AAV variants for treatment of complement disorders

Abstract

A recombinant AAV (rAAV) comprising a variant adeno-associated virus (AAV) capsid and a transgene encoding a human factor H variant is provided. Also provided are methods of delivering a transgene to the retina, as well as methods of treating dry age-related macular degeneration and map-like atrophy secondary to an age-related macular degeneration disorder by contacting retinal cells with rAAV.

Description

[0001] Cross-reference with related applications This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 494,925, filed April 7, 2023, and 63 / 586,227, filed September 28, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence list submission via EFS-WEB A computer-readable XML file named “090400-5022-WO-Sequence-Listing”, created on April 2, 2024, with a file size of approximately 87,900 bytes, contains the sequence list of this application and is incorporated herein by reference in its entirety. Background of the Invention Many diseases are caused by complement dysregulation, leading to complement-mediated damage to autologous tissues. Complement dysregulation can originate from somatic or germline mutations in complement regulators or related genes, causing these regulators to malfunction. In particular, there are common and rare human diseases caused by excessive complement activation resulting from dysregulation of the complement activation cascade.

[0004] Current treatment approaches focus on the development of agents such as monoclonal antibodies (mAbs), peptides, or other small molecules that bind to and block specific alternative or terminal complement pathway components. A clinically validated example is eculizumab, a humanized mAb targeting complement C5, which has been approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Other described approaches include mAbs targeting factor B (fB), factor D (fD), or properin (fP), as well as cyclic peptides that bind to and inhibit C3. The limitation of these approaches is that they require repeated and inconvenient intravenous (IV) administration to patients. Furthermore, because these treatments block alternative or terminal pathways, they pose a risk of impairing host defenses. In fact, patients receiving eculizumab therapy must be vaccinated against bacterial strains that cause fatal meningitis, and these patients also receive prophylactic antibiotic therapy before treatment with the approved mAb.

[0005] Other approaches have tested recombinant regulatory proteins such as soluble DAF, CR1, CRIg, and proteins containing the minimal domains of the liquid-phase regulator fH (N-terminal short shared repeats [SCR] 1–5 and C-terminal SCR 19–20), or fusion proteins between fH and CR2 (TT30). However, large-scale heterologous expression of such proteins as therapeutic agents requires significant effort, and animal studies have shown rapid in vivo clearance after administration, making such therapeutic strategies cumbersome and less feasible due to the need for multiple and frequent administrations of these protein drugs.

[0006] There remains a need in the art for compositions that can be used to treat complement-mediated diseases with greater and more lasting efficacy. Invention Overview This document describes recombinant AAV (rAAV) viral particles comprising a variant AAV capsid sequence encapsulating a heterologous nucleic acid containing a modified human complement regulator H (fH) gene operatively linked to an expression control sequence, wherein the human fH (hfH) gene encodes a soluble hfH protein variant that retains complement regulatory function, wherein the fH variant comprises short common repeats (SCRs) 1, 2, 3, 4, 19, and 20. In some embodiments, rAAV comprises a heterologous nucleic acid containing an hfH gene that encodes a soluble hfH protein variant that retains complement regulatory function, wherein the fH variant comprises or is composed of or is primarily composed of SCRs, wherein the SCRs are selected from SCR1, SCR2, SCR3, SCR4, SCR6, SCR7, SCR8, SCR17, SCR18, SCR19, and SCR20.

[0008] In a related aspect, rAAV viral particles are provided, comprising a variant AAV capsid sequence encapsulating a heterologous nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding a modified hfH variant, the modified hfH variant comprising a leader sequence and a human complement receptor SCR, wherein the SCR is selected from the following: (a) SCR1-4, 7, and 19-20; (b) SCR1-4, 6, 7, and 19-20; (c) SCR1-4, 7, 8, and 19-20; (d) SCR1-4, 6, 7, 8, and 19-20; (e) SCR1-4, 17, and 19-20; (f) SCR1-4 and 18-20; (g) SCR1-4 and 17-20; (h) SCR1-4, 7, and 18-20; (i) SCR1-4, 6, 7, and 18-20; (j) SCR1-4, 7, 8, and 18-20; (k) SCR1-4, 6, 7, 8, and 18-20; (l) SCR1-4, 7, and 17-20; (m) SCR1-4, 6, 7, and 17-20; (n) SCR1-4, 7, 8, and 17-20; or (o) SCR1-4, 6, 7, 8, and 17-20. Optionally, at least one glycosylation site is modified into at least one SCR.

[0009] The rAAV variant AAV capsid protein comprises, relative to the corresponding parental AAV capsid protein, a capsid protein containing a peptide insert (“heteropeptide” or “peptide insert”) of about 7 to 20 amino acids in the GH ring of the capsid protein, preferably in the surface exposed region of the GH ring, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO:1). In some preferred aspects, the peptide insert has 1 to 3 amino acid spacer amino acids (Y1-Y3) at the N-terminus and / or C-terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1), wherein Y1-Y3 are each independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In some embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In a particularly preferred embodiment, the peptide insert comprises, is substantially composed of, or is composed of the amino acid sequence LAISDQTKHA (SEQ ID NO:2). In some preferred embodiments, the insertion site is located between amino acids 587 and 588 of VP1 in AAV2, or between amino acids 588 and 589 in AAV2, or at a corresponding position in the capsid protein of another AAV serotype. In some embodiments, the capsid protein further comprises one or more amino acid substitutions relative to the VP1 capsid of AAV2, or one or more corresponding substitutions in another AAV serotype, preferably wherein the capsid protein further comprises a P34A amino acid substitution relative to the VP1 capsid of AAV2, or a corresponding substitution in another AAV serotype.

[0010] In other embodiments, a method is provided for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a soluble hfH protein variant as described herein to a mammalian subject, the method comprising administering to the mammal an effective amount of rAAV as described herein or a pharmaceutical composition comprising therein, preferably wherein said rAAV or pharmaceutical composition is administered via intravitreal injection. In some aspects, the heterologous nucleic acid is delivered to the subject's retinal cells, such as the subject's photoreceptor cells (e.g., rod cells; cone cells), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelium (RPE) cells.

[0011] In some embodiments, after rAAV administration to a subject, detectable plasma levels of the hfH variant are present in the subject for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, or at least six months. In a particularly preferred embodiment, rAAV is administered to the subject via intravitreal administration.

[0012] In other embodiments, pharmaceutical compositions are provided comprising rAAV as described herein and pharmaceutically acceptable excipients.

[0013] In other respects, methods are provided for treating complement-related conditions by delivering rAAV or pharmaceutical compositions containing rAAV as described herein to a subject. Complement-related conditions that can be treated include, but are not limited to, membranoproliferative glomerulonephritis, atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), geographic atrophy secondary to AMD, microangiopathic hemolytic anemia, thrombocytopenia, acute renal failure, paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or bacterial infections caused by recruitment of bacterial pathogens.

[0014] In a further aspect, a method is provided for treating dry age-related macular degeneration (AMD) (e.g., advanced dry AMD) in a subject with this need by delivering an effective amount of rAAV as described herein or a pharmaceutical composition containing rAAV to the patient. In a related aspect, the rAAV or pharmaceutical composition is administered to the subject for the treatment of geographic atrophy secondary to AMD. Preferably, the rAAV or pharmaceutical composition is administered to the subject via intravitreal injection. Brief description of the attached diagram Figure 1 This is a schematic diagram of a transgenic cassette containing 5' and 3' AAV inverted terminal repeats (ITRs) from the AAV2 genome, a polyadenylation signal (SV40 late poly-A), and a nucleotide sequence encoding a codon-optimized human fH variant containing SCRs 1-4, 6-8, and 17-20 (“miniCFH”) operatively linked to the CAG promoter.

[0016] Figure 2A -F. Figure 2A -F indicates the use of the included... Figure 1 The expression, activity, and function of miniature CFH in human cells after transfection with the AAV plasmid of the transgenic cassette are shown. Figure 2A-2B This shows the effect of ELISA ( Figure 2A ) and protein blotting ( Figure 2B Dose-dependent expression of miniature and full-length CFH in protein lysates from the supernatant of plasmid-transfected HEK293T cells. Figure 2C C3b binding was demonstrated: pre-incubation with a CFH-blocking antibody, rather than with a control antibody or simulated incubation, reduced CFH and miniature CFH signals, indicating that miniature CFH directly binds to C3b. Figure 2DHeparin binding was demonstrated: pre-incubation with a CFH-blocking antibody reduced recombinant CFH and miniature CFH signaling, indicating that miniature CFH binding preserved heparin binding activity. Figure 2E The results of a C3b cleavage assay, performed to confirm the functional activity of miniature CFH expressed in HEK293T cells, are shown. The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form a C3 convertase upon binding to factor B, or, in the presence of CFH as a cofactor, can be degraded into smaller, inactive fragments by complement factor I (CFI), causing disruption of the alternative complement cascade. These smaller fragments resulting from C3b degradation were observed by Western blotting. Figure 2F Complement inhibition (Wieslab® Complement System Alternative Pathway Assay) was demonstrated, confirming that the supernatant from AAV plasmid cassette-transfected cells resulted in inhibition of membrane attack complex (MAC) formation, similar to the full-length CFH and anti-C5 antibody controls. NT = Untransfected Figure 3A -C illustrates the expression, activity, and function of miniature CFH in human RPE cells. IPSC-derived retinal pigment epithelial (RPE) cells were transduced with rAAV comprising (i) a variant AAV capsid protein containing the amino acid sequence SEQ ID NO:42, and (ii) a protein containing... Figure 1 The nucleic acids of the expression cassette are shown below. RPE was transduced at different multiples of infection (MOI; vector genome (vg) / cell). Culture medium was harvested 4 days after transduction and 7 days after transduction, and the expression of secreted miniature CFH was measured by ELISA. Figure 3A ), and the complement inhibitory activity was determined using the Wieslab® complement system alternative pathway assay. Figure 3B Complement inhibition was also evaluated using ICC. Figure 3C On day 6 post-transduction, alternative complement in iPSC-derived RPEs was activated by adding 1% NHS and 0.5 mg / ml yeast polysaccharide to the culture medium. Twenty-four hours later, on day 7 post-transduction, cells were fixed and stained with the first MAC antibody IgG2aκ mouse anti-human (Abcam catalog #59835) or allotype control mouse IgG2aκ (Invitrogen catalog #14-4724-82), and the nuclei were stained with the second A488 goat anti-mouse (Invitrogen catalog #A11001) and DRAQ5 (ThermoFisher catalog #62251). Images were acquired using a fluorescence microscope. MAC formation in the culture was inhibited by cell-expressed microCFH at all MOIs. Anti-C5 antibody was used as a positive control for complement inhibition and MAC formation inhibition. NT = Untransduced Figure 4 The LCMS quantification of short CFH (sCFH) concentration in aqueous humor (AH) samples of NHP following intravitreal (bilateral) administration of a specified dose of rAAV, comprising (i) a variant AAV capsid protein containing the amino acid sequence SEQ ID NO:42, and (ii) a protein containing... Figure 1 The nucleic acid of the expression cassette is shown here.

[0017] Figure 5 The expression of sCFH RNA in untransfected HEK 293T cells and HEK 293T cells after transfection with rAAV is shown (analyzed by in situ hybridization), wherein the rAAV comprises (i) a variant AAV capsid protein containing the amino acid sequence of SEQ ID NO:42, and (ii) a protein containing... Figure 1 The nucleic acid of the expression cassette is shown here.

[0018] Figure 6 The results of in situ hybridization analysis of ocular tissue from non-human primates following intravitreal (bilateral) administration of a specified dose of rAAV comprising (i) a variant AAV capsid protein containing the amino acid sequence SEQ ID NO:42, and (ii) a protein containing... Figure 1 The nucleic acid of the expression cassette is shown here.

[0019] Figure 7A -F Figure 7A This is a Western blot, showing the detection of CFH protein after transfection with either CFH or mini CFH constructs. No bands were shown in the analyzed NT cell supernatant, which served as a negative control. Recombinant full-length CFH (250 kDa, lane 5) served as a positive control. The arrows indicate the theoretical molecular weight (MW) of mini CFH (80 kDa) and full-length CFH (139 kDa). The ladder is indicated on the upper left. Figure 7B The concentrations of CFH protein in the supernatant of HEK293T cells transfected with 0.125 or 0.5 μg of CFH or a miniature CFH construct are shown, as determined by human factor H ELISA, with ****p<0.0001 compared to NT; and ^p=0.0003 compared between 0.125 μg DNA and 0.5 μg DNA samples. Figure 7C The supernatant from HEK cells transfected with miniature CFH DNA (0.5 µg DNA / well, 15 µl supernatant) is shown, which resulted in complement inhibition comparable to the positive control eculizumab (6 µg), **p=0.0028, ***p=0.0002 compared to NT. Figure 7DThis is a Western blot of six reactions for the various components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, C3b) and supernatant (CFH supernatant) after transfection are shown. Cleavage products are highlighted with arrows. Increased cleavage products were observed after the addition of the supernatant following micro-CFH transfection, compared to the NT supernatant (lane 3) (lane 2). Lane 4 shows the positive control reaction with all three recombinant proteins (CFH, CFI, C3b), and lanes 5-7 show the negative controls. Figure 7E The study showed that micro-CFH cells in the supernatant after transfection bound to C3b and were deactivated after incubation with CFH antibody. The *p=0.05 compared to CFH antibody and the **p=0.01 compared to CFH antibody. Figure 7F The study showed that micro-CFH cells in the supernatant after transfection bound to heparin and were deactivated after incubation with CFH antibody, *p=0.05 compared to CFH antibody, **p=0.01 compared to CFH antibody. NT = untransfected. Error bars ± standard deviation; n=3 replicates; one-way ANOVA, Tukey post-hoc. Figure 8 Transduction of iPSC-RPE cells leads to the expression of the miniature CFH protein. The concentration [nM] of CFH in the supernatant of RPE cells transduced with rAAV (including the capsid protein containing the amino acid sequence shown in SEQ ID NO:42, and the heteronucleotide encoding a shortened form of complement factor H (“miniature CFH”) having the amino acid sequence shown in SEQ ID NO:34) at different MOIs is shown, illustrating the dose-response in CFH expression. Any signal beyond that visible in NT cells is then correlated with miniature CFH expressed by rAAV. MOI = multiplicity of infection; NT = untransduced. Error bars ± standard deviation; n = 4 replicates. Significance relative to NT, **p = 0.0071 and ***p = 0.0007. Significance for comparisons between MOIs 5,000 and 50,000, ^p = 0.0477. Statistical methods: one-way ANOVA, Tukey post-hoc.

