. See, for example, Sola and Griebenow, 2009, J Pharm Sci., 98(4):1223-1245.

[0177] In another specific embodiment, the benefit confederated by N-glycosylation is modified binding affinity. It is known in the art that the presence of an N-glycosylation site within the variable domain of an antibody can increase the antibody's affinity for its antigen. See, for example, Bovenkamp et al., 2016, J.Immunol.196:1435-1441. Assays for measuring antibody binding affinity are known in the art. See, for example, Wright et al., 1991, EMBO J.10:2717-2723, and Leibiger et al., 1999, Biochem.J.338:529-538.

[0178] 5.3.2 Tyrosine Sulfation Tyrosine sulfated tyrosine (Y) residues containing glutamate (E) or aspartate (D) occur within the +5 to -5 position of Y, where the -1 position of Y is a neutral or acidically charged amino acid, but not a basic amino acid such as arginine (R), lysine (K), or histidine (H), which exclude sulfated amino acids. The HuPTM scFv described herein contains a tyrosine sulfated site.

[0179] Importantly, tyrosine sulfated antigen-binding fragments cannot be produced in E. coli, which naturally lacks the enzymes necessary for tyrosine sulfated cell formation. Furthermore, CHO cells are deficient in tyrosine sulfated cell formation and are not secretory cells, possessing limited capacity for post-translational tyrosine sulfated cell formation. See, for example, Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537. Advantageously, the method provided herein requires the expression of HuPTM scFv in secretory and tyrosine sulfated human cells.

[0180] Tyrosine sulfate is advantageous for several reasons. For example, tyrosine sulfate of antigen-binding fragments of therapeutic antibodies against a target has been shown to dramatically increase their affinity for the antigen and its activity. See, for example, Loos et al., 2015, PNAS 112:12675-12680 and Choe et al., 2003, Cell 114:161-170. Assays for detecting tyrosine sulfate are known in the art. See, for example, Yang et al., 2015, Molecules 20:2138-2164.

[0181] Amino acid and nucleotide sequences In certain embodiments, the anti-C5 antigen-binding fragment transgene comprises nucleotide sequences encoding six C5-A-mab CDRs that encode the antigen-binding fragment and are underlined in the heavy and light chain variable domain sequences in Figures 2B and 2C. Table 5 provides the Fab, scFv, and full-length heavy and light chain amino acid sequences of the anti-C5 antibody, as well as the expression product of the transgene including the signal sequence and linker. Table 6 provides the Fab, and full-length heavy and light chain encoding sequences of the antibodies, transgene coding sequences, and artificial genomes disclosed herein. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 8-1] Table 8-2 Table 8-3 Table 8-4 Table 8-5 Table 8-6 Table 8-7 Table 8-8 Table 8-9 Table 8-10 Table 8-11 Table 8-12 Table 8-13 Table 8-14 Table 8-15 Table 8-16 Table 8-17 Table 8-18 Table 8-19 Table 8-20 Table 8-21 Table 8-22 Table 8-23 Table 8-24 Table 8-25 Table 8-26 Table 8-27 Table 8-28 Table 8-29 Table 8-30 Table 8-31 Table 8-32 Table 8-33 Table 8-34 Table 8-35 Table 8-36 Table 8-37 Table 8-38 Table 8-39 Table 8-40 Table 8-41 Table 8-42 Table 8-43 Table 8-44 Table 8-45 Table 8-46 Table 8-47 Table 8-48 Table 8-49 Table 8-50 Table 8-51 Table 8-52 Table 8-53 Table 8-54 Table 8-55 Table 8-56 Table 8-57 Table 8-58 Table 8-59 Table 8-60 Table 8-61 Table 8-62 Table 8-63 Table 8-64 Table 8-65 Table 8-66 Table 8-67 Table 8-68 Table 8-69 [Table 8-70] [Table 8-71] [Table 8-72] [Table 8-73] [Table 8-74] [Table 8-75] [Table 8-76]

[0182] 5.6. Monitoring of effectiveness The compositions and methods described herein may be evaluated for efficacy using any method for evaluating efficacy in the treatment, prevention, or improvement of atrophic AMD. Evaluation may be determined in animal models or human subjects. Efficacy for visual impairment may be measured by evaluating the best corrected visual acuity (BCVA), e.g., the increase in the number of letters or lines, and efficacy may be evaluated as an increase in two or more ETDRS lines or a reduction in geographic atrophy including the fovea, as assessed by visual examination.

[0183] The compositions and methods described herein may be evaluated for efficacy using any method for evaluating efficacy in the treatment, prevention, or improvement of atrophic AMD. Evaluation may be determined in animal models or human subjects. Efficacy for visual impairment may be measured by evaluating the best corrected visual acuity (BCVA), e.g., the number of letters or lines, and efficacy may be evaluated as an increase of two or more ETDRS lines or an increase in logMAR. Physical changes to the eye, including changes in geographic atrophy, may be measured by optical coherence tomography using methods known in the art.

[0184] The compositions and methods described herein may be evaluated for efficacy using in vitro complement inhibition assays such as membrane invasion complex ("MAC") formation, C5a generation, and hemolysis. Complement inhibition assays can be performed on any suitable cell type, such as ARPE19 cells (MAC and C5a assay), iPSC-derived RPE cells (MAC and C5a assay), or sheep / rabbit erythrocytes (hemolysis assay). MAC formation assays measure MAC deposition on the surface of RPE cells (relative inhibition of MAC formation %). C5a generation assays measure the ability of a C5 antibody to prevent C5 cleavage (less C5 cleavage = less C5a). Hemolysis assays allow for comparison of complement inhibition between different complement inhibitors (50% complement inhibition dose (ng / mL) (CH)). 50 ;AH 50 ).

[0185] Animal models may be used to evaluate recombinant vectors encoding anti-C5 antibodies for expression, therapeutic effects, and adverse effects. Animal models may include humanized C5 rodent models (Example 9) or NaIO3-induced rat or mouse models (see also Example 9). Animals may be administered the vectors described herein, for example, subretinal or choroidal, and then evaluated for geographic atrophy (or changes thereof) by OCT, retinal pathology (damage to RPE), and other assessments of atrophic AMD pathology, as well as other markers of C5a reduction, C5 cleavage, or complement activation.

[0186] Endpoints include, but are not limited to, the following: the mean change in geographic atrophy in the study eye from baseline to 12, 16, 20, 24, or 28 weeks, or at the time of administration; earlier, the proportion of responders in the study eye at 12, 16, 20, 24, or 28 weeks; the mean change in best-corrected visual acuity from baseline to 12, 16, 20, 24, or 28 weeks; the change from baseline in quality of life / patient-reported outcome assessments; and the mean change in visual acuity from baseline to 12, 16, 20, 24, or 28 weeks. [Examples]

[0187] 6 Examples 6.1 Example 1: C5-D-mab scFv cDNA-based vector C5-D-mab scFv includes a transgene containing a nucleotide sequence encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of C5-D-mab (amino acid sequences are SEQ ID NOs. 170 and 171, respectively) linked by a flexible linker (e.g., GGGGSGGGGSGGGGS (SEQ ID NO. 41) or nucleotide sequence Table 4), A cDNA-based vector was constructed. The scFv has a structure VH-linker-VL or VL-linker-VH, and may have the amino acid sequence of SEQ ID NO: 178 or 179, respectively. The scFv encoded by the transgene may further contain a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), at its N-terminus, and may have the 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 also includes a constitutive promoter CAG (SEQ ID NO: 45) operably linked to the nucleotide sequence encoding the scFv. This includes the following. Alternatively, other constitutive promoters may be used, such as mU1a, EF1a, CB7, CB or CB long promoter, tissue-specific promoters such as the eye tissue-specific promoter, in particular the GRK1 promoter (SEQ ID NO: 58), or the BEST1 / GRK1 tandem promoter (SEQ ID NO: 144), or inductive promoters such as the hypoxia-inducible promoter. The artificial genome from 5'ITR to 3'ITR has the nucleotide sequence of SEQ ID NO: 212 or 214. Schematic diagrams of various anti-C5 inhibitors are shown in Figure 28. The nucleotide coding sequence of the C5-D-mab scFv(HL) cassette, which includes the CAG promoter, signal sequence, scFv transgene, and poly(A) sequence, has the nucleotide sequence of SEQ ID NO: 186.

