AAV vector for the treatment of complement bypass-related diseases

Abstract

A CR2-FH fusion protein, the polynucleotide encoding it, a carrier, a pharmaceutical composition thereof, and its uses are provided, which can be used to treat complement bypass-related diseases such as dry AMD.

Description

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 2023105884953, filed on May 19, 2023. The entire contents of Chinese application No. 2023105884953 are incorporated herein by reference for all purposes. Technical Field

[0003] This application relates to fusion proteins, constructs, and uses thereof for treating complement bypass-related diseases. Specifically, this application relates to a CR2-FH molecule for treating complement bypass-related diseases, a construct encoding the CR2-FH molecule, and uses thereof. Background Technology

[0004] The complement system is an important host defense system, playing a crucial role in regulating humoral and cellular immunity, catabolizing immune complexes, and clearing apoptotic cells. The complement system is composed of various soluble protein molecules, including intrinsic complement components, multiple 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 at C5 to produce the membrane attack complex (MAC), which exerts a cell-lysing effect and participates in immunity, forming an important natural immune barrier in the human body. 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. Aberrant complement activation is associated with 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, anaphylatoxins C3a and C5a, and other acute-phase reactant proteins have been shown to be present in drusen deposits in the eyes of patients. In AMD patients, plasma levels of C3a, C3d, Bb, and C5a are elevated, all of which suggest that complement activation plays a role in the pathogenesis of AMD. In addition, complement bypass-related diseases include rheumatoid arthritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGNII), 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 apheresis, leukocyte apheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiation-induced allergic reactions.

[0005] Mid-Age Disorder (AMD) is a degenerative and blinding disease affecting 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 prevalent blinding disease in people over 65 years of age, affecting approximately 9% of the global population. AMD is mainly divided into two types: dry and wet. Dry AMD accounts for more than 80% of cases and can develop into geographic atrophy (GA) in its later stages. The typical characteristic of dry AMD is the formation of drusen (a type of retinal pigment epithelium) associated with degenerative changes in retinal pigment epithelium (RPE) cells. A visible area of ​​pigmentation appears in the central macula, along with the loss of photoreceptor cells—rods and cones. In the later stages of dry AMD, extensive atrophy of the RPE and choroidal vessels 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. It consists of 20 short consensus repeats (SCRs) and is a key inhibitor of the alternative pathway. FH dysfunction 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 trace amounts of C3b and C3bBb (C3 convertases). These C3 convertases are rapidly acted upon by FH and cannot reactivate C3 and subsequent complement components. Under pathological conditions, FH's control over C3 convertases is insufficient. C3 convertases hydrolyze the C3 protein into C3b molecules, simultaneously producing the anaphylactic toxin C3a. This leads to a shift in the complement cascade to its terminal cleavage pathway, producing the anaphylactic toxins C5a and MAC, both of which cause strong inflammatory signals. Previous studies have demonstrated that in dry AMD, retinal homeostasis is impaired due to RPE cell dysfunction, and reduced FH leads to C3 accumulation at both 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 damage. Furthermore, FH possesses a single nucleotide polymorphism (SNP), making it the first complement SNP discovered to be associated with dry AMD. Since complement plays a crucial role in host defense and immune complex catabolism, targeting the complement inhibitor FH to complement activation and disease sites may enhance its efficacy while reducing the side effects of complement inhibition. Complement receptor 2 (CR2) is a complement receptor, a member of the C3-binding protein family, composed of 15 or 16 SCR domains. CR2's natural ligands are iC3b, C3dg, and C3d, which are catabolite fragments of C3. C3 cleavage 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, especially when deposited on the host surface containing complement activating regulators. Even in the absence of membrane-bound complement regulators, high 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, the C3 ligand of CR2, once produced, is relatively long-lived and presents at high concentrations at complement activation sites. CR2 can thus act as an effective targeting carrier to deliver molecules to complement activation sites.Although significant progress has been made in the treatment of AMD, particularly with the use of VEGF inhibitors, with antibody drugs and gene therapy products for wet AMD, no gene therapy drugs for dry AMD have been approved in China to date. Therefore, there is an urgent need for new treatments to address this issue. Based on the advantage of gene therapy products' sustained effects, we aim 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 subsequently found in human tissues. Due to its good safety profile, broad host cell range, low immunogenicity, and efficient long-term expression of exogenous genes, it has become an important tool for gene delivery. Currently, cutting-edge AAV genome design can design self-complementary sequences for the single-stranded DNA carried in the capsid. The advantage of this sequence is that it can be transcribed without the step of single-stranded DNA replication into double-stranded DNA, 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 globally, including Novartis' Zolgensma (AAV9), UniQure's Glybera (AAV1), and Spark Therapeutics' Luxturna (AAV2). This demonstrates the enormous potential of AAV gene therapy, making gene therapy a core technology for overcoming the dilemma of drugs having a sustained effect but limited duration. Summary of the Invention

[0008] This disclosure provides, in one aspect, a complement receptor 2 (CR2)-factor H (FH) fusion protein (hereinafter referred to as CR2-FH fusion protein), comprising: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion contains the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 46); and the FH portion contains the first four N-terminal short homologous repeat (SCR) domains of FH (e.g., FH(SCR1-SCR4)). In some embodiments, a separate 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 linked by an amino acid linker sequence. The linker sequence comprises the sequence shown as (G4S)n and / or an endogenous linker sequence, where n is an integer greater than 0.

[0009] "Separated" substances refer to substances or components that have been artificially obtained and exist in a sufficiently pure state. In some embodiments, the purity of the fusion protein is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99%, determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reversed-phase HPLC).

[0010] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion contains the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46); the FH portion comprises two or more FH fragments, each FH fragment containing the first four N-terminal SCR domains of FH (e.g., FH(SCR1-SCR4)), preferably, the two or more FH fragments are linked by a linker sequence, more preferably, the linker sequence comprises (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0011] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46); the FH portion comprises the first four N-terminal SCR domains of FH (e.g., FH(SCR1-SCR4)), the eighth N-terminal SCR domain of FH (e.g., FH(SCR8)), and the nineteenth to twentieth N-terminal SCR domains of FH (e.g., FH(SCR19-SCR20)).

[0012] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown 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 eighteenth to twentieth N-terminal SCR domains of FH (e.g., FH(SCR18-SCR20)).

[0013] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown 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 eighteenth N-terminal SCR domain of FH (e.g., FH(SCR18)), and the twentieth N-terminal SCR domain of FH (e.g., FH(SCR20)).

