AAV variant for the treatment of complement imbalance

Abstract

Recombinant AAV (rAAV) comprising a variant adeno-associated virus (AAV) capsid and a transgene encoding a human factor H variant is provided. Also provided are a method for delivering the transgene to the retina, and a method for treating dry age-related macular degeneration and geographic atrophy secondary to age-related macular degeneration by contacting retinal cells with rAAV. The variant AAV capsid protein may contain a peptide insertion ("heterogeneous peptide" or "peptide insertion") of about 7 to 20 amino acids within the GH loop of the capsid protein, preferably within the surface-exposed region of the GH loop, compared to the corresponding parental AAV capsid protein.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits under U.S. Provisional Patent Application No. 63 / 494,925, filed on April 7, 2023, and U.S. Provisional Patent Application No. 63 / 586,227, filed on September 28, 2023, the entire contents of each of these applications are incorporated herein by reference.

[0002] Submission of sequence listings via EFS-WEB A computer-readable XML file titled "090400-5022-WO-Sequence-Listing," created on April 2, 2024, with a file size of approximately 87,900 bytes, contains the sequence listing of this application, and the entire file is incorporated herein by reference. [Background technology]

[0003] Background of the Invention Many human diseases are caused by complement dysregulation, leading to complement-mediated autotissue damage. Complement dysregulation can result from mutations in complement regulators or their related genes, either somatically or germline-wise, causing these regulators to no longer function properly. In particular, there are common and rare human diseases 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), peptides, or other small molecules that bind to and block specific alternative or terminal complement pathway components. A clinically validated example is eculizumab, a humanized mAb against complement C5, 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 propagin (fP), and cyclic peptides that bind to and inhibit C3. Limitations of these approaches include the need for repeated and inconvenient intravenous (IV) administration by the patient. Furthermore, these treatments risk compromising host defense because they block alternative or terminal pathways. In fact, patients receiving eculizumab treatment must be vaccinated against bacterial strains that cause fatal meningitis, and these patients also receive prophylactic antibiotic treatment before being treated with approved mAb drugs.

[0005] Other approaches have explored recombinant regulatory proteins such as soluble DAF, CR1, and CRIg, as well as proteins containing minimal domains of the liquid phase regulator fH (N-terminal short consensus repeats [SCR]1-5 and C-terminal SCR19-20), or fusion proteins between fH and CR2 (TT30). However, large-scale heterologous expression of such proteins as therapeutic agents requires considerable effort, animal studies have shown their rapid in vivo clearance rates after administration, and such therapeutic strategies are cumbersome and impractical due to the need for multiple and frequent administrations of such protein drugs.

[0006] There is still a need in the art for compositions useful in treating complementarity-mediated diseases with longer-lasting efficacy. [Overview of the Initiative]

[0007] Summary of the Invention This specification describes recombinant AAV (rAAV) virions comprising a variant AAV capsid sequence that encapsulates a heterogeneous nucleic acid containing a modified human complement regulator H(fH) gene operably linked to an expression control sequence, wherein the human fH(hfH) gene encodes a soluble hfH protein variant that retains complement regulatory function, and the fH variant comprises short consensus repeats (SCRs) 1, 2, 3, 4, 19, and 20. In some embodiments, the rAAV comprises a heterogeneous nucleic acid containing an hfH gene encoding a soluble hfH protein variant that retains complement regulatory function, and the fH variant comprises, consists of, or essentially consists of SCRs, where the SCRs are selected from the group consisting of SCR1, SCR2, SCR3, SCR4, SCR6, SCR7, SCR8, SCR17, SCR18, SCRsCR19, and SCR20.

[0008] In a related embodiment, an rAAV virion is provided comprising a variant AAV capsid sequence for encapsulating heterologous nucleic acids, wherein the nucleic acid comprises a nucleotide sequence encoding a modified hfH variant comprising a leader sequence and human complement receptor SCRs, the SCRs being (a) SCR1-4, 7 and 19-20; (b) SCR1-4, 6, 7, 19-20; (c) SCR1-4, 7, 8, 19-20; (d) SCR1-4, 6, 7, 8 and 19-20; (e) SCR1-4, 17 and 19- Selected from the group consisting of 20; (f) SCR1-4, 18-20; (g) SCR1-4, 17-20; (h) SCR1-4, 7 and 18-20; (i) SCR1-4, 6, 7, 18-20; (j) SCR1-4, 7, 8 and 18-20; (k) SCR1-4, 6, 7, 8 and 18-20; (l) SCR1-4, 7, 17-20; (m) SCR1-4, 6, 7, 17-20; (n) SCR1-4, 7, 8 and 17-20; or (o) SCR1-4, 6, 7, 8 and 17-20. If necessary, at least one glycosylation site is modified to at least one SCR.

[0009] The rAAV variant AAV capsid protein contains a peptide insertion ("heterogeneous peptide" or "peptide insertion") of about 7 to 20 amino acids within the GH loop of the capsid protein, preferably within the surface-exposed region of the GH loop, compared to the corresponding parental AAV capsid protein, wherein the peptide insertion contains the amino acid sequence ISDQTKH (SEQ ID NO: 1). In some preferred embodiments, the peptide insertion has 1 to 3 amino acid spacer amino acids (Y1 to Y3) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 1), each of Y1 to Y3 independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. In some embodiments, the peptide insertion contains 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In a particularly preferred embodiment, the peptide insertion contains, essentially consists 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 VP1 of AAV2, or between amino acids 588 and 589 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. In some embodiments, the capsid protein further comprises one or more amino acid substitutions for the VP1 capsid of AAV2 or one or more corresponding substitutions in another AAV serotype, and preferably, the capsid protein further comprises a P34A amino acid substitution for the VP1 capsid of AAV2 or a corresponding substitution in another AAV serotype.

[0010] In other embodiments, a method is provided for delivering heterologous nucleic acids comprising a nucleotide sequence encoding a soluble hfH protein variant described herein to a mammalian subject, comprising administering an effective amount of rAAV described herein or a pharmaceutical composition comprising the same to the mammal, preferably administered by intravitreal injection. In some embodiments, the heterologous nucleic acids are delivered to target retinal cells, e.g., target photoreceptor cells (e.g., rod cells, cone cells), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia cells), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelial (RPE) cells.

[0011] In some embodiments, detectable plasma levels of the hfH variant are present in the subject for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 6 months after administration of rAAV to the subject. In a particularly preferred embodiment, rAAV is administered to the subject by intravitreal administration.

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

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

[0014] In a further embodiment, a method is provided for treating subjects requiring treatment for dry age-related macular degeneration (AMD) (e.g., late dry AMD) by delivering rAAV or an effective amount of a pharmaceutical composition containing rAAV described herein to the subject. In a related embodiment, rAAV or the pharmaceutical composition is administered to the subject for the treatment of geographic atrophy secondary to AMD. Preferably, rAAV or the pharmaceutical composition is administered to the subject by intravitreal injection. Brief explanation of the drawing [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a schematic diagram of a transgene cassette containing nucleotide sequences encoding codon-optimized human fH variants, including 5' and 3' AAV reverse terminal repeats (ITRs) derived from the AAV2 genome, a polyadenylation signal (SV40 late polyA), and SCR1-4, 6-8, and 17-20 ("miniCFH") operably linked to the CAG promoter.

[0016] [Figure 2-1]Figures 2A–2F show the expression, activity, and function of miniCFH in human cells after transfection with an AAV plasmid containing the transgene cassette shown in Figure 1. Figures 2A–2B show dose-dependent miniCFH and full-length CFH expression in protein lysates from the supernatant of plasmid-transfected HEK293T cells, as measured by ELISA (Figure 2A) and Western blotting (Figure 2B). Figure 2C shows C3b binding: pre-incubation with a blocking CFH antibody reduces CFH and miniCFH signals, while pre-incubation with a control antibody or mock incubation does not reduce CFH and miniCFH signals, indicating that miniCFH directly binds to C3b. Figure 2D shows heparin binding: pre-incubation with a blocking CFH antibody reduces recombinant CFH and miniCFH signals, indicating that miniCFH binds and retains heparin-binding activity. 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 a C3 convertase on binding factor B in the presence of CFH as a cofactor, or it can be degraded by complement factor I (CFI) to become a smaller inactive fragment that disrupts the alternative complement cascade. These smaller fragments resulting from C3b degradation are observed by Western blotting. Figure 2F shows complement inhibition (Wieslab® Complement system Alternative pathway assay), demonstrating that the supernatant from cells transfected with an AAV plasmid cassette resulted in similar inhibition of membrane attack complex (MAC) formation as with full-length CFH and anti-C5 antibody controls. NT = Untransfected [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0017] [Figure 3-1]Figures 3A-3C show the expression, activity, and function of miniCHH in human RPE cells. Retinal pigment epithelial (RPE) cells derived from iPSCs were transduced with (i) a variant AAV capsid protein containing the amino acid sequence of SEQ ID NO: 42 and (ii) rAAV containing nucleic acid with the expression cassette shown in Figure 1. RPE cells were transduced at different MOIs (metabolic units of the vector genome per cell (vg)). The culture medium was collected 4 and 7 days after transduction, and secreted miniCHH expression was assayed by ELISA (Figure 3A). Complement inhibitory activity was assayed by the Wieslab® Complement system Alternative pathway assay (Figure 3B). Complement inhibition was also evaluated by ICC (Figure 3C). Alternative complement was activated in iPSC-derived RPE cells 6 days post-transduction by adding zymosan at a final concentration of [1%] NHS and [0.5 mg / ml] NHS to the culture medium. Cells were fixed 24 hours and 7 days after transduction and stained with primary MAC antibody IgG2aKappa mouse anti-human (Abcam catalog number 59835) or isotype control mouse IgG2aKappa (Invitrogen catalog number 14-4724-82). The nuclei were stained with secondary A488 goat anti-mouse (Invitrogen catalog number A11001) and DRAQ5 (ThermoFisher catalog number 62251). Images were taken using a fluorescence microscope. miniCFH expressed by cells at all MOIs inhibited MAC formation in the culture. Anti-C5 antibody was used as a positive control for complement inhibition and prevention of MAC formation. NT = not transduced [Figure 3-2] Same as above.

[0018] [Figure 4] Figure 4 shows the LCMS quantification of short CFH (sCFH) concentrations in aqueous humor (AH) samples from NHP after intravitreous (bilateral) administration of a specified dose of rAAV containing (i) a variant AAV capsid protein with the amino acid sequence of SEQ ID NO: 42 and (ii) nucleic acid containing the expression cassette shown in Figure 1 to NHP.

[0019] [Figure 5] Figure 5 shows sCFH RNA expression (by in situ hybridization analysis) in untransfected HEK 293T cells and HEK 293T cells after transfection with rAAV containing (i) a variant AAV capsid protein containing the amino acid sequence of SEQ ID NO: 42 and (ii) nucleic acid containing the expression cassette shown in Figure 1.

[0020] [Figure 6] Figure 6 shows the results of in situ hybridization analysis of eye tissue from non-human primates after intravitreous (bilateral) administration of a specified dose of rAAV containing (i) a variant AAV capsid protein with the amino acid sequence of SEQ ID NO: 42 and (ii) nucleic acid containing the expression cassette shown in Figure 1.