[0188] 6.2 Example 2: C5-A-mab scFv cDNA-based vector A C5-A-mab scFv cDNA-based vector was constructed containing a transgene containing a nucleotide sequence encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of C5-A-mab) linked by a flexible linker (e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 41) or a nucleotide sequence encoding Table 4). Each scFv has a structure VH-linker-VL or VL-linker-VH, and may have the amino acid sequence of SEQ ID NO: 180 or 181, respectively. The scFv encoded by the transgene may further contain a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), at its N-terminus, and may have the 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 also contains the nucleotide sequence encoding the scFv. It includes a constitutive promoter CAG (SEQ ID NO: 45) operably linked to it. Alternatively, other constitutive promoters may be used, such as tissue-specific promoters like mU1a, EF1a, CB7, CB or CB long promoter, eye tissue-specific promoters, in particular the GRK1 promoter (SEQ ID NO: 48), or the BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or inductive promoters like the hypoxia-inducible promoter. The artificial genome includes or completes the nucleotide sequences of SEQ ID NOs: 233-236.

[0189] 6.3 Example 3: C5-C-mab Fab cDNA-based vector C5-C-mab scFv includes a transgene containing a nucleotide sequence encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of C5-C-mab (amino acid sequences are SEQ ID NOs. 174 and 175, respectively), linked by a flexible linker (e.g., GGGGSGGGGSGGGGS (SEQ ID NO. 41) or nucleotide sequence Table 4). A cDNA-based vector was constructed. Each scFv has a structure VH-linker-VL or VL-linker-VH, and may have the amino acid sequences of SEQ ID NOs. 265 and 266, respectively. The scFv encoded by the transgene may further contain a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), at its N-terminus, and may have the 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 a constitutive promoter CAG (SEQ ID NO: 45) operably ligated to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, e.g., mU1a, EF1a, CB7, CB or CB long promoter, tissue-specific promoters such as the eye tissue-specific promoter, particularly the GRK1 promoter (SEQ ID NO: 48), or the BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or inductive promoters such as the hypoxia-inducible promoter may be used.

[0190] 6.4 Example 4: C5-B-mab scFv cDNA-based vector C5-B-mab scFv includes a transgene containing a nucleotide sequence encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of C5-B-mab (amino acid sequences are SEQ ID NOs. 176 and 177, respectively), linked by a flexible linker (e.g., GGGGSGGGGSGGGGS (SEQ ID NO 41) or nucleotide sequence Table 4), A cDNA-based vector was constructed. Each scFv has a structure VH-linker-VL or VL-linker-VH, and may have the amino acid sequences of SEQ ID NOs. 187 and 188, respectively. The scFv encoded by the transgene may further contain a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), at its N-terminus, and may have the 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 a constitutive promoter CAG (SEQ ID NO: 45) operably ligated to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, e.g., mU1a, EF1a, CB7, CB or CB long promoter, tissue-specific promoters such as the eye tissue-specific promoter, in particular the GRK1 promoter (SEQ ID NO: 48), or the BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or inductive promoters such as the hypoxia-inducible promoter may be used.

[0191] 6.5 Example 5: BB5.1 scFv cDNA-based vector BB5.1 scFv includes a transgene containing a nucleotide sequence encoding a heavy chain variable domain (VH) and a light chain variable domain (VL of C5-D-mab (amino acid sequences are SEQ ID NOs. 191 and 192, respectively), linked by a flexible linker (e.g., GGGGSGGGGSGGGGS (SEQ ID NO 41) or nucleotide sequence 4) or Table 4). A cDNA-based vector was constructed. Each scFv has a structure VH-linker-VL or VL-linker-VH, and may have the amino acid sequence of SEQ ID NO: 25 or 26, respectively. The scFv encoded by the transgene may further contain a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 56), at its N-terminus, and may have the 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 a constitutive promoter CAG (SEQ ID NO: 45) operably ligated to the nucleotide sequence encoding the scFv. Alternatively, other constitutive promoters, e.g., mU1a, EF1a, CB7, CB or CB long promoter, tissue-specific promoters such as the eye tissue-specific promoter, in particular the GRK1 promoter (SEQ ID NO: 48), or the BEST1 / GRK1 tandem promoter (SEQ ID NO: 186), or inductive promoters such as the hypoxia-inducible promoter may be used.

[0192] 6.7 Example 7: NaIO3-induced mouse model We evaluate anti-C5 antibody AAV constructs using a NaIO3-induced model (inducing RPE damage) of atrophic AMD in rodents. The AAV8 constructs, AAV8.CAG.C5-D-mab.Fab (SEQ ID NO: 33), AAV8.CAG.C5-D-mab.full (SEQ ID NO: 34), AAV8.C5-A-mab.Fab.IgG1 (SEQ ID NO: 35), AAV8.C5-A-mab.Fab.IgG2 (SEQ ID NO: 36), AAV8.C5-A-mab.full (SEQ ID NO: 37), and AAV8.CAG.BB5.1 (SEQ ID NO: 38) are administered subretinal or suprachoroidally to humanized C5- mice at doses of 1E7, 1E8, or 1E9. After 28 days, NaIO3 is administered to the mice to induce geographic atrophy. After one week, the mice's eyes were evaluated by fundus examination and for visual impairment. Subsequently, they were sacrificed, and their eyes were evaluated for RPE damage and inhibition of photoreceptor loss, as well as for transgene and C5 levels.

[0193] 6.8 Example 8: Hemolysis assay All C5 inhibitor expression cassettes used in this study were constructed using a CAG promoter and rabbit beta-globin polyA. All transgenes were codon-optimized and CpG depleted. Cisplasmids were first screened in post-transfection assays in 293T cells and then packaged as AAV8 viral vectors (including scAAV8 vectors) for further study.

[0194] The classical pathway of complement activation (CP) is primarily initiated by immune complexes. A standard assay for the overall functional activity of this pathway is CH50. This assay uses sheep erythrocytes coated with rabbit antibodies (called EAs) to activate the complement system.

[0195] Classical complement pathway-related hemolysis inhibition assays were utilized using supernatants collected from plasmids transfected into HEK293T cells (encoding complement inhibitors as described herein). Supernatants (containing media with or without complement inhibitors, or negative controls containing vectorized antibodies against non-complement-related targets) were collected and 5 × 10⁶ units were added to gelatin-veronal-buffered saline (GVB++ buffer) in the wells of an assay plate. 8 The test was applied to sheep erythrocytes coated with an optimal level of rabbit anti-sheep erythrocyte IgM antibody, suspended at a cell / mL concentration. The hemolysis percentage was compared to a positive test hemolysis solution containing normal human serum titrated to a maximum of 50% hemolysis. The hemolysis percentage was calculated as follows: Hemolysis percentage = (Test sample hemolysis (OD405) - Background hemolysis (OD405)) / (Maximum hemolysis (OD405) - Background hemolysis (OD405)) × 100.

[0196] Pure supernatants collected from HEK293 cells transfected with vectorized C5-D-mab or BB5.1 plasmids (different vectorization forms: IgG, Fab, or ScFv) or recombinant protein C5 inhibitors were assayed for the percentage of EA lysis as an indicator of complement activation inhibition (Figures 4A–7B). Purified C5 inhibitor proteins were tested in series concentration (nM) to determine the minimum concentration required to lyse 50% of cells (1 CH50 units) (Figures 5A–5F). Activation of the classical pathway requires calcium and magnesium ions. The initial reaction involves C1 binding, as well as activation of C2 and C4, forming C3 convertase. This enzyme cleaves C5 and C3, which facilitates the activation of the membrane invasion pathway (proteins C5, C6, C7, C8, and C9). These five components are assembled in the membrane of sheep erythrocytes, lysing the cells. Next, the release of hemoglobin is quantified to measure the total complement activity present in the sample.

[0197] C5 inhibitors expressed in HEK293 cells suppress complement pathway activation to varying degrees in hemolysis inhibition assays. The scFv format showed strong complement inhibition (Figure 4A-B). Recombinant purified forms of each C5 inhibitor showed potent inhibition of complement activation in classical and alternative hemolysis assays (Figure 5A-F).

