VARIANTS OF AAV FOR THE TREATMENT OF COMPLEMENT DISORDERS

Description

1 / 102 “AAV VARIANTS FOR TREATMENT OF COMPLEMENT DISORDERS DESCRIPTIVE REPORT REFERENCE TO RELATED REQUESTS

[0001] This Application claims the benefit of the Patent Applications U.S. Provisional Serial Numbers 63 / 494,925, filed April 7, 2023, and 63 / 586,227, filed September 28, 2023, the full contents of which are incorporated herein by reference. Sending a list of sequences via EFS-Web

[0002] A machine-readable XML file, titled “0904005022-WO-Sequence-Listing”, created on April 2, 2024, with a file size of approximately 87,900 bytes, contains the sequence listing for this application and is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Several human diseases are caused by complement dysregulation, resulting in autologous tissue injury mediated by complement. Complement dysregulation can arise from somatic or germline mutations in complement-regulating or regulatory genes, such that these regulators no longer function normally. In particular, there are common and rare human diseases that are caused by excessive complement activation resulting from dysregulation of the complement activation cascade.

[0004] Current therapeutic approaches focus on the development of reagents such as monoclonal antibodies (mAbs), Petition 870250109947, dated 01 / 12 / 2025, page 5 / 129 2 / 102 peptides or other small molecules that bind to and block specific components of the alternative or terminal complement pathway. A clinically validated example is Eculizumab, a humanized mAb against complement C5 that has been approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS). Other described approaches include mAbs against factor B (fB), factor D (fD), or properdin (fP), and a cyclic peptide that binds to and inhibits C3. A limitation of these approaches is that they require repeated intravenous (IV) doses, which are inconvenient for patients. Furthermore, because these treatments block either the alternative or terminal pathway, they risk compromising host defense. In fact, patients on Eculizumab therapy need to be vaccinated against bacterial strains that cause lethal meningitis and also receive prophylactic antibiotic therapy before being treated with the approved mAb medication.

[0005] In other approaches, recombinant regulatory proteins, such as soluble DAF, CR1, CRIg, and proteins comprising minimal domains of fluid-phase regulatory fH (short consensus repeat [SCR] 1-5 N-terminal and SCR 19-20 C-terminal) or fH-CR2 fusion proteins (TT30) have been tested. However, large-scale heterologous expression of these proteins as therapeutic drugs requires significant effort, and animal studies have shown that their in vivo clearance rate after administration is rapid, making these therapeutic strategies cumbersome and less practical, as multiple and frequent administrations of these protein drugs would be necessary.

[0006] The need for compositions useful for treating complement-mediated diseases with greater and longer-lasting efficacy remains in the field. SUMMARY OF THE INVENTION Petition 870250109947, dated 01 / 12 / 2025, page 6 / 129 3 / 102

[0007] Recombinant AAV virions (rAAV) comprising an AAV variant capsid sequence encapsulating a heterologous nucleic acid comprising an operatively engineered human complement regulatory factor H (fH) gene linked to an expression control sequence are described herein, wherein the human fH gene (hfH) encodes a variant of the soluble hfH protein that retains complement regulatory function, wherein said fH variant comprises short consensus repeats (SCRs) 1, 2, 3, 4, 19 and 20. In some embodiments, the rAAV comprises a heterologous nucleic acid comprising an hfH gene encoding a variant of the soluble hfH protein that retains complement regulatory function, wherein said fH variant comprises or consists of or consists essentially of SCRs, wherein the SCRs are selected from the group consisting of SCR1, SCR2, SCR3, SCR4, SCR6, SCR7, SCR8, SCR17, SCR18, SCRSCR19 and SCR20.

[0008] In related aspects, an rAAV virion comprising an AAV variant capsid sequence encapsulating a heterologous nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding an engineered hfH variant comprising a leader sequence and human complement receptor SCRs, wherein the SCRs are selected from the group consisting of: (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) SCR14 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) SCR14, 6, 7, 8 and 17-20. Optionally, at least one glycosylation site is designed in at least one of the SCRs.

[0009] The rAAV variant AAV capsid protein comprises a capsid protein comprising a peptide insertion of about 7 to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH loop of the capsid protein, preferably in a Petition 870250109947, dated 01 / 12 / 2025, page 7 / 129 4 / 102 region exposed to the surface of the GH loop, relative to a corresponding parental AAV capsid protein, 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 spacers (Y1-Y3) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1), wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In some embodiments, the peptide insert comprises 2 amino acid spacers at the N-terminus and 1 amino acid spacer at the C-terminus. In particularly preferred embodiments, the peptide insert comprises, consists essentially of, or consists of the amino acid sequence LAISDQTKHA (SEQ ID NO:2).In certain preferred embodiments, the insertion site is between amino acids 587 and 588 of the AAV2 VP1 or between amino acids 588 and 589 of AAV2 or the corresponding positions 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 AAV2 VP1 capsid 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 AAV2 VP1 capsid or the corresponding substitution in another AAV serotype.

[00010] In other embodiments, a method is provided for administering a heterologous nucleic acid comprising a nucleotide sequence encoding a variant of the soluble hfH protein, as described in this document, to a mammalian subject, comprising the method administering to the mammal an effective amount of a rAAV, as described in this document, or a pharmaceutical composition comprising it, preferably wherein the rAAV or the pharmaceutical composition is administered by intravitreal injection. In some aspects, the heterologous nucleic acid is administered to a retinal cell of the subject, for example, a cell Petition 870250109947, dated 01 / 12 / 2025, page 8 / 129 5 / 102 photoreceptor (e.g., rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g., a Müllerian glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, and / or a retinal pigment epithelium (RPE) cell from the subject.

[00011] In some embodiments, detectable plasma levels of the hfH variant are present in an individual for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, or at least 6 months after administration of rAAV to the individual. In particularly preferred embodiments, rAAV is administered to an individual by intravitreal injection.

[00012] In other embodiments, a pharmaceutical composition is provided comprising a rAAV as described herein and a pharmaceutically acceptable excipient.

[00013] In other respects, a method is provided for treating a complement-related disorder by administering to the individual a rAAV as described herein or a pharmaceutical composition comprising the rAAV. Complement-related disorders that may be treated include, but are not limited to, membranoproliferative glomerulonephritis, atypical hemolytic-uremic syndrome (aHUS), age-related macular degeneration (AMD), geographic atrophy secondary to microangiopathic hemolytic anemia AMD, thrombocytopenia, acute renal failure, paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or bacterial infections caused by the introduction of bacterial pathogens.

[00014] In a further aspect, a method is provided for treating dry age-related macular degeneration (DAM) (e.g., advanced-stage dry AMD) in an individual in need thereof, by administering to the individual an effective amount of a rAAV, as described herein, or a pharmaceutical composition that Petition 870250109947, dated 01 / 12 / 2025, page 9 / 129 6 / 102 comprises rAAV. In related aspects, rAAV or the pharmaceutical composition is administered to an individual for the treatment of geographic atrophy secondary to AMD. Preferably, rAAV or the pharmaceutical composition is administered to the individual by intravitreal injection. DESCRIPTION OF THE DRAWINGS

[00015] FIG. 1 is a schematic of a transgenic cassette comprising terminal repeats (ITRs) of 5' and 3' AAVs from the AAV2 genome, a polyadenylation signal (late polyA of SV40) and a nucleotide sequence encoding a codon-optimized human fH variant containing SCRs 1-4, 6-8 and 17-20 (“miniCFH”), operatively linked to a CAG promoter.

[00016] FIG. 2A illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Fig. 2B). Fig. 2C illustrates C3b binding: pre-incubation with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Fig. 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds and retains heparin binding activity. Fig.Figure 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells. The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the complement cascade. Petition 870250109947, dated 01 / 12 / 2025, page 10 / 129 7 / 102 alternative. These smaller fragments resulting from C3b degradation are observed by Western blot. Figure 2F illustrates complement inhibition (Wieslab® Complement System Alternative Pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of membrane attack complex (MAC) formation similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected. Figure 2B illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Figure 2B). Figure 2C illustrates C3b binding: pre-incubation with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Figure 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds and retains heparin binding activity. Fig. 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells.The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the alternative complement cascade. These smaller fragments resulting from C3b degradation are observed by Western blot. Figure 2F illustrates complement inhibition (Wieslab® Complement System alternative pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of complement formation. Petition 870250109947, dated 01 / 12 / 2025, page 11 / 129 8 / 102 membrane attack complex (MAC) similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected. Figure 2C illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Figure 2B). Figure 2C illustrates C3b binding: pre-incubation with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Figure 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds and retains heparin binding activity. Fig. 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells.The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the alternative complement cascade. These smaller fragments resulting from C3b degradation are observed by Western blot. Figure 2F illustrates complement inhibition (Wieslab® Complement System alternative pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of membrane attack complex (MAC) formation similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected. Figure 2D illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid. Petition 870250109947, dated 01 / 12 / 2025, page 12 / 129 9 / 102 comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Fig. 2B). Fig. 2C illustrates C3b binding: pre-incubation with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Fig. 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds and retains heparin binding activity. Fig. 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells. The C3 protein is cleaved into C3a and C3b fragments.The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the alternative complement cascade. These smaller fragments resulting from C3b degradation are observed by Western blot. Figure 2F illustrates complement inhibition (Wieslab® Complement System alternative pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of membrane attack complex (MAC) formation similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected. Figure 2E illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Figure 2B). Figure 2C illustrates the binding of C3b: pre-incubation. Petition 870250109947, dated 01 / 12 / 2025, page 13 / 129 10 / 102 with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Fig. 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds and retains heparin-binding activity. Fig. 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells. The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the alternative complement cascade. These smaller fragments resulting from the degradation of C3b are observed by Western blot.Figure 2F illustrates complement inhibition (Wieslab® Complement System Alternative Pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of membrane attack complex (MAC) formation similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected. Figure 2F illustrates the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid comprising the transgenic cassette illustrated in Figure 1. Figures 2A-2B illustrate the dose-dependent expression of miniCFH and full-length CFH in protein lysates from supernatants of plasmid-transfected HEK293T cells by ELISA (Figure 2A) and western blot (Figure 2B). Figure 2C illustrates C3b binding: pre-incubation with CFH-blocking antibodies, not with control antibodies or sham incubation, reduces the signal of CFH and miniCFH, indicating that miniCFH binds directly to C3b. Figure 2D illustrates heparin binding: pre-incubation with CFH-blocking antibodies reduces the signal of recombinant CFH and miniCFH, indicating that miniCFH binds Petition 870250109947, dated 01 / 12 / 2025, page 14 / 129 11 / 102 and retains heparin-binding activity. Fig. 2E shows the results of a C3b cleavage assay performed to confirm the functional activity of miniCFH expressed in HEK293T cells. The C3 protein is cleaved into C3a and C3b fragments. The C3b fragment can form C3 convertase upon binding to factor B or can be degraded by complement factor 1 (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments, causing disruption in the alternative complement cascade. These smaller fragments resulting from C3b degradation are observed by Western blot. Figure 2F illustrates complement inhibition (Wieslab® Complement System alternative pathway assay), demonstrating that the supernatant from cells transfected with AAV plasmid cassette resulted in inhibition of membrane attack complex (MAC) formation similar to full-length CFH controls and anti-C5 antibodies. NT = not transfected.

[00017] FIG. 3A illustrates the expression, activity, and function of miniCHF in human RPE cells. Retinal pigment epithelial cells (RPEs) derived from IPSCs were transduced with an rAAV comprising (i) a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1. RPEs were transduced at different multiplicities of infection (MOI; vector genomes (vg) per cell). Four days later, seven days after transduction, the medium was collected and analyzed for secreted miniCHF expression by ELISA (Fig. 3A) and complement inhibition activity by the alternative pathway complement system assay (Fig. 3B). Complement inhibition was also assessed by ICC (Fig. 3C).Alternative complement was activated in iPSC-derived RPE on day 6 post-transduction by adding NHS at a final concentration [1%] and Zymosan at a final concentration [0.5 mg / ml NHS]. 24 hours later, 7 days post-transduction, cells were fixed and stained with mouse anti-human MAC IgG2aKappa primary antibody (Abcam). Petition 870250109947, dated 01 / 12 / 2025, page 15 / 129 12 / 102 cat#59835) or mouse IgG2aKappa isotype control (Invitrogen cat#14-4724-82) and stained with goat A488 anti-mouse secondary antibody (Invitrogen cat#Al1001) and DRAQ5 (ThermoFisher cat#62251) for nuclei. Images were obtained using a fluorescence microscope; miniCFH expressed by cells in all MOIs prevented MAC formation in cultures. An anti-C5 antibody was used as a positive control for complement inhibition and prevention of MAC formation. NT = non-transduced Figure 3B illustrates the expression, activity, and function of miniCHF in human RPE cells. Retinal pigment epithelial cells (RPEs) derived from IPSCs were transduced with an rAAV comprising (i) a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1. RPEs were transduced at different multiplicities of infection (MOI; vector genomes (vg) per cell). Four days later, seven days after transduction, the medium was collected and analyzed for secreted miniCHF expression by ELISA (Fig. 3A) and complement inhibition activity by the alternative pathway complement system assay (Fig. 3B). Complement inhibition was also assessed by ICC (Fig. 3C). The alternative complement was activated in iPSC-derived RPE on day 6 post-transduction by adding NHS to the culture medium at a final concentration of [1%] and Zymosan at a final concentration of [0.5 mg / ml NHS].Twenty-four hours later, seven days after transduction, cells were fixed and stained with mouse anti-human MAC IgG2aKappa primary antibody (Abcam cat#59835) or mouse isotype control IgG2aKappa (Invitrogen cat#14-4724-82) and stained with goat anti-mouse A488 secondary antibody (Invitrogen cat#Al1001) and DRAQ5 (ThermoFisher cat#62251) for nuclei. Images were obtained using a fluorescence microscope; miniCFH expressed by cells in all MOIs prevented MAC formation in cultures. Petition 870250109947, dated 01 / 12 / 2025, p. 16 / 129 13 / 102 anti-C5 antibody was used as a positive control for complement inhibition and prevention of MAC formation. NT = not transduced Figure 3C illustrates the expression, activity, and function of miniCHF in human RPE cells. Retinal pigment epithelial cells (RPEs) derived from IPSCs were transduced with an rAAV comprising (i) a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1. RPEs were transduced at different multiplicities of infection (MOI; vector genomes (vg) per cell). Four days later, seven days after transduction, the medium was collected and analyzed for secreted miniCHF expression by ELISA (Fig. 3A) and complement inhibition activity by the alternative pathway complement system assay (Fig. 3B). Complement inhibition was also assessed by ICC (Fig. 3C). The alternative complement was activated in iPSC-derived RPE on day 6 post-transduction by adding NHS to the culture medium at a final concentration of [1%] and Zymosan at a final concentration of [0.5 mg / ml NHS].Twenty-four hours later, seven days after transduction, cells were fixed and stained with mouse anti-human MAC IgG2aKappa primary antibody (Abcam cat#59835) or mouse isotype control IgG2aKappa (Invitrogen cat#14-4724-82) and stained with goat anti-mouse A488 secondary antibody (Invitrogen cat#Al1001) and DRAQ5 (ThermoFisher cat#62251) for nuclei. Images were obtained using a fluorescence microscope; miniCFH expressed by cells in all MOIs prevented MAC formation in cultures. An anti-C5 antibody was used as a positive control for complement inhibition and prevention of MAC formation. NT = not transduced.

[00018] FIG. 4 illustrates the LCMS quantification of short CFH (sCFH) concentrations in aqueous humor (AH) samples from NHPs after intravitreal (bilateral) administration of the specified dose of rAAV. Petition 870250109947, dated 01 / 12 / 2025, p. 17 / 129 14 / 102 comprising (i) a variant AAV capsid protein comprising the amino acid sequence of SEQ TD NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1 for the NHPs.

[00019] FIG. 5 illustrates the expression of sCFH RNA (by in situ hybridization analysis) in untransfected HEK 293T cells and in HEK 293T cells after transfection with rAAV, comprising (i) an AAV variant capsid protein comprising the amino acid sequence of SEQ ID NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1.

[00020] FIG. 6 illustrates the results of in situ hybridization analysis of non-human primate ocular tissue after intravitreal (bilateral) administration of the specified dose of rAAV, comprising (i) an AAV variant capsid protein comprising the amino acid sequence of SEQ ID NO:42 and (ii) a nucleic acid comprising the expression cassette illustrated in Figure 1.

[00021] FIG. 7A. is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant showed no band, serving as a negative control. The full-length recombinant CFH (250 kDa, band 5) served as a positive control. The arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of the CFH protein in the supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Ap<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples.Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg of DNA / well, 15 µIU of supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the control. Petition 870250109947, dated 01 / 12 / 2025, page 18 / 129 15 / 102 negative (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows. After adding the miniCFH transfection supernatant to the reaction, an increase in cleavage products (Lane 2) was observed compared to the negative control supernatants (Lane 3). The positive control reaction with the three recombinant proteins (CFH, CFI, and C3b) is in Lane 4, and the negative controls are in Lanes 5 to 7. Figure 7E shows the miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies.Figure 7F shows miniCFH in the supernatants after transfection bound to heparin, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. NT: not transfected. Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. Figure 7B is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant showed no band, serving as a negative control. The full-length recombinant CFH (250 kDa, band 5) served as a positive control. The arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of the CFH protein in the supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Ap<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples. Petition 870250109947, dated 01 / 12 / 2025, page 19 / 129 16 / 102 Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg DNA / well, 15 µIU supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the negative control (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows. After adding the miniCFH transfection supernatant to the reaction, an increase in cleavage products (Lane 2) was observed compared to the negative control supernatants (Lane 3). The positive control reaction with the three recombinant proteins (CFH, CFI and C3b) is in Range 4 and the negative controls are in Ranges 5 to 7.Figure 7E shows miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. Figure 7F shows miniCFH in the supernatants after transfection bound to heparin, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. NT: not transfected. Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. Figure 7C is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant showed no band, serving as a negative control. The full-length recombinant CFH (250 kDa, band 5) served as a positive control. The arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of the CFH protein in Petition 870250109947, dated 01 / 12 / 2025, page 20 / 129 17 / 102 Supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Λρ<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples. Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg DNA / well, 15 µIU supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the negative control (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows.After adding the supernatant from the miniCFH transfection to the reaction, an increase in cleavage products (Range 2) was observed compared to the negative control supernatants (Range 3). The positive control reaction with the three recombinant proteins (CFH, CFI, and C3b) is in Range 4, and the negative controls are in Ranges 5 to 7. Figure 7E shows the miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. Figure 7F shows the miniCFH in the supernatants after transfection bound to heparin, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. NT: not transfected. Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. Figure 7D is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant did not show a band, serving as a negative control. The recombinant CFH of full length (250 Petition 870250109947, dated 01 / 12 / 2025, page 21 / 129 18 / 102 kDa, band 5) served as a positive control. Arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of CFH protein in the supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Ap<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples. Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg DNA / well, 15 µIU supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the negative control (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image.The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows. After adding the miniCFH transfection supernatant to the reaction, an increase in cleavage products (Range 2) was observed compared to the negative control supernatants (Range 3). The positive control reaction with the three recombinant proteins (CFH, CFI, and C3b) is in Range 4, and the negative controls are in Ranges 5 to 7. Figure 7E shows the miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. Figure 7F shows miniCFH in the supernatants after transfection bound to heparin, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. NT: not transfected.Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. Petition 870250109947, dated 01 / 12 / 2025, p. 22 / 129 19 / 102 Figure 7E is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant showed no band, serving as a negative control. The full-length recombinant CFH (250 kDa, band 5) served as a positive control. The arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of the CFH protein in the supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Ap<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples.Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg DNA / well, 15 µIU supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the negative control (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows. After adding the miniCFH transfection supernatant to the reaction, an increase in cleavage products (Lane 2) was observed compared to the negative control supernatants (Lane 3). The positive control reaction with the three recombinant proteins (CFH, CFI and C3b) is in Range 4 and the negative controls are in Ranges 5 to 7.Figure 7E shows miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. Figure 7F shows miniCFH in the supernatants after transfection bound to heparin, which was eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01. Petition 870250109947, dated 01 / 12 / 2025, page 23 / 129 20 / 102 for anti-CFH antibodies. NT: not transfected. Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. Figure 7F is a Western blot showing the detection of the CFH protein after transfection with the CFH or miniCFH constructs. The analyzed NT cell supernatant showed no band, serving as a negative control. The full-length recombinant CFH (250 kDa, band 5) served as a positive control. The arrows indicate the theoretical molecular weight of miniCFH (80 kDa) and full-length CFH (139 kDa). The molecular weight marker is shown on the left. Figure 7B shows the concentrations of the CFH protein in the supernatants of HEK293T cells transfected with 0.125 or 0.5 µg of the CFH or miniCFH construct, determined by ELISA for human Factor H. ****p<0.0001 compared to the negative control; Ap<0.0003 compared between the 0.125 µg DNA and 0.5 µg DNA samples.Figure 7C shows the supernatants of HEK cells transfected with miniCFH DNA (0.5 µg DNA / well, 15 µIU supernatant), which led to complement inhibition comparable to the positive control eculizumab (6 µg), **p = 0.0028, ***p = 0.0002 compared to the negative control (NT). Figure 7D is a 6-reaction Western blot, including several components identified in the legend at the top of the image. The purified recombinant protein components (purified CFH, CFI, and C3b) and the supernatants after transfection (CFH supernatant) are shown. Cleavage products are highlighted with arrows. After adding the miniCFH transfection supernatant to the reaction, an increase in cleavage products (Lane 2) was observed compared to the negative control supernatants (Lane 3). The positive control reaction with the three recombinant proteins (CFH, CFI and C3b) is in Range 4 and the negative controls are in Ranges 5 to 7.Figure 7E shows miniCFH in the supernatants after transfection bound to C3b, which was eliminated after incubation with an antibody. Petition 870250109947, dated 01 / 12 / 2025, page 24 / 129 21 / 102 anti-CFH. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. Figure 7F shows miniCFH in the supernatants after transfection bound to heparin and eliminated after incubation with an anti-CFH antibody. *p = 0.05 for anti-CFH antibodies **p = 0.01 for anti-CFH antibodies. NT: not transfected. Error bars + standard deviation; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test.

