AAV vectors for treatment of complement bypass related diseases

Abstract

The invention provides a CR2-FH fusion protein, polynucleotide for coding the CR2-FH fusion protein, a carrier, a pharmaceutical composition and application of the CR2-FH fusion protein. The CR2-FH fusion protein can be used for treating complement bypass related diseases such as dry AMD.

Description

[Corrected 20.06.2024 in accordance with Regulation 26] AAV vectors for the treatment of complement pathway-related diseases

[0001] priority

[0002] This application claims the benefit of and priority to Chinese application No. 2023105884953, filed May 19, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present application relates to fusion proteins, constructs and uses thereof for treating diseases associated with the complement alternative pathway. Specifically, the present application relates to CR2-FH molecules for treating diseases associated with the complement alternative pathway, constructs encoding the CR2-FH molecules and uses thereof. Background Art

[0004] The complement system is a crucial host defense system, playing a crucial role in regulating humoral and cellular immunity, decomposing immune complexes, and clearing apoptotic cells. The complement system is composed of multiple soluble protein molecules, including intrinsic complement components, various regulatory factors, and complement receptors. The complement cascade has three activation pathways: the classical pathway, the alternative pathway, and the lectin pathway. These three pathways converge downstream at C3 and are ultimately activated by C5 to produce the membrane attack complex (MAC), which exerts a cytolytic effect and participates in immunity, serving as a crucial innate immune barrier. However, inappropriate complement activation and its deposition on host cells can lead to complement-mediated target cell lysis and tissue destruction due to the production of potent inflammatory mediators. Abnormal complement activation has been implicated in the pathogenesis of age-related macular degeneration (AMD). Genome-wide studies have shown that genetic variations in multiple components of the complement cascade are associated with an increased risk of AMD. C3, C5, the anaphylatoxins C3a and C5a, and other acute-phase reactant proteins have been shown to be present in drusen deposits in patients' eyes. Plasma levels of C3a, C3d, Bb, and C5a are elevated in AMD patients, suggesting a role for complement activation in the pathogenesis of AMD. In addition, diseases associated with the complement alternative pathway include rheumatoid arthritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), factor H-related hemolytic uremic syndrome (HUS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), lupus nephritis, stroke, myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, burns, inflammation associated with cardiopulmonary bypass and hemodialysis, plasmapheresis, platelet separation, leukocyte separation, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiocontrast-induced anaphylaxis.

[0005] AMD is a degenerative, blinding disease that affects the macula or central area of ​​the retina and is the leading cause of irreversible vision loss in the elderly. In developed countries, AMD is a highly prevalent blinding disease in people over 65 years old, affecting approximately 9% of the population worldwide. AMD is mainly divided into dry and wet forms, of which dry AMD accounts for more than 80% and can develop into geographic atrophy (GA) in the late stages. The typical feature of dry AMD is the formation of drusen associated with degenerative changes in the retinal pigmented epithelium (RPE) cells, a visible pigmented area in the center of the macula, and loss of photoreceptor cells - rods and cones. In the late stages of dry AMD, large areas of RPE atrophy and choroidal blood vessels atrophy lead to permanent central vision loss.

[0006] Factor H (FH) is a single-chain glycoprotein composed of 1231 amino acids with a molecular weight of 155 kDa and composed of 20 short consensus repeats (SCRs). It is a key inhibitor of the alternative pathway. FH deficiency or insufficient levels may promote complement activation, thereby increasing the risk of local tissue damage. Under normal physiological conditions, C3 interacts with factors B (FB) and D (FD) to produce minimal amounts of C3b and C3bBb (C3 convertase). C3 convertase is rapidly activated by FH and is unable to activate C3 and subsequent complement components. In pathological conditions, FH fails to adequately control C3 convertase, which hydrolyzes C3 into C3b and produces the anaphylatoxin C3a. This shifts the complement cascade to its terminal cleavage pathway, which generates the anaphylatoxins C5a and MAC, both of which induce potent inflammatory signals. Studies have demonstrated that in dry AMD, retinal homeostasis is impaired due to RPE cell dysfunction. Reduced FH leads to C3 accumulation at the RNA and protein levels, making RPE cells more susceptible to oxidative stress damage. Endogenous FH helps regulate transcriptional and metabolic homeostasis and protects RPE cells from oxidative stress. Furthermore, FH harbors single nucleotide polymorphisms (SNPs), the first complement SNPs discovered to be associated with dry AMD. Because complement plays a crucial role in host defense and immune complex catabolism, targeting the complement inhibitor FH to sites of complement activation and disease may improve its efficacy while reducing the side effects of complement inhibition. Complement receptor 2 (CR2) is a complement receptor and member of the C3-binding protein family. It is composed of 15 or 16 SCR domains. The natural ligands for CR2 are iC3b, C3dg, and C3d, which are cleavage fragments of C3. Cleavage of C3 initially leads to the production and deposition of C3b on the surface of activated cells. The C3b fragment participates in the production of enzyme complexes that amplify the complement cascade. On the cell surface, C3b is rapidly converted to inactive iC3b, particularly when deposited on host surfaces containing complement-activating regulators. Even in the absence of membrane-bound complement regulators, significant levels of iC3b are formed due to the action of FH. Subsequently, iC3b is digested by Factor I (FI) and other proteases into membrane-bound fragments C3dg and C3d, but this process is relatively slow. Therefore, once produced, the C3 ligand for CR2 is relatively long-lived and present in high concentrations at sites of complement activation. CR2 can therefore serve as an effective targeting vehicle for molecules to sites of complement activation.While significant progress has been made in the treatment of AMD, particularly with the use of VEGF inhibitors, including antibody-based drugs and gene therapy products for wet AMD, there are currently no approved gene therapy drugs for dry AMD in China. Therefore, new therapeutics are urgently needed to address this issue. Given the advantage of sustained action of gene therapy products, we are hoping to develop highly effective treatments for dry AMD.

[0007] Adeno-associated virus (AAV) was first discovered in the mid-1960s from laboratory adenovirus (AdV) preparations and was soon found in human tissue. Its safety, broad host cell range, low immunogenicity, and ability to efficiently and long-term express foreign genes have made it a valuable tool for gene delivery. Currently, cutting-edge AAV genome design allows the single-stranded DNA carried within the capsid to be engineered to contain self-complementary sequences. The advantage of this sequence is that it does not require the replication of single-stranded DNA into double-stranded DNA for transcription, resulting in faster and higher gene expression compared to traditional single-stranded AAV genomes. To date, six gene therapy drugs using recombinant AAV vectors have been approved for marketing worldwide: in addition to Novartis's Zolgensma (AAV9), UniQure's Glybera (AAV1), and Spark Therapeutics' Luxturna (AAV2). From this we can see that AAV gene therapy has great potential, and gene therapy has become a core technology to solve the dilemma of sustained and limited drug effects.

[0008] Summary of the Invention

[0009] In one aspect, the present disclosure provides a complement receptor 2 (CR2)-factor H (FH) fusion protein (hereinafter referred to as a CR2-FH fusion protein), comprising: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); and the FH portion comprises the first four N-terminal short homology repeat (SCR) domains of FH (e.g., FH (SCR1-SCR4)). In some embodiments, an isolated CR2-FH fusion protein is provided. In some embodiments, the CR2 portion and the FH portion are fused directly or indirectly to each other in the form of a fusion protein. In some embodiments, the CR2 portion and the FH portion are covalently linked. In some embodiments, the CR2 portion and the FH portion are optionally linked by a linker sequence. In some embodiments, the CR2 portion and the FH portion are connected via an amino acid linker sequence comprising a sequence represented by (G4S)n and / or an endogenous linker sequence, wherein n is an integer greater than 0.