[0198] 6.9 Example 9: Binding kinetics and affinity of recombinant purified forms of each C5 inhibitor The recombinant purified proteins produced by each C5 inhibitor (expressed in HEK293 cells as described 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 monkey, and mouse C5 were measured using the OctetRED384 system as follows.

[0199] a. Affinity and kinetics of human complement C5: The assay was performed at 30°C and 1000 rpm. Biotinylated human complement C5 protein was first immobilized on an SA biosensor. C5-D-mab IgG and Ab fragments, as well as Coversin, were applied as analytes for the association and dissociation steps. [Table 9]

[0200] b. Affinity and kinetics of complement C5 in cynomolgus monkeys: The assay was performed at 30°C and 1000 rpm. Biotinylated cyno C5 antigen was first immobilized on an SA biosensor. C5-D-mab IgG and Ab fragments, as well as recombinant C5 inhibitor proteins, were applied as analytes for the association and dissociation steps. [Table 10]

[0201] c. Affinity and kinetics of mouse complement C5: The assay was performed at 30°C and 1000 rpm. Biotinylated mouse C5 antigen was first immobilized on an SA biosensor. BB5.1 IgG and Ab fragments were applied as analytes for the association and dissociation steps. [Table 11]

[0202] The assay was performed at 30°C and 1000 rpm. Biotinylated mouse C5 antigen was first immobilized on an SA biosensor. C5-D-mab IgG and Ab fragments, as well as coprazine, were applied as analytes for the association and dissociation steps. [Table 12]

[0203] All three formats of the anti-human C5 inhibitor (C5-D-mab) demonstrated KD values ​​for human and cyno C5 in the low to high picomolar range; however, the C5 inhibitors did not bind very strongly at low nanomolar affinity constants. Anti-mouse (BB5.1) and anti-human (C5-D-mab) C5 inhibitors with the same vectorized antibody format showed comparable affinity to mouse C5. See Table 11. [Table 13]

[0204] 6.10 Example 10: Evaluation of AAV expression C5 inhibitors in iPSC-derived RPE Membrane invasion complex (MAC) formation was measured. Briefly, ARPE19 cells or fully mature polarized iPSC-RPE (iRPE cells, Cellular Dynamics International catalog number: R1102) monolayers aged 4-5 weeks were used in the study. ARPE19 or iRPE cells were treated with purified recombinant C5 inhibitor in culture medium for 24 hours, followed by a second treatment with purified recombinant C5 inhibitor in culture medium for 48 hours, along with 5% CC-HS (NHS, complement technology) or 5% CI-HS (thermally inactivated CC-HS). Cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes and immunostained with antibodies or cell stains against C5b-9 (Invitrogen, MA5-28502), ZO-1 (Invitrogen, REF 40220), phalloidin (AF-568 phalloidin: Invitrogen A12380) and DAPI.

[0205] C5 inhibitors prevented C5 cleavage and reduced membrane invasion complex (MAC) formation (Figures 6A-C, ARPE19, Figures 6D-6H, iPSC-derived RPE). iPSC-derived RPE transduced with AAV anti-hC5 scFV (C5-D-mab scFv) at increased MOI showed dose-dependent increases in transgene product (TP) levels in the apical and basal compartments (Figure 6G). TP levels (Figure 6G) were consistent with AAV mRNA / cDNA measured by ddPCR (Figure 6H).

[0206] 6.11 Example 11: Evaluation of AAV expression C5 inhibitors in vivo C5 inhibitors encoding AAV8 were injected into the eyes of wild-type mice via subretinal (SR) administration at doses of 1E8 and 3E8vg / eye. Anti-C5 scFV (anti-hC5:C5-D-mab or anti-mC5:BB5.1) delivered via subretinal AAV showed TP levels more than 10 times higher than IgG (full-length antibody) and Fab form (Figure 7A = ng / eye; Figure 7B = pmol / eye), and similar levels to purified anti-mC5 IgG delivered once daily intraperitoneally (IP) (data not shown). AAV.anti-h5.IgG and AAV.anti-hC5.Fab SR delivery showed similar TP levels in the retina and RPE / choroid / sclera, but AAV.anti-hC5.scFV had higher levels of TP in the RPE / choroid / sclera, suggesting better penetration of anti-hC5.scFV into the outer layers of the eye (Figures 7C and 7D and Table 12). A single subretinal delivery of AAV resulted in high levels of TP in mouse eyes.

[0207] The scFv antibody fragment transgene product showed improved expression and distribution to the outer ocular layer after subretinal delivery in mice. [Table 14]

[0208] 6.12 Example 12: CAG Promoter Study CAG deletion mutants Del5, DelM, and Del3, operably linked to C5-D-mab scFv, were constructed using standard molecular biology techniques. See Figure 8. CAG-Delm has approximately 60% promoter strength as a full-length CAG. See Figure 9.

[0209] Either a full-length CAG or a CAGdelm promoter controlling C5-D-mab scFv expression was packaged in an AAV8 capsid. HEK293T cells were transduced using the resulting AAV8 particles at MOIs of 5e4, 1.5e5, or 5e5, and C5-D-mab scFv expression was measured on days 1, 2, 3, and 6 post-transduction (ng). See Figures 10A–10D. The CAGdel promoter had approximately 60% of the activity of the full-length CAG, but when combined with the scFV format mAb in the condensed scAAV vector, the expression level increased compared to ssAAV.

[0210] 6.13 Example 13: Investigation of AAV test substance after choroidal administration in NHP The objective of this study was to evaluate the ocular tropism of up to two different adeno-associated virus (AAV) pools / libraries after intrachoroidal administration to female cynomolgus monkeys. See Table 13. After administration on day 1, animals were observed for 3–12 weeks for in vivo distribution sample collection to investigate the transduction protocol. [Table 15]

[0211] procedure Administer 100 μl / eye to the animals via intrachoroidal injection on day 1 of the treatment period. Administer to the right eye first. All post-treatment collection timings are based on the administration timing of the left eye.

[0212] Ophthalmic examination Ophthalmological examinations should be performed at least twice before medication is prescribed. During the medication period, ophthalmological examinations should be performed on days 3, 8, 15, 17, 29, 31, 42, 57, 59, and 85.

[0213] In short, anesthetize the animal with ketamine. Examine the animal with a slit-lamp biomicroscope and an inverted ophthalmoscope. Examine the adnexa and anterior portion of both eyes using the slit-lamp biomicroscope. Examine the fundus of both eyes using an inverted ophthalmoscope (if visible). Dilate the pupils with a mydriatic agent (e.g., 1% tropicamide) before examining with the inverted ophthalmoscope.

[0214] Intraocular pressure measurement Intraocular pressure (IOP) measurement is performed in conjunction with ophthalmic examination (OE). On the day of OE, IOP measurement is performed on eyes that have previously been dilated. IOP measurement is performed at least twice before medication is administered. During the medication period, eye examinations are performed on days 3, 8, 15, 17, 29, 31, 42, 57, 59, and 85.

[0215] In short, anesthetize the animal with ketamine. IOP measurement is performed using an applanation tonometer. A local anesthetic (e.g., 0.5% proparacaine) is applied before IOP measurement.

[0216] Spectral domain optical coherence tomography Spectral domain optical coherence tomography (OCT or sdOCT) is performed at least once before medication is administered. During the medication period, OCT is performed once at weeks 2, 4, 8, and 12.

[0217] The animals are fasted for at least 10 hours before the procedure. The animals are anesthetized with ketamine and maintained with sevoflurane. The pupils are dilated with a mydriatic agent. OCT is performed and the data are evaluated. Imaging is performed to obtain an axial image of the retinal surface of the posterior fundus. The device is set to perform a standard retinal scan (macular volume scan and / or line scan and / or circular scan). Additional methods or scans may be used. A 55-degree lens may be used if necessary.

[0218] Eye photography - fundus Eye photographs will be taken at least once before medication is administered. During the medication period, eye photographs will be taken once each at weeks 2, 4, 8, and 12.

[0219] Fast the animals for at least 10 hours before the procedure. Anesthetize the animals with ketamine and dexmedetomidine. Dilate the pupils with a mydriatic agent. Take photographs with a wide-angle lens and a digital fundus camera. Take color photographs of each eye to include stereoscopic images of the posterior pole and non-stereoscopic images of the two intermediate peripheral fields (temporal and nasal). If possible, take additional images of the supratemporal side as well.