[00022] FIG. 8 illustrates the transduction of iPSC-RPE cells resulting in the expression of the miniCFH protein. CFH concentration [nM] in the supernatant of RPE cells transduced with rAAV (comprising a capsid protein comprising the amino acid sequence established in SEQ ID NO:42 and a heterologous nucleic acid encoding a shortened form of complement factor H (“miniCFH”) having the amino acid sequence established in SEQ m NO:34) at different MOIs, which showed a dose response in CFH expression. Any signal beyond that seen in NT cells would then correlate with miniCFH expressed from rAAV. MOI = multiplicity of infection; NT = non-transduced. Error bars ± standard deviation; n = 4 replicates of the experiment. Significance relative to NT, **p = 0.0071 and ***p = 0.0007. Significance: Ap = 0.0477 for comparison between MOI 5,000 and MOI 50,000. Statistics: One-way ANOVA, Tukey post-hoc test.

[00023] FIG. 9A illustrates the inhibition of the alternative complement pathway by miniCFH expressed by RPE cells derived from iPSCs transduced with rAAV comprising a capsid protein comprising 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, operatively linked to a CAG promoter. Cell supernatants from rAAV-transduced RPE cells were analyzed for complement inhibitory activity by two methods: Wieslab assay (Fig. 9A) and soluble C5b-9 ELISA (Fig. 9B). Fig. 9A shows the inhibition of alternative complement activity. Petition 870250109947, dated 01 / 12 / 2025, page 25 / 129 22 / 102 resulting from the addition of the specified samples to a Wieslab assay with nominal human serum. The activity reading with the addition of NT supernatants is defined as 0% inhibition. A dose response was observed, with higher MOI doses of rAAV resulting in increased complement inhibition. Error bars ± standard deviation; n=3 replicates of the experiment. Significance relative to NT, unless otherwise indicated. *p = 0.0342, **p = 0.0026, ****p < 0.0001, AAp = 0.0078 for comparison between MOI 5,000 and MOI 50,000. Fig. 9B shows the concentration of soluble C5b-9 in samples with rAAV-transduced RPE supernatant at different MOIs, showing an inverted dose response. The concentration of soluble C5b-9 was nominalized, with C5b-9 concentration in NT supernatant = 1. Error bars ± standard deviation; significance with respect to NT, **p=0.0071 and ****p<0.0001. ANOVA, Tukey post-hoc test. NT, non-transduced; MOI, multiplicity of infection. Figure 9B illustrates the inhibition of the alternative complement pathway by miniCFH expressed by iPSC-derived RPE cells transduced with rAAV comprising a capsid protein comprising 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, operatively linked to a CAG promoter. Cellular supernatants from rAAV-transduced RPE were analyzed for complement inhibitory activity by two methods: Wieslab assay (Fig. 9A) and soluble C5b-9 ELISA (Fig. 9B). Fig. 9A shows the inhibition of alternative complement activity resulting from the addition of the specified samples to a Wieslab assay with nominal human serum. The activity reading with the addition of NT supernatants is defined as 0% inhibition. A dose response was observed, with higher MOI doses of rAAV resulting in increased complement inhibition.Error bars ± standard deviation; n=3 replicates of the experiment. Significance relative to the NT, unless otherwise indicated. Petition 870250109947, dated 01 / 12 / 2025, page 26 / 129 23 / 102 contrary. *p = 0.0342, **p = 0.0026, ****p < 0.0001, ΛΛρ = 0.0078 for comparison between MOI 5,000 and MOI 50,000. Fig. 9B shows the concentration of soluble C5b-9 in samples with RPE supernatant transduced with rAAV at different MOIs, showing a dose-reversed response. The concentration of soluble C5b-9 was nominalized, with C5b-9 concentration in NT supernatant = 1. Error bars ± standard deviation; significance relative to NT, **p=0.0071 and ****p<0.0001. ANOVA, Tukey post-hoc test. NT, non-transduced; MOI, multiplicity of infection.

[00024] FIG. 10 shows the acceleration of C3 convertase degradation activity by miniCFH expression. C3b cleavage products in purified C3b samples incubated with purified CFI and in purified supernatant or full-length CFH samples were detected by Western blot. Samples without CFI and CFH show no α-chain cleavage of C3b, while samples with either CFI or purified CFH show low levels of cleavage products. Incubation of C3b with CFI and miniCFH results in extensive cleavage, with a marked reduction in the α-chain and an increase in the abundance of cleavage products. MiniCFH from cell supernatants, as a substitute for purified CFH, recapitulates these effects. NT, non-transduced; MOI, multiplicity of infection. Representative blot, n=4 replicates of the experiment.

[00025] FIG. 11A shows that transduction of RPE with rAAV (comprising a capsid protein with the amino acid sequence defined in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence defined in SEQ ID NO:34, operably linked to a CAG promoter) prior to complement activation induction in cultures protected RPE from MAC deposition. Figure 11A shows immunocytochemical (ICC) analysis. Representative images, n = 4 experimental replicates. Scale bar: 200 gm. Figure 11B shows flow cytometry analysis. The percentage of Petition 870250109947, dated 01 / 12 / 2025, page 27 / 129 24 / 102 total RPE cells with MAC deposition in different MOIs showed a dose-dependent response. Error bars ± standard deviation; n = 4 experimental replicates. Significance relative to NT, ****p < 0.0001. Statistics: One-way ANOVA, Tukey post-hoc test. NT, non-transduced; MOI, multiplicity of infection. MAC = Membrane Attack Complex. Figure 11B shows that transduction of RPE with rAAV (comprising a capsid protein with the amino acid sequence defined in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence defined in SEQ ID NO:34, operably linked to a CAG promoter) prior to complement activation induction in cultures protected RPE from MAC deposition. Figure 11A shows immunocytochemistry (ICC) analysis. Representative images, n = 4 experimental replicates. Scale bar: 200 gm. Figure 11B shows flow cytometry analysis. The percentage of total RPE cells with MAC deposition at different MOIs showed a dose-dependent response. Error bars ± standard deviation; n = 4 experimental replicates. Significance relative to NT, ****p < 0.0001. Statistics: One-way ANOVA, Tukey post-hoc test. NT, non-transduced; MOI, multiplicity of infection. MAC = Membrane Attack Complex.

[00026] FIG. 12 shows the alternative complement inhibitory activity of rAAV (comprising a capsid protein comprising the amino acid sequence presented in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence presented in SEQ ID NO:34, operatively linked to a CAG promoter) in an in vitro RPE disease model. The concentration of iC3b in a supernatant of the rAAV-transduced RPE disease model showed a decrease compared to NT. The iC3b concentration was normalized, with the iC3b concentration in the supernatant Petition 870250109947, dated 01 / 12 / 2025, page 28 / 129 25 / 102 NT. NT, not transduced; MOT, multiplicity of infection; RPE, retinal pigment epithelial cells. Error bars ± standard deviation; n = 4 replicates of the experiment. Significance with respect to NT, **p = 0.0021 and ****p < 0.0001. Statistics; one-way ANOVA, post-hoc test of Tukey.

[00027] FIG. 13 shows the concentrations of miniCFH in the aqueous humor of non-human primates after intravitreal administration of a rAAV comprising a capsid protein 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, operatively linked to a CAG promoter. The expression of the miniCFH protein in the aqueous humor was measured by LC-MS. Fluid expression is shown in ng / mL. Data are summarized from 3-4 animals. Mean + standard deviation.

[00028] FIG. 14 shows miniCFH concentrations in retinal tissue and RPE / choroid in non-human primates after intravitreal administration of a rAAV comprising a capsid protein comprising 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, operably linked to a CAG promoter. miniCFH protein expression in aqueous humor (AH), retinal tissue (Ret), and RPE / choroid (RPE / C) of NHP eyes after rAAV IVT at (left) 5x10¹¹ vg / eye and (right) 1x10¹⁰ vg / eye measured by LC-MS at terminal necropsy time points. Data are summarized from 3-4 animals. Fluid expression is shown in ng / mL. Tissue expression is shown in ng / g. Mean plus standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[00029] Definitions

[00030] As used herein, the term “functional variant of fH” includes variants of fH that are characterized by having activity Petition 870250109947, dated 01 / 12 / 2025, page 29 / 129 26 / 102 complement regulator (cofactor activity) located in SCR1-4 and, optionally, a functional capacity for binding to C3b and GAG (located in SCR7 and SCR1 9-20 of wild type) characteristic of wild-type fH. In some embodiments, the modified fH variants exhibit more than 100% of the cofactor activity of wild-type fH and / or GAG binding capacity. In another embodiment, the modified fH variant exhibits less than about 95% to about 100% of the functional activity of wild-type fH. For example, an engineered fH variant may have at least 50% of the cofactor activity present in the functional wild-type fH and, more desirably, 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 designed fH variant may alternatively or additionally have at least 50% of the GAG-binding capacity of functional fH and, more desirably, 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 increase in circulating lifetime compared to hfH proteins are known in the art.

[00031] As used herein, when referring to SCR #-##, the domains include the periods and are the same as “SCR#, ... SCR##”. In certain embodiments, periods are used between the domains. For example, SCR1-4 refers to “SCR1, SCR2, SCR3 and SCR4” and is the same as “SCR1,2,3,4” or “SCR1 .2.3.4”. SCR19-20 refers to SCR19 and SCR20 and is the same as “SCR19. 20”. For example, “SCR6-8”, “SCR6. 7.8” and “SCR6,7,8” refer to the same domains.

[00032] The term “isolate” designates a biological material (cell, nucleic acid, or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in its natural state in a plant or animal is not Petition 870250109947, dated 01 / 12 / 2025, p. 30 / 129 27 / 102 is isolated; however, the same polynucleotide, separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated".

[00033] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not normally translated into an amino acid, it can be considered part of a coding region, but any flanking sequences, for example, promoters, ribosome binding sites, transcription terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by an initiation codon at the 5' end, which codes for the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which codes for the carboxyl terminus of the resulting polypeptide.Two or more coding regions may be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. It follows, therefore, that a single vector may contain only a single coding region or comprise two or more coding regions.

[00034] As used herein, the term “regulatory region” refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and loop-and-stem structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and a transcription termination sequence Petition 870250109947, dated 01 / 12 / 2025, p. 31 / 129 28 / 102 will generally be located 3' relative to the coding sequence.

[00035] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.

[00036] A polynucleotide encoding a gene product, for example, a polypeptide, may include a promoter and / or other transcription or translation control elements operatively associated with one or more coding regions. In an operable association, a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory regions so as to place the expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are “operatively associated” if induction of promoter function results in transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the promoter’s ability to direct gene product expression or interfere with the ability of the DNA template to be transcribed.Other elements of transcription control, besides a promoter, for example, enhancers, operators, repressors, and transcription termination signals, can also be operatively associated with a coding region to direct gene product expression.

[00037] “Transcriptional control sequences” refer to regulatory DNA sequences, such as promoters, enhancers, terminators, and the like, that provide the expression of a coding sequence in a host cell. A variety of transcriptional control regions are known to those skilled in the art. These include, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments of cytomegalovirus (the immediate initial promoter, together with intron-A), simian virus 40 (the initial promoter), and retroviruses (such as Rous sarcoma virus). Other regions of Petition 870250109947, dated 01 / 12 / 2025, page 32 / 129 29 / 102 transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., interferon- or interleukin-inducible promoters).

[00038] Similarly, a variety of translation control elements are known to those with common knowledge in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and picornavirus-derived elements (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[00039] The term “expression,” as used herein, refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes, without limitation, the transcription of the 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 an mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product may be a nucleic acid, for example, a messenger RNA produced by the transcription of a gene, or a polypeptide that is translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, for example, polyadenylation or splicing, or polypeptides with post-translational modifications, for example, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[00040] “Promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, Petition 870250109947, dated 01 / 12 / 2025, p. 33 / 129 30 / 102 a coding sequence is located 3' relative to a promoter sequence. Promoters may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types are commonly called “constitutive promoters”. Promoters that cause a gene to be expressed in a specific cell type are commonly called “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly called "developmental-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed after exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or similar that induces the promoter are commonly called "inducible promoters" or "regulatable promoters." It is widely recognized that, as in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[00041] The term “plasmid” represents an extrachromosomal element, frequently carrying a gene that is not part of the cell's central metabolism, and generally in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integration sequences, phage or nucleotide sequences, linear, circular or supercoiled, of single- or double-stranded DNA or RNA, derived Petition 870250109947, dated 01 / 12 / 2025, page 34 / 129 31 / 102 from any source, in which several nucleotide sequences have been joined or recombined into a single construct that is capable of introducing a promoter fragment and a DNA sequence for a selected gene product, along with an appropriate 3' untranslated sequence into a cell.

[00042] A polynucleotide or polypeptide has a certain percentage of “sequence identity” with another polynucleotide or polypeptide, meaning that when aligned, this percentage of bases or amino acids is the same when comparing the two sequences. Sequence similarity can be determined in several ways. To determine sequence identity, sequences can be aligned using computer methods and programs, including BLAST, available online at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetic Computing Group (GCG) package, Madison, Wisconsin, USA. Other alignment techniques are described in Methods in Enzymology, vol. 266: Computational Methods for Sequence Analysis. Macromoleculars (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that allow gaps in the sequence. The Smith-Waterman algorithm is an example that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Additionally, the GAP program, using the Needleman and Wunsch alignment method, can be used to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[00043] The term “amino acid substitution” and its synonyms described above are intended to encompass the modification of an amino acid sequence by replacing one amino acid with another substitute amino acid. The substitution can be a conservative substitution. It can also be a non-conservative substitution. The term “conservative,” when referring to two amino acids, is intended to mean that the amino acids share a common property recognized by an expert in the field. For example, amino acids with side chains Petition 870250109947, dated 01 / 12 / 2025, p. 35 / 129 32 / 102 non-acidic hydrophobic amino acids, amino acids with acidic hydrophobic side chains, amino acids with non-acidic hydrophilic side chains, amino acids with acidic hydrophilic side chains, and amino acids with basic hydrophilic side chains. Common properties may also include amino acids with hydrophobic side chains, amino acids with hydrophobic aliphatic side chains, amino acids with hydrophobic aromatic side chains, amino acids with neutral polar side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically basic side chains. Naturally occurring and non-naturally occurring amino acids are known in MT and can be used as substitute amino acids in some modalities.Methods for replacing an amino acid are well known to experts in the field and include, but are not limited to, mutations of the nucleotide sequence that codes for the amino acid sequence. The reference to "one or more" here is intended to encompass individual modalities of: for example, 1, 2, 3, 4, 5, 6 or more.

[00044] As used herein, the terms “treatment”, “treating” and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or symptom thereof and / or may be therapeutic in terms of partial or complete cure of a disease and / or adverse effect attributable to the disease.“Treatment,” as used herein, encompasses any treatment of a disease in a mammal, particularly in a human being, and includes: (a) preventing the disease (and / or symptoms caused by the disease) from occurring in a subject who may be predisposed to the disease or at risk of acquiring it, but who has not yet been diagnosed as a carrier; (b) inhibiting the disease (and / or symptoms caused by the disease), that is, halting its development; and (c) alleviating the disease (and / or symptoms caused by the disease), that is, causing regression of the disease (and / or symptoms caused by the disease), that is, improving the disease and / or one or more symptoms of the disease. Petition 870250109947, dated 01 / 12 / 2025, page 36 / 129 33 / 102

[00045] As used in this document, the term “treatment of complement factor H disorders” may encompass the relief, reduction, and / or improvement of symptoms, and / or the prevention of the development of additional symptoms associated with complement factor H disorder, which can manifest in several different phenotypes, including recurrent asymptomatic bacterial infections and renal failure. Typically, it is characterized by decreased serum levels of factor H, complement component C3, and other terminal complement components, indicating activation of the alternative complement pathway. This disorder is associated with several renal diseases with variable clinical presentation and progression, including C3 glomerulopathy and atypical hemolytic-uremic syndrome.Also presented here are compositions and methods for the treatment of one or more of the following conditions: dry age-related macular degeneration (AMD), geographic atrophy secondary to AMD, atypical hemolytic uremic syndrome (including, for example, microangiopathic hemolytic anemia, thrombocytopenia, acute renal failure), paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, ischemic stroke, and / or prevention or treatment of bacterial infections caused by the recruitment of bacterial pathogens (e.g., Aspergillus spp.; Borrelia burgdorferi; B. duttonii; B. recurrentis; Candida albicans; Francisella tularensis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes or one of the five B. burgdorferi factor H-binding proteins (CRASP-1, CRASP-2, CRASP-3, CRASP-4 or CRASP-5), among others).

[00046] As used herein, the term “treatment of complement-associated disorders” includes relieving, reducing and / or improving symptoms of both the complement factor H disorders identified above and other disorders associated with uncontrolled complement regulation via the alternative pathway.

[00047] “Complement-mediated disorders” may encompass symptoms associated with complement dysregulation, which may be Petition 870250109947, dated 01 / 12 / 2025, page 37 / 129 34 / 102 manifest in various phenotypes, including asymptomatic recurrent bacterial infections and various tissue lesions, including, among others, kidney disease. Unless otherwise indicated, both homozygous and heterozygous individuals are included in this definition. Complement dysregulation is typically caused by loss-of-function mutations or autoantibodies against complement regulatory proteins, including, among others, factor H (fH), factor I (fI), and membrane cofactor protein (MCP), or by gain-of-function mutations in other complement proteins, including, among others, 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 a decrease in other terminal complement components, indicating activation of the alternative and / or terminal complement pathway.Complement-mediated pathologies that can be treated by the present invention of composition and method include, but are not limited to, the following diseases with variable clinical presentation and progression: C3 glomerulopathy (formally called membranoproliferative glomerulonephritis type II or MPGNII), of which there are two known forms - dense deposit disease (DDD) and C3 glomerulonephritis (C3GN); thrombotic microangiopathy (TMA), including, but not limited to, atypical hemolytic uremic syndrome (aHUS), HUS due to E.Shiga toxin-producing E. coli (STECHUS) and thrombotic thrombocytopenic purpura (TTP); Degenerative retinal diseases, including age-related macular degeneration (AMD), RPE (retinal pigment epithelium) degeneration, chorioretinal degeneration, photoreceptor degeneration, paroxysmal nocturnal hemoglobinuria (PNH), all-organ and all-context ischemia-reperfusion injury, rheumatoid arthritis, hemodialysis, diabetic nephropathy, diabetic vasculopathy, asthma, systemic lupus erythematosus (SLE), ischemic stroke, abdominal aortic aneurysm (AAA), antineutrophil cytoplasmic antibody (ANCA)-mediated vasculitis, and hemorrhagic lung injury and disease. Petition 870250109947, dated 01 / 12 / 2025, page 38 / 129 35 / 102 ANCA, ANCA glomerulonephritis, graft-versus-host disease (GvHD), acute or late graft rejection in organ transplantation, Crohn's disease, psoriasis, multiple sclerosis, antiphospholipid syndrome, pre-eclampsia, atherosclerosis, neuromyelitis optica (NMO), autoimmune bullous skin disease, bullous pemphigoid (BP), Alzheimer's disease (AD), as well as bacterial infections caused by the recruitment of bacterial pathogens (e.g., Aspergillus spp.; Borrelia burgdorferi; B. duttonii; B. recurrentis; Candida albicans; Fr. sella tularensis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes).

[00048] The terms “individual”, “host”, “subject” and “patient” are used interchangeably herein and refer to a mammal, including but not limited to humans; non-human primates, including apes; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[00049] The term “effective amount,” as used herein, refers to an amount sufficient to produce beneficial or desired clinical results. “An effective amount may be administered in one or more administrations.” rAAV virion that is capable of effectively delivering a heterologous nucleic acid to a target cell (or target cells) of the individual. Effective amounts can be determined preclinically, for example, by detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques well understood in the art, for example, RT-PCR, western blotting, ELISA, fluorescence or other reporter readouts, and the like.They can be determined clinically, for example, by detecting a change in the onset or progression of the disease using methods known in the art, for example, the 6-minute walk test, left ventricular ejection fraction, hand dynamometry, the Vignos Scale and the like, as described herein and known in the art. Petition 870250109947, dated 01 / 12 / 2025, page 39 / 129 36 / 102 technical. Detailed description

[00050] Novel rAAV virions encoding modified factor H (fH) genes and protein variants are described here. These rAAV virions are characterized by durable and robust fH protein expression in the retina and by increased efficacy in treating conditions associated with factor H and other complement disorders.

[00051] The administration of these rAAV virions to individuals who require them can be carried out via various routes, preferably by intravitreal administration. Methods for using these rAAV virions in regimens for the treatment of factor H-associated disorders, particularly dry AMD and geographic atrophy secondary to AMD, are also provided.

