AAV-mediated ocular gene therapy

Abstract

The present disclosure provides a method of reducing the levels of preexisting neutralizing antibodies against adeno-associated viruses (AAV) prior to an AAV-mediated gene therapy to treat an ocular disease.

Description

AAV-MEDIATED OCULAR GENE THERAPY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from 63 / 694,636, filed September 13, 2024, and U.S. Provisional Application No.63 / 803,510, filed on May 9, 2025, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on September 11, 2025, is named “122548.WO019.xml” and is 45,875 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[0003] Adeno-associated viral (AAV) vectors are important tools for gene therapy as they are non-pathogenic, replication-deficient, and non-integrating. A significant hurdle in AAV- mediated gene therapy is the prevalence of preexisting neutralizing antibodies (nAbs) in human populations (Naso et al., Mol Ther. (2020) 28:723-46; Ros-Ganan et al., Clinical & Translational Immunology (2022); e1375). These antibodies neutralize the AAV vectors, severely limiting their ability to transduce target cells and express the payload genes.

[0004] Preexisting anti-AAV nAbs have been found in the interior of the eye, despite the eye’s immune privilege. The presence of these nAbs has been linked to weak or no transgene expression following intravitreal injection of AAV in monkeys (Kotterman et al., Gene Ther. (2015) 22(2):116-26). Furthermore, there has been evidence that AAV-based gene therapy can induce capsid-specific T cell responses and new production of anti-AAV antibodies, further limiting the therapeutic potential of such therapy (Bucher et al., Prog Retin Eye Res. (2021) 83:100915). Thus, there remains a need for improved AAV-mediated ocular gene therapy in AAV-seropositive patients.SUMMARY OF THE INVENTION

[0005] The present disclosure provides a method of delivering a therapeutic protein to a diseased eye in a subject in need thereof through ocular gene therapy, comprising: injecting intravitreally to the diseased eye a composition comprising an IgG-degrading enzyme (e.g., Ides) and then injecting intravitreally about 1E10 to about 1E12 vector genomes of a recombinant adeno-associated virus (rAAV) whose genome comprises a coding sequence for the therapeutic protein.

[0006] In another aspect, the present disclosure also provides a method of reducing anti- drug immune response in ocular gene therapy in a subject in need thereof (e.g., for four or more weeks), comprising: injecting intravitreally to a diseased eye of the subject a composition comprising an IgG-degrading enzyme (e.g., Ides) and then injecting intravitreally about 1E10 to about 1E12 vector genomes of a recombinant adeno-associated virus (rAAV) whose genome comprises a coding sequence for a therapeutic protein.

[0007] In some embodiments, the first and second injecting steps are separated by no more than five days (e.g., by 1-3 days). In some embodiments, the composition comprises 10-50 µg of IdeS.

[0008] In some embodiments, the rAAV is of AAV2 serotype.

[0009] In some embodiments, the diseased eye has age-related macular degeneration (AMD). In further embodiments, the diseased eye has wet AMD and the therapeutic protein comprises Ig-like domain 2 of VEGFR-1 (Flt-1).

[0010] In some embodiments, the therapeutic protein comprises SEQ ID NO:3, optionally wherein the rAAV comprises a nucleic acid comprising SEQ ID NO:2. In some embodiments, the therapeutic protein comprises SEQ ID NO:5, optionally wherein the rAAV comprises a nucleic acid comprising SEQ ID NO:4.

[0011] In some embodiments, the diseased eye has dry AMD and the rAAV comprises a nucleic acid encoding a first antibody fragment specific for activated complement subcomponent C1s (aC1s) and a second antibody fragment specific for complement factor Bb, wherein the first antibody fragment comprises heavy chain CDR (HCDR) 1-3 comprising SEQ ID NOs:6-8, respectively, and light chain CDR (LCDR) 1-3 comprising SEQ ID NOs:9- 11, respectively, and the second antibody fragment comprises HCDR1-3 comprising SEQ ID NOs:15-17, respectively, and LCDR1-3 comprising SEQ ID NOs:18-20, respectively.

[0012] In some embodiments, the first antibody fragment comprises a heavy chain variable domain (VH) comprising SEQ ID NO:12 and a light chain variable domain (VL) comprising SEQ ID NO:13, and the second antibody fragment comprises a VH comprisingSEQ ID NO:21 and a VL comprising SEQ ID NO:22. In some embodiments, the first and second antibody fragments are two separate single-chain Fab proteins, the first antibody fragment comprising SEQ ID NO:14 and the second antibody fragment comprising SEQ ID NO:23, optionally wherein the rAAV comprises a genome comprising SEQ ID NO:24.

[0013] In some embodiments, the first and second antibody fragments are fused through a peptide linker to form a fusion protein. In further embodiments, the fusion protein comprises SEQ ID NO:25, optionally the rAAV comprises a genome comprising SEQ ID NO:26. In other embodiments, the fusion protein comprises SEQ ID NO:27, optionally wherein the rAAV comprises a genome comprising SEQ ID NO:28.

[0014] In some embodiments, the subject has been determined to be seropositive for AAV prior to the first injecting step.

[0015] Also provided herein are recombinant AAV pharmaceutical compositions herein for use in any of the methods provided herein, as well as a use of the recombinant AAV for the manufacture of a medicament for use in any of the methods provided herein.

[0016] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGs.1A-E show the impact of systemic administration of an IgG-degrading enzyme (IdeS) on nonhuman primates (NHPs) treated with gene therapy vector AAVGMU037-sFLT02. FIG.1A is a diagram showing the timeline of the study. The figure shows when IdeS and AAVGMU037-sFLT02 were administered systemically and when serum was obtained from non-human primates (NHPs) during the study (at day 0). The figure also shows that NHPs were sacrificed at the end of the in-life study (day 90). Droplets in the figure indicate when bleeds were taken. FIGs.1B and 1C are dot plots showing the serum neutralizing antibody (nAb) titers in seropositive NHPs not pre-treated or pre-treated with IdeS, respectively, two days prior to AAVGMU037 -sFLT02 dosing; each dot represents a test animal. A titer of 0 on the graph indicates a titer <4, which is the lower limit of detection (LLOD) of the assay. FIG.1D is a bar graph showing vector genome (vg) levels in the liver of NHPs 90 days after AAVGMU037-sFLT02 dosing; the bars represent, from left to right: seronegative NHPs, seropositive NHPs that did not receive IdeS, and seropositiveNHPs that received intravenous (IV) injection of IdeS prior to AAVGMU037-sFLT02 dosing. FIG.1E is a line graph showing sFLT02 expression out to 90 days after AAVGMU037-sFLT02 dosing. The lines represent: seronegative NHPs (black line); seropositive NHPs that did not receive IdeS (purple line); seropositive NHPs that received an IV injection of IdeS (blue line) prior to AAVGMU037-sFLT02 dosing.

[0018] FIGs.2A and 2B are graphs showing vg levels in the NHP retina five weeks after AAV2-sFLT02 dosing. The graphs show median with 95% confidence interval. * P<0.05 as determined by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. In FIG.2A, the bars represent, from left to right: seronegative NHP receiving intravitreal (IVT) injection of PBS; seropositive NHP receiving IVT injection of PBS; seropositive NHP receiving IVT injection of IdeS; and seropositive NHP receiving IV injection of IdeS. In FIG.2B, left panel, the bars represent, from left to right: seronegative NHP receiving IVT injection of PBS; seropositive NHP receiving IVT injection of PBS; and seropositive NHP receiving IVT injection of IdeS, prior to intravitreal dosing of AAV2- sFLT02. In FIG.2B, middle and right panels, the dot plots show pre-existing serum nAb (day 1) vs. retina vector genome levels (middle panel) and day 4 serum nAb titers vs. retina vector genome levels (right panel). Data is from OS (left) eye.

[0019] FIG.3 is a panel of graphs showing transgene transcript expression in the retina (left panel) and sFLT02 levels in the vitreous (right panel). Data is from OS eye. The bars represent, from left to right: seronegative NHP receiving IVT injection of PBS; seropositive NHP receiving IVT injection of PBS; and seropositive NHP receiving IVT injection of IdeS prior to IVT dosing of AAV2 sFLT02; n = 3 for “seropositive + PBS” transcript data due to insufficient RNA from one retina.

[0020] FIG.4A- 4C are graphs showing AAV2 nAb titers in serum (FIG.4A), vitreous humor (VH) (FIG.4B), and aqueous humor (AH) (FIG.4C) as a function of time (at days 4, 18, or 39) after IVT AAV administration in NHPs. Boxplots show distribution of nAb titers within each treatment group and for each time point. Groups of bars, representing treatment groups, from left to right: seronegative NHP receiving IVT injection of PBS; seropositive NHP receiving IVT injection of PBS; and seropositive NHP receiving IVT injection of IdeS. Day 1: IdeS or PBS; Day 4: IVT AAV2-sFLT02; Day 18: 2 weeks post AAV administration; Day 39: end of study. VH day 18, OS eye only. All other AH and VH samples are from both eyes. * VH (all groups) not collected on day 4.