[0010] "Isolated" material refers to a substance or component that has been artificially obtained to be "separated" and exists in a sufficiently pure state. In certain embodiments, the purity of the fusion protein is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reversed-phase HPLC).

[0011] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) a FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46); and the FH portion comprises two or more FH fragments, each comprising the first four N-terminal SCR domains of FH (e.g., FH (SCR1-SCR4)). Preferably, the two or more FH fragments are connected by a linker sequence, more preferably, the linker sequence comprises (G4S). n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0012] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) a FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); and the FH portion comprises the first four N-terminal SCR domains of FH (e.g., FH (SCR1-SCR4)), the N-terminal eighth SCR domain of FH (e.g., FH (SCR8)), and the N-terminal nineteenth to twentieth SCR domains of FH (e.g., FH (SCR19-SCR20)).

[0013] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); wherein the FH portion comprises the first four N-terminal SCR domains of FH (e.g., FH (SCR1-SCR4)) and the N-terminal eighteenth to twentieth SCR domains of FH (e.g., FH (SCR18-SCR20)).

[0014] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) a FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); wherein the FH portion comprises the first four N-terminal SCR domains of FH (e.g., FH (SCR1-SCR4)), the N-terminal eighteenth SCR domain of FH (e.g., FH (SCR18)), and the N-terminal twentieth SCR domain of FH (e.g., FH (SCR20)).

[0015] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); wherein the FH portion comprises the first four N-terminal SCR domains of two FH (e.g., FH (SCR1-SCR4)) and the N-terminal seventh SCR domain of FH (e.g., FH (SCR7)).

[0016] In some embodiments, the linker sequence comprises (G4S) n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0017] In some embodiments, the domains are connected by a linker sequence comprising (G4S) n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0018] In some embodiments, the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence shown in SEQ ID NO:2.

[0019] In some embodiments, the N-terminal eighth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:3.

[0020] In some embodiments, the N-terminal nineteenth to twentieth SCR domains of FH comprise the amino acid sequence shown in SEQ ID NO:4.

[0021] In some embodiments, the N-terminal eighteenth to twentieth SCR domains of FH comprise the amino acid sequence shown in SEQ ID NO:7.

[0022] In some embodiments, the N-terminal eighteenth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:39.

[0023] In some embodiments, the N-terminal twentieth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:40.

[0024] In some embodiments, the N-terminal seventh SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:43.

[0025] In some embodiments, the CR2 portion or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46.

[0026] In some embodiments, the CR2 portion and the FH portion are connected by a linker sequence, preferably, the linker sequence is (G4S) n As shown, where n is an integer greater than 0.

[0027] In some embodiments, the CR2-FH fusion protein comprises the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55.

[0028] In some embodiments, the CR2-FH fusion protein comprises a signal peptide sequence. Preferably, the signal peptide sequence is located at the N-terminus. More preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17.

[0029] In some embodiments, the CR2-FH fusion protein comprises the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 or SEQ ID NO:56.

[0030] In another aspect, the present disclosure provides polynucleotides encoding the CR2-FH fusion proteins described herein. In some embodiments, the polynucleotide sequence encoding the first four N-terminal short homology repeat (SCR) domains of FH comprises the sequence set forth in SEQ ID NOs: 19, 28, or 29. In some embodiments, the polynucleotide sequence encoding the eighth N-terminal SCR domain of FH comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the polynucleotide sequence encoding the nineteenth to twentieth N-terminal SCR domains of FH comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the polynucleotide sequence encoding the eighteenth to twentieth N-terminal SCR domains of FH comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the polynucleotide sequence encoding the CR2 portion comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the eighteenth N-terminal SCR domain of FH comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the N-terminal twentieth SCR domain encoding the FH comprises the sequence shown in SEQ ID NO: 58. In some embodiments, the N-terminal seventh SCR domain encoding the FH comprises the amino acid sequence shown in SEQ ID NO: 61.

[0031] In some embodiments, the nucleotide sequence encoding the CR2-FH fusion protein comprises the sequence shown in SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, or SEQ ID NO:74.

[0032] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein comprises a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide is located at the 5' end of the polynucleotide sequence encoding the CR2-FH fusion protein. More preferably, the polynucleotide sequence encoding the signal peptide is as shown in SEQ ID NO: 37 or 38.

[0033] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein comprises the sequence shown in SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:73 or SEQ ID NO:75.

[0034] In another aspect, the present disclosure provides a vector encoding the polynucleotide described in the present disclosure. In some embodiments, the vector is selected from at least one of an adeno-associated virus AAV vector, an adenovirus vector, an RNA virus vector, a lentivirus vector, and a vaccinia virus vector.

[0035] In another aspect, the present disclosure provides a host cell comprising a polynucleotide as described in the present disclosure or a vector as described in the present disclosure.

[0036] In another aspect, the present disclosure provides an AAV particle comprising an AAV vector as described in the present disclosure.

[0037] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one of the CR2-FH fusion protein described in the present disclosure, the polynucleotide described in the present disclosure, the vector described in the present disclosure, the host cell described in the present disclosure, and the AAV particle described in the present disclosure.

[0038] and a pharmaceutically acceptable carrier.

[0039] In some embodiments, the composition is suitable for intraocular, intravenous, intraarterial, subcutaneous, intratracheal, or inhalation administration.

[0040] In another aspect, the present disclosure provides use of the CR2-FH fusion protein described herein, the polynucleotide described herein, the vector described herein, the host cell described herein, the AAV particle described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating a complement alternative pathway-associated disease in a subject.

[0041] The present disclosure provides a method for treating a complement alternative pathway-associated disease in a subject in need thereof, wherein an effective amount of the CR2-FH fusion protein described in the disclosure, the polynucleotide described in the disclosure, the vector described in the disclosure, the host cell described in the disclosure, the AAV particle described in the disclosure, or the pharmaceutical composition described in the disclosure is administered.

[0042] In some embodiments, the complement alternative pathway-associated disease is an inflammatory disease or an autoimmune disease.

[0043] In some embodiments, the complement alternative pathway-associated disease is age-related macular degeneration, preferably dry age-related macular degeneration.

[0044] In some embodiments, the complement alternative pathway-associated disease is a symptom of microangiopathic hemolytic anemia, thrombocytopenia, or acute renal failure.

[0045] In some embodiments, the complement alternative pathway-associated disease is selected from macular degeneration, ischemia-reperfusion, organ transplant rejection, drusen-associated disease, pregnancy-related disease, adverse drug reaction, and post-cardiopulmonary bypass complications.

[0046] In some embodiments, the complement alternative pathway-associated disease is selected from age-related macular degeneration (AMD), rheumatoid arthritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), factor H-related hemolytic uremic syndrome (HUS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), lupus nephritis, stroke, myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, burns, inflammation associated with cardiopulmonary bypass and hemodialysis, plasmapheresis, platelet separation, leukocyte separation, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiocontrast-induced anaphylaxis. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG1 shows a schematic diagram of the structures of XMDC025, XMDC026, XMDC029, CR2-FH (1-4) and CR2-FH (1-5).

[0048] 2A-E show the vector information of ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4) and ssAAV-CR2-FH(1-5).