[00052] Heterologous Nucleic Acid

[00053] The novel rAAV virions described here comprise a heterologous nucleic acid encoding a variant of human factor H (hfH), operably linked to an expression control sequence. Typically, the heterologous nucleic acid includes an AAV genome with the rep and cap genes deleted and / or replaced by the hfH sequence and its associated expression control sequences. The hfH sequence is typically inserted adjacent to one or two (i.e., flanked by) AAV TRs or TR elements suitable for viral replication (Xiao et al., 1997, J. Virol. 71(2): 941-948), in place of the nucleic acid encoding the viral rep and cap proteins. Other regulatory sequences suitable for facilitating tissue-specific expression of the hfH gene sequence in the target cell (e.g., a retinal cell) may also be included.

[00054] In some respects, the rAAV virion comprises a heterologous nucleic acid comprising (a) a terminal AAV2 repeat (b) a transcription control sequence (c) a sequence of Petition 870250109947, dated 01 / 12 / 2025, page 40 / 129 37 / 102 nucleotides encoding an hfH variant as described herein (d) a polyadenylation sequence and (e) a terminal AAV2 repeat.

[00055] hfH genes

[00056] The amino acid sequence of mature human complement factor H “wild type” (isoform 1) is provided at www.uniprot.org / uniprot / P08603 and serves as a reference for the amino acid numbering of hfH isoform I [see also SEQ ID NO: 39 of US Patent No. 10,988,519, the full content of which is incorporated herein by reference]: MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKC RPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGN VFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPE NGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEK PKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTES GWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGN TAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYY CDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNHGRK FVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVKTCSKSSIDIE NGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIK SCDIPVFMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNG WSDLPICYERECELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNS VQCYHFGLSPDLPICKEQVQSCGPPPELLNGNVKEKTKEEYGHSEVVEYY CNPRFLMKGPNKIQCVDGEWTTLPVCIVEESTCGDIPELEHGWAQLSSPPY YYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQCVAIDKLKKCKSSNLIIL EEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNCSMAQIQLC PPPPQIPNSHNMTITLNYRDGEKVSVLCQENYLIQEGEEITCKDGRWQSIPL CVEKIPCSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTC YMGKWSSPPQCEGLPCK.SPPEISHGVVAHMSDSYQYGEEVTYKCFEGFG IDGPAIAKCLGEKWSHPPSCIKTDCLSLPSFENAIPMGEKKDVYKAGEQVTY TCATYYKMDGASNVTCINSRWTGRPTCRDTSCVNPPTVQNAYIVSRQMSKY PSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPID. Petition 870250109947, dated 01 / 12 / 2025, p. 41 / 129 38 / 102 NGDITSFPLSVYAPASSVEYQCQNLYOLEGNKRITCRNGQWSEPPKCLHPC VISREIMENYNIALRWTAKQKILYSRTGESVEFVCKRGYRLSSRSHTLRTTC WDGKLEYPTCAKR (SEQ ID NO:3)

[00057] The leader sequence (MRLLAKIICLMLWAICVA; SEQ ID NO:4) is located in amino acids 1 to 18 of factor H, with reference to SEQ ID N0:3. The mature (secreted) hfH protein is located in amino acids 19 to 1231 of SEQ ID N0:3. There are alternative methods for determining the location of the 20 short complement repeats (SCRs). The location of the domains mentioned here is based on the numbering used in C. Estaller et al, Eur J Immunol. 1991 Mar; 21 (3):799-802.

[00058] The amino acid sequence of each of the 20 hfH SCRs is provided in Table 1 below (see also the identification numbers SEQ: 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 38 of US Patent No. 10,988,519): SCR Amino Acid Sequence SCR Amino Acid Sequence SCR1 EDCNELPPRRNTEILTGSWSD QTYPEGTQATYKCRRSLGNIIM VCRICGEWVNPLRKC (SEQ ID NO: 5) SCR 11 CGPPPELLNGNVKEKTKEEY GHSEWEYYCNPRFLMKGPN KIQCVDGEWTTLPVC (SEQ ID NO: 15) SCR2 CGHPGDTPFGTFTLTGGNVF EYGVKAVYTCNEGYQLLGEI NYRECDTDGWTNDIPI (SEQ ID NO: 6) SCR12 CGDIPELEHGWAQLSSPPYYY GDSVEFNCSESFTMIGHRSIT CIHGVWTQLPQC (SEQ ID NO: 16) SCR3 CLPVTAPENGKJVSSAMEP DREYHFGQAVRFVCNSGYK IEGDEEMHCSDDGFWSKEK PKC (SEQ ID NO:7) SCR13 CKSSNLIILEEHLKNKKEFDH NSNIRYRCRGKEGWIHTVCIN GRWDPEVNC (SE ID NO: 17) SCR4 CKSPDVINGSPISQKIIYKEN ERFQYKCNMGYEYSERGDA SCR14 CPPPPQIPNSHNMTTTLNYRD GEKVSVLCQENYLIQEGEEIT Petition 870250109947, dated 01 / 12 / 2025, page 42 / 129 39 / 102 VCTESGWRPLPSC (SEQ ID NO:8) CKDGRWQSIPLC (SE ID NO:18) SCR5 CDNPYIPNGDYSPLRIKHRT GDEITYQCRNGFYPATRGNT AKCTSTTGWIPAPRC (SEQ ID NO:9) SCR15 CSQPPQIEHGTINSSRSSQES YAHGTKLSYTCEGGFRISEEN ETTCYMGKWSSPPQC SCR7 (SE ID NO:19) CYFPYLENGYNQNYGRKFV QGKSIDVACHPGYALPKAQT TVTCMENGWSPTPRC (SEQ ID NO:11) SCR17 CLSLPSFENAIPMGGEKKDVY KAGEQVTYTCATYYKMDGAS NVTCINSRWTGRPTC (SEQ ID NO:21) SCR8 CSKSSIDIENGFISESQYTYA LKEKAKYQCKLGYVTADGE TSGSITCGKDGWSAQPTC SCR19 (SEQ ID NO:13) CGPPPPIDNGDITSFPLSVYAP ASSVEYQCQNLYQLEGNKRIT CRNGQWSEPPKC (SEQ ID NO: 23) SCR10 CELPKIDVHLVPDRKKDQY KVGEVLKFSCKPGFTIVGPN SVQCYHFGLSPDPIC (SEQ ID NO:14) SCR20 CVISREIMENYNIALRWTAKQ KLYSRTHESVEFVCKRYRLSS RSHTLRTTCWDGKLEYPTCAKR (SEQ ID NO:24)

[00059] Optionally, a modified hfH variant, as described herein, may have a heterologous leader sequence replaced by the native hfH leader sequence. Additionally, or optionally, another hfH isoform (e.g., isoform 2), whose sequence is available, for example, at http: / / www.uniprot.org / uniprot / P08603, Petition 870250109947, dated 01 / 12 / 2025, p. 43 / 129 40 / 102 and / or one of the naturally occurring amino acid variants present therein that are not associated with a disorder. See SEQ ID NO: 40 of US Patent No. 10,988,519. In the following descriptions, substitutions may be written as (first amino acid identified by a one-letter code) - residue position no. - (second amino acid identified by a one-letter code) wherein the first amino acid is the substituted amino acid and the second amino acid is the substitute amino acid at the specified position with reference to isoform 1; However, by means of conventional alignment steps, the corresponding amino acid residues identified herein with respect to the isoform 1 numbering may be located in isoform 2 and in naturally occurring non-disease-causing variants of fH isoform 1 or 2 SCRs.

[00060] A rAAV, as described in this document, comprises a heterologous nucleic acid encoding a functional variant of fH. Examples of functional variants of fH include those possessing the SCR1-4 and SCR19-20 domains of the fH protein, with one or more SCR7, SCR17, or SCR18 domains. Other variants include those possessing one or more SCR6, SCR8, SCR16, SCR17, SCR18 domains, or fragments thereof, and combinations thereof. For example, such variants may include, for instance, fH SCRI-4, 6-8, 19-20; fH SCRI-4, 6-8, 18-20; fH SCRI-4, 6-8, 17-20; fH SCRI-4, 6-7, 19-20; fH SCR1-4, 67, 18-20; fH SCR1-4, 6-7, 17-20; fH SCRI-4, 7-8, 19-20; fH SCRI-4, 7-8, 18-20; fH SCRI-4, 7-8, 17-20; fH SCRI-4, 7, 19-20; fH SCR1-4, 7, 18-20; fH SCR1-4, 7, 17-20; SCRI-4, 17, 19-20; SCRI-4, 18-20; SCRI-4, 17-20 and / or fH SCR1-4, 7, 16-20, among others. In certain embodiments, the hfH variant also includes additional hfH SCRs, for example, SCR 6, SCR8, SCR16 or combinations thereof.In preferred embodiments, hfH SCR5 is absent. However, in certain embodiments, hfH SCR5 may be present in full or in fraction. In certain embodiments, hfH SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, and / or SCR15 are absent or at least functionally deleted. Optionally, one or more of the SCRs in these variants may be one. Petition 870250109947, dated 01 / 12 / 2025, p. 44 / 129 41 / 102 “functional fragment” of SCRs, instead of a full-length SCR. By “functional fragment” we mean an amino acid sequence (or corresponding coding sequence) shorter than the full-length SCR, characterized by having one or more of the following features: complement inhibitory activity, heparin binding capacity and / or C3b binding activity.

[00061] With hfH variants, domains may be located immediately adjacent to one another (for example, the carboxyl terminus of one domain may immediately follow the amino terminus of the preceding domain). Alternatively, one or more SCR domains may have a linker composed of one to about 12 to 18 amino acids located between them. 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 the symbol () indicates an optional component. “L” refers to a linker, which may be absent or independently selected from an amino acid sequence of about 1 to about 12-18 amino acids. In other words, when a variant contains multiple ligands, each of them can have the same sequence or a different sequence.In certain embodiments, a variant contains at least one, at least two, at least three, at least four, at least five, and at least six ligands. Examples of suitable ligands include the natural ligands described herein or artificial ligands. Each of these wild-type ligands may be located in its native position. Alternatively, one or more of these wild-type ligands may be used in a different ligand position or in multiple different ligand positions.

[00062] Optionally, one or more of these ligands may be fH sequences and are selected independently. Alternatively, one or more of the ligands may be heterologous to fH, for example, from a different source, whether official, synthetic, or from a different protein that confers adequate flexibility to the fH variant. Petition 870250109947, dated 01 / 12 / 2025, page 45 / 129 42 / 102 Examples of other suitable linkers may include, for example, a polyglyceride linker and other linkers that provide adequate flexibility (e.g., http: / / parts.igem.org / Protein_domains / Linker), which is incorporated by reference herein. In certain embodiments, the linkers do not possess any fH function.

[00063] Some suitable and representative fH bonding sequences are provided in Table 2 below. Linking Sequence Example Linking 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

[00064] In some preferred embodiments, rAAV comprises a heterologous nucleic acid encoding a variant of hfH with the following structure: SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6(L5)-SCR7-(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20.

[00065] In a particularly preferred embodiment, the heterologous nucleic acid encodes a variant of fH with the following amino acid sequence: MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKC RPGYRSLGNIIMVCRKGEWVALNPLRKCOKRPCGHPGDTPFGTFTLTGGN Petition 870250109947, dated 01 / 12 / 2025, p. 46 / 129 43 / 102 VFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPE NGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEK PKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTES GWRPLPSCEEKSTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCD EHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFV QGKSIDVACHPGYALPKAQTTVICMENGWSPTPRCIRVKTCSKSSIDIENGF ISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSIKT DCLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRW TGRPTCRDTSCVNPPTVQNAYIVSROMSKYPSGERVRYOCRSPYEMFGDE EVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQC QNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKL YSRTGESVEFVCKRGYRLSSRSSHTLRTTCWDGKLEYPTCAKR (SEQ JD NO:34)

[00066] There are different modifications, and the rAAV comprehende and the different nuclei code that codecs some variants of hfH com in the settings. SCR1-(U)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8(L7')-SCR19-(L10)-SCR20.

[00067] These and other variants may include other fH sequences. For example, the coding sequence of the fH variant may also include a leader sequence. Such a leader sequence may be an fH leader sequence (e.g., MRLLAKIICLMLWAICVA; SEQ ID NO:4). Optionally, the leader sequence may be from another source, for example, an IL-2 leader sequence, [see, for example, the index of identified mammalian leader sequences at www.signalpeptide.de / ], incorporated by reference herein. In one embodiment, the selected leader sequence is less than about 26 amino acids in length (e.g., from about 1 to about 26 amino acids), more preferably less than 20 amino acids (from about 1 to about 20 amino acids), and most preferably less than about 18 amino acids in length (from about 1 to about 18 amino acids). By “functional deletion” is meant an amino acid sequence (or sequence Petition 870250109947, dated 01 / 12 / 2025, page 47 / 129 44 / 102 encoding the same) which does not have complement inhibitory activity, C3b binding activity and, optionally, also does not have heparin binding activity.

[00068] In addition to the fH protein variants provided here, nucleic acid sequences encoding these fH protein variants are provided. The coding sequences for these variants may be wild-type sequences of the leader sequence and / or one or more SCRs of isoform 1, isoform 2, or non-disease-associated variants. Alternatively or additionally, web-based or commercially available computer programs, as well as service companies, may be used to backtranslate the amino acid sequences of the leader sequence and / or one or more SCRs into nucleic acid coding sequences, including RNA and / or cDNA. See, for example, backtranseq by EMBOSS, Gene Infinity (www.geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (www.expasy.org / tools / )

[00069] In one embodiment, the RNA and / or cDNA coding sequences are designed for optimal expression in human cells.

[00070] Codon-optimized coding regions can be designed by several different methods. This optimization can be performed using methods available online, published methods, or a company that provides codon optimization services. One codon optimization method is described, for example, in document WO 2015 / 012924 A2, which is incorporated by reference in this document. Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Ideally, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. Using one of these methods, one can apply the frequencies to any polypeptide sequence and produce a nucleic acid fragment of a codon-optimized coding region encoding the polypeptide. Petition 870250109947, dated 01 / 12 / 2025, page 48 / 129 45 / 102

[00071] In another embodiment, at least one glycosylation site is designed on at least one of the SCRs present in the hfH variant, at least two of the SCRs present in the hfH variant, at least three of the SCRs present in the hfH variant, or more. For example, the glycosylation site may be designed on 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 embodiment, SCR4, 17 and 18 are glycosylated. In a preferred embodiment, the hfH variant comprises, consists of or essentially consists of SCR1-4, SCR6-8 and SCR17-19, and SCR17 and SCR18 are glycosylated. In certain embodiments, a glycosylation site may be transformed into a ligand. However, in this case, the ligand preferably has at least six amino acids in length up to about 18 amino acids in length, for example, 8-18, 10-15 or 12 amino acids.

[00072] As used herein, a glycosylation site refers to the point of attachment of oligosaccharides to a carbon atom (C-link), nitrogen atom (N-link), or oxygen atom (O-link), or glycation (non-enzymatic attachment of reducing sugars to the nitrogen atom of a protein (e.g., the nitrogen atom of an asparagine side chain (Asn) that is part of an Asn-XSer / Thr, where X is any amino acid except Pro). In certain embodiments, N-glycosylation sites are desirable. Several techniques are known in the field for the engineering of N-glycosylation sites. See, for example, Y Liu et al, Biotech Prog 2009 September-October; 25(5): 1468-1475; Sala RJ, Griebenos K. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010 Feb. 1; 24(1): 9-21.