[0220] Fundus autofluorescence imaging Fundus autofluorescence imaging should be performed at least once before drug administration. During the drug treatment period, fundus autofluorescence imaging should be performed once at weeks 2, 4, 8, and 12.

[0221] Fast the animals for at least 10 hours before the procedure. Anesthetize the animals with ketamine and dexmedetomidine or ketamine and maintain them on sevoflurane, if applicable. Dilate the pupils with a mydriatic agent. Take images using a Heidelberg SPECTRALIS® device. Fundus autofluorescence images to include posterior poles and non-stereoscopic images of two intermediate peripheral fields (temporal and nasal, if possible). Take additional images of the superior temporal side, if possible.

[0222] Anti-AAV2 and AAV8 neutralizing antibody (NAM) analysis Anti-AAV2 and AAV8 neutralizing antibody analysis should be performed within 5 days of animal transport prior to drug administration. During the drug administration period, anti-AAV2 and AAV8 neutralizing antibody analysis should be performed on day 1 before drug administration and on each scheduled day of slaughter.

[0223] Collect 2.4 mL of blood from the femoral vein. Alternative sites may be used if necessary, and the blood collection site will be documented. Keep the blood sample at room temperature and allow it to coagulate before centrifugation. Centrifuge the sample within 1 hour of collection. Collect serum into two nearly equal aliquots. After collection, place the samples on dry ice until stored in a freezer.

[0224] Anti-AAV9 total antibody (TAB) analysis Total anti-AAV9 antibody analysis should be performed within 5 days of animal transport prior to drug administration.

[0225] Collect 2.4 mL of blood from the femoral vein. Alternative sites may be used if necessary, and the blood collection site will be documented. Keep the blood sample at room temperature and allow it to coagulate before centrifugation. Centrifuge the sample within 1 hour of collection. Collect serum into two nearly equal aliquots. After collection, place the samples on dry ice until stored in a freezer.

[0226] Isolation of peripheral blood mononuclear cells for ELISPOT Peripheral blood mononuclear cells (PBMCs) are isolated for ELISPOT at least once before drug administration. During the drug administration period, PBMCs are isolated on days 15, 29, 57, and 85. Briefly, a 3 mL blood sample is collected from the femoral vein. Alternative sites may be used if necessary. Whole blood collection

[0227] Collect whole blood during the medication period on days 3, 8, 15, 29, 57, and 85. In short, collect blood samples from the femoral vein. Alternative sites may be used if necessary.

[0228] Whole blood: Blood samples for whole blood collection are kept on moist ice or a cooling cryolack after collection. Whole blood is collected and transferred to three nearly equal aliquots.

[0229] Serum: Keep blood samples at room temperature for serum collection and allow them to coagulate before centrifugation. Centrifuge the samples within one hour of collection and collect the serum into three nearly equal aliquots. After collection, place the whole blood and serum samples on dry ice until stored in the freezer.

[0230] Collection of aqueous humor Collect aqueous humor once before administering medication. Collect aqueous humor once on days 15, 22, 29, 57, and 85. Briefly, collect a blood sample from the femoral vein. Alternative sites may be used if necessary.

[0231] Aqueous humor samples from each eye are placed in separate tubes bearing Watson barcode labels, flash-frozen in liquid nitrogen, and then placed on dry ice until stored in a freezer. The aqueous humor samples are then analyzed for introduced gene products.

[0232] Blood collection for clinical chemistry Collect blood at least twice before administering the medication. Collect blood during the medication period on days 8, 15, 29, 57, and 85. Briefly, collect blood samples from the femoral vein. Alternative sites may be used if necessary. Collect 1 mL for hematology, 1.8 mL for coagulation, and 1 mL for clinical chemistry. Test results are provided in Table 14. [Table 16]

[0233] Collection of ocular fluid and frozen ocular tissue for in vivo distribution and analysis of transgene products Collect the following ocular fluid / tissue from the left eye of two of the three animals in groups 1 and 2, and the following ocular fluid / tissue from both eyes of one of the three animals in groups 1 and 2. Enucleate each eye (if applicable). Immediately after enucleation, collect a sample of aqueous humor and divide it into two nearly equal aliquots. Then collect the vitreous fluid and ocular tissue. Collect the vitreous fluid and divide it into two nearly equal aliquots. See Table 15.

[0234] The anterior segment of the eye is removed, and the eye is divided into four nearly equal quadrants (superitemporal [including the area of ​​the drug administration site], superior nasal, inferior temporal, and inferior nasal). Two nearly equal fragments (located distal and proximal to the optic disc) are collected from each quadrant.

[0235] All other eye tissues are collected as single samples (one sample / tube). If necessary, rinse the tissues with saline, wipe them dry, and then place them in separate tubes. After collection, flash freeze the samples in liquid nitrogen and store them on dry ice (unless storing them immediately in a freezer).

[0236] To minimize the risk of potential contamination, eye fluid and tissues are collected using ultra-clean procedures in accordance with Labcorp SOPs. In addition, any work surfaces and non-disposable tools used are washed between animals with DNA Away surface decontamination agent (Thermo Scientific, catalog no. 7010 or equivalent) and RNAse decontamination solution (Invitrogen RNaseZap or equivalent). [Table 17]

[0237] Frozen whole-body tissue collection for in vivo distribution and analysis of transgene products For analysis of in vivo distribution and transgene product analysis, the following tissues (see Table 16) should be collected and frozen. [Table 18]

[0238] 6.14 Examples 14A, 14B, and 14C: NaIO3-induced mouse models A. The anti-mouse C5 (mC5) antibody BB5.1 was evaluated using a NaIO3-induced model of atrophic AMD in rodents (sodium iodate-induced retinal degeneration model). Recombinant anti-mC5 antibody BB5.1 (group 1) and an unrelated isotype-matched (mouse IgG1) antibody (group 2) were administered intraperitoneally (ip) at a dose of 1 mg daily to C57BL / 6J mice. Table 17. Mice were evaluated on day 0 for baseline electroretinography (ERG), body weight (BW), and optical coherence to thrombocytometry (OCT). Then, each was administered daily from day 1 to day 9. On day 3, NaIO3 was administered intravenously to each mouse to induce intraocular inflammatory events. Mice were re-evaluated for ERG / OCT on days 6 and 10, and then sacrificed on day 10 for tissue collection. Without being bound by any particular theory, inflammation induced by NaIO3 injection leads to degeneration of the retinal pigment epithelium (RPE) layer of the eye, causing loss of RPE, and also thinning of the photoreceptor layer, the outer nucleus layer (ONL), which mainly contains photoreceptors. The thickness of the ONL can be visualized by optical coherence tomography (OCT) or immunohistochemical staining of the tissue.

[0239] n=8 eyes / groups were treated for hematoxylin & eosin (H&E) histopathology, n=8 eyes / groups were treated for retinal pigment epithelium (RPE) / choroidal flat-mount immunohistochemistry (IHC), n=8 whole spheres / groups were flash-frozen, and n=8 whole spheres were fixed in 4% paraformaldehyde (PFA). [Table 19]

[0240] In Example 13, antibody product levels were detected in the eye after evaluation following intraperitoneal injection of recombinant anti-C5 BB5.1 antibody (Figures 7A-7B). This NaIO3 experiment demonstrated that intraperitoneal injection of recombinant anti-C5 antibody (BB5.1) had a protective effect against ONL degeneration in the eyes of treated mice (Figures 11A-11C). The BB5.1 antibody significantly mitigated ONL and retinal thinning, as measured by OCT 3 and 7 days after NaIO3 induction (days 6 and 10, respectively).

[0241] ERGs were performed on both eyes using a Diagnosys ERG system. Animals were dark-adapted for at least 12 hours prior to the ERG. Under dark adaptation, the eyes were dilated using a cocktail of 1% tropicamide HCl and 2.5% phenylephrine HCl. Before recording the ERG, pupil dilation was checked to ensure complete dilation. The animals were placed on the ERG machine, then 0.5% propalacaine HCl and GenTeal were applied to the eyes, followed by the application of electrode contact leads and reference leads. To control body temperature, the animals were placed on a warm water blanket and contact lens leads were placed in the eyes. A reference subcutaneous lead was placed on the head and an earth lead was placed near the animal's tail. A light-adapted ERG with a 30 cd / m2 background light followed immediately after the dark-adapted series. There was a 5-minute light-adaptation period before the light-adaptation signal was collected. The background light was always used for all light-adaptation measurements.