[0021] [Figure 7-1]Figure 7A is a Western blot showing the detection of CFH protein after transfection with CFH or miniCFH constructs. The analyzed NT cell supernatant did not show a band as a negative control. Recombinant full-length CFH (250 kDa, lane 5) was used as a positive control. Arrows indicate the theoretical MW of miniCFH (80 kDa) and full-length CFH (139 kDa). The ladder is shown on the left. Figure 7B shows the CFH protein concentrations in the supernatant of HEK293 T cells transfected with 0.125 or 0.5 μg of CFH or miniCFH constructs, determined using human H factor ELISA, with ****p < 0.0001 compared to NT. ^p = 0.0003 (comparison between 0.125 μg DNA sample and 0.5 μg DNA sample). Figure 7C shows the supernatant from HEK cells transfected with miniCFH DNA (0.5 μg DNA / well, 15 μl supernatant), which resulted in complement inhibition comparable to the positive control eculizumab (6 μg) compared to NT, with **p=0.0028 and ***p=0.0002. Figure 7D is a Western blot of six reactions containing various components identified in the legend at the top of the image. It shows the purified recombinant protein components (purified CHF, CFI, C3b) and the supernatant after transfection (CFH supernatant). Cleavage products are highlighted with arrows. When the supernatant was added to the reaction after transfection with miniCFH, an increase in cleavage products was observed compared to the NT supernatant (lane 3) (lane 2). Positive control reactions using all three recombinant proteins (CFH, CFI, C3b) are shown in lane 4, and negative controls are shown in lanes 5-7. Figure 7E shows miniCFH in the supernatant after transfection that bound to C3b and disappeared after CFH antibody incubation. *p=0.05 for CFH antibody, **p=0.01 for CFH antibody. Figure 7F shows miniCFH in the supernatant after transfection that bound to heparin and disappeared after CFH antibody incubation. *p=0.05 for CFH antibody, **p=0.01 for CFH antibody. NT = error bar ± standard deviation of untransfected cells; n = 3 experimental replicates; one-way ANOVA, Tukey's post-hoc test. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0022] [Figure 8] Figure 8 shows the transduction of iPSC-RPE cells resulting in miniCFH protein expression. CFH concentrations [nM] in the supernatant from RPE cells transduced with rAAV (a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding a truncated form of complement factor H ("miniCFH") with the amino acid sequence shown in SEQ ID NO: 34) at different MOIs showed a dose-response in CFH expression. Subsequently, signals exceeding those observed from NT cells would correlate with miniCFH expressed from rAAV. MOI = multiplicity of infection; NT = untransduced. Error bars ± standard deviation; n = 4 experimental repeats. Significance relative to NT, **p=0.0071 and ***p=0.0007. Significance for comparison between MOI 5,000 and MOI 50,000, ^p=0.0477. Statistics; one-way ANOVA, Tukey's post-hoc test.

[0023] [Figure 9]Figures 9A and 9B show inhibition of the surrogate complement pathway by miniCFH expressed in iPSC-derived RPE cells transduced with rAAV, which contains a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter. Cell supernatants from RPE cells transduced with rAAV were assayed for complement inhibitory activity using two methods: Wieslab assay (Figure 9A) and soluble C5b-9 ELISA (Figure 9B). Figure 9A shows inhibition of surrogate complement activity resulting from the addition of a specific sample to the Wieslab assay using normal human serum. The reading of activity upon addition of NT supernatant is defined as 0% inhibition. A dose-response was observed, with higher MOI doses of rAAV increasing complement inhibition. Error bars ± standard deviation; n=3 experimental repeats. Significance relative to NT unless otherwise specified. For comparison between MOI 5,000 and MOI 50,000, *p=0.0342, **p=0.0026, ****p<0.0001, ^^p=0.0078. Figure 9B shows the soluble C5b-9 concentrations in samples where supernatants from RPE transduced with rAAV at different MOIs showed an inverse dose-response. The concentration of soluble C5b-9 was normalized, with C5b-9 concentration in NT supernatant = 1. Error bars ± standard deviation; n=4 experimental replicates. Significance relative to NT, **p=0.0071 and ****p<0.0001. Significance for comparison between MOI 5,000 and MOI 50,000, ^p=0.0272. Statistics; one-way ANOVA, Tukey's post-hoc test. NT, untransduced; MOI, infection multiplicity.

[0024] [Figure 10]Figure 10 shows the acceleration of C3 convertase degradation activity by expressed miniCFH. C3b cleavage products were detected by Western blotting in samples of purified C3b incubated with purified CFI and supernatant or purified full-length CFH. Samples lacking both CFI and CFH showed no cleavage of the C3b α chain, while samples with either or the other of purified CFI or CFH showed low levels of cleavage products. Incubation of C3b with both CFI and miniCFH resulted in extensive cleavage, a significant reduction in the α chain, and an increased abundance of cleavage products. MiniCFH from cell supernatant instead of purified CFH reproduced these effects. NT, untransduced; MOI, infection multiplicity. Representative blot, n=4 experimental replicates.

[0025] [Figure 11-1] Figures 11A and 11B show that transduction of RPE with rAAV (a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42, and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter) before induction of complement activation in culture protects RPE from MAC deposition. Figure 11A shows immunocytochemistry (ICC) analysis. Representative image, n=4 experimental replicates. Scale bar = 200 μm. Figure 11B shows flow cytometry analysis. The percentage of total RPE cells with MAC deposition at different MOIs shows the dose-response. Error bars ± standard deviation; n=4 experimental replicates. Significance relative to NT, ****p<0.0001. Statistics; one-way ANOVA, Tukey's post-hoc test. NT, untransduced; MOI, infection multiplicity. MAC = membrane attack complex. [Figure 11-2] Same as above.

[0026] [Figure 12]Figure 12 shows the surrogate complement inhibitory activity of rAAV (containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter) in an in vitro RPE disease model. iC3b concentrations in the supernatant of disease models derived from RPE transduced with rAAV were decreased compared to NT. iC3b concentrations were normalized, with iC3b concentration in NT supernatant = 1. NT, untransduced; MOI, infection multiplicity; RPE, retinal pigment epithelial cells. Error bars ± standard deviation; n = 4 experimental replicates. Significance relative to NT, **p = 0.0021 and ****p < 0.0001. Statistics; one-way ANOVA, Tukey's post-hoc test.

[0027] [Figure 13] Figure 13 shows the concentration of miniCFH in the aqueous humor of non-human primates after intravitreous administration of rAAV containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to a CAG promoter. MiniCFH protein expression in the aqueous humor was measured by LC-MS. Fluid expression is expressed as ng / mL. Data from 3-4 animals are summarized. Mean + standard deviation.

[0028] [Figure 14] Figure 14 shows the concentrations of miniCFH in retinal tissue and RPE / choroidal tissue (RPE / Choroid) in non-human primates after intravitreal administration of rAAV containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous 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 / choroidal tissue (RPE / C) of NHP eyes after IVT of rAAV at (left) 5 × 10¹¹ vg / eye and (right) 1 × 10¹⁰ vg / eye, measured by LC-MS at end-necropsy. Data from 3-4 animals are summarized. Fluid expression is shown as ng / mL. Tissue expression is shown as ng / g. Mean + standard deviation. [Modes for carrying out the invention]

[0029] Detailed description of the invention definition

[0030] As used herein, the term “functional fH variant” includes fH variants characterized by having complement-modulating activity (cofactor activity) located in SCR1-4, and, optionally, functional C3b-binding and GAG-binding activity characteristic of wild-type fH (located in wild-type SCR7 and SCR19-20). In some embodiments, the modified fH variant has more than 100% of the wild-type fH cofactor activity and / or GAG-binding activity. In other embodiments, the modified fH variant has less than about 95% to about 100% of wild-type functional fH. For example, the modified fH variant may have at least 50%, more preferably at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the cofactor activity present in functional wild-type fH. In another embodiment, the modified fH variant may alternatively or additionally have at least 50%, more preferably at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the GAG ​​binding capacity of the functional fH. Methods for determining cofactor activity, binding, and / or increased circulating half-life compared to the hfH protein are known in the art.

[0031] As used herein, when referring to SCR#-##, the domain includes the endpoint and is the same as "SCR#,...SCR##". In certain embodiments, dots are used between 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 domain.

[0032] The term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally exists). For example, a polynucleotide present in the natural state of a plant or animal is not isolated, but the same polynucleotide separated from a naturally occurring adjacent nucleic acid is considered "isolated."

[0033] As used herein, “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are typically not translated into amino acids but can be considered part of a coding region, while any adjacent sequences, such as promoters, ribosome-binding sites, transcription terminators, or introns, are not part of a coding region. The boundaries of a coding region are typically determined by a 5' start codon encoding the amino terminus of the resulting polypeptide and a 3' translation stop codon encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions may exist in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Thus, a single vector may contain only one coding region, or it may contain two or more coding regions.

[0034] As used herein, the term “regulatory region” refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that affects 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 stem-loop structures. When a coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located 3' of the coding sequence.

[0035] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule,” and refers to polymers of nucleotides.

[0036] A gene product, such as a polynucleotide encoding a polypeptide, may include promoters and / or other transcriptional or translational regulatory elements that are operably associated with one or more coding regions. In an operable association, the coding region of a gene product, such as a polypeptide, associates with one or more regulatory regions to place the expression of the gene product under the influence or control of the regulatory regions. For example, if the induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and the nature of the linkage between the promoter and the coding region does not interfere with the promoter's ability to direct the expression of the gene product or the ability of the DNA template to be transcribed, then the coding region and promoter are "operably associated". Other transcriptional regulatory elements other than promoters, such as enhancers, operators, repressors, and transcription termination signals, can also operably associate with coding regions to direct gene product expression.

[0037] "Transcriptional regulatory sequences" refer to DNA regulatory sequences such as promoters, enhancers, and terminators that provide expression for coding sequences in host cells. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, e.g., cytomegalovirus (pre-initial promoter in combination with intron-A), Simianvirus 40 (initial promoter), and promoter and enhancer segments derived from retroviruses (such as Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, e.g., actin, heat shock proteins, bovine growth hormone, and rabbit betaglobin, as well as other sequences that can regulate gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0038] Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, elements derived from ribosome binding sites, translation start and termination codons, and picornaviruses (in particular, internal ribosome entry sites or IRESs, also known as CITE sequences).

[0039] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of mRNA into polypeptides. Expression produces a “gene product.” As used herein, a gene product may be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0040] "Promoter" and "promoter sequence" refer to interchangeable DNA sequences that can control the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. A promoter may be entirely derived from a native gene, or it may consist of different elements derived from different naturally occurring promoters, or it may contain synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. In most cases, promoters that express a gene in most cell types are generally called "constitutive promoters." Promoter that expresses a gene in a specific cell type are generally called "cell-specific promoters" or "tissue-specific promoters." Promoter that expresses a gene at a specific stage of development or cell differentiation are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoter that is induced and expresses a gene after exposure or treatment of cells with promoter-inducing drugs, biological molecules, chemicals, ligands, light, etc. are generally called "inducible promoters" or "modulatory promoters." In most cases, the precise boundaries of regulatory sequences are not fully defined, and it is further recognized that DNA fragments of different lengths can have the same promoter activity.

[0041] The term "plasmid" refers to extrachromosomal elements that often carry genes that are not part of the cell's central metabolism, and typically take the form of a circular double-stranded DNA molecule. Such elements can be single-stranded or double-stranded DNA or RNA autonomous replication sequences, genomic integration sequences, phages or nucleotide sequences originating from any source, and can be linear, circular or supercoiled, where some nucleotide sequences are ligated or recombined into a unique construct that can introduce promoter fragments and DNA sequences for selected gene products into the cell, along with appropriate 3' untranslated sequences.

[0042] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids in the two sequences is the same when compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCC) package from Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth.Mol.Biol.70:173-187(1997). Additionally, the GAP program, which uses the Needleman and Wunsch alignment methods, can be used to align sequences. See J.Mol.Biol.48:443-453(1970).

[0043] The term "amino acid substitution" and its synonyms used above are intended to encompass the modification of an amino acid sequence by substituting one amino acid with another. Substitutions may be conservative or non-conservative. When referring to two amino acids, the term "conservative" is intended to mean that the amino acids share common properties recognized by those skilled in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. Common properties may also include amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and can be used as amino acid substitutions in embodiments. Methods for substituting amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. In this specification, references to “one or more” are intended to encompass, for example, individual embodiments 1, 2, 3, 4, 5, 6, or more.

[0044] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes (a) preventing the development of the disease in subjects who are susceptible to the disease (and / or symptoms caused by the disease) or at risk of acquiring the disease but have not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., stopping its development; and (c) reducing the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., remission of one or more symptoms of the disease.

[0045] As used herein, the term “treating complement factor H impairment” may encompass alleviating, reducing, and / or improving symptoms associated with complement factor H impairment, which can manifest as several different phenotypes, including asymptomatic, recurrent bacterial infections, and renal failure, and / or preventing the development of further symptoms. This typically features decreased serum levels of factor H, complement component C3, and other terminal complement components, and indicates activation of alternative complement pathways. This impairment is associated with several renal diseases with varying clinical manifestations and progressions, including C3 glomerulopathy and atypical hemolytic uremic syndrome. This specification provides compositions and methods for treating one or more of the following conditions: dry age-related macular degeneration (AMD), geographic atrophy secondary to AMD, atypical hemolytic uremic conditions (e.g., including syndrome microangiogenic hemolytic anemia, thrombocytopenia, and acute renal failure), paroxysmal nocturnal hemoglobinuria (PNH), schizophrenia, and ischemic stroke; and / or compositions and methods for preventing or treating bacterial infections caused by the recruitment of bacterial pathogens such as Aspergillus species (e.g., Aspergillus species; Borrelia burgdorferi; B. duttonii B. recurrentis; Candida albicans; Francisella tularensis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes, or one of the five factor H-binding proteins of B. burgdorferi (CRASP-1, CRASP-2, CRASP-3, CRASP-4, or CRASP-5).