[0021] FIGs.5A to 5D are graphs showing AAV2 nAb titers in serum (FIG.5A), VH (FIG.5B), and AH (FIG.5C) five weeks after IVT AAV administration. Boxplots showdistribution of nAb titers within each treatment group. FIG.5D is a combination of FIGs. 5A to 5C organized by treatment groups. VH and AH include separate measurements for each eye. Bars or groups of bars (each group representing a treatment group), from left to right: seronegative NHP receiving IVT injection of PBS; seropositive NHP receiving IVT injection of PBS; and seropositive NHP receiving IVT injection of IdeS. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present inventors have discovered that intravitreal delivery of an IgG- degrading enzyme (e.g., IdeS) reduces the adverse impact of preexisting anti-AAV neutralizing antibodies (nAbs) on viral transduction in AAV-mediated ocular gene therapy. The present inventors have also discovered that contrary to intravitreal delivery, systemic (e.g., intravenous) delivery of the same enzyme does not have the same mitigating effect. Accordingly, the present disclosure provides methods of improved ocular AAV-mediated gene therapy in patients with preexisting anti-AAV nAbs by pretreating the patients with intravitreal injection of an IgG-degrading enzyme (e.g., IdeS). The methods lead to increased viral transduction in targeted ocular cells, higher and more persistent transgene expression, and reduced development of anti-drug immune response against the AAV vector used in the gene therapy. I. Pretreatment with IgG-Degrading Enzymes

[0023] Recombinant AAV is a widely used viral vector for delivering a payload (e.g., a coding sequence for a therapeutic protein, or a nucleic acid such as donor DNA for gene editing) to targeted human cells. AAV-mediated ocular gene therapy has been explored for treating congenital ocular diseases (e.g., retinitis pigmentosa) and degenerative ocular diseases (e.g., aged-related macular degeneration or “AMD”). To improve the efficacy and safety of AAV-mediated ocular gene therapy in patients who are seropositive for anti-AAV nAbs, the present invention utilizes pretreatment of the diseased eye with intravitreal injection of an IgG-specific protease. The term “seropositive” refers to the presence of antibodies against AAV in the blood of a subject. Some of such antibodies may be neutralizing antibodies (nAbs).

[0024] In some embodiments, the IgG-specific protease is a protein belonging to the family of proteases called immunoglobulin-degrading enzymes and produced by bacteria to evade a host’s immune system. Members of this family, which can be used in the present methods as an IgG-degrading enzyme, include, without limitation: IdeS (Von Pawel-Rammingen et al., EMBO J (2002) 21(7):1607-15), SpeB (ibid.), IdeZ (Hulting et al., FEMS Microbiol Lett. (2009) 298(1):44-50, and Elmore et al., JCI Insight. (2020) 5(19):e139881), IdeE (Lannergard and Guss, FEMS Microbiol Lett. (2006) 262(2):230-5), IceMG (Smith et al., Molecular Therapy (2024) 32(7):2080-93), and IdeP (Rungelrath et al., Vet Microbiol. (2017) 201:42-8).

[0025] In some embodiments, the IgG-specific protease used herein is IdeS derived from Streptococcus pyogenes. IdeS cleaves IgG at a single site below the hinge region, yielding F(ab’)2 and Fc fragments. IdeS can cleave all four subclasses of human IgG antibodies. In some embodiments, the IdeS is a recombinant protein produced in E. coli host cells with an amino acid sequence shown below: MDSFSANQEI RYSEVTPYHV TSVWTKGVTP PANFTQGEDV FHAPYVANQG WYDITKTFNG KDDLLCGAAT AGNMLHWWFD QNKDQIKRYL EEHPEKQKIN FNGEQMFDVK EAIDTKNHQL DSKLFEYFKE KAFPYLSTKH LGVFPDHVID MFINGYRLSL TNHGPTPVKE GSKDPRGGIF DAVFTRGDQS KLLTSRHDFK EKNLKEISDL IKKELTEGKA LGLSHTYANV RINHVINLWG ADFDSNGNLK AIYVTDSDSN ASIGMKKYFV GVNSAGKVAI SAKEIKEDNI GAQVLGLFTL STGQDSWNQT N (SEQ ID NO:29)

[0026] In some embodiments, the IgG-specific protease used in the present method is IdeZ derived from Streptococcus equi. Like IdeS, IdeZ specifically cleaves IgG molecules below the hinge region, yielding F(ab’)2and Fc fragments.

[0027] In some embodiments, the IdeS is injected intravitreally in an ophthalmological formulation at a dose of about 8 to about 100 µg per eye. In some embodiments, the dose is 8-50, 50-100, or 25-75 µg per eye. In some embodiments, the dose is 8-20, 15-45, 20-30, 20- 40, 25-35, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 µg per eye. In some embodiments, the IdeS is provided at a dose of about 30 µg.

[0028] In some embodiments, the ophthalmological formulation comprises a phosphate- buffered solution, with a pH of about 6.5 to about 7.5, e.g., around pH 7.

[0029] In some embodiments, the IgG-specific protease (e.g., IdeS) is administered within 5 days (within 2 or 3 days) before the administration of the AAV drug. In some embodiments, the protease is administered 1, 2, 3, 4 or 5 days prior to the AAV administration. In some embodiments, the protease is administered 2 or 3 days prior to the AAV administration.

[0030] The pretreatment with an IgG-degrading enzyme such as IdeS may be given to a patient with or without pre-screening the patient for the presence of anti-nAbs. Whether to pre-screen or not may depend on the serotype of the AAV vector to be used in the genetherapy and the prevalence of seropositivity for that AAV serotype among the population in the geographic region where the patient is located. If pre-screening is to be conducted, the pretreatment is given to only patients who are tested positive for anti-AAV nAbs. Pre- screened may be performed 1-10 weeks (e.g., 4-8 weeks) prior to pretreatment. In some embodiments, the patient is pre-screened for serum anti-AAV nAbs with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks prior to pretreatment. In some embodiments, the patient is pre-screened for serum anti-AAV neutralizing antibodies within 6 weeks prior to pretreatment.

[0031] Anti-AAV nAbs can be assayed by well-known methods in the art. For example, nAbs may be detected and quantified using enzyme-linked immunosorbent assay (ELISA), or cell-based assays in which an AAV vector expressing a reporter gene (e.g., a β-galactosidase reporter gene; see Examples below) is used. The presence and titers of anti-AAV nAbs may be assayed at the serum level, as serum anti-AAV nAb levels have been found to correlate the anti-AAV nAb levels. Alternatively, the anti-AAV nAb presence and titers may be assayed in ocular biofluids (e.g., aqueous humor and vitreous humor).

[0032] In some embodiments, the pretreatment with IdeS may lead to more than 5-to 10- fold increase in viral transduction and gene expression in the eye treated with rAAV- mediated gene therapy. In some embodiments, the pretreatment leads to a significant reduction of anti-drug immune response as measured by anti-AAV nAb titers post rAAV injection and this anti-inflammatory effect may last for more than one month (i.e., for longer than four or five weeks). II. AAV-Mediated Ocular Gene Therapy

[0033] Recombinant AAV of any suitable AAV serotype may be used as a vector for delivering a payload of interest to the eye. For example, the rAAV may be AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, or AAVrh10, or of a pseudotype or a serotype that is a mutant, variant or derivative of one of the AAV serotypes listed herein (i.e., AAV derived from multiple serotypes). The AAV may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans or nonhuman primates.

[0034] In some embodiments, the rAAV is of AAV2.7m8, AAV5, AAV8, AAV9, AAVR100, AAVPHP.eB, AAVPHP.B, AAVrh78R, or AAV.ANC80 serotype. In particular embodiments, the rAAV is of a serotype with tropism for the eye. In certain embodiments, the rAAV is of AAV2 or AAV5 serotype. In some embodiments, the rAAV has a wildtype capsid, such as a capsid of wildtype AAV2 or AAV5 serotype. In further embodiments, therAAV has a wildtype AAV2 capsid and its recombinant genome comprises AAV2 inverted terminal repeats (ITR) flanking the payload nucleotide sequence.

[0035] In some embodiments, the rAAV herein has an AAV2 capsid. In particular embodiments, the AAV2 capsid is a wildtype AAV2 capsid. In other embodiments, the AAV2 capsid contains mutations that improve the rAAV2’s potency and production yield.

[0036] Viral vectors described herein may be produced using methods known in the art. Any suitable permissive or packaging cells may be employed to produce the viral particles. For example, mammalian (e.g., 293 or HeLa) or insect (e.g., Sf9) cells may be used as the packaging cell line. Recombinant AAV vectors can be replicated and packaged into infectious viral particles when introduced into host cells that have been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and capsid proteins). See, e.g., U.S. Pat. 11,261,463. Recombinant AAVs may then be isolated from the producing cells, purified, and formulated into ophthalmological compositions suitable for intravitreal delivery to the eye.

[0037] The ophthalmological compositions may comprise pharmacologically, especially ophthalmologically, acceptable carriers, diluents, and / or excipients. For example, the composition may comprise a tonicity agent (e.g., sodium chloride, amino acids, sugars, or combinations thereof), a surfactant (e.g., polysorbate 20 or polysorbate 80), and / or a stabilizer (e.g., a methionine).

[0038] The pharmaceutical compositions may be delivered by intravitreal injection (e.g., front, mid or back vitreous injection).

[0039] The present pharmaceutical compositions may be delivered in a therapeutically effective amount to treat an ocular condition. An “therapeutically effective amount” means a dosage sufficient to produce a desired result, e.g., amelioration of one or more symptoms of the disease to be treated (e.g., macular edema, growth of retinal lesions or destruction of retinal layer, photoreceptor cell death, in the case of wet AMD; and growth of GA lesions, retinal lesions, destruction of retinal layer, or photoreceptor cell death in the case of dry AMD), and / or slowing progression of the disease. A desired result may also include improvement in one or more functional symptoms; for example, the desired result may be reduction of visual distortions, improved central vision, improved vision in low light settings, and / or reduced blurriness. By “treat” is meant amelioration of one or more symptoms of the disease and / or slowing of the progress of the disease.

[0040] The rAAV may be delivered to the eye at a dose of about 1E7 to about 1E15 vector genomes (vg), for example, about 1E10 to about 1E12 vg, per eye. In some embodiments, the dosage of rAAV injected into the eye is 1E8 to 1E14, 10E9 to 10E13, 10E9 to 10E12 vg.