[0014] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown 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 seventh N-terminal SCR domain of FH (e.g., FH(SCR7)).

[0015] In some implementations, the connector sequence includes (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0016] In some implementations, the domains are connected by a connector sequence, the connector sequence comprising (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0017] In some embodiments, the first four N-terminal short homologous repeat (SCR) domains of the FH contain an amino acid sequence as shown in SEQ ID NO: 2.

[0018] In some embodiments, the N-terminal eighth SCR domain of the FH contains an amino acid sequence as shown in SEQ ID NO: 3.

[0019] In some embodiments, the nineteenth to twentieth SCR domains at the N-terminus of the FH contain an amino acid sequence as shown in SEQ ID NO: 4.

[0020] In some embodiments, the eighteenth to twentieth SCR domains at the N-terminus of the FH contain an amino acid sequence as shown in SEQ ID NO: 7.

[0021] In some embodiments, the N-terminal eighteenth SCR domain of the FH contains an amino acid sequence as shown in SEQ ID NO: 39.

[0022] In some embodiments, the twentieth SCR domain at the N-terminus of the FH comprises an amino acid sequence as shown in SEQ ID NO: 40.

[0023] In some embodiments, the N-terminal seventh SCR domain of the FH contains an amino acid sequence as shown in SEQ ID NO: 43.

[0024] In some embodiments, the CR2 portion or a variant thereof comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 46.

[0025] In some implementations, the CR2 portion and the FH portion are connected by a connector sequence, preferably such as (G4S). n As shown, n is an integer greater than 0.

[0026] 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.

[0027] In some embodiments, the CR2-FH fusion protein includes a signal peptide sequence, preferably located at the N-terminus, and more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17.

[0028] 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.

[0029] In another aspect, this disclosure provides a polynucleotide encoding the CR2-FH fusion protein described herein. In some embodiments, the polynucleotide sequence encoding the first four N-terminal short homologous repeat (SCR) domains of the FH comprises the sequence shown in SEQ ID NO: 19, 28, or 29. In some embodiments, the polynucleotide sequence encoding the eighth N-terminal SCR domain of the FH comprises the sequence shown in SEQ ID NO: 20. In some embodiments, the polynucleotide sequence encoding the nineteenth to twentieth N-terminal SCR domains of the FH comprises the sequence shown in SEQ ID NO: 21. In some embodiments, the polynucleotide sequence encoding the eighteenth to twentieth N-terminal SCR domains of the FH comprises the sequence shown in SEQ ID NO: 24. In some embodiments, the polynucleotide sequence encoding the CR2 portion comprises the sequence shown in SEQ ID NO: 18. In some embodiments, the eighteenth N-terminal SCR domain of the FH comprises the sequence shown in SEQ ID NO: 57. In some embodiments, the twentieth SCR domain encoding the FH at the N-terminus comprises the sequence shown in SEQ ID NO: 58. In some embodiments, the seventh SCR domain at the N-terminus of the FH comprises the amino acid sequence shown in SEQ ID NO: 61.

[0030] In some embodiments, the nucleotide sequence encoding the CR2-FH fusion protein comprises the sequences 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.

[0031] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein includes 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.

[0032] 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.

[0033] In another aspect, this disclosure provides a vector encoding the polynucleotides described herein. In some embodiments, the vector is selected from at least one of adeno-associated virus (AAV) vectors, adenovirus vectors, RNA virus vectors, lentivirus vectors, and vaccinia virus vectors.

[0034] In another aspect, this disclosure provides a host cell that contains polynucleotides as described in this disclosure or a vector as described in this disclosure.

[0035] In another aspect, this disclosure provides AAV particles that comprise the AAV carrier as described in this disclosure.

[0036] In another aspect, this disclosure provides a pharmaceutical composition comprising at least one of the following: the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, and the AAV particles described in this disclosure.

[0037] And pharmaceutically acceptable carriers.

[0038] In some embodiments, the composition is suitable for intraocular, intravenous, intra-arterial, subcutaneous, intratracheal, or inhalation administration.

[0039] In another aspect, this disclosure provides the use of the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, the AAV particles described in this disclosure, or the pharmaceutical composition described in this disclosure in the preparation of a medicament for treating complement bypass-related diseases in a subject.

[0040] This disclosure provides a method for treating complement bypass-related disease in desired subjects, wherein an effective amount of the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, the AAV particles described in this disclosure, or the pharmaceutical composition described in this disclosure is administered.

[0041] In some implementations, the complement bypass-related disease is an inflammatory disease or an autoimmune disease.

[0042] In some embodiments, the complement bypass disease is age-related macular degeneration, preferably dry age-related macular degeneration.

[0043] In some implementations, the complement bypass-related disease is a symptom of microangiopathic hemolytic anemia, thrombocytopenia, or acute renal failure.

[0044] In some embodiments, the complement bypass-related diseases are selected from macular degeneration, ischemia-reperfusion, organ transplant rejection, drusen-related diseases, pregnancy-related diseases, adverse drug reactions, and complications after cardiopulmonary bypass.

[0045] In some embodiments, the complement bypass-related diseases are 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 apheresis, leukocyte apheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiation-induced allergic reactions. Attached Figure Description

[0046] Figure 1 The structural schematic diagrams of XMDC025, XMDC026, XMDC029, CR2-FH(1-4) and CR2-FH(1-5) are shown.

[0047] Figure 2 AE shows the carrier information for ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4) and ssAAV-CR2-FH(1-5).

[0048] Figure 3 AB showed 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 outer nuclear layer of the retina in PEG-400-induced dry AMD model mice.

[0049] Figure 4AB showed the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5) on the nuclear density of the outer nuclear layer of the retina in PEG-400-induced dry AMD model mice.

[0050] Figure 5 AB shows HE-stained images of the retina of PEG-400-induced dry AMD model mice after injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5).

[0051] Figure 6 AB showed 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 cone cells in PEG-400-induced dry AMD model mice.

[0052] Figure 7 AB shows immunofluorescence staining images of cone cells in PEG-400-induced dry AMD model mice after injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5).

[0053] Figure 8 AB showed the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) on the extracellular segment thickness of the retinal rod cells in PEG-400-induced dry AMD model mice.

[0054] Figure 9 AB shows immunofluorescence staining images of retinal rod cells in PEG-400-induced dry AMD model mice by AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5).

[0055] Figures 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 of PEG-400-induced dry AMD model mice.