[0046] As used herein, the term “treat complement-related disorders” includes not only both of the complement factor H disorders identified above, but also alleviating, reducing, and / or improving the symptoms of other disorders related to uncontrolled alternative pathway complement regulation.

[0047] "Complement-mediated disorders" may encompass conditions associated with complement dysregulation that can manifest as several distinct phenotypes, including asymptomatic recurrent bacterial infections and various tissue injuries, including but not limited to kidney disease. Unless otherwise specified, both homozygous and heterozygous subjects are included in this definition. Complement dysregulation is typically caused by loss of functional mutations in complement regulatory proteins, including but not limited to fH, factor I (fI), and membrane cofactor proteins (MCP), or by autoantibodies against complement regulatory proteins, or by gain-of-function mutations in other complement proteins, including but not limited to C3 and factor B (fB). Complement dysregulation is typically, but not necessarily, characterized by decreased serum levels of factor H, complement component C3, fB, and other terminal complement components, and by activation of alternative and / or terminal complement pathways. Complement-mediated lesions that can be treated by the compositions and methods of the present invention include the following diseases with various clinical symptoms and progression: C3 glomerulosis (formally called membranoproliferative glomerulonephritis type II or MPGNII), which includes two known forms - high-density glomerulonephrosis (DDD) and C3 glomerulonephritis (C3GN).Thrombotic microangiopathy (TMA), including but not limited to atypical hemolytic uremic syndrome (aHUS), Shiga-like toxin-producing Escherichia coli HUS (STEC-HUS), and thrombotic thrombocytopenic purpura (TTP); retinal degenerative eye diseases, such as age-related macular degeneration (AMD), RPE degeneration, chorioretinal degeneration, photoreceptor degeneration, paroxysmal nocturnal hemoglobinuria (PNH), ischemia-reperfusion injury of all organs and the environment, rheumatoid arthritis, hemodialysis, diabetic nephropathy, diabetic vascular disease, asthma, systemic lupus erythematosus (SLE), ischemic stroke, abdominal aortic aneurysm (A This includes, but is not limited to, AA, anti-neutrophil cytoplasmic antibody (ANCA)-mediated vasculitis (ANCA vasculitis), ANCA-mediated hemorrhagic lung injury and disease, ANCA glomerulonephritis, graft-versus-host disease (GvHD), acute or delayed graft rejection in organ transplantation, Crohn's disease, psoriasis, multiple sclerosis, antiphospholipid syndrome, pre-eclampsia, atherosclerosis, neuromyelitis optica (NMO), autoimmune cutaneous bullous diseases, bullous pemphigoid (BP), Alzheimer's disease (AD), and bacterial infections caused by the mobilization of bacterial pathogens (e.g., Aspergillus species; Borrelia burgdorferi; B. duttonii B. recurrentis; Candida albicans; Fr and sella tularensis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pyogenes).

[0048] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to, but refer to: humans; mammals, including non-human primates such as monkeys; mammalian sports animals (e.g., horses); mammalian domestic animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0049] As used herein, the term “effective dose” is a quantity sufficient to produce a beneficial or desired clinical outcome. An effective dose may be administered in one or more doses. For the purposes of this disclosure, an effective dose of a compound (e.g., infectious rAAV virion) is a quantity sufficient to alleviate, improve, stabilize, reverse, prevent, delay or slow the progression (and / or associated symptoms) of a particular medical condition (e.g., a disorder associated with complement dysfunction). Thus, an effective dose of infectious rAAV virion is the amount of infectious rAAV virion that can effectively deliver heterologous nucleic acid to target cells (or target cells) of an individual. An effective dose may be determined preclinically by detecting the gene product (RNA, protein) encoded by the heterologous nucleic acid sequence within a cell or tissue using, for example, techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescence or other reporter readout. The effective dose can be clinically determined, for example, by detecting changes in disease onset or progression using methods known in the art, such as the 6-minute walk test described herein and known in the art, left ventricular ejection fraction, handheld dynamometry, and the Vignos scale.

[0050] Detailed explanation Novel rAAV virions encoding modified factor H (fH) gene and protein variants are described herein. These rAAV virions are characterized by persistent and robust expression of the fH protein in the retina, as well as increased efficacy in treating symptoms associated with factor H and other complement disorders.

[0051] The delivery of these rAAV virions to subjects requiring them can be achieved via several routes, preferably by intravitreal administration. Methods for using these rAAV virions in regimens for treating factor H-related disorders, particularly dry AMD and geographic atrophy secondary to AMD, are also provided.

[0052] heterologous nucleic acid

[0053] The novel rAAV virions described herein include heterogeneous nucleic acids encoding human factor H (hfH) variants operably linked to expression regulatory sequences. Typically, the heterogeneous nucleic acids include an AAV genome in which the rep and cap genes are deleted and / or replaced by the hfH sequence and its associated expression regulatory sequences. The hfH sequence is typically inserted adjacent to (i.e., sandwiched between) one or two AAV TR or TR elements suitable for viral replication, in place of the nucleic acids encoding the viral rep and cap proteins (Xiao et al., 1997, J. Virol. 71(2):941-948). Other regulatory sequences suitable for use in promoting tissue-specific expression of the hFH gene sequence in target cells (e.g., retinal cells) may also be included.

[0054] In some embodiments, the rAAV virion comprises a heterogeneous nucleic acid including (a) an AAV2-terminal repeat sequence, (b) a transcriptional regulatory sequence, (c) a nucleotide sequence encoding an hfH variant as described herein, (d) a polyadenylated sequence, and (e) an AAV2-terminal repeat sequence.

[0055] hfH gene

[0056] The amino acid sequence of mature "wild-type" human complement factor H (isotype 1) is available at www.uniprot.org / uniprot / P08603 and serves as a reference for amino acid numbering of hfH isotype 1 [see also Sequence ID No. 39 of U.S. Patent No. 10,988,519, whose entire content is incorporated herein by reference]: [ka] [ka]

[0057] The leader sequence (MRLLAKIICLMLWAICVA; SEQ ID NO: 4) is located at amino acids 1 through 18 of factor H, with reference to SEQ ID NO: 3. The mature (secreted) hfH protein is located at amino acids 19 through 1231 of SEQ ID NO: 3. There is another method for determining the locations of 20 short complementary repeat sequences (SCRs). The domain locations referred to herein are based on the numbering used by C. Estaller et al., Eur J Immunol. 1991 Mar;21(3):799-802.

[0058] Table 1 below provides the amino acid sequences of each of the 20 SCRs of hfH (see also SEQ ID NOs. 3, 5, 7, 9, 11, 13, 14, 16, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 38 of U.S. Patent No. 10,988,519): [Table 1-1] [Table 1-2]

[0059] If necessary, the modified hfH variants described herein may have heterologous leader sequences substituted for the natural hfH leader sequence. In addition, or if necessary, the sequence may be another hfH isoform (e.g., isoform 2), which is available, for example, from http: / / www.uniprot.org / uniprot / P08603, and / or one of the natural amino acid variants therein that are not associated with the disorder. See Sequence ID No. 40 of U.S. Patent No. 10,988,519. In the following description, substitutions may be denoted as (first amino acid identified by a single-letter code)-residue position#-(second amino acid identified by a single-letter code), where the first amino acid is the substituted amino acid and the second amino acid is the substituted amino acid at the position specified with respect to isoform 1. However, by conventional alignment processes, the corresponding amino acid residues identified herein with respect to the numbering of isoform 1 may be located in isoform 2 and the natural variants of the SCR of isoform 1 or 2 of fH that do not cause the disorder.

[0060] The rAAVs described herein include heterogeneous nucleic acids encoding functional fH variants. Examples of functional fH variants include those having one or more SCR7, SCR17, or SCR18 domains, or those having SCR1-4 and 19-20 of the fH protein. Further variants include those having one or more SCR6, SCR8, SCR16, SCR17, SCR18, or fragments thereof, and combinations thereof. For example, such variants include, for example, fH SCR1~4, 6~8, 19~20; fH SCR1~4, 6~8, 18~20; fH SCR1~4, 6~8, 17~20; fH SCR1~4, 6~7, 19~20; fH SCR1~4, 6~7, 18~20; fH SCR1~4, 6~7, 17~20; fH SCR1~4, 7~8, 19~20; fH SCR1~4, 7~8, 18~20; fH SCR1~4, 7~8, 17~20; fH SCR1~4, 7, 19~20; fH SCR1~4, 7, 18~20; fH This may include SCR1-4, 7, 17-20; SCR1-4, 17, 19-20; SCR1-4, 18-20; SCR1-4, 17-20 and / or fH SCR1-4, 7, 16-20, etc. In certain embodiments, the hfH variant further includes additional hfH SCRs, e.g., SCR6, SCR8, SCR16, or combinations thereof. In preferred embodiments, hfHSCR5 is absent. However, in certain embodiments, hfHSCR5 may be present in whole or in part. In certain embodiments, hfHSCR9, SCR10, SCR11, SCR12, SCR13, SCR14, and / or SCR15 are absent or at least functionally removed. If necessary, one or more SCRs in these variants may be "functional fragments" of SCRs rather than full-length SCRs. A "functional fragment" means a sub-full-length SCR amino acid sequence (or its coding sequence) characterized by having one or more of the following: complement inhibitory activity, binding ability, heparin binding activity, and / or C3b binding activity.

[0061] In hfH variants, domains may be located directly adjacent to each other (for example, the carboxyl terminus of one domain may immediately follow the amino terminus of a preceding domain). Alternatively, one or more of the SCR domains may have a linker consisting of 1 to about 12 to 18 amino acids located between them. For example, the variant may contain SCR1-(L)-SCR2-(L)-SCR3-(L)-SCR4-(L)-(SCR6-(L))-SCR7-(L)-(SCR8-(L))-(SCR16-(L))-(SCR17-(L))-(SCR18-(L5))-SCR19-(L)-SCR20, where () indicates an optional component, and "L" refers to a linker that may not be present or may be independently selected from an amino acid sequence of about 1 to about 12 to 18 amino acids. In other words, if a variant includes multiple linkers, each linker may have the same or different arrangement. In certain embodiments, a variant includes at least one, at least two, at least three, at least four, at least five, or at least six linkers. Examples of suitable linkers include the natural or artificial linkers described herein. Each of these wild-type linkers may be located in its natural position. Alternatively, one or more of these wild-type linkers may be used in different linker positions, or in multiple different linker positions.

[0062] If necessary, one or more of these linkers may be fH sequences and are selected independently. Alternatively, one or more linkers may be heterogeneous to fH, for example, derived from artificial, synthetic, or different proteins that confer appropriate flexibility to the fH variant. Examples of other suitable linkers include, for example, the polyGly linker and other linkers that provide appropriate flexibility (e.g., http: / / parts.igem.org / Protein_domains / Linker) (which is incorporated herein by reference). In certain embodiments, the linker lacks fH function.

[0063] Several representative and suitable fH linker sequences are shown in Table 2 below. [Table 2]

[0064] In some preferred embodiments, rAAV comprises heterologous nucleic acids encoding an hfH variant having the following structure: SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20.

[0065] In a particularly preferred embodiment, the heterologous nucleic acid encodes an fH variant having the following amino acid sequence. [ka]

[0066] In other embodiments, rAAV comprises heterologous nucleic acids encoding an hfH variant having the following SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7')-SCR19-(L10)-SCR20.