[00073] Exemplary hfH variant nucleotide sequences encoding the amino acid sequence of SEQ ID NO:34 are provided below: ATGCGCCTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCG Petition 870250109947, dated 01 / 12 / 2025, p. 49 / 129 46 / 102 TGGCCGAGGACTGTAACGAGCTGCCCCCTCGGGAGAAATACAGAGATCCT GACCGGCTTTGGAGCGATCAGACCTACCCAGAGGCACACAGGCCAT CTACAAGTGCAGCCCGGCCTATCGTCTCCCTGGGCAATATCATCATGGTG TGCAGGAAGGGAGAGTGGGCCCTGAACCCACTGAGAAAGTGCCAG AAGAGGCCATGTTGGACACCCAGGCGCACACCTTTCGGCACCTTTACAC TGACCGGCGGCAACGTGTTTCGAGTACCGGCGTGAAGGCCGGTGTATACCT GCAATGAGGGCTACCACCAGCTGCTGGGCGGAGAATCAACTACCCGGAGTGTG ACACAGATGGCTGGACCAATGATATCCCCATCTGCGAGGTGGTGAAGTG TCTGCCAGTGACCCCCCAGAACGGCAAGATCGTGAGCTCGCGCAT GGAGCCTGACAGAGAGAGAGAGAGATATCACTTCGGOCAGGCGTGAGGTTCGTGTGTG CAATAGCGGCTACAAGATCGAGGGCGATGGAGAGAATGCACTGTTCGCGA GATGGCTTCTGTGTCAAGGAAGCCCAAGTGCGTGGAGAATCTOCTGTA AGTCTCCTGACGTGATTAACGGCAGCCCAATCTCCCCAGAAGATCATCTAT AAGGAGAATGAGCGGTTTCAGTACAAGTGCAACATGGGCTACGAGTATT CCGAGAGAGGCGATGCCGTGTGCACAGAGTCTGGATGGAGGCCCTGC CTAGCTGCGAGAGAAGTCCACCCTGAAGCCCTTGTGACTATCCAGATAT CAAGCACGGCGGCCTGTATCACGAGAATATGAGGCGCCCCTACTTCCCA GTGGCCGTGGGCAAGTACTATTCCTACTATTGCGACGAGCACTTTGAGA CACCATCCGGCTCTTACTGGGACACATCCACTGTACCCAGGATGGCTG GTCTCCCCGCGCGTGCCTTGCCTGGAGAAAGTGTTACTTCCCCTATCTGGGAAACGGCTACAACCAGAATTATGGCAGGAAGTTTGTGCAGGGCAAGTCCA TCGACGTGGCATGCCACCCAGGATACGCACTGCCTAAGGCACAGACCA CAGTGACCTGTATGGAGAATGGCTGGAGCCCAACACCCAGCOGTGCAT CCGCGTGAAGACCTGTTCCAAGAGCAGCATCGACATCGAGAACGGCTTC ATCACAGCG.GAGTCCCAGTACACCTATGCCCTGAAGGAGAAGGCCAAG TATCAGTGCAAGCTGGGATACGTGACOGCAGACGGAGAAACATCTGGCA GCATCACCTGCGGCAAGGATGGCTGGTCTGCCCAGCCCACATGCATCAA GAGCATCAAGACCGACTGTCTGTCTCTGCCCAGCTTTGAGAATGCCATC CCTATGGGCGAGAAGAAGGACGTGTACAAGGCCGGCGAGCAGGTGACA TACACCTGCGCCACATACTATAAGATGGACGGCGCCAGCAACGTGACCT GTATCAATTCCCGGTGGACAGGCAGACCTACCTGCAGGGATACAAGCTG CGTGAACCCACCCACCGTGCAGAATGCCTATATCGTGTCCCGCCAGATG Petição 870250109947, de 01 / 12 / 2025, pág. 50 / 129 47 / 102 TCTAAGTACCCTAGCGGCGAGCGGGTGAGATATCAGTGCCGGTCCCCAT ACGAGATGTTCGGCGACGAGGAAGTGATGTGCCTGAACGGCAATTGGA CAGAGCCTCCACAGTGCAAGGATAGCACCGGCAAGTGTGGACCACCTC CACCAATCGACAACGGCGATATCACATCCTITCCACTGAGCGTGTACGCC CCCGCCTCCTCTGTGGAGTATCAGTGCCAGAACCTGTACCAGCTGGAGG GCAATAAGCGCATCACCTGTCGGAACGGCCAGTGGTCTGAGCCTCCAAA GTGCCTGCACCCTTGCGTGATCTCCAGAGAGATCATGGAGAACTATAAT ATCGCCCTGCGCTGGACAGCCAAGCAGAAGCTGTACTCTOGGACCGGC GAGAGCGTGGAGTTCGTGTGCAAGCGGGGCTATAGACTGAGCTCCCGC AGCCACACACTGCGGACCACATGCTGGGACGGCAAGCTGGAGTACCCA ACCTGTGCCAAGAGGTGA (SEQ ID NO:35) ATGAGGCTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGTG TGGCTGAGGACTGTAATGAGCTGCCCCCTAGGAGAAATACAGAGATCCT GACTGGCTCTTGGTCTGATCAGACCTACCCAGAGGGCACACAGGCCATC TACAAGTGCAGGCCTGGCTATAGATCCCTGGGCAATATCATCATGGTGT GCAGGAAGGGAGAGTGGGTGGCCCTGAACCCACTGAGAAAGTGCCAGA AGAGGCCATGTGGACACCCAGGAGACACACCTTTTGGCACCTTTACACT GACAGGGGGCAATGTGTTTGAGTATGGTGTGAAGGCAGTGTATACCTGC AATGAGGGCTACCAGCTGCTGGGAGAGATCAACTACAGGGAGTGTGACA CAGATGGCTGGACCAATGATATCCCCATCTGTGAGGTGGTGAAGTGTCT GCCAGTGACTGCCCCTGAGAATGGCAAGATAGTGAGCTCTGCCATGGAG CCTGACAGAGAGTATCACTTTGGCCAGGCTGTGAGGTTTGTGTGCAATA GTGGCTACAAGATTGAGGGGGATGAGGAGATGCACTGTTCAGATGATGG CTTCTGGTCTAAGGAGAAGCCCAAGTGTGTGGAGATCTCCTGTAAGTCT CCTGATGTGATTAATGGCAGCCCAATCTCCCAGAAGATCATCTATAAGGA GAATGAGAGATTTCAGTACAAGTGCAACATGGGCTATGAGTATTCAGAGA GAGGTGATGCTGTGTGCACAGAGTCTGGATGGAGGCCCCTGCCTAGCT GTGAGGAGAAGTCCACCCTGAAGCCTTGTGACTATCCAGATATCAAGCA TGGAGGCCTGTATCATGAGAATATGAGGAGGCCTTACTTCCCAGTGGCT GTGGGCAAGTACTATTOCTACTATTGTGATGAGCACTTTGAGACACCATC TGGCTCTTACTGGGACCACATCCACTOTACCCAGGATGGCTGGTCTCCT。 Petition 870250109947, dated 01 / 12 / 2025, p. 51 / 129 48 / 102 GCTGTGCCTTGCCTGAGAAAGTGTACTTCCCCTATCTGGAGAATGGCTA CAACCAGAATTATGGCAGGAAGTTTGTGCAGGCAAGTCCATTGATGTG GCATGCCACCCAGGATATGCACTGCCTAAGGCACAGACCACAGTGACCT GTATGGAGAATGGCTGGAGCCCAACACCCAGGTGCATCAGGGTGAAGA CCTGTTCCAAGAGCAGCATTGACATAGAGAATGGCTTCATCTCAGAGTCC CAGTAACACCTATGCCTGAAGGAGAAGGCCAAGTATCAGTGCAAGCTG GGATATGTGACAGCAGATGGAGAAACATCTGGCAGCATCACCTGTGGCA AGGATGGCTGGTCTGCCCAGCCACATGCATCAAGAGCATCAAGACTGA CTGTCTGTCTCTGCCCAGCTTGGAGAATGCCATCCCTATGGTGGAGAAG AAGGATGTGTACAAGGCTGGAGAGCAGGTGACATACACCTGTCCCACAT ACTATAAGATGGATGGAGCCAGCAATGTGACCTGTATCAATTCCAGATGG ACAGGCAGACCTACCTGCAGGATACAAGCTGTGTAACCCACCCACTG TGCAGAATGCCTATATTGTGTCCAGGCAGATGTCTAAGTACCTICTGGA GAGAGAGTGAGATATCAGTGCAGATCCCCATATGAGAGTGTTGGGGATG AGGAAGTGATGTGCCTGAATGGCAATTOGACAGOGOCTCCCACAGGTGC AAGGATAGCACTGGCAAGTGTGGACCACCTCCCACCAATTGACAATGGAG ATATCACATCCTTTCACTGTCAGTGTATGCCCCTGCTCCTCTTGGAG TATCAGTGCCAGAACCTGTACCAGCTGGAGGGCAATAAGAGAATCACCT GTAGAAATGGCCAGTGGTCGTCTGAGCCTCCAAAGTGCCTGCACCCTTGGTGTGATCTCCAGAGAGAGATCATGGAGAACTATAATATAGCCCTGAGGTGGACA GCCAAGCAGAAGCTGTACTCTAGGACTGGGGAGTCAGTGGAGTTTGTGT GCAAGAGAGGCTATAGACTGAGCTCCAGAAGCCACACACTGAGGACCAC ATGCTGOGATGGCAAGCTGGAGTACCCAAGTGACCAGGTTGAGGACTGGCAAGCTGGAGAGTGACCAGGTTGACTGACTGACTGACTGACTGACTGAGGGAGGAGGACTGACTGACTGACTGACTGAGGAGGAGGACCAC (GGCAAGCTGGAGGAGGACCAGGTTGACTGACTGACTGACTGACTGACTGACTGAGGAGGAGGACCAC) NO: ATGAGACTTCTAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGTA GCAGAAGATTGCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCTGAC AGGTTCCTGGTCTGACCAAACATATCCAGAAGGCACCCAGOCTATCTATA AATGCAGGCCTGGATATAGATCATCAGGATTGGATTGATGAT AAGGGAGAATGGGTTGCTCTTAATCCATTAAGGAAATGTCAGAAAAGGC CCTGTGGACATCCTGGAGATACTCCTTTTGGTACTTTTACCCTTACAGGA GGAAATGTGTTTGAATATGGTGTAAAAGCTGTGTATACATGTAATGAGGG Petition 870250109947, of 01 / 12 / 2025, p. 52 / 129 49 / 102 GTATCAATTGCTAGGTGAGATTAATTACAGGGAATGTGACACACAGAATGGAT GGACCAATGATATTCCTATATGTGAAGTTGTGAAGTOTITACCAGTGACAG CACCAGAGAATGGAAAAATTGTCAGTAGTGCAATGGAACCAGATAGAGA ATACCATTTTGGACAAGCAGTAAGATTTOTATGTAACTCAGGCTACAAGA TTGAAGGAGATGAAGAAATOCATTOTTCAGATGATGGTTTTTGGAGTAAA GAGAAACCAAAGTGTGTGGAAATTTCATGCAAATCCCCAGATGTTATAAA TGGATCTCCTATATCTCAGAAGATTATTTATAAGGAGAATGAAAGATTTCA ATATAAATGTAACATGGGTTATGAATACAGTGAAAGAGGAGATGCTGTAT GCACTGAATCTGGATGGAGACCTTTGCCTTCATGTGAAGAAAAATCAACC TTGAAACCTTGTGATTATCCAGACATTAAACATGGAGGTCTATATCATGAG AATATGAGAAGACCATACTITCCAGTAGCTGTAGGAAAATATTACTCCTAT TACTGTGATGAACATTITGAGACTCCTTCAGGAAGTTACTGGGATCACATT CATTGCACACAAGATGGATGGAGTCCAGCAGTACCATGCCTCAGAAAAT GTTATTTTCCTTATTTGGAAAATGGATATAATCAAAATTATGGAAGAAAGTT TGTACAGGGTAAATCTATAGATGTTGCCTGCCATCCTGGCTATGCTCTTC CAAAAGCTCAGACCACAGTTACATGTATOGAGAATGGCTGGTCTCCTACT CCCAGATGCATCAGAGTCAAAACATGTTCCAAATCAAGTATAGATATTGA GAATGGGTTTATTTCTGAAATCTCAGTATACATATGCCTTAAAAGAAAAAG CTAAATATCAATGCAAACTAGGATATGTAACAGCAGAATGGTGAAACATCAGGATCAATTACATGTGGGAAAGATGGATGGTCAGCTCAACCCACCTGCA TTAAATCTATAAAAACAGATTGTCTCAGTTTACCTAGCTTTGAAAATGCCA TACCCATGGGAGAGAAGAAGGATGTGTATAAGGCTGGTGAGCAAGTGAC GCATTAATAGCAGATGGACAGGAAGGCCAACATGCAGAGACACCTCCTG TGTGAATCCTCCCACAGTACAAAATGCTTATATAGTGAGTAGACAGATGA GTAAATATCCATCTGGTGAGAGTAAGATATCAATGTAGGAGCCCTTAT GAAATGTTTGGGGATGAAGAAGTGGAGGAGTTGACTGACTGA ACCACCTCAATGCAAAGATTCTACAGGAAAATGGGGCCCCCTCCACCT ATTGACAATGGGGACATTCATTCCCCTTGTCAGTATATGCTOCAGC TTCATCAGTTGAGTACCAATGCCAGAACTTOTATCAACTTGAGGGTAACA AGAGGATAACATGTAGAAATGGACAATGACCAGCAGACCATGACCAT CATCCCTGTGTAATATCCAGGGAAATTATGGAAAATTAACATAGCATTA Petition 870250109947, of 01 / 12 / 2025, p. 53 / 129 50 / 102 AGGTGGACAGCCAAACAGAAGCTITATTCTAGAACAGGTGAATCAGTTGA atttgtgtgtaaaagaggatatagactttcatcaaggtctcacacattga GGACAACATGTTGGGATGGGAAACTGGAGTATCCAACTTGTGCAAAAAG ATGA (SEQ ID NO:37)

[00074] ITRs

[00075] Inverted terminal repeats (ITR(s)) selected for use in the rAAV virion are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. ITRs can be synthetic sequences that function as terminal repeats investigated by AAV, such as the “double-D sequence,” as described in U.S. Patent No. 5,478,745 by Samulski et al., the full description of which is incorporated herein by reference. Typically, but not necessarily, the TRs are from the same parvovirus; for example, both ITR sequences are from AAV2.

[00076] In some respects, the hierologous encapsulated nucleotide by the rAAV virion comprises a 5' ITR with the following sequence: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTOGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CT (SEQ ID NO:38)

[00077] In related aspects, the heterologous nucleic acid encapsulated by the rAAV virion comprises a 3' ITR with the following sequence: AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTTGCGCGCTCGCT CGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTT GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGC CAA (SEQ lD NO:39)

[00078] Expression control sequences Petition 870250109947, dated 01 / 12 / 2025, p. 54 / 129 51 / 102

[00079] The hFH gene is operationally linked to at least one transcription control sequence, preferably a transcription control sequence heterologous to the nucleic acid. In some respects, the transcription 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 G protein-coupled receptor-specific rhodopsin kinase 1 (hGRK) promoter or a human interreceptor retinoid-binding protein (IRBP) promoter. In other respects, the transcription control sequence comprises a constitutive promoter that results in a similar level of nucleic acid expression in many cell types.Suitable constitutive promoters include the CAG promoter comprising (C) immediate-early enhancer element of cytomegalovirus (CMV), (A) first exon and first intron of chicken beta-actin gene and (G) splicing acceptor of rabbit beta-globin gene (see Miyazaki et al. (1989) Gene 79(2): 269-277), cytomegalovirus (CMV) promoter (Stinski et al., (1985) Journal of Virology 55(2): 431-441), human elongation factor 1a (EF1a) promoter (Kim et al. (1990) Gene 91(2): 217-223), human phosphoglycerate kinase (PGK) promoter (Singer-Sam et al. (1984) Gene 32(3): 409-417), mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518), and ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105).

[00080] In a preferred aspect, the hfH gene is operatively linked to a CAG promoter. In a particularly preferred embodiment, the CAG promoter comprises the sequence of SEQ ID NO:40 or comprises a sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to it: ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCOTTACATAACTTACGGTAAATGGCCCGCCTGOCTGAC CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT Petition 870250109947, dated 01 / 12 / 2025, p. 55 / 129 52 / 102 AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC GGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCT CCCCATCTCCCCCCCCTOCCCCACCCCAATTTIGTATTTATTTATTTTTT AATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG CCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCT TTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCG CGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGC TCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTA CTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAAT TAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAG CCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGG GGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCC GCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTG TGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCC CGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGG TGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGC TGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGG CTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTG CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCC GGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTT TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGG CGCGGGGCGAAGCGGTGCGGCGCGCAGGAAGGAAATGGCGGGGG AGGGCCTTCGTGCGTCGCCGCGCCGCCCCCCTTCCCTCTCCAGGC CTCGGGGCT CAGGGCGGGGTTCCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTC TGCTAACCATGTTCATGCCTTCTTCTITTTCCTACAG (SEQ ID NO:40) Petition 870250109947, of 01 / 12 / 2025, p. 56 / 129 53 / 102

[00081] In some aspects, the heterologous nucleic acid encapsulated by the rAAV virion comprises an SV40 polyadenylation sequence, the following sequence: GGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAAC TAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGC TTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG CATTCATTTTATGTTTCAGGTTCAGGGAGGTTGGGAGGGAG CAAGTAAAACCTCTACA AATGTGGTATGGCTGATTATGATCA (SEQ lD NO:41)

[00082] In some respects, heterologous nucleic acid comprises the following nucleotide sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to it: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTATCGATTGAATTCCCCGGGGATCCACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGG TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAG GTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTITGTGCAGCGATGGGGGC GGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGA GGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAG AGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGG CGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGA Petição 870250109947, de 01 / 12 / 2025, pág. 57 / 129 54 / 102 CGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCC GCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGA CGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTT GTTTCTTTTCTGTGGCTOCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCC CTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGC GTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCG CTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGA GGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAG GGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCT CCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTAC GGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAG GTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCT CGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGG CGCGGCGAGCCGCAGCCATTGCCTITTATGGTAATCGTGCGAGAGGGCG CAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCC GGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCG CCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGG CTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCITCTTIT TCCTACAGTCTAGAGTCGACCTGCAGGTGGATATCTTGCTAGCACGCCA CCATGCGCCTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTG CGTGGCCGAGGACTGTAACGAGCTGCCCCCTCGGAGAAATACAGAGAT CCTGACCGGCTCTTGGAGCGATCAGACCTACCCAGAGGGCACACAGGC CATCTACAAGTOCAGGCCCGGCTATCGCTCCCTGGGCAATATCATCATG GTGTGCAGGAAGGGAGAGTGGGTGGCCCTGAACCCACTGAGAAAGTGC CAGAAGAGGCCATGTGGACACCCAGGCGACACACCTTTCGGCACCTTTA CACTTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTAT ACCTGCAATGAGGGCTACCAGCTGCTGGGCGAGATCAACTACCGGGAG TGTGACACACAGATATGGCTGGACCAATGATATCCCCATCTGCGAGGTT GGTGAAGTGGTCTGCCACAGTGACCGGCCCCCGAGAACGGCAAGATCG Petição 870250109947, de 01 / 12 / 2025, pág. 58 / 129 55 / 102 TGAGCTCCGCOCCATGGAGCOCCTGACAGGAGAGTCAATCACTGGCC AGGCCGTGAGGTTCGTGTGCAATAGCGGCTACAAGATCGAGGGCGATG AGGAGATGCACTGTTCCGACGATGGGCTTCTGGTCAAGGAGAAGCCCAA GTGCGTGGAGATCTCCTGTAAGTCTCCTGACGTGATTAACGGCAGCCCA ATTCCCCAGAAGATCATCTATAAGGAGAATGAGCGGTTTCAGTACAAGTG CAACATGGGCTACGGAGAGAAGTCCACCCTGAA GCCTTGTGACTATCCAGATATCAAGCACGGCGGCCTGTTATCACGAGAAT ATGAGGCGCCCTACTTCCCAGTGGGCCGTGGGCAAGTACTATTCCTACT ATTGCGACGAGCACTTTGAGACACCATCCGGCTCTTACTGGGACACAT CCACTGTACCCAGGATGGGCTGTTCCCGCCGTCTGCCTGGAGAAAG TGTTACTTCCCCTATCTGAGAACGGCTACAACCAGAATTATGGCAGGAA GTTTGTGCAGGGCAAGTCCATCGACGTGGCATGCCACCCAGGATACGCA CTGCCTAAGGCACAGACCACAGTGACCTGTGAGAGAATGGCTGGAGCC CAACACCCAGGTGCATCCGCGTGAAGACCTOTTCCAAGAGCAGCATCGA CATCGAGAACGGCTTCATCAGCGAGTCCCAGTACACCTATGCCCTGAAG GAGAAGGCCAAGTATCAGIGCAAGCTGGGATACGTGACCGCAGACGGA GAAACATCTGGCAGCATCACCTGCGGCAAGGATGGCTGGTCTGCCCA CCCACATGCATCAAGAGCATCAAGACCGACTGTCTGTCTCTGCCCAGCT TTGAGAATGCCATCCCTATGGGCGAGAAGAAGGACGTOTACAAGGCCGGCGAGCAGGTGACATACACCTGCGCCACATACTATAAGATGGACGGCGC CAGCAACGTGACCTGTATCAATTCCCGGTGGACAGGCAGACCTACCTGC AGGGATACAAGCTGCGTGAACCCACCCACCGTGCAGAATGCCTATATCG TGTCCCGCCAGATGTCTAAGTACCCTAGCOGCGAGCGGGTGAGATATCA GTGCCGGTCCCCATACGAGATGTTCGGCGACGAGGAAGTGATGTGCCT GAACGGCAATTGGACAGAGCCTCCACAGTGCAAGGATAGCACCGGCAA GTGTGGACCACCTCCACATCGACAACGGCGATATCACATCCTTTCCA CTGAGCGTGTACGCCCCCGCCTCCTCTGTGGAGTATCAGTGCCAGAACC TGTACCAGCTGGAGGGCAATAAGCGCATCACCTGTCGGAACGGCCAGT GGTCTGAGCCTCCAAAGTGCCTGCACCCTTGCGTGATCTCCAGAGAGAT CATGGAGAACTATAATATCGCCCTGCGCTGGACAGCCAAGCAGAAGCTG TACTCTCGGACCGGCGAGAGCGTGGAGTTCGTGTGCAAGCGGGGCTAT Petition 870250109947, dated 01 / 12 / 2025, page 59 / 129 56 / 102 AGACTGAGCTCCCGCAGCCACACACTGCGGACCACATGCTGGGACGGC AAGCTGGAGTACCCAACCTGTGCCAAGAGGTGAGCCACCGCGTAACACGT GCATGCGAGAGATCTGCGGCCGCGAGCTCGGGGATCCAGACATGATAA GATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAA TGCTITATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGG TTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAA TGTGGTATGGCTGATTATGATCAATGCATCCTAGCCGGAGGAACCCCTA GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCC TCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO:43)

[00083] In related respects, heterogeneous nucleic acid comprises the following nucleotide sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to it: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTOGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTATCGATTGAACCAGGCGCGCCCAGTTTAAACAGTCCGGAACTAGTTAT TAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTC CGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT TACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCC CCCCCCTCCCCACCCCCAATTITGTATTTATTTATTITITAATTATTITGTGCA GCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG Petição 870250109947, de 01 / 12 / 2025, pág. 60 / 129 57 / 102 GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCG GCAGCCAATCAGAGCGGCGCGCTCCGAAAGTITCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGG GAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCC TCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTG AGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCT CCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCG TGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGC GGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAG TGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGG GGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGG GGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCC TGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGG GGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGG GTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCC GGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCG GCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTG CGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAA ATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCG TCGCCGCGCCGCCGTCCCCTTCTCCCTCTCOAGCCTCGGGGCTGTCCG CGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTOCTAACCATGTTCA TOCCTTCTTCTITTTCCTACAGTCTAGAGTCGACGGTCACCGGTCAGCTA GCACGCCACCATGCGCCTGCTGGCCAAGATCATCTGCCTGATGCTGTGG GCCATCTGCGTGGCCGAGGACTOTAACGAGCTGCCCCCTCGGAGAAAT ACAGAGATCCTGACCGGCTCTTGGAGCGATCAGACCTACCCAGAGGGC ACACAGGCCATCTACAAGTGCAGGCCCGGCTATCGCTCCCTGGGCAATA TCATCATGGTGTGCAGGAAGGGAGAGTGGGTGGCCCTGAACCCACTGA GAAAGTGCCAGAAGAGGCCATGTGGACACCCAGGCGACACACCTTTCG GCACCTTTACACTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGG Petição 870250109947, de 01 / 12 / 2025, pág. 61 / 129 58 / 102 CCGTGTATACCTGCAATGAGGGCTACCAGCTGTGGGCGAGATCAACTA CCGGGAGTGTGACACAGATGGCTGGACCAATGATATCCCCATCTGCGAG GTGGTGAAGTGTCTGCCAGTGACCGCCCCGAGAACGGCAAGATCGTG AGCTCCGCCATGGAGCCTGACAGAGAGTATCACTTCGGCCAGGCCGTG AGGTTCGTGTGCCAATAGCGGCTACAAGATCGAGGGCGATGAGGAGATG CACTGTTCCGACGATGGCTTCTGGTCTAAGGAGAAGCCCAAGTGCGTGG AGATCTCCTGTAAGTCTCCTGACGTGATTAACGGCAGCCCAATCTCCCA GAAGATCATCTATAAGGAGAATGGAGCGGTTTITCAGTACAAGTGCAACA TGGGCTACGAGTATTCCGAGAGAGGCGATGCCGTGTGCACACAGAGTCT GGATGGAGGCCCCTGCCTAGCTGCGAGAGAAGTCCAACCCTGAAGOG CCTTGTGACTATCCAGATATCAAGCACGGCGGCCTTGTATCACGAGAATAT GAGOGGCGCCCTTTACTICCCAGTGGCGGCAAGTACTATTCCTACT ATTGOGCGACGAGCACTTTGAGACACCATCCGGCCTCTTACTGGGACCA CATCCACTGTACCCAGGATGGTGGTCTCCCGCGTGCTTGCCTGAGA AAGTGTTACTTCCCCTATCTGGGAGAACGGCTACAACCAGAATTATGGCAG GAAGTTTGTGCAGGCAAGTCCCATCGACGTOTOGCATGCCACCCAGG AGATACGCACTGCCTAAGGCACAGACCACAGTGGACCCTGTATOTOGAGA ​​ATGGGCTGGGAGCCCCAACACCCAGGTGCATCCGCGTGAAGACCCTGTT CCAACAGAGCAGCATCGACATCGAGAACGGCTTCATCAGCGAGCTCCAGTCCAG TACACCTATGCCCTGAAGGAGGAAGGCOCCAAGTATCAGTGCAAGCTGGGATACGTGACCGCAGACGGAGAAACATCTGGCAGCATCACCTGCGGCA AGGATGGCTGGTCTGCCCAGCCCACATGCATCAAGAGCATCAAGACCGA CTGTCTGTCTCTGCCCAGCTTTGAGAATGCCATCCCTATGGGCGAGAAG AAGGACGTGTACAAGGCCGGAGCAGGCCGACCGCCCA TACTATAAGATGGACGGCGCCAGCAACGTGACCTGTATCAATTCCCGGT GGACAGGCAGACCTACCTGCAGGGATACAAGCTGCGTGAACCCACCCA CCGTGCAGAATGCCTATATCGTGTCCCGCCAGATGTCTAAGTACCCTAG CGGCGAGCGGGTGAGATATCAGGTCCAGGTCCCTAGTC CGACGAGGAAGTGATGTGCCTGAACGGCAATTGGACAGAGCCTCCACA GTGCAAGGATAGCACCGGCAAGTGTGGACCACCTCCACCAATCGACAAC GGCGATATCACATCCTTTCCACTGAGCGT Petition 870250109947, of 01 / 12 / 2025, p. 62 / 129 59 / 102 CACCTGTCGGAACGGCCAGTGGTCTGAGCCTCCAAGTGCCTGCACCCT TGCGTGATCTCCAGAGAGATCATGGAGAACTATAATATCGCCCTGCGCT GGACAGCCAAGCAGAAGCTGTACTCTCGGACCGGCGAGAGCGTGGAGT TCGTGTGCAAGCGGGGCTATAGACTGAGCCCACCACCTGGACTGACTGGAGAGCCCACT GGACCACATGCTGGGACGGCAAGCTGGAGTACCCAACCTGTGCCAAGA GGTGAGCCTAGGACTGTACAGTACGCGTAACGTGGGGGATCCAGAC ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTG AAAAAATGCTTTTTTTGAATTATTTTGTTTTGTTAACCAGAC ATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTTGT TTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTAAAGCAAGTAAAACCT CTACAAAATGTGGTATGGCTGATTATGATCATGCATGGTCCCAGCCTTC ATTGAGCCTATTGGTCCCACCCACCCGACCTAGGACCTAGGGGCTTTTAAGCAAGTAAAACCT ATGCTCTTTGAGACACCACTGICTCCCTCAAAGTCAGGAGGCCCCCATTA ATTAAGAGAGAGTGGGAGCAGGAGGCCTGTCAGATGGCTACTCTTAAGT GGAGTACTGCCCAGAGGGCTGCTCAGAGTGGGTGGCTGTAACCCTGCA GGGGCTGACAGAGAGCACCACTGACTGACTGGACTGACTGACTGAGTCAGATGGCTACTCTTAAGT GGCCAGACTGGCCGGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACT CCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCC GGGCGTCGGGCGACCTTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC GCGCAGAGAGGGAGTGGGGCAA (SEQ4) ID NO:4:

