Protein co-expression vector system containing bidirectional promoter, and application thereof

Abstract

Provided is a protein co-expression vector system. The protein co-expression vector system comprises nucleic acids of two target genes and a bidirectional promoter, and further comprises the following structure: gene B-promoter B-enhancer-promoter A-gene A. The protein co-expression vector enables expression levels of different target genes to be relatively balanced, and provides a new strategy for multiple gene co-expression.

Description

A protein co-expression vector system containing a bidirectional promoter and its application Technical Field

[0001] This application relates to biotechnology, specifically to a protein co-expression vector system containing a bidirectional promoter and its applications. Background Technology

[0002] Gene therapy is an innovative medical approach that involves introducing normal genes into cells with genetic defects to correct or compensate for diseases caused by abnormal genes, thereby achieving the goal of treatment.

[0003] Vectors used for gene therapy can be divided into viral vectors and non-viral vectors. Among viral vectors, the most commonly used ones include adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors.

[0004] Adeno-associated virus (AAV) vectors have been widely used in gene therapy due to their advantages such as good safety, low immunogenicity, and stable expression. However, AAV vectors still have some limitations in gene therapy, such as low transduction efficiency, small vector capacity, weak targeting, and host immune response. These limitations affect the widespread application of AAV vectors. To overcome these limitations, researchers have developed a variety of optimization strategies, including improving vector transduction efficiency, reducing the host's immune response, improving targeting, expanding vector capacity, and dual-target or multi-target therapy.

[0005] Dual-target / multi-target adeno-associated virus (AAV) vector systems are a gene therapy strategy that can simultaneously intervene on two therapeutic targets. Through AAV vector-mediated gene expression, multiple molecular pathways of the disease are affected simultaneously, thereby enhancing the therapeutic effect.

[0006] A core issue in dual-target / multi-target adeno-associated virus (AAV) vector systems involves the co-expression or balanced expression of two or more target proteins. Protein co-expression vector systems are a molecular biology technique that allows the simultaneous expression of multiple proteins or their encoding genes within the same host cell. Commonly used protein co-expression strategies include: 1) Multi-promoter expression vectors: Multiple independent transcription units are inserted into the same vector, each with its own promoter and ORF. Disadvantages include unbalanced gene expression levels, interference between promoters in some cell lines, and even gene rearrangement. The number of transcription units is also limited. 2) Splitting vectors: A single promoter is used to form a complete transcript. The primary transcript breaks at the splice signal to form independent secondary mRNAs, which are translated into different proteins. The splice signal mechanism is unclear and difficult to control, making this method less common. 3) Fusion proteins: Multiple proteins are sequentially linked and transcribed under the same promoter to form a complete transcript, which is then translated into a fusion protein. The disadvantage is that because the product is a fusion protein, it may affect the function of one of the proteins. 4) Insertion of protease cleavage sites. Furin is a highly conserved protease in eukaryotic cells, located on the Golgi apparatus, and specifically recognizes the Arg-Xaa-Lys / Arg-Arg site. Utilizing this property, this protease recognition site can be added to the protein-protein junction, allowing the fusion protein to be cleaved by Furin at the Golgi apparatus after translation. The drawback is that, because Furin is located on the Golgi apparatus, proteins processed by the endoplasmic reticulum and Golgi apparatus are typically secretory and membrane-bound proteins. A significant number of proteins exist in the cytoplasm and do not necessarily require processing by the endoplasmic reticulum and Golgi apparatus, greatly limiting the application of this method. 5) Adding IRES. Different ORFs linked by the same promoter are translated into multiple proteins via IRES. The drawback is that the translation efficiency upstream of the IRES is often higher than that downstream. The commonly used EMCV IRES length is approximately ~570 bp, which is relatively large and also limits the application of this method. 6) Self-cleaving polypeptide 2A. The 2A peptide has been found in various viruses, typically 18-22 amino acids in length. It encodes a highly conserved shared motif at its C-terminus (Asp-Val / Ile-Glu-X-Asn-Pro-Gly- / / -Pro), with the cleavage site located between Gly- / / -Pro. The cleavage mechanism involves the higher-order structure of 2A creating steric hindrance at the ribosomal peptidyl transferase center, preventing the formation of a peptide bond between Gly and Pro. However, downstream translation can still proceed via the ribosome, and the entire process does not require the participation of other proteases. Commonly used 2A sequences include FMDV, ERAV, PTV-1, and TaV.

[0007] Therefore, how to provide a co-expression vector system with a relatively balanced gene expression level and high expression efficiency has become an urgent problem to be solved in this field, which is of great significance for the large-scale production and clinical application of gene therapy drugs. Summary of the Invention

[0008] To provide a co-expression vector system with relatively balanced gene expression levels and high expression efficiency, this invention provides a protein co-expression vector system, which contains nucleic acids of two target genes and a bidirectional promoter, and has the following structure: gene B - promoter B - enhancer - promoter A - gene A.

[0009] In some embodiments, gene A and gene B may be the same or different. In some preferred embodiments, gene A and gene B are different.

[0010] In other implementations, promoter A and promoter B may be the same or different.

[0011] In some implementations, gene A or gene B encodes complement inhibitors or complement regulatory proteins, neurotrophic factors, anti-vascular endothelial growth factor (VEGF), endostatin, anti-angiogenic factors, etc.