[0242] Dark adaptation ERG: Processes 1-9: Photopic ERG at 0.001-10 cd*s / m2; each process advances by half a logarithmic unit in light intensity. Both ERG traces and rhythmic small waves were collected. Step 10: Scotopic 150cd / m2 photopic C-wave measurement.

[0243] Bright Adaptation ERG: Processes 1-3: Photopic ERG at 1-10 cd*s / m2, each process advances half a logarithmic unit of light intensity. Both ERG traces and rhythmic small waves are collected. Processes 4-7: Photopic ERG flicker ERG at 10-40Hz with a frequency of 3.0c cd*s / m2, with each process advancing by 10Hz.

[0244] Following ERG and OCT procedures, the animals were administered atipamezole (0.1-1 mg / kg SQ) to reverse the xylazine effect and restore them to normal from the procedure.

[0245] During scotopic α-wave measurements, the α-wave amplitudes for both groups were approximately 20–50 μV at all time points, ranging from -3.00 cd*s / m² to -1.52 cd*s / m² (Figure 3). At -1.52 cd*s / m²s, the baseline α-wave amplitudes for both groups increased to approximately 220–240 μV at 1.00 cd*s / m². On day 6, the α-wave amplitude of group 1 began to increase to a similar amplitude after -1.00 cd*s / m². At -1.00 cd*s / m² on day 6, the α-wave amplitude of group 2 increased to approximately 87 μV at 1.00 cd*s / m². On day 10, the α-wave amplitudes for both groups remained generally within the range of 30–40 μV at all light intensities.

[0246] Compared to baseline recordings, dark-adapted ERG recordings performed 3 and 7 days after NaIO3 administration showed that IP delivery of BB5.1 significantly improved a and b wave amplitudes 3 days after NaIO3 administration, indicating improved rod function (Figures 15A-15F).

[0247] H&E histopathology was performed on n=8 eyes / group. Generally, in group 1, retinal lesions were multifocal and mild, while in group 2, they were moderate to severe and diffuse. Furthermore, severe retinal photoreceptor damage and diffuse retinal thinning were observed in eyes in group 2, which were not observed in group 1. Eyes in group 1 were characterized in 3 out of 4 animals by mild multifocal areas of RPE proliferation with migration to the retina, mild thinning of the photoreceptor layer, and collapse of the outer retinal nucleus layer. All eyes in group 2 had moderate to severe diffuse areas of RPE proliferation with moderate to severe photoreceptor layer damage, collapse of the outer nucleus layer, and diffuse retinal thinning. The lesions observed in group 1 were multifocal and mild, while the lesions observed in group 2 were diffuse and moderate to severe. In addition, eyes in group 2 showed more severe retinal photoreceptor destruction and diffuse retinal thinning that were not observed in eyes in group 1.

[0248] B. Next, an atrophic AMD NaIO3 induction model was performed to evaluate AAV vector-expressed anti-C5 antibodies. AAV8.CAG.anti-hC5-D-mab.scFv.HL and AAV8.CAG.anti-mC5-BB5.1.IgG were administered intravenously (IV) to C57BL / 6J mice at a dose of 3E13. Briefly, the mice were evaluated for ERG / body weight (BW) / OCT in a manner similar to Example 16A, and then each was administered IV with the test AAV vector at a dose of 3E13vg / kg (Table 18). On day 28, the mice were evaluated again for OCT / fundus / ERG and NaIO3, and on day 29, each mouse was administered intravenously to induce intraocular inflammatory events. [Table 20]

[0249] Several days after NaIO3 administration (approximately day 32), the eyes of the mice were evaluated (OCT imaging, ERG, etc.), then sacrificed, and the eyes were further evaluated for RPE damage and inhibition of photoreceptor loss, as well as vector genome (DNA and RNA) and transgene product (TP) levels.

[0250] NaIO3 Research Design C In short, male C57Bl / 6J mice were intravenously injected with AAV8 vectors encoding anti-C5-D-mab.scFv (anti-hC5-scFv01), anti-mC5-IgG01 (mC5-targeting mouse full-length mAb, positive control), and anti-NS-scFv01 (non-specific scFv, negative control) at a dose of 3e13 GC / kg body weight. The vectors were injected on day 1, and NaIO3 was injected on day 29. Serum was collected on days 0, 29, and 35 using sensitized sheep erythrocytes to assess protein expression levels and hemolytic function. Eyes were collected by necropsy, and anti-C5-D-mab.scFv levels in whole eye lysates and serum were measured by ELISA. Eyes designated for histological analysis were collected in Davidson fixative and processed into paraffin blocks. ONL thickness on H&E-stained slides and photoreceptor layer measurement from OCT images were performed using ImageJ.

[0251] Dark-adapted ERG recordings after NaIO3 administration, compared to baseline recordings, showed that delivery of anti-C5 scFv via AAV 29 days prior to injury significantly improved wave a and b amplitudes after NaIO3 administration, indicating improved rod function (Figures 29A-29B). OCT imaging, retinal outer layer measurements, and ONL thickness after NaIO3 administration showed that AAV8.CAG.C5-D-mab.scFv (AAV.anti-hC5-scFv01) was comparable to anti-mC5-IgG in reducing photoreceptor cell degeneration and thus reducing the structural integrity of the retinal outer layer compared to the nonspecific antibody anti-NS-scFv01 (Figures 29C-29I).

[0252] 6.15 Example 15: Administration of anti-C5 antibody expression vector to non-human primate (NHP) SCS Two cohorts of three female cynomolgus monkeys were administered supratemporalally in the superior temporal quadrant using a custom MedOne transscleral needle, either 1) AAV8.CAG.C5-D-mab.scFv.HL (n=5 eyes administered at 3e12 GC / eye in 100uL; both eyes of two animals were administered, but the third animal was administered to the right eye only, with its left eye not administered and used as a negative control) or 2) AAV3B.CAG.C5-D-mab.scFv.HL (n=5 eyes administered at 3e12 GC / eye in 100uL, and one eye administered at approximately 2e12 GC / eye in 70uL). (Table 19.) Animals administered with AAV8 were negative for anti-AAV8 neutralizing antibody (Nab) and total antibody (Tab), and animals administered with AAV3B were negative for anti-AAV3B Tab. Ophthalmic examinations, fundus photography, optical coherence tomography, and fundus autofluorescence imaging were performed, intraocular pressure was measured, and aqueous humor was collected periodically by puncture. The animals were euthanized on day 85 of the study. [Table 21]

[0253] Two eyes were administered AAV8, and two eyes were administered AAV3B. One untreated eye was fixed and processed for histopathological analysis, and tissue from the remaining eye was frozen and collected for DNA / RNA biodistribution and transgene product analysis. These tissues included the 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 optic cup into quadrants and collecting distal and proximal fragments from each quadrant. Each fragment was then separated into the retina, RPE / choroid, and sclera for analysis. In addition to sample 5 containing the macula, distal and proximal samples from the temporal quadrant were pooled (samples 3 and 4, respectively) for transgene product analysis (by ELISA). Nasal samples were collected for biodistribution, and both DNA and RNA were extracted from each sample. Peripheral tissues, including the liver, heart, kidneys, and spleen, were also collected from each animal by freezing for in vivo distribution. Serum was also collected throughout the study to evaluate transgene product levels and anti-transgene product antibodies (ATPA).

[0254] AAV8 anti-C5-ScFv vector results: Vector genome copy levels were assessed by ddPCR using primers / probes for poly(A). For the retina, RPE / choroid, and sclera, each data point represents a single sample from a single tissue sample from a single animal, and for other ocular tissues, each data point represents a single extraction from that tissue. Vector genome copy levels ranged from approximately 1e2–1e7 GC / ug of DNA (Figure 13A). In the posterior segment, as is typical for AAV delivery to the SCS, vector genome copy levels were highest in the sclera, followed by the RPE / choroid and then the retina. Vector DNA was also detected in several other ocular tissues, but not always resulting in detectable RNA expression. RNA expression in the retina and RPE / choroid was similar (Figure 13B). The in vivo distribution in the left eye of animal 1 was judged to be significantly lower than expected. The in vivo distribution of the vector genome was also evaluated in peripheral tissues, and the vector genome was detected in the spleens of all three AAV8-treated animals, in the livers of two of the three animals, and in the kidney of one of the three animals (Figure 13C).