[00084] In related respects, heterologous nucleic acid comprises the following nucleotide sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to it: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTATCGATTGAACCAGGCGCGCCCAGTTTAAACAGTCCGGAACTAGTTAT TAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTC CGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG Petition 870250109947, dated 01 / 12 / 2025, page 63 / 129 60 / 102 ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT TACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCC CCCCCCTCCCCACCCCCAATTITGTATTTATTTATTTTTTAATTATTITGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGC GGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTITCCTTTTATGGCGAGG CGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCG CCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTOTAATTAGCGCTTGG TTTAATGACGGCTIGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGG CTCCGGGAGGGCCCTTTGTGCGGDGGGAGCGGCTCGGGGGGTGCGTG CGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCG GCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGC AGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCG GGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTG GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCC CCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTG CGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGG GGGGTOGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGG GCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCG GCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCG AAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCG TGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCOCAGCCTCGGGGCT GTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGG GTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCAT Petição 870250109947, de 01 / 12 / 2025, pág. 64 / 129 61 / 102 GTTCATGCCTTCTTCTITTTCCTACAGTCTAGAGTCGACGGTCACCGGTCA GCTAGCACGCCACCATGAGGCTGCTGGCCAAGATCATCTGCCTGATGCT GTGGGCCATCTGTGTGGCTGAGGACTGTAATGAGCTGCCCCCTAGGAG AAATACAGAGATCCTGACTGGCTCTTGGTCTGATCAGACCTACCCAGAG GGCACACAGGCCATCTACAAGTGCAGGCCTGGCTATAGATCCCTGGGCA ATATCATCATGGTGTGCAGGAAGGGAGAGTGGGTGGCCCTGAACCCACT GAGAAAGTGCCAGAAGAGGCCATGTGGACACCCAGGAGACACACCTTTT GGCACCTTTACACTGACAGGGGGCAATGTGTTTGAGTATGGTGTGAAGG CAGTGTATACCTGCAATGAGGGCTACCAGCTGCTGGGAGATCAACTA CAGGGAGTGTGACACAGATGGCTGGACCAATGATATCCCCATCTGTGAG GTGGTGAAGTGTCTGCCAGTGACTGCCCCTGAGAATGGCAAGATAGTGA GCTCTGCCATGGAGCCTGACAGAGAGTATCACTTTGGCCAGGCTGTGAG GTITGTGTGCAATAGTGGCTACAAGATTGAGGGGGATGAGGAGATGCAC TGTTCAGATGATGGCTTCTGGTCTAAGGAGAAGCCCAAGTGTGTGGAGA TCTCCTGTAAGTCTCCTGATGTGATTAATGGCAGCCCAATCTCCCAGAAG ATCATCTATAAGGAGAATGAGAGATTTCAGTACAAGTGCAACATGGGCTA TGAGTATTCAGAGAGAGGTGATGCTGTGTGCACAGAGTCTGGATGGAGG CCCCTGCCTAGCTGTGAGGAGAAGTCCACCCTGAAGCCTTGTGACTATC CAGATATCAAGCATGGAGGCCTGTATCATGAGAATATGAGGAGGCCTTACTTCCCAGTGGCTGTGGGCAAGTACTATTCCTACTATTGTGATGAGCACT TTGAGACACCATCTGGCTCTTACTGGGACCACATCCACTGTACCCAGGA TGGCTGGTCTCCTGCTGTGCCTTGCCTGAGAAAGTGTTACTTCCCCTATC TGGAGAATGGCTACAACCAGAATTATGGCAGGAAGTTTGTGCAGGGCAA GTCCATTGATGTGGCATGCCACCCAGGATATGCACTGCCTAAGGCACAG ACCACAGTGACCTGTATGGAGAATGGCTGGAGCCCAACACCCAGGTGCA TCAGGGTGAAGACCTGTTCCAAGAGCAGCATTGACATAGAGAATGGCTT CATCTCAGAGTCCCAGTACACCTATGCCCTGAAGGAGAAGGCCAAGTAT CAGTGCAAGCTGGGATATGTGACAGCAGATGGAGAAACATCTGGCAGCA TCACCTGTGGCAAGGATGGCTGGTCTGCCCAGCCCACATGCATCAAGAG CATCAAGACTGACTGTCTGTCTCTGCCCAGCTTTGAGAATGCCATCCCTA TGGGTGAGAAGAAGGATGTGTACAAGGCTGGAGAGCAGGTGACATACA CCTGTGCCACATACTATAAGATGGATGGAGCCAGCAATGTGACCTGTAT Petição 870250109947, de 01 / 12 / 2025, pág. 65 / 129 62 / 102 CAATTCCAGATGGACAGGCAGACCTACCTGCAGGGATACAAGCTOTGTG AACCCACCCACTGTGCAGAATGCCTATATTGTGTCCAGGCAGATGTCTAA GTACCCTTCTGGAGAGAGAGTGAGATATCAGTGCAGATCCCCATATGAG ATGTTTGGGGATGAGGAAGTGATGCCTGAATGGCAATTGGACAGAGC CTCCACAGTGCAAGGATAGCACTGGCAAGTGTGGACCACCTCCACCAAT TGACAATGGAGATATCACATCCTTTCCACTGTCAGTGTATGCCCTGCCT CCCTGTGGGAGCTCCAGAAGCCACACACTGAGGACCACATGCTGGGAT GGCAAGCTGGAGTAAGTATCAGTGCCAGACCTGTCACAGCTGGAGGG CATAAGAGAATCACCTGTAGAAATGGCCAGTGGTCTGAGCCTCCAAAG TGCCTGCACCCTTGTGTGATCTCCAGAGAGATCATGGAGAACTATAATAT AGCCCTGAGGTGGACAGCCAAGCAGAAGCTGTACTCTAGGACTOGGGA GTCAGTGGAGTTTGTGTGCAAGAGAGGCTATAGACTCCCCACCTGTGC AAGAGGTGAGCCTAGGACTGTACAGTACGCGTAACACGTGGGGGATCC AGACATGATAAGATACATTGATGAGTTTGGGACAAACCACAACTAGAATGC AGTGAAAAAAATGCTTTATTTGTGGAAATTTGTGGATGCTATTGCTTTATTGT AACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTT TATGTTCAGGTTCAGGGGAGGTGTGGAGGTTITTTAAAGCAAGTAAA ACCTCTACAAATGTGGTATGGCCTGATTATGATCAATGCATGGTCCCAGCC TTCATTGGAGCCTATTGGTCCCCCTCAGAACCCACCCACCCCGGCAGTAGAGGATGTCTCTTGAGACACCACTGTCTCCCTCAAGTTCAGGAGGCCCCC ATTAATTAAGAGAGAGTGGGAGCAGGAGGCCTGTCAGATGGCTACTCTT AAGTGGAGTACTGCCCAGAGGGCTGCTCAGAGTGGGTGGCTGTAACCC TGCAGGGGCTGACAGAGCACACATCCATACTGGTGAAGGACCTGTGACC CACTGGCCAGACTGGCCGGCCGGAGGAACCCCTAGTGATGGAGTTGGC CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGC GAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO:45)

[00085] In related respects, heterologous nucleic acid comprises the following nucleotide sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to it: Petition 870250109947, dated 01 / 12 / 2025, p. 66 / 129 63 / 102 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTATCGATTGAACCAGGCGCGCCCAGTTTAAACAGTCCGGAACTAGTTAT TAATAGTAATCAATTACGGGGTCATTAGTTCATAGOCCATATATGGAGTTC CGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCACAGTACATATCTACGTATTAGTCATCG CTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTOCACACTCTCCC CATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTA TTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAG GCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC GGGCGGGGAGTCGCTGCOICGACGCTGCSCCTTCGCCCCGTGCCCCGC TCCGCCGCCGCCTCGCGCCGCCCGCCCCGOGGCTCTGACTCTGACCG CGTGTTACTCCOCCACAGGTGAGCGGGCGGGACGGCCCTTOTOCTOCCGGGCTGTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTG GCTGCGTGTGAAAGCOCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCG GGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGC GCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCG CGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGG CCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAA AGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGG GCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGC GCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGC CGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGG GGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAG Petição 870250109947, de 01 / 12 / 2025, pág. 67 / 129 64 / 102 CCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTT CCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGC ACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAA GGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCC TTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGC GGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACA GTCTAGAGTCGACGGTCACCGGTCAGCTAGCACGCCACCATGAGACTTC TAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGTAGCAGAAGATT GCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCTGACAGGTTCCTGG TCTGACCAAACATATCCAGAAGGCACCCAGGCTATCTATAAATGCAGGC CTGGATATAGATCTCTTGGAAATATCATAATGGTATGCAGGAAGGGAGAA TGGGTTGCTCTTAATCCATTAAGGAAATGTCAGAAAAGGCCCTGTGGACA TCCTGGAGATACTCCTTTTGGTACTTTTACCCTTACAGGAGGAAATGTGT TTGAATATGOTGTAAAAGCTGTGTATACATGTAATGAGGGGTATCAATTG CTAGGTGAGATTAATTACAGGGAATGTGACACAGATGGATGGACCAATG ATATICCTATATGTGAAGTTGTGAAGTGTTTACCAGTGACAGCACCAGAGA ATGGAAAAATTGTCAGTAGTGCAATGGAACCAGATAGAGAATACCATTTT GGACAAGCAGTAAGATTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAATGCATTGTTCAGATGATGGTTTTTGGAGTAAAGAGAAACCAA AGTGTGTGGAAATTTCATGCAAATCCCCAGATGTTATAAATGGATCTCCT ATATCTCAGAAGATTATTTATAAGGAGAATGAAAGATTTCAATATAAATGT AACATGGGTTATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATC TGGATGGAGACCTTTGCCTTCATGTGAAGAAAAATCAACCTTGAAACCTT GTGATTATCCAGACATTAAACATGGAGGTCTATATCATGAGAATATGAGA AGACCATACTTTCCAGTAGCTGTAGGAAAATATTACTCCTATTACTGTGAT GAACATTTTGAGACTCCTTCAGGAAGTTACTGGGATCACATTCATTGCAC ACAAGATGGATGGAGTCCAGCAGTACCATGCCTCAGAAAATGTTATTTTC CTTATTTGGAAAATGGATATAATCAAAATTATGGAAGAAAGTTTGTACAGG GTAAATCTATAGATGTTGCCTGCCATCCTGGCTATGCTCTTCCAAAAGCT CAGACCACAGTTACATGTATGGAGAATGGCTGGTCTCCTACTCCCAGAT GCATCAGAGTCAAAACATGTTCCAAATCAAGTATAGATATTGAGAATGGG Petição 870250109947, de 01 / 12 / 2025, pág. 68 / 129 65 / 102 TTTATTTCTGAATCTCAGTATACATATGCCTTAAAAGAAAAAGCTAAATATC AATGCAAACTAGGATATGTAACAGCAGATGGTGAAACATCAGGATCAATT ACATGTGGGAAAAGATGGATGGTCAGCTCAACCCACCTGCATTAAATCTAT AAAAAACAGATTGTCCAGTTTACCTAGOTTIGAAAATGCCATACCCATGGG AGAGAAAGGAAGGATGIGTATAAGGCTGGTGAGCAAGTGACCTTACACTTGT GCAACATATTACAAAATGGATGGAGCCAGTAATGTAACATGCATTAATAG CAGATGGACAGGAAGGCAACATGCAGAGACCCTCCTGTGTGAATCC CCCACAGTACAAAATGCTTATATAGTAGAGACAGTAAATATCC ATCTGGTGAGAGAGTAAGATATCAATGTAGGAGCCCCTATGAAATGTTTG GGGATGAAGAAGTGATGTTTTTAAATGGAAACTGGGACAGAACCACCTCCA A TGCAAAGATTTACAGGAAAATGTGGCCCCCCCCCCCCCCCCATTATTGACAATG GGGACATTACTTCATTCCCCCTTGTCAGTATATGCTCAGCCTCATCAGTT GAGTACCAATGCCAGAACTTGTATCAACTTGAGGGTAACAAGAGGATAAC ATGTAGAAATGGACAATGGTCAGAACCACCAAAATGCTTACATCCCTGTG TAATATCCAGGGAAATTATGGAAATTATAACATAGCATTAAGGTGGACA GCCAAACAGAAGCTTTTATTCTAGAACAGGTAGAATCAGTTGAATTTGTGTG TAAAAAGAGGATATAGACTTTCATCAAGGTCTCACACATTGAGGACAACAT GTTGGGGAATGGAAACTGGAGTATCCAACTTGTGCAAAAAGATGAGC GGACTGTACAGTACGCGTAACACGTGGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAATOCAGTGAAAAAAAATGCT TTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CAATAAACAAGTTAACAACAACAATTGCATTCATTTATGTTTCAGGTTCA GGGGGAGGTGTGGAGGAGCAGACTTACTTACTTA GGTATGGCTGATTATGATCAATGCATGGTCCCAGCCTTCATTGAGCCTAT TGGTCCCCCCTCAGAACCCACCCACCTGGCAGTAGAGGATGTCTCTTTGA GACACCACTGTCTOCCTCAAGTTCAGGAGGCCCCCATTAATAGAGAG AGTGGGAGCAGGAGGCCTGTCAGCTTAGGCTTAGGCTGAGGGAG CCAGAGGGCTGCTCAGAGTGGGTGGCTGTAACCCTGCAGGGGCTGACA GAGCACACATCCATACTGGTGAAGGACCTGTGACCCACTGGCCAGACTG GCCGGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCCTG CGCGCTCGCTCGCTCACTGACCCGGGCGCCG GGCGACCTTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG Petition 870250109947, of 01 / 12 / 2025, p. 69 / 129 66 / 102 AGGGAGTGGCCAA (SEQ ID NO:46)

[00086] AAV capsid

[00087] The variant AAV capsid of rAAV – encapsulating the heterologous nucleic acid encoding a variant hFh – comprises a variant AAV capsid protein comprising an insertion of about 7 to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) into the GH loop of a parental AAV capsid protein, wherein the peptide comprises the amino acid sequence ISDQTKH (SEQ ID NO: 1). Preferably, the variant capsid protein, when present in an AAV virion, confers greater infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parenteral capsid protein.

[00088] By “GH loop”, or IV loop, of the AAV capsid protein, we mean the solvent-accessible portion referred to in the literature as the GH loop, or IV loop, of the AAV capsid protein. For the GH loop / IV loop 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. Thus, for example, the insertion site may be located between amino acids 570-611 of VP1 of AAV2.

[00089] In some embodiments, the peptide insert has 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of the amino acid sequence 1SDQTKH (SEQ ID NO: 1). Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and praline (P). In certain embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In preferred embodiments, the peptide insert comprises or consists of the amino acid sequence LAISDQTKHA Petition 870250109947, dated 01 / 12 / 2025, pp. 70 / 129 67 / 102 (SEQ TD NO:2).

[00090] In some respects, the variant AAV capsid protein comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ 1D NO: 1) and further comprises one or more amino acid substitutions relative to a 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.

[00091] In some preferred embodiments, the AAV variant capsid protein comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:1) and comprises a P34A amino acid substitution relative to the VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.

[00092] In other respects, the variant capsid protein may comprise one or more features disclosed in U.S. Patent No. 11,576,983, in particular, one or more features disclosed from column 26, line 66 to column 29, line 50 of U.S. Patent No. 11,576,983.

[00093] In a particularly preferred embodiment, the variant capsid protein comprises the following amino acid sequence or comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDS Petição 870250109947, de 01 / 12 / 2025, pág. 71 / 129 68 / 102 EYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEY FPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRIN TPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSE YSWTGATKYHLNGRDSL.VNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQ AATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFG LKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:42).

[00094] The AAV capsid protein variant of SEQ ID NO:42 contains the following modifications relative to the native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, which is located within the assembled capsid (VP1 protein only), and (ii) a 10-amino acid insertion (leucine-alanine-isoleucineserine-aspartic acid-glutamine-threonine-lysine-histidinealanine / LAISDQTKHA (SEQ ID NO:2)) at amino acid position 588, which is present in VP1, VP2, and VP3. In some embodiments, the capsid comprises a variant capsid protein comprising a sequence with at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:42 and comprising a P34A substitution and an insertion of the LAISDQTKHA peptide (SEQ ID NO:2) at amino acid position 588.

[00095] Packaging cells, which are encompassed by “host cells,” are also provided herein, which can be cultured to produce packaged viral vectors of the invention. The packaging cells of the invention generally include cells with (1) heterologous viral vector function(s), (2) packaging function(s), and (3) auxiliary function(s). Each of these component functions is discussed in the following sections.

[00096] Initially, vectors can be produced by various methods known to qualified technicians (see, for example, WO Petition 870250109947, dated 01 / 12 / 2025, page 72 / 129 69 / 102 (2013 / 063379). A preferred method is described in Grieger et al., 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, where an adherent HEK293 cell line from a qualified master clinical cell bank is used to grow under animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production. Using the triple transfection method (e.g., WO 96 / 40240), the HEK293 cell line in suspension yields more than 10⁵ vector genome (vg)-containing particles / cell or more than 10¹⁴ vg / L of cell culture when harvested 48 hours after transfection.More specifically, triple transfection refers to the packaging cell being 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 Ela, Elb, E2a, E4, and VA RNA), and another plasmid encodes the transgene and its various control elements (e.g., modified GLA gene and CAG promoter).

[00097] To achieve the desired yields, several variables are optimized, such as the selection of a compatible, serum-free suspension medium that supports both growth and transfection, the selection of a transfection reagent, the transfection conditions, and cell density. A universal purification strategy, based on ion-exchange chromatography methods, was also developed, resulting in high-purity vector preparations of serotypes 1-6, 8, 9, and several chimeric capsids. This user-friendly process can be completed in one week, results in high filled-to-empty particle ratios (>90% filled particles), provides post-purification yields (>1χ10Λ13 vg / L) and adequate purity for clinical applications, being universal with respect to all serotypes and chimeric particles. This scalable manufacturing technology was used to fabricate clinical vectors of T-phase GMP AAVs for retinal neovascularization (AAV2). Petition 870250109947, dated 01 / 12 / 2025, page 73 / 129 70 / 102 hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which were administered to patients. Additionally, a minimum 5-fold increase in overall vector production was achieved by implementing a perforation method involving the collection of rAAV from the culture medium at multiple time points after transfection.

[00098] 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, with rep and cap deleted and replaced by the modified GLA sequence and its associated expression control sequences. Viral vector functions include sufficient expression control sequences to result in viral vector replication for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the transgene and its associated expression control sequences. The transgene is typically flanked by two AAV TRs, in place of the deleted rep and cap viral ORFs.Appropriate expression control sequences are included, such as a tissue-specific promoter and other regulatory sequences suitable for use in facilitating tissue-specific expression of the transgene in the target cell. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or a marker polypeptide.