[0020] Figures 9A-9BThis study demonstrates an alternative complement pathway inhibition via miniature CFH expressed in iPSC-derived RPE cells transduced via rAAV, which comprises a capsid protein operatively linked to a CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34. Complement inhibitory activity was determined in cell supernatants from rAAV-transduced RPE cells using two methods: Wieslab assay (…). Figure 9A ) and soluble C5b-9 ELISA ( Figure 9B ). Figure 9A The inhibition of alternative complement activity induced by the addition of specified samples to Wieslab assays containing normal human serum is shown. An activity readout with output from NT supernatant is defined as 0% inhibition. Dose-response was observed, with higher MOI doses of rAAV leading to increased complement inhibition. Error bars ± standard deviation; n = 3 replicates. Significance relative to NT is not specified unless otherwise stated. For comparisons between MOI 5,000 and MOI 50,000, *p = 0.0342, **p = 0.0026, ****p < 0.0001, ^^p = 0.0078. Figure 9B The concentration of soluble C5b-9 in samples with supernatant from an RPE used for rAAV transduction at different MOIs is shown to demonstrate a reverse dose-response. The concentration of soluble C5b-9 was normalized, with C5b-9 concentration in the NT supernatant = 1. Error bars ± standard deviation; n = 4 replicates. Significance relative to NT: **p = 0.0071 and ****p < 0.0001. Significance for comparisons between MOIs 5,000 and 50,000: ^p = 0.0272. Statistical methods: one-way ANOVA, Tukey post-hoc. NT, untransduced; MOI, multiplicity of infection.

[0021] Figure 10 This study demonstrated that expressed miniature CFH accelerates the decay activity of C3 convertase. C3b cleavage products were detected by Western blotting in purified C3b samples and supernatant samples incubated with purified CFI or purified full-length CFH. Samples lacking both CFI and CFH did not show cleavage of the C3b α-chain, while samples with purified CFI or CFH showed low levels of cleavage products. Incubating C3b with both CFI and miniature CFH resulted in extensive cleavage, a significant reduction in α-chain cleavage, and an increase in the abundance of cleavage products. Miniature CFH from cell supernatant, instead of purified CFH, reproduced these effects. NT, untransduced; MOI, multiplicity of infection. Representative blots, n=4 replicates.

[0022] Figure 11A-11B This demonstrates how rAAV (comprising a capsid protein operatively linked to the CAG promoter containing the amino acid sequence shown in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34) can protect RPE from MAC deposition prior to complement activation induction in cultures. Figure 11A Immunocytochemical (ICC) analysis is shown. Representative images, n = 4 replicates. Scale bar = 200 µm. Figure 11B Flow cytometry analysis is shown. Dose response is shown as the percentage of total RPE cells with MAC deposition at different MOIs. Error bars ± standard deviation; n = 4 replicates. Significance relative to NT, ****p < 0.0001. Statistical methods: one-way ANOVA, Tukey post-hoc. NT, untransduced; MOI, multiplicity of infection. MAC = membrane attack complex.

[0023] Figure 12 Alternative complement inhibitory activity of rAAV (comprising a capsid protein operatively linked to the CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34) was demonstrated in an in vitro RPE disease model. The concentration of iC3b from the disease model supernatant transduced with rAAV showed a reduction compared to NT. iC3b concentrations were normalized, where iC3b concentration in the NT supernatant = 1. NT, untransduced; MOI, multiplicity of infection; RPE, retinal pigment epithelial cells. Error bars ± standard deviation; n = 4 replicates. Significance relative to NT, **p = 0.0021 and ****p < 0.0001. Statistical methods: one-way ANOVA, Tukey post-hoc.

[0024] Figure 13 The concentration of miniature CFH in the aqueous humor of nonhuman primates following intravitreal administration of rAAV, which comprises a capsid protein operatively linked to a CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heteronucleotide encoding the amino acid sequence shown in SEQ ID NO:34. Miniature CFH protein expression in the aqueous humor was measured by LC-MS. Fluid expression is shown in ng / mL. Data are generalized from 3–4 animals. Mean + Standard Deviation Figure 14The image shows micro CFH concentrations in retinal tissue and RPE / choroid in non-human primates following intravitreal administration of rAAV, which comprises a capsid protein operatively linked to a CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34. (Left) 5x10 11 vg / eye and (right) 1x10 10 Following intravitreal transplantation (IVT) of vg / eye, miniature CFH protein expression in the aqueous humor (AH), retinal tissue (Ret), and RPE / choroidal tissue (RPE / C) of NHP eyes was measured by LC-MS at the final necropsy time point. Data are generalized from 3–4 animals. Fluid expression is shown as ng / mL. Tissue expression is shown as ng / g. Mean + standard deviation. Invention Details definition As used herein, the term "functional fH variant" includes an fH variant characterized by complement regulatory activity (cofactor activity) located in SCR1-4, and optionally functional C3b binding and GAG binding capacities (located within wild-type SCR7 and SCR19-20) characteristic of wild-type fH. In some embodiments, the modified fH variant has more than 100% of the cofactor activity and / or GAG binding capacity of wild-type fH. In another embodiment, the modified fH variant has less than about 95% to about 100% of the wild-type functional fH. For example, the modified fH variant may have at least 50% of the cofactor activity present in functional wild-type fH, and more preferably at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment, the modified fH variant may alternatively or additionally have at least 50% of the GAG-binding capacity of functional fH, and more preferably at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. Methods for determining cofactor activity, binding, and / or determining the increased circulating half-life compared to the hfH protein are known in the art.

[0026] As used herein, when referring to SCR#-##, the domain includes the endpoints and is identical to "SCR#, ... SCR##". In some implementations, periods are used between domains. For example, SCR1-4 refers to "SCR1, SCR2, SCR3, and SCR4" and is identical to "SCR1,2,3,4" or "SCR1.2.3.4." SCR19-20 refers to SCR19 and SCR20 and is identical to "SCR19,20". For example, "SCR6-8", "SCR6.7.8", and "SCR6,7,8" refer to the same domain.

[0027] The term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally exists). For example, a polynucleotide that exists naturally in a plant or animal is not isolated; however, the same polynucleotide isolated from a neighboring nucleic acid in which it naturally exists is considered "isolated."

[0028] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While a “stop codon” (TAG, TGA, or TAA) is not typically translated into amino acids, it can be considered part of a coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. The boundaries of a coding region are typically defined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the amino terminus of the resulting polypeptide). Two or more coding regions can exist in a single polynucleotide construct, e.g., on a single vector; or in separate polynucleotide constructs, e.g., on separate (different) vectors. Therefore, a subsequent single vector may contain only a single coding region or contain two or more coding regions.

[0029] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding region that influences transcription, RNA processing, stability, or translation of the relevant coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located at the 3' end of the coding sequence.

[0030] As used in this article, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and refers to a polymer of nucleotides.

[0031] Polynucleotides encoding gene products, such as polypeptides, may include promoters and / or other transcriptional or translational regulatory elements that are operatively bound to one or more coding regions. In operative binding, the coding region of a gene product, such as a polypeptide, binds to one or more regulatory regions in such a manner to place the expression of the gene product under the influence or control of the regulatory regions. For example, the coding region and the promoter are “operatively bound” if the induction of promoter function leads to the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the binding between the promoter and the coding region does not interfere with the promoter’s ability to direct the expression of the gene product or the ability to transcribe the DNA template. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, may also be operatively bound to coding regions to direct the expression of gene products.

[0032] "Transcriptional control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, etc., that provide for the expression of coding sequences in host cells. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (an immediate early promoter that binds to intron A), simian virus 40 (an early promoter), and retroviruses (e.g., Raoult's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0033] Similarly, various translation control elements are known to those skilled in the art. These elements include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from microRNAs (particularly internal ribosome entry sites, or IRES, also known as CITE sequences).

[0034] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or a polypeptide. It includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product can be a nucleic acid, such as messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. Gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, lipid addition, binding to other protein subunits, or proteolytic cleavage).

[0035] "Promoter" and "promoter sequence" are used interchangeably and refer to the DNA sequence that controls the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at the 3' end of the promoter sequence. A promoter can be derived entirely from a natural gene, or composed of different elements derived from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. Promoters that induce gene expression in most cell types most of the time are generally called "constitutive promoters." Promoters that induce gene expression in specific cell types are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that induce gene expression at specific developmental stages or cell differentiation stages are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and induce gene expression after exposure or treatment of cells with promoter-inducing agents, biomolecules, chemicals, ligands, light, etc., are generally called "inducible promoters" or "regulatory promoters." It was further recognized that, since the exact boundaries of the regulatory sequence are not fully defined in most cases, DNA fragments of different lengths can have the same promoter activity.

[0036] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the cell's central metabolic processes, and is typically in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences derived from any source, genome-integrated sequences, bacteriophage or nucleotide sequences, linear, circular or supercoiled single-stranded or double-stranded DNA or RNA, in which multiple nucleotide sequences have been linked or rearranged into a unique structure capable of introducing promoter fragments and the DNA sequence of selected gene products, along with appropriate 3' untranslated sequences, into the cell.

[0037] A certain percentage of “sequence identity” between a polynucleotide or polypeptide and another polynucleotide or polypeptide means the percentage of identical bases or amino acids when comparing two sequences during alignment. Sequence similarity can be determined in a variety of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) software package from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), edited by Doolittle, Academic Press, Inc. Of particular interest are alignment procedures that allow for gaps in sequences. Smith-Waterman is one type of algorithm that allows for gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Alternatively, the GAP procedure, which uses the Needleman and Wunsch alignment method, can be used to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0038] The term "amino acid substitution" and its synonyms described above are intended to cover amino acid sequence modifications by replacing an amino acid with another substituted amino acid. Substitution can be conservative substitution. It can also be non-conservative substitution. The term "conservative" when referring to two amino acids is intended to mean that the amino acids share common properties recognized by those skilled in the art. For example, amino acids having hydrophobic non-acidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic non-acidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties can also include amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids having polar neutral side chains, amino acids having charged side chains, amino acids having charged acidic side chains, and amino acids having charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and can be used as substituted amino acids in embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. The use of “one or more” in this document is intended to cover, for example, 1, 2, 3, 4, 5, 6 or more individual implementations.

[0039] As used herein, the terms “treatment,” “treating,” etc., refer to achieving the desired pharmacological and / or physiological effect. The effect may be preventative in the complete or partial prevention of the disease or its symptoms, and / or therapeutic in the partial or complete cure of the disease and / or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease (and / or symptoms caused by the disease) in a subject who may be susceptible to the disease or at risk of acquiring the disease but has not yet been diagnosed with it; (b) suppressing the disease (and / or symptoms caused by the disease), i.e., preventing its development; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., promoting the resolution of the disease (and / or symptoms caused by the disease), i.e., improving the disease and / or one or more symptoms of the disease.

[0040] As used herein, the term "treatment of complement factor H syndrome" can encompass the reduction, decrease, and / or improvement of symptoms, and / or prevention of the development of additional symptoms associated with complement factor H syndrome, which can manifest in several different phenotypes, including asymptomatic, recurrent bacterial infections, and renal failure. This is typically characterized by reduced serum levels of factor H, complement component C3, and other terminal complement components, indicating activation of the alternative complement pathway. This syndrome is associated with a variety of renal diseases with variable clinical presentation and progression, including C3 glomerulonephropathy and atypical hemolytic uremic syndrome. This article also provides compositions and methods for treating one or more of the following: dry age-related macular degeneration (AMD), geographic atrophy secondary to AMD, atypical hemolytic uremic syndromes (including, for example, microangiopathic hemolytic anemia syndrome, thrombocytopenia, acute renal failure), paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or prevention or treatment, especially of bacterial pathogens (e.g., Aspergillus species; Borrelia burgdorferi). Borrelia burgdorferi ); Borrelia d'Arc ( B. duttonii ); Regressive thermophobic spirochetes ( B. recurrentis ); Candida albicans ( Candida albicans ); Tulafrancsis ( Francisella tularensis ); Haemophilus influenzae ( Haemophilus influenzae ); Neisseria meningitidis ( Neisseria meningitidis ); Streptococcus pyogenes ( Streptococcus pyogenesCompositions and methods for recruiting bacteria that cause infection, such as borborygmus factor H binding proteins (CRASP-1, CRASP-2, CRASP-3, CRASP-4 or CRASP-5).

[0041] As used in this article, the term “treatment of complement-related disorders” includes alleviating, reducing and / or improving symptoms of both the complement factor H disorders identified above and other disorders associated with uncontrolled alternative pathway complement regulation.