[0067] These and other variants may include other fH sequences. For example, the coding sequence of an fH variant may also include a leader sequence. Such a leader sequence may be an fH leader (e.g., MRLLAKIICLMLWAICVA; SEQ ID NO: 4). If necessary, the leader sequence may be from another source, e.g., an IL-2 leader [see, e.g., the index of mammalian leader sequences identified at www.signalpeptide.de / ], which is incorporated herein by reference. In one embodiment, the selected leader sequence is less than about 26 amino acids long (e.g., less than about 1 to about 26 amino acids), more preferably less than 20 amino acids long (less than about 1 to about 20 amino acids), and most preferably less than about 18 amino acids long (less than about 1 to about 18 amino acids). "Functional deletion" means an amino acid sequence (or its coding sequence) that lacks complement inhibitory activity, C3b binding activity, and optionally further lacks heparin binding activity.

[0068] In addition to the fH protein variants provided herein, nucleic acid sequences encoding these fH protein variants are also provided. These variant coding sequences may be derived from the wild-type sequences of the leader sequence and / or isoform 1, isoform 2, or one or more SCRs of non-disease-associated variants. Alternatively or additionally, web-based or commercially available computer programs, as well as service-based companies, can be used to back-translate the amino acid sequences of the leader sequence and / or one or more SCRs into nucleic acid coding sequences containing both RNA and / or cDNA. For example, see backtranseq by EMBOSS, Gene Infinity (www.geneinfinity.org / sms- / sms_backtranslation.html), and ExPasy (www.expasy.org / tools / ).

[0069] In one embodiment, RNA and / or cDNA coding sequences are designed for optimal expression in human cells.

[0070] Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be carried out using methods available online, published methods, or by using companies that provide codon optimization services. One codon optimization method is described, for example, in International Publication 2015 / 012924A2 (which is incorporated herein by reference). Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Preferably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only fragments of the ORF may be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence to produce nucleic acid fragments of a codon-optimized coding region encoding that polypeptide.

[0071] In another embodiment, at least one glycosylation site is modified to include at least one SCR present in the hfH variant, at least two SCR present in the hfH variant, at least three SCR present in the hfH variant, or more. For example, the glycosylation site may be modified to 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 further embodiments, SCR4, 17, and 18 are glycosylated. In a preferred embodiment, the hfH variant includes, consists of, or is essentially composed of SCR1-4, SCR6-8, and SCR17-19, and SCR17 and SCR18 are glycosylated. In a particular embodiment, the glycosylation site may be modified to a linker. However, in such examples, the linker is preferably at least 6 amino acids long and up to about 18 amino acids long, for example, 8-18, 10-15, or 12 amino acids long.

[0072] As used herein, a glycosylation site refers to a point where an oligosaccharide is bonded to a carbon atom (C bond), a nitrogen atom (N bond), or an oxygen atom (O bond), or to glycation (the non-enzymatic bonding of a reducing sugar to a nitrogen atom of a protein (e.g., the nitrogen atom of the asparagine (Asn) side chain, which is part of Asn-X-Ser / Thr, where X is any amino acid other than Pro)). In certain embodiments, an N-glycosylation site is preferred. Various techniques for modifying N-glycosylation sites are known in the art. See, for example, Y Liu et al., Biotech Prog 2009 September-October;25(5):1468-1475; Sala RJ, Griebenos K. Glycoslylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010 Feb.1;24(1):9-21.

[0073] An exemplary hfH variant nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34 is provided below. [ka] [ka] [ka] [ka] [ka] [ka]

[0074] ITR

[0075] The reverse terminal repeat sequence (ITR) selected for use in rAAV virions is preferably an AAV sequence, with serotypes 1, 2, 3, 4, 5, and 6 preferred. The ITR may be a synthetic sequence that functions as an AAV reverse terminal repeat, such as a “double D sequence” as described in U.S. Patent No. 5,478,745 by Samulski et al., the entire disclosure of which is incorporated herein by reference in whole. Typically, however not necessarily, the TRs originate from the same parvovirus, for example, both ITR sequences originate from AAV2.

[0076] In some embodiments, the heterologous nucleic acid encapsulated by the rAAV virion contains a 5'ITR having the following sequence: [ka]

[0077] In a related embodiment, the heterologous nucleic acid encapsulated by the rAAV virion includes a 3'ITR having the following sequence: [ka]

[0078] Expression regulatory sequences

[0079] The hFH gene is operably ligated to at least one transcriptional regulatory sequence, preferably a nucleic acid and a heterogeneous transcriptional regulatory sequence. In some embodiments, the transcriptional regulatory sequence includes a cell or tissue-specific promoter that results in cell-specific expression of the nucleic acid in photoreceptor cells, such as the human rod cell photoreceptor-specific human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter or the human photoreceptor-inter-retinoid-binding protein (IRBP) promoter. In other embodiments, the transcriptional regulatory sequence includes a constitutive promoter that results in similar levels of expression of the nucleic acid in many cell types. Appropriate constitutive promoters include the CAG promoter, which contains (C) the cytomegalovirus (CMV) pre-early enhancer element, (A) the first exon and first intron of the chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene (see Miyazaki et al. (1989) Gene 79(2):269-277), the cytomegalovirus (CMV) promoter (Stinski et al., (1985) Journal of Virology 55(2):431-441), the human elongation factor 1α promoter (EF1α) (Kim et al., (1990) Gene 91(2):217-223), the human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al., (1984) Gene 32(3):409-417), and the mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS). Examples include 109(17):6513-6518) and the ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261:101-105).

[0080] In a preferred embodiment, the hfH gene is operably ligated to a CAG promoter. In a particularly preferred embodiment, the CAG promoter contains the sequence of SEQ ID NO: 40, or contains a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto: [ka] [ka]

[0081] In some embodiments, the heterologous nucleic acid encapsulated in the rAAV virion contains an SV40 polyadenylated sequence of the following sequence: [ka]

[0082] In some embodiments, heterologous nucleic acids include the following nucleotide sequences or nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka]

[0083] In a relevant embodiment, the heterologous nucleic acid includes 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 thereto: [ka] [ka]

[0084] In a relevant embodiment, the heterologous nucleic acid includes 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 thereto: [ka] [ka]

[0085] In a relevant embodiment, the heterologous nucleic acid includes 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 thereto: [ka] [ka] [ka]

[0086] AAV Capsid

[0087] A variant AAV capsid of rAAV—encapsulating a heterologous nucleic acid encoding the hFh variant—contains a variant AAV capsid protein with an insertion of approximately 7 to 20 amino acids ("heterologous peptide" or "peptide insertion") within the GH loop of the parent AAV capsid protein, the peptide containing the amino acid sequence ISDQTKH (Sequence ID 1). Preferably, when the variant capsid protein is present in an AAV virion, it increases the infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent capsid protein.

[0088] The "GH loop" or loop IV of the AAV capsid protein refers in this art to the solvent-accessible portion of the AAV capsid protein known as the GH loop or loop IV. For more information on the GH loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Therefore, for example, the insertion site may be within the range of approximately amino acids 570-611 of AAV2 VP1.

[0089] In some embodiments, the peptide insertion has 1 to 3 spacer amino acids (Y1 to Y3) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 1). Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, the peptide insertion has 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insertion has 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In preferred embodiments, the peptide insertion includes or is derived from the amino acid sequence LAISDQTKHA (SEQ ID NO: 2).

[0090] In some embodiments, the variant AAV capsid protein comprises a peptide insertion containing the amino acid sequence ISDQTKH (SEQ ID NO: 1) and further comprises one or more amino acid substitutions compared to the corresponding parent AAV capsid protein. Typical examples of amino acid substitutions are described, 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.

[0091] In some preferred embodiments, the variant AAV capsid protein comprises a peptide insertion containing the amino acid sequence ISDQTKH (SEQ ID NO: 1), and further comprises a P34A amino acid substitution for the VP1 capsid of AAV2 or a corresponding substitution in another AAV serotype.

[0092] In other embodiments, the variant capsid protein may include one or more features disclosed in U.S. Patent No. 11,576,983, particularly one or more features disclosed in columns 26, lines 66-29, and line 50 of U.S. Patent No. 11,576,983.

[0093] 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: [ka]

[0094] The variant AAV capsid protein of SEQ ID NO: 42 includes the following modifications to the native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34 located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO: 2)) at amino acid position 588 present in VP1, VP2, and VP3. In some embodiments, the capsid includes a variant capsid protein having a sequence at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 42, with the P34A substitution and LAISDQTKHA (SEQ ID NO: 2) peptide insertion at amino acid position 588.

[0095] Packaging cells, which are contained within “host cells,” that can be cultured to produce the packaged viral vectors of the present invention, are also provided herein. Packaging cells of the present invention generally include cells having heterologous (1) viral vector function, (2) packaging function, and (3) helper function. Each of these component functions is described in the following sections.

[0096] First, the vector can be prepared by several methods known to those skilled in the art (see, for example, International Publication No. 2013 / 063379). A preferred method is described in Grieger et al., 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. Briefly, using efficient transfection of HEK293 cells as a starting point, adherent HEK293 cell lines from a qualified clinical master cell bank are grown under animal component-free suspension conditions in shaking flasks and WAVE bioreactors, enabling rapid and scalable rAAV production. Using a triple transfection method (e.g., International Publication No. 96 / 40240), the suspension HEK293 cell line, when recovered 48 hours after transfection, yields 10 5 More than 10 vector genome-containing particles (vg) / cell or 10 14 This generates cell cultures exceeding vg / L. More specifically, triple transfection refers to introducing three plasmids into packaging cells. One plasmid encodes the AAV rep and cap genes, another encodes various helper functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, VA RNA, etc.), and yet another plasmid encodes the transgene and its various regulatory elements (e.g., modified GLA gene and CAG promoter).

[0097] To achieve the desired yield, many variables are optimized, including the selection of suitable serum-free suspension media to support both proliferation and transfection, the selection of transfection reagents, transfection conditions, and cell density. A universal purification strategy based on ion exchange chromatography has also been developed, resulting in high-purity vector preps for AAV serotypes 1-6, 8, 9, and various chimeric capsids. This user-friendly process can be completed within one week, yields a high perfect-to-air particle ratio (>90% perfect particles), provides a post-purification yield (>1 × 10^13 vg / L) and purity suitable for clinical use, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been utilized in the production of GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), and is being administered to patients. Furthermore, by implementing a perfusion method involving the collection of rAAV from the culture medium at multiple time points after transfection, overall vector production was increased by at least 5 times.

[0098] Packaging cells include viral vector functionality along with packaging and vector functionality. Viral vector functionality typically includes a portion of a parvovirus genome, such as the AAV genome, in which rep and cap are deleted and replaced by a modified GLA sequence and its associated expression regulatory sequences. Viral vector functionality includes sufficient expression regulatory sequences to result in replication of the viral vector for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as the AAV genome, in which rep and cap are deleted and replaced by a transgene and its associated expression regulatory sequences. The transgene typically occupies two AAV TRs in place of the ORFs of the deleted viral rep and cap. Appropriate expression regulatory sequences are included, e.g., tissue-specific promoters and other regulatory sequences suitable for use in promoting tissue-specific expression of the transgene in target cells. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or marker polypeptide.

[0099] The terminal repeat sequences (TR(s)) (degradable and non-degradable) selected for use in viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 preferred. Degradable AAV TRs do not need to have a wild-type TR sequence (e.g., a wild-type sequence can be modified by insertion, deletion, cleavage, or missense mutation) as long as the TR mediates the desired function, such as packaging, incorporating, and / or rescuing the provirus. The TR is a synthetic sequence that functions as an AAV reverse terminal repeat sequence, such as the “double D sequence” described in U.S. Patent No. 5,478,745 by Samulski et al., the disclosure of which is incorporated herein by reference in whole. Typically, though not necessarily, the TRs originate from the same parvovirus, for example, both TR sequences originate from AAV2.

[0100] The packaging function includes variant capsid components, as described above.

[0101] The packaged viral vector includes a variant hfH transgene and expression regulatory sequence adjacent to the TR element, referred herein as the “transgene” or “transgene expression cassette,” sufficient to package the vector DNA and subsequently result in the expression of the gene sequence in transduced cells. The viral vector function may be supplied to cells, for example, as a component of a plasmid or amplicon. The viral vector function may be extrachromosomal within a cell line and / or incorporated into the cell’s chromosomal DNA.

[0102] Any method for introducing a nucleotide sequence having viral vector functionality into a cell host for replication and packaging, such as but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals, may be used. In embodiments where viral vector functionality is provided by transfection using the viral vector, standard methods for inducing viral infection may be used.