[0041] In some embodiments, the patient is treated, before, during, and / or after the rAAV injection, with an anti-inflammatory agent (e.g., a corticosteroid) to prevent or ameliorate potential immune response against the rAAV, in addition to pretreatment with an IgG- degrading enzyme as described herein. In some embodiments, the anti-inflammatory agent is a corticosteroid selected from methylprednisolone, difluprednate, triamcinolone, dexamethasone, etc. Other corticosteroids that are readily known and available in the art may be used in the methods disclosed herein. The anti-inflammatory agent may be administered locally (e.g., through eye drops) or systematically. In some embodiments, the agent may be administered intraocularly (e.g., intravitreally), orally, intravenously, intramuscularly, or subcutaneously.

[0042] The anti-inflammatory agent may be administered prior to AAV injection and may be administered following AAV injection as deemed necessary by a clinician.

[0043] AAV-mediated ocular gene therapies may be used to treat congenital conditions such as retinitis pigmentosa and Stargardt disease and acquired conditions such as retinoschisis and AMD. Additional exemplary diseases for gene therapy may include glaucoma, diabetic retinopathy, and macular edema. Exemplary therapies are described below. A. AAV-Based Gene Therapy for Wet AMD

[0044] The present disclosure provides ocular gene therapy for wet AMD with improved efficacy and safety. In some embodiments, the present methods comprise intravitreal injection of an IgG-degrading enzyme as described above, followed by intravitreal injection of a composition comprising an rAAV for delivering to an eye inflicted with wet AMD a soluble protein comprising at least an extracellular Ig-like domain of VEGFR-1 (Flt-1). An exemplary amino acid sequence of human Flt-1 is shown in SEQ ID NO:1, wherein amino acids 1-26 span the signal sequence and the extracellular region spans amino acids 27-758. Flt-1’s extracellular portion includes seven Ig-like domains. Ig-like domain 2 encompasses amino acids 151-214 of SEQ ID NO:1. In some embodiments, the therapeutic protein delivered by the rAAV comprises amino acids 151-214 of SEQ ID NO:1 or an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to this stretch of sequence. In further embodiments, the therapeutic protein comprises amino acids residues131-226 of SEQ ID NO:1 or an amino acid sequence that is at least 95% (e.g., at least 96, 97, 98, or 99%) identical to this stretch of sequence.

[0045] The percent identity of two amino acid sequences (or of two nucleic acid sequences) may be obtained by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence.

[0046] In some embodiments, the therapeutic protein comprises a multimerization (e.g., dimerization) domain linked to the Flt-1 sequence, e.g., an Flt-1 Ig-like domain 2 sequence. For example, the dimerization domain may be a human IgG (e.g., IgG1) Fc sequence comprising a partial or complete hinge region, a CH2 domain, and a CH3 domain. In another example, the dimerization domain may contain just a human IgG (e.g., IgG1) CH3 domain.

[0047] In the therapeutic protein, the Flt-1 sequence may be linked to the multimerization domain directly or through a peptide linker. Exemplary peptide linkers are flexible peptide linkers such as glycine / serine-rich linkers (i.e., more than half of the residues in the linker are glycine and / or serine residues). In some embodiments, the peptide linker comprises 2-20 amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids. In further embodiments, the linker comprises nine glycine residues.

[0048] In certain embodiments, the therapeutic protein comprises SEQ ID NO:3. In further embodiments, the rAAV for delivering this therapeutic protein has a genome comprising SEQ ID NO:2 and operably linked transcription regulatory elements such as a promoter (e.g., a hybrid chicken β-actin promoter).

[0049] In certain embodiments, the therapeutic protein comprises SEQ ID NO:5. In further embodiments, the rAAV for delivering this therapeutic protein has a genome comprising SEQ ID NO:4 and operably linked transcription regulatory elements such as a promoter (e.g., a hybrid chicken β-actin promoter).

[0050] In some embodiments, the patients have choroidal neovascularization (CNV) secondary to wet AMD. B. AAV-Based Gene Therapy for Dry AMD

[0051] The present disclosure provides ocular gene therapy for dry AMD, including associated geographic atrophy, with improved efficacy and safety. In some embodiments, the present methods comprise intravitreal injection of an IgG-degrading enzyme as described above, followed by intravitreal injection of a composition comprising an rAAV for deliveringto an eye inflicted with dry AMD dual inhibitors of the complement classical and alternative pathways.

[0052] In some embodiments, the rAAV introduces both an inhibitor for activated complement subcomponent C1s (aC1s) and an inhibitor for complement Bb to the diseased eye of a patient. The aC1s inhibitor inhibits the initiation of the classical complement pathway and the Bb inhibitor inhibits the initiation of the alternative complement pathway. The two inhibitors are expressed either as two separate proteins or linked in a fusion protein,

[0053] In some embodiments, the aC1s inhibitor is an antibody fragment such as a single- chain Fab (scFab) or a single-chain Fv (scFv) whose heavy chain complementarity-determine regions (HCDR) 1-3 comprise SEQ ID NOs:6, 7, and 8, respectively, and whose light chain CDR (LCDR) 1-3 comprise SEQ ID NOs:9, 10, and 11, respectively. In further embodiments, the anti-aC1s scFv or scFab comprises a VH comprising SEQ ID NO:12 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; and a VLcomprising SEQ ID NO:13 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In certain embodiments, the anti-aC1s scFv or scFab comprises a peptide linker, such as a flexible linker, e.g., a linker comprising (G4S)n(SEQ ID NO:30), where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the VHand the VLof the scFv or the heavy and light chains of the scFab. In particular embodiments, the anti-aC1s scFab comprises SEQ ID NO:14 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.

[0054] In some embodiments, the rAAV has a genome comprising a bi-directional expression cassette for expressing the present anti-aC1s scFab and the present anti-Bb scFab as two separate proteins. In further embodiments, the rAAV genome comprises SEQ ID NO:24.

[0055] In other embodiments, the rAAV has a genome comprising an expression cassette for expressing the present anti-aC1s scFab or scFv and the present anti-Bb scFab or scFv as a single fusion protein. For example, the fusion protein may comprise SEQ ID NO:25, where the rAAV genome may comprise, e.g., SEQ ID NO:26. In another example, the fusion protein may comprise SEQ ID NO:27, where the rAAV genome may comprise, e.g., SEQ ID NO:28.

[0056] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in thepractice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0057] The terms “subject,” “individual” or “patient” are used interchangeably herein and refer to a vertebrate, e.g., a mammal. Mammals include, but are not limited to, rodents (e.g., mice and rats), simians, humans, farm animals, sport animals, and pets.

[0058] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any compound disclosed herein can be used in any of the treatment method here, wherein the individual to be treated is as defined anywhere herein. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0059] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLES Example 1: Systemic administration of an IgG-degrading enzyme (IdeS) depletes serum nAbs and enables AAV transduction

[0060] This example illustrates the rationale for pre-treatment with an IgG-degrading enzyme before administration of AAV gene therapy vector so as to increase the efficacy of ocular gene therapy in patients with preexisting AAV-neutralizing antibodies (nAb). A. Study Design

[0061] A NHP study was conducted (1) to determine the impact of serum nAb on AAV- mediated vector transduction of the liver, and (2) to measure the improvement in AAV-based viral transduction and transgene expression in AAV-seropositive animals that had been pre- treated with intravenous IdeS.

[0062] NHPs were assigned to three groups, (1) seronegative NHPs, (2) seropositive NHPs that did not receive IdeS treatment and (3) seropositive NHPs that received an IV administration of IdeS two days prior to administration of AAVGMU037-sFLT02. Serum nAb titers were measured on serum collected on study day 0 immediately before PBS or IdeS dosing, and again on days 3 and 7, following AAV administration on day 2. Levels of liver transduction and transgene expression of AAV-seronegative, AAV-seropositive, and AAV- seropositive animals that had been pre-treated with intravenous IdeS were assessed.

[0063] On study day 0, the NHPs were administered either PBS or sterile-filtered IdeS (0.5 mg / kg) through intravenous injection (FIG.1A). On day 2, the NHPs were dosed with AAVGMU037-sFLT02 (5x1012vg / kg) through IV injections.

[0064] Serum nAb titers were tested on serum collected on study day 0 prior to PBS or IdeS dosing, and again on study days 3 and 7 following AAV administration on day 2. Levels of liver transduction and transgene expression of AAV-seronegative, AAV- seropositive, and AAV-seropositive animals that had been pre-treated with intravenous IdeS were assessed. B. Results

[0065] Neutralizing antibodies to the recombinant AAVGMU037 vector were quantified. It was observed that seropositive NHPs that received IV PBS (no IdeS treatment) had no decrease in serum nAb titers 3 days later. These NHPs mounted a robust AAV nAb response five days post AAV administration, which was detectable in serum (with titers ranging from 16 – 128 on day 3 to 8000 by day 7) (FIG.1B). When AAV was given to seropositive NHPs that were pre-treated with IdeS, serum nAb titers were completely suppressed (not detectable) on day 3. This suppression would allow AAVGMU037 transduction of its target cells (FIG. 1C). The nAb response was restored to levels comparable to when PBS (no IdeS) was added by day 7 (FIG.1B). Indeed, when the recombinant AAVGMU037 vector vg levels in the NHP liver were quantified using quantitative PCR, seropositive animals pre-treated with IdeSshowed vg levels that were nearly 1,000-times higher than seropositive animals pre-treated with PBS and were comparable to seronegative animals (FIG.1D).

[0066] Similar results were seen when the levels of vector-derived sFLT02 protein levels in the serum were quantified throughout the 90-day study using an ELISA kit (R&D Systems). As shown in FIG.1E, seropositive NHPs pre-treated with IdeS displayed sFLT02 protein levels that were approximately two orders of magnitude higher than seropositive NHPs pre-treated with PBS and were comparable to seronegative NHPs. Specifically, in seronegative NHPs pre-treated with PBS (serum nAb titers <4), AAVGMU037-sFLT02 transduction of the liver achieved approximately 1x106vector genomes (vg) per 500 ng genomic DNA. In seropositive NHPs that received PBS treatment, vector transduction was an approximate 2 logs lower than what was detected in seronegative animals.