[0056] Figures 11A-B show F-actin staining images of RPE cells from PEG-400-induced dry AMD model mice after injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5).

[0057] Figure 12 shows the structural schematic diagrams of XMDC061, XMDC062, XMDC025-(CR2-R36A K41A K67A), XMDC026-(CR2-R36A K41A K67A), XMDC029-(CR2-R36A K41A K67A), XMDC061-(CR2-R36A K41A K67A), and XMDC062-(CR2-R36A K41A K67A).

[0058] Figures 13A-G show the carrier information for ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36A K41AK67A), 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).

[0059] 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) carrier.

[0060] 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 outer nuclear layer thickness of the retina in PEG-400-induced dry AMD model mice.

[0061] 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 of the outer nuclear layer of the retina in PEG-400-induced dry AMD model mice.

[0062] Figure 17 shows HE staining images of the retina of PEG-400-induced dry AMD model mice after injection of 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-R36AK41A K67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5).

[0063] 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 cone cells in PEG-400-induced dry AMD model mice.

[0064] Figure 19 shows immunofluorescence staining images of cone cells in PEG-400-induced dry AMD model mice after injection of 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-R36AK41A K67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5).

[0065] 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 extracellular segment thickness of the retinal rod cells in PEG-400-induced dry AMD model mice.

[0066] Figure 21 shows 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).

[0067] 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 of PEG-400-induced dry AMD model mice.

[0068] Figure 23 shows F-actin staining images of RPE cells from PEG-400-induced dry AMD model mice after injection of 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-R36AK41A K67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5). Invention Details

[0070] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this 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 this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0071] This disclosure provides, in one aspect, a complement receptor 2 (CR2)-factor H (FH) fusion protein (hereinafter referred to as CR2-FH fusion protein), comprising: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion contains the first four N-terminal SCR domains of CR2 or variants thereof; and the FH portion contains the first four N-terminal short homologous repeat (SCR) domains of FH. In some embodiments, a separate 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 by an amino acid linker sequence. In some embodiments, the first four N-terminal short homologous repeat (SCR) domains of the FH contain the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the CR2 portion contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46. In some embodiments, the CR2 portion and the FH portion are connected by a connector sequence, preferably, the connector sequence includes (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0072] As used herein, a "fusion protein" refers to two or more peptides, polypeptides, or proteins that are operatively linked to each other. In some embodiments, the CR2 and FH moieties in a CR2-FH fusion protein are directly fused together. In some embodiments, the CR2 and FH moieties 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 "native" linker sequences found between different domains of complement factors, also referred to as endogenous linker sequences. The order of the CR2 and FH moieties in a fusion protein can vary. For example, in some embodiments, the C-terminus of the CR2 moieties is fused (directly or indirectly) to the N-terminus of the FH moieties 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.

[0073] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion contains the first four N-terminal SCR domains of CR2; the FH portion comprises two or more FH fragments, each containing the first four N-terminal SCR domains of FH; preferably, the two or more FH fragments are linked by a linker sequence; more preferably, the linker sequence comprises, for example, (G4S). n The sequences shown are and / or endogenous sequences, where n is an integer greater than 0. In some embodiments, the first four N-terminal short homologous repeat (SCR) domains of the FH comprise the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 46.

[0074] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing 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 homologous repeat (SCR) domains of FH comprise the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence shown 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 shown in SEQ ID NO: 3. In some embodiments, the nineteenth to twentieth N-terminal SCR domains of FH comprise the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the CR2 portion and the FH portion are connected by a connector sequence, preferably, the connector sequence includes, for example, (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0075] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing 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 homologous repeat (SCR) domains of the FH comprise the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the CR2 portion comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the eighteenth to twentieth N-terminal SCR domains of the FH comprise the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the CR2 portion and the FH portion are linked by a linker sequence, preferably, the linker sequence comprises (G4S) n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0076] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequences shown 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 eighteenth N-terminal SCR domain of FH (e.g., FH(SCR18)), and the twentieth N-terminal SCR domain of FH (e.g., FH(SCR20)). In some embodiments, the eighteenth N-terminal SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the twentieth N-terminal SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the CR2 portion and the FH portion are connected by a connector sequence, preferably, the connector sequence includes, for example, (G4S). n The sequence and / or endogenous connection sequence shown, where n is an integer greater than 0.

[0077] In some embodiments, the provided CR2-FH fusion protein comprises: a) a CR2 portion containing a CR2 fragment, and b) an FH portion containing an FH fragment, wherein the CR2 portion comprises the first four N-terminal SCR domains of CR2 or variants thereof (e.g., CR2(SCR1-SCR4) or variants thereof, such as the amino acid sequence shown 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 seventh N-terminal SCR domain of FH (e.g., FH(SCR7)). In some embodiments, the seventh N-terminal SCR domain of FH comprises the amino acid sequence shown in SEQ ID NO: 43.

[0078] In some implementations, the connector sequence includes (G4S). n The sequence shown is the FH endogenous connection sequence, where n is an integer greater than 0.

[0079] In some implementations, the domains are connected by a connector sequence, the connector sequence comprising (G4S). n The sequence shown is the FH endogenous connection sequence, where n is an integer greater than 0.

[0080] 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, or an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 10, wherein the CR2-FH fusion protein has a dual function of binding to the CR2 ligand and inhibiting complement activation in the bypass pathway. The CR2-FH fusion protein can bind to the CR2 ligand with a binding affinity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the CR2 protein. The 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 also inhibits complement activation via the alternative pathway, possessing complement inhibitory activity of approximately 50%, 60%, 70%, 80%, 90%, or 100% or higher of the complement inhibitory activity of the FH protein.

[0081] In some embodiments, an isolated CR2-FH fusion protein is provided. In some embodiments, the CR2-FH fusion protein forms a dimer or a multimer.

[0082] In some embodiments, the CR2-FH fusion protein includes a signal peptide sequence, preferably located at the N-terminus, and more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17.

[0083] In some embodiments, the CR2-FH fusion protein comprises an amino acid sequence as 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, or an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 11.