[0049] 3A-B show the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5) on the thickness of the retinal outer nuclear layer in PEG-400-induced dry AMD model mice.

[0050] 4A-B show the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5) on the nuclear density in the outer nuclear layer of the retina of PEG-400-induced dry AMD model mice.

[0051] 5A-B show HE staining images of the retinas of PEG-400-induced dry AMD model mice injected with AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0052] Figures 6A-B show the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5) on the number of retinal cones in PEG-400-induced dry AMD model mice.

[0053] 7A-B show images of immunofluorescence staining of cone cells in PEG-400-induced dry AMD model mice injected with AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0054] Figures 8A-B show the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5) on the thickness of retinal rod outer segments in PEG-400-induced dry AMD model mice.

[0055] Figures 9A-B show images of immunofluorescence staining of retinal rod cells in PEG-400-induced dry AMD model mice using AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5).

[0056] 10A-B show the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) on RPE cells in PEG-400-induced dry AMD model mice.

[0057] 11A-B show images of F-actin staining of RPE cells in PEG-400-induced dry AMD model mice injected with AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5).

[0058] Figure 12 shows a schematic structural diagram of XMDC061, XMDC062, XMDC025-(CR2-R36A K41A K67A), XMDC026-(CR2-R36A K41A K67A), XMDC029-(CR2-R36A K41A K67A), XMDC061-(CR2-R36AK41A K67A) and XMDC062-(CR2-R36A K41A K67A).

[0059] Figures 13A-G show the vector information of ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36A K41A K67A), ssAAV-XMDC026-(CR2-R36A K41A K67A), ssAAV-XMDC029-(CR2-R36A K41A K67A), ssAAV-XMDC061-(CR2-R36A K41A K67A) and ss-AAVXMDC062-(CR2-R36A K41A K67A).

[0060] Figures 14A-B show a schematic diagram of the CR2-FH (SCR1-5+1-5) structure and an information diagram of the ssAAV-CR2-FH (SCR1-5+1-5) vector.

[0061] Figure 15 shows the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on the thickness of the retinal outer nuclear layer in PEG-400-induced dry AMD model mice.

[0062] Figure 16 shows the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on the nuclear density in the outer nuclear layer of the retina of mice with dry AMD model induced by PEG-400.

[0063] Figure 17 shows HE staining images of the retinas of PEG-400-induced dry AMD model mice injected with AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41AK67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41AK67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5).

[0064] Figure 18 shows the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on the number of retinal cones in PEG-400-induced dry AMD model mice.

[0065] Figure 19 shows images of immunofluorescence staining of cone cells in PEG-400-induced dry AMD model mice injected with AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41AK67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41AK67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5).

[0066] Figure 20 shows the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on the thickness of retinal rod outer segments in PEG-400-induced dry AMD model mice.

[0067] Figure 21 shows the immunofluorescence staining images of retinal rod cells in PEG-400-induced dry AMD model mice using AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5).

[0068] Figure 22 shows the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41AK67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41AK67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on RPE cells in mice with dry AMD model induced by PEG-400.

[0069] Figure 23 shows F-actin staining images of RPE cells in PEG-400-induced dry AMD model mice injected with AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41AK67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41AK67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5).

[0070] Detailed Description of the Invention

[0071] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0072] In one aspect, the present disclosure provides a complement receptor 2 (CR2)-factor H (FH) fusion protein (hereinafter referred to as a CR2-FH fusion protein), comprising: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof; and the FH portion comprises the first four N-terminal short homology repeat (SCR) domains of FH. In some embodiments, an isolated CR2-FH fusion protein is provided. In some embodiments, the CR2 portion and the FH portion are fused directly or indirectly to each other in the form of a fusion protein. In some embodiments, the CR2 portion and the FH portion are covalently linked. In some embodiments, the CR2 portion and the FH portion are linked via an amino acid linker sequence. In some embodiments, the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46. In some embodiments, the CR2 portion and the FH portion are connected by a linker sequence, preferably, the linker sequence comprises (G4S) n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0073] As used herein, "fusion protein" refers to two or more peptides, polypeptides, or proteins that are operably linked to one another. In some embodiments, the CR2 portion and the FH portion of the CR2-FH fusion protein are directly fused to one another. In some embodiments, the CR2 portion and the FH portion are linked by an amino acid linker sequence. Examples of linker sequences are known in the art and include, for example, (Gly4Ser), (Gly4Ser)2, (Gly4Ser)3, (Gly3Ser)4, (SerGly4), (SerGly4)2, (SerGly4)3, and (SerGly4)4. Linker sequences may also include "natural" linker sequences found between different domains of complement factors, also known as endogenous linker sequences. The order of the CR2 and FH portions in the fusion protein may vary. For example, in some embodiments, the C-terminus of the CR2 portion is fused (directly or indirectly) to the N-terminus of the FH portion of the molecule. In some embodiments, the N-terminus of the CR2 portion is fused (directly or indirectly) to the C-terminus of the FH portion of the molecule.

[0074] In some embodiments, a CR2-FH fusion protein is provided comprising: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2; the FH portion comprises two or more FH fragments, each comprising the first four N-terminal SCR domains of FH, preferably, the two or more FH fragments are connected by a linker sequence, more preferably, the linker sequence comprises (G4S) n In some embodiments, the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46.

[0075] In some embodiments, a CR2-FH fusion protein is provided comprising: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2; and the FH portion comprises the first four N-terminal SCR domains of FH, the eighth N-terminal SCR domain of FH, and the nineteenth to twentieth N-terminal SCR domains of FH. In some embodiments, the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the CR2 portion comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:46. In some embodiments, the eighth N-terminal SCR domain of FH comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the nineteenth to twentieth N-terminal SCR domains of FH comprise the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the CR2 portion and the FH portion are connected via a linker sequence. Preferably, the linker sequence comprises (G4S) n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0076] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2; and wherein the FH portion comprises the first four N-terminal SCR domains of FH and the eighteenth to twentieth N-terminal SCR domains of FH. In some embodiments, the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the eighteenth to twentieth N-terminal SCR domains of FH comprise the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the CR2 portion and the FH portion are connected by a linker sequence, preferably, the linker sequence comprises (G4S). n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0077] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2 (SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); wherein the FH portion comprises the first four N-terminal SCR domains of FH (e.g., FH (SCR1-SCR4)), the N-terminal eighteenth SCR domain of FH (e.g., FH (SCR18)), and the N-terminal twentieth SCR domain of FH (e.g., FH (SCR20)). In some embodiments, the N-terminal eighteenth SCR domain of FH comprises the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the N-terminal twentieth SCR domain of FH comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the CR2 portion and the FH portion are connected via a linker sequence. Preferably, the linker sequence comprises (G4S) n The sequence shown and / or the endogenous linked sequence, wherein n is an integer greater than 0.

[0078] In some embodiments, a CR2-FH fusion protein is provided that comprises: a) a CR2 portion comprising a CR2 fragment, and b) an FH portion comprising an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof (e.g., CR2(SCR1-SCR4) or a variant thereof, such as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 46); wherein the FH portion comprises the first four N-terminal SCR domains of two FHs (e.g., FH(SCR1-SCR4)) and the N-terminal seventh SCR domain of FH (e.g., FH(SCR7)). In some embodiments, the N-terminal seventh SCR domain of FH comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0079] In some embodiments, the linker sequence comprises (G4S) n The sequence shown and / or the FH endogenous connection sequence, wherein n is an integer greater than 0.