[0042] "Complement-mediated symptoms" can encompass symptoms associated with complement dysregulation, which can manifest in several different phenotypes, including asymptomatic recurrent bacterial infections and various tissue damages, including but not limited to kidney disease. Unless otherwise stated, this definition includes both homozygous and heterozygous subjects. Complement dysregulation is typically caused by loss-of-function mutations in complement regulatory proteins (including but not limited to fH, factor I (fI), and membrane cofactor protein (MCP)) or by autoantibodies against complement regulatory proteins, or by gain-of-function mutations in other complement proteins (including but not limited to C3 and factor B (fB)). Complement dysregulation is typically (though not always) characterized by reduced serum levels of factor H, complement component C3, fB, and other terminal complement components, indicating activation of alternative and / or terminal complement pathways. Complement-mediated pathological conditions treatable by the compositions and methods of the present invention include, but are not limited to, the following diseases with different clinical presentations and progressions: C3 glomerulonephropathy (formally known as membranoproliferative glomerulonephritis type II or MPGNII), in which two known forms exist – dense deposit disease (DDD) and C3 glomerulonephritis (C3GN); thrombotic microangiopathy (TMA), including but not limited to atypical hemolytic uremic syndrome (aHUS) and Shiga toxin-producing Escherichia coli HUS. (STEC-HUS) and thrombotic thrombocytopenic purpura (TTP); retinal degenerative eye diseases, including age-related macular degeneration (AMD), RPE degeneration, choroidal retinal degeneration, photoreceptor cell degeneration, paroxysmal nocturnal hemoglobinuria (PNH), ischemia-reperfusion injury of all organs and environments, rheumatoid arthritis, hemodialysis, diabetic nephropathy, diabetic vascular disease, asthma, systemic lupus erythematosus (SLE), ischemic stroke, abdominal aortic aneurysm (AAA), antineutrophil cytoplasmic antibody (ANCA)-mediated vasculitis (ANCA vasculitis), and ANCA-mediated hemorrhagic lung injury. And diseases, ANCA glomerulonephritis, graft-versus-host disease (GvHD), acute or delayed graft rejection in organ transplantation, Crohn's disease, psoriasis, multiple sclerosis, antiphospholipid syndrome, preeclampsia, atherosclerosis, neuromyelitis optica (NMO), autoimmune cutaneous bullous disease, bullous pemphigoid (BP), Alzheimer's disease (AD), and bacterial infections caused by the recruitment of bacterial pathogens (e.g., Aspergillus species; Borrelia burgdorferi; Borrelia davidiana; Borrelia relapsingis; Candida albicans; Tula Francisella; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes).

[0043] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to humans; non-human primates, including apes; mammalian locomotion animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0044] As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. An effective amount may be administered in one or more doses. For the purposes of this disclosure, an effective amount of a compound (e.g., infectious rAAV viral particles) is an amount sufficient to mitigate, improve, stabilize, reverse, prevent, slow, or delay the progression of a particular disease state (e.g., a condition associated with complement dysfunction) (and / or associated symptoms). Therefore, an effective amount of infectious rAAV viral particles is an amount of infectious rAAV viral particles capable of effectively delivering heterologous nucleic acids to an individual's target cells (or multiple target cells). An effective amount can be clinically determined, for example, by detecting a gene product (RNA, protein) encoded by a heterologous nucleic acid sequence in cells or tissues using techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescence, or other reporter gene readouts. An effective dose can be determined clinically, for example, by detecting changes in the onset or progression of disease using methods known in the art, such as the 6-minute walk test, left ventricular ejection fraction, handheld dynamometer, Vignos scale, etc., as described herein and as known in the art. Invention Details This article describes novel rAAV viral particles encoding modified factor H (fH) gene and protein variants. These rAAV viral particles are characterized by persistent and stable expression of the fH protein in the retina, and increased efficacy in treating factor H-related conditions and other complement disorders.

[0046] These rAAV viral particles can be delivered to subjects in need via various routes, preferably via intravitreal administration. Methods for using these rAAV viral particles in treatment regimens for H factor-related conditions, particularly dry AMD and geographic atrophy secondary to AMD, are also provided.

[0047] Heterologous nucleic acids The novel rAAV viral particles described herein comprise a heterologous nucleic acid operatively linked to an expression control sequence encoding a variant of human factor H (hfH). Typically, the heterologous nucleic acid comprises the AAV genome with the rep and cap genes deleted and / or replaced by the hfH sequence and its associated expression control sequence. The hfH sequence is typically inserted adjacent to one or two (i.e., flanked by) AAV TR or TR elements sufficient for viral replication (Xiao et al., 1997, J. Virol. 71(2): 941-948), replacing the nucleic acid encoding the viral rep and cap proteins. Additional regulatory sequences suitable for promoting tissue-specific expression of the hFH gene sequence in target cells (e.g., retinal cells) may also be included.

[0048] In some respects, the rAAV viral particle contains a heterologous nucleic acid comprising (a) an AAV2 terminal repeat, (b) a transcriptional control sequence, (c) a nucleotide sequence encoding the hfH variant as described herein, (d) a polyadenylated sequence and (e) an AAV2 terminal repeat.

[0049] hfH gene The amino acid sequence of mature “wild-type” human complement factor H (isotype 1) is available at www.uniprot.org / uniprot / P08603 and serves as a reference for the amino acid numbering of hfH isotype 1 [see also SEQ ID NO:39 of U.S. Patent No. 10,988,519, the entire contents of which are incorporated herein by reference]: The leader sequence (MRLLAKIICLMLWAICVA; SEQ ID NO:4) is located at amino acids 1 to 18 of factor H, see SEQ ID NO:3. The mature (secretory) hfH protein is located at amino acids 19 to 1231 of SEQ ID NO:3. Alternative methods exist for determining the location of the 20 short complement repeats (SCRs). The domain locations mentioned in this paper are based on the numbering used in C. Estaller et al., Eur J Immunol. 1991 Mar; 21 (3):799-802.

[0050] The amino acid sequences of the 20 SCRs of hfH are provided in Table 1 below (see also SEQ ID NO: 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 38 of U.S. Patent No. 10,988,519):

[0051] Optionally, the modified hfH variants described herein may have a heterologous leader sequence that replaces the native hfH leader sequence. Additionally or optionally, another hfH isotype (e.g., isotype 2), whose sequence is available, for example, from http: / / www.uniprot.org / uniprot / P08603, and / or one of the disease-independent native amino acid variants thereof. See SEQ ID NO:40 of U.S. Patent No. 10,988,519. In the following description, substitution may be written as (first amino acid identified by a single-letter code) - residue position # - (second amino acid identified by a single-letter code), whereby the first amino acid is the substituted amino acid, and the second amino acid is the substituted amino acid with respect to isotype 1 at the specified position; however, by conventional alignment procedures, the corresponding amino acid residue identified herein with respect to isotype 1 can be located in isotype 2 and in non-pathogenic native variants of isotype 1 or 2 of fH.

[0052] As described herein, rAAV contains a heterologous nucleic acid encoding a functional fH variant. Examples of functional fH variants include those with SCR1-4 and 19-20 containing an fH protein, having one or more of the SCR7, SCR17, or SCR18 domains. Further variants include those having one or more of SCR6, SCR8, SCR16, SCR17, SCR18, or fragments thereof, or combinations thereof. For example, such variants may include, in particular, fH SCR1-4, 6-8, 19-20; fH SCR1-4, 6-8, 18-20; fH SCR1-4, 6-8, 17-20; fH SCR1-4, 6-7, 19-20; fH SCR1-4, 6-7, 18-20; fH SCR1-4, 6-7, 17-20; fH SCR1-4, 7-8, 19-20; fH SCR1-4, 7-8, 18-20; fH SCR1-4, 7-8, 17-20; fHSCR1-4, 7, 19-20; fH SCR1-4, 7, 18-20; fH SCR1-4, 7, 17-20; SCR1-4, 17, 19-20; SCR1-4, 18-20; SCR1-4, 17-20 and / or fH SCR1-4, 7, 16-20. In some embodiments, the hfH variants further include additional hfH SCRs, such as SCR 6, SCR8, SCR16 or combinations thereof. In a preferred embodiment, hfHSCR5 is absent. However, in some embodiments, hfH SCR5 may be present in its entirety or as a portion thereof. In some embodiments, hfH SCR9, SCR10, SCR11, SCR12, SCR13, SCR14 and / or SCR15 are absent, or at least functionally missing. Optionally, one or more SCRs in these variants may be “functional fragments” of an SCR, rather than full-length SCRs. "Functional fragment" refers to an amino acid sequence (or its coding sequence) smaller than the full-length SCR, characterized by having one or more of complement-inhibiting activity, heparin-binding ability, and / or C3b-binding activity.

[0053] For the hfH variant, the domains can be positioned adjacent to each other (e.g., the carboxyl terminus of one domain may immediately follow the amino terminus of a previous domain). Alternatively, one or more of the SCR domains may have a linker consisting of 1 to about 12 to 18 amino acids positioned therebetween. For example, a variant may contain SCR1-(L)-SCR2-(L)-SCR3-(L)-SCR4-(L)-(SCR6-(L))-SCR7-(L)-(SCR8-(L))-(SCR16-(L))-(SCR17-(L))-(SCR18-(L5))-SCR19-(L)-SCR20, where () indicates an optional component, and "L" indicates a linker, which may be absent or independently selected from an amino acid sequence of about 1 to about 12 to 18 amino acids. In other words, when a variant contains multiple linkers, the linkers may each have the same sequence or different sequences. In some embodiments, the variant contains at least one, at least two, at least three, at least four, at least five, or at least six joints. Examples of suitable joints include the natural or artificial joints described herein. These wild-type joints can each be located in their natural location. Alternatively, one or more of these wild-type joints can be used in different joint locations, or multiple different joint locations.

[0054] Optionally, one or more of these linkers may be fH sequences and are chosen independently. Alternatively, one or more linkers may be heterologous to fH, for example, from different sources, whether artificial, synthetic, or from different proteins that confer suitable flexibility to fH variants. Examples of other suitable linkers may include, for example, polygly linkers and other linkers that provide suitable flexibility (e.g., http: / / parts.igem.org / Protein_domains / Linker), which are incorporated herein by reference. In some embodiments, the linkers lack any fH functionality.

[0055] Table 2 below provides some representative suitable fH connector sequences. Connector sequence Exemplary bonding QKRP (SEQ ID NO:25) SCR1 - SCR2 EVVK (SEQ ID NO:26) SCR2 – SCR3 VEIS (SEQ ID NO:27) SCR3 – SCR4 EEKSTLKP (SEQ ID NO:28) SCR4 – SCR6 LRK SCR6 – SCR7 IRVKT (SEQ ID NO:29) SCR7 – SCR8 IKTD (SEQ ID NO:30) SCR8 – SCR17 IKSKDSTGK (SEQ ID NO:31) SCR8 – SCR19 RDTS (SEQ ID NO:32) SCR17 – SCR18 KDSTGK (SEQ ID NO:33) SCR18 – SCR19 LHP SCR19 – SCR20

[0056] In some preferred embodiments, rAAV comprises a heterologous nucleic acid encoding an hfH variant having the following structure: SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20.

[0057] In a particularly preferred embodiment, the heterologous nucleic acid encodes an fH variant having the following amino acid sequence: MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVKTCSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSIKTDCLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTCRDTSCVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR (SEQ ID NO:34) In other embodiments, the rAAV comprises a heterologous nucleic acid encoding an hfH variant having: SCR1-(Ll)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7’)-SCR19-(L10)-SCR20.

[0058] These and other variants may include other fH sequences. For example, the coding sequence of an fH variant may also include a leader sequence. Such a leader sequence may be an fH leader region (e.g., MRLLAKIICLMLWAICVA; SEQ ID NO:4). Optionally, the leader sequence may be derived from another source incorporated herein by reference, such as an IL-2 leader region [see, for example, an index of mammalian leader sequences identified at www.signalpeptide.de / ]. In one embodiment, the selected leader sequence is less than about 26 amino acids in length (e.g., about 1 to about 26 amino acids), more preferably less than 20 amino acids (about 1 to about 20 amino acids), and most preferably less than about 18 amino acids (about 1 to about 18 amino acids). “Loss of function” means the absence of an amino acid sequence (or its coding sequence) lacking complement-inhibiting activity, C3b-binding activity, and optionally further lacking heparin-binding activity.

[0059] In addition to the fH protein variants provided herein, nucleic acid sequences encoding these fH protein variants are provided. The coding sequences for these variants may be derived from the wild-type sequence of the leader sequence and / or one or more SCRs of isotype 1, isotype 2, or non-disease-related variants. Alternatively or additionally, web-based or commercially available computer programs and service-based companies may be used to reverse translate the amino acid sequences of the leader sequence and / or one or more SCRs into nucleic acid coding sequences, including both RNA and / or cDNA. See, for example, backtranseq via EMBOSS, Gene Infinity (www.geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (www.expasy.org / tools / ).

[0060] In one implementation, the RNA and / or cDNA coding sequences are designed for optimal expression in human cells.

[0061] Codon-optimized coding regions can be designed using a variety of different methods. This optimization can be performed using methods available online, publicly available methods, or by companies that provide codon optimization services. One codon optimization method is described, for example, in WO 2015 / 012924 A2, which is incorporated herein by reference. In short, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Appropriately, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF can be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence, and nucleic acid fragments encoding codon-optimized coding regions of the polypeptide are produced.