[0067] These data demonstrate that preexisting AAV nAb seropositivity lowers the efficacy of liver transduction following intravenous AAV dosing, and that intravenous administration of an IgG-degrading enzyme is an effective approach to increase AAV- mediated vector transduction in the liver, even in highly seropositive NHPs. This finding led us to further explore whether IdeS treatment would improve retinal AAV transduction and transgene expression in seropositive NHPs. Example 2: Delivery of IdeS to NHP Eyes

[0068] This example illustrates an approach to increasing the efficacy of AAV-mediated gene therapy in the eye in patients with preexisting AAV-neutralizing antibodies (nAb) by pre-treating the eye intravitreally with an IgG-degrading enzyme. A. Study Design

[0069] A non-human primate (NHP) study was conducted (1) to determine the impact of serum nAb on AAV-mediated vector transduction of the retina, (2) to measure the improvement in AAV-based viral transduction and transgene expression in the eyes of AAV- seropositive animals that had been pre-treated with intravitreal IdeS, and (3) to assess the tolerability of intravitreal IdeS treatment. IdeS was purchased from Promega.

[0070] Cynomolgus macaques were pre-screened for titers of serum anti-AAV2 nAb 6 weeks prior to the study start and assigned to five treatment groups, with a range of preexisting nAb titers distributed between the seropositive groups that received intravitreal PBS or IdeS intravitreally or intravenously. Serum nAb titers were retested on serum collected on study day 1 immediately prior to PBS or IdeS dosing, and one NHP in the vehicle control group was found to have lower nAbs than identified in the pre-screen, whileone NHP in the intravitreal (IVT) IdeS treatment group and two NHPs in the intravenous (IV) IdeS treatment group were found to have higher nAb titers than identified in the pre-screen. The treatment groups are summarized in table 1 below. Table 1. Serum nAb Titers in NHPs

[0071] On study day 1, the NHPs were administered either PBS or sterile-filtered IdeS (15 µg / eye) through bilateral intravitreal injections, or sterile-filtered IdeS (0.5 mg / kg) through intravenous injection (Table 1). Three days later (study day 4), the NHPs were dosed with AAV2-sFLT02 (2x1010vg / eye) through bilateral intravitreal injections. NHPs in the control group received no ocular treatment on day 1 and were administered formulation buffer on study day 4. All NHPs received prophylactic steroids (40 mg methylprednisolone through weekly intramuscular injection) beginning on Day -3 and continuing weekly through the duration of the study, which concluded after 5 weeks of in-life exposure to AAV2- sFLT02. B. Results 1. Overall Tolerability

[0072] Intravitreal administration of PBS, IdeS, and AAV2-sFLT02 were well tolerated in cynomolgus macaques. There was no mortality and no test article-related clinical signs, effects on body weight, or changes among clinical pathology parameters, and no macroscopic observations. One eye of one seronegative animal that received intravitreal injection of PBS on Day 1 and AAV2-sFLT02 on Day 4 developed delayed intraocular inflammation on Day 26 that persisted through the end of the study. The eye also had a vitreous humor collection performed on Day 18 prior to development of inflammation observed on Day 26. The observed inflammation may have been secondary to the vitreous humor collection procedure;this is a known risk of the procedure. Due to the inflammation in this eye and the impact on retinal integrity, samples from this eye were excluded from analyses of vector transduction and transgene expression. 2. Vector Transduction

[0073] Vector genome levels in the NHP retina were quantified using quantitative PCR analyses of DNA purified from the retinas of the left eyes. Comparable DNA input across samples was confirmed using TUBB as a reference gene. In seronegative NHPs (serum nAb titers <4), AAV2-sFLT02 transduction of the retina achieved approximately 4x104vector genomes (vg) per 500 ng genomic DNA. In seropositive NHPs that received IVT PBS treatment, vector transduction was an order of magnitude lower than what was detected in the seronegative animals, averaging 4x103vg per 500 ng genomic DNA (FIGs.2A and 2B). Within the seropositive NHPs, there was no correlation between serum nAb titers and levels of transduction, as similar levels of transduction were detected in the context of serum nAb titers ranging from 8 to 512. In contrast to the seropositive NHPs that received PBS treatment, seropositive NHPs that received IVT IdeS treatment prior to AAV2 administration had levels of vector transduction comparable to the seronegative NHPs (approximately 6x104vg per 500 ng genomic DNA). In the IVT IdeS-treated seropositive group, IdeS treatment was effective in NHPs with Day 1 preexisting nAb titers up to 2048. In contrast to seropositive animals that received intravitreal IdeS, seropositive animals administered IdeS intravenously had vector genome levels similar to seropositive animals that did not receive IdeS.

[0074] These data demonstrate that preexisting AAV nAb seropositivity lowers the efficacy of retinal transduction following intravitreal AAV dosing, and that intravitreal (but not intravenous) administration of an IgG-degrading enzyme is an effective approach to increasing AAV-mediated vector transduction in the retina, even in highly seropositive NHPs. 3. Transgene Expression

[0075] Vector-derived transcript levels in the NHP retina were quantified using quantitative reverse transcriptase (RT)-PCR analyses of RNA purified from the retinas of the left eye. Comparable RNA input across samples was confirmed using HPRT1 as a reference gene, and transcript levels in each sample were corrected for any non-specific signal detected in control reactions processed without reverse transcriptase. Similar to what was detected for vector transduction, the median sFLT02 transcript levels were an order of magnitude lower in seropositive NHPs that received IVT PBS treatment (~3.4x103transcripts per 500 ng RNA)compared to seronegative NHPs (~3.9x104transcripts per 500 ng RNA), and seropositive NHPs that received IVT IdeS treatment prior to AAV2 administration have transcript levels comparable to the seronegative NHPs (~2.8x104transcripts per 500 ng RNA) (FIG.3, left panel). sFLT02 protein levels in the vitreous from the left eye also were quantified. It was observed that sFLT02 levels were increased in seropositive NHPs treated with IVT IdeS as compared to seropositive NHPs treated with IVT PBS (FIG.3, right panel). 4. Neutralizing Antibody Response

[0076] Neutralizing antibodies to AAV2 were quantified using a cell-based assay and an AAV2 vector expressing a β-galactosidase reporter gene (AAV2CMVLacZ). HeLa cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin (DMEM complete media) at 37oC and 5% CO2. HeLa cells were seeded into 96-well plates at a density of 2×104cells / well. Twenty-four hours after seeding, the cells were infected with Ad5ts149 at a multiplicity of infection (MOI) of 2 for 4 hours at 39°C. Serum, aqueous humor (AH), and vitreous humor (VH) samples were heat-inactivated at 56oC for 30 minutes prior to the assay. Two-fold serial dilutions of the serum, AH, VH, or positive control IVIG (Sigma G4386 lot #SLBM0524V) in DMEM complete media were prepared in triplicate (or duplicate if sample was limiting) in a 96-well round-bottom plate. AAV2CMVLacZ was added to the test samples at a MOI of 200 in an equal volume of DMEM complete media, incubated at 37oC for 1 hour, and then the test samples were added to the Ad5ts149-infected HeLa cells (after replacing the Ad5ts149-containing media with fresh media). Cells were incubated with the test samples at 39oC for 72 hours. AAV transduction was measured by quantifying β-galactosidase activity using the Galacto-StarTMkit (Tropix) and measuring relative light units on a BioTek luminometer. The neutralizing titer was calculated relative to AAV2CMVLacZ-infected HeLa cells in DMEM complete media alone (without serum, AH, or VH) and is reported as the reciprocal of the dilution of the test sample that decreases β- galactosidase expression by 50% or more. Cells in media without AAV2CMVLacZ served as the negative control in the assay. Any sample with a neutralizing titer >4 is considered nAb positive.

[0077] In seronegative NHPs (preexisting serum nAb titers <4), the majority of NHPs exhibited minimal to no increase in nAb titer five weeks after IVT AAV administration (Day 39) (Table 2 and FIGs.4A-4C, and 5A-5D). In contrast, seropositive NHPs that received IVT PBS mounted a robust AAV nAb response, which was detectable in serum (titers ranging from 512-4096), VH (titers ranging from 32-2048) and AH (titers ranging from 8-256) on day 39. However, seropositive NHPs that received an IVT dose of IdeS three days prior to AAV administration had lower levels of nAbs in serum, AH, and VH compared to seropositive NHPs that did not receive IdeS. In the eye, the majority of IVT IdeS-treated eyes had undetectable nAbs in the AH or VH. Table 2. Serum nAb Titers Post-AAV Treatment