[0255] Except for animals in which low-vector transduction was established based on in vivo distribution data, anti-C5-scFv was detected in aqueous humor from all treated eyes throughout the study (Figures 14A and 14B). Expression was mostly stable, with an average anti-C5-scFv level of 0.68 ug in AH on D29, except for the right eye of animal 1, which showed elevated levels throughout the study (Figure 14C). Except for the left eye of animal 1 (low-vector level), the average anti-C5-scFv level in vitreous fluid (VH) on day 85 was 1.2 ug / mL (Figures 15A and 15B). Anti-C5-scFv was also detected in the retina, RPE / choroid, and sclera of animals 2 and 3, with the highest transgene product level detected in the RPE / choroid, achieving a maximum of 6 ng of anti-C5-scFv / mg in the tissue (Figure 16). Transgene product (TP) levels correlated with vector genome levels in the retina and RPE / choroid, but not in the sclera. The lowest levels of anti-C5-scFv were detected in the serum of animal 3 at any given time. Serum aC5-scFv increased over time in both animals 1 and 2, decreased by day 85, and correlated well with the extent of its biodistribution in the liver in these animals (Figures 17A-17C).

[0256] Ophthalmic examinations revealed that AAV8.CAG.aC5-scFv was well-tolerated in this study, with one eye from a single animal exhibiting mild (0.5+) vitreous cell presence on D29. No treatment-related changes in intraocular pressure were observed, and OCT assessments also showed no treatment-related changes in ocular structure throughout the study. Histopathological analysis revealed slight inflammation in the sclera of half of the eyes, which was judged to be related to the test sample. All other histopathological findings were considered spontaneous and were not considered harmful, as there was no evidence of retinal degeneration or clinical findings affecting ocular function.

[0257] Results of the AAV3B-anti-C5-scFv vector:Vector genome copy levels ranged from approximately 1e2–1e6 GC / ug of DNA (based on ddPCR using primers / probes for poly(A); for the retina, RPE / choroid, and sclera, each data point represents a single sample from a single fragment from a single animal; for other ocular tissues, each data point represents a single extraction from that tissue) (Figures 21A–21C). In the posterior segment, as is typical for AAV delivery to the SCS, vector genome copy levels were highest in the sclera, followed by the RPE / choroid and then the retina. The vector genome levels in the posterior segment of P0102-OS suggest insufficient transduction, and the transduction gene product data indicates + / - data points from this eye. Vector DNA was also detected in several other ocular tissues. RNA expression was highest in the retina and RPE / choroid, with similar transcriptional copies, followed by the iris / ciliary body and trabecular meshwork. The vector genome was detected in the spleen of all three AAV3B-treated animals. Since the data points did not provide a signal greater than five times the background, the in vivo distribution in the heart, kidneys, and liver was considered undetectable.

[0258] Except for P0102-OS (established low-vector transduction based on in vivo distribution data), anti-C5-scFv was detected in aqueous humor from all treated eyes throughout day 29 of the study (Figures 19A-19B), with an average aC5-scFv / mL AH of 1.5 ug. After D29, anti-C5-scFv levels continued to rise in 3 out of 5 eyes, but AH levels in both eyes from P0101 decreased to undetectable levels, correlated with the onset of ATPA (Figure 19C). Except for one eye with poor transduction from the animal that developed ATPA and the other eye, the anti-C5-scFv level in vitreous fluid on D85 was an average of 16.5 ug / mL (Figures 20A-20B). Except for one eye with poor transduction from the ATPA-developing animal and the other eye, aC5-scFv was detected in the retina, RPE / choroid, and sclera of the remaining treated eyes. The highest transgene product levels were detected in the RPE / choroid, achieving a tissue level of up to 225 ng of aC5-scFv / mg (Figures 21A-21H). Transgene product levels correlated with vector genome levels in the retina and RPE / choroid, but not in the sclera. Anti-C5-scFv was either minimal or undetectable in serum at all time points in all three animals. The low levels of detected anti-C5-scFv in serum at D85 correlated well with the extent of its biodistribution in the liver of those animals (Figures 22A-22C). One of the three animals (P0101) expressed ATPA, which was first detected on D57 and correlated with decreased AH, VH, and detectable anti-C5-scFv levels in ocular tissue at D85.

[0259] Ophthalmic examinations and imaging revealed that AAV3B.CAG.anti-C5-D-mab.scFv was initially well tolerated until week 8, D57, when the retinas of both eyes P0101 and P0103 exhibited a mottled appearance. Both eyes of P0101 showed perivascular sheath at D57, decreasing in intensity by D85, and minimal or no vitreous cells at D57 / D85, correlating with the onset of ATPA. No treatment-related changes in intraocular pressure were observed. Histopathological analysis confirmed the presence of choroidal infiltration in P0101-OD and P0103-OD, as well as scleral inflammation in P0101-OD, which was considered related to the test substance. All other histopathological findings were considered spontaneous and were not considered harmful, as there was no evidence of retinal degeneration or clinical findings affecting ocular function.

[0260] Choroidal delivery of AAV vectors encoding complement inhibitors optimized for ocular expression results in localized and sustained expression of bioactive proteins, thus representing a minimally invasive approach to treating atrophic AMD, reducing the treatment burden and directly delivering therapeutic molecules to the pathogenic site of AMD.

[0261] In vivo expression in NHP In short, cynomolgus monkeys were administered an AAV vector expressing anti-C5-D-mab.scFv at a rate of 3e12 GC / eye in two different studies. In the 3-month study, the AAV8 vector was administered, and anesthetic hematologic hilum (AH) was collected by puncture on days 15, 29, 57, and 85. Eye tissue was collected from two eyes and dissected according to the scheme in Figure 29C, separating the retina, RPE-choroid, and sclera. Anti-C5-D-mab.scFv (anti-hC5-scFv01) levels were measured from temporal tissue by ELISA, while ddPCR was performed on nasal tissue to measure the AAV vector genome. Both eyes were collected in Davidson fixative and processed into paraffin blocks for histological analysis. Anti-C5-D-mab.scFv levels were detected in aqueous humor and vitreous fluid of NHP patients at days 15, 29, 57, and 85 (Figures 29A-29B). In particular, anti-C5-D-mab.scFv was mainly detected in the distal and proximal regions of RPE-C tissue (Figure 29D). Finally, genomic copies (DNA) were more abundant in RPE-C and scleral tissue compared to the retina after SCS delivery (Figure 29E). The vector was well tolerated throughout, as shown by SD-OCT, fundus autofluorescence imaging, and histological analysis (data not shown).

[0262] 6.16 Example 16: Subretinal administration of NHP Four cynomolgus monkeys were each administered subretinal (SR) via either AAV8.CAG.C5-D-mab.scFv.HL or vehicle (n = 1 eye administered via vehicle; n-3 eyes administered with 1e10 GC / eye; n=4 eyes administered with 1e11 GC / eye). One animal developed cataracts due to the injection procedure (not TA-related) and was replaced. Several animals were also administered an AAV8 vector expressing nonspecific (NS)scFv using the same scFv framework.

[0263] Four punches were taken from posterior ocular tissue, and the single punch with the highest biodistribution (BD) was plotted (assumed administration site). Vector DNA was measured in ocular tissue 4 weeks after SR injection. Similar results for vector transduction were observed with anti-C5 versus nonspecific scFv (Figure 23). Insufficient transduction of scleral tissue was expected and confirmed.

[0264] Aqueous humor (AH) was collected on days 15 and 29 (2 weeks and 4 weeks after drug administration, respectively). TP (ScFv level) was measured by ELISA using antigen-coated plates (coated with C5), and the detection of TP by the protein-L HRP subset was confirmed by bioanalysis by mass spectrometry, which shows that "free" and "total" TP levels are similar. TP expression is variable but mainly dose-dependent for both scFvs. (Figures 27A-27B) Two animals (non-specific scFV group) had detectable ATPA in their serum but still showed increased TP from D15 to D29. ATPA was not detected in anti-C5 treated animals.