[0103] Packaging functions include genes for viral vector replication and packaging. Therefore, for example, packaging functions may include, as needed, functions required for viral gene expression, viral vector replication, rescue of the viral vector from its integrated state, viral gene expression, and packaging of the viral vector into viral particles. Packaging functions can be supplied together or separately to packaging cells using gene constructs such as plasmids or amplicons, baculoviruses, or HSV helper constructs. Packaging functions may be extrachromosomal within packaging cells, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

[0104] Helper functions include helper viral elements necessary to establish active infection of packaging cells 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 E1a, E1b, E2a, E4, and VA RNA. Packaging functions can be supplied by infecting packaging cells with the required virus. Packaging functions can be supplied to packaging cells together or separately using gene constructs such as plasmids or amplicons. See, for example, the pXR helper plasmid described by Rabinowitz et al., 2002, J. Virol. 76:791, and the pDG plasmid described by Grimm et al., 1998, Human Gene Therapy 9:2745-2760. Packaging functions may be extrachromosomal within packaging cells, but are preferably incorporated into the cell's chromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

[0105] Any suitable helper virus function can be used. For example, if the packaging cells are insect cells, baculoviruses can act as helper viruses. Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV Rep proteins can favorably facilitate more scalable AAV vector production schemes.

[0106] Any method can be used to introduce a nucleotide sequence with helper function into a cell host for replication and packaging (including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals). In embodiments where the helper function is provided by transfection using a viral vector or infection using a helper virus, standard methods for inducing viral infection can be used.

[0107] Any suitable permissible or packaging cells known in the art can be used to produce packaged viral vectors. Mammalian cells or insect cells are preferred. Examples of cells useful for producing packaging cells in the implementation of the present invention include, for example, human cell lines such as VERO, WI38, MRC5, A549, HEK 293 cells (expressing functional adenovirus E1 under the control of a constitutive promoter), B-50, or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one embodiment, the packaging cells can be grown in suspension culture, and more preferably, the cells can be grown in serum-free culture. In one embodiment, the packaging cells are HEK293 cells grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Patent No. 9,441,206 and deposited as ATCC number PTA13274. Numerous rAAV packaging cell lines are known in the art, including but not limited to those disclosed in International Publication No. 2002 / 46359. In another embodiment, the packaging cells are cultured in the form of cell stacks (e.g., a 10-layer cell stack seeded with HEK293 cells).

[0108] Cell lines for use as packaging cells include insect cell lines. Any insect cell that can enable AAV replication and be maintained during culture can be used according to the present invention. Examples include Spodoptera frugiperda, e.g., Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus-derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings regarding the use of insect cells for heterologous polypeptide expression, methods for introducing nucleic acids into such cells, and methods for maintaining such cells during 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. Acad. Sci. USA 88:4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

[0109] The viral capsid according to the present invention can be produced by expression from, for example, a baculovirus (Brown et al., (1994) Virology 198:477-488) using any method known in the art. As a further alternative, the viral vector of the present invention can be produced in insect cells using a baculovirus vector to deliver the rep / cap gene and rAAV template, for example, as described by Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0110] In another embodiment, the present invention provides a method for rAAV production in insect cells, in which a baculovirus packaging system or vector can be constructed to carry the AAV Rep and Cap coding regions by modifying these genes into the polyhedrin coding region of a baculovirus vector and producing a viral recombinant by transfection into a host cell. In particular, when baculovirus production is used for AAV, preferably the AAV DNA vector product is a self-complementary AAV-like molecule that does not use mutations to AAV ITR. This appears to be a byproduct of inefficient AAV Rep nicking in insect cells, resulting in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell is either a baculovirus-infected cell or has additional nucleic acids that encode baculovirus helper functions introduced into it, or contains these baculovirus helper functions. These baculoviruses can express AAV components and subsequently promote capsid production.

[0111] During production, packaging cells generally contain one or more viral vector functions, along with helper and packaging functions sufficient to result in viral vector replication and packaging. These various functions may be supplied together or separately to the packaging cells using gene constructs such as plasmids or amplicons, may be extrachromosomal within the cell line, or may be incorporated into the cell's chromosomes.

[0112] Cells may be supplied with one or more of the above-mentioned functions already incorporated, for example, a cell line having one or more vector functions incorporated extrachromosomally or incorporated into the cell's chromosomal DNA, a cell line having one or more packaging functions incorporated extrachromosomally or incorporated into the cell's chromosomal DNA, or a cell line having helper functions incorporated extrachromosomally or incorporated into the cell's chromosomal DNA.

[0113] rAAV vectors 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 those described in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Patent No. 6,566,118 and International Publication No. 98 / 09657.

[0114] How to deliver the nucleic acid encoding hfH to the retina

[0115] In some embodiments, methods for delivering heterologous nucleotide sequences encoding hfH to the retina are provided using rAAV as described herein. rAAV can be used to deliver nucleotide sequences encoding hfH to retinal cells in vitro, for example, to produce hfH polypeptides or nucleic acids in vitro for ex vivo gene therapy. rAAV is even more useful in methods for delivering nucleotide sequences to subjects requiring rAAV, such as expressing hfH in subjects requiring rAAV, for example, humans with dry AMD or humans with geographic atrophy. Thus, in this way, hfH can be produced in vivo in subjects to restore complement regulation.

[0116] Accordingly, one embodiment provides a method for delivering a nucleic acid encoding an hfH variant to retinal cells, comprising contacting the retinal cells with the rAAV virions described herein.

[0117] In another embodiment, a method is provided for delivering a nucleic acid encoding an hfH variant to retinal cells of a mammalian subject, comprising the step of administering an effective amount of the rAAV virion described herein or a pharmaceutical formulation comprising the same to the mammalian subject.

[0118] rAAV can be administered to the retina of the subject by any suitable route. In preferred embodiments, rAAV is administered intraocularly to the subject, preferably by subretinal, suprachoroidal and / or intravitreal injection. In some particularly preferred embodiments, rAAV is administered to the subject by intravitreal injection, more preferably by a single intravitreal injection.

[0119] Treatment method

[0120] In certain embodiments, a method is provided for treating dry AMD in a subject requiring treatment of dry AMD, comprising administering recombinant adeno-associated virus (rAAV) to a subject, comprising a heterologous nucleic acid comprising (a) a variant AAV capsid protein having a heterologous peptide insertion of 7 to 20 amino acids in length covalently inserted into the GH loop of an AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 1), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding the hfH variant described herein, which is operably linked to a promoter; or administering a pharmaceutical composition comprising rAAV and a pharmaceutically acceptable carrier, preferably 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 rAAV in the manufacture of pharmaceuticals for the treatment of dry AMD is also provided.

[0121] In related embodiments, a method is provided for treating geographic atrophy in a subject requiring treatment for geographic atrophy, comprising the steps of: administering a recombinant adeno-associated virus (rAAV) virion comprising a heterologous nucleic acid comprising (a) a variant AAV capsid protein (SEQ ID NO: 1) having a heterologous peptide insertion of 7 to 20 amino acids in length covalently inserted into the GH loop of the AAV capsid protein, and (ii) a nucleotide sequence encoding the hfH variant described herein, operably linked to a promoter; or administering a pharmaceutical composition comprising the rAAV virion and a pharmaceutically acceptable carrier, preferably by intravitreal injection. The use of rAAV or a pharmaceutical composition containing the same for treating geographic atrophy is also provided. The use of rAAV in the manufacture of pharmaceuticals for treating geographic atrophy is also provided.

[0122] In some embodiments, the variant AAV 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%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 42, and includes a P34A substitution and a LAISDQTKHA (SEQ ID NO: 2) peptide insertion at amino acid position 588.

[0123] In some preferred embodiments, the nucleotide sequence encoding the hfH variant encodes an hfH variant having the structure SCR1-(L1)-SCR2-(L2)-SCR3-(L3)-SCR4-(L4)-SCR6-(L5)-SCR7-(L6)-SCR8-(L7)-SCR17-(L8)-SCR18-(L9)-SCR19-(L10)-SCR20, wherein each of SCR1-4, 6-8, and 17-20 contains the amino acid sequence according to Table 1, and each of L1-L10 contains the amino acid sequence according to Table 2. In related embodiments, the nucleotide sequence encoding the hfH variant contains the amino acid sequence of SEQ ID NO: 34, or encodes an hfH variant containing an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 34. In other related embodiments, the nucleotide sequence encoding the hfH variant contains the nucleotide sequence shown as any one of SEQ ID NOs: 35-37.

[0124] In some embodiments, rAAV is administered to a subject having dry AMD and / or geographic atrophy by periorbital, intravitreal, suprachoroidal, and / or subretinal injection, preferably intravitreal injection, at a dose of about 1×10 8 vector genomes (vg) / eye to about 1×10 13 vg / eye, about 1×10 8 vg / eye to about 1×10 12 vg / eye, about 1×10 9 vg / eye to about 1×10 12 vg / eye, about 1×10 9 vg / eye to about 1×10 11 vg / eye or about 6×10 9 vg / eye to about 6×10 10 vg / eye. In some embodiments, rAAV is administered to a subject having dry AMD and / or geographic atrophy by periorbital, intravitreal, suprachoroidal, and / or subretinal injection, preferably intravitreal injection, at a dose of about 1×10 8 vg / eye, about 2×10 8 vg / eye, about 3×10 8 vg / eye, about 4×10 8vg / eye, approximately 5×10 8 vg / eye, approximately 6×10 8 vg / eye, approximately 7×10 8 vg / eye, approximately 8×10 8 vg / eye, approx. 10 8 vg / eye, approximately 1×10 9 vg / eye, 2×10 9 vg / eye, approximately 3×10 9 vg / eye, approximately 4×10 9 vg / eye, approximately 5×10 9 vg / eye, approximately 6×10 9 vg / eye, approximately 7×10 9 vg / eye, approximately 8×10 9 vg / eye, approximately 9×10 9 vg / eye, approximately 1×10 10 vg / eye, approximately 2×10 10 vg / eye, approximately 3×10 10 vg / eye, approximately 4×10 10 vg / eye, approximately 5×10 10 vg / eye, approximately 6×10 10 vg / eye, approximately 7×10 10 vg / eye, approximately 8×10 10 vg / eye, approximately 9×10 10 vg / eye, approximately 1×10 11 vg / eye, approximately 2×10 11 vg / eye, approximately 3×10 11 vg / eye, approximately 4×10 11 vg / eye, approximately 5×10 11 vg / eye, approximately 6×10 11 vg / eye, approximately 7×10 11 vg / eye, approximately 8×10 11 vg / eye, approximately 9×10 11 vg / eye, または approx. 1×10 12 The dosage of vg / eye is not the same as the dosage.

[0125] In some embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects requiring treatment for dry AMD and / or geographic atrophy, comprising administering to a subject by periorbital, intravitreous, suprachoroidal and / or subretinal injection an effective amount of rAAV virion comprising (i) a capsid protein comprising an amino acid sequence shown as SEQ ID NO: 42, and (ii) a heterogeneous nucleic acid comprising 5'-3':(a) an AAV2 terminal repeat sequence, (b) a CAG or CMV promoter, (c) a nucleotide sequence selected from SEQ ID NOs: 35-37, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat sequence.

[0126] In related embodiments, a method is provided for treating dry AMD and / or geographic atrophy in subjects requiring treatment for dry AMD and / or geographic atrophy, comprising administering to a subject by periorbital, intravitreal, suprachoroidal and / or subretinal injection a pharmaceutical composition comprising: (i) a capsid protein comprising an amino acid sequence shown as SEQ ID NO: 42; and (ii) a heterogeneous nucleic acid comprising 5'-3':(a) an AAV2 terminal repeat sequence, (b) a CAG or CMV promoter, (c) a nucleotide sequence selected from SEQ ID NOs: 35-37, (d) a polyadenylated sequence, and (e) an AAV2 terminal repeat sequence. In some embodiments, the pharmaceutical composition comprises about 1 × 10⁻¹⁶ 8 vg~approx. 1×10 13 vg, approx. 1×10 9 vg~approx. 1×10 12 vg, approx. 1×10 9 vg~approx. 1×10 11 VG or approximately 6 x 10 9 vg~approx. 6×10 10 Contains vg. In other embodiments, the pharmaceutical composition contains about 1 × 10 8 vg, approx. 2×10 8 vg, approx. 3×10 8 vg, approx. 4×10 8 vg, approx. 5×10 8 vg, approx. 6×10 8 vg, approx. 7×10 8 vg, approx. 8×10 8vg, approximately 9×10 8 vg, approximately 1×10 9 vg, 2×10 9 vg, approximately 3×10 9 vg, approximately 4×10 9 vg, approximately 5×10 9 vg, approximately 6×10 9 vg, approximately 7×10 9 vg, approximately 8×10 9 vg, approximately 9×10 9 vg, approximately 1×10 10 vg, approximately 2×10 10 vg, approximately 3×10 10 vg, approximately 4×10 10 vg, approximately 5×10 10 vg, approximately 6×10 10 vg, approximately 7×10 10 vg, approximately 8×10 10 vg, approximately 9×10 10 vg, approximately 1×10 11 vg, approximately 2×10 11 vg, approximately 3×10 11 vg, approximately 4×10 11 vg, approximately 5×10 11 vg, approximately 6×10 11 vg, approximately 7×10 11 vg, approximately 8×10 11 vg, approximately 9×10 11 vg, or approximately 1×10 12 contains vg.