[0062] In another embodiment, at least one glycosylation site is modified into at least one SCR, at least two SCRs, at least three SCRs, or more SCRs present in the hfH variant. For example, the glycosylation site may be modified into one or more of SCR1, SCR2, SCR3, SCR4, SCR19, and / or SCR20. In another embodiment, SCR17 and / or SCR18 are additionally or alternatively glycosylated. In yet another further embodiment, SCR4, 17, and 18 are glycosylated. In a preferred embodiment, the hfH variant comprises or is composed of or substantially comprises SCR1-4, SCR6-8, and SCR17-19, and SCR17 and SCR18 are glycosylated. In some embodiments, the glycosylation site may be modified into the linker. However, in this case, the linker is preferably at least six amino acids long up to about 18 amino acids long, such as 8-18, 10-15, or 12 amino acids.

[0063] As used herein, a glycosylation site refers to the attachment point of an oligosaccharide to a carbon atom (C-linked), nitrogen atom (N-linked), or oxygen atom (O-linked), or to glycosylation (the non-enzymatic attachment of a reducing sugar to the nitrogen atom of a protein (e.g., the asparagine (Asn) side chain of the Asn-X-Ser / Thr moiety, where X is any amino acid other than Pro). In some embodiments, an N-glycosylation site is required. Various techniques for modifying N-glycosylation sites are known in the art. See, for example, Y Liu et al., Biotech Prog 2009 Sep-October; 25(5): 1468-1475; Sala RJ, Griebenos K. Glycoslylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010 Feb 1; 24(1): 9-21.

[0064] The following provides an exemplary hfH variant nucleotide sequence encoding the amino acid sequence of SEQ ID NO:34: ITR The preferred inverted terminal repeat (ITR) for use in rAAV viral particles is an AAV sequence, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. The ITR can be a synthetic sequence that serves as an AAV inverted terminal repeat, such as the “double-D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al., the entire contents of which are incorporated herein by reference. Typically, but not necessarily, the TRs originate from the same parvovirus; for example, both ITR sequences may be derived from AAV2.

[0065] In some respects, the heterologous nucleic acid encapsulated by rAAV viral particles contains a 5' ITR with the following sequence: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 38) In related aspects, the heterologous nucleic acid encapsulated by rAAV viral particles contains a 3' ITR with the following sequence: AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO: 39) Expression control sequence The hFH gene is operatively linked to at least one transcriptional control sequence, preferably a transcriptional control sequence heterologous to the nucleic acid. In some aspects, the transcriptional control sequence comprises a cell- or tissue-specific promoter that results in cell-specific expression of the nucleic acid, for example, in photoreceptor cells, such as the human rod photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter or the human photoreceptor intercellular retinol-binding protein (IRBP) promoter. In other aspects, the transcriptional control sequence comprises a constitutive promoter that results in similar expression levels of the nucleic acid in multiple cell types. Suitable constitutive promoters include the CAG promoter, which contains (C) cytomegalovirus (CMV) immediate early enhancer elements, (A) chicken... β-actin The first exon and first intron of the gene, and (G) rabbit β-globinGene splice acceptors (see Miyazaki et al. (1989)) Gene 79(2): 269-277), Cytomegalovirus promoter (CMV) (Stinski et al., (1985) Journal of Virology 55(2): 431-441), Human extension factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2): 217-223), Human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984)) Gene 32(3): 409-417), Mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518), and ubiquitous promoters (Wulff et al. (1990)). FEBS Letters 261: 101-105).

[0066] In a preferred aspect, the hfH gene is operatively linked to the CAG promoter. In a particularly preferred embodiment, the CAG promoter comprises the sequence of SEQ ID NO:40, or comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. In some respects, the heterologous nucleic acid encapsulated by rAAV viral particles contains SV40 polyadenylated sequences of the following: GGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCA (SEQ ID NO:41) In some respects, heterologous nucleic acids contain the following nucleotide sequences, or nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with them: In this regard, the heterologous nucleic acid comprises the following nucleotide sequences, or nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it: In this regard, the heterologous nucleic acid comprises the following nucleotide sequences, or nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it: In this regard, the heterologous nucleic acid comprises the following nucleotide sequences, or nucleotide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it: AAV capsid The rAAV variant AAV capsid—encapsulating a heterologous nucleic acid encoding the hFh variant—contains a variant AAV capsid protein containing an insert of about 7 to about 20 amino acids (“heteropeptide” or “peptide insert”) within the GH ring of the parental AAV capsid protein, wherein the peptide contains the amino acid sequence ISDQTKH (SEQ ID NO:1). Preferably, when present in AAV viral particles, the variant capsid protein confers increased retinal cell infectivity compared to the infectivity of retinal cells from AAV viral particles containing the corresponding parental capsid protein.

[0067] The “GH ring” or ring IV of an AAV capsid protein refers to the solvent-accessible portion, and is referred to in the art as the GH ring or ring IV of the AAV capsid protein. For more information on the GH ring / ring IV of the AAV capsid, see, for example, van Vliet et al. (2006). Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol.Ther. 15:1955. Therefore, for example, the insertion site could be located within approximately amino acid 570-611 of AAV2 VP1.

[0068] In some embodiments, the peptide insert has 1 to 3 spacer amino acids (Y1-Y3) at the N-terminus and / or C-terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1). Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In some embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In a preferred embodiment, the peptide insert comprises or consists of the amino acid sequence LAISDQTKHA (SEQ ID NO:2).

[0069] In some aspects, the variant AAV capsid protein includes a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:1), and further comprises one or more amino acid substitutions relative to the corresponding parental AAV capsid protein. Representative examples of amino acid substitutions can be found, for example, in column 26, lines 40-65 of U.S. Patent No. 11,576,983, the entire contents of which are incorporated herein by reference.

[0070] In some preferred embodiments, the variant AAV capsid protein includes a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO:1), and further includes a P34A amino acid substitution relative to the VP1 capsid of AAV2, or a corresponding substitution in another AAV serotype.

[0071] In other respects, the variant capsid protein may include one or more features disclosed in U.S. Patent No. 11,576,983, particularly one or more features disclosed in columns 26, lines 66 through 29, and line 50 of U.S. Patent No. 11,576,983.

[0072] In a particularly preferred embodiment, the variant capsid protein comprises the following amino acid sequence, or comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:42).

[0073] The variant AAV capsid protein of SEQ ID NO:42 contains the following modifications relative to the native AAV2 capsid protein: (i) a proline (P) to alanine (A) mutation at amino acid position 34, the position being located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids at amino acid position 588 (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO:2)), the position being present in VP1, VP2, and VP3. In some embodiments, the capsid comprises a variant capsid protein containing a sequence having at least 90%, at least 95%, at least 98%, and at least 99% identity with SEQ ID NO:42, and containing a P34A substitution at amino acid position 588 and a LAISDQTKHA (SEQ ID NO:2) peptide insertion fragment.

[0074] This document also provides packaging cells, which are encompassed by “host cells”, which can be cultured to produce the packaging viral vectors of the present invention. The packaging cells of the present invention generally comprise cells having heterologous (1) viral vector function, (2) packaging function, and (3) helper function. These component functions are each discussed in the following sections.

[0075] Initially, the vector can be prepared by several methods known to those skilled in the art (see, for example, WO 2013 / 063379). A preferred method is described in Grieger, et al. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. In short, efficient transfection of HEK293 cells serves as a starting point, in which adherent HEK293 cell lines from qualified clinical master cell banks are used for growth in shake flasks and WAVE bioreactors under suspension conditions without animal components, allowing for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), at harvest time 48 hours post-transfection, suspension HEK293 cell lines yielded greater than 10 5 One particle (vg) containing the vector genome per cell or more than 10 14 Cell cultures in vg / L. More specifically, triple transfection refers to the fact that packaging cells are transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various auxiliary functions (e.g., adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNA), and a third plasmid encodes the transgene and its various control elements (e.g., modified GLA genes and CAG promoters).

[0076] To achieve the desired yield, multiple variables were optimized, such as selecting a serum-free suspension medium compatible with both growth and transfection, and choosing transfection reagents, transfection conditions, and cell density. A universal purification strategy based on ion-exchange chromatography was also developed, resulting in high-purity formulations of AAV serotypes 1–6, 8, and 9, as well as various chimeric capsids. This user-friendly process can be completed within one week, resulting in a high complete particle / empty particle ratio (>90% complete particles), providing purified yields (>1 x 10^13 vg / L) and purity suitable for clinical applications, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been used to manufacture GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered to patients. In addition, by implementing the perfusion method, a minimum 5-fold increase is achieved in overall vector production, which allows for the harvesting of rAAV from the culture medium at multiple time points after transfection.

[0077] Packaging cells include viral vector functions, along with packaging and vector functions. Viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, where the rep and cap are deleted and replaced with modified GLA sequences and their associated expression control sequences. Viral vector functions include sufficient expression control sequences to result in replication of the viral vector used for packaging. Typically, viral vectors include a portion of a parvovirus genome, such as an AAV genome, where the rep and cap are deleted and replaced with transgenes and their associated expression control sequences. Transgenes are typically flanked by two AAV TRs, replacing the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as tissue-specific promoters and other regulatory sequences suitable for promoting tissue-specific expression of the transgene in target cells. Transgenes are typically nucleic acid sequences that can be expressed to produce therapeutic peptides or marker peptides.

[0078] The terminal repeats (TRs) (distinguished and indistinguishable) used in the viral vector are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. Distinguished AAV TRs do not need to have a wild-type TR sequence (e.g., wild-type sequences can be altered by insertion, deletion, truncation, or missense mutations), as long as the TR mediates the desired function, such as viral packaging, integration, and / or provirus rescue. TRs can be synthetic sequences that act as inverted terminal repeats of AAVs, such as the “double-D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al., the entire contents of which are incorporated herein by reference. Typically, but not necessarily, TRs originate from the same parvovirus; for example, both TR sequences may be from AAV2.

[0079] The packaging features include the various coating components described above.

[0080] The packaged viral vector, comprising a variant hfH transgene and an expression control sequence flanked by TR elements, referred herein as a “transgene” or “transgene expression cassette,” is sufficient to result in the packaging of the vector DNA and subsequent expression of the gene sequence in transduced cells. The viral vector function can be provided to the cell, for example, as a component of a plasmid or amplicon. The viral vector function can exist extrachromosomally within the cell line and / or can integrate into the cell’s chromosomal DNA.

[0081] Any method can be used to introduce a nucleotide sequence carrying viral vector function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where viral vector function is provided by transfection with a viral vector, standard methods for producing viral infection can be used.

[0082] Packaging functions include genes for viral vector replication and packaging. Therefore, for example, packaging functions may include, as needed, functions required for viral gene expression, viral vector replication, recovery of the viral vector from its integrated state, viral gene expression, and packaging of the viral vector into viral particles. Packaging functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicones, baculoviruses, or HSV helper constructs. Packaging functions may exist extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

[0083] Accessory functions include helper viral elements required to establish active infection of packaging cells, which are essential for initiating viral vector packaging. Examples include functions derived from adenoviruses, baculoviruses, and / or herpesviruses sufficient to cause viral vector packaging. For example, adenoviral accessory functions typically include adenoviral components E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be provided by infecting packaging cells with the desired virus. Packaging functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicon. See, for example, the pXR helper plasmid described in Rabinowitz et al., 2002, J. Virol. 76:791, and the pDG plasmid described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760. Packaging functions can exist extrachromosomally within packaging cells, but are preferably integrated into the cell's chromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

[0084] Any suitable helper virus function can be employed. For example, baculoviruses can act as helper viruses when the packaging cells are insect cells. Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybridized herpesviruses encoding the AAV Rep protein can advantageously facilitate more scalable AAV vector production protocols.

[0085] Any method can be used to introduce a nucleotide sequence carrying an accessory function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In embodiments where the accessory function is provided by transfection with a viral vector or infection with a viral vector, standard methods for producing viral infections can be used.

[0086] Any suitable permissive or packaging cells known in the art can be used for the production of packaged viral vectors. Mammalian or insect cells are preferred. Examples of cells that can be used for the production of packaging cells in the practice of this invention include, for example, human cell lines such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one aspect, the packaging cells are capable of growth in suspension culture, more preferably, the cells are capable of growth in serum-free medium. In one embodiment, the packaging cells are HEK293 cells grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Patent No. 9,441,206 and deposited as ATCC number PTA 13274. Numerous rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002 / 46359. In another respect, packaging cells are cultured in the form of cell stacks (e.g., a 10-layer cell stack seeded with HEK293 cells).

[0087] Cell lines used as packaging cells include insect cell lines. Any insect cell line that allows AAV replication and can be maintained in culture can be used according to the present invention. Examples include fall armyworm (Spodoptera frugiperda) cell lines such as Sf9 or Sf21, Drosophila species cell lines, or mosquito cell lines such as those derived from Aedes albopictus. A preferred cell line is the fall armyworm Sf9 cell line. The following references, concerning their teachings on the use of insect cells for the expression of heterologous peptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture, are incorporated into this article: Methods in Molecular Biology, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88: 4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

[0088] The viral capsid according to the invention can be produced using any method known in the art, for example, by expression from baculoviruses (Brown et al., (1994) Virology 198:477-488). As a further alternative, the viral vector of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap gene and rAAV template, as described, for example, by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0089] In another aspect, the present invention provides a method for the production of rAAV in insect cells, wherein a baculovirus packaging system or vector carrying AAV Rep and Cap coding regions can be constructed by: modifying these genes into the polyhedral protein coding region of the baculovirus vector; and generating a viral recombinant by transfection into a host cell. Notably, when using baculoviruses for AAV production, the AAV DNA vector product is preferably a self-complementary AAV-like molecule without requiring mutations regarding the AAV TR. This appears to be a byproduct of ineffective AAV Rep cleavage in insect cells, resulting in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell is a baculovirus-infected cell, or a cell in which additional nucleic acids encoding baculovirus helper functions have been introduced, or in which these baculovirus helper functions are included. These baculoviruses can express AAV components and subsequently promote capsid production.