[0080] In some embodiments, the domains are connected by a linker sequence comprising (G4S) n The sequence shown and / or the FH endogenous connection sequence, wherein n is an integer greater than 0.

[0081] In some embodiments, the CR2-FH fusion protein comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55, or an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, wherein the CR2-FH fusion protein has the dual function of binding to CR2 ligand and inhibiting complement activation of the alternative pathway. The CR2-FH fusion protein can bind to the CR2 ligand with an affinity that is any one of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of the CR2 protein. Binding affinity can be determined by any method known in the art, including, for example, surface plasmon resonance, calorimetric titration, ELISA, and flow cytometry. The CR2-FH fusion protein can also inhibit complement activation via the alternative pathway, exhibiting complement inhibitory activity that is any one of about 50%, 60%, 70%, 80%, 90%, or 100% or greater of the complement inhibitory activity of the FH protein.

[0082] In some embodiments, isolated CR2-FH fusion proteins are provided. In some embodiments, the CR2-FH fusion protein forms dimers or multimers.

[0083] In some embodiments, the CR2-FH fusion protein comprises a signal peptide sequence. Preferably, the signal peptide sequence is located at the N-terminus. More preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17.

[0084] In some embodiments, the CR2-FH fusion protein comprises the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56, or an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:11.

[0085] CR2 part

[0086] The CR2 portion described herein comprises CR2 or a fragment thereof. CR2 is a transmembrane protein primarily expressed on mature B cells and follicular dendritic cells. CR2 is a member of the C3-binding protein family. The natural ligands for CR2 are iC3b, C3dg, and C3d, which are cleavage fragments of C3. Cleavage of C3 initially leads to the production and deposition of C3b on the surface of activated cells. C3b fragments participate in the production of enzyme complexes that amplify the complement cascade. On the cell surface, C3b is rapidly converted to inactive iC3b, particularly when deposited on host surfaces containing complement regulators. Even in the absence of membrane-bound complement regulators, significant levels of iC3b are formed due to the action of FH. Subsequently, iC3b is digested by Factor I (FI) and other proteases into the membrane-bound fragments C3dg and C3d, but this process is relatively slow. Therefore, once produced, the C3 ligands for CR2 are relatively long-lived and present in high concentrations at sites of complement activation. CR2 can therefore act as an efficient targeting vector to bring molecules to the site of complement activation. CR2 contains an extracellular portion with 15 or 16 repeating units called short homologous repeats (SCR domains). The SCR domain has a typical framework of highly conserved residues, which include four cysteines, two prolines, one tryptophan, and several other partially conserved glycine and hydrophobic residues. The SCR1-4 domains are located at amino acid positions 23-271 in the human CR2 protein sequence. In some embodiments of the present disclosure, the CR2 portion comprises the first four N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises the amino acid sequence set forth in SEQ ID NO: 1. Variants of the CR2 portion may also include some mutation sites, and variants of the CR2 portion comprise the amino acid sequence set forth in SEQ ID NO: 46.

[0087] Factor H fraction (FH fraction)

[0088] The FH portion of the CR2-FH fusion protein described herein comprises FH or a fragment thereof. Complement factor H (FH) is a single polypeptide chain plasma glycoprotein. The protein is composed of 20 repeated SCR domains of approximately 60 amino acids arranged in a continuous manner like a string of 20 beads. Factor H binds to C3b, accelerates the decay of the alternative pathway C3 convertase (C3Bb), and acts as a cofactor for the proteolytic inactivation of C3b. In the presence of factor H, C3b proteolysis leads to the cleavage of C3b. The SCR1-4 domains are located at amino acids 21-262 of the human complement factor H protein sequence. In some embodiments of the present disclosure, the FH portion comprises two or more FH fragments, each comprising the first four N-terminal SCR domains of FH. Preferably, the two or more FH fragments are connected by a linker sequence, more preferably, the linker sequence is such as (G4S) n , wherein n is an integer greater than 0. In some embodiments, the first four N-terminal short homology repeat (SCR) domains of the FH comprise the amino acid sequence shown in SEQ ID NO: 2. In some embodiments of the present disclosure, the FH portion comprises the first four N-terminal SCR domains of FH, the eighth N-terminal SCR domain of FH, and the nineteenth to twentieth N-terminal SCR domains of FH. In some embodiments of the present disclosure, the FH portion comprises the first four N-terminal SCR domains of FH and the eighteenth to twentieth N-terminal SCR domains of FH. In some embodiments, FH is wild-type FH. In some embodiments, the FH portion is a fragment of wild-type FH or a plurality of fragments directly or indirectly connected.

[0089] In another aspect, the present disclosure provides polynucleotides encoding the CR2-FH fusion proteins described herein. In some embodiments, the polynucleotide sequence encoding the first four N-terminal short homology repeat (SCR) domains of FH comprises the sequence set forth in SEQ ID NOs: 19, 28, or 29. In some embodiments, the polynucleotide sequence encoding the eighth N-terminal SCR domain of FH comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the polynucleotide sequence encoding the nineteenth to twentieth N-terminal SCR domains of FH comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the polynucleotide sequence encoding the eighteenth to twentieth N-terminal SCR domains of FH comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the polynucleotide sequence encoding the CR2 portion comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the eighteenth N-terminal SCR domain of FH comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the polynucleotide encoding the 20th N-terminal SCR domain of FH comprises the polynucleotide sequence shown in SEQ ID NO: 58. In some embodiments, the polynucleotide encoding the 7th N-terminal SCR domain of FH comprises the polynucleotide sequence shown in SEQ ID NO: 61.

[0090] In some embodiments, the nucleotide sequence encoding the CR2-FH fusion protein comprises the sequence set forth in SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, or SEQ ID NO:74, or a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, or SEQ ID NO:74.

[0091] In some embodiments, the polynucleotide sequence is a DNA or RNA, such as an mRNA sequence.

[0092] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein comprises a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide is located at the 5' end of the polynucleotide sequence encoding the CR2-FH fusion protein. More preferably, the polynucleotide sequence encoding the signal peptide is as shown in SEQ ID NO: 37 or 38.

[0093] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein comprises the sequence set forth in SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:73, or SEQ ID NO:75, or a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:73, or SEQ ID NO:75.

[0094] In another aspect, the present disclosure provides a vector encoding the polynucleotide described in the present disclosure. In some embodiments, the vector is selected from at least one of an adeno-associated virus AAV vector, an adenovirus vector, an RNA virus vector, a lentivirus vector, and a vaccinia virus vector.

[0095] In another aspect, the present disclosure provides a host cell comprising a polynucleotide as described in the present disclosure or a vector as described in the present disclosure.

[0096] In another aspect, the present disclosure provides AAV particles comprising an AAV vector as described herein. In some embodiments, the AAV particles are produced by a production cell line comprising one or more nucleic acids encoding an AAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus functions.

[0097] In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the AAV vector comprises one or more AAV inverted terminal repeat (ITR) sequences flanking the heterologous nucleic acid. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are ITRs of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITRs and capsids of the AAV particles are derived from the same AAV serotype. In some embodiments, the ITRs and capsids are derived from AAV2. In other embodiments, the ITRs and capsid of the AAV viral particle are derived from different AAV serotypes.

[0098] In some embodiments, the AAV vector comprises one or more promoters, enhancers, or polyadenylation signals.

[0099] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one of the CR2-FH fusion protein described in the present disclosure, the polynucleotide described in the present disclosure, the vector described in the present disclosure, the host cell described in the present disclosure, and the AAV particle described in the present disclosure.