[0084] CR2 part

[0085] The CR2 section described in this article includes CR2 or fragments 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 of CR2 are iC3b, C3dg, and C3d, which are fragments of C3. C3 cleavage initially leads to the production and deposition of C3b on the surface of activated cells, and 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, especially when deposited on the host surface containing complement activating regulators. Even in the absence of membrane-bound complement regulators, large 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, the C3 ligands of CR2 are relatively long-lived once produced and are present in high concentrations at complement activation sites. CR2 can therefore serve as an efficient targeting vector to deliver molecules to complement activation sites. CR2 contains an extracellular portion having 15 or 16 repeating units called short homologous repeat (SCR domains). The SCR domains have a typical framework of highly conserved residues, including four cysteine ​​residues, two proline residues, one tryptophan residue, and several other partially conserved glycine residues and hydrophobic residues. The SCR1-4 domains are located at amino acids 23-271 of the human CR2 protein sequence. In some embodiments of this 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 shown in SEQ ID NO: 1. Variants of the CR2 portion may also contain mutation sites, and variants of the CR2 portion comprise the amino acid sequence shown in SEQ ID NO: 46.

[0086] H-factor part (FH part)

[0087] The FH portion of the CR2-FH fusion protein described herein comprises FH or fragments thereof. Complement factor H (FH) is a single-chain plasma glycoprotein. This protein consists of 20 repeating SCR domains of approximately 60 amino acids arranged in a continuous 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 hydrolytic inactivation of C3b. In the presence of factor H, C3b hydrolysis 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 this disclosure, the FH portion comprises two or more FH fragments, each FH fragment containing the first four N-terminal SCR domains of FH. Preferably, the two or more FH fragments are linked by a linker sequence, more preferably, the linker sequence is such as (G4S). n As shown, n is an integer greater than 0. In some embodiments, the first four N-terminal short homologous repeat (SCR) domains of the FH comprise the amino acid sequence shown in SEQ ID NO: 2. In some embodiments of this disclosure, the FH portion comprises the first four N-terminal SCR domains of the FH, the eighth N-terminal SCR domain of the FH, and the nineteenth to twentieth N-terminal SCR domains of the FH. In some embodiments of this disclosure, the FH portion comprises the first four N-terminal SCR domains of the FH and the eighteenth to twentieth N-terminal SCR domains of the FH. In some embodiments, the FH is wild-type FH. In some embodiments, the FH portion is a fragment of wild-type FH or several fragments directly or indirectly linked together.

[0088] In another aspect, this disclosure provides a polynucleotide encoding the CR2-FH fusion protein described herein. In some embodiments, the polynucleotide sequence encoding the first four N-terminal short homologous repeat (SCR) domains of the FH comprises the sequence shown in SEQ ID NO: 19, 28, or 29. In some embodiments, the polynucleotide sequence encoding the eighth N-terminal SCR domain of the FH comprises the sequence shown in SEQ ID NO: 20. In some embodiments, the polynucleotide sequence encoding the nineteenth to twentieth N-terminal SCR domains of the FH comprises the sequence shown in SEQ ID NO: 21. In some embodiments, the polynucleotide sequence encoding the eighteenth to twentieth N-terminal SCR domains of the FH comprises the sequence shown in SEQ ID NO: 24. In some embodiments, the polynucleotide sequence encoding the CR2 portion comprises the sequence shown in SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the eighteenth N-terminal SCR domain of the FH comprises the polynucleotide sequence shown in SEQ ID NO: 57. In some embodiments, the twentieth SCR domain encoding the FH at the N-terminus comprises a polynucleotide sequence as shown in SEQ ID NO: 58. In some embodiments, the seventh SCR domain at the N-terminus of the FH comprises a polynucleotide sequence as shown in SEQ ID NO: 61.

[0089] 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, or a sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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.

[0090] In some implementations, the polynucleotide sequence is DNA or RNA, such as an mRNA sequence.

[0091] In some embodiments, the polynucleotide sequence encoding the CR2-FH fusion protein includes 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.

[0092] 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, or a sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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.

[0093] In another aspect, this disclosure provides a vector encoding the polynucleotides described herein. In some embodiments, the vector is selected from at least one of adeno-associated virus (AAV) vectors, adenovirus vectors, RNA virus vectors, lentivirus vectors, and vaccinia virus vectors.

[0094] In another aspect, this disclosure provides a host cell that contains polynucleotides as described in this disclosure or a vector as described in this disclosure.

[0095] In another aspect, this disclosure provides AAV particles comprising the AAV vector as described herein. In some embodiments, the AAV particles are produced via 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 viral functions.

[0096] 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 includes one or more AAV inverted terminal repeat (ITR) sequences flanking it. In some embodiments, the heterologous nucleic acid flanking it is two AAVITRs. In some embodiments, the AAV ITR is an ITR of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the AAVITR is an AAV2 ITR. In some embodiments, the ITR and capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the ITR and capsid are derived from AAV2. In other embodiments, the ITR and capsid of the AAV viral particles are derived from different AAV serotypes.

[0097] In some embodiments, the AAV vector contains one or more promoters, enhancers, or polyadenylation signals.

[0098] In another aspect, this disclosure provides a pharmaceutical composition comprising at least one of the following: the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, and the AAV particles described in this disclosure.

[0099] And pharmaceutically acceptable carriers.

[0100] In some embodiments, the composition is suitable for intraocular, intravenous, intra-arterial, subcutaneous, intratracheal, or inhalation administration.

[0101] In another aspect, this disclosure provides the use of the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, the AAV particles described in this disclosure, or the pharmaceutical composition described in this disclosure in the preparation of a medicament for treating complement bypass-related diseases in a subject.

[0102] This disclosure provides a method for treating complement bypass-related disease in desired subjects, wherein an effective amount of the CR2-FH fusion protein described in this disclosure, the polynucleotide described in this disclosure, the carrier described in this disclosure, the host cell described in this disclosure, the AAV particles described in this disclosure, or the pharmaceutical composition described in this disclosure is administered.

[0103] In some implementations, the complement bypass-related disease is an inflammatory disease or an autoimmune disease.

[0104] In some embodiments, the complement bypass disease is age-related macular degeneration, preferably dry age-related macular degeneration.

[0105] In some implementations, the complement bypass-related disease is a symptom of microangiopathic hemolytic anemia, thrombocytopenia, or acute renal failure.

[0106] In some embodiments, the complement bypass-related diseases are selected from macular degeneration, ischemia-reperfusion, organ transplant rejection, drusen-related diseases, pregnancy-related diseases, adverse drug reactions, and complications after cardiopulmonary bypass.

[0107] In some embodiments, the complement bypass-related diseases are 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 apheresis, leukocyte apheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal LDL precipitation (HELP), and radiation-induced allergic reactions.

[0108] In some implementations, the subject is a mammal, preferably a human.

[0109] This disclosure will now be described in further detail. However, the implementation of this disclosure is not limited to the embodiments described below.