[0090] During production, packaging cells typically include one or more viral vector functions, along with auxiliary and packaging functions sufficient to lead to viral vector replication and packaging. These various functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicones, and they can exist extrachromosomally within the cell line or be integrated into the cell's chromosome.

[0091] Cells can provide any one or more of the aforementioned functions that have been incorporated, such as cell lines with one or more carrier functions that are incorporated into or integrated into the chromosomal DNA of the cell, cell lines with one or more packaging functions that are incorporated into or integrated into the chromosomal DNA of the cell, or cell lines with auxiliary functions that are incorporated into or integrated into the chromosomal DNA of the cell.

[0092] The rAAV vector can be purified using methods standard in the art, such as column chromatography or cesium chloride gradient. Methods for purifying the rAAV vector are known in the art and include those described in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0093] Methods for delivering nucleic acids encoding hfH to the retina In several embodiments, methods are provided for delivering heterologous nucleotide sequences encoding hfH to the retina using rAAV as described herein. rAAV can be used to deliver nucleotide sequences encoding hfH to retinal cells in vitro, for example, to produce hfH peptides or nucleic acids in vitro for ex vivo gene therapy. rAAV can also be used in methods for delivering nucleotide sequences to subjects in need, for example, to express hfH in subjects in need, such as those with dry AMD or geographic atrophy. In this way, hfH can thus be generated in vivo in the subject to restore complement regulation.

[0094] Therefore, in one aspect, a method for delivering nucleic acids encoding the hfH variant to retinal cells is provided, the method comprising contacting the retinal cells with rAAV viral particles as described herein.

[0095] In another aspect, a method is provided for delivering a nucleic acid encoding an hfH variant to retinal cells in a mammalian subject, the method comprising administering an effective amount of rAAV viral particles, as described herein, or a pharmaceutical preparation containing such particles, to the mammalian subject.

[0096] rAAV can be administered to the subject's retina via any suitable route. In a preferred embodiment, rAAV is preferably administered intraocularly via subretinal, suprachoroidal, and / or intravitreal injection. In some particularly preferred embodiments, rAAV is administered via intravitreal injection, more preferably via a single intravitreal injection.

[0097] Treatment In some embodiments, a method is provided for treating dry AMD in a subject requiring such treatment, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) a variant AAV capsid protein comprising a heterologous peptide insert of 7 to 20 amino acids covalently inserted into the GH ring of the AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO:1), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a nucleotide sequence of the hfH variant as described herein, said nucleotide sequence being operatively linked to a promoter, or administering to the subject a pharmaceutical composition comprising said rAAV and a pharmaceutically acceptable carrier, preferably wherein said rAAV or said pharmaceutical composition is administered to the subject via intravitreal injection. Use of said rAAV or pharmaceutical compositions comprising thereof for the treatment of dry AMD is also provided. Use of said rAAV in the manufacture of a medicament for the treatment of dry AMD is also provided.

[0098] In related embodiments, a method for treating geographic atrophy in subjects requiring such treatment is provided, the method comprising administering to the subject recombinant adeno-associated virus (rAAV) viral particles comprising: (i) a variant AAV capsid protein containing a heterologous peptide insert of 7 to 20 amino acids covalently inserted into the GH ring of the AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO:1), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding the hfH variant as described herein, the nucleotide sequence being operatively linked to a promoter, or administering to the subject a pharmaceutical composition comprising the rAAV viral particles and a pharmaceutically acceptable carrier, preferably wherein the rAAV or the pharmaceutical composition is administered to the subject via intravitreal injection. Use of the rAAV or pharmaceutical compositions comprising therein for treating geographic atrophy is also provided. Use of the rAAV in the manufacture of medicaments for treating geographic atrophy is also provided.

[0099] In some respects, the variant AAV capsid protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and comprises a P34A substitution at amino acid position 588 and a LAISDQTKHA (SEQ ID NO:2) peptide insertion fragment.

[0100] In some preferred aspects, the nucleotide sequence encoding the hfH variant encodes an hfH variant having the following structure: SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20, wherein SCR1-4, 6-8, and 17-20 each comprise the amino acid sequence according to Table 1, and wherein L1-L10 each comprise the amino acid sequence according to Table 2. In related aspects, the nucleotide sequence encoding the hfH variant encodes an hfH variant that comprises the amino acid sequence of SEQ ID NO:34, or comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:34. In other relevant respects, the nucleotide sequence encoding the hfH variant comprises the nucleotide sequence shown in any of SEQ ID No: 35-37.

[0101] In some respects, rAAV is administered via periocular, intravitreal, suprachoroidal, and / or subretinal injection, preferably via intravitreal injection, at a dose of approximately 1 × 10⁻⁶ for subjects with dry AMD and / or geographic atrophy. 8 One carrier composition (vg) / eye to approximately 1×10 13 vg / eye, approximately 1×10 8 vg / eye to approximately 1×10 12 vg / eye, approximately 1×10 9 vg / eye to approximately 1×10 12 vg / eye, approximately 1x10 9 vg / eye to approximately 1x10 11 vg / eye, or approximately 6×10 9 vg / eye to approximately 6×10 10 vg / eye. In some embodiments, rAAV is administered via periocular, intravitreal, suprachoroidal, and / or subretinal injection, preferably via intravitreal injection, at a dose of approximately 1 x 10⁻⁶ for subjects with dry AMD and / or geographic atrophy. 8 vg / eye, approximately 2x10 8 vg / eye, approximately 3x10 8 vg / eye, approximately 4x10 8 vg / eye, approximately 5x10 8 vg / eye, approx. 6x10 8 vg / eye, approximately 7x10 8 vg / eye, approximately 8x10 8 vg / eye, approx. 10 8 vg / eye, approximately 1x10 9 vg / eye, 2x10 9 vg / eye, approximately 3x10 9 vg / eye, approximately 4x10 9 vg / eye, approximately 5x10 9 vg / eye, approx. 6x10 9 vg / eye, approximately 7x10 9 vg / eye, approximately 8x10 9 vg / eye, approx. 9x10 9 vg / eye, approximately 1x10 10 vg / eye, approximately 2x10 10 vg / eye, approximately 3x10 10 vg / eye, approximately 4x10 10 vg / eye, approximately 5x10 10 vg / eye, approx. 6x10 10 vg / eye, approximately 7x10 10 vg / eye, approximately 8x10 10 vg / eye, approx. 9x1010 vg / eye, approximately 1x10 11 vg / eye, approximately 2x10 11 vg / eye, approximately 3x10 11 vg / eye, approximately 4x10 11 vg / eye, approximately 5x10 11 vg / eye, approx. 6x10 11 vg / eye, approximately 7x10 11 vg / eye, approximately 8x10 11 vg / eye, approx. 9x10 11 vg / eye or approximately 1x10 12 vg / eye.

[0102] In some embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering to the subject an effective amount of rAAV viral particles comprising: (i) a capsid comprising a capsid protein comprising the amino acid sequence shown in SEQ ID NO:42; and (ii) a heterologous nucleic acid comprising from 5' to 3' the following: (a) an AAV2 terminal repeat; (b) a CAG or CMV promoter; (c) a nucleotide sequence selected from SEQ ID No: 35-37; (d) a polyadenylated sequence; and (e) an AAV2 terminal repeat.

[0103] In relevant implementations, a method is provided for treating dry AMD and / or geographic atrophy in subjects with this need, comprising administering a pharmaceutical composition to the subject via periocular, intravitreal, suprachoroidal, and / or subretinal injection, comprising a pharmaceutically acceptable carrier and rAAV viral particles comprising: (i) a capsid protein comprising the amino acid sequence shown in SEQ ID NO:42, and (ii) a heterologous nucleic acid from 5' to 3' comprising: (a) an AAV2 terminal repeat, (b) a CAG or CMV promoter, (c) a nucleotide sequence selected from SEQ ID No: 35-37, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat. In some aspects, the pharmaceutical composition comprises about 1 × 10 8 vg to approximately 1×10 13 vg, approximately 1×10 9 vg to approximately 1×10 12 vg, approximately 1×10 9 vg to approximately 1×10 11 vg or approximately 6×10 9 vg to approximately 6×10 10 vg. In other respects, the pharmaceutical composition contains about 1x10 8vg, approximately 2x10 8 vg, approximately 3x10 8 vg, approximately 4x10 8 vg, approximately 5x10 8 vg, approximately 6x10 8 vg, approximately 7x10 8 vg, approximately 8x10 8 vg, approximately x10 8 vg, approximately 1x10 9 vg, 2x10 9 vg, approximately 3x10 9 vg, approximately 4x10 9 vg, approximately 5x10 9 vg, approximately 6x10 9 vg, approximately 7x10 9 vg, approximately 8x10 9 vg, approximately 9x10 9 vg, approximately 1x10 10 vg, approximately 2x10 10 vg, approximately 3x10 10 vg, approximately 4x10 10 vg, approximately 5x10 10 vg, approximately 6x10 10 vg, approximately 7x10 10 vg, approximately 8x10 10 vg, approximately 9x10 10 vg, approximately 1x10 11 vg, approximately 2x10 11 vg, approximately 3x10 11 vg, approximately 4x10 11 vg, approximately 5x10 11 vg, approximately 6x10 11 vg, approximately 7x10 11 vg, approximately 8x10 11 vg, approximately 9x10 11 vg or approximately 1x10 12 vg.

[0104] Pharmaceutical compositions comprising rAAV as described herein are provided. In some embodiments, the pharmaceutical composition comprises about 1 x 10 8 To approximately 1 x 10 14 One vector particle or vector genome, approximately 1 x 10 8 To approximately 1 x 10 13 One vector particle or vector genome, approximately 1 x 10 9 To approximately 1 x 10 12 One vector particle or vector genome, or approximately 1x10 8Approximately 2x10 8 Approximately 3x10 8 Approximately 4x10 8 Approximately 5x10 8 Approximately 6x10 8 Approximately 7x10 8 Approximately 8x10 8 Approximately 9x10 8 Approximately 1 x 10 9 Approximately 2 x 10 9 Approximately 3x10 9 Approximately 4 x 10 9 Approximately 5 x 10 9 Approximately 6 x 10 9 Approximately 7 x 10 9 Approximately 8 x 10 9 Approximately 9 x 10 9 Approximately 1 x 10 10 Approximately 2 x 10 10 Approximately 3 x 10 10 Approximately 4 x 10 10 Approximately 5 x 10 10 Approximately 6 x 10 10 Approximately 7 x 10 10 Approximately 8 x 10 10 Approximately 9 x 10 10 Approximately 1 x 10 11 Approximately 2 x 10 11 Approximately 3 x 10 11 Approximately 4 x 10 11 Approximately 5 x 10 11 Approximately 6 x 10 11 Approximately 7 x 10 11 Approximately 8 x 10 11 Approximately 9 x 10 11 Or approximately 1 x 10 12 One carrier particle or carrier genome. In some aspects, the drug composition contains about 1 × 102 9 To approximately 1×10 11 vg, and preferably contains about 6×10 9 vg to approximately 6×10 10 vg. In some preferred embodiments, the pharmaceutical composition is administered via intravitreal injection to a person suffering from dry AMD and / or geographic atrophy. Example

[0105] The following examples illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention in any way. While the invention has been described with respect to its preferred embodiments, various modifications thereto will be apparent to those skilled in the art upon reading this application.

[0106] Example 1 Recombinant AAV (rAAV) viral particles were constructed, carrying a transgene encoding a shortened form of human complement factor H. Figure 1 Complement factor H (CFH) is a natural inhibitor of the alternative complement pathway, and therefore rAAV can be used, in particular, to treat geographic atrophy secondary to age-related macular degeneration. Endogenous CFH is a 155 kDa protein containing 20 short common repeat (SCR) units. The N-terminus is a key site for cofactor activity / decay-accelerating activity, and the C-terminus is used for cellular regulation (see, for example, de Córdoba SR, de Jorge EG. Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H. Clin Exp Immunol. Jan 2008; 151(1):1-13).

[0107] rAAV comprises a capsid protein operatively linked to a CAG promoter, containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding a shortened form of complement factor H (“mini CFH”) having the amino acid sequence shown in SEQ ID NO:34. The mini CFH transgene contains SCR1-4, 6-8, and 17-20.

[0108] rAAV underwent in vitro characterization by: (a) transfecting the rAAV plasmid cassette into human HEK293T cells, and (b) transducing human iPSC-derived retinal pigment epithelial (RPE) cells in vitro with rAAV.

[0109] Expression and activity of miniature CFH in HEK293T cells To confirm the in vitro function of the miniature CFH transgene utilized in rAAV, human cells were transfected with an rAAV plasmid cassette. HEK293T cells were transfected with either low (0.125 µg) or high (0.5 µg) plasmid DNA encoding either a miniature CFH or a full-length CFH, each driven by a CAG promoter. Dose-dependent expression and adequately sized proteins were observed in the cell supernatant. Figure 2A and 2BMiniature CFHs from cell supernatant showed direct binding to C3b and heparin, similar to full-length CFHs. Figure 2C and 2D These data confirmed that the mini CFH transgene was expressed and secreted by the rAAV plasmid cassette and retained complement and heparin binding properties. Next, the activity and functionality of the mini CFH transgene were evaluated. Complement 3 (C3) protein was cleaved into C3a and C3b fragments, and in the presence of CFH as a cofactor, the C3b fragment was further degraded into smaller, inactive fragments by complement factor I (CFI) to disrupt the alternative complement cascade. Protein cleavage products from rAAV plasmid cassette-transfected cells confirmed that mini CFH mediated C3b degradation. Figure 2E Furthermore, miniature CFHs from transfected cells confirmed complete inhibition of MAC formation. Figure 2F In summary, these results confirm that transfection with miniature CFH cDNA leads to the expression and secretion of functional CFH proteins.