[0100] and a pharmaceutically acceptable carrier.

[0101] In some embodiments, the composition is suitable for intraocular, intravenous, intraarterial, subcutaneous, intratracheal, or inhalation administration.

[0102] In another aspect, the present disclosure provides use of the CR2-FH fusion protein described herein, the polynucleotide described herein, the vector described herein, the host cell described herein, the AAV particle described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating a complement alternative pathway-associated disease in a subject.

[0103] The present disclosure provides a method for treating a complement alternative pathway-associated disease in a subject in need thereof, wherein an effective amount of the CR2-FH fusion protein described in the disclosure, the polynucleotide described in the disclosure, the vector described in the disclosure, the host cell described in the disclosure, the AAV particle described in the disclosure, or the pharmaceutical composition described in the disclosure is administered.

[0104] In some embodiments, the complement alternative pathway-associated disease is an inflammatory disease or an autoimmune disease.

[0105] In some embodiments, the complement alternative pathway-associated disease is age-related macular degeneration, preferably dry age-related macular degeneration.

[0106] In some embodiments, the complement alternative pathway-associated disease is a symptom of microangiopathic hemolytic anemia, thrombocytopenia, or acute renal failure.

[0107] In some embodiments, the complement alternative pathway-associated disease is selected from macular degeneration, ischemia-reperfusion, organ transplant rejection, drusen-associated disease, pregnancy-related disease, adverse drug reaction, and post-cardiopulmonary bypass complications.

[0108] In some embodiments, the complement alternative pathway-associated disease is selected from age-related macular degeneration (AMD), rheumatoid arthritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), factor H-related hemolytic uremic syndrome (HUS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), lupus nephritis, stroke, myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, burns, inflammation associated with cardiopulmonary bypass and hemodialysis, plasmapheresis, platelet separation, leukocyte separation, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiocontrast-induced anaphylaxis.

[0109] In some embodiments, the subject is a mammal, preferably a human.

[0110] The present disclosure will be described in further detail below. However, the implementation of the present disclosure is not limited to the following embodiments.

[0111] Example 1: Construction of AAV plasmid vector expressing CR2 and FH segment genes

[0112] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2), SCR8 amino acid sequence (SEQ ID NO: 3), and SCR19-SCR20 amino acid sequence (SEQ ID NO: 4) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and connected with (G4S)2 and an endogenous linker linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR8)+linker+FH(SCR19-SCR20)-+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR I restriction enzyme site was introduced at the 3' end. The V restriction enzyme cleavage site was named XMDC025 (the whole gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 1.

[0113] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2) and SCR18-SCR20 amino acid sequence (SEQ ID NO: 7) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the mixture was connected with an endogenous linker using (G4S)2 to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR18-SCR20)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end, and an EcoR I restriction enzyme site was introduced at the 3' end. The V restriction enzyme cleavage site was named XMDC026 (the whole gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 1.

[0114] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and connected with (G4S)2 and an endogenous linker linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR1-SCR4)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. The composition was named XMDC029 (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural schematic diagram is shown in Figure 1.

[0115] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the mixture was connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp + linker + CR2 (SCR1-SCR4) + (G4S)2 + linker + FH (SCR1-SCR4) + linker-. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. The composition was named CR2-FH (1-4) (the whole gene was synthesized by Shanghai Sangon Biotech Co., Ltd.). A schematic diagram of its structure is shown in Figure 1.

[0116] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR5 amino acid sequence (SEQ ID NO: 14) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the mixture was connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp + linker + CR2 (SCR1-SCR4) + (G4S)2 + linker + FH (SCR1-SCR5) + linker-. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. The composition was named CR2-FH (1-5) (the whole gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd.). A schematic diagram of its structure is shown in Figure 1.

[0117] XMDC025, XMDC026, XMDC029, CR2-FH(1-4), CR2-FH(1-5), and ssAAV plasmids were digested with BamH I / EcoR V. The ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4), and ssAAV-CR2-FH(1-5) vectors were constructed through conventional molecular biology procedures such as ligation, transformation, and clone screening and identification. The vector information is shown in Figure 2. High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for future use.

[0118] The endogenous linker in the above structure is the endogenous linker sequence in the CR2 or FH structure. Therefore, those skilled in the art will understand that the linker sequence in the structure is determined by the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0119] Table 1. Sequence information

[0120] Example 2: Preparation and identification of recombinant AAV virus

[0121] Recombinant AAV viruses were prepared using a three-plasmid packaging system. The helper plasmid (phelper), AAV Cap and Rep protein expression plasmids, and the expression vector target plasmids (ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4), ssAAV-CR2-FH(1-5)) were prepared at a mass ratio of 2:1:1 and PEI promoter to form a transfection complex. HEK293T cells were transfected for AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) virus packaging. Supernatants were collected twice, on days 3 and 7 after transfection, to obtain AAV virus particles containing the target gene. Density gradient centrifugation (Beckman ultracentrifuge) with different gradients of iodixanol (15%, 25%, 40%, and 60%) was performed to obtain purified AAV virus. The purified AAV virus was identified by transmission electron microscopy and the AAV virus titer was quantified by qPCR.

[0122] Example 3: PEG-400-induced dry AMD mouse model and injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5)

[0123] Using polyethylene glycol (PEG) to induce a dry AMD mouse model, PEG-treated mice develop retinal pathological changes similar to the clinical pathological features of dry AMD, such as retinal structural destruction, RPE cell damage, and photoreceptor loss. The specific method is as follows:

[0124] Seventy SPF 4-week-old male C57BL / 6J mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), weighing approximately 14 g, were housed under a 12-hour light and dark alternation environment according to the mouse circadian rhythm.

[0125] Since it takes a certain amount of time for the expression of the target gene to reach a stable level after AAV injection, the drug was injected into the vitreous cavity of both eyes on the first day, and PEG-400 was injected into the subretinal space of both eyes on the 22nd day to establish the model. On the 27th day, the eyeballs were removed for frozen sectioning and staining to observe the retinal and RPE structures. The treatments are shown in Table 2:

[0126] Table 2. Drug administration by intravitreal injection in both eyes of mice on day 1

[0127] The specific steps are as follows:

[0128] Intravitreal injection: 5% mydriatic solution was instilled into each eye to dilate the pupils of the mouse. Anesthetized mice were anesthetized by intraperitoneal injection of 10 mL / kg of 5% chloral hydrate (Shenggong). The anesthetized animals were placed in lateral recumbency on the operating table. The injection site (Hamilton needle, 7632-01) was selected, 1-2 mm posterior to the limbus, either temporal or nasal to the eye. Care was taken to avoid damaging the posterior lens capsule and other retinal areas. The needle was inserted into the vitreous cavity and the injection solution was injected. After a 10-second pause, the needle was slowly withdrawn. Erythromycin eye ointment was applied and the animals were returned to their cages.

[0129] Example 4: Hematoxylin and eosin (HE) staining to evaluate the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5) on the outer nuclear layer thickness and nuclear density of the retina in PEG-400-induced dry AMD model mice

[0130] The eyes of the mice in Example 3 were enucleated, and one eye was fixed in 4% paraformaldehyde (biosharp) at 4°C overnight. After washing four times with PBS (biosharp), the cornea and lens were peeled off, and the eye cup, including the retina, choroid, and sclera, was dehydrated with 30% sucrose (Shenggong) until it sank to the bottom. After that, 10 μm thick frozen sections were made and HE staining was performed. After staining, the outer nuclear layer (ONL) thickness and nuclear density of the retinal sections were measured and analyzed using Image J.