[0110] Example 1: Construction of AAV plasmid vector expressing CR2 and FH fragment genes

[0111] Based on the SCR1-SCR4 amino acid sequences (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequences (SEQ ID NO: 2), SCR8 amino acid sequences (SEQ ID NO: 3), and SCR19-SCR20 amino acid sequences (SEQ ID NO: 4) of FH (Gene ID: 3075) published on NCBI, the secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR8)+linker+FH(SCR19-SCR20)-+linker. Nucleotide sequences were designed according to human codon preferences, and BamH was introduced at the 5' end. An EcoR V restriction site was introduced at the 3' end, and the gene was named XMDC025 (the whole gene was synthesized by Sangon Biotech Shanghai Co., Ltd.). Its structural diagram is shown below. Figure 1 .

[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) 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 linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure 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 preferences, and a BamH I restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end. This sequence was named XMDC026 (the complete gene synthesized by Sangon Biotech Shanghai Co., Ltd.), and its structural diagram is shown below. Figure 1 .

[0113] Based on the SCR1-SCR4 amino acid sequences of CR2 (Gene ID: 1380) (SEQ ID NO: 1) and 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 linked with (G4S)2 and an endogenous linker to form an open reading frame with the structure 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 preferences, 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 resulting gene was named XMDC029 (the complete gene synthesized by Suzhou Genewise Biotechnology Co., Ltd.). Its structural diagram is shown below. Figure 1 .

[0114] Based on the SCR1-SCR4 amino acid sequences of CR2 (Gene ID: 1380) (SEQ ID NO: 1) and 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 linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker-. The nucleotide sequence was designed according to human codon preferences, and a BamHI restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end, named CR2-FH(1-4) (the whole gene was synthesized by Sangon Biotech Shanghai Co., Ltd.). Its structural diagram is shown below. Figure 1 .

[0115] Based on the SCR1-SCR4 amino acid sequence of CR2 (Gene ID: 1380) (SEQ ID NO: 1) and the SCR1-SCR5 amino acid sequence of FH (Gene ID: 3075) (SEQ ID NO: 14), the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR5)+linker-. The nucleotide sequence was designed according to human codon preferences, and a BamHI restriction site was introduced at the 5' end and an EcoR V restriction site was introduced at the 3' end, named CR2-FH(1-5) (the whole gene was synthesized by Sangon Biotech Shanghai Co., Ltd.). Its structural diagram is shown below. Figure 1 .

[0116] XMDC025, XMDC026, XMDC029, CR2-FH(1-4), CR2-FH(1-5), and ssAAV plasmids were digested with BamH I / EcoR V. Following routine molecular biology procedures such as ligation, transformation, and cloning screening, vectors ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4), and ssAAV-CR2-FH(1-5) were constructed. Vector information is available in [link to relevant documentation]. Figure 2 High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for later use.

[0117] In the above structure, the intrinsic linker is an intrinsic connection sequence within the CR2 or FH structure. Therefore, those skilled in the art should understand that the linker sequence in the structure is determined based on the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0118] Table 1. Sequence Information

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Example 2: Preparation and Identification of Recombinant AAV Virus

[0129] Recombinant AAV virus was prepared using a three-plasmid packaging system. Phelper plasmids, AAV Cap and Rep protein expression plasmids, and target expression vector plasmids (ssAAV-XMDC025, ssAAV-XMDC026, ssAAV-XMDC029, ssAAV-CR2-FH(1-4), ssAAV-CR2-FH(1-5)) were mixed with PEI transfection agent at a mass ratio of 2:1:1 to form a transfection complex. This complex was then transfected into HEK293T cells to package AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) viruses. The supernatant was collected twice, on days 3 and 7 post-transfection, yielding AAV virus particles containing the target gene. AAV virus was purified by density gradient centrifugation (Beckman ultracentrifuge) using different gradients of iodixanol (15%, 25%, 40%, and 60%). The purified AAV virus was then characterized by transmission electron microscopy (TEM) and its titer was quantified by qPCR.

[0130] Example 3: PEG-400-induced dry AMD mouse model and administration of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) by injection

[0131] A dry AMD mouse model was induced using polyethylene glycol (PEG). Mice treated with PEG exhibited retinal pathological changes similar to the clinicopathological features of dry AMD, such as retinal structural damage, RPE cell injury, and photoreceptor loss. The specific method is as follows:

[0132] Seventy SPF-grade 4-week-old male C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing approximately 14g, were housed in an alternating 12-hour light and dark environment according to the mouse's circadian rhythm.

[0133] Because it takes time for the expression of the target gene to reach a stable level after AAV injection, intravitreal injections were performed bilaterally on day 1, and subretinal injections of PEG-400 were performed bilaterally on day 22 to establish the retinal model. On day 27, the eyeballs were enucleated for frozen sectioning and staining to observe the retinal and RPE structures. Treatment details are shown in Table 2.

[0134] Table 2. Bilateral intravitreal injection administration of drugs to mice on day 1

[0135]

[0136] The specific operating steps are as follows:

[0137] Intravitreal injection administration: Dilate the pupils of both eyes with 5% mydriatic solution, and anesthetize the mice with an intraperitoneal injection of 10 mL / kg of 5% chloral hydrate (Sangoku). Place the anesthetized animal on its side on the operating table. Select an injection site 1-2 mm posterior to the limbus of the temporal or nasal superior angle of the eye (syringe: Hamilton needle, 7632-01), taking care to avoid damage to the posterior capsule of the lens and other retinal areas. Insert the needle into the vitreous cavity, inject the solution, pause for 10 seconds after injection, slowly withdraw the needle, apply erythromycin eye ointment, and return the mouse to its cage.

[0138] Example 4: Evaluation of the effects of hematoxylin and eosin (HE) staining on the thickness and nuclear density of the outer nuclear layer of the retina in PEG-400-induced dry AMD model mice: 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.

[0139] In Example 3, the mice had their eyes removed, and one eye was fixed overnight in 4% paraformaldehyde (BioSharp) at 4°C. After washing four times with PBS (BioSharp), the cornea and lens were separated, and the retina, choroid, sclera, etc., were retained in the eye cups. The eyes were then dehydrated with 30% sucrose (BioSharp) until they settled. After that, 10 μm thick frozen sections were prepared 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 ImageJ.