[0110] In vitro transduction of human iPSC-derived retinal pigment epithelial (RPE) cells To confirm the in vitro expression and function of the mini CFH transgene after rAAV transduction, human iPSC-derived RPE cells were transduced with rAAV at different multiplici of infection (MOI). Transduction with AAV resulted in dose-dependent secretory protein expression of the mini CFH transgene. Figure 3A Furthermore, the inhibition of the alternative complement pathway confirms the suppression of MAC formation. Figure 3B and 3C In summary, these in vitro data confirmed the expression, secretion, and function of the miniature CFH transgene derived from rAAV.

[0111] Materials and Methods Miniature CFH Expression (ELISA, WB). HEK293T cells (CRL-3216 from ATCC) were transfected with plasmid DNA encoding either a miniature CFH driven by the CAG promoter or a full-length CFH. The plasmid DNA was transfected at 0.125 µg or 0.5 µg DNA / 12 well (2e5 cells / well) using Fugene HD reagent (Promega Corporation catalog # E2311). Cells were incubated for 48 hours in 1 ml DMEM medium containing 1% penicillin / streptomycin and 10% heat-inactivated serum. After the initial 48 hours, the medium was replaced with 0.35 ml serum-free medium (DMEM medium containing 1% penicillin / streptomycin), and cells were incubated for 24 hours. Transgenic expression was assessed in the serum-free supernatant by ELISA and Western blotting.

[0112] C3b cleavage assays were performed to confirm the functional activity of CFH expressed in 293T cells. The C3 protein consists of an α-chain and a β-chain. The C3 protein is cleaved into C3a and C3b fragments. C3a is a small fragment with an approximate molecular weight of 9 kDa and 77 amino acids. The C3b fragment (containing the remaining α-chain and β-chain) can form a C3 convertase upon binding factor B, or it can be degraded into smaller inactive fragments by complement factor I (CFI) in the presence of CFH as a cofactor. The latter case disrupts the alternative complement cascade. These smaller fragments resulting from C3b degradation can be detected by Western blotting. Serum-free culture supernatant from CFH-transfected cells (or untransfected cells as a negative control) was incubated at 37°C for 1 hour with 1 µg of purified C3b (Complement Technology Inc. catalog # A114) and 1 µg of purified CFI (Complement Technology Inc. catalog # A138). As a positive control, we used 0.5 µg of purified CFH (Complement Technology Inc., Catalogue # A114) mixed with 1 µg of purified C3b (Complement Technology Inc. Catalogue # A138) in PBS and incubated at 37°C for 1 hour. Additional negative controls were included, in which CFH or CFI was omitted from the recombinant protein mixture.

[0113] Complement Inhibition Assay. The Wieslab® Complement System Alternative Pathway Kit (Eagle Biosciences Inc. Catalog #COMPL AP 330) is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum. This assay combines the principles of a hemolysis assay for complement activation with the use of labeled antibodies specific to neoantigens generated due to complement activation. The amount of neoantigen generated is proportional to the functional activity of the complement pathway. The wells of a 96-well plate are coated with a specific activator of the alternative pathway (bacterial lipopolysaccharide, LPS). Normal human serum is diluted to 5% in a diluent containing a specific inhibitor to ensure activation of only the alternative pathway. Complement is activated by LPS during incubation of the diluted serum in the wells. The wells are then washed, and C5b-9 is detected using an alkaline phosphatase-labeled antibody specific to the neoantigen expressed during MAC formation. Specific antibody detection is obtained by incubation with an alkaline phosphatase substrate solution after a further washing step. The amount of complement activation is correlated with color intensity and is measured as absorbance (optical density (OD)). Adding complement inhibitors such as eculizumab (C5 blocking antibody), CFH from CFH-transfected cell culture supernatant, or recombinant CFH protein to 5% normal human serum can partially or completely inhibit the reaction, as demonstrated by reduced absorbance signal.

[0114] C3b binding ELISA. The C3b binding ELISA is an internal ELISA modified by Nichols et al. ( / / pubmed.ncbi.nlm.nih.gov / 26221753 / ). The assay measures whether CpG-free CpG-free CpG-free micro-CFH retains direct C3b binding ability by using a monoclonal anti-CFH blocking antibody to prevent the binding of micro-CFH to a C3b-coated plate. The assay was performed using both full-length recombinant CFH as a proof-of-concept and supernatant from 293T cells transfected with CpG-free micro-CFH. As a control, we included antibody-free CFH and simulated incubation conditions with transfected cell supernatant.

[0115] Heparin binding ELISA. The heparin binding ELISA is an internal ELISA modified by Nichols et al. ( / / pubmed.ncbi.nlm.nih.gov / 26221753 / ). The assay measures whether CpG-free microCFH retains heparin binding capacity by using a monoclonal anti-CFH blocking antibody to prevent the binding of microCFH to a heparin-coated plate. The assay was performed using both full-length recombinant CFH as a control and supernatant from 293T cells transfected with CpG-free microCFH. As a control, we also included supernatant from transfected cells not treated with the blocking antibody. The results showed that pre-incubation with the blocking CFH antibody reduced the signal of both recombinant CFH and CpG-free microCFH, indicating that the CpG-free microCFH construct retained heparin binding activity.

[0116] Transduction of human RPE cells. iPSC-derived RPEs were transduced using the capsid of SEQ ID NO:42, which delivers a payload encoding a miniature CFH driven by the CAG promoter. The RPEs were previously internally derived and cryopreserved. RPEs were seeded onto plates coated with hESC-qualified Matrigel™ (Corning catalog # 354277) for 30 ± 5 days prior to transduction and maintained in serum-free X-VIVO-10 medium (Lonza catalog # BEBP02-055Q) containing 1% penicillin / streptomycin and 10 μM of the RHO / ROCK pathway inhibitor Y-27632 (STEMCELL Technologies catalog # 72304) for 30 ± 5 days. The medium was changed every 2–3 days. RPEs were transduced at 5,000; 20,000; and 50,000 multiples of infection (MOI, vector genome (vg) / cell). Cell counting was performed on the day of transduction and immediately prior to transduction; an appropriate volume of virus was mixed with fresh culture medium and added to the cells. For the first 3 days after transduction, the culture medium was removed and fresh medium was added. Four days later, and seven days after transduction, the culture medium was harvested and mini-CFH expression and activity were measured.

[0117] Example 2 High-performance liquid chromatography / mass spectrometry (LC-MS) was used to confirm the presence of the miniature CFH gene product (short CFH or sCFH) in non-human primates (NHPs) following in vivo administration of rAAV (comprising a capsid protein containing the amino acid sequence shown in SEQ ID NO:42 operatively linked to the CAG promoter and a heteronucleotide encoding a shortened form of complement factor H (“miniature CFH”) having the amino acid sequence shown in SEQ ID NO:34) operatively to the vitreous body of Example 1.

[0118] Materials and Methods Aqueous humor (AH) and vitreous humor (VH) samples were collected from NHP treated with rAAV (intravitreal) therapy, flash-frozen, and stored for analysis. An sCFH LCMS method was developed and validated to identify peptide markers specific to sCFM encoded by rAAV. These specific peptide markers were then quantified against a standard curve to determine the sCFH protein concentration in each sample. Each experimental run included a quality control sample containing known levels of sCFH. The standard deviation acceptance criterion for replicate tests was ≤20%. The LLOQ / BLQ for the assay was determined to be ~50 ng / mL, and samples were run in triplicate.

[0119] result Table 3 below summarizes the LCMS quantitative results of sCFH concentrations in AH samples containing NHP with intravitreal administration of specified doses (1e10, 5e10, 5e11 vg / eye) of rAAV (see also: Figure 4 ): Table 3

[0120] Table 4 below summarizes the LCMS quantitative results of sCFH concentration in VH samples of NHP treated with a specified dose of rAAV: Table 4

[0121] in conclusion At each time point and dose, the sCFH gene product encoded by rAAV was detected in VH and AH (Tables 3 and 4). Figure 4 Based on the measured AH level of sCFH and previous studies / models using rAAV containing the capsid protein of SEQ ID NO:42 to explore the relative concentration of AH / VH / retina, the retinal sCFH concentration was predicted to be within the normal / therapeutic range (see Table 5 below).

[0122] Table 5 dose Average observed AH Predicted peak AH concentration (ng / ml) (all PK regressions) Predicted peak AH concentration (ng / ml) (low PK regression) Predicted R / C concentration (ng / g) Predicted R / C sCFH concentration (nM) 1e12 --- 373 2232 15,624 195 nM 5e11 1767 (87) 187 1116 13,606 170 nM 1E11 -- 37 223 1719 21.5 nM 5e10 10 (48) 19 112 800 10 nM 3.3e10 -- 12.3 74 570 7 nM 1e10 58 (35) 3.7 22.3 447 5.6 nM 6e9 -- 2.2 13 103 1.29 nM 3e9 -- 1 6.7 52 0.65 nM 1e9 -- 0.4 2.2 17 0.213 nM Endogenous CFH AH: 50-350 ng / ml Endogenous CFH predicted retinal / choroidal lesions: 3-70 nM.

[0123] An in situ hybridization (ISH) method was developed to evaluate sCFH expression using transfected 293T cells to confirm specificity and optimize assay conditions. The ISH method was then used to detect the presence of sCFH RNA in NHP eye tissue following intravitreal injection with rAAV (comprising a capsid protein operatively linked to a CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding a miniature CFH having the amino acid sequence shown in SEQ ID NO:34).

[0124] Materials and Methods Ocular tissue samples (whole eyeball samples) were collected from NHP treated with rAAV at a dose of 5E10 vg / eye, fixed, and processed for analysis.

[0125] sCFH ISH assays were performed using a custom-designed, proprietary 20ZZ probe for sCFH mRNA (designed to be non-cross-reactive with endogenous cynomolgus monkey or human FLCFH). sCFH ISH was performed on the Roche Discovery ULTRA Autostainer platform using the Roche DISC. mRNA Probe AMP. Kit RUO, RochemRNA Sample Prep Kit RUO, DISC. mRNA DAB Detection RUO, blueing reagent and hematoxylin, and the ACDRNAscope® VS Universal HRP Reagent Kit. Full slide image scans were captured using a Zeiss Axioscan instrument, and representative images were captured and reported using Zeiss Zen-lite imaging software.

[0126] The results of the ISH analysis show that Figure 5 and 6 middle. Figure 5 This study confirmed that sCFH RNA was present in transfected HEK293T cells compared to untransfected (negative control) HEK293T cells. Figure 6 Extensive sCFH RNAISH signaling was confirmed in the retina and macular region.

[0127] Example 3 Further in vitro studies were conducted to confirm the in vitro functional activity of the mini CFH protein expressed after transfection into human HEK293T cells.

[0128] HEK293T cells were transfected with low-level (0.125 μg) or high-level (0.5 μg) rAAV plasmid cassettes of Example 1 (comprising a heterologous nucleic acid operably linked to a CAG promoter encoding a shortened form of complement factor H (“mini CFH”) having the amino acid sequence shown in SEQ ID NO:34), or rAAV plasmid cassettes encoding the full-length CFH protein. Mini CFH or full-length CFH protein secreted into the supernatant was visualized by Western blotting. Dose-dependent expression and appropriately sized protein ( ) were observed in the supernatant from transfected cells. Figure 7A The expression of CFH protein secreted after transfection with the rAAV plasmid cassette from Example 1 was quantified by ELISA. Figure 7B Transfection resulted in higher secretion and detectable CFH expression than the untransfected control. Figure 7B ).

[0129] In addition to protein expression, the activity of mini-CFH secreted from HEK293T cells transfected with the mini-CFH construct was evaluated. Complement factor H is an essential complement regulator for controlling the alternative pathway and, in turn, negatively regulates the formation of the C5b-9 complex (also known as the membrane attack complex (MAC)), the endpoint of the complement cascade. To test whether the mini-CFH was functional, alternative complement inhibitory activity was measured using a modified form of the Wieslab® Complement System Alternative Pathway Assay. Supernatant from cells transfected with the rAAV plasmid cassette of Example 1 showed significant inhibition of alternative complement system activity. Figure 7C Recombinant eculizumab antibody, a C5 inhibitor, was used as a positive control and showed strong inhibition of the alternative complement pathway in this assay. Figure 7C ).

[0130] In addition to determining the effect of miniature CFH on the endpoint of complement activation (MAC formation), the upstream mechanism of CFH was assessed via a C3b cleavage activity assay. The upstream complement protein C3 can be hydrolyzed into C3a and C3b fragments upon complement activation. The C3b fragment contains both α and β chains and can form C3 convertase upon binding factor B, or be degraded into smaller, inactive cleavage products by complement factor I (CFI) in the presence of CFH as a cofactor. The degradation of the C3b β chain disrupts the complement cascade and is a marker of complement pathway inhibition. These smaller fragments resulting from C3b cleavage can be detected by Western blotting. Supernatants from cells transfected with the rAAV plasmid cassette of Example 1 or untransfected cells were incubated with recombinant proteins CFI and C3b, and analyzed by Western blotting. Figure 7DThe membrane was probed with an anti-C3 antibody to detect all C3 protein products, including the C3 α chain, C3 β chain, and three β chain cleavage products. All three C3b cleavage products were observed in the reaction incubated with cell supernatant transfected with the rAAV plasmid cassette of Example 1, similar to the positive control, but not detected in the NT reaction. Additionally, the C3b α chain band was significantly reduced under these conditions, similar to the positive control. These data support the upstream function of the miniature CFH generated by the rAAV plasmid cassette of Example 1 to aid in CFI during C3b β chain cleavage, ultimately inhibiting the progression of the complement cascade.