[00099] Terminal repeats (TRs) (resolvable and non-resolvable) selected for use in viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. Resolvable AAV TRs do not need to have a wild-type TR sequence (e.g., a wild-type sequence can be altered by insertion, deletion, truncation, or nonsense mutations), as long as the TR mediates the desired functions, e.g., viral packaging, provirus integration and / or rescue, and the like. TRs can be synthetic sequences that function as repeats. Petition 870250109947, dated 01 / 12 / 2025, page 74 / 129 71 / 102 inverted AVV terminals, such as the “double-D sequence”, as described in U.S. Patent No. 5,478,745 with Samulski et al., the full discussion of which is incorporated herein by reference. Typically, but not necessarily, the TRs are of the same parvovimos, for example, both TR sequences are of AAV2. [000100] Packaging functions include variant capsid components, as described above. [000101] The packaged viral vector includes a variant hfH transgene and expression control sequences flanked by TR elements, herein referred to as the “transgene” or “transgene expression cassette,” sufficient to result in the packaging of the vector DNA and subsequent expression of the gene sequence in the transduced cell. The functions of the viral vector may, for example, be provided to the cell as a component of a plasmid or an amplicon. The functions of the viral vector may exist extrachromosomally within the cell lineage and / or may be integrated into the chromosomal DNA of the cell. [000102] Any method of introducing the nucleotide sequence carrying the viral vector functions into a host cell for replication and packaging may be employed, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments where the viral vector functions are provided by transfection using a viral vector, standard methods for producing viral infection may be used. [000103] Packaging functions include genes for viral vector replication and packaging. Thus, for example, packaging functions may include, as needed, functions required for viral gene expression, viral vector replication, rescue of the viral vector from the integrated state, viral gene expression, and packaging of the viral vector into a viral particle. Packaging functions may be provided together or separately to the cell. Petition 870250109947, dated 01 / 12 / 2025, page 75 / 129 72 / 102 Packaging using a genetic construct, such as a plasmid or an amplicon, a baculovirus, or an HSV helper construct. Packaging functions can exist extrachromosomally within the packaging cell, but are preferentially integrated into the cell's chromosomal DNA. Examples include genes encoding the AAV Rep and Cap proteins. [000104] Helper functions include auxiliary viral elements necessary to establish active infection of the packaging cell, which is required to initiate viral vector packaging. Examples include functions derived from adenovirus, baculovirus, and / or herpesvirus, sufficient to result in viral vector packaging. For example, adenovirus helper functions typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA. Packaging functions can be provided by infecting the packaging cell with the necessary virus. Packaging functions can be provided jointly or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. See, for example, pXR helper plasmids, as described in Rabinowitz et al., 2002, l Virol. 76:791, and pDG plasmids described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760.Packaging functions can exist extrachromosomally within the packaging cell, but are preferentially integrated into the cell's chromosomal DNA (e.g., El or E3 in BEK 293 cells). [000105] Any suitable helper virus functions can be used. For example, when the packaging cells are insect cells, baculovirus can serve as a helper virus. Herpesvirus can also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV Rep protein(s) can advantageously facilitate more scalable AAV vector production schemes. [000106] Any method of introducing the sequence of Petition 870250109947, dated 01 / 12 / 2025, pp. 76 / 129 73 / 102 nucleotides containing the auxiliary functions in a host cell for replication and packaging can be employed, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments where the auxiliary functions are provided by transfection using a viral vector or infection using a helper virus, standard methods for producing viral infection can be used. [000107] Any suitable permissive or packaging cell known in the art may be employed in the production of the packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines such as VERO, WI38, MRCS, A549, HEK 293 cells (which express functional adenoviral cells under the control of a constitutive promoter), B-50 cells or any other HeLa cells, HepG2, Saos-2, HuH7 and HT1080 cell lines. In one aspect, the packaging cell is capable of growing in suspension culture; more preferably, the cell is capable of growing in serum-free culture. In one embodiment, the packaging cell is an HEK293 growing in suspension in serum-free medium. In another embodiment, the packaging cell is the HEK293 cell described in the US Patent. No. 9,441,206 and deposited as ATCC No. 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 aspect, the packaging cell is grown in the form of a cell stack (e.g., a 10-layer cell stack seeded with HEK293 cells). [000108] Cell lines for use as packaging cells include insect cell lines. Any insect cell that allows AAV replication and can be maintained in culture can be used according to the present invention. Examples include Petition 870250109947, dated 01 / 12 / 2025, page 77 / 129 74 / 102 Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, for example, cell lines derived from Aedes albopictus. A preferred cell line is the Sf9 cell line of Spodoptera frugiperda. The following references are incorporated here for their teachings regarding the use of insect cells for the expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. 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. Academic. Science. 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., Pat. No. 6,204,059. [000109] The viral capsids according to the invention can be produced using any method known in the art, for example, by expression from baculovirus (Brown et al., (1994) Virology 198:477-488). As a further alternative, the virus vectors of the invention can be produced in insect cells using baculovirus vectors to provide the rep / cap genes and the rAAV template, as described, for example, by Urabe et al., 2002, Human Gene Therapy 13:1935-1943. [000110] In another aspect, the present invention provides a method for producing AAVs in insect cells whereby a baculovirus packaging system or vectors can be constructed to carry the Rep and Cap coding region by engineering these genes into the polyhedrin coding region of a baculovirus vector and producing recombinant viruses by transfection into a host cell. Notably, when using baculovirus production for AAVs, preferably the AAV DNA vector product is an AAV-like self-complementary molecule, without the use of murination for ITR AAVs. This appears to be a byproduct of the cutting process. Petition 870250109947, dated 01 / 12 / 2025, pp. 78 / 129 75 / 102 inefficient AAV replication in insect cells, resulting in a self-complementary DNA molecule due to the absence of functional activity of the Rep enzyme. The host cell is either a baculovirus-infected cell or has introduced baculovirus auxiliary functions that encode additional nucleic acids or includes these baculovirus auxiliary functions. These baculoviruses can express AAV components and subsequently facilitate capsid production. [000111] During production, packaging cells typically include one or more viral vector functions, along with auxiliary and packaging functions sufficient to result in viral vector replication and packaging. These various functions may be provided together or separately to the packaging cell using a genetic construct, such as a plasmid or an amplicon, and may exist extrachromosomally within the cell lineage or integrated into the cell's chromosomes. [000112] Cells may be provided with any one or more of the aforementioned functions already incorporated, for example, a cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, or a cell line with auxiliary functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA. [000113] The rAAV vector can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods 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 No. 6,566,118 and WO 98 / 09657. [000114] Methods of administering nucleic acids that encode Petition 870250109947, dated 01 / 12 / 2025, pp. 79 / 129 76 / 102 hfH for the retina [000115] In various embodiments, methods are provided for the delivery of heterologous nucleotide sequences encoding hfH to the retina using a rAAV, as described herein. The rAAV can be employed to deliver a nucleotide sequence encoding hfH to a retinal cell in vitro, for example, to produce an hfH polypeptide or nucleic acid in vitro for ex vivo gene therapy. rAAVs are additionally useful in a method for delivering a nucleotide sequence to an individual in need of it, for example, to express hfH in an individual in need of it, such as a human with dry AMD or a human with geographic atrophy. In this way, hfH can be produced in vivo in the individual to restore complement regulation. [000116] Thus, in one aspect, a method is provided for delivering a nucleic acid encoding an hfH variant to a retinal cell, the method comprising contacting the retinal cell with an rAAV virion, as described herein. [000117] In another aspect, a method is provided for administering a nucleic acid encoding an hfH variant to a retinal cell in a mammalian individual, the method comprising administering an effective amount of the rAAV virion, as described in this document, or a pharmaceutical formulation comprising it, to a mammalian individual. [000118] rAAV can be administered to an individual's retina by any suitable route. In preferred modalities, rAAV is administered to the individual intraocularly, preferably by subretinal, suprachoroidal and / or intravitreal injection. In some particularly preferred modalities, rAAV is administered to an individual by intravitreal injection, with a greater preference for a single intravitreal injection. [000119] Treatment methods [000120] In certain modalities, a method is provided for the Petition 870250109947, dated 01 / 12 / 2025, pages 80 / 129 77 / 102 treatment of dry AMD in an individual requiring such treatment, comprising the method of administering to the individual 13. recombinant adeno-associated virus (rAAV) comprising: (a) an AAV variant capsid protein comprising a heterologous peptide insert with a length of 7 to 20 amino acids covalently inserted into the GH loop of the AAV capsid protein, wherein the peptide insert comprises the amino acid sequence lSDQTKH (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding an hfH variant as described herein, said nucleotide sequence being operatively linked to a promoter, or administering to the individual a pharmaceutical composition comprising said rAAV or a pharmaceutically acceptable carrier, preferably wherein said rAAV or said pharmaceutical composition is administered to the individual by intravitreal injection.The use of said rAAV or a pharmaceutical composition comprising it for the treatment of dry AMD is also provided. The use of said rAAV in the manufacture of a medicament for the treatment of dry AMD is also provided. [000121] In related embodiments, a method is provided for the treatment of geographic atrophy in a subject requiring such treatment, the method being administered to the subject by administering to the subject a recombinant adeno-associated virion (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insert of 7 to 20 amino acids in length covalently inserted into the GH loop 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 an hfH variant as described herein, said nucleotide sequence operatively linked to a promoter, or by administering to the subject a pharmaceutical composition comprising said rAAV virion and a pharmaceutically acceptable carrier, preferably wherein said rAAV or said pharmaceutical composition is Petition 870250109947, dated 01 / 12 / 2025, page 81 / 129 78 / 102 administered to the subject by intravitreal injection. The use of said rAAV or a pharmaceutical composition comprising the same for the treatment of geographic atrophy is also provided. The use of said rAAV in the manufacture of a medicament for the treatment of geographic atrophy is also provided. [000122] In some respects, the AAV variant capsid protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical or 100% identical to the amino acid sequence presented as SEQ ID NO:42 and comprises a P34A substitution and a LAISDQTKHA peptide insertion (SEQ ID NO:2) at amino acid position 588. [000123] In some preferred aspects, the nucleotide sequence encoding the hfH variant encodes an h:iH variant with the structure SCRI-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20, wherein each of SCRs 1-4, 6-8 and 17-20 comprises the amino acid sequence according to Table 1 and wherein each of L1-L10 comprises the amino acid sequence according to Table 2. In related aspects, the nucleotide sequence encoding the hfH variant encodes an hfH variant comprising the amino acid sequence of SEQ ID NO:34 or comprising an amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown as SEQ ID NO:34. In other related respects, the nucleotide sequence encoding the hfH variant comprises the nucleotide sequence shown as any of the SEQ ID Nos: 35-37. [000124] In some respects, rAAV is administered by periocular, intravitreal, suprachoroidal and / or subretinal injection, preferably by intravitreal injection, to a subject with dry AMD and / or geographic atrophy at a dose of approximately 1χ10⁸ vector genomes (vg) / eye to approximately 1χ10¹³vg / eye, from approximately 1χ10⁸vg / eye to approximately Petition 870250109947, dated 01 / 12 / 2025, page 82 / 129 79 / 102 1x1012vg / eye, from about 1*109vg / eye to about 1*1012vg / eye, from about 1x109vg / eye to about 1X1011vg / eye or from about 6*109vg / eye to about 6χ1010vg / eye. In some modalities, rAAV is administered by periocular, intravitreal, suprachoroidal, and / or subretinal injection, preferably by intravitreal injection, to an individual with dry AMD and / or geographic atrophy, at a dose of approximately 1x10¹²vg / eye, approximately 2x10²vg / eye, approximately 3x10¹²vg / eye, approximately 4x10¹²vg / eye, approximately 5x10⁸vg / eye, approximately 6x10²vg / eye, approximately 7x10¹²vg / eye, approximately 8x10²vg / eye, approximately 1x10³vg / eye, approximately 1x10⁹vg / eye, 2x10⁹vg / eye, approximately 3x10⁹vg / eye, approximately 4x10⁹vg / eye, approximately 5x10⁹ vg / eye, approximately 6x109 vg / eye, approximately 7x109 vg / eye, approximately 8x1010 vg / eye, approximately 9x1010 vg / eye, approximately 1x1010 vg / eye, approximately 2x1010 vg / eye,approximately 3x10¹⁰vg / eye, approximately 4x10¹⁰vg / eye, approximately 5x10¹⁰vg / eye, approximately 6x10¹⁰vg / eye, approximately 7x10¹⁰vg / eye, approximately 8x10¹⁰vg / eye, approximately 9x10¹⁰vg / eye, approximately 1x10¹¹vg / eye, approximately 2x10¹¹vg / eye, approximately 3x10¹⁰vg / eye, approximately 4x10¹⁰vg / eye, approximately 5x10¹¹vg / eye, approximately 6x10¹⁰vg / eye, approximately 7x10¹¹vg / eye, approximately 8x10¹¹vg / eye, approximately 9x10¹¹vg / eye, or approximately 1x10² vg / eye.., [000125] In some embodiments, a method is provided for treating dry AMD and / or geographic atrophy in a subject in need thereof, comprising administering to the subject by periocular, intravitreal, suprachoroidal and / or subretinal injection, an effective amount of an rAAV virion comprising (i) a capsid comprising a capsid protein comprising the amino acid sequence established as SEQ ID NO:42 and (ii) a heterologous nucleic acid comprising from 5' to 3': (a) a terminal AAV2 repeat (b) a CAG or CMV promoter (c) a nucleotide sequence selected from SEQ ID Nos:35-37 (d) a polyadenylation sequence and (e) a Petition 870250109947, dated 01 / 12 / 2025, page 83 / 129 80 / 102 repeat terminal AAV2. [000126] In related embodiments, a method is provided for treating dry AMD and / or geographic atrophy in a subject in need thereof, comprising administering to the subject by periocular, intravitreal, suprachoroidal and / or subcutaneous injection, a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and an rAAV virion comprising (i) a capsid protein comprising the amino acid sequence presented as SEQ ID NO:42 and (ii) a heterologous nucleic acid comprising from 5' to 3': (a) a terminal AAV2 repeat (b) a CAG or CMV promoter (c) a nucleotide sequence selected from SEQ ID Nos:35-37 (d) a polyadenylation sequence and (e) a terminal AAV2 repeat. In some aspects, the pharmaceutical composition comprises between approximately 1x10⁸vg and approximately 1x10¹³vg, between approximately 1x10⁹vg and approximately 1x10¹²vg, between approximately 1x10⁹vg and approximately 1x10¹¹vg, or between approximately 6x10⁹vg and approximately 6x10¹⁰vg.In other respects, the pharmaceutical composition comprises approximately 1x10⁸vg, approximately 2x10⁸vg, approximately 3x10⁸vg, approximately 4x10⁸vg, approximately 5x10⁸vg, approximately 6x10⁸vg, approximately 7x10⁸vg, approximately 8x10⁸vg, approximately x10⁸vg, approximately 1x10⁹vg, 2x10⁹vg, approximately 3x10⁹vg, approximately 4x10⁹vg, approximately 5x10⁹vg, approximately 6x10⁹vg, approximately 7x10⁹vg, approximately 8x10⁹vg, approximately 9x10⁹vg, approximately 1x10¹⁰vg, approximately 2x10¹⁰vg, approximately 3x10¹⁰vg, approximately 4x1010vg, approximately 5x1010vg, approximately 6x1010vg, approximately 7x1010vg, approximately 8x1010vg, approximately 9x1010vg, approximately 1x1011vg, approximately 2x1011vg, approximately 3x1011vg, approximately 4x1011vg, approximately 5x1011vg, approximately 6x1011vg, approximately 7x1011vg, approximately 8x1011vg, approximately 9x1011vg, or approximately 1x1012vg. [000100] Pharmaceutical compositions comprising a vector-derived anaerobic substance (rAAV) as described in this document are presented. In some embodiments, the pharmaceutical composition comprises approximately 1 x 10⁵ to approximately 1 x 10¹⁴ vector particles or genomes, approximately 1 x 10⁸ to approximately 1 x 10¹³ vector particles or genomes, approximately 1 x Petition 870250109947, dated 01 / 12 / 2025, page 84 / 129 81 / 102 109a about 1 x 10 vector particles or genomes, or about 1 x 105, about 2 x 105, about 3 x 105, about 4 x 105, about 5 x 105, about 6 x 105, about 7 x 105, about 8 x 105, about 9 x 105, about of 1 x 109, about 2 x 109, about 3 x 109, about 4 x 109, about 5 x 109, about 6 x 109, about 7 x 109, about 8 x 109, about 9 x 109, about 1 x 101°, about 2 x 101°, about 3 x 101°, about 4 x 101°, approximately 5 x 101°, approximately 6 x 101°, approximately 7 x 101°, approximately 8 x 101°, approximately 9 x 101°, approximately 1 x 1011, approximately 2 x 1011, approximately 3 x 1011, approximately 4 x 1011, approximately 5 x 1011, approximately 6 x 1011, approximately 7 x 1011, approximately 8 x 1011, approximately 9 x 1011 or approximately 1 x 1012 vector particles or vector genomes. In some aspects, the pharmaceutical composition comprises approximately 1 x 109a approximately 1 x 1011vg and preferably comprises approximately 6 x 109vg to approximately 6 x 101°vg.In some preferred embodiments, the pharmaceutical composition is administered to a human being with dry AMD and / or geographic atrophy by intravitreal injection. EXAMPLES [000127] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. Although this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to a person skilled in the art from reading this Application. Example 1 [000128] A recombinant AAV virion (rAAV) was constructed carrying a transgene encoding a shortened form of human complement factor H (Fig. 1). Complement factor H (CFH) is a natural inhibitor of the alternative complement pathway and, as such, rAAV is useful for the treatment of, among other things, geographic atrophy secondary to age-related macular degeneration. Endogenous CFH Petition 870250109947, dated 01 / 12 / 2025, page 85 / 129 82 / 102 is a 155 kDa protein composed of 20 short consensus repeat (SCR) units. The N-terminal end is the critical site for cofactor activity / degradation acceleration activity and the C-terminal end for cellular regulation (see, for example, de Cordoba SR, de Jorge EG. Series of mini-translational reviews on complement factor H: genetics and disease associations of human complement factor H. Clin Exp Immunol. 2008 Jan;151(1):1-13). [000129] rAAV comprises a capsid protein with the amino acid sequence defined at SEQ ID NO:42 and a heterologous nucleic acid encoding a shortened form of complement factor H (“miniCFH”) with the amino acid sequence defined at SEQ ID NO:34, operatively linked to a CAG promoter. The miniCFH transgene contains SCRsl-4, 6-8, and 17-20. [000130] rAAV was subjected to in vitro characterization by (a) transfection of the rAAV plasmid cassette into human HEK293T cells and (b) in vitro transduction of retinal pigment epithelial cells (RPEs) derived from human induced pluripotent stem cells (iPSCs) with rAAV. [000131] Expression and Activity of miniCFH in HEK293T Cells [000132] To demonstrate the in vitro function of the miniCFH transgene used in rAAV, human cells were transfected with the rAAV plasmid cassette. HEK293T cells were transfected with low (0.125 μg) or high (0.5 μg) concentrations of plasmid DNA encoding a miniCFH or CFH (full length), each under the control of a CAG promoter. Dose-dependent expression and appropriately sized protein were observed in the cell supernatants (Figures 2A and 2B). The miniCFH present in the cell supernatants was shown to bind directly to C3b and heparin, similarly to the full-length CFH (Figures 2C and 2D). These data confirm that the miniCFH transgene is expressed from the rAAV plasmid cassette, secreted, and retains complement- and heparin-binding characteristics. Next, the activity of the miniCFH transgene was evaluated and... Petition 870250109947, dated 01 / 12 / 2025, page 86 / 129 83 / 102 its functionality confirmed. Complement protein 3 (C3) is cleaved into C3a and C3b fragments, and the C3b fragment is subsequently degraded by complement factor I (CFI), in the presence of CFH as a cofactor, into smaller, inactive fragments, interrupting the alternative complement cascade. Protein lysates from cells transfected with the rAAV plasmid cassette demonstrate that miniCFH mediated the degradation of C3b (Figure 2E). Furthermore, miniCFH from transfected cells demonstrated complete inhibition of MAC formation (Figure 2F). Overall, these results demonstrate that transfection with miniCFH cDNA leads to the expression and secretion of functional CFH protein. [000133] In vitro transduction of human iPSC-derived retinal pigment epithelial cells (RPEs) [000134] To demonstrate in vitro expression and confirm the function of the miniCFH gene after transduction with rAAV, human iPSC-derived RPE cells were transduced with rAAV at different multiplicities of infection (MOI). Transduction of cells with rAAV resulted in dose-dependent expression of the secreted miniCFH transgene protein (Figure 3A). Furthermore, the alternative component pathway was inhibited and demonstrated inhibition of MAC formation (Figures 3B and 3C). Together, these in vitro data confirm the expression, secretion, and functionality of the rAAV miniCFH gene. [000135] Materials and Methods [000136] miniCFH Expression (ELISA, WB). HEK293T cells (ATCC CRL-32l6) were transfected with plasmid DNA encoding a miniCFH or CFH (full length) controlled by the CAG promoter. Plasmid DNA was transfected at 0.125 µg or 0.5 µg DNA / 12 wells (2 and 5 cells / well) with Fugene HD reagent (Promega Corporation cat# E23l1). Cells were incubated for 48 h in 1 ml DMEM medium with 1% penicillin / streptomycin and 10% heat-inactivated serum. After the initial 48 h, the medium was changed to 0.35 ml of serum-free medium (DMEM medium with 1% penicillin / streptomycin) and the cells were incubated for 24 h. The serum-free supernatants were Petition 870250109947, dated 01 / 12 / 2025, page 87 / 129 84 / 102 tested for transgenic expression by ELISA and Western blot. [000137] The C3b cleavage assay was performed to confirm the functional activity of CFH expressed in 293T cells. The C3 protein is composed of alpha and beta chains. The C3 protein is cleaved into C3a and C3b fragments. C3a is a small fragment, with 77 amino acids, with an approximate molecular weight of 9 kDa. The C3b fragment (containing the remaining alpha and beta chains) can form the C3 convertase by binding to factor B, or it can be degraded by complement factor I (CFI), in the presence of CFH as a cofactor, into smaller inactive fragments. This latter event interrupts the alternative complement cascade. These smaller fragments resulting from C3b degradation can be detected by Western blot. The serum-free supernatant of CFH-transfected cells (or non-transfected cells in the negative control) was incubated with 1 µg of purified C3b (Complement Technology, catalog no. A114) and 1 µg of purified CFI (Complement Technology Inc.)