[0127] There is provided a pharmaceutical composition comprising the rAAV described herein. In some embodiments, the pharmaceutical composition is about 1×10 8 ~ about 1×10 14 vector particles or vector genomes, about 1×10 8 ~ about 1×10 13 vector particles or vector genomes, about 1×10 9 ~ about 1×10 12 vector particles or vector genomes, or about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×108 , about 8×10 8 , about 9×10 8 , about 1×10 9 , about 2×10 9 , about 3×10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , or approximately 1 x 10 12 The vector particles or vector genome are included. In some embodiments, the pharmaceutical composition comprises about 1 × 10⁻¹⁶ particles. 9 ~Approx. 1×10 11 Contains vg, preferably about 6 × 10 9 vg~approx. 6×10 10 Contains vg. In some preferred embodiments, the pharmaceutical composition is administered by intravitreal injection to a person having dry AMD and / or geographic atrophy. [Examples]

[0128] Examples The following embodiments illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. While the present invention has been described in relation to its preferred embodiments, various modifications will be apparent to those skilled in the art from reading this application.

[0129] Example 1 We constructed a recombinant AAV (rAAV) virion containing a transgene encoding a truncated form of human complement factor H (Figure 1). Complement factor H (CFH) is a natural inhibitor of alternative complement pathways, and therefore rAAV is useful, in particular, for treating geographic atrophy secondary to age-related macular degeneration. Endogenous CFH is a 155 kD protein containing 20 short consensus repeat (SCR) units. The N-terminus is a key site for cofactor activity / degradation-promoting activity, and the C-terminus is a key site for cell regulation (see, e.g., de Cordoba SR, de Jorge EG. Translational mini-review series on complement factor H: genesics and disease associations of human complement factor H. Clin Exp Immunol. 2008 Jan;151(1):1-13).

[0130] rAAV comprises a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding a truncated form of complement factor H ("miniCFH") having the amino acid sequence shown in SEQ ID NO: 34, operably linked to the CAG promoter. The miniCFH transgene includes SCR1-4, 6-8, and 17-20.

[0131] rAAV was subjected to in vitro characterization by (a) transfection of human HEK293T cells with an rAAV plasmid cassette, and (b) in vitro transduction of human iPSC-derived retinal pigment epithelium (RPE) cells using rAAV.

[0132] miniCFH expression and activity in HEK293T cells

[0133] To demonstrate the in vitro function of the miniCFH transgene used in rAAV, human cells were transfected with an rAAV plasmid cassette. HEK293T cells were transfected with low (0.125 μg) or high (0.5 μg) plasmid DNA encoding either miniCFH or CFH (full length), respectively, driven by the CAG promoter. Dose-dependent expression and appropriately sized proteins were observed in the cell supernatant (Figures 2A and 2B), and miniCFH from the cell supernatant was shown to bind directly to C3b and heparin, similar to full-length CFH (Figures 2C and 2D). These data confirm that the miniCFH transgene is expressed, secreted, and retains complement and heparin-binding properties from the rAAV plasmid cassette. Next, the activity of the miniCFH transgene was evaluated to confirm its functionality. Complement 3 (C3) protein is cleaved into C3a and C3b fragments, and the C3b fragment is further degraded by complement factor I (CFI) in the presence of CFH as a cofactor to smaller, inactive fragments that block the alternative complement cascade. Protein lysates from cells transfected with the rAAV plasmid cassette demonstrate that miniCFH mediated C3b degradation (Figure 2E). Furthermore, miniCFH from transfected cells showed 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.

[0134] In vitro transduction of human iPSC-derived retinal pigment epithelium (RPE) cells

[0135] To demonstrate in vitro expression of the miniCFH transgene after transduction with rAAV and to confirm its function, human iPSC-derived RPE cells were transduced with rAAV at different MOIs. Transduction of cells with rAAV resulted in dose-dependent secretion of the miniCFH transgene (Figure 3A). Furthermore, the surrogate complement pathway was inhibited, demonstrating inhibition of MAC formation (Figures 3B and 3C). These in vitro data confirm the expression, secretion, and functionality of the miniCFH transgene from rAAV.

[0136] material and method

[0137] miniCFH expression (ELISA, WB). HEK293T cells (CRL-3216 from ATCC) were transfected with plasmid DNA encoding miniCFH or CFH (full length) driven by the CAG promoter. Plasmid DNA was transfected with 0.125 μg or 0.5 μg DNA / 12 wells (2e5 cells / well) using Fugene HD reagent (Promega Corporation catalog number E2311). Cells were incubated for 48 hours in 1 ml of DMEM medium containing 1% penicillin / streptomycin and 10% thermoactivated serum. After the first 48 hours, the medium was replaced with 0.35 ml of serum-free medium (DMEM medium containing 1% penicillin / streptomycin), and cells were incubated for 24 hours. Transgene expression was tested for in serum-free supernatant by ELISA and Western blotting.

[0138] A C3b cleavage assay was performed to confirm the functional activity of CFH expressed in 293T cells. The C3 protein consists of an alpha chain and a beta chain. The C3 protein is cleaved into C3a and C3b fragments. C3a is a small 77-amino acid fragment with a molecular weight of approximately 9 kDa. The C3b fragment (including the remaining alpha and beta chains) can form a C3 convertase when bound to factor B in the presence of CFH as a cofactor, or it can be degraded into smaller inactive fragments by complement factor I (CFI). The latter event disrupts the alternative complement cascade. These smaller fragments resulting from C3b degradation can be detected by Western blotting. Serum-free medium supernatant from CFH-transfected cells (or negative control non-transfected cells) was incubated at 37°C for 1 hour with 1 μg of purified C3b (Complement Technology Inc. catalog no. A114) and 1 μg of purified CFI (Complement Technology Inc. catalog no. A138). As a positive control, the inventors used 0.5 μg of purified CFH (Complement Technology Inc. catalog number A137) mixed with 1 μg of purified C3b (Complement Technology Inc. catalog number A114) and 1 μg of purified CFI (Complement Technology Inc. catalog number A138) in PBS and incubated at 37°C for 1 hour. An additional negative control was included in which either CFH or CFI was omitted from the recombinant protein mixture.

[0139] Complement Inhibition Assay. The Wieslab® Complement System Alternative Pathway Kit (Eagle Biosciences Inc., catalog number COMPL AP 330) is an enzyme immunoassay for the qualitative determination of functional alternative complement pathways in human serum. This assay combines the principle of a hemolysis assay for complement activation with the use of a labeled antibody specific to the neoantigen produced as a result of complement activation. The amount of neoantigen produced is proportional to the functional activity of the complement pathway. Wells of a 96-well plate are coated with a specific activator of the alternative pathway (bacterial lipopolysaccharide, LPS). Normal human serum is diluted to 5% with 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 LPS. The wells are then washed, and C5b-9 is detected with a specific alkaline phosphatase-labeled antibody against the neoantigen expressed during MAC formation. After further washing, detection of the specific antibody is obtained by incubation with an alkaline phosphatase substrate solution. The amount of complement activation correlates with color intensity and is measured in terms of absorbance (optical density (OD)). Addition of complement inhibitors such as eculizumab (C5 blocking antibody), CFH from CFH-transfected cell culture supernatant, or recombinant CFH protein to 5% normal human serum can partially or completely inhibit the response, as evidenced by a decrease in the absorbance signal.

[0140] C3b-binding ELISA. The C3b-binding ELISA is an in-house ELISA adapted by Nichols et al. ( / / pubmed.ncbi.nlm.nih.gov / 26221753 / ). The assay tests whether CpG-free miniCFH retains direct C3b-binding ability by utilizing a monoclonal anti-CFH blocking antibody to prevent binding of miniCFH to a C3b-coated plate. The assay was performed using both full-length recombinant CFH as proof of concept and supernatant from 293T cells transfected with CpG-free miniCFH. As a control, the inventors included mock incubation conditions for transfected cell supernatants without CFH and antibodies.

[0141] Heparin-binding ELISA. The heparin-binding ELISA is an in-house ELISA adapted by Nichols et al. ( / / pubmed.ncbi.nlm.nih.gov / 26221753 / ). The assay tests whether CpG-free miniCFH retains heparin-binding ability by preventing binding of miniCFH to heparin-coated plates using a monoclonal anti-CFH blocking antibody. The assay was performed using both full-length recombinant CFH as a control and supernatant from 293T cells transfected with CpG-free miniCFH. As a control, the inventors also included transfected cell supernatant that was not treated with the blocking antibody. The results show that pre-incubation with the blocking CFH antibody reduces the signals of recombinant CFH and CpG-free miniCFH, indicating that the CpG-free miniCFH construct binds and retains heparin-binding activity.

[0142] Transduction of human RPE cells. IPSC-derived RPE cells were transduced with the capsid of SEQ ID NO: 42, which delivers a payload encoding miniCFH driven by a CAG promoter. RPE cells were previously induced and cryopreserved in-house. 30+ / -5 days prior to transduction, RPE cells were seeded on plates coated with hESC-qualified Matrigel® (Corning catalog no. 354277) and maintained for 30+ / -5 days in serum-free X-VIVO-10 medium (Lonza catalog no. BEBP02-055Q) containing 1% penicillin / streptomycin and the RHO / ROCK pathway inhibitor Y-27632 (STEMCELL Technologies catalog no. 72304) at 10 μM. The medium was changed every 2-3 days. RPE was introduced at infection multiplicity (MOI, vector genome per cell (vg)) of 5,000, 20,000, and 50,000. Cell counts were determined on the same day, immediately before transduction. An appropriate amount of virus was mixed with fresh medium and added to the cells. Three days after transduction, the medium was removed and fresh medium was added. Four and seven days after transduction, the medium was collected and assayed for miniCFH expression and activity.

[0143] Example 2 High-performance liquid chromatography / mass spectrometry (LC-MS) was used to confirm the presence of the miniCFH gene product (short CFH or sCFH) in non-human primates (NHPs) after intravitreous administration of rAAV of Example 1 (containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42, and a heterogeneous nucleic acid encoding a truncated form of complement factor H ("miniCFH") having the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter).

[0144] material and method

[0145] Aqueous humor (AH) and vitreous humor (VH) samples were collected from (intravitreal) NHP treated with rAAV, rapidly frozen, and stored for analysis. The sCFH LC-MS method was developed and validated to identify the sCFH-specific peptide signature encoded by rAAV. These specific peptide mass signatures were then quantified against a standard curve to determine the concentration of sCFH protein in each sample. A quality control sample containing a known level of sCFH was included in each experimental run. The standard deviation acceptance criterion for repeated sample testing was ≤20%. The LLOQ / BLQ for the assay was determined to be approximately 50 ng / mL, and samples were run in triplicates.