[0131] Furthermore, as expected, the miniature CFH in the cell supernatant from cells transfected with the rAAV plasmid cassette of Example 1 binds directly to both C3b and heparin, similar to the full-length CFH. Figure 7E and Figure 7F These data confirm that the mini CFH transgene can be expressed and secreted, and retains the correct complement and heparin binding properties.

[0132] Further experiments were performed to confirm the expression of the mini-CFH gene product and the functional activity of the expressed mini-CFH protein in vector-transduced human iPSC-derived RPE cells following transduction at different multiples of infection (MOIs). iPSC-derived RPE cells were transduced with rAAV at several different MOIs: 5,000; 20,000; and 50,000 vg / cell (for experiments evaluating the expression and functional activity of mini-CFH secreted into the cell supernatant), and MOIs of 800; 2,000; and 5,000 vg / cell (for disease model experiments). rAAV comprises a capsid protein operatively linked to the CAG promoter, containing the amino acid sequence shown in SEQ ID NO:42, and a heteronucleotide encoding a shortened form of complement factor H (“mini-CFH”) having the amino acid sequence shown in SEQ ID NO:34. Following transduction, the viral inoculum was removed and the culture medium replaced approximately 72 hours later. Seven days after transduction, the cell supernatant was collected, aliquoted, and stored at -80°C, and the cells were fixed.

[0133] To confirm that rAAV transduction of iPSC-derived RPE cells resulted in the expression of functional proteins, the concentration of secreted miniature CFH in the cell supernatant was quantified by ELISA 7 days post-transduction. ELISA detected both endogenous full-length CFH and rAAV-encoded miniature CFH. Signals exceeding those visible in untransduced cells were associated with rAAV-expressed miniature CFH. Figure 8As shown, untransduced RPE cells secreted low levels of endogenous CFH, and the supernatant from RPE cells transduced at MOIs of 20,000 and 50,000 showed statistically higher CFH concentrations. Furthermore, the CFH concentration was significantly higher in the supernatant from RPE cells transduced at MOI 50,000 compared to MOI 5,000. This data confirms an in vitro dose-dependent response.

[0134] Subsequently, the ability of secreted miniature CFH protein to inhibit the alternative complement pathway was evaluated in rAAV-transduced RPE cells. The rAAV comprises a capsid protein operatively linked to the CAG promoter, containing the amino acid sequence shown in SEQ ID NO:42, and a heteronucleotide encoding the amino acid sequence shown in SEQ ID NO:34. Alternative complement inhibitory activity was measured in supernatants from transduced cells using two different methods: 1) a modified form of the Wieslab® alternative complement pathway assay, and 2) quantification of soluble C5b-9. To evaluate the functional inhibitory effect of miniature CFH on the Wieslab® alternative complement pathway, supernatant samples from rAAV-transduced RPE cells were mixed with serum and applied to a Wieslab assay plate. Inhibition of the complement pathway resulted in reduced MAC signaling.

[0135] Supernatants from cells transduced with rAAV at 20,000 MOI and 50,000 MOI produced statistically significant complement inhibition compared to untransduced RPE cell samples. As a positive control, RPE cells were treated with 200 nM recombinant CFH, a concentration comparable to the highest levels of expressed miniature CFH observed in this study. Dose-dependent complement inhibitory activity was observed, and treatment of RPE cells with high concentrations of eculizumab (a known complement inhibitor targeting C5) also resulted in near-complete complement inhibition. Figure 9A ) Since the Wieslab assay is not a true quantitative measure of complement activation, a second, more quantitative method was used to assess the complement-inhibitory activity of the expressed miniature CFH. The concentration of soluble C5b-9 was measured using the SC5b ELISA kit. Samples from the supernatant of RPE transduced with rAAV showed a significantly reduced SC5b-9 formation compared to samples with supernatant from untransduced RPE. Figure 9BFurthermore, a dose-dependent effect on soluble C5b-9 formation was observed. As expected, untransduced cell samples supplemented with eculizumab showed minimal C5b-9 formation. This reduction in soluble C5b-9 confirms functional inhibition of the alternative complement pathway. Overall, these results confirm that miniature CFH secreted by RPE cells transduced with rAAV can inhibit the alternative complement pathway in a dose-dependent manner.

[0136] In addition to characterizing the functional role of expressed miniature CFH in complement activation, the upstream mechanisms of miniature CFH were assessed via a C3 convertase decay acceleration activity assay. To determine whether miniature CFH acts as a cofactor for complement factor I (CFI) to promote C3b degradation in the same manner as full-length CFH, supernatant from rAAV-transduced cells was mixed with purified C3b and CFI (instead of purified full-length CFH). Figure 10 (lanes 5-9). Compared to untransduced samples ( Figure 10 Compared to lane 5, samples from transduced RPE ( Figure 10 Lanes 6-8 lead to greater C3b α-chain cleavage, with samples at higher MOIs producing more cleavage than those at lower MOIs. In the absence of CFI, miniature CFH generated via RPE produces minimal C3b cleavage, similar to purified full-length CFH. Figure 10 Lane 9) supports its role as a cofactor in the decomposition of C3b.

[0137] In addition to these findings, the efficacy of rAAV, comprising a capsid protein containing the amino acid sequence shown in SEQ ID NO:42 operatively linked to a CAG promoter, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34, was examined in an in vitro human RPE disease model. For this purpose, a model of retinal complement activation was established in human iPSC-derived RPE cultures. To simulate the disease state, serum and the alternative complement pathway activator yeast glycan were introduced into the culture medium, resulting in MAC deposition on the RPE over the next 24 hours, as determined by immunocytochemistry (ICC) and flow cytometry.

[0138] As shown in Figure 11 below, rAAV treatment of the RPE 6 days prior to complement activation induced a dose-dependent reduction in MAC deposition on the RPE triggered by the presence of yeast glycans. As expected, eculizumab exerted a similar protective effect when added to the culture concurrently with complement activation. Flow cytometry quantification of MAC deposition ( Figure 11BThe study revealed that RPE cells transduced with rAAV and RPE cells supplemented with eculizumab exhibited significantly reduced MAC formation compared to untransduced RPE. Furthermore, MAC formation was significantly reduced in RPE cells transduced at MOIs of 2,000 and 5,000 compared to MOI 800, indicating a dose-dependent response. Overall, these immunocytochemical (ICC) and flow cytometry data confirm that, in addition to complement inhibition in cell-free assays, miniature CFH secreted by RPE transduced with rAAV products also protects RPE from MAC deposition in a disease-like context.

[0139] Finally, the upstream function of miniature CFH in this in vitro model was evaluated. Activation of the alternative complement pathway led to a significant increase in C3b, which was converted to iC3b. As discussed earlier, CFH is a cofactor in the cleavage of C3b into iC3b; however, because this cleavage inactivates C3 convertase, the production of iC3b ultimately resulted in the production of less C3b, and therefore less iC3b in the system. iC3b is commonly used as a readout for C3b because it has a significantly longer half-life compared to C3b. Therefore, iC3b concentration was measured in the disease model supernatant as a substitute for C3b levels. Figure 12 Compared to the supernatant from untransduced RPE cells, the supernatant from RPE cells transduced with rAAV showed reduced levels of iC3b (). Figure 12 However, as expected, there was no difference in iC3b concentration between untransduced RPE supernatant and untransduced RPE supernatant supplemented with eculizumab, because eculizumab regulates the complement system further downstream in the protein cascade.

[0140] In summary, human iPSC-derived RPE cells transduced with rAAV (comprising a capsid protein operatively linked to the CAG promoter containing the amino acid sequence shown in SEQ ID NO:42, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34) expressed and secreted miniature CFH in a dose-dependent manner. The expressed miniature CFH exhibited functional alternative complement inhibitory activity and specific C3 convertase decay-accelerating activity, consistent with the functional activity of endogenous CFH. Furthermore, when the complement pathway was subsequently activated, rAAV transduction of RPE resulted in a protective effect on RPE cultures, as evaluated by the levels of C5b-9 / MAC deposition and iC3b levels on the RPE. MAC is the terminal complex in the complement cascade and a major effector of complement-mediated cell death. iC3b is a degradation product of C3b and a non-terminal readout of alternative complement activation. Ultimately, soluble iC3b levels were reduced in cultures transduced with rAAV compared to untransduced cultures, as were MAC deposits on RPEs visualized by ICC and quantified by flow cytometry.

[0141] Example 4 In vivo ocular pharmacodynamic studies of rAAV expressing micro-CFH in non-human primates The safety and expression of the mini CFH transgenic protein of rAAV (comprising a capsid protein containing the amino acid sequence shown in SEQ ID NO:42 operably linked to the CAG promoter, and a heteronucleotide encoding the amino acid sequence shown in SEQ ID NO:34) were evaluated in male cynomolgus monkeys following a single intravitreal (IVT) dose to both eyes, followed by a 6 or 12-week observation period. Male cynomolgus monkeys were administered rAAV once per eye via IVT in both eyes. Groups 1 and 2 consisted of 2 males per group, and were administered 5 × 10⁻⁶ doses of rAAV to each eye. 11 and 5×10 10 The drugs were administered via vg / eye and were sacrificed 6 weeks after the dose for tissue collection. Groups 3, 4, and 5 were administered 5 × 10⁻⁶ g / eye. 11 5×10 10 and 1×10 10 rAAV IVT was administered once per eye (bilateral) and observed for 12 weeks prior to terminal sacrifice.

[0142] Following in vitro transfusion (IVT) administration of rAAV, the concentration of miniature CFH protein was examined in aqueous humor, retinal tissue, and serum samples by LC-MS. Treatment-related dose-response regarding miniature CFH protein expression was observed in the aqueous humor. Figure 13 ) The levels of the miniature CFH transgenic protein were higher in the retina and RPE / choroid, accompanied by relatively lower concentrations in the aqueous humor. Figure 14 Given the retinal orientation of the capsid and the fact that retinal tissue is likely the dominant source of micro CFH protein secretion, this distribution pattern was expected.

[0143] Miniature CFH protein was only present at 5 × 10⁻⁶ on day 15. 11 The presence of detectable LLOQ values ​​in serum of the vg / ocular dose group, along with extremely low values ​​close to 5 ng / mL, confirms that the miniature CFH protein expressed by rAAV is largely contained within the eye after IVT administration and is not distributed into the systemic circulation at a meaningful level (see Table 6 below).

[0144] Table 6. Mini CFH levels in aqueous humor, vitreous humor, and serum of rAAV-treated cynomolgus monkey samples. BQL: Below the quantifiable limit Limit of quantification: 5 ng / mL.

[0145] No significant macroscopic findings were observed in animals euthanized at week 6 or 12, or any significant changes in clinicopathology, hematology, or coagulation findings across rAAV dose groups. No treatment-related changes were observed with respect to any clinicopathological parameters following rAAV treatment, and minor fluctuations were generally attributable to the immunosuppressive regimen.

[0146] Microscopic findings observed after 6 weeks of rAAV treatment included minimal, non-adverse perivascular mononuclear cell infiltration in the retina. Findings at 12 weeks were comparable and included minimal to mild, non-adverse mononuclear cell infiltration in the ciliary body, minimal retinal degeneration and condensation cells of undetermined significance in one eye, and minimal perivascular mononuclear cell infiltration in the optic nerve head, none of which were considered adverse. No systemic organ findings were observed microscopically in any dose group. IVT treatment with rAAV resulted in the production of both carrier capsid antibodies and anti-micro CFH antibodies in most animals by week 12. No antigen-specific IFN-γ T cell responses against the carrier capsid or CFH peptide library, as measured by ELISPOT, were detected in NHP eyes treated with rAAV at 6 or 12 weeks post-dose. Overall, rAAV showed up to 5 × 10⁻⁶ in male cynomolgus monkeys. 11 vg / e eye (HED 1×10) 12 A single bilateral IVT dose of (vg / eye) was well tolerated.

[0147] Single intravitreal dose ocular evaluation study in cynomolgus monkeys Another study was initiated to comprehensively evaluate ocular structural and functional parameters in a group of male and female cynomolgus nonhuman primates following a single unilateral IVT dose of rAAV (including a capsid protein containing the amino acid sequence shown in SEQ ID NO:42, operably linked to a CAG promoter, and a heterologous nucleic acid encoding the amino acid sequence shown in SEQ ID NO:34), and to follow up for 12 weeks post-dose.

[0148] Two groups of cynomolgus monkeys, each consisting of one male and two females, were used at a dose of 2.8 × 10⁻⁶ on day 1 of the study. 10 vg / eye or 1.5×10 11 The rAAV dose was administered once in the right eye at a dose of vg / eye. The left eye of the animal was simultaneously treated with the medium. The dose volume was 50 μL / eye for both the left and right eyes.

[0149] Viviparous parameters included daily mortality / cage-side assessment, during week -1, before dose, and at 1, 2, 4, and 8 hours post-dose on day 1 of the study, and on days 2–7, followed by weekly detailed clinical observation for the remainder of the study. Body weight was assessed before administration and weekly during the study. Ophthalmological examinations were performed on all animals before testing and on days 2, 4 (±1), and 7, and during weeks 2, 3, 4, 5, 6, 9, and 12.

[0150] Intraocular pressure was assessed before the study and at days 2, 4 (±1), and 7, and at weeks 2, 3, 4, 5, 6, 9, and 12. Fluorescein angiography, optical coherence tomography (OCT), and wide-angle color fundus imaging were evaluated before the study and at weeks 3, 6, and 12. Finally, electroretinal regography (ERG) was performed before drug administration and at weeks 6 and 12, prior to final sacrifice.