[0140] See results Figure 3 , Figure 4 , Figure 5Compared with the control group, the thickness and nuclear density of the outer nuclear layer of the retina in the model group mice 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) could significantly increase the thickness and nuclear density of the outer nuclear layer and improve the structural damage of the retina. Injection of AAV-CR2-FH (1-5) could only significantly increase the thickness of the outer nuclear layer. AAV-XMDC029 had a better effect on improving retinal thickness than AAV-CR2-FH (1-4), and a better effect on improving retinal nuclear density than AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0141] Example 5: Immunofluorescence staining assessment of 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.

[0142] The frozen sections from Example 4 were air-dried at room temperature in a slide holder. The tissue on the slides was circled using an immunohistochemical pen (Vectorlabs). Poly-L-lysine slides (Style) were placed in a humidified chamber, and the tissue was washed with PBS and incubated at room temperature for 5 minutes. The PBS was discarded, and this washing process was repeated 5 times, 10 minutes each time. 500 μL of blocking buffer (5% goat serum (Beyotime) + 0.5% Triton 100 (Sangon Biotech)) was added per slide, and the slides were incubated at room temperature for 45 minutes. The blocking buffer was discarded, and 200 μL of a primary antibody mixture (Cone Arresting-1 (EMD Millipore Corp.) and Rodopsin-1 (Santa Cruz)) was added per slide. The slides were incubated overnight at 4°C. The next day, the slides were washed 5 times with PBS at room temperature for 5 minutes each time. A secondary antibody mixture (Anti-rabbit IgG Alexa Fluor) was added. Counterstaining was performed using 555 (CellSignaling) and Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen) diluted 1:500, and the slides were incubated in a humidified chamber at room temperature in the dark for 2.5 hours. After this step, the slides were protected from light. The secondary antibody was removed, and the slides were washed five times with PBS at room temperature for 5 minutes each time. DAPI staining solution (Sigma) was added, 50 μL / slide, and the slides were incubated at room temperature for 10 minutes, followed by three washes with PBS for 5 minutes each time. The slides were then mounted and observed and photographed under a fluorescence microscope.

[0143] See results Figure 6 , Figure 7 , Figure 8 and Figure 9 Compared with the control group, the number of retinal cone cells in the model group was reduced, the thickness of the outer segment of rod cells was narrowed, and 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 of cone cells and rod cells. AAV-XMDC029 and AAV-CR2-FH(1-4) had a better effect on improving cone cell damage than AAV-CR2-FH(1-5).

[0144] Example 6: F-actin staining to assess the effects of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, AAV-CR2-FH(1-4), and AAV-CR2-FH(1-5) on PEG-400-induced stem AMD model mouse RPE cells.

[0145] The other eye removed from the mouse in Example 3 was fixed overnight in 4% paraformaldehyde at 4°C. The cornea, lens, muscle, and retina were removed. The sclera, choroid, and RPE complex were fixed in 4% paraformaldehyde for 2 hours at room temperature, washed three times with PBS for 5 minutes each time. The sclera, choroid, and RPE complex were then blocked in blocking solution (5% goat serum + 0.5% Triton X-100) at room temperature for 1 hour. 594 phalloidin (Jackson ImmunoResearch) (1:40 methanol stock solution) was stained on a shaker at room temperature for 40 minutes, then washed 6 times with PBS for 5 minutes each time on the shaker. It was then stained with DAPI at room temperature for 30 minutes, and then washed 3 times with PBS for 5 minutes each time. The sclera, choroid, and RPE complex were transferred to a glass slide, cut into about 8 pieces, mounted, and observed and photographed under a fluorescence microscope.

[0146] The results are shown in Figures 10 and 11. Compared with the control group, the RPE cells in the model group were significantly larger and significantly damaged. Compared with the model group, injection of AAV-XMDC025, AAV-XMDC026, AAV-XMDC029, and AAV-CR2-FH (1-4) significantly reduced the RPE cell area and improved RPE cell damage. Injection of AAV-CR2-FH (1-5) had no effect on improving RPE cell damage. The effect of AAV-XMDC029 on improving RPE cell damage was better than that of AAV-CR2-FH (1-4) and AAV-CR2-FH (1-5).

[0147] Based on HE staining, immunofluorescence staining, and F-actin staining results, injection of AAV-XMDC029 showed better improvement in retinal outer nuclear layer nuclear density and RPE cell damage in PEG-400-induced dry AMD mouse models than AAV-CR2-FH(1-4) and AAV-CR2-FH(1-5), better improvement in outer nuclear layer thickness than AAV-CR2-FH(1-4), and better improvement in cone cells than AAV-CR2-FH(1-5).

[0148] Example 7: Construction of other plasmid vectors

[0149] Option 1

[0150] Based on the SCR1-SCR4 amino acid sequences (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequences (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 secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker to form an open reading frame with the structure 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 preferences, and a BamH I restriction site was introduced at the 5' end and an EcoR restriction site was introduced at the 3' end. The V restriction site was named XMDC061 (the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0151] Fang An 2

[0152] Based on the SCR1-SCR4 amino acid sequences (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR4 amino acid sequences (SEQ ID NO: 2) and SCR7 amino acid sequences (SEQ ID NO: 43) of FH (Gene ID: 3075) published on NCBI, the secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure 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. Nucleotide sequences were designed according to human codon preferences, and a BamH I restriction site was introduced at the 5' end and an EcoR restriction site was introduced at the 3' end. The V restriction site was named XMDC062 (the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0153] Option 3

[0154] Based on the amino acid sequences 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), SCR8 (SEQ ID NO: 3), and SCR19-SCR20 (SEQ ID NO: 4) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 4) was added to the N-terminus of the composition. NO: 17), connected by (G4S)2 and an endogenous linker, formed an open reading frame with the structure 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. It was named XMDC025-(CR2-R36A K41A K67A) (the whole gene was synthesized by Sangon Biotech Shanghai Co., Ltd.). Its structural diagram is shown in Figure 12.

[0155] Option 4

[0156] Based on the amino acid sequences of the R36A K41A K67A mutant of SCR1-SCR4 from CR2 (Gene ID: 1380) (SEQ ID NO: 46) and the amino acid sequences of SCR1-SCR4 from FH (Gene ID: 3075) (SEQ ID NO: 2) and SCR18-SCR20 (SEQ ID NO: 7), the secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker, forming an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR18-SCR20)+linker. Nucleotide sequences were designed according to human codon preferences, and a BamH I restriction site was introduced at the 5' end and an EcoR restriction site was introduced at the 3' end. The V restriction site was named XMDC026-(CR2-R36A K41A K67A) (the whole gene was synthesized by Sangon Biotech Shanghai Co., Ltd.), and its structural diagram is shown in Figure 12.