[0078] These data demonstrate that intravitreal delivery of IdeS prior to AAV administration reduces neutralizing antibodies in the eye and systemically at 5 weeks after AAV dosing.SEQUENCES SEQ ID NO:1 – Full-length human Flt-1 amino acid sequence (signal sequence: boldface; Ig-like domain 2: underline) MVSYWDTGVL LCALLSCLLL TGSSSGSKLK DPELSLKGTQ HIMQAGQTLH LQCRGEAAHK WSLPEMVSKE SERLSITKSA CGRNGKQFCS TLTLNTAQAN HTGFYSCKYL AVPTSKKKET ESAIYIFISD TGRPFVEMYS EIPEIIHMTE GRELVIPCRV TSPNITVTLK KFPLDTLIPD GKRIIWDSRK GFIISNATYK EIGLLTCEAT VNGHLYKTNY LTHRQTNTII DVQISTPRPV KLLRGHTLVL NCTATTPLNT RVQMTWSYPD EKNKRASVRR RIDQSNSHAN IFYSVLTIDK MQNKDKGLYT CRVRSGPSFK SVNTSVHIYD KAFITVKHRK QQVLETVAGK RSYRLSMKVK AFPSPEVVWL KDGLPATEKS ARYLTRGYSL IIKDVTEEDA GNYTILLSIK QSNVFKNLTA TLIVNVKPQI YEKAVSSFPD PALYPLGSRQ ILTCTAYGIP QPTIKWFWHP CNHNHSEARC DFCSNNEESF ILDADSNMGN RIESITQRMA IIEGKNKMAS TLVVADSRIS GIYICIASNK VGTVGRNISF YITDVPNGFH VNLEKMPTEG EDLKLSCTVN KFLYRDVTWI LLRTVNNRTM HYSISKQKMA ITKEHSITLN LTIMNVSLQD SGTYACRARN VYTGEEILQK KEITIRDQEA PYLLRNLSDH TVAISSSTTL DCHANGVPEP QITWFKNNHK IQQEPGIILG PGSSTLFIER VTEEDEGVYH CKATNQKGSV ESSAYLTVQG TSDKSNLELI TLTCTCVAAT LFWLLLTLFI RKMKRSSSEI KTDYLSIIMD PDEVPLDEQC ERLPYDASKW EFARERLKLG KSLGRGAFGK VVQASAFGIK KSPTCRTVAV KMLKEGATAS EYKALMTELK ILTHIGHHLN VVNLLGACTK QGGPLMVIVE YCKYGNLSNY LKSKRDLFFL NKDAALHMEP KKEKMEPGLE QGKKPRLDSV TSSESFASSG FQEDKSLSDV EEEEDSDGFY KEPITMEDLI SYSFQVARGM EFLSSRKCIH RDLAARNILL SENNVVKICD FGLARDIYKN PDYVRKGDTR LPLKWMAPES IFDKIYSTKS DVWSYGVLLW EIFSLGGSPY PGVQMDEDFC SRLREGMRMR APEYSTPEIY QIMLDCWHRD PKERPRFAEL VEKLGDLLQA NVQQDGKDYI PINAILTGNS GFTYSTPAFS EDFFKESISA PKFNSGSSDD VRYVNAFKFM SLERIKTFEE LLPNATSMFD DYQGDSSTLL ASPMLKRFTW TDSKPKASLK IDLRVTSKSK ESGLSDVSRP SFCHSSCGHV SEGKRRFTYD HAELERKIAC CSPPPDYNSV VLYSTPPI SEQ ID NO:2 - sFLT01 nucleic acid sequence atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc acaggatctg gtagaccttt cgtagagatg tacagtgaaa tccccgaaat tatacacatg actgaaggaa gggagctcgt cattccctgc cgggttacgt cacctaacat cactgttact ttaaaaaagt ttccacttga cactttgatc cctgatggaa aacgcataat ctgggacagt agaaagggct tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca aaccggtgga ggtggaggtg gaggtggagg tcctaaatct tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcctctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatag SEQ ID NO:3 - sFLT01 amino acid sequence (signal sequence: boldface; Gly9 linker linking an Flt-1 extracellular region sequence to human IgG1 hinge, CH2and CH3: italic) MVSYWDTGVL LCALLSCLLL TGSGRPFVEM YSEIPEIIHM TEGRELVIPC RVTSPNITVT LKKFPLDTLI PDGKRIIWDS RKGFIISNAT YKEIGLLTCE ATVNGHLYKT NYLTHRQTGG GGGGGGGPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK SEQ ID NO:4 – sFLT02 nucleic acid sequence atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc acaggatctg gtagaccttt cgtagagatg tacagtgaaa tccccgaaat tatacacatg actgaaggaa gggagctcgt cattccctgc cgggttacgt cacctaacat cactgttact ttaaaaaagt ttccacttga cactttgatc cctgatggaa aacgcataat ctgggacagt agaaagggct tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca aaccggtgga ggtggaggtg gaggtggagg tcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat ag SEQ ID NO:5 - sFLT02 amino acid sequence (signal sequence: boldface; Gly9 linker linking an Flt-1 extracellular region sequence to human IgG1CH3: italic) MVSYWDTGVL LCALLSCLLL TGSGRPFVEM YSEIPEIIHM TEGRELVIPC RVTSPNITVT LKKFPLDTLI PDGKRIIWDS RKGFIISNAT YKEIGLLTCE ATVNGHLYKT NYLTHRQTGG GGGGGGGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO:6 – HCDR1 of anti-C1s antibody DDYIH SEQ ID NO:7 – HCDR2 of anti-C1s antibody RIDPADGHTK YAPKFQV SEQ ID NO:8 – HCDR3 of anti-C1s antibodyYGYGREVFDY SEQ ID NO:9 – LCDR1 of anti-C1s antibody KASQSVDYDG DSYMN SEQ ID NO:10 – LCDR2 of anti-C1s antibody DASNLES SEQ ID NO:11 – LCDR3 of anti-C1s antibody QQSNEDPWT SEQ ID NO:12 – VHof anti-C1s antibody QVQLVQSGAE VKKPGASVKL SCTASGFNIK DDYIHWVKQA PGQGLEWIGR IDPADGHTKY APKFQVKVTI TADTSTSTAY LELSSLRSED TAVYYCARYG YGREVFDYWG QGTTVTVSS SEQ ID NO:13 – VLof anti-C1s antibody DIVLTQSPDS LAVSLGERAT ISCKASQSVD YDGDSYMNWY QQKPGQPPKI LIYDASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAIY YCQQSNEDPW TFGGGTKVEI K SEQ ID NO:14 – αC1s scFab DIVLTQSPDS LAVSLGERAT ISCKASQSVD YDGDSYMNWY QQKPGQPPKI LIYDASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAIY YCQQSNEDPW TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGECGG GGSGGGGSGG GGSGGGGSGG GGSGGGGSGG GGSQVQLVQS GAEVKKPGAS VKLSCTASGF NIKDDYIHWV KQAPGQGLEW IGRIDPADGH TKYAPKFQVK VTITADTSTS TAYLELSSLR SEDTAVYYCA RYGYGREVFD YWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV SEQ ID NO:15 – HCDR1 of anti-Bb antibody NYAMS SEQ ID NO:16 – HCDR2 of anti-Bb antibody TISNRGSYTY YPDSVKG SEQ ID NO:17 – HCDR3 of anti-Bb antibody ERPMDY SEQ ID NO:18 – LCDR1 of anti-Bb antibody KASQDVGTAV A SEQ ID NO:19 – LCDR2 of anti-Bb antibody WASTRHT SEQ ID NO:20 – LCDR3 of anti-Bb antibodyHQHSSNPLT SEQ ID NO:21 – VHof anti-Bb antibody EVQLVESGGG LVKPGGSLRL SCAASGFTFS NYAMSWVRQA PGKRLEWVAT ISNRGSYTYY PDSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCARER PMDYWGQGTL VTVSS SEQ ID NO:22 – VLof anti-Bb antibody DIQMTQSPST LSASVGDRVT ITCKASQDVG TAVAWYQQKP GKAPKLLIYW ASTRHTGVPD RFSGSGSGTD FTLTISSLQA EDFAVYFCHQ HSSNPLTFGQ GTKLEIK SEQ ID NO:23 – αBb scFab DIQMTQSPST LSASVGDRVT ITCKASQDVG TAVAWYQQKP GKAPKLLIYW ASTRHTGVPD RFSGSGSGTD FTLTISSLQA EDFAVYFCHQ HSSNPLTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSE VQLVESGGGL VKPGGSLRLS CAASGFTFSN YAMSWVRQAP GKRLEWVATI SNRGSYTYYP DSVKGRFTIS RDNAKNSLYL QMNSLRAEDT ALYYCARERP MDYWGQGTLV TVSSASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTKTYTCNVD HKPSNTKVDK RV SEQ ID NO:24 - Nucleotide sequence of AAV2#9 (5’ ITR boldfaced; bGH polyA signal underlined; reverse complement of αC1s scFab coding sequence italicized; IgG kappa signal coding sequence italicized and underlined; Kozak sequence boxed; CBA promoter(reverse) bolded and underlined; CBA promoter boxed and underlined; CMV enhancer boldfaced and italicized; αBb scFab boldfaced, italicized, and underlined; and 3’ ITR boxed and italicized) TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGCCCGGGC AAAGCCCGGG CGTCGGGCGA CCTTTGGTCG CCCGGCCTCA GTGAGCGAGC GAGCGCGCAG AGAGGGAGTG GCCAACTCCA TCACTAGGGG TTCCTTACAA TTCTAGTTCC CCAGCATGCC TGCTATTGTC TTCCCAATCC TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGGTTTA AACCCTGCAG GGAGCTCTCA CACCCGCTTA TCCACCTTGG TGTTGCTGGG CTTGTGGTCC ACGTTGCAGG TGTAGGTCTT TGTGCCCAGG CTAGAGCTAG GCACTGTCAC GACAGAGGAC AGAGAGTACA GGCCGCTGCT CTGCAGCACG GCGGGGAAGG TGTGCACCCC GCTTGTCAGG GCTCCGCTGT TCCAGGACAC GGTCACAGGC TCAGGGAAAT AATCCTTGAC CAGGCAGCCC AGAGCAGCCG TGCTCTCTGA GGTACTTCTG CTACAAGGAG CCAGTGGGAA CACGCTAGGG CCCTTTGTGC TGGCGGACGA CACGGTCACT GTTGTGCCCT GTCCCCAGTA GTCGAACACT TCTCTGCCGT AGCCGTATCT GGCGCAGTAG TACACAGCGG TGTCCTCGGA TCTAAGGCTG CTCAGTTCCA GATAAGCTGT AGAGGTGCTG GTATCGGCGG TGATGGTGAC TTTCACCTGG AACTTAGGGG CGTACTTTGT GTGGCCGTCG GCAGGGTCGA TTCTGCCGAT CCACTCCAGT CCCTGGCCGG