[0151] rAAV at 2.8×10 10 Or 1.5×10 11 Intravitreal administration of a dose of vg / eye up to 12 weeks post-injection was well tolerated in cynomolgus monkeys, as supported by mortality, clinical observation, ophthalmological, clinicopathological, macroscopic pathological, and organ weight parameters. No intraocular inflammation, rAAV-related changes in IOP values ​​or ocular structures as identified by fluorescein angiography or anterior and posterior segment OCT, or therapeutically relevant effects on retinal function as identified by ERG assessment were observed at any time point.

[0152] At rAAV of 2.8×10 10 Or 1.5×10 11Intravitreal administration of vg / eye up to day 80 showed no definitive therapeutic effect of rAAV on clinicopathological parameters in either sex. Minor fluctuations in hematological and clinical chemistry findings are likely attributable to methylprednisolone administration and are not considered to be related to rAAV treatment.

[0153] Overall, rAAV levels are as high as 1.5 × 10⁻⁶ in non-human primates. 11 A single intravitreal dose of vg / eye was well tolerated without any treatment-related effects or changes in ocular structure or function.

[0154] While the materials and methods of the present invention have been described according to preferred embodiments, variations may be applied to the methods described herein without departing from the concept, spirit, and scope of the invention, as will be apparent to those skilled in the art. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention.

Claims

1. A recombinant adeno-associated virus (rAAV) comprising: (i) a variant AAV capsid protein comprising a heteropeptide of 7, 8, 9, 10, or 11 amino acids covalently inserted into the GH ring of the capsid protein relative to the corresponding parental AAV capsid protein, wherein the peptide insertion fragment comprises the amino acid sequence ISDQTKH (SEQ ID NO:1), and (ii) a heteronucleic acid comprising a nucleotide sequence encoding a complement regulatory factor H (CFH) protein or a fragment thereof.

2. The rAAV according to claim 1, wherein the insert peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino terminus and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1), preferably wherein the insert peptide is LAISDQTKHA (SEQ ID NO:2).

3. The rAAV according to claim 1 or 2, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO:47), or at a corresponding position in the capsid protein of another AAV serotype.

4. The rAAV according to any one of claims 1 to 3, wherein the capsid protein comprises relative to AAV2 (SEQ ID NO: 1). One or more amino acid substitutions in VP1 of NO:47), or one or more corresponding substitutions in the capsid protein of another AAV serotype, preferably one or more of the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L and L735Q, more preferably P34A amino acid substitution.

5. The rAAV of claim 4, wherein the capsid protein comprises a P34A amino acid substitution relative to VP1 of AAV2, and comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% identity with the entire length of the amino acid sequence shown in SEQ ID NO:42, preferably wherein the capsid protein comprises the amino acid sequence shown in SEQ ID NO:

42.

6. The rAAV according to any one of claims 1 to 5, wherein the rAAV exhibits increased infectivity to retinal cells, preferably at least 2-fold increased infectivity, compared to the infectivity of AAV containing the corresponding parental AAV capsid protein.

7. The rAAV according to any one of claims 1 to 6, wherein the variant AAV capsid protein comprises an amino acid sequence having 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

42.

8. The rAAV according to any one of claims 1 to 7, wherein the rAAV comprises a heterologous acid from 5' to 3' comprising: (a) an inverted terminal repeat, (b) a promoter, (c) a nucleotide sequence encoding a CFH protein or a fragment thereof, (d) a polyadenylated sequence and / or a WPRE sequence, and (e) an inverted terminal repeat.

9. The rAAV of claim 8, wherein the reverse end repetition in the rAAV is an AAV2 reverse end repetition.

10. The rAAV according to any one of claims 1 to 9, wherein the CFH protein or a fragment thereof comprises one or more short concordant repeats (SCRs).

11. The rAAV of claim 10, wherein the CFH protein fragment comprises the following: SCRs: 1, 2, 3, 4, 19, 20 and one or more of SCRs 7, 17 and / or 18, and optionally a leader sequence and one or more connector sequences.

12. The rAAV of claim 10, wherein the CFH protein fragment comprises the following: SCRs: 1, 2, 3, 4, 19, 20 and one or more of SCRs 7, 17 and / or 18 and one or more of SCRs 5, SCR 6, SCR 8, SCR 16, and optionally a leader sequence and one or more connector sequences.

13. The rAAV according to any one of claims 11-13, wherein the CFH protein fragment lacks at least SCR5, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15 and / or SCR16.

14. The rAAV according to any one of claims 10 to 13, wherein the CFH protein fragment is composed of an SCR domain selected from one or more of the following: (a) SCR1, 2, 3, 4, 7 and 19-20; (b) SCR1-4, 6, 7 and 19-20; (c) SCR1-4, 7, 8 and 19-20; (d) SCR1-4, 6, 7, 8 and 19-20; (e) SCR1-4, 17, 19-20; (f) SCR1-4 and l8-20; (g) SCR1-4 and 17-20; (h) SCR1-4, 7 and 18-20; (i) SCR1-4, 6, 7 and 18-20; (j) SCR1-4, 7, 8 and 18-20; (k) SCR1-4, 6-8 and 18-20, (1) SCR1-4, 7 and 17-20; (m) SCR1-4, 6, 7 and 17-20; (n) SCR1-4, 7, 8 and 17-20; or (o) SCR1-4, 6-8 and 17-20, And optionally a leader sequence and one or more connector sequences.

15. The rAAV according to any one of claims 10 to 14, wherein the CFH protein fragment comprises SCR1-4, 6-8 and 17-20, preferably wherein the CFH protein fragment does not comprise SCR5 and SCR9-16.

16. The rAAV according to any one of claims 10 to 15, wherein the CFH protein fragment comprises a linker of at least 1 to about 18 amino acids located between one or more SCRs.

17. The rAAV according to any one of claims 10 to 16, wherein the CFH protein fragment comprises SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-(SCR6-(L4'))-SCR7-(L5)-(SCR8-(L5'))-(SCR16-(L5"))-(SCR17-(L5"'))-(SCR18-(L5""))-SCR19-(L6)-SCR20, wherein () indicates optional components, "L" indicates a linker, and L1, L2, L3, L4, L4', L5, L5', L5", L5'", L5"" and L6 may each be absent or independently selected from an amino acid sequence of about 1 to about 12 to about 18 amino acids.

18. The rAAV according to any one of claims 1 to 17, wherein the CFH protein fragment comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

34.

19. The rAAV according to any one of claims 1 to 18, wherein the CFH protein fragment comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

34.

20. The rAAV according to any one of claims 1 to 19, wherein the CFH protein fragment comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

34.

21. The rAAV according to any one of claims 1 to 20, wherein the CFH protein fragment comprises an amino acid sequence having 100% identity with the amino acid sequence shown in SEQ ID NO:

34.

22. The rAAV according to any one of claims 10 to 21, wherein the CFH protein fragment comprises at least one glycosylation site in one or more SCRs.

23. The rAAV of claim 22, wherein the glycosylation site is modified into one or more of SCR1, SCR2, SCR3, SCR4, SCR17, SCR18, SCR19 and / or SCR20.

24. The rAAV of claim 23, wherein the glycosylation site is modified into one or more of SCR17 and / or SCR18.

25. The rAAV of claim 25, wherein the glycosylation sites are modified into SCR17 and SCR18.

26. The rAAV according to any one of claims 22 to 25, wherein the CFH protein fragment comprises SCR1-4, 6-8 and 17-20, and wherein the glycosylation site is modified into SCR17 and SCR18, preferably wherein the CFH protein fragment does not contain SCR5 and SCR9-16.

27. The rAAV according to any one of claims 22 to 26, wherein the CFH protein fragment comprises an amino acid sequence having 100% sequence identity with the sequence shown in SEQ ID NO: 34, and wherein the glycosylation site is modified into SCR17 and SCR18.

28. The rAAV according to any one of claims 8 to 27, wherein the promoter is a ubiquitous promoter.

29. The rAAV according to any one of claims 8 to 27, wherein the promoter is a tissue-specific promoter.

30. The rAAV of claim 28, wherein the promoter is a CAG promoter.

31. The rAAV according to any one of claims 8 to 30, wherein the rAAV comprises a heterologous nucleic acid, the heterologous nucleic acid comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO:43-46.

32. The rAAV according to any one of claims 8 to 31, wherein the rAAV comprises a heterologous nucleic acid, the heterologous nucleic acid comprising a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO:43-46.

33. The rAAV according to any one of claims 8 to 32, wherein the rAAV comprises a heterologous nucleic acid, the heterologous nucleic acid comprising a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO:43-46.

34. The rAAV according to any one of claims 8 to 33, wherein the rAAV comprises a heterologous nucleic acid, the heterologous nucleic acid comprising the nucleotide sequence shown in any one of SEQ ID NO:43-46.

35. A host cell comprising rAAV according to any one of claims 8 to 34.

36. A pharmaceutical composition comprising rAAV according to any one of claims 8 to 34 and a pharmaceutically acceptable carrier, diluent, excipient or buffer.

37. A method for treating dry age-related macular degeneration (dry AMD) in a subject with this need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 8 to 34 or the pharmaceutical composition according to claim 36.

38. The method of claim 37, wherein the rAAV or pharmaceutical composition is administered to the subject via periocular, intravitreal, suprachoroidal, or subretinal administration in a dose of approximately 10 8 One vector genome (vg) / eye to approximately 10 13 vg / eye, preferably about 6×10 9 vg / eye to approximately 6×10 10 A dose of vg / eye, or approximately 1 x 10 8 vg / eye, approximately 2x10 8 vg / eye, approximately 3x10 8 vg / eye, approximately 4x10 8 vg / eye, approximately 5x10 8 vg / eye, approx. 6x10 8 vg / eye, approximately 7x10 8 vg / eye, approximately 8x10 8 vg / eye, approx. 10 8 vg / eye, approximately 1x10 9 vg / eye, 2x10 9 vg / eye, approximately 3x10 9 vg / eye, approximately 4x10 9 vg / eye, approximately 5x10 9 vg / eye, approx. 6x10 9 vg / eye, approximately 7x10 9 vg / eye, approximately 8x10 9 vg / eye, approx. 9x10 9 vg / eye, approximately 1x10 10 vg / eye, approximately 2x10 10 vg / eye, approximately 3x10 10 vg / eye, approximately 4x10 10 vg / eye, approximately 5x10 10 vg / eye, approx. 6x10 10 vg / eye, approximately 7x10 10 vg / eye, approximately 8x10 10 vg / eye, approx. 9x10 10 vg / eye, approximately 1x10 11 vg / eye, approximately 2x10 11 vg / eye, approximately 3x10 11 vg / eye, approximately 4x10 11 vg / eye, approximately 5x10 11 vg / eye, approximately 6x10 11 vg / eye, approx. 7x10 11 vg / eye, approximately 8x10 11 vg / eye, approx. 9x10 11 vg / eye or approximately 1x10 12 The dose is vg / eye.

39. The method of claim 38, wherein the rAAV or pharmaceutical composition is administered intravitreally at a dose of about 1 x 10⁻⁶. 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

40. A method for treating geographic atrophy (GA) in a subject with this need, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 8 to 34 or the pharmaceutical composition according to claim 36.

41. The method of claim 40, wherein the rAAV or pharmaceutical composition is administered to the subject via periocular, intravitreal, suprachoroidal, or subretinal administration in a dose of approximately 10 8 One vector genome (vg) / eye to approximately 10 13 vg / eye, preferably about 6×10 9 vg / eye to approximately 6×10 10 A dose of vg / eye, or approximately 1 x 10 8 vg / eye, approximately 2x10 8 vg / eye, approximately 3x10 8 vg / eye, approximately 4x10 8 vg / eye, approximately 5x10 8 vg / eye, approximately 6x10 8 vg / eye, approx. 7x10 8 vg / eye, approximately 8x10 8 vg / eye, approx. 10 8 vg / eye, approximately 1x10 9 vg / eye, 2x10 9 vg / eye, approximately 3x10 9 vg / eye, approximately 4x10 9 vg / eye, approximately 5x10 9 vg / eye, approximately 6x10 9 vg / eye, approx. 7x10 9 vg / eye, approximately 8x10 9 vg / eye, approx. 9x10 9 vg / eye, approximately 1x10 10 vg / eye, approximately 2x10 10 vg / eye, approximately 3x10 10 vg / eye, approximately 4x10 10 vg / eye, approximately 5x10 10 vg / eye, approximately 6x10 10 vg / eye, approx. 7x10 10 vg / eye, approximately 8x10 10 vg / eye, approx. 9x10 10 vg / eye, approximately 1x10 11 vg / eye, approximately 2x10 11 vg / eye, approximately 3x10 11 vg / eye, approximately 4x10 11 vg / eye, approximately 5x10 11 vg / eye, approximately 6x10 11 vg / eye, approx. 7x10 11 vg / eye, approximately 8x10 11 vg / eye, approx. 9x10 11 vg / eye or approximately 1x10 12 The dose is vg / eye.

42. The method of claim 41, wherein the rAAV or pharmaceutical composition is administered intravitreally at a dose of about 1 x 10⁻⁶. 9 vg / eye to approximately 1 x 10 10 A dose of vg / eye was administered to the subject.

43. A method for delivering rAAV according to any one of claims 8 to 34 or a pharmaceutical composition according to claim 36 to the eye of a subject, wherein the rAAV or pharmaceutical composition is administered to the subject via periocular, intravitreal, suprachoroidal, or subretinal administration.

44. The method of claim 43, wherein the rAAV or pharmaceutical composition is administered to the subject's eye via intravitreal administration.

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