[0265] TP expression was measured by ELISA in vitreous fluid (VH) using antigen-coated plates (C5) and protein-L HRP to detect TP (Figures 25A-25B). Subsets of values ​​were confirmed by mass spectrometry, as before, to show similar "free" and "total" TP levels. TP expression was variable but dose-dependent for both scFvs. Two animals (non-specific scFV group) had detectable ATPA in serum but exhibited TP expression within the range of other samples, and ATPA was not detected in anti-C5 treated animals. Vectorized scFv expression data were compared with non-specific vectorized IgG (full-length) antibody. Note that IgG data were obtained from different studies and different assays were used to measure non-specific (non-C5) TP. However, the study designs were largely the same and performed in the same laboratory (Figures 26A-26B). In this model, vectorized scFv was expressed more highly in AH than in vectorized IgG format. Because IgG was not well tolerated at doses higher than 1E10, the amount of data available to compare expression at equivalent doses was limited, except for the higher-expression scFv 1E10 dose.

[0266] TP expression was also measured in various eye tissues. Four punch samples were analyzed from tissue after in vivo vector distribution (DNA and RNA), and the remaining posterior tissue from these eyes was separated into three tissue layers and homogenized for TP expression. TP was measured using the same ELISA method as for AH and VH. Subretinal delivery of the AAV-anti-C5-scFv vector resulted in high anti-C5 scFv (TP) expression in the retina and RPE-choroid (Figures 27A-27B).

[0267] Expression and tolerability in NHP after injection either subretinal (1-month study period) or suprachoroidal (3-month study period, Example 17, hereafter) were tested. Tolerability, evaluated by in vivo imaging and histopathology, was determined to be well-tolerated, resulting in high expression of bioactive C5 inhibitors in ocular fluid and tissues (including RPE / choroid), and minimal levels of C5 inhibitors in serum. In vivo-expressed C5 inhibitors present in NHP vitreous fluid (from eyes injected subretinally) were also examined and found to be bioactive.

[0268] 6.17 Example 17: One-month evaluation of the expression of formulated AAV vector administered via choroidal administration to Yucatan miniature pigs. This example relates to the evaluation of the in vivo distribution and expression of AAV transgene products from different formulations of AAV vectors carrying transgenes (e.g., AAV.ScFv antibody) in animals (e.g., miniature pigs such as Yucatan miniature pigs) after a single intrachoroidal injection. Briefly, three pigs receive each test substance formulation via bilateral intrachoroidal injection using a 29-gauge needle approximately 1110 μm long (performed once). [Table 22]

[0269] Test substances 3-5 were provided as follows: Test substance 3: AAV8.CAG.C5-D-mab.scFv.HL is 3 × 10 13 Provided at concentrations of GC / mL (frozen (-80℃)) as the following pre-formulated solutions: 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL anhydrous disodium hydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, and 0.001% (0.01 mg / mL) poloxamer 188 (formulation 1). Test substance 4: AAV8.CAG.C5-D-mab.scFv.HL is 3 × 10 13The following pre-formulated solutions are provided at concentrations of GC / mL (frozen (-80℃)): 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL anhydrous disodium hydrogen phosphate, 25.0 mg / mL (2.5% w / v) sucrose, 0.002% (0.02 mg / mL) poloxamer 188, and 0.7% hyaluronic acid (formulation 2+HA). Test substance 5: AAV8.CAG.C5-D-mab.scFv.HL is 3 × 10 12 The following pre-formulated solutions are provided at concentrations of GC / mL (frozen (-80℃)): 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL anhydrous disodium hydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, and 0.001% (0.01 mg / mL) poloxamer 188 (formulation 1). [Table 23-1] [Table 23-2]

[0270] Choroidal administration (Day 1): The animals are fasted the night before administration. Approximately 15 minutes before anesthesia, 1.0% tropicamide HCl is applied topically to the ocular surface to induce pupillary dilation, and atropine (0.05 mg / kg) IM or glycopyrrolate (0.01 mg / kg) IM is administered to reduce the risk of aspiration during sedation. The animals receive a single dose of buprenorphine (0.01-0.05 mg / kg) IM as an analgesic, and the animals are anesthetized according to IACUC. The area around both eyes, including the eyelids, is washed and sterilized (including the ocular surface). The animals are then placed under a surgical microscope, with the first eye facing upward, and a surgical drape and eyelid retractor are positioned. The test substance is administered by suprachorionic injection over 5–10 seconds using a 29-gauge needle approximately 1100 μm in length, provided by the sponsor, delivered to the superior temporal quadrant 4 mm from the edge, between 10 and 11 o'clock in the right eye and between 1 and 2 o'clock in the left eye. After injection, the needle is held in the eye for approximately 5 seconds before withdrawal. After withdrawing the needle, a cotton swab is placed over the injection site for approximately 10 seconds. A topical drop of antibiotic ophthalmic solution is applied to the ocular surface. After administration, the animals undergo post-administration OCT (to examine the injection site and the nasal-temporal region near the injection site) and fundus imaging. The animals are weighed before the start of administration, weekly, and at euthanasia. Ophthalmic examination (OE), optical coherence tomography (OCT), color fundus imaging, blood collection (for serum, plasma, and whole blood), and tissue collection are performed according to the experimental design.

[0271] Biological activity of introduced gene products: In short, aqueous humor was collected by aspiration from the eyes of Yucatan miniature pigs administered via SCS using the AAV8.CAG.C5-D-mab.scFv (AAV8-anti-hC5-scFv01) vector in 3e12 GC / eye, before administration and on days 15 and 29 after administration. Vitreous fluid and ocular tissue were collected by necropsy, and the expression of anti-C5-D-mab.scFv was measured by ELISA. Vitreous fluid from animals with appropriate expression levels was used to test the bioactivity in hemolysis assays. Miniature pigs administered with AAV.anti-C5-D-mab.scFv (anti-hC5-scFv01) showed stronger inhibition of complement pathway activation (classical pathway hemolysis) compared to controls (Figure 31).

[0272] 6.18 Example 18: Transduction and evaluation of the bioactivity of scFV-AAV in iPSC-derived RPE cells Retinal pigment epithelial cells (RPEs) function as barrier and homeostatic regulators in the retina and can be destroyed in response to inflammation. Briefly, we cultured human induced pluripotent stem cell (iPSC)-derived RPEs and maintained them in porous membrane inserts for up to 60 days, allowing the cells to express characteristic transepithelial electrical resistance (TER) and observe changes induced by the expression of C5 inhibitors by the cells.

[0273] Once the RPE culture conditions and readout measurements were established (see Example 12 below), the RPEs were transduced with an AAV expressing anti-human C5 scFv. The iPSC-derived RPEs cultured in the inserts have particular potential as a platform for the development of human-specific ocular gene therapies for therapeutic targets such as C5 inhibition.

[0274] Immunohistochemistry was used to evaluate the inhibition of membrane invasion complexes (MACs) in iPSC-derived RPE cells transduced with AAV vectors expressing C5 inhibitors.

[0275] Equivalents While the present invention is described in detail with reference to its specific embodiments, it will be understood that functionally equivalent modifications fall within the scope of the invention. Indeed, in addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the accompanying claims. Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein by mere conventional experimentation. Such equivalents are intended to be covered by the following claims.

[0276] All publications, patents, and patent applications referenced herein are incorporated by reference, each individual publication, patent, or patent application, to the same extent as specifically and individually.

Claims

1. A composition comprising an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector is (a) A viral AAV capsid that is tropic towards ocular tissue cells, (b) an artificial genome comprising an expression cassette adjacent to an AAV reverse terminal repeat (ITR), wherein the expression cassette comprises a transgene encoding a single-stranded variable fragment (scFv) that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker. i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence that is 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence that is 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence that is 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence that is 90% identical thereto The composition wherein the introduced gene is operably linked to one or more regulatory sequences that promote the expression of the introduced gene in human eye tissue cells.

2. The virus capsid is 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 rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu. The composition according to claim 1, comprising a VP1 capsid protein whose amino acid sequence is at least 95% identical to that of the VP1 capsid protein of serotype 37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu12 (AAV.hu12), or serotype hu26 (AAV.hu26).

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

4. The composition according to any one of claims 1 to 3, wherein the human eye tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells).

5. The composition according to any one of claims 1 to 4, wherein one or more regulatory sequences include regulatory sequences from Table 1 or Table 1a.