., catalog no. A138) for 1 hour at 37 °C. As a positive control, we used 0.5 µg of purified CFH (Complement Technology Inc., catalog no. A137) mixed with 1 µg of purified C3b (Complement Technology Inc., catalog no. A114) and 1 µg of purified CFI (Complement Technology Inc., catalog no. A138) in PBS and incubated for 1 hour at 37 °C, where CFH or CFI were omitted from the recombinant protein mixture. [000138] Complement inhibition assay. The Wieslab® Complement system Alternative pathway kit (Eagle Biosciences Inc. cat #COMPL AP 330) is an enzyme immunoassay for the qualitative determination of the alternative complement pathway in human serum. The assay combines the principles of the hemolytic assay for complement activation with the use of labeled antibodies specific for neoantigens produced as a result of complement activation. The amount of neoantigen generated is proportional to the functional activity of the complement pathways. The wells of the 96-well plate are coated with a specific activator of the alternative pathway (lipopolysaccharides). Petition 870250109947, dated 01 / 12 / 2025, pp. 88 / 129 85 / 102 bacterial, EPS). Normal human serum is diluted to 5% in a diluent containing a specific blocker to ensure that only the alternative pathway is activated. During incubation of the diluted serum in the wells, complement is activated by EPS. The wells are then washed and C5b9 is detected with a specific antibody labeled with alkaline phosphatase for the neoantigen expressed during MAC formation. After an additional washing step, detection of specific antibodies is obtained by incubation with alkaline phosphatase substrate solution. The amount of complement activation correlates with the color intensity and is measured in terms of absorbance (optical density (OD)).The addition of complement inhibitors, such as eculizumab (a C5 blocking antibody), CFH from supernatants of cell cultures transfected with CFH, or recombinant CFH protein to 5% normal human serum, can partially or completely inhibit the reaction, as evidenced by a decrease in the absorbance signal. [000139] C3b Binding ELISA. The C3b binding ELISA is an internally developed ELISA adapted from Nichols et al. ( / / pubmed.ncbi.nl.nih.gov / 26221753 / ). The assay tests whether CpG-free miniCFH retains the ability to directly bind to C3b, using blocking anti-CFH monoclonal antibodies to prevent miniCFH binding to a C3b-coated plate. The assay was performed with full-length recombinant CFH as a proof of concept, and with supernatants from 293T cells transfected with CpG-free miniCFH. As a control, we included sham incubation conditions of CFH and transfected cell supernatant without antibodies. [000140] Heparin-binding ELISA. The heparin-binding ELISA is an internally developed ELISA adapted from Nichols et al. ( / / pubmed.ncbi.nlm.nih.gov / 26221753 / ). The assay tests whether CpG-free miniCFH retains heparin-binding capacity using blocking anti-CFH monoclonal antibodies to prevent miniCFH from binding to a heparin-coated plate. The assay was performed with Petition 870250109947, dated 01 / 12 / 2025, pages 89 / 129 86 / 102 Full-length recombinant CFH was used as a control, along with supernatants from 293T cells transfected with CpG-free miniCFH. Supernatants from transfected cells not treated with blocking antibodies were also included as controls. The results show that pre-incubation with blocking antibodies reduces the signal of both recombinant CFH and CpG-free miniCFH, indicating that CpG-free miniCFH constructs maintain heparin-binding activity. [000141] Transduction of human RPE cells. IPSC-derived RPE cells were transduced with a capsid of SEQ ID NO:42, which provides a payload encoding miniCFH under the control of a CAG promoter. RPE cells were previously obtained and cryopreserved internally. Approximately 30 ± 5 days before transduction, RPE cells were seeded onto hESC-qualified Matrigel™ coated plates (Corning cat# 354277) and maintained in serum-free XVIVO-10 medium (Lonza cat# BEBP02-055Q) with 1% penicillin / streptomycin and the RHO / ROCK pathway inhibitor Y-27632 (STEMCELL Technologies cat# 72304) at 10 μM for 30 ± 5 days. The medium was changed every 2-3 days. RPE cells were transduced with multiplicity of infection (MOI, vector genomes (vg) per cell) of 5,000, 20,000, and 50,000. Cell counting was performed on the same day and immediately before transduction; the appropriate volume of virus was mixed with fresh medium and added to the cells.After the initial 3 days following transduction, the culture medium was removed and replaced with fresh medium. Four days later, that is, seven days after transduction, the medium was collected and analyzed for miniCFH expression and activity. Example 2 [000142] High-performance liquid chromatography / mass spectrometry (LC-MS) was employed to confirm the presence of the product of Petition 870250109947, dated 01 / 12 / 2025, pages 90 / 129 87 / 102 gene miniCFH (short CFH or sCFH) in non-human primates (NHPs) after intravitreal administration of the rAAV of Example 1 (comprising a capsid protein comprising the amino acid sequence defined in SEQ ID NO:42 and a heterologous nucleic acid encoding a shortened form of complement factor H (“miniCFH”) with the amino acid sequence defined in SEQ ID NO:34, operable and linked to a CAG promoter). [000143] Materials and methods [000144] Aqueous humor (AH) and vitreous humor (VH) samples were collected from non-human primates (NHPs) treated (intravitreally) with rAAV, flash-frozen, and stored for analysis. An LCMS method for sCFH was developed and qualified to identify rAAV-encoded sCFH-specific peptide signatures. These specific peptide mass signatures were then quantified against a standard curve to determine the sCFH protein concentration in each sample. Quality control samples containing known levels of sCFH were included in each experimental run. The standard deviation acceptance criterion for repeated sample testing is <20%. The LLOQ / BLQ for the assay was set at ~50 ng / mL, and samples were analyzed in triplicate. [000145] Results [000146] Table 3 below contains a summary of the LCMS quantification results of sCFH concentrations in HA samples of intravitreal NHPs delivered with the specified dose (1e10, 5e10, 5e11 vg / eye) of rAAV (see also FIG. 4): Table 3 Sample type, dose (vg / eye), time point (days), mean mini-CFH ng / mL SD, Frequency BLQ Petition 870250109947, dated 01 / 12 / 2025, pp. 91 / 129 88 / 102 AH 1e10 15 58.27 34.80 0 / 6 BLQ AH 1e10 29 52.83 22.74 0 / 6 BLQ AH 1e10 43 66.16 34.83 1 / 6 BLQ AH 1e10 58 94.00 0.85 4 / 6 BLQ AH 1e10 71 87.30 3.25 4 / 6 BLQ AH 1e10 85 85.10 25.31 4 / 6 BLQ AH 5e10 15 104.40 48.37 0 / 8 BLQ AH 5e10 29 164.62 139.38 2 / 8 BLQ AH 5e10 43 65.53 19.63 2 / 6 BLQ AH 5e10 58 43.30 5.37 2 / 4 BLQ AH 5e10 71 39.95 0.78 2 / 4 BLQ AH 5e10 85 44.50 4.53 2 / 4 BLQ AH 5e11 15 176.95 87.40 2 / 6 BLQ AH 5e11 29 118.53 104.92 0 / 6 BLQ AH 5e11 43 107.02 97.77 0 / 6 BLQ AH 5e11 58 146.00 63.64 0 / 2 BLQ AH 5e11 71 85.15 2.19 0 / 2 BLQ AH 5e11 85 39.30 1.27 0 / 2 BLQ [000147] Table 4, below, contains a summary of the LCMS quantification results of sCFH concentrations in VH samples of NHPs treated with a specified dose of rAAV: Table 4 sample type dose (vg / eye) time point (days) mean miniCFH ng / mL SD Frequency BLQ VH 1e10 85 213.00 NA 2 / 3 BLQ VH 5e10 43 309.00 NA 1 / 2 BLQ VH 5e10 85 141.00 NA 1 / 2 BLQ Petition 870250109947, dated 01 / 12 / 2025, pages 92 / 129 89 / 102 VH 5e11 42 120.57 52.97 0 / 3 BLQ VH 5e11 86 80.10 NA 0 / 1 BLQ [000148] Conclusion [000149] The sCFH gene product encoded by rAAV was detected in VH and AH at each time point and dose (Tables 3 and 4; FIG. 4). Based on measured sCFH AH levels and previous studies / models with rAAV comprising a capsid protein of SEQ ID NO:42 exploring relative AH / VH / retina concentrations, retinal sCFH concentrations were predicted to be within the normal / therapeutic range (see Table 5 below). Table 5 Average Observed Dose of AH Predicted Peak AH Concentration (ng / ml) (regression of all pharmacokinetics) Predicted Peak AH Concentration (ng / ml) (low PK regression) Predicted R / C Cone (ng / g) Predicted sCFH R / C Cone (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 Petition 870250109947, dated 01 / 12 / 2025, pp. 93 / 129 90 / 102 Predicted endogenous CFH for retina / choroid: 3-70 nM [000150] An in situ hybridization (ISH) method was developed to evaluate sCFH expression using 293T transfected cells to confirm specificity and optimize assay conditions. The ISH method was then used to detect the presence of sCFH RNA in NHP ocular tissue after intravitreal injection with rAAV (comprising a capsid protein comprising the amino acid sequence presented in SEQ ID NO:42 and a heterologous nucleic acid encoding miniCFH with the amino acid sequence presented in SEQ ID NO:34, operatively linked to a CAG promoter). [000151] Materials and methods [000152] Ocular tissue samples (whole eyeball sample) were collected from NHPs treated with rAAV at 5E10 vg / eye, fixed, and processed for analysis. [000153] The ISH assay for sCFH was performed using a custom-designed, proprietary 20ZZ probe for sCFH mRNA (designed to be non-cross-reactive with endogenous macaque cinnamolgus or full-length human CFH), targeting amino acids 195-1318. The ISH for sCFH was performed on the Roche Discovery ULTRA Autostainer platform using the Roche DISC. mRNA Probe AMP. Kit RUO, Roche mRNA Sample Prep Kit RUO, DISC. mRNA DAB Detection RUO, bluing reagent and hematoxylin, as well as the ACD RNAscope® VS Universal HRP reagent kit. Full slide images were captured using the Zeiss Axioscan instrument, and representative images were captured and reported using Zeiss Zen-lite imaging software. The results of the ISH analysis are shown in Figures 5 and 6. Figure 5 demonstrates the sCFH RNA in transfected HEK293T cells compared with non-transfected HEK293T cells (control). Petition 870250109947, dated 01 / 12 / 2025, pages 94 / 129 91 / 102 negative). Figure 6 demonstrates the ISH signal of sCFH RNA disseminated in the retinal and macular regions. Example 3 [000154] Additional in vitro studies were performed to demonstrate the in vitro functional activity of the miniCFH protein expressed after transfection of human HEK293T cells. [000155] HEK293T cells were transfected with either a low dose (0.125 µg) or a high dose (0.5 µg) of the rAAV plasmid cassette from Example 1 (comprising a heterologous nucleic acid encoding a shortened form of complement factor H (“miniCFH”) with the amino acid sequence shown in SEQ ID NO:34, operatively linked to a CAG promoter) or an rAAV plasmid cassette encoding the full-length CFH protein. The miniCFH or full-length CFH protein secreted in the supernatant was visualized by Western blot. Dose-dependent expression and appropriately sized protein were observed in the supernatants of transfected cells (Figure 7A). The expression of the secreted CFH protein was quantified after transfections with the rAAV plasmid cassette from Example 1 by ELISA (Figure 7B). Transfection resulted in detectable expression of secreted CFH compared to the non-transfected control (Figure 7B). [000156] In addition to protein expression, the activity of miniCFH secreted by HEK293T cells transfected with the miniCFH construct was evaluated. Complement factor H is an essential regulator for the control of the alternative pathway and, in turn, negatively regulates the formation of the C5b-9 complex, also called the Membrane Attack Complex (MAC), an endpoint of the complement cascade. To test whether miniCFH was functional, alternative complement inhibitory activity was measured using a modified version of the Wieslab® Complement System Alternative Pathway Assay. The supernatants of Petition 870250109947, dated 01 / 12 / 2025, pages 95 / 129 92 / 102 cells transfected with the rAAV plasmid cassette from Example 1 showed significant inhibition of alternative complement system activity (Figure 7C). The recombinant antibody Eculizumab, a C5 inhibitor, was used as a positive control and shows strong inhibition of the alternative complement pathway in this assay (Figure 7C). [000157] In addition to evaluating the effect of miniCFH on the complement activation endpoint, MAC formation, an upstream CFH mechanism, was assessed using the C3b cleavage activity assay. An upstream complement protein C3 can be hydrolyzed into C3a and C3b fragments after complement activation. The C3b fragment contains an alpha and beta chain and can form C3 convertase upon binding to factor B or can be degraded by complement factor I (CFI), in the presence of CFH as a cofactor, into smaller inactive cleavage products. Degradation of the C3b beta chain disrupts the alternative complement cascade and is a marker of complement pathway inhibition. These smaller fragments resulting from C3b cleavage can be detected by Western blot. The supernatant from cells transfected with the rAAV plasmid cassette from Example 1 or from untransfected cells was incubated with recombinant CFI and C3b proteins and analyzed by Western blot. (Figure 7D).The membrane was probed with an anti-C3 antibody to detect all C3 protein products, including the alpha chain of C3, the beta chain of C3, and three beta chain cleavage products. In the reaction incubated with supernatants from cells transfected with the rAAV plasmid cassette of Example 1, all three C3b cleavage products could be observed, similar to the positive control, but not detected in the NT reaction. Furthermore, the u chain band of C3b was depleted under this condition, similar to the positive control. These data corroborate the upstream function of the miniCFH produced from the rAAV plasmid cassette of Example 1 to assist CFI in cleaving the beta chain of C3b to ultimately inhibit the progression of the complement cascade. Petition 870250109947, dated 01 / 12 / 2025, pages 96 / 129 93 / 102 [000158] Furthermore, as expected, the miniCFH from cell supernatants of cells transfected with the rAAV plasmid cassette from Example 1 bound directly to C3b and heparin, similarly to the full-length CFH (Figure 7E and Figure 7F). These data confirmed that the miniCFH transgene can be expressed, secreted, and retain appropriate complement- and heparin-binding characteristics. [000159] Other experiments were performed to demonstrate the expression of the miniCFH gene product, as well as the functional activity of the miniCFH protein expressed in vector-transduced human iPSC-derived RPE cells after transduction at different multiplicities of infection (MOI). iPSC-derived RPE cells were transduced with rAAV at several different MOTs: 5,000; 20,000; and 50,000 vg / cell for experiments to evaluate the expression and functional activity of miniCFH secreted in cell supernatants, and MOIs of 800; 2,000; and 5,000 vg / cell for experiments with disease models. The rAAV comprises a capsid protein comprising the amino acid sequence shown in SEQ ID NO: 42 and a heterologous nucleic acid encoding a shortened form of complement factor H (“miniCFH”) with the amino acid sequence shown in SEQ ID NO: 34, operationally linked to a CAG promoter.After transduction, the viral inoculum was removed after approximately 72 hours and the medium was changed. Seven days after transduction, cell supernatants were collected, aliquoted, and stored at -80 °C, and the cells were fixed. [000160] To demonstrate that transduction of iPSC-derived RPE cells with rAAV results in the expression of a functional protein, the concentration of miniCFH secreted in cell supernatants was quantified by ELISA seven days after transduction. ELISA detects both endogenous follicular-length CFH and rAAV-encoded miniCFH. The signal beyond that observed in non-transduced cells correlates with rAAV-expressed miniCFH. As Petition 870250109947, dated 01 / 12 / 2025, pp. 97 / 129 94 / 102 shown in Figure 8, non-transduced RPE cells secrete low levels of endogenous CFH, and supernatants from RPEs transduced into MOTs of 20,000 and 50,000 showed statistically higher CFH concentrations. Furthermore, the CFH concentration was significantly higher in supernatants from RPE cells transduced into MOT 50,000 compared to MOI 5,000. These data demonstrate a dose-dependent response in vitro. [000161] Subsequently, the ability of the secreted miniCFH protein to inhibit the alternative complement pathway was also evaluated in RPE cells transduced with rAAV, comprising a capsid protein comprising the amino acid sequence presented in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence presented in SEQ ID NO:34, operatively linked to a CAG promoter. The alternative complement inhibitory activity was measured in supernatants from transduced cells using two different methods: 1) A modified version of the Wieslab® Alternative Complement System Pathway assay and 2) Quantification of soluble C5b-9. To evaluate the functional inhibitory effect of miniCFH on the alternative pathway of the Wieslab® Complement System, supernatant samples from RPE cells transduced with rAAV were mixed with serum and the samples were applied to Wieslab assay plates.Inhibition of the complement pathway resulted in a decrease in the MAC signal. [000162] Supernatants from cells transduced with rAAV at 20,000 MOI and 50,000 MOI produced statistically significant complement inhibition compared to non-transduced RPE cell samples. As a positive control, RPE cells were treated with 200 nM recombinant CHI, a concentration comparable to the highest levels of expressed miniCFH observed in this study. Dose-dependent complement inhibitory activity was observed, and treatment of RPE cells with high-concentration eculizumab, a known complement inhibitor that targets Petition 870250109947, dated 01 / 12 / 2025, pages 98 / 129 95 / 102 C5 also resulted in almost complete inhibition of complement. (Figure 9A) [000163] Since the Wieslab assay is not a truly quantitative measure of complement activation, the complement inhibitory activity of expressed rAAV was assessed using a second, more quantitative method. The concentration of soluble C5b-9 was measured using an SC5b ELISA kit. RPE supernatant samples transduced with rAAV showed significantly reduced SC5b-9 formation compared to samples with non-transduced RPE supernatant (Figure 9B). Furthermore, a dose-dependent effect on soluble C5b-9 formation was also observed. As expected, non-transduced cell samples supplemented with Eculizumab showed minimal C5b-9 formation. This decrease in soluble C5b-9 demonstrates functional inhibition of the alternative complement pathway. Taken together, these results demonstrated that miniCFH secreted by rAAV-transduced RPE cells was able to inhibit the alternative complement pathway in a dose-dependent manner. [000164] In addition to characterizing the functional effect of miniCFH expressed on complement activation, an upstream mechanism of miniCFH was evaluated by means of an assay of accelerated C3 convertase decay activity. To determine whether miniCFH acts as a cofactor with Complement Factor I (CFI) to promote C3b degradation in the same way as full-length CFH, supernatants from cells transduced with rAAV were combined with purified C3b and CFI instead of purified full-length CFH (Figure 10, lanes 5-9). Compared to non-transduced samples (Figure 10, lane 5), transduced RPE samples (Figure 10, lanes 6-8) resulted in greater cleavage of the alpha chain of C3b, with samples with higher MOI producing more cleavage than samples with lower MOI.In the absence of CFI, the miniCFH produced by EPR produced minimal C3b cleavage, similar to purified full-length CFH (Figure 10, band 9), reinforcing its role as a cofactor in the degradation of. Petition 870250109947, dated 01 / 12 / 2025, pages 99 / 129 96 / 102 C3b. [000165] In addition to these findings, the efficacy of rAAV, composed of a capsid protein with the amino acid sequence described in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence described in SEQ ID NO:34, operatively linked to a CAG promoter, was examined in an in vitro model of human RPE disease. For this, a retinal complement activation model was established in RPE cultures derived from human iPS cells. To simulate the disease state, serum and the alternative complement pathway activator, Zymosan, were introduced into the culture medium, resulting in the deposition of membrane attack complex (MAC) in the RPE over the following 24 hours, as determined by immunocytochemistry (ICC) and flow cytometry. [000166] As shown in Figure 11, treatment with rAAV of the RPE six days prior to complement activation elicited a dose-dependent decrease in MAC deposition in the RPE, triggered by the presence of Zymosan. Eculizumab, when added to cultures simultaneously with complement activation, exerted a similar protective effect, as expected. Flow cytometric quantification of MAC deposition (Figure 11B) revealed that RPE cells transduced with rAAV and RPE supplemented with Eculizumab exhibited significantly reduced MAC formation compared to non-transduced RPE. Furthermore, MAC formation was significantly lower in RPE cells transduced at MOI 2,000 and 5,000 compared to MOI 800, indicating a dose-dependent response.Together, these immunocytochemistry (TCC) and flow cytometry data demonstrated that miniCFH secreted by the RPE transduced with the rAAV product, in addition to its complement inhibition in cell-free assays, also protected the RPE from MAC deposition in a disease-like setting. [000167] Finally, the upstream function of miniCFH in this in vitro model was evaluated. Activation of the alternative complement pathway. Petition 870250109947, dated 01 / 12 / 2025, pages 100 / 129 97 / 102 results in a large increase in C3b, which is converted to iC3b. As discussed earlier, CFH is a cofactor in the cleavage of C3b to iC3b; however, since this cleavage inactivates C3 convertase, the creation of iC3b ultimately results in less C3b being produced and therefore less iC3b in the system. iC3b is commonly used as an indicator of C3b because it has a significantly longer half-life compared to C3b. Therefore, the concentration of iC3b was measured in the supernatants of the disease model as a surrogate for C3b levels (Figure 12). Supernatants from RPE cells transduced with rAAV showed a reduced level of iC3b compared to the supernatant from non-transduced RPE cells (Figure 12).However, as expected, there was no difference in iC3b concentration between the non-transduced EPR supernatant and the non-transduced RPE supernatant supplemented with eculizumab, since eculizumab regulates the complement system further downstream in the protein cascade. [000168] In summary, human iPSC-derived EPR cells transduced with rAAV (comprising a capsid protein with the amino acid sequence established at SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence established at SEQ ID NO:34, operatively linked to a CAG promoter) expressed and secreted miniCFH in a dose-dependent manner. The expressed miniCFH exhibited functional activity of alternative complement inhibition and specific activity of accelerating C3 convertase degradation, consistent with the functional activity of endogenous CFH.Furthermore, transduction of RPEs with rAAV resulted in a protective effect on RPE cultures when the complement pathway was subsequently activated, as assessed by the level of C5b-9 / MAC deposition in the RPE, as well as by iC3b levels. MAC is the terminal complex in the complement cascade and a primary effector of complement-mediated cell death. iC3b is a degradation product of C3b and does not represent an endpoint marker of alternative complement activation. Ultimately, the levels of soluble iC3b... Petition 870250109947, dated 01 / 12 / 2025, pages 101 / 129 98 / 102 were reduced in cultures transduced with rAAV compared to non-transduced cultures, as was MAC deposition in the RPE, visualized by immunocytochemistry and quantified by flow cytometry analysis. Example 4 [000169] In Vivo Ocular Pharmacodynamic Study with rAAV expressing miniCFH m in Non-Human Primates [000170] The safety and expression of the miniCFH transgenic protein of a Raav (comprising a capsid protein comprising the amino acid sequence presented in SEQ ID NO: 42 and a heterologous nucleic acid encoding the amino acid sequence presented in SEQ ID NO: 34, operatively linked to a CAG promoter) in male cynomolgus monkeys after a single intravitreal (IVT) dose in both eyes, followed by a 6 or 12-week observation period, were evaluated. Male cynomolgus monkeys were dosed once by IVT in both eyes with the rAAV at a dose volume of 50 gL / eye. Groups 1-2 consisted of 2 males per group and received doses of 5x1011 and 5x1010vg / eye, respectively, and were sacrificed 6 weeks post-dose for tissue collection. Groups 3, 4, and 5 received single doses of rAAV of 5x1011, 5x1010, and 1x1010vg / eye (bilaterally), respectively, and were observed for 12 weeks before terminal sacrifice. [000171] After rAAV administration, aqueous humor, retinal tissue, and serum samples were examined for miniCFH protein concentrations by LC-MS. A treatment-related dose response for miniCFH protein expression was observed in the aqueous humor. [000172] Levels of the transgenic protein miniCFH were higher in the retina and choroid of the retinal pigment epithelium (RPE), with comparatively low concentrations in the aqueous humor (Figure 14). Petition 870250109947, dated 01 / 12 / 2025, pages 102 / 129 99 / 102 This distribution pattern was expected, given the retinotropic nature of the capsid and the fact that retinal tissues are likely a predominant source of miniCFH protein secretion. [000173] The miniCFH protein was detectable only in the serum of the 5x1011 vg / eye dose groups on Day 15, with very low values ​​close to the LLOQ of 5 ng / mL, demonstrating that the miniCFH protein expressed from rAAV is largely contained within the eye after IVT administration and is not distributed to the systemic circulation at significant levels (see Table 6 below). Table 6. Levels of miniCFH in aqueous humor, vitreous humor, and serum in samples from cynomolgus monkeys treated with rAAV. Treatment Group Day 15 Serum miniCFH levels (ng / mL) High dose (5x10¹¹ vg / eye) 6.07 0 BQL of 3 Medium dose (5x10¹⁰ vg / eye) All BQL Low dose (1x10¹⁰ vg / eye) All BQL BQL: Below the quantifiable limit Lower limit of quantification: 5 ng / mL [000174] No significant macroscopic changes were observed in animals sacrificed at weeks 6 or 12, nor were there significant changes in clinical pathology, hematology, or coagulation between the rAAV dose groups. No changes were observed. Petition 870250109947, dated 01 / 12 / 2025, pages 103 / 129 100 / 102 related to treatment in no clinical pathology parameter after treatment with rAAV, and the small fluctuations were generally attributed to the immunosuppression regimen. [000175] Microscopic findings observed after rAAV treatment at 6 weeks included minimal, non-adverse perivascular mononuclear cell infiltrates in the retina. Findings at week 12 were comparable and included minimal to mild non-adverse mononuclear cell infiltrates in the ciliary body, minimal retinal degeneration and pyknotic cells of uncertain significance in one eye, and minimal perivascular mononuclear cell infiltrates in the optic nerve head, none of which were considered adverse. Systemic organs showed no microscopic findings in either dose group. rAAV treatment resulted in the generation of antibodies against the vector capsid and anti-miniCFH antibodies in most animals at week 12. No antigen-specific IFNγ T cell responses, measured by ELISPOT, were detected against the vector capsid or CFH peptide pools in rAAV-treated NHP eyes 6 or 12 weeks post-dose.Overall, rAAV was well tolerated after a single bilateral dose of IVT in male cynomolgus monkeys, at doses up to 5x1011-vg / eye (HED 1xJ012 vgieye). [000100] Study of Ocular Evaluation with Single Intravitreal Dose in Cynomolgus Monkeys. [000176] Another study was initiated to comprehensively evaluate ocular structure and function parameters in small groups of male and female cynomolgus non-human primates after a single unilateral dose of rAAV (comprising a capsid protein comprising the amino acid sequence defined in SEQ ID NO:42 and a heterologous nucleic acid encoding the amino acid sequence defined in SEQ ID NO:34, operatively linked to a CAG promoter) and follow-up for 12 weeks after the dose. Petition 870250109947, dated 01 / 12 / 2025, pages 104 / 129 101 / 102 [000177] Two groups of cynomolgus monkeys, consisting of 1 male and 2 females per group, received a single dose on Day 1 of the study in the right eye with rAAV at 2.8x1010vg / eye or 1.5x1011vg / eye. The left eyes of the animals were treated concomitantly with the vehicle. The dose volume was 50 gL / eye for both the right and left eyes. [000178] In-life parameters included daily mortality / interval assessments, detailed clinical observations once before dosing during Week -1, before dosing and 1, 2, 4, and 8 hours after dosing on Day 1 of the Study, and on days 2 to 7, and then weekly for the remainder of the study. Body weights were assessed before administration and weekly during the study. Ophthalmological examinations were performed before testing for all animals and on days 2, 4 (± 1), 7 and during weeks 2, 3, 4, 5, 6, 9, and 12. [000179] Intraocular pressure was assessed before the study and on days 2, 4 (± 1), 7 and during weeks 2, 3, 4, 5, 6, 9 and 12. Fluorescein angiography, optical coherence tomography (OCT) and color video-field fundus imaging were assessed before the study and during weeks 3, 6 and 12. Finally, electroretinography (ERG) assessments were performed before administration and during weeks 6 and 12, prior to terminal sacrifice. [000180] Intravitreal administration of rAAV doses of 2.8 x 1010 or 1.5 x 1011vg / eye was well tolerated in cynomolgus monkeys for 12 weeks after irrigation, as evidenced by mortality, clinical observation, ophthalmological pathology, clinical pathology, macroscopic pathology, and organ weight parameters. There were no findings of intraocular inflammation at any time, nor were there any rAAV-related changes in IOP values ​​or ocular structure, as identified by fluorescein angiography or anterior and posterior segment OCT, nor were there any treatment-related effects on retinal function identified by ERG assessment. [000181] There were no definitive treatment-related effects with rAAV on clinical pathology parameters in either sex after the Petition 870250109947, dated 01 / 12 / 2025, pages 105 / 129 102 / 102 intravitreal administration of rAAV at 2.8x1010 or 1.5x1011 vg / eye until day 80. Small fluctuations in hematological and clinical chemistry findings were probably attributable to methylprednisolone administration and were not considered related to rAAV treatment. [000182] Overall, rAAV was well tolerated after a single intravitreal dose in non-human primates of up to 1.5x1011 vg / eye, with no definite treatment-related effects or changes in ocular structure or function. [000183] Although the materials and methods of this invention have been described in terms of preferred embodiments, it will be evident to those skilled in the art that variations can be applied to the method described herein without departing from the concept, spirit, and scope of the invention. All such substitutes and similar modifications, apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the invention. Petition 870250109947, dated 01 / 12 / 2025, pp. 106 / 129