GGGCCTGCTT CACCCAGTGG ATGTAATCGT CCTTGATATT GAAGCCGCTG GCGGTGCAGC TCAGCTTAAC ACTAGCGCCA GGCTTTTTCA CCTCGGCTCC GCTCTGCACC AGCTGCACCT GGGATCCGCC GCCGCCGCTG CCGCCTCCGC CGCTGCCGCC TCCGCCGCTT CCGCCTCCCC CAGAGCCGCC GCCACCGCTG CCTCCTCCGC CGGAGCCGCC GCCGCCGCAC TCGCCCCGGT TGAAGCTTTT GGTCACAGGA GAGGACAGGC CCTGATGTGT CACTTCACAG GCGTACACCT TGTGCTTCTC GTAGTCGGCC TTGCTCAAGG TCAGGGTGCT GGACAGGCTG TATGTTGAGT CCTTGCTGTC CTGCTCGGTC ACGCTCTCTT GGCTGTTGCC GCTTTGCAGG GCGTTGTCAA CTTTCCATTG GACCTTTGCC TCTCTGGGGT AGAAGTTATTCAGCAGGCAC ACCACAGAGG CGGTTCCGCT CTTCAGCTGC TCGTCGCTTG GAGGGAAGAT AAAGACAGAA GGGGCGGCCA CGGTGCGCTT GATTTCCACC TTGGTGCCGC CTCCAAAGGT CCAGGGGTCC TCGTTGCTCT GCTGGCAGTA GTAGATGGCA AAATCCTCGG GTTCCAGAGA AGAAATTGTC AGGGTGAAAT CAGTGCCAGA GCCGCTGCCG CTGAATCTGG CGGGGATGCC GCTTTCCAGA TTGCTGGCGT CGTAGATCAG GATTTTTGGA GGCTGGCCGG GTTTCTGCTG GTACCAGTTC ATGTAGCTGT CGCCGTCATA GTCCACGCTC TGAGAGGCTT TACAGCTGAT TGTGGCCCGT TCGCCGAGGC TCACGGCCAG GCTATCAGGG CTCTGCGTCA GCACGATATC GCCGGTGGTG TCAGGCAGCC ACAGGAGCAG CAGGAACAGC AGCTGGGCAG GGGCTTCCAT GGTGGGCTCT GGCGCCCGCC GCGCGCTTCG CTTTTTATAG GGCCGCCGCC GCCGCCGCCT CGCCATAAAA GGAAACTTTC GGAGCGCGCC GCTCTGATTG GCTGCCGCCG CACCTCTCCG CCTCGCCCCG CCCCGCCCCT CGCCCCGCCC CGCCCCGCCT GGCGCGCGCC CCCCCCCCCC CCCCGCCCCC ATCGCTGCAC AAAATAATTA AAAAATAAAT AAATACAAAA TTGGGGGTGG GGAGGGGGGG GAGATGGGGA GAGTGAAGCA GAACGTGGGG CTCACCTCGC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTACTCAGCTGCT GTTCCTGCTG CTGCTGTGGC TGCCTGACAC CACCGGCGAC ATCCAGATGA CACAGAGCCC TAGCACCCTG AGCGCCTCCG TGGGGGACAG AGTGACAATC ACATGTAAAG CCTCCCAGGA CGTGGGCACT GCCGTGGCCT GGTACCAGCA AAAACCGGGA AAAGCCCCTA AGCTGCTGAT CTACTGGGCC AGCACCAGAC ACACCGGCGT CCCCGATAGA TTCAGCGGCT CTGGCAGCGG AACTGATTTC ACCCTGACCA TTTCTTCTCT GCAGGCCGAG GACTTCGCCG TGTACTTTTG CCACCAGCAC AGCAGCAACC CTCTGACCTT CGGACAGGGC ACAAAGCTGG AAATCAAGCG GACAGTGGCT GCTCCTTCTG TGTTCATCTT TCCACCTAGC GACGAGCAGC TGAAGAGCGG CACCGCCTCT GTGGTGTGCC TGCTGAACAA CTTCTACCCC AGAGAAGCCA AAGTGCAGTG GAAGGTGGAC AACGCCCTGC AATCTGGCAA CAGCCAGGAG AGCGTGACGG AACAAGATAG CAAGGACAGC ACCTACTCCC TGAGCAGCAC ACTGACCTTG TCCAAGGCAG ATTACGAGAA GCACAAGGTG TACGCCTGCG AGGTGACCCA CCAGGGACTG AGCAGCCCAG TGACCAAGAG CTTCAACAGA GGAGAGTGCG GCGGCGGCGG AAGCGGAGGC GGAGGCAGCG GCGGCGGCGG CAGTGGAGGC GGCGGCTCTG GCGGAGGGGG CAGTGGCGGT GGCGGATCCG GCGGCGGCGG CAGCGAGGTG CAGCTTGTGG AATCCGGCGG CGGCCTGGTG AAGCCCGGCG GTAGCCTGAG ACTGTCTTGT GCCGCCTCTG GCTTCACCTT TAGCAATTAC GCCATGAGCT GGGTGCGGCA GGCTCCCGGC AAAAGACTGG AATGGGTCGC CACCATCAGC AACCGGGGAT CATATACCTA CTACCCTGAT AGCGTGAAAG GCAGGTTCAC AATCAGCCGG GACAATGCCA AGAACAGCCT GTACCTGCAG ATGAACTCAC TGCGGGCCGA GGACACCGCC CTGTATTACT GCGCCAGAGA GAGACCTATG GACTACTGGG GCCAGGGCAC CCTGGTGACC GTTTCCTCCG CCAGCACCAA GGGCCCTAGC GTGTTCCCTC TGGCCCCATG CAGCAGAAGC ACATCTGAGA GCACCGCCGC TCTGGGCTGC CTGGTGAAGG ACTACTTCCC CGAGCCTGTG ACAGTGAGCT GGAACTCCGG CGCCCTGACC AGCGGCGTGC ACACATTTCC AGCTGTGCTG CAGTCTAGCG GCCTGTACAG CCTGAGCAGC GTTGTGACAG TGCCTTCTAG CAGCCTCGGC ACCAAGACCT ACACCTGTAA CGTGGATCAT AAGCCTTCTA ATACCAAGGT TGACAAGAGA GTGTGAGAGC TCCCTGCAGG GTTTAAACCT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATTCT GGGGGGTGGG GTGGGGCAGGSEQ ID NO:25 [αBb scFab – (G4S)3- αC1s scFab-CM] amino acid sequence (construct #12) (signal peptide boldfaced) MEAPAQLLFL LLLWLPDTTG DIQMTQSPST LSASVGDRVT ITCKASQDVG TAVAWYQKKP GKAPKLLIYW ASTRHTGVPD RFSGSGSGTD FTLTISSLQA EDFAVYFCHQ HSSNPLTFGQ GTKLEIKRTV AAPAVFIFPP SDEQLKSGTA SVVCLLKNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSE VQLVESGGGL VKPGGSLRLS CAASGFTFSN YAMSWVREAP GKRLEWVATI SNRGSYTYYP DSVKGRFTIS RDNAKNSLYL QMNSLRAEDT ALYYCARERP MDYWGQGTLV TVSSASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVEVPSSSL GTKTYTCNVD HKPSNTKVDK RVGGGGGSGG GGSGGGGSDI VLTQSPDSLA VSLGERATIS CKASQSVDYD GDSYMNWYQE KPGQPPKILI YDASNLESGI PARFSGSGSG TDFTLTISSL EPEDFAIYYC QQSNEDPWTF GGGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGECGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SQVQLVQSGA EVKKPGASVK LSCTASGFNI KDDYIHWVKK APGQGLEWIG RIDPADGHTK YAPKFQVKVT ITADTSTSTA YLELSSLRSE DTAVYYCARY GYGREVFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV* SEQ ID NO:26 - Nucleotide sequence of AAV2#12 (5’ ITR boldfaced; minCBA promoter (which comprises a CMV enhancer, a CBA promoter, and a truncated chimeric intron) underlined; Kozak sequence boxed; IgG kappa signal coding sequence italicized; αBb scFab coding sequence bolded and underlined; (G4S)7linker coding sequence in lower case, boldfaced and italicized; (G4S)3linker coding sequence boxed and italicized; αC1s scFab coding sequence boldfaced and italicized; bGH polyA italicized and underlined; and 3’ ITR boxed and boldfaced) TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGCCCGGGC AAAGCCCGGG CGTCGGGCGA CCTTTGGTCG CCCGGCCTCA GTGAGCGAGC GAGCGCGCAG AGAGGGAGTG GCCAACTCCA TCACTAGGGG TTCCTTACCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC CAGTGAATTC GGACCGAGAT CTGAATTCGG TACCTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT CGAGGTGAGC CCCACGTTCT GCTTCACTCT CCCCATCTCC CCCCCCTCCC CACCCCCAAT TTTGTATTTA TTTATTTTTT AATTATTTTG TGCAGCGATG GGGGCGGGGG GGGGGGGGGG GCGCGCGCCA GGCGGGGCGG GGCGGGGCGA GGGGCGGGGC GGGGCGAGGC GGAGAGGTGC GGCGGCAGCC AATCAGAGCG GCGCGCTCCG AAAGTTTCCT TTTATGGCGA GGCGGCGGCG GCGGCGGCCC TATAAAAAGC GAAGCGCGCG GCGGGCGGGA GTCGCTGCGC GCTGCCTTCG CCCCGTGCCC CGCTCCGCCG CCGCCTCGCG CCGCCCGCCC CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC TTCTCCTCCG GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC TCCGGGAGCT AGAGCCTCTG CTAACCATGT TCATGCCTTC TTCTTTTTCC TACAGCTCCT GGGCAACGTG CTGGTTATTG TGCTGTCTCA TCATTTTGGC AAAGAATTCC TCGAAGATCC GGTACCCAAT TGCCCACCAT GGAAGCCCCT GCCCAGCTGC TGTTCCTGCT GCTACTGTGG CTGCCTGATA CCACCGGCGA TATCCAGATG ACGCAGAGTC CCAGCACCCT GAGCGCCTCT GTGGGCGACC GGGTGACCAT CACCTGTAAA GCCTCCCAGG ACGTGGGCAC AGCTGTTGCT TGGTATCAGAAAAAGCCTGG CAAGGCCCCT AAGCTGCTGA TCTACTGGGC CAGCACAAGA CACACAGGAG TGCCTGACAG ATTCAGCGGC AGCGGCTCTG GGACTGATTT CACCTTGACA ATCAGCTCTC TGCAGGCCGA GGACTTTGCC GTGTACTTCT GCCACCAACA CAGTTCTAAC CCCCTGACCT TCGGCCAAGG AACCAAGCTG GAAATCAAGC GGACCGTGGC CGCTCCTGCC GTGTTCATCT TCCCTCCAAG CGATGAGCAG CTGAAAAGCG GCACCGCGTC CGTCGTGTGC CTGCTGAAGA ACTTCTACCC GAGAGAAGCG AAGGTGCAGT GGAAAGTCGA CAACGCCCTG CAGAGCGGAA ATAGCCAGGA GAGCGTGACC GAACAAGACT CTAAGGACAG CACCTACTCG CTGTCCTCCA CGCTGACTCT GTCTAAGGCC GACTATGAGA AGCACAAGGT GTACGCCTGC GAGGTGACCC ACCAGGGCCT GAGCAGCCCC GTTACCAAGA GCTTCAACAG AGGAGAATGC ggcggaggtg gcagcggcgg cggcgggagc ggcggcggcg gctcaggcgg agggggaagt ggcggcggcg gcagcggcgg cggaggcagc ggcggtggcg gctctGAGGT GCAACTGGTG GAATCTGGGG GCGGACTGGT GAAGCCTGGC GGCAGTCTGA GACTGAGCTG TGCCGCTTCC GGATTCACCT TTAGCAATTA CGCCATGAGC TGGGTGCGGG AGGCCCCTGG AAAGCGGCTG GAATGGGTTG CTACAATCAG CAATAGAGGC AGCTACACAT ACTACCCCGA CAGTGTCAAA GGCCGGTTTA CAATCAGCCG CGACAACGCC AAAAACAGCC TGTACCTGCA GATGAACTCC CTGCGGGCTG AGGATACAGC CCTCTACTAC TGTGCCAGAG AACGTCCAAT GGACTATTGG GGCCAAGGCA CACTGGTGAC CGTGAGCAGC GCGTCTACCA AGGGCCCTTC TGTTTTCCCT CTGGCCCCCT GCAGCAGAAG CACGAGCGAG AGCACCGCTG CCCTGGGCTG