[0146] result

[0147] Table 3 below contains a summary of the LC-MS quantification results of sCFH concentrations in AH samples from NHP patients who received intravitreal doses of rAAV (see also Figure 4) at specified doses: Table 3 [Table 3]

[0148] Table 4 below contains a summary of the results of LCMS quantification of sCFH concentration in VH samples of NHP treated with a specified dose of rAAV: Table 4 [Table 4]

[0149] conclusion

[0150] The sCFH gene product encoded by rAAV was detected in VH and AH at each time point and dose (Tables 3 and 4; Figure 4). Based on previous studies / models using rAAV containing the SEQ ID NO: 42 capsid protein to explore the measured sCFH AH levels and the relative AH / VH / retinal concentrations, retinal sCFH concentrations were predicted to be within the normal / therapeutic range (see Table 5 below). Table 5 [Table 5] Endogenous CFH AH:50~350ng / ml Endogenous CFH prediction retina / choroid: 3-70 nM

[0151] Using transfected 293T cells, we evaluated sCFH expression to confirm its specificity and developed an in situ hybridization (ISH) method to optimize assay conditions. We then used the ISH method to detect the presence of sCFH RNA in NHP ocular tissue after intravitreous injection of rAAV (containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding miniCFH with the amino acid sequence shown in SEQ ID NO: 34, operably linked to a CAG promoter).

[0152] material and method

[0153] Ocular tissue samples (whole eyeball samples) were collected from NHP treated with 5E10vg / eye rAAV, fixed, and processed for analysis.

[0154] The sCFH ISH assay was performed using a custom, proprietary 20ZZ probe targeting sCFH mRNA (designed to avoid cross-reactivity with endogenous cynomolgus monkey or human FL CFH) between amino acids 195–1318. (sCFH ISH 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, and ACD RNAscope® VS Universal HRP Reagent Kit were used. Full slide image scans were captured using a 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 sCFH RNA in transfected HEK293T cells compared to untransfected (negative control) HEK293T cells. Figure 6 demonstrates widespread sCFH RNA ISH signaling in the retinal and macular regions.

[0155] Example 3 Further in vitro studies were conducted to demonstrate the in vitro functional activity of the miniCFH protein expressed after transfection of human HEK293T cells.

[0156] HEK293T cells were transfected with either a low (0.125 μg) or high (0.5 μg) level of the rAAV plasmid cassette from Example 1 (containing heterogeneous nucleic acid encoding a truncated form of complement factor H ("miniCFH") having the amino acid sequence shown in SEQ ID NO: 34, operably ligated to the CAG promoter) or an rAAV plasmid cassette encoding full-length CFH protein. The miniCFH or full-length CFH protein secreted into the supernatant was visualized by Western blotting. Dose-dependent expression and appropriately sized proteins were observed in the supernatant from transfected cells (Figure 7A). Secreted CFH protein expression was quantified by ELISA after transfection with the rAAV plasmid cassette from Example 1 (Figure 7B). Transfection resulted in detected secreted CFH expression compared to the untransfected control (Figure 7B).

[0157] In addition to protein expression, the activity of miniCFH secreted from HEK293T cells transfected with the miniCFH construct was evaluated. Complement factor H is an essential complement regulator for controlling alternative pathways and similarly negatively regulates the formation of the C5b-9 complex, also known as the membrane attack complex (MAC), which is the endpoint of the complement cascade. To test whether miniCFH is functional, alternative complement inhibitory activity was measured using a modified version of the Wieslab® Complement System Alternative Pathway Assay. Supernatant from cells transfected with the rAAV plasmid cassette of Example 1 showed significant inhibition of alternative complement system activity (Figure 7C). Recombinant eculizumab antibody, a C5 inhibitor, was used as a positive control, and this assay showed potent inhibition of the alternative complement pathway (Figure 7C).

[0158] In addition to assaying the effects of miniCFH on complement activation and MAC formation endpoints, the upstream mechanism of CFH was evaluated by a C3b cleavage activity assay. The upstream complement protein C3 can be hydrolyzed into C3a and C3b fragments upon complement activation. The C3b fragment contains alpha and beta chains and, in the presence of CFH as a cofactor, can form a C3 convertase upon binding to factor B or be degraded by complement factor I (CFI) to 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 blotting. Supernatants from cells transfected with the rAAV plasmid cassette of Example 1 or from untransfected cells were incubated with recombinant proteins CFI and C3b and analyzed by Western blotting (Figure 7D). The membrane was probed with an anti-C3 antibody to detect all C3 protein products, including the C3 alpha chain, C3 beta chain, and three beta chain cleavage products. In the reaction, incubated with the supernatant of cells transfected with the rAAV plasmid cassette from Example 1, all three C3b cleavage products were observed, similar to the positive control but not detected in the NT reaction. Furthermore, the C3b α chain band was depleted under these conditions, similar to the positive control. These data support the upstream function of miniCFH produced from the rAAV plasmid cassette from Example 1, which assists CFI in C3b beta chain cleavage, ultimately inhibiting complement cascade progression.

[0159] Furthermore, as expected, miniCFH from the cell supernatant of cells transfected with the rAAV plasmid cassette of Example 1 directly bound to both C3b and heparin, similar to full-length CFH (Figures 7E and 7F). These data confirm that the miniCFH transgene can be expressed, secreted, and retain appropriate complement and heparin binding properties.

[0160] Further experiments were conducted to demonstrate the expression of the miniCFH gene product and the functional activity of the expressed miniCFH protein in vector-transduced human iPSC-derived RPE cells after transduction at different MOIs (Moritomotor Infection). The MOIs after rAAV transduction into iPSC-derived RPE cells were 5,000, 20,000, and 50,000 vg / cell in experiments evaluating the expression and functional activity of miniCFH secreted into the cell supernatant, and 800, 2,000, and 5,000 vg / cell in disease model experiments. rAAV consists of a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding a truncated form of complement factor H ("miniCFH") having the amino acid sequence shown in SEQ ID NO: 34, operably linked to the CAG promoter. After transduction, the viral inoculation material was removed approximately 72 hours later, and the culture medium was replaced. Seven days after transduction, the cell supernatant was collected, divided into aliquots, and stored at -80°C to fix the cells.

[0161] To demonstrate that transduction of iPSC-derived RPE cells with rAAV results in the expression of functional proteins, the concentration of secreted miniCFH in the cell supernatant was quantified by ELISA 7 days after transduction. ELISA detects both endogenous full-length CFH and miniCFH encoded by rAAV. Signals exceeding those observed in untransduced cells correlate with miniCFH expressed by rAAV. As shown in Figure 8, untransduced RPE cells secreted low levels of endogenous CFH, while supernatants from RPE cells transduced at MOIs of 20,000 and 50,000 showed statistically higher CFH concentrations. Furthermore, CFH concentrations were significantly higher in the supernatant from RPE cells transduced at MOI 50,000 compared to MOI 5,000. This data demonstrates an in vitro dose-dependent response.

[0162] Next, the ability of the secreted miniCFH protein to inhibit the alternative complement pathway was also evaluated in RPE cells transduced with rAAV containing a capsid protein with 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 the CAG promoter. Alternative complement inhibitory activity was measured in the supernatant from transduced cells by two different methods: 1) a modified version of the Wieslab® Complement System Alternative Pathway assay and 2) quantification of soluble C5b-9. To evaluate the functional inhibitory effect of miniCFH on the Wieslab® Complement System Alternative pathway, supernatant samples from rAAV-transduced RPE cells were mixed with serum, and the samples were applied to Wieslab assay plates. Complement pathway inhibition resulted in a decrease in MAC signaling.

[0163] Supernatants from cells transduced with rAAV at 20,000 MOI and 50,000 MOI resulted in statistically significant complement inhibition compared to untransduced RPE cell samples. As a positive control, RPE cells were treated with 200 nM recombinant CFH, a concentration comparable to the highest levels of miniCFH expression observed in this study. Dose-dependent complement inhibitory activity was observed, and treatment of RPE cells with high concentrations of eculizumab, a known C5-targeting complement inhibitor, also resulted in near-complete complement inhibition. (Figure 9A)

[0164] Since the Wieslab assay is not a truly quantitative measure of complement activation, the complement inhibitory activity of expressed miniCFH was evaluated using a second, more quantitative method. The concentration of soluble C5b-9 was measured using the SC5b ELISA kit. Supernatant samples from RPE cells transduced with rAAV showed a significant reduction in SC5b-9 formation compared to samples with supernatant from untransduced RPE cells (Figure 9B). Furthermore, a dose-dependent effect on soluble C5b-9 formation was also observed. As expected, untransduced cell samples supplemented with eculizumab showed minimal C5b-9 formation. This reduction in soluble C5b-9 demonstrates functional inhibition of the alternative complement pathway. In summary, these results demonstrate that miniCFH secreted by RPE cells transduced with rAAV can dose-dependently inhibit the alternative complement pathway.

[0165] In addition to characterizing the functional effect of expressed miniCFH on complement activation, the upstream mechanism of miniCFH was evaluated via a C3 convertase disintegration-promoting activity assay. To determine whether miniCFH acts as a cofactor with complement factor I (CFI) to promote C3b degradation, similar to full-length CFH, supernatant from rAAV-transduced cells was combined with purified C3b and CFI instead of purified full-length CFH (Figure 10, lanes 5-9). Compared to untransduced samples (Figure 10, lane 5), samples derived from transduced RPE (Figure 10, lanes 6-8) resulted in larger C3b α-chain cleavage, and higher MOI samples resulted in larger cleavage than lower MOI samples. In the absence of CFI, miniCFH produced by RPE resulted in minimal C3b cleavage, similar to purified full-length CFH (Figure 10, lane 9), supporting its role as a cofactor in C3b degradation.

[0166] In addition to these findings, the efficacy of rAAV, which includes a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter, was investigated in an in vitro human RPE disease model. For this purpose, a model of retinal complement activation was established in human iPSC-derived RPE cultures. To mimic the disease state, serum and zymosan, an alternative complement pathway activator, were introduced into the culture medium, resulting in MAC deposition on the RPE over the following 24 hours, as determined by immunocytochemistry (ICC) and flow cytometry.

[0167] As shown in Figure 11 below, rAAV treatment of RPE 6 days prior to complement activation induced a dose-dependent reduction in MAC deposition on RPE due to the presence of zymosan. Eculizumab, when added to the culture simultaneously with complement activation, exerted a similar protective effect, as expected. Flow cytometry quantification of MAC deposition (Figure 11B) revealed that RPE cells transduced with rAAV and RPE supplemented with eculizumab showed significantly reduced MAC formation compared to untransduced RPE. Furthermore, MAC formation was significantly lower in RPE cells transduced at MOI 2,000 and 5,000 compared to MOI 800, showing a dose-dependent response. In summary, these immunocytochemistry (ICC) and flow cytometry data demonstrate that, in addition to complement inhibition in cell-free assays, miniCFH secreted from RPE transduced with rAAV products also protected RPE from MAC deposition in disease-like conditions.

[0168] Finally, we evaluated the upstream function of miniCFH in this in vitro model. Activation of the surrogate complement pathway results in a significant increase in C3b 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 generation of iC3b ultimately reduces the amount of C3b produced, and therefore reduces the amount of iC3b in the system. Since iC3b has a significantly longer half-life compared to C3b, it is commonly used as a readout for C3b. Therefore, iC3b concentration was measured as a surrogate for C3b levels in the disease model supernatant (Figure 12). Supernatant from RPE cells transduced with rAAV showed a decrease in iC3b levels compared to supernatant from untransduced RPE cells (Figure 12). However, since eculizumab modulates the complement system further downstream in the protein cascade, as expected, there was no difference in iC3b concentration between the untransduced RPE supernatant supplemented with eculizumab and the untransduced RPE supernatant.

[0169] In summary, human iPSC-derived RPE cells transduced with rAAV (a capsid protein containing the amino acid sequence shown in SEQ ID NO: 42, and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34 operably linked to the CAG promoter) expressed and secreted miniCFH in a dose-dependent manner. The expressed miniCFH exhibited functional surrogate complement inhibitory activity and specific C3 convertase degradation-promoting activity, consistent with the functional activity of endogenous CFH. Furthermore, transduction of RPE with rAAV resulted in a protective effect on RPE cultures if the complement pathway was subsequently activated, as assessed by the levels of C5b-9 / MAC deposition and iC3b levels on RPE. MAC is the terminal complex of the complement cascade and the primary effector of complement-mediated cell death. iC3b is the degradation product of C3b and a non-endpoint readout of surrogate complement activation. Ultimately, cultures introduced with rAAV showed reduced soluble iC3b levels compared to cultures without rAAV, and MAC deposition on RPE was similarly reduced, as visualized by ICC and quantified by flow cytometry.

[0170] Example 4 In vivo ocular pharmacodynamic studies using rAAV expressing miniCFH in non-human primates.