[0157] Option 5

[0158] Based on the amino acid sequences of the R36A K41A K67A mutant of SCR1-SCR4 from CR2 (Gene ID: 1380) (SEQ ID NO: 46) and the amino acid sequences of SCR1-SCR4 from FH (Gene ID: 3075) (SEQ ID NO: 2), the secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition. This was then linked by (G4S)2 and an endogenous linker, forming an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+(G4S)2+linker+FH(SCR1-SCR4)+linker. A nucleotide sequence was designed according to human codon preferences, with a BamHI restriction site introduced at the 5' end and an EcoR V restriction site introduced at the 3' end, named XMDC029-(CR2-R36AK41A). K67A (the whole genome synthesized by Suzhou Genewise Biotechnology Co., Ltd.), its structural diagram is shown in Figure 12.

[0159] Option 6

[0160] Based on the amino acid sequences 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 secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker to form an open reading frame with the structure CD5-sp+linker+CR2(SCR1-SCR4)+(G4S)2+linker+FH(SCR1-SCR4)+linker+FH(SCR18)+linker+FH(SCR20)+linker. Nucleotide sequences were designed according to human codon preferences, and BamH was introduced at the 5' end. The I restriction site was introduced at the 3' end with an EcoR V restriction site, named XMDC061-(CR2-R36AK41A K67A) (the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 12.

[0161] Option 7

[0162] Based on the amino acid sequences 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) and SCR7 (SEQ ID NO: 43) of FH (Gene ID: 3075) published on NCBI, the secretion signal peptide CD5-sp amino acid sequence (SEQ ID NO: 43) was added to the N-terminus of the composition. NO: 17), connected by (G4S)2 and an endogenous linker, formed an open reading frame with the structure 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 BamHI 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 K41AK67A) (the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.). Its structural diagram is shown in Figure 12.

[0163] In the above structure, the intrinsic linker is an intrinsic connection sequence within the CR2 or FH structure. Therefore, those skilled in the art should understand that the linker sequence in the structure is determined based on the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0164] XMDC061, XMDC062, XMDC025-(CR2-R36A K41A K67A), XMDC026-(CR2-R36A K41A K67A), XMDC029-(CR2-R36A K41A K67A), XMDC061-(CR2-R36AK41A K67A), XMDC062-(CR2-R36A K41A K67A), and ssAAV plasmids were digested with BamHI / EcoRV. Following routine molecular biology procedures such as ligation, transformation, and cloning screening, plasmids ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36A K41A K67A), and ssAAV-XMDC026-(CR2-R36AK41A) were constructed. The vectors ssAAV-XMDC029-(CR2-R36AK41A K67A), ssAAV-XMDC061-(CR2-R36AK41A K67A), and ssAAV-XMDC062-(CR2-R36A K41AK67A) are shown in Figure 13. High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for later use.

[0165] Option 8

[0166] Based on the SCR1-SCR4 amino acid sequences (SEQ ID NO: 1) of CR2 (Gene ID: 1380) and the SCR1-SCR5 amino acid sequences (SEQ ID NO: 14) of FH (Gene ID: 3075) published on NCBI, the secretory signal peptide CD5-sp amino acid sequence (SEQ ID NO: 17) was added to the N-terminus of the composition and linked with (G4S)2 and an endogenous linker to form an open reading frame with the structure 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 preferences, and a BamH I restriction site was introduced at the 5' end and an EcoR restriction site was introduced at the 3' end. The V restriction site was named CR2-FH(SCR1-5+1-5) (the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.), and its structural diagram is shown in Figure 14.

[0167] In the above structure, the intrinsic linker is an intrinsic connection sequence within the CR2 or FH structure. Therefore, those skilled in the art should understand that the linker sequence in the structure is determined based on the different CR2 or FH structure sequences connected to its C-terminus or N-terminus.

[0168] The CR2-FH(SCR1-5+1-5) and ssAAV plasmids were double-digested with BamHI / EcoR V. Following standard molecular biology procedures such as ligation, transformation, and cloning screening, the ssAAV-CR2-FH(SCR1-5+1-5) vector was constructed. Vector information is shown in Figure 14. High-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for later use.

[0169] Table 3. Sequence Information

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Example 8: Preparation and identification of other recombinant AAV viruses

[0190] Recombinant AAV virus was prepared using a three-plasmid packaging system, including a helper plasmid, AAV Cap and Rep protein expression plasmids, and expression vector target plasmids (ssAAV-XMDC061, ssAAV-XMDC062, ssAAV-XMDC025-(CR2-R36AK41A K67A), ssAAV-XMDC026-(CR2-R36A K41A K67A), ssAAV-XMDC029-(CR2-R36A K41AK67A), ssAAV-XMDC061-(CR2-R36A K41A K67A), and ssAAV-XMDC062-(CR2-R36AK41A). A transfection complex was formed by mixing 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) at a mass ratio of 2:1:1 with PEI transfection agent, and then transfected into HEK293T cells to package the viruses AAV-XMDC061, AAV-XMDC062-(CR2-R36A K41AK67A), and AAV-CR2-FH(SCR1-5+1-5). Supernatant was collected twice, on days 3 and 7 post-transfection, yielding AAV viral particles containing the target gene. The purified AAV virus was obtained by density gradient centrifugation (Beckman ultracentrifuge) using different concentrations of iodixanol (15%, 25%, 40%, and 60%). The purified AAV virus was then characterized for quality assessment using transmission electron microscopy and its titer was quantified by qPCR.

[0191] Example 9: PEG-400-induced dry AMD mouse model and 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-R36AK41AK67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5) by injection

[0192] The specific method is as follows:

[0193] Eighty-eight SPF-grade male C57BL / 6J mice, aged 4-5 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing approximately 14-18g, were housed in an alternating 12-hour light and dark environment according to the mouse's circadian rhythm.

[0194] Because it takes time for the expression of the target gene to reach a stable level after AAV injection, intravitreal injections were performed in both eyes on day 1, and subretinal injections of PEG-400 were performed in both eyes on day 22 to establish the retinal model. On day 27, the eyeballs were enucleated for frozen sectioning and staining to observe the retinal and RPE structures. Treatment details are shown in Table 4.

[0195] Table 4. Dosing Regimen

[0196]

[0197] The specific injection procedure is the same as in Example 3.