[0131] The results are shown in Figures 3, 4, and 5. Compared with the control group, the thickness and nuclear density of the outer nuclear layer of the retina of the mice in the model group were significantly reduced, and the retinal structure was significantly damaged. Compared with the model group, injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, and AAV-CR2-FH (1-4) significantly increased the thickness and nuclear density of the outer nuclear layer and improved the structural damage of the retina. Injection of AAV-CR2-FH (1-5) only significantly increased the thickness of the outer nuclear layer. AAV-XMDC029 had a better effect on improving retinal thickness than AAV-CR2-FH (1-4), and had a better effect on improving retinal nuclear density than AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0132] Example 5: Immunofluorescence staining to evaluate the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5) on retinal photoreceptor cells in PEG-400-induced dry AMD model mice

[0133] The frozen sections in Example 4 were air-dried at room temperature in a slide box. The tissue on the slide was circled using an immunohistochemistry pen (Vectorlabs). Polylysine slides (Sita) were placed in a humidified chamber, and the tissue on the slide was washed with PBS and incubated at room temperature for 5 minutes. The PBS on the slide was discarded, and the washing process was repeated five times, each for 10 minutes. Blocking solution (5% goat serum (Biyuntian) + 0.5% Triton 100 (Sangong)) was added at 500 μL / slide, and the slides were incubated at room temperature for 45 minutes. The blocking solution on the slides was discarded, and a primary antibody mixture (Cone Arresting-1 (EMD Millipore Corp) for cone photoreceptors and Rodopsin-1 (Santa Cruz)) was added dropwise at 200 μL / slide, and the slides were incubated in a humidified chamber at 4°C overnight. The next day, the slides were rinsed five times with PBS at room temperature for 5 minutes each. A secondary antibody mixture (Anti-rabbit IgG Alexa Fluor) was added. 555 (Cell Signaling) and Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen) were counterstained at a dilution of 1:500 and incubated in a humidified chamber at room temperature in the dark for 2.5 hours. All sections needed to be protected from light after this step. The secondary antibody was removed and the sections were rinsed with PBS five times at room temperature for 5 minutes each. DAPI staining solution (Sigma) was added at 50 μL / slide and incubated at room temperature for 10 minutes. The sections were then rinsed with PBS three times for 5 minutes each. The sections were mounted and photographed under a fluorescence microscope.

[0134] The results are shown in Figures 6, 7, 8 and 9. Compared with the control group, the number of retinal cones in the model group was reduced, the thickness of the outer segments of rods was narrowed, and the photoreceptor cells were significantly damaged. Compared with the model group, injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5) could significantly improve the damage to cones and rods. AAV-XMDC029 and AAV-CR2-FH (1-4) had better improvement effects on cone damage than AAV-CR2-FH (1-5).

[0135] Example 6: F-actin staining to evaluate the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH (1-4), and AAV-CR2-FH (1-5) on RPE cells in mice with dry AMD induced by PEG-400

[0136] The other eye of the mouse removed in Example 3 was fixed in 4% paraformaldehyde at 4°C overnight; the cornea, lens, muscle and retina were removed, and the sclera, choroid and RPE complex were fixed in 4% paraformaldehyde at room temperature for 2 hours and washed with PBS three times for 5 minutes each time; the sclera, choroid and RPE complex were blocked in blocking solution (5% goat serum + 0.5% TritonX-100) at room temperature for 1 hour and washed with PBS for 3 hours. The cells were stained with 594 phalloidin (Jackson ImmunoResearch) (1:40 methanol stock solution) on a shaker at room temperature for 40 minutes, then washed six times with PBS on a shaker for 5 minutes each, stained with DAPI for 30 minutes at room temperature, and then washed three times with PBS for 5 minutes each. The sclera, choroid, and RPE complex were transferred to a slide, cut into about 8 petals, mounted, and observed and photographed under a fluorescence microscope.

[0137] The results are shown in Figures 10 and 11. Compared with the control group, the RPE cells in the model group were significantly enlarged and the RPE cells were obviously damaged. Compared with the model group, the injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, and AAV-CR2-FH (1-4) can significantly reduce the RPE cell area and improve RPE cell damage. Injection of AAV-CR2-FH (1-5) has no effect on improving RPE cell damage. The improvement effect of AAV-XMDC029 on RPE cell damage is better than that of AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0138] Combined with the results of HE staining, immunofluorescence staining and F-actin staining, injection of AAV-XMDC029 had a better effect on improving the nuclear density of the outer nuclear layer of the retina and RPE cell damage in the PEG-400-induced dry AMD mouse model than AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5), and had a better effect on improving the thickness of the outer nuclear layer and cone cells than AAV-CR2-FH(1-4).

[0139] Example 7: Construction of other plasmid vectors

[0140] Solution 1

[0141] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2), SCR18 amino acid sequence (SEQ ID NO: 39), and SCR20 amino acid sequence (SEQ ID NO: 40) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and connected with (G4S)2 and an endogenous linker linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR18)+linker+FH(SCR20)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR I restriction enzyme site was introduced at the 3' end. V restriction enzyme cleavage site, named XMDC061 (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural schematic diagram is shown in Figure 12.

[0142] Option 2

[0143] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequence (SEQ ID NO: 2) and SCR7 amino acid sequence (SEQ ID NO: 43) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the mixture was connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR7)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR I restriction enzyme site was introduced at the 3' end. The V restriction enzyme cleavage site was named XMDC062 (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0144] Option 3

[0145] According to the amino acid sequence of the R36A K41A K67A mutant of SCR1-SCR4 of CR2 (Gene ID: 1380) published on NCBI (SEQ ID NO: 46) and the amino acid sequence of SCR1-SCR4 (SEQ ID NO: 2), SCR8 (SEQ ID NO: 3), and SCR19-SCR20 (SEQ ID NO: 4) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 5) was added to the N-terminus of the composition. NO: 17), and connected with (G4S)2 and endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR8)+linker+FH(SCR19-SCR20)-+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. The gene was named XMDC025-(CR2-R36A K41A K67A) (the whole gene was synthesized by Shanghai Sangon Biotech Co., Ltd.). The schematic diagram of its structure is shown in Figure 12.

[0146] Option 4

[0147] Based on the amino acid sequence of the R36A K41A K67A mutant of SCR1-SCR4 of CR2 (Gene ID: 1380) (SEQ ID NO: 46) and the amino acid sequence of SCR1-SCR4 (SEQ ID NO: 2) and SCR18-SCR20 (SEQ ID NO: 7) of FH (Gene ID: 3075), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR18-SCR20)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR I restriction enzyme site was introduced at the 3' end. V restriction enzyme cleavage site, named XMDC026-(CR2-R36A K41A K67A) (the whole gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd.), and its structural schematic diagram is shown in Figure 12.