[0171] The safety of rAAV (containing a capsid protein with the amino acid sequence shown in SEQ ID NO: 42 and a heterogeneous nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 34, operably linked to a CAG promoter) and miniCFH transgene protein expression were evaluated after a single intravitreous (IVT) administration to both eyes of male cynomolgus monkeys. Male cynomolgus monkeys received a single IVT administration of rAAV at a dose of 50 μL / eye to both eyes. Groups 1 and 2 consisted of 2 males per group, each group comprising 5 × 10⁶ individuals. 11 and 5×10 10 The patients were administered vg / eye and euthanized 6 weeks after administration for tissue collection. Groups 3, 4, and 5 each received rAAV at 5 × 10⁶ doses. 11 , 5×10 10 and 1 × 10 10 One dose of IVT was administered to each eye (vg / eye, bilateral), and the patients were observed 12 weeks before final euthanasia.

[0172] After in vitro administration of rAAV, the concentration of miniCFH protein was examined in aqueous humor, retinal tissue, and serum samples by LC-MS. A treatment-related dose-response for miniCFH protein expression was observed in aqueous humor (Figure 13).

[0173] miniCFH transgene protein levels were higher in the retina and RPE / choroid and relatively lower in the aqueous humor (Figure 14). This distribution pattern was expected given the retinotropic properties of the capsid and the fact that retinal tissue is likely the primary source of miniCFH protein secretion.

[0174] miniCFH protein was 5 × 10 on day 15. 11It was only detectable in the serum of the vg / eye dose group, at a very low value close to the LLOQ of 5 ng / mL, demonstrating that the miniCFH protein expressed from rAAV was mostly contained within the eye after IVT administration and was not distributed in the systemic circulation at significant levels (see Table 6 below). Table 6. miniCFH levels in aqueous humor, vitreous humor, and serum of rAAV-treated cynomolgus monkey samples

Table 6

[0175] There were no significant gross findings in animals euthanized at week 6 or 12, or significant changes in clinical pathology, hematology, or coagulation findings across the rAAV dose groups. No treatment-related changes were observed in any clinical pathology parameters after rAAV treatment, and minor fluctuations were generally attributable to the immunosuppressive regimen.

[0176] Microscopic findings noted after rAAV treatment at 6 weeks included minimal non-harmful perivascular mononuclear cell infiltration in the retina. Findings at 12 weeks were equivalent and included minimal to mild non-harmful mononuclear cell infiltration in the ciliary body, minimal retinal degeneration and nuclear condensation cells of unknown significance in one eye, and minimal perivascular mononuclear cell infiltration at the optic nerve head, none of which were considered harmful. No microscopic findings were present in the systemic organs in any of the dose groups. IVT treatment with rAAV resulted in the production of both vector capsid antibodies and anti-miniCFH antibodies in most animals by 12 weeks. Antigen-specific IFN-γ T cell responses measured by ELISPOT were not detected against either the vector capsid or CFH peptide pool in rAAV-treated NHP eyes at either 6 or 12 weeks post-administration. Overall, rAAV was well tolerated after a single bilateral IVT dose in male cynomolgus monkeys at doses up to 5×10 11 vg / eye (HED 1×10 12 vg / eye).

[0177] Single-dose intravitreal administration ocular evaluation study in cynomolgus monkeys

[0178] Another study was initiated to comprehensively evaluate the ocular structural and functional parameters in a small group of male and female cynomolgus non-human primates after single-dose unilateral intravitreal injection (IVT) of rAAV (containing 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), and the animals were followed up for 12 weeks after administration.

[0179] Two groups of cynomolgus monkeys, each consisting of 1 male and 2 females per group, were administered rAAV once into the right eye at either 2.8×10 10 vg / eye or 1.5×10 11 vg / eye on Day 1 of the study. The left eyes of the animals were treated with vehicle simultaneously. The dose was 50 μL / eye for both the left and right eyes.

[0180] Survival parameters included daily mortality / cage-side evaluation, once before dosing in Week 1, before dosing and at 1, 2, 4 and 8 hours after dosing on Day 1 of the study, and then daily until Day 7, and then weekly for the remainder of the study period. Body weight was evaluated weekly before dosing and during the study period. Ocular examinations were performed on all animals before the test, on Day 2, Day 4 (±1), Day 7, and at Week 2, Week 3, Week 4, Week 5, Week 6, Week 9 and Week 12.

[0181] Intraocular pressure was evaluated before the study, on Day 2, Day 4 (±1), Day 7, and at Week 2, Week 3, Week 4, Week 5, Week 6, Week 9 and Week 12. Fluorescein angiography, optical coherence tomography (OCT) and wide-field color fundus imaging were evaluated before the study and at Weeks 3, 6 and 12. Finally, electroretinogram (ERG) evaluations were performed before dosing and at Weeks 6 and 12 before final euthanasia.

[0182] 2.8×10 10 or 1.5×10 11Intravitreal administration of rAAV at a dose of vg / ocular was well tolerable in cynomolgus monkeys throughout 12 weeks post-injection, as supported by mortality, clinical findings, ocular, clinicopathological, gross pathological, and organ weight parameters. There were no signs of intraocular inflammation at any point in time, no rAAV-related changes in IOP values ​​or ocular structures identified by fluorescein angiography or anterior and posterior OCT, and no treatment-related effects on retinal function identified by ERG assessment.

[0183] 2.8 x 10 up to day 80 10 or 1.5 × 10 11 Following intravitreal administration of vg / ocular rAAV, there were no definitive rAAV-related effects on clinicopathological parameters in either sex. Minor variations in hematological and clinical chemistry findings may have been attributable to methylprednisolone administration and were not considered to be related to rAAV treatment.

[0184] Overall, rAAV is 1.5 × 10⁻⁶ in non-human primates. 11 The patient was well-tolerated after a single intravitreal administration of up to vg / eye, and no clear procedure-related effects or changes in the structure or function of the eye were observed.

[0185] While the materials and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods described herein without departing from the concept, spirit, and scope of the present invention. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to fall within the spirit, scope, and concept of the present invention.

Claims

1. (i) a variant AAV capsid protein comprising a heterologous peptide having a length of 7, 8, 9, 10, or 11 amino acids compared to the corresponding parent AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 1), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding the complement regulator H (CFH) protein or a fragment thereof, comprising a recombinant adeno-associated virus (rAAV).

2. The inserted peptide has 1 to 3 spacer amino acids (Y) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO: 1). １ ~Y ３ The rAAV according to claim 1, which has ) and preferably the inserted peptide is LAISDQTKHA (SEQ ID NO: 2).

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

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

5. The rAAV according to claim 4, wherein the capsid protein contains a P34A amino acid substitution to VP1 of AAV2 and comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the full-length amino acid sequence shown in SEQ ID NO: 42, and preferably the capsid protein comprises the amino acid sequence shown in SEQ ID NO:

42.

6. The rAAV according to any one of claims 1 to 5, wherein the rAAV exhibits an increase in the infectivity of retinal cells, preferably at least a twofold increase in infectivity, compared to the infectivity of retinal cells by an AAV containing the corresponding parent AAV capsid protein.

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

42.

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

9. The rAAV according to claim 8, wherein the reverse terminal repeat sequence is an AAV2 reverse terminal repeat sequence.

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

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

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

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

14. The CFH protein fragment mentioned above (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 A combination of SCR domains selected from one or more of the following, If necessary, a leader array and one or more linker arrays The rAAV according to any one of claims 10 to 13, comprising:

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

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

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

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

34.

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

34.

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

34.

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

34.

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

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

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

25. The rAAV according to claim 25, wherein the glycosylation sites are modified to SCR17 and SCR18.

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

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

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

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

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

31. The rAAV according to any one of claims 8 to 30, comprising a heterogeneous nucleic acid having a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence shown in any one of sequence numbers 43 to 46.

32. The rAAV according to any one of claims 8 to 31, comprising a heterogeneous nucleic acid having a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any one of sequence numbers 43 to 46.

33. The rAAV according to any one of claims 8 to 32, comprising a heterogeneous nucleic acid having a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of sequence numbers 43 to 46.

34. The rAAV according to any one of claims 8 to 33, comprising a heteronucleotide containing the nucleotide sequence shown in any one of sequence numbers 43 to 46.

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

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

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

38. The rAAV or the pharmaceutical composition is about 10 ８ vector genomes (vg) / eye to about 10 １３ vg / eye, preferably about 6×10 ９ vg / eye to about 6×10 １０ vg / eye, or about 1×10 ８ vg / eye, about 2×10 ８ vg / eye, about 3×10 ８ vg / eye, about 4×10 ８ vg / eye, about 5×10 ８ vg / eye, about 6×10 ８ vg / eye, about 7×10 ８ vg / eye, about 8×10 ８ vg / eye, about ×10 ８ vg / eye, about 1×10 ９ vg / eye, 2×10 ９ vg / eye, about 3×10 ９ vg / eye, about 4×10 ９ vg / eye, about 5×10 ９ vg / eye, about 6×10 ９ vg / eye, about 7×10 ９ vg / eye, about 8×10 ９ vg / eye, about 9×10 ９ vg / eye, about 1×10 １０ vg / eye, about 2×10 １０ vg / eye, about 3×10 １０ vg / eye, about 4×10 １０ vg / eye, about 5×10 １０ vg / eye, about 6×10 １０ vg / eye, about 7×10 １０ vg / eye, about 8×10 １０ vg / eye, about 9×10 １０ vg / eye, about 1×10 １１ vg / eye, about 2×10 １１ vg / eye, about 3×10 １１ vg / eye, about 4×10 １１ vg / eye, about 5×10 １１ vg / eye, about 6×10 １１ vg / eye, about 7×10 １１ vg / eye, about 8×10 １１ vg / eye, about 9×10 １１ vg / eye or about 1×10 １２ The method according to claim 37, wherein the drug is administered to the subject at a dose of vg / ocular by periorbital, intravitreous, suprachoroidal, or subretinal administration.

39. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 38, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / ocular.

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

41. The rAAV or the pharmaceutical composition is administered periophthalmos, intravitreous, suprachoroidal, or subretinally, resulting in approximately 10 ８ Vector genome (vg) / eye ~ approximately 10 １３ At a dose of vg / eye, preferably about 6 x 10 ９ vg / eye ~ approx. 6 x 10 １０ At a dose of vg / ocular, or approximately 1 x 10 ８ vg / eye, approx. 2 x 10 ８ vg / eye, approx. 3 x 10 ８ vg / eye, approx. 4 x 10 ８ vg / eye, approx. 5 x 10 ８ vg / eye, approx. 6 x 10 ８ vg / eye, approximately 7x10 ８ vg / eye, approx. 8 x 10 ８ vg / eye, approx. x 10 ８ vg / eye, approximately 1×10 ９ vg / eye, 2×10 ９ vg / eye, approx. 3 x 10 ９ vg / eye, approx. 4 x 10 ９ vg / eye, approx. 5 x 10 ９ vg / eye, approx. 6 x 10 ９ vg / eye, approximately 7x10 ９ vg / eye, approx. 8 x 10 ９ vg / eye, approx. 9 x 10 ９ vg / eye, approximately 1×10 １０ vg / eye, approx. 2 x 10 １０ vg / eye, approx. 3 x 10 １０ vg / eye, approx. 4 x 10 １０ vg / eye, approx. 5 x 10 １０ vg / eye, approx. 6 x 10 １０ vg / eye, approximately 7x10 １０ vg / eye, approx. 8 x 10 １０ vg / eye, approx. 9 x 10 １０ vg / eye, approximately 1×10 １１ vg / eye, approx. 2 x 10 １１ vg / eye, approx. 3 x 10 １１ vg / eye, approx. 4 x 10 １１ vg / eye, approx. 5 x 10 １１ vg / eye, approx. 6 x 10 １１ vg / eye, approximately 7x10 １１ vg / eye, approx. 8 x 10 １１ vg / eye, approx. 9 x 10 １１ vg / eye or approximately 1 x 10⁻⁶ １２ The method according to claim 40, wherein the subject is administered a dose of vg / ocular.

42. The rAAV or the pharmaceutical composition contains about 1 × 10 ９ vg / eye ~ approx. 1 x 10 １０ The method according to claim 41, wherein the drug is administered to the subject by intravitreous administration at a dose of vg / ocular.

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

44. The method according to claim 43, wherein the rAAV or the pharmaceutical composition is administered to the target eye by intravitreal administration.

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