[0198] Example 10: Evaluation of the effects of hematoxylin and eosin (HE) staining on the outer nuclear layer thickness and nuclear density of the retina in PEG-400-induced dry AMD model mice: 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)

[0199] In Example 9, the mice had their eyes removed, and one eye was frozen sectioned and stained with hematoxylin and eosin (HE). After staining, the retinal sections were photographed and the thickness and nuclear density of the outer nuclear layer (ONL) were measured and analyzed using ImageJ. The specific operation steps were the same as in Example 4.

[0200] The results are shown in Figures 15, 16, and 17. Compared with the control group, the thickness and density of the outer nuclear layer of the retina in the model group mice 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-R36AK41A K67A), AAV-XMDC029, and AAV-CR2-FH(SCR1-5+1-5) all significantly increased the thickness and density of the outer nuclear layer and improved the retinal structural damage.

[0201] Example 11: Immunofluorescence staining assessment of the effects of AAV-XMDC061, AAV-XMDC062, AAV-XMDC025-(CR2-R36A K41A K67A), AAV-XMDC026-(CR2-R36A K41A K67A), AAV-XMDC(129-(CR2-R36A K41A K67A), AAV-XMDC061-(CR2-R36A K41A K67A), AAV-XMDC062-(CR2-R36A K41A K67A), AAV-XMDC(129), and AAV-CR2-FH(SCR1-5+1-5) on retinal photoreceptor cells in PEG-400-induced dry AMD model mice.

[0202] The frozen sections from Example 9 were subjected to immunofluorescence staining and observed and photographed under a fluorescence microscope. The specific operating steps were the same as in Example 5.

[0203] The results are shown in Figures 18, 19, 20 and 21. Compared with the control group, the number of retinal cone cells in the model group was reduced, the thickness of the outer segment of rod cells was narrowed, and there was significant damage to both cone and rod cells. Compared with the model group, injection of AAV-XMDC029 can significantly improve the damage caused by modeling and effectively protect the thickness of the outer segment of cone and rod cells.

[0204] Example 12: F-actin staining assessment of 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 PEG-400-induced stem AMD model mouse RPE cells.

[0205] The other eye of the mouse removed in Example 9 was stained with F-actin and observed and photographed under a fluorescence microscope. The specific operation steps were the same as in Example 6.

[0206] The results are shown in Figures 22 and 23. Compared with the control group, the RPE cells in the model group were significantly larger and 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) significantly reduced the RPE cell area and significantly improved RPE cell damage.

[0207] Based on 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), and AAV-XMDC062-(CR2-R36A K41A) were injected. K67A), AAV-XMDC029, and AAV-CR2-FH (SCR1-5+1-5) all significantly improved the damage to the outer nuclear layer structure of the retina and RPE cells induced by PEG-400 in a dry AMD mouse model. Among them, AAV-XMDC029 can also effectively improve the damage to the outer segment thickness of cone cells and rod cells.

[0208] Statistical analysis

[0209] Data processing and statistical analysis were performed using GraphPad Prism 8.0 software. The statistical significance level was set at 5% or p≤0.05. The mean and standard error (Mean±SEM) of each analytical indicator were calculated, and p≤0.05 was considered statistically significant.

[0210] This disclosure is not limited to the embodiments described above. Any modifications, alterations, substitutions, combinations, or simplifications made without departing from the spirit and principles of this disclosure are equivalent technical solutions and are included within the protection scope of this disclosure.

Claims

1. A Complement Receptor 2 (CR2)-Factor H (FH) fusion protein comprising: a) a CR2 portion containing the CR2 fragment, and b) an FH portion containing the FH fragment, wherein the CR2 portion and the FH portion are connected by a linker sequence. The CR2 portion consists of the first four N-terminal short common repeat (SCR) domains of CR2, and the FH portion consists of two FH segments, each consisting of the first four N-terminal SCR domains of an FH segment. The two FH segments are connected by a linker sequence. The amino acid sequence of the CR2-FH fusion protein is shown in SEQ ID NO:

10.

2. A Complement Receptor 2 (CR2)-Factor H (FH) fusion protein comprising: a) a CR2 portion containing the CR2 fragment, and b) an FH portion containing the FH fragment, wherein the CR2 portion and the FH portion are linked by a linker sequence, and the CR2-FH fusion protein further comprises a signal peptide sequence. The CR2 portion consists of the first four N-terminal SCR domains of CR2, and the FH portion consists of two FH segments, each consisting of the first four N-terminal SCR domains of an FH segment. The two FH segments are connected by a linker sequence. The amino acid sequence of the CR2-FH fusion protein is shown in SEQ ID NO:

11.

3. A polynucleotide encoding the CR2-FH fusion protein as described in claim 1 or 2.

4. The polynucleotide of claim 3, wherein the polynucleotide sequence encoding the CR2-FH fusion protein is the nucleotide sequence shown in SEQ ID NO: 30 or 31.

5. A carrier comprising the polynucleotide as described in claim 3 or 4.

6. The vector of claim 5, wherein the vector is an adeno-associated virus (AAV) vector.

7. The vector of claim 5, wherein the vector is selected from at least one of adenovirus vectors, RNA virus vectors, lentivirus vectors, and vaccinia virus vectors.

8. A host cell comprising the polynucleotide as described in claim 3 or 4 or the AAV vector as described in claim 6.

9. A host cell comprising the vector as described in claim 5 or 7.

10. AAV particles comprising the AAV carrier as described in claim 6.

11. A pharmaceutical composition comprising at least one of the following: the CR2-FH fusion protein as claimed in claim 1 or 2, the polynucleotide as claimed in claim 3 or 4, the AAV carrier as claimed in claim 6, the host cell as claimed in claim 8, and the AAV particles as claimed in claim 10. And pharmaceutically acceptable carriers.

12. A pharmaceutical composition comprising the carrier as described in claim 5 or 7, or the host cell as described in claim 9. And pharmaceutically acceptable carriers.

13. The pharmaceutical composition of claim 11 or 12, wherein the composition is suitable for intraocular, intravenous, intra-arterial, subcutaneous or inhalation administration.

14. The pharmaceutical composition of claim 11 or 12, wherein the composition is suitable for intratracheal administration.

15. Use of the CR2-FH fusion protein of claim 1 or 2, the polynucleotide of claim 3 or 4, the AAV vector of claim 6, the host cell of claim 8, the AAV particles of claim 10, or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating complement bypass-related diseases in a subject; The complement bypass disease mentioned above is dry age-related macular degeneration.

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