6. The composition according to claim 5, wherein the one or more regulatory sequences include a CAG promoter (SEQ ID NO: 44), a mutant 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 NO: 47 or 139), a mouse pyramidal arrestin (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 Best1 / GRK1 tandem promoter (SEQ ID NO: 143).

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

8. The composition according to any one of claims 1 to 7, wherein the introduced gene encodes a signal sequence at the N-terminus of the scFv that is directed toward secretion and post-translational modification in the human eye tissue cells.

9. The composition according to claim 8, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (Sequence ID 55) or a signal sequence from Table 2.

10. The composition according to claim 9, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).

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

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

13. The composition according to any one of claims 1 to 12, wherein the linker is GGGGS (SEQ ID NO: 38), GGGGSGGGGGS (SEQ ID NO: 39), GGGGSGGGGGGGGGSGGGGGS (SEQ ID NO: 40), GGGGSGGGGGGGGGGGGGSGGGGGS (SEQ ID NO: 41), or GGGGSGGGGGGGGGGGGGGGSGGGGGS (SEQ ID NO: 42).

14. The composition according to claim 13, wherein the linker is GGGGGSGGGGGGGGGS (Sequence ID 42).

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

16. The composition according to any one of claims 1 to 15, wherein the introduced gene encodes a polypeptide having the 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 according to claim 16, wherein the introduced gene encodes a polypeptide having amino acid sequence number 180.

18. The composition according to any one of claims 1 to 17, wherein the artificial genome comprises the 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.

19. The composition according to claim 18, wherein the artificial genome comprises a nucleotide sequence encoding C5-D-mab.scFv (sequence number 207, or sequence number 208, or sequence number 209).

20. A pharmaceutical composition for use in treating age-related macular degeneration (AMD) in human subjects requiring treatment, comprising an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector is (a) A viral capsid that is tropic towards ocular tissue cells, (b) an artificial genome comprising an expression cassette adjacent to an AAV reverse terminal repeat (ITR), wherein the expression cassette comprises a transgene encoding a single-stranded variable fragment (scFv) that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker. i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence that is 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence that is 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence that is 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence that is 90% identical thereto The transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells. The pharmaceutical composition wherein the AAV vector is formulated for subretinal, intravitreous, intranasal, intrachorionic, suprachoroidal, or systemic administration to a human subject.

21. The virus capsid is 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 rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu. The pharmaceutical composition according to claim 20, comprising a VP1 capsid protein whose amino acid sequence is at least 95% identical to that of the VP1 capsid protein of serotype 37 (AAVhu.37), serotype rh73 (AAVrh73), or serotype rh74 (AAVrh74), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu12 (AAV.hu12), or serotype hu26 (AAV.hu26).

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

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

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

25. The pharmaceutical composition according to claim 24, wherein the one or more regulatory sequences include a CAG promoter (SEQ ID NO: 44), a mutant 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 NO: 47 or 139), a mouse pyramidal arrestin (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 Best1 / GRK1 tandem promoter (SEQ ID NO: 143).

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

27. The pharmaceutical composition according to any one of claims 20 to 26, wherein the introduced gene encodes a signal sequence at the N-terminus of the scFv that is directed toward secretion and post-translational modification in the human eye tissue cells.

28. The pharmaceutical composition according to claim 27, wherein the signal sequence is MYRMQLLLLIALSLALVTNS (Sequence ID 55) or a signal sequence from Table 2.

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

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

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

32. The pharmaceutical composition according to any one of claims 20 to 31, wherein the linker is GGGGS (SEQ ID NO: 39), GGGGSGGGGGS (SEQ ID NO: 40), GGGGSGGGGGGGGGSGGGGGS (SEQ ID NO: 41), GGGGSGGGGGGGGGGGGGSGGGGGS (SEQ ID NO: 42), or GGGGSGGGGGGGGGGGGGGGSGGGGGS (SEQ ID NO: 43).

33. The pharmaceutical composition according to claim 32, wherein the linker is GGGGGSGGGGGGGS (Sequence ID 42).

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

35. The pharmaceutical composition according to any one of claims 20 to 34, wherein the introduced gene encodes a polypeptide having the 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 according to claim 35, wherein the introduced gene encodes a polypeptide having amino acid sequence number 180.

37. The pharmaceutical composition according to any one of claims 20 to 36, wherein the artificial genome comprises the 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 according to claim 37, wherein the artificial genome comprises the nucleotide sequence of SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO:

209.

39. The pharmaceutical composition according to any one of claims 20 to 38, wherein the scFv bound to C5 inhibits both the classical complement pathway and the alternative complement pathway.

40. The pharmaceutical composition according to any one of claims 20 to 39, wherein the scFv bound to C5 inhibits the formation of a membrane invasion complex (MAC).

41. A method for producing recombinant AAV, (a) (i) A cis-expression cassette adjacent to the AAV ITR, wherein the cis-expression cassette includes a transgene encoding scFv that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker. i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence 90% identical thereto, and the transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells, an artificial genome comprising the cis-expression cassette, (ii) A trans-expression cassette lacking AAV ITR, wherein the trans-expression cassette encodes the AAV rep and the AAV capsid protein, which are operably linked to an expression control element that drives the expression of AAV rep and the AAV capsid protein in host cells in a culture and supplies the AAV rep and the AAV capsid protein in trans, and the capsid has ocular tissue cell tropism, (iii) Culturing host cells containing adenovirus helper function sufficient to enable replication and packaging of the artificial genome by the AAV capsid protein, (b) The method comprising recovering recombinant AAVs that form a capsid of the artificial genome from a cell culture.

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

43. The method according to claim 41 or 42, wherein the ocular tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells).

44. The method according to any one of claims 41 to 43, wherein the artificial genome comprises the 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. It is a host cell, A cis-expression cassette adjacent to the AAV ITR, wherein the cis-expression cassette includes a transgene encoding scFv that binds to C5, The scFv bound to C5 includes a variable heavy domain (VH) and a variable light domain (VL) covalently bonded by a polypeptide linker. i) The VH contains the amino acid sequence of SEQ ID NO: 170 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 171 or a sequence that is 90% identical thereto, or ii) The VH contains the amino acid sequence of SEQ ID NO: 172 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 173 or a sequence that is 90% identical thereto, or iii) The VH contains the amino acid sequence of SEQ ID NO: 174 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 175 or a sequence that is 90% identical thereto, or iv) The VH contains the amino acid sequence of SEQ ID NO: 176 or a sequence that is 90% identical thereto, and the VL contains the amino acid sequence of SEQ ID NO: 177 or a sequence that is 90% identical thereto The host cell comprising a plasmid comprising the cis-expression cassette, wherein the transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in human eye tissue cells.

46. The host cell according to claim 45, wherein the ocular tissue cells are retinal cells, RPE choroidal tissue cells, BrM epithelial cells, choroidal capillary epithelial cells, or photoreceptor cells (rods, cones, and / or retinal ganglion cells).

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

48. The host cell according to any one of claims 45 to 47, wherein the introduced gene encodes a polypeptide having the amino acid sequence 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 according to claim 48, wherein the artificial genome comprises the 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.

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

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

52. The composition or pharmaceutical composition according to any one of claims 1 to 19, wherein the composition or pharmaceutical composition comprises about 0.2 mg / mL of potassium chloride, about 0.2 mg / mL of potassium dihydrogen phosphate, about 5.84 mg / mL of sodium chloride, about 1.15 mg / mL of anhydrous disodium hydrogen phosphate, about 25.0 mg / mL (2.5% w / v) of sucrose, about 0.002% (0.02 mg / mL) of poloxamer 188, and about 0.7% w / v of hyaluronic acid.

53. The composition or pharmaceutical composition according to any one of claims 1 to 19, wherein the composition or pharmaceutical composition comprises about 0.2 mg / mL of potassium chloride, about 0.2 mg / mL of potassium dihydrogen phosphate, about 5.84 mg / mL of sodium chloride, about 1.15 mg / mL of anhydrous disodium hydrogen phosphate, about 40.0 mg / mL (4% w / v) of sucrose, about 0.001% (0.01 mg / mL) of poloxamer 188, and about 1% of high viscosity grade carboxymethylcellulose (CMC).

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