TCTGGTGAAG GATTATTTCC CTGAGCCTGT GACCGTGTCT TGGAATAGCG GAGCCCTGAC CAGCGGAGTG CATACATTCC CTGCTGTGCT GCAGTCTAGT GGGCTGTACA GCCTGTCTTC CGTTGTGGAA GTCCCTAGCA GCAGCCTGGG CACCAAGACC TACACCTGCA ACGTGGATCA TAAGCCAAGC AACACCAAGG TGGATAAGAG AGTGGGCGGT GGCGGAGGCT CGGGCGGCGG CGGCAGCGGC GGCGGCGGCA GCGACATCGT GCTGACCCAG TCTCCAGATT CTCTGGCCGT GTCACTGGGA GAGAGAGCCA CCATTAGCTG CAAGGCCTCT CAGAGCGTAG ACTACGACGG CGACTCCTAC ATGAACTGGT ACCAGGAAAA GCCTGGCCAG CCTCCTAAGA TCTTGATCTA CGATGCCTCC AATCTGGAGA GCGGGATCCC CGCTAGATTC AGCGGGTCTG GAAGTGGAAC CGACTTCACA CTGACCATCT CTAGCCTGGA GCCCGAGGAC TTTGCCATCT ACTACTGCCA GCAGAGCAAC GAGGACCCCT GGACATTCGG CGGCGGCACA AAGGTTGAGA TCAAGAGAAC CGTTGCCGCT CCTAGCGTGT TTATCTTCCC TCCCTCTGAC GAGCAGCTGA AGAGCGGCAC AGCCTCCGTG GTGTGCCTGC TGAACAACTT CTACCCCAGA GAGGCCAAGG TCCAGTGGAA GGTCGACAAT GCCCTTCAGA GCGGCAACAG CCAGGAGTCC GTGACCGAGC AGGATAGCAA GGACTCTACC TACAGCCTGT CCTCTACGCT GACCCTGAGC AAAGCCGATT ACGAAAAGCA CAAAGTGTAC GCCTGTGAAG TGACACACCA GGGCCTGTCT AGCCCTGTGA CAAAGAGCTT TAACCGGGGC GAGTGCggcg gcggtggaag cggaggtgga ggttcaggag gcggcggaag cggaggcgga ggcagtgggg gcggcggctc cggcggcggc ggcagcggag gcggcggttc ccAAGTGCAG CTCGTGCAGA GCGGCGCCGA GGTGAAAAAG CCCGGAGCCA GCGTGAAGCT GTCTTGCACC GCCTCCGGAT TCAACATCAA AGACGACTAC ATCCACTGGG TCAAGAAAGC CCCAGGGCAG GGGCTGGAGT GGATCGGCAG GATCGACCCT GCTGATGGCC ACACCAAATA CGCCCCAAAG TTCCAGGTGA AAGTGACAAT TACCGCAGAT ACCTCCACCA GCACCGCTTA TCTGGAACTG AGCTCTCTGC GGAGCGAGGA CACAGCCGTG TACTACTGCG CCAGATACGG CTACGGCAGA GAAGTGTTCG ACTACTGGGG CCAGGGCACC ACAGTGACAG TGAGCTCTGC CAGCACAAAG GGCCCCAGCG TGTTTCCTCT GGCCCCTTGC AGCAGAAGCA CCAGCGAGAG CACCGCCGCC CTGGGCTGCC TGGTGAAGGA CTACTTCCCT GAACCCGTGA CCGTCTCCTG GAACAGTGGC GCCTTGACCT CTGGCGTGCA CACCTTCCCC GCCGTGCTGC AGAGCTCCGG CCTGTACAGC CTGTCTAGCG TGGTGACCGT GCCTAGCTCG AGCCTGGGCA CAAAGACATA TACCTGTAAC GTGGACCACA AGCCCAGCAA CACGAAGGTG GACAAGCGAG TGTGAGTTTA AACCTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGGSEQ ID NO:27 – [αC1s scFab – (G4S)2– αBb scFv-CM] amino acid sequence (construct #14) (signal peptide boldfaced)MEAPAQLLFL LLLWLPDTTG DIVLTQSPDS LAVSLGERAT ISCKASQSVD YDGDSYMNWY QEKPGQPPKI LIYDASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAIY YCQQSNEDPW TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGECGG GGSGGGGSGG GGSGGGGSGG GGSGGGGSGG GGSQVQLVQS GAEVKKPGAS VKLSCTASGF NIKDDYIHWV KKAPGQGLEW IGRIDPADGH TKYAPKFQVK VTITADTSTS TAYLELSSLR SEDTAVYYCA RYGYGREVFD YWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV GGGGSGGGGS EVQLVESGGG LVKPGGSLRL SCAASGFTFS NYAMSWVREA PGKRLEWVAT ISNRGSYTYY PDSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCARER PMDYWGQGTL VTVSSGGGGS GGGGSGGGGS DIQMTQSPST LSASVGDRVT ITCKASQDVG TAVAWYQKKP GKAPKLLIYW ASTRHTGVPD RFSGSGSGTD FTLTISSLQA EDFAVYFCHQ HSSNPLTFGQ GTKLEIK* SEQ ID NO:28 – Nucleotide sequence of AAV2#14 (3’ ITR boldfaced; minCBA promoter underlined; Kozak sequence boxed; IgG kappa signal sequence italicized; αC1s scFab coding sequence boldfaced and underlined; (G4S)2linker coding sequence boxed and italicized; (G4S)7coding sequence in lower case, boldfaced, and italicized; αBb scFv coding sequence boldfaced and italicized; bGH polyA signal italicized and underlined; and 5’ ITR boxed and boldfaced) TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG AGAGGGAGTG GCCAACTCCA TCACTAGGGG TTCCTAATTT GATCTGAATT CGGTACCTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTATT TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA GCCAATCAGA GCGGCGCGCT CCGAAAGTTT CCTTTTATGG CGAGGCGGCG GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG GGAGTCGCTG CGCGCTGCCT TCGCCCCGTG CCCCGCTCCG CCGCCGCCTC GCGCCGCCCG CCCCGGCTCT GACTGACCGC GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA ATTAGCGCTT GGTTTAATGA CGGCTTGTTT CTTTTCTGTG GCTGCGTGAA AGCCTTGAGG GGCTCCGGGA GCTAGAGCCT CTGCTAACCA TGTTCATGCC TTCTTCTTTT TCCTACAGCT CCTGGGCAAC GTGCTGGTTA TTGTGCTGTC TCATCATTTT GGCAAAGAAT TCCTCGAAGA TCCGGTACCC AATTGCCACC ATGGAAGCCC CCGCCCAGCT GCTGTTCCTG CTGCTCCTGT GGCTGCCTGA TACCACCGGC GATATCGTCC TGACCCAGAG CCCTGATAGC CTGGCCGTTT CACTGGGCGA GCGGGCCACA ATCTCCTGCA AGGCCTCTCA GTCTGTTGAC TACGACGGCG ACAGCTACAT GAACTGGTAC CAGGAGAAAC CCGGCCAACC TCCAAAGATC CTGATCTACG ACGCCTCTAA TCTGGAGAGC GGCATCCCCG CCCGGTTCAG CGGGTCCGGC AGCGGCACCG ACTTTACCCT GACCATCTCT AGCCTGGAGC CTGAGGACTT CGCCATCTAC TACTGTCAGC AGAGCAACGA GGATCCTTGG ACCTTTGGCG GCGGCACAAA GGTGGAAATC AAGCGGACCG TCGCCGCTCC ATCCGTGTTT ATCTTCCCTC CTTCCGACGA GCAGCTCAAG AGCGGTACCG CCAGCGTGGT GTGCCTGCTG AACAACTTCT ACCCCAGAGA GGCCAAGGTG CAGTGGAAGG TAGACAACGC CTTGCAGAGC GGCAACTCTC AAGAGAGCGT GACAGAGCAG GACTCTAAGG ACAGCACATA CAGCCTAAGC TCCACCCTGA CCCTCAGCAA GGCCGACTAC GAGAAGCACA AGGTGTACGC CTGTGAAGTT ACACACCAGG GCCTGAGCAG TCCGGTGACC AAGTCCTTCA ACAGAGGCGA ATGCggcggaggaggctctg gcggcggcgg cagcggcgga ggcggcagcg gcggcggagg ctctggcggc ggtggcagcg gaggcggcgg aagcggcgga ggtggcagcC AGGTGCAGCT GGTGCAGAGC GGTGCTGAAG TGAAGAAACC CGGCGCTTCC GTGAAACTGA GCTGCACCGC CAGCGGATTT AACATCAAGG ACGACTACAT TCACTGGGTG AAAAAGGCCC CTGGCCAGGG CCTGGAATGG ATCGGGAGAA TCGACCCCGC CGATGGCCAT ACCAAGTACG CTCCTAAGTT CCAGGTGAAA GTGACCATCA CCGCTGATAC AAGCACCTCT ACAGCCTACC TGGAGCTGAG CTCCCTGCGG TCTGAGGACA CCGCCGTGTA CTACTGCGCC AGATACGGCT ACGGCAGAGA GGTGTTCGAC TACTGGGGAC AGGGCACTAC AGTCACCGTG TCTAGTGCTA GCACGAAGGG CCCTAGCGTG TTCCCTCTGG CTCCATGTAG CAGAAGCACC AGCGAAAGCA CAGCTGCTCT GGGCTGCCTG GTGAAAGACT ACTTCCCCGA GCCTGTGACC GTCAGCTGGA ACTCCGGCGC CCTGACCAGC GGAGTGCACA CCTTTCCTGC TGTGCTGCAA TCCTCTGGCC TGTACTCTCT GAGCTCTGTT GTGACAGTGC CTTCTAGCAG CCTGGGAACC AAGACCTACA CCTGCAACGT GGACCACAAG CCCAGCAACA CCAAGGTGGA TAAGCGCGTG GGCGGCGGCG GATCTGGCGG AGGCGGCAGC GAGGTGCAGC TGGTGGAAAG CGGCGGCGGC CTGGTGAAGC CTGGCGGCTC ACTGAGACTG AGCTGTGCCG CCAGCGGCTT CACCTTCTCC AACTACGCCA TGAGCTGGGT GCGGGAAGCC CCAGGAAAGC GCCTGGAGTG GGTCGCCACC ATCAGCAATA GAGGCTCGTA TACATATTAC CCTGATTCCG TCAAAGGCAG ATTCACCATC TCTAGAGATA ATGCCAAGAA CAGCCTGTAC CTGCAGATGA ACTCCCTCAG AGCCGAGGAT ACAGCCCTGT ATTACTGCGC CAGAGAACGG CCTATGGACT ACTGGGGCCA AGGCACTCTG GTGACAGTGA GCAGCGGCGG CGGTGGTTCC GGCGGCGGAG GCTCTGGAGG AGGCGGCAGC GACATCCAGA TGACCCAGAG CCCTAGCACC CTGTCCGCCA GCGTGGGAGA TAGAGTGACC ATTACCTGTA AAGCGAGCCA GGATGTGGGC ACCGCCGTGG CCTGGTATCA GAAGAAGCCT GGCAAGGCCC CTAAGCTGCT GATCTACTGG GCCTCTACCC GGCACACAGG CGTGCCCGAC AGATTCTCCG GCTCCGGTTC TGGAACAGAC TTCACACTGA CCATCAGCTC TCTTCAGGCC GAGGACTTCG CCGTGTACTT CTGCCACCAG CACAGCTCTA ATCCTCTGAC ATTCGGCCAA GGCACAAAGC TGGAAATCAA GTGAGTTTAA ACCTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC AATAGCAGGC ATGCTGGGGA ACTAGGTAAG GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG CGGCCTCAGT GAGCGAGCGA GCGCGCAGAG AGGGAGTGGC CAA SEQ ID NO:29 – IdeS amino acid sequence MDSFSANQEI RYSEVTPYHV TSVWTKGVTP PANFTQGEDV FHAPYVANQG WYDITKTFNG KDDLLCGAAT AGNMLHWWFD QNKDQIKRYL EEHPEKQKIN FNGEQMFDVK EAIDTKNHQL DSKLFEYFKE KAFPYLSTKH LGVFPDHVID MFINGYRLSL TNHGPTPVKE GSKDPRGGIF DAVFTRGDQS KLLTSRHDFK EKNLKEISDL IKKELTEGKA LGLSHTYANV RINHVINLWG ADFDSNGNLK AIYVTDSDSN ASIGMKKYFV GVNSAGKVAI SAKEIKEDNI GAQVLGLFTL STGQDSWNQT N