Claims

1 / 10 CLAIMS 1. Recombinant Adeno-Associated Virus, (rAAV), comprising (i) a variant AAV capsid protein comprising a heterologous peptide with a length of 7, 8, 9, 10 or 11 amino acids covalently inserted into the GH loop of the capsid protein relative to a corresponding parental AAV capsid protein, characterized in that the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO:1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a complement regulatory factor H (CFH) protein or a fragment thereof.

2. Recombinant Adeno-Associated Virus, according to Claim 1, characterized in that the insertion peptide has 1 to 3 amino acid spacers (Y1-Y3) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1), preferably in that the insertion peptide is LAISDQTKHA (SEQ ID NO:2).

3. Recombinant Adeno-Associated Virus, according to Claim 1 or 2, characterized in that the insertion site is located between the amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO:47) or the corresponding position in the capsid protein of another AAV serotype.

4. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 3, characterized in that the capsid protein comprises one or more amino acid substitutions with respect to VP1 of AAV2 (SEQ ID 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, Petition 870250091693, dated 07 / 10 / 2025, p. 189 / 199 2 / 10 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, with a greater preference for a P34A amino acid substitution.

5. Recombinant Adeno-Associated Virus, according to Claim 4, characterized in that the capsid protein comprises a P34A amino acid substitution relative to VP1 of AAV2 and comprises an amino acid sequence at least 90% identical, at least 95% identical, at least 98% identical or 100% identical along the entire length of the amino acid sequence presented as SEQ ID NO:42, preferably wherein the capsid protein consists of the amino acid sequence shown as SEQ ID NO:

42.

6. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 5, characterized in that the rAAV has an increased infectivity, preferably at least twice as high, of a retinal cell compared with the infectivity of the retinal cell by an AAV comprising the capsid protein of the corresponding parental AAV.

7. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 6, characterized in that the capsid protein of the variant AAV comprises an amino acid sequence with 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

42.

8. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 7, characterized in that the rAAV comprises a heterologous acid comprising from 5' to 3': (a) an inverted terminal repeat (b) a promoter (c) a nucleotide sequence encoding the CFH protein or a fragment thereof (d) a polyadenylation sequence and / or a WPRE sequence and (e) an inverted terminal repeat.

9. Recombinant Adeno-Associated Virus, according to Claim 8, characterized in that the rAAV in which the inverted terminal repeat is an inverted terminal repeat of AAV2.

10. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 9, characterized in that the CFH protein or a fragment thereof comprises one or more short consensus repeats (SCRs).

11. Recombinant Adeno-Associated Virus, according to Claim 10, characterized in that the CFH protein fragment consists of SCRs: 1, 2, 3, 4, 19, 20 and one or more of SCR 7, 17 and / or 18, and optionally a leader sequence and one or more linking sequences.

12. Recombinant Adeno-Associated Virus, according to Claim 10, characterized in that the CFH protein fragment consists of SCRs: 1, 2, 3, 4, 19, 20 and one or more of SCR 7, 17 and / or 18 and one or more of SCR5, SCR6, SCR8, SCR16 and, optionally, a leader sequence and one or more linking sequences.

13. Recombinant Adeno-Associated Virus, according to any one of Claims 11 to 12, characterized in that the CFH protein fragment no longer has at least SCR5, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15 and / or SCR16.

14. Recombinant Adeno-Associated Virus, according to any one of Claims 10 to 13, characterized in that the CFH protein fragment consists of a combination of SCR domains selected from one or more of: Petition 870250091693, dated 10 / 07 / 2025, page 191 / 199 4 / 10 (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 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, 6-8, 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, 6-8, and 17-20, and optionally a leader sequence and one or more linking sequences.

15. Recombinant Adeno-Associated Virus, according to any one of Claims 10 to 14, characterized in that the CFH protein fragment comprises SCR1-4, 6-8 and 17-20, preferably in Petition 870250091693, dated 07 / 10 / 2025, page 192 / 199 5 / 10 that the CFH protein fragment no longer comprises SCR5 and SCR916.

16. Recombinant Adeno-Associated Virus, according to any one of Claims 10 to 15, characterized in that the CFH protein fragment comprises at least one linker of 1 to 18 amino acids located between one or more of the SCRs.

17. Recombinant Adeno-Associated Virus, according to any one of Claims 10 to 16, characterized in that 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, where () indicates optional component(s), “L” refers to a ligand, and each of L1, L2, L3, L4, L4', L5, L5', L5”, L5''', L5'''' and L6 may be absent or selected. regardless of an amino acid sequence of 1 to 12 to 18 amino acids.

18. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 17, characterized in that the CFH protein fragment comprises an amino acid sequence with at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

34.

19. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 18, characterized in that the CFH protein fragment comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

34.

20. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 19, characterized in that the CFH protein fragment comprises an amino acid sequence with at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

34.

21. Recombinant Adeno-Associated Virus, according to any one of Claims 1 to 20, characterized in that the CFH protein fragment comprises an amino acid sequence that is 100% identical to the amino acid sequence shown in SEQ ID NO:

34.

22. Recombinant Adeno-Associated Virus, according to any one of Claims 10 to 21, characterized in that the CFH protein fragment comprises at least one glycosylation site in one or more SCRs.

23. Recombinant Adeno-Associated Virus, according to Claim 22, characterized in that the glycosylation site is modified in one or more of SCR1, SCR2, SCR3, SCR4, SCR17, SCR18, SCR19 and / or SCR20.

24. Recombinant Adeno-Associated Virus, according to Claim 23, characterized in that the glycosylation site is modified in one or more of SCR17 and / or SCR18.

25. Recombinant Adeno-Associated Virus, according to Claim 23, characterized in that the glycosylation site is modified in SCR17 and SCR18.

26. Recombinant Adeno-Associated Virus, according to any one of Claims 22 to 25, characterized in that the CFH protein fragment comprises SCR1-4, 6-8 and 17-20 and in that the glycosylation site is modified in SCR 17 and SCR18, preferably in that the CFH protein fragment no longer comprises SCR5 and SCR9-16.

27. Recombinant Adeno-Associated Virus, according to any one of Claims 22 to 26, characterized in that the CFH protein fragment comprises an amino acid sequence with 100% sequence identity to the sequence established in SEQ ID NO: 34 and in which the glycosylation site is modified in SCR17 and SCR18.

28. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 27, characterized in that the promoter is a ubiquitous promoter.

29. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 27, characterized in that the promoter is a tissue-specific promoter.

30. Recombinant Adeno-Associated Virus, according to Claim 28, characterized in that the promoter is a CAG promoter.

31. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 30, characterized in that rAAV comprises a heterologous nucleic acid comprising a nucleotide sequence with at least 80% sequence identity to the nucleotide sequence shown in any one of the SEQ ID Nos: 43-46.

32. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 31, characterized in that rAAV comprises a heterologous nucleic acid comprising a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence shown in any one of the SEQ ID Nos: 43-46.

33. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 32, characterized in that the rAAV comprises a heterologous nucleic acid comprising a nucleotide sequence with at least 95% identity of Petition 870250091693, dated 10 / 07 / 2025, page 195 / 199 8 / 10 sequence with the nucleotide sequence shown in any one of SEQ ID Nos: 43-46.

34. Recombinant Adeno-Associated Virus, according to any one of Claims 8 to 33, characterized in that the rAAV comprises a heterologous nucleic acid comprising the nucleotide sequence set forth in any one of the SEQ ID Nos: 43-46.

35. Host Cell, characterized in that it comprises the rAAV, as defined in any one of Claims 8 to 34.

36. Pharmaceutical Composition, characterized in that it comprises rAAV, as defined in any one of Claims 8 to 34, and a pharmaceutically acceptable carrier, diluent, excipient or buffer.

37. Method for Treating Dry Age-Related Macular Degeneration (dry AMD) in an individual in need thereof, characterized in that it comprises administering to the individual a therapeutically effective amount of rAAV, as defined in any of Claims 8 to 34, or of the Pharmaceutical Composition, as defined in Claim 36.

38. Method for Treating Dry Age-Related Macular Degeneration, according to Claim 37, characterized in that the rAAV or pharmaceutical composition is administered to the individual by periocular, intravitreal, suprachoroidal or subretinal administration at a dosage of 10⁸ vector genomes (vg) / eye to 10¹³ vg / eye, preferably at a dosage of 6x10⁹ vg / eye to 6x10¹⁰ vg / eye, or at a dosage of 1x10⁸ vg / eye, 2x10⁸ vg / eye, 3x10⁸ vg / eye, 4x10⁸ vg / eye, 8x10⁸ vg / eye, 3x10⁹ vg / eye, 5x10⁸ vg / eye, x10⁸ vg / eye, 4x10⁹ vg / eye, 6x10⁸ vg / eye, 1x109 vg / eye, 5x109 vg / eye, 7x108 vg / eye 2x109 vg / eye 6x109 vg / eye Petition 870250091693, dated 07 / 10 / 2025, page. 196 / 199 9 / 10 7x109 vg / eye, 2x1010 vg / eye, 6x1010 vg / eye, 1x1011 vg / eye, 5x1011 vg / eye, 9x1011 vg / eye 8x109 vg / eye, 3x1010 vg / eye, 7x1010 vg / eye, 2x1011 vg / eye, 6x1011 vg / eye, 1x1012 vg / eye.9x109 vg / eye, 4x1010 vg / eye, 8x1010 vg / eye, 3x1011 vg / eye, 7x1011 vg / eye, 1x1010 vg / eye, 5x1010 vg / eye, 9x1010 vg / eye, 4x1011 vg / eye, 8x1011 vg / eye.

39. Method for Treating Dry Age-Related Macular Degeneration, according to Claim 38, characterized in that the rAAV or the pharmaceutical composition is administered to the individual by intravitreal administration at a dosage of 1x 109 vg / eye to 1x 1010 vg / eye.

40. Method for Treating Geographic Atrophy (GA) in an individual in need thereof, characterized in that it comprises administering to the individual a therapeutically effective amount of rAAV, as defined in any one of Claims 8 to 34, or of the Pharmaceutical Composition, as defined in Claim 36.

41. Method for Treating Geographic Atrophy, according to Claim 40, characterized in that the rAAV or the pharmaceutical composition is administered to the individual by periocular, intravitreal, suprachoroidal or subretinal administration, at a dosage of 10⁸ vector genomes (vg) / eye to 10¹³ vg / eye, preferably at a dosage of 6x10⁹ vg / eye to 6x10¹⁰ vg / eye, or at a dosage of 1x10⁸ vg / eye, 2x10⁸ vg / eye, 3x10⁸ vg / eye, 4x10⁸ vg / eye, 5x10⁸ vg / eye, 6x10⁸ vg / eye, 7x10⁸ vg / eye, 8x10⁸ vg / eye, x10⁸ vg / eye, 1x10⁹ vg / eye, 2x109 vg / eye, 3x109 vg / eye, 4x109 vg / eye, 5x109 vg / eye, 6x109 vg / eye, 7x109 vg / eye, 8x109 vg / eye, 9x109 vg / eye, 1x1010 vg / eye, 2x1010 vg / eye, 3x1010 vg / eye, 4x1010 vg / eye, 5x1010 vg / eye, 6x1010 vg / eye, 7x1010 vg / eye, 8x1010 vg / eye, 9x1010 vg / eye, 1x1011 vg / eye, 2x1011 vg / eye, 3x1011 vg / eye, 4x1011 vg / eye, 5x1011 vg / eye, Petition 870250091693, dated 07 / 10 / 2025, page.197 / 199 10 / 10 6x1011 vg / olho, 7x1011 vg / olho, 8x1011 vg / olho, 9x1011 vg / olho or 1x1012 vg / olho.

42. Method for Treating Geographic Atrophy, according to Claim 41, characterized in that the rAAV or the pharmaceutical composition is administered to the individual by intravitreal administration at a dosage of 1 x 109 vg / eye to 1 x 1010 vg / eye.

43. Method for administering rAAV, as defined in any of Claims 8 to 34, or the pharmaceutical composition, as defined in Claim 36, into the eye of an individual, characterized in that the rAAV or the pharmaceutical composition is administered to the individual by periocular, intravitreal, suprachoroidal, or subretinal administration.

44. Method for administering rAAV or the pharmaceutical composition, according to Claim 43, characterized in that the rAAV or the pharmaceutical composition is administered to the individual's eye by intravitreal administration.

45. Use of Recombinant Adeno-Associated Virus, as defined in Claims 1 to 34, or of Pharmaceutical Composition, as defined in Claim 36, characterized in that it is in the manufacture of medicaments to treat complement-related disorders. Petition 870250091693, dated 10 / 07 / 2025, pp. 198 / 199

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