[0148] Option 5

[0149] Based on the amino acid sequence of the R36A K41A K67A mutant of SCR1-SCR4 of CR2 (Gene ID: 1380) (SEQ ID NO: 46) and the amino acid sequence of SCR1-SCR4 of FH (Gene ID: 3075) (SEQ ID NO: 2), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the composition was connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR1-SCR4)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. The composition was named XMDC029-(CR2-R36A K41A K67A) (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0150] Option 6

[0151] Based on the amino acid sequence of the R36A K41A K67A mutant of SCR1-SCR4 of CR2 (Gene ID: 1380) (SEQ ID NO: 46) and the amino acid sequences of SCR1-SCR4 (SEQ ID NO: 2), SCR18 (SEQ ID NO: 39), and SCR20 (SEQ ID NO: 40) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp + linker + CR2 (SCR1-SCR4) + (G4S)2 + linker + FH (SCR1-SCR4) + linker + FH (SCR18) + linker + FH (SCR20) + linker. The nucleotide sequence was designed according to human codon preference, and BamH was introduced at the 5' end. I restriction enzyme site, and an EcoR V restriction enzyme site was introduced at the 3' end, and the product was named XMDC061-(CR2-R36A K41A K67A) (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0152] Option 7

[0153] According to the amino acid sequence of the R36A K41A K67A mutant of SCR1-SCR4 of CR2 (Gene ID: 1380) published on NCBI (SEQ ID NO: 46) and the amino acid sequence of SCR1-SCR4 of FH (Gene ID: 3075) (SEQ ID NO: 2), SCR7 amino acid sequence (SEQ ID NO: 43), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 43) was added to the N-terminus of the composition. NO: 17), and connected with (G4S)2 and endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR7)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. It was named XMDC062-(CR2-R36A K41A K67A) (full gene synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural schematic diagram is shown in Figure 12.

[0154] The endogenous linker in the above structure is the endogenous linker sequence in the CR2 or FH structure. Therefore, those skilled in the art will understand that the linker sequence in the structure is determined by the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0155] XMDC061, XMDC062, XMDC025-(CR2-R36A K41A K67A), XMDC026-(CR2-R36A K41A K67A), XMDC029-(CR2-R36A K41A K67A), XMDC061-(CR2-R36A K41A K67A), XMDC062-(CR2-R36A K41A K67A) and ssAAV plasmids were digested with BamH I / EcoR V. Then ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36A K41A K67A), ssAAV-XMDC026-(CR2-R36A K41A K67A) The vectors were ssAAV-XMDC029-(CR2-R36A K41A K67A), ssAAV-XMDC061-(CR2-R36AK41A K67A), and ssAAV-XMDC062-(CR2-R36A K41A K67A). The vector information is shown in Figure 13. High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN).

[0156] Option 8

[0157] Based on the SCR1-SCR4 amino acid sequence (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR5 amino acid sequence (SEQ ID NO: 14) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition, and the mixture was connected with (G4S)2 and an endogenous linker to form an open reading frame with the structure of CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR5)+linker+(G4S)2+linker+FH(SCR1-SCR5)+linker. The nucleotide sequence was designed according to human codon preference, and a BamH I restriction site was introduced at the 5' end, and an EcoR I restriction enzyme site was introduced at the 3' end. The V restriction enzyme cleavage site was named CR2-FH (SCR1-5+1-5) (the whole gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 14.

[0158] The endogenous linker in the above structure is the endogenous linker sequence in the CR2 or FH structure. Therefore, those skilled in the art will understand that the linker sequence in the structure is determined by the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0159] The CR2-FH (SCR1-5+1-5) and ssAAV plasmids were double-digested with BamH I / EcoR V, and the ssAAV-CR2-FH (SCR1-5+1-5) vector was constructed through conventional molecular biology procedures such as ligation, transformation, and clone screening and identification. The vector information is shown in Figure 14. High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for future use.

[0160] Table 3. Sequence information

[0161] Example 8: Preparation and identification of other recombinant AAV viruses

[0162] Recombinant AAV virus was prepared using a three-plasmid packaging system, including helper plasmids, AAV Cap and Rep protein expression plasmids, and expression vector destination plasmids (ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36A K41A K67A), ssAAV-XMDC026-(CR2-R36A K41A K67A), ssAAV-XMDC029-(CR2-R36A K41A K67A), ssAAV-XMDC061-(CR2-R36A K41A K67A), ssAAV-XMDC062-(CR2-R36A K41A K67A), ssAAV-XMDC063-(CR2-R36A K41A K67A), ssAAV-XMDC064-(CR2-R36A K41A K67A), ssAAV-XMDC065-(CR2-R36A K41A K67A), ssAAV-XMDC066-(CR2-R36A K41A K67A), ssAAV-XMDC067-(CR2-R36A K41A K67A), ssAAV-XMDC068-(CR2-R36A K41A K67A), ssAAV-XMDC069-(CR2-R36A K41A K67A), ssAAV-XMDC070-(CR2-R36A K41A K67A), ssAAV-XMDC071-(CR2-R36A K41A K67A), ssAAV-XMDC072-(CR2- K67A), ssAAV-CR2-FH (SCR1-5+1-5)) were mixed with PEI promoter at a mass ratio of 2:1:1 to form a transfection complex, and HEK293T cells were transfected for AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), and AAV-CR2-FH (SCR1-5+1-5) virus packaging. Supernatants were collected twice, on days 3 and 7 after transfection, to obtain AAV viral particles containing the target gene. Density gradient centrifugation (Beckman ultracentrifuge) using varying gradients of iodixanol (15%, 25%, 40%, and 60%) was performed to obtain purified AAV virus. The purified AAV virus was then analyzed for quality by transmission electron microscopy and quantified by qPCR.

[0163] Example 9: PEG-400-induced dry AMD mouse model and injection administration of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41AK67A), AAV-XMDC029, and AAV-FH (CR2-R36A K41A K67A).

[0164] The specific method is as follows:

[0165] 88 SPF male C57BL / 6J mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) weighing approximately 14-18 g were housed under a 12-hour light-dark cycle according to the mouse circadian rhythm.

[0166] Since it takes a certain amount of time for the expression of the target gene to reach a stable level after AAV injection, the drug was injected into the vitreous cavity of both eyes on the first day, and PEG-400 was injected into the subretinal space of both eyes on the 22nd day to establish the model. On the 27th day, the eyeballs were removed for frozen sectioning and staining to observe the retinal and RPE structures. The treatments are shown in Table 4:

[0167] Table 4. Dosage regimen

[0168] The specific operation steps of injection administration are the same as those in Example 3.

[0169] Example 10: Hematoxylin and eosin (HE) staining was used to evaluate the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on the outer nuclear layer thickness and nuclear density of the retina in PEG-400-induced dry AMD model mice

[0170] The eyes of the mice in Example 9 were enucleated, and one eye was frozen and sectioned and stained with HE. After staining, the retinal sections were photographed and the outer nuclear layer (ONL) thickness and nuclear density were measured and analyzed using Image J. The specific operating steps were the same as those in Example 4.

[0171] The results are shown in Figures 15, 16, and 17. Compared with the control group, the thickness and nuclear density of the retinal outer nuclear layer of the mice in the model group were significantly reduced, and the retinal structure was significantly damaged. Compared with the model group, injection of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5) could significantly increase the thickness and nuclear density of the outer nuclear layer and improve the retinal structural damage.

[0172] Example 11: Immunofluorescence staining to evaluate the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on retinal photoreceptor cells in PEG-400-induced dry AMD model mice

[0173] The frozen sections in Example 9 were subjected to immunofluorescence staining and observed and photographed under a fluorescence microscope. The specific operating steps were the same as those in Example 5.

[0174] The results are shown in Figures 18, 19, 20 and 21. Compared with the control group, the number of retinal cones in the model group decreased, the thickness of the outer segments of rods narrowed, and the cones and rods were significantly damaged. Compared with the model group, the injection of AAV-XMDC029 can significantly improve the damage caused by modeling and effectively protect the thickness of the outer segments of cones and rods.