Claims

CLAIMS 1. A method of delivering a therapeutic protein to a diseased eye in a subject in need thereof through ocular gene therapy, comprising: injecting intravitreally to the diseased eye a composition comprising an IgG- degrading enzyme, optionally Ides, and then injecting intravitreally about 1E10 to about 1E12 vector genomes of a recombinant adeno-associated virus (rAAV) whose genome comprises a coding sequence for the therapeutic protein.

2. A method of reducing anti-drug immune response in ocular gene therapy in a subject in need thereof, optionally for four or more weeks, comprising: injecting intravitreally to a diseased eye of the subject a composition comprising an IgG-degrading enzyme, optionally Ides, and then injecting intravitreally about 1E10 to about 1E12 vector genomes of a recombinant adeno-associated virus (rAAV) whose genome comprises a coding sequence for a therapeutic protein.

3. The method of claim 1 or 2, wherein the first and second injecting steps are separated by no more than five days, optionally by 1-3 days.

4. The method of any one of claims 1-3, wherein the composition comprises 10-50 µg of IdeS.

5. The method of any one of claims 1-4, wherein the rAAV is of AAV2 serotype.

6. The method of any one of claims 1-5, wherein the diseased eye has age-related macular degeneration (AMD).

7. The method of claim 6, wherein the diseased eye has wet AMD and the therapeutic protein comprises Ig-like domain 2 of VEGFR-1 (Flt-1).

8. The method of claim 7, wherein the therapeutic protein comprises SEQ ID NO:3, optionally wherein the rAAV comprises a nucleic acid comprising SEQ ID NO:2.

9. The method of claim 7, wherein the therapeutic protein comprises SEQ ID NO:5, optionally wherein the rAAV comprises a nucleic acid comprising SEQ ID NO:

4.

10. The method of claim 6, wherein the diseased eye has dry AMD and the rAAV comprises a nucleic acid encoding a first antibody fragment specific for activated complement subcomponent C1s (aC1s) and a second antibody fragment specific for complement factor Bb, wherein the first antibody fragment comprises heavy chain CDR (HCDR) 1-3 comprising SEQ ID NOs:6-8, respectively, and light chain CDR (LCDR) 1-3 comprising SEQ ID NOs:9-11, respectively, and the second antibody fragment comprises HCDR1-3 comprising SEQ ID NOs:15-17, respectively, and LCDR1-3 comprising SEQ ID NOs:18-20, respectively.

11. The method of claim 10, wherein the first antibody fragment comprises a heavy chain variable domain (VH) comprising SEQ ID NO:12 and a light chain variable domain (VL) comprising SEQ ID NO:13, and the second antibody fragment comprises a VH comprising SEQ ID NO:21 and a VL comprising SEQ ID NO:

22.

12. The method of claim 11, wherein the first and second antibody fragments are two separate single-chain Fab proteins, the first antibody fragment comprising SEQ ID NO:14 and the second antibody fragment comprising SEQ ID NO:23, optionally wherein the rAAV comprises a genome comprising SEQ ID NO:

24.

13. The method of claim 11, wherein the first and second antibody fragments are fused through a peptide linker to form a fusion protein.

14. The method of claim 13, wherein the fusion protein comprises SEQ ID NO:25, optionally the rAAV comprises a genome comprising SEQ ID NO:

26.

15. The method of claim 13, wherein the fusion protein comprises SEQ ID NO:27, optionally wherein the rAAV comprises a genome comprising SEQ ID NO:28.

16. The method of any one of the preceding claims, wherein the subject has been determined to be seropositive for AAV prior to the first injecting step.

17. A recombinant adeno-associated virus for use in the method of any one of claims 1- 16.

18. Use of a recombinant adeno-associated virus in the manufacture of a medicament for use in the method of any one of claims 1-16.

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