[0175] Example 12: F-actin staining to evaluate the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) on RPE cells in mice with dry AMD induced by PEG-400

[0176] The other eye of the mouse enucleated in Example 9 was stained for F-actin and observed and photographed under a fluorescence microscope. The specific operating steps were the same as those in Example 6.

[0177] The results are shown in Figures 22 and 23. Compared with the control group, the RPE cells in the model group were significantly enlarged and the RPE cells were significantly damaged. Compared with the model group, injection of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36AK41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC029-(CR2-R36AK41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36AK41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) can significantly reduce the RPE cell area, and RPE cell damage is significantly improved.

[0178] Combined with the results of HE staining, immunofluorescence staining and F-actin staining, AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41AK67A), AAV-XMDC029-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41AK67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) all significantly improved the retinal outer nuclear layer structural damage and RPE cell damage in the PEG-400-induced dry AMD mouse model. Among them, AAV-XMDC029 can also effectively improve the damage to the outer segment thickness of cones and rods.

[0179] Statistical analysis

[0180] GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was set at 5% or p ≤ 0.05, and the mean and standard error (mean ± SEM) of each analysis indicator were calculated. A p ≤ 0.05 was considered statistically significant.

[0181] The present disclosure is not limited to the above-mentioned embodiments. Any changes, modifications, substitutions, combinations, and simplifications made without violating the spirit and principles of the present disclosure are equivalent technical solutions of the present disclosure and are included in the scope of protection of the present disclosure.

Claims

1. A complement receptor 2 (CR2)-factor H (FH) fusion protein, include: a) a CR2 portion comprising a CR2 fragment, and b) a FH portion comprising a FH fragment, wherein the CR2 portion and the FH portion are optionally connected by a linker sequence, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or a variant thereof, and the FH portion comprises the first four N-terminal short homology repeat (SCR) domains of FH.

2. The CR2-FH fusion protein of claim 1, wherein the FH portion comprises two or more FH fragments, each of which comprises the first four N-terminal SCR domains of FH, and the two or more FH fragments are optionally connected by a linker sequence.

3. The CR2-FH fusion protein of claim 1, wherein the FH portion comprises the first four N-terminal SCR domains of FH, the N-terminal eighth SCR domain of FH, and the N-terminal nineteenth to twentieth SCR domains of FH.

4. The CR2-FH fusion protein of claim 1, wherein the FH portion comprises the first four N-terminal SCR domains of FH and the N-terminal eighteenth to twentieth SCR domains of FH.

5. The CR2-FH fusion protein of claim 1, wherein the FH portion comprises the first four N-terminal SCR domains of FH, the N-terminal eighteenth SCR domain of FH, and the N-terminal twentieth SCR domain of FH.

6. The CR2-FH fusion protein of claim 1, wherein the FH portion comprises the first four N-terminal SCR domains of two FHs and the N-terminal seventh SCR domain of FH.

7. The CR2-FH fusion protein of any one of claims 1 to 6, wherein the linker sequence comprises (G 4 S) n The sequence shown and / or the endogenous connection sequence, wherein n is an integer greater than 0.

8. The CR2-FH fusion protein according to any one of claims 1 to 6, wherein the domains are connected by a linker sequence, and the linker sequence comprises (G 4 S) n The sequence shown and / or the endogenous connection sequence, wherein n is an integer greater than 0.

9. The CR2-FH fusion protein of any one of claims 1 to 8, wherein the first four N-terminal short homology repeat (SCR) domains of FH comprise the amino acid sequence shown in SEQ ID NO:

2.

10. The CR2-FH fusion protein of claim 3, wherein the N-terminal eighth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:

3.

11. The CR2-FH fusion protein of claim 3, wherein the N-terminal nineteenth to twentieth SCR domains of FH comprise the amino acid sequence shown in SEQ ID NO:

4.

12. The CR2-FH fusion protein of claim 4, wherein the N-terminal eighteenth to twentieth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:

7.

13. The CR2-FH fusion protein of claim 5, wherein the N-terminal eighteenth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:

39.

14. The CR2-FH fusion protein of claim 5, wherein the N-terminal twentieth SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:

40.

15. The CR2-FH fusion protein of claim 6, wherein the N-terminal seventh SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO:

43.

16. The CR2-FH fusion protein of any one of claims 1 to 15, wherein the CR2 portion or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

46.

17. The CR2-FH fusion protein of any one of claims 1 to 16, wherein the CR2-FH fusion protein comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO:

55.

18. The CR2-FH fusion protein according to any one of claims 1 to 17, wherein the CR2-FH fusion protein comprises a signal peptide sequence, preferably, the signal peptide sequence is located at the N-terminus, more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

17.

19. The CR2-FH fusion protein of claim 18, wherein the CR2-FH fusion protein comprises the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 or SEQ ID NO:

56.

20. A polynucleotide encoding the CR2-FH fusion protein according to any one of claims 1 to 19.

21. The polynucleotide according to claim 1, wherein the polynucleotide sequence encoding the CR2-FH fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 22, 23, 25, 26, 30, 31, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71, 72, 73, 74 or 75.

22. A vector encoding the polynucleotide according to claim 20 or 21.

23. The vector of claim 22, wherein the vector is selected from at least one of an adeno-associated virus AAV vector, an adenovirus vector, an RNA virus vector, a lentivirus vector, and a vaccinia virus vector.

24. A host cell comprising the polynucleotide of claim 20 or 21 or the vector of claim 23.

25. An AAV particle comprising the AAV vector of claim 23.

26. A pharmaceutical composition comprising at least one of the CR2-FH fusion protein of any one of claims 1 to 19, the polynucleotide of claim 20 or 21, the vector of claim 22 or 23, the host cell of claim 24, and the AAV particle of claim 25, and a pharmaceutically acceptable carrier.

27. The composition of claim 26, wherein the composition is suitable for intraocular, intravenous, intraarterial, subcutaneous, intratracheal or inhalation administration.

28. Use of the CR2-FH fusion protein of any one of claims 1 to 19, the polynucleotide of claim 20 or 21, the vector of claim 22 or 23, the host cell of claim 24, the AAV particle of claim 25, or the pharmaceutical composition of claim 26 in the preparation of a medicament for treating a complement alternative pathway-associated disease in a subject.

29. The use according to claim 28, wherein the complement alternative pathway-associated disease is an inflammatory disease or an autoimmune disease.

30. The use according to claim 28, wherein the complement alternative pathway-related disease is age-related macular degeneration, preferably dry age-related macular degeneration.

31. The use of claim 28, wherein the complement alternative pathway-associated disease is a symptom of microangiopathic hemolytic anemia, thrombocytopenia, or acute renal failure.

32. The method of claim 28, wherein the complement alternative pathway-associated disease is selected from macular degeneration, ischemia-reperfusion, organ transplant rejection, drusen-associated disease, pregnancy-associated disease, adverse drug reactions, and post-cardiopulmonary bypass complications.

33. The method of claim 28, wherein the complement pathway-associated disease is selected from age-related macular degeneration (AMD), rheumatoid arthritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), factor H-associated hemolytic uremic syndrome (HUS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), lupus nephritis, stroke, myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, burns, inflammation associated with cardiopulmonary bypass and hemodialysis, plasmapheresis, platelet separation, leukocyte separation, extracorporeal membrane oxygenation (ECMO), heparin-induced in vitro LDL precipitation (HELP), and radiocontrast-induced anaphylaxis.

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