[0012] In some preferred embodiments, the complement factors or complement regulatory proteins include complement factor I, complement factor H, complement factor C3, complement factor C5, complement factor B, complement factor D, complement regulatory protein CD59, etc. Complement factors are a group of proteins involved in the activation and regulation of the complement system, playing a crucial role in innate immunity. The complement system is activated through three pathways: the classical pathway, the alternative pathway, and the lectin pathway. These pathways ultimately converge on the activation of the C3 complement component, triggering a series of cascade reactions that produce various biological effects.

[0013] In some preferred embodiments, the neurotrophic factors include, but are not limited to, AADC (aromatic L-amino acid decarboxylase), GDNF (glial cell-derived neurotrophic factor), the NT family of neurotrophic factors, GBA1 (glucocerebrosidase), CNTF (ciliary neurotrophic factor), PEDF (human pigment epithelium-derived factor), and RdCVF (cone cell growth factor). Generally, neurotrophic substances and growth factors that regulate nerve cell survival are collectively referred to as neurotrophic factors.

[0014] The promoter comprises a promoter sequence or a functional fragment thereof. The promoter is specific to eukaryotic or mammalian cells. In this invention, promoter A or promoter B is selected from: β-actin promoter (CB), cytomegalovirus promoter (CMV), elongation factor 1α promoter (EF1α), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, artificially spliced ​​promoter (CASS), and pMNTC promoter. Preferably, the promoter comprises the CB promoter, CMV promoter, CAG promoter, or a truncated form thereof, such as miniCMV.

[0015] An "enhancer" is defined as a sequence that enhances promoter activity (i.e., increases the transcription rate of sequences downstream of the promoter). This sequence, unlike the promoter, lacks promoter activity and can generally function regardless of its position relative to the promoter (i.e., upstream or downstream). Enhancer elements are well known in the art, and non-limiting examples of enhancer elements (or portions thereof) that can be used in this invention include baculovirus enhancers and enhancer elements found in insect cells. Exemplary examples of enhancers include, but are not limited to, the CAG enhancer, CMV enhancer, UBC enhancer, SV40 enhancer, EF1a enhancer, etc.

[0016] The bidirectional promoter described in this invention has the structure "promoter B-enhancer-promoter A", where two promoters share a single enhancer. Promoter A and promoter B are located on opposite sides of the promoter and their transcription directions are opposite. The enhancer's function is independent of the forward or reverse direction of its sequence, but the presence of a promoter is required for it to function. Furthermore, this invention is not limited to the CMV enhancer; other enhancers such as SV40 or EF1a can also be used, and they will still function.

[0017] In some preferred embodiments, the enhancer is a CMV enhancer, and promoter A and promoter B are selected from: CB promoter, CMV promoter, CAG promoter, or truncated versions thereof, and promoter A and promoter B are not identical. In some specific embodiments, the "promoter B-enhancer-promoter A" relationship is miniCMV promoter-CMV enhancer-CMV promoter. In some specific embodiments, the "promoter B-enhancer-promoter A" relationship is CMV promoter-CMV enhancer-miniCMV promoter. In some specific embodiments, the "promoter B-enhancer-promoter A" relationship is CMV promoter-CMV enhancer-CB promoter. In some specific embodiments, the "promoter B-enhancer-promoter A" relationship is CB promoter-CMV enhancer-CMV promoter. In some specific embodiments, the "promoter B-enhancer-promoter A" relationship is CAG promoter-CMV enhancer-CMV promoter. In some specific implementations, the “promoter B-enhancer-promoter A” is a CMV promoter-CMV enhancer-CAG promoter.

[0018] In some embodiments, the expression vector of the present invention further includes other expression regulatory elements. An expression regulatory element is a nucleic acid sequence that regulates the expression of a nucleotide sequence operatively linked to it. The expression regulatory element is operatively linked to the nucleotide sequence when it controls and regulates the transcription and / or translation of the nucleotide sequence. Therefore, the expression regulatory element may include an internal ribosome entry site (IRES), a transcription terminator, a start codon preceding a protein-coding gene, an intron splicing signal, and a stop codon. It may also include a nucleic acid sequence design that removes unwanted potential splicing sites. It may also include a sequence that guides the addition of polyadenylation or polyA.

[0019] In some specific embodiments, the gene B-promoter B or promoter A-gene A of the present invention further includes an intron. In some cases, an intron may refer to any sequence that can be transcribed but not translated. In some cases, an intron may refer to any sequence that has been transcribed and removed from the mature RNA transcript in the cell. In some exemplary embodiments, the intron is selected from PI introns, VH4 introns, SV40 introns, Chi introns, U12 introns, RHD introns, MBL introns, etc.

[0020] In some specific embodiments, the expression regulatory element of the present invention further includes a 5” untranslated region (5”UTR). In some embodiments, the 5”UTR sequence is heterologous to the promoter sequence. In some such embodiments, the 5”UTR includes a sequence selected from the group consisting of: UTR1, UTR2, an enhancer element from an adenovirus major late promoter (eMLP), and a triplet leader sequence (TPL sequence) from adenovirus. In some embodiments, the 5”UTR includes the UTR1 sequence. In one embodiment, the 5”UTR includes the UTR2 sequence. In a preferred embodiment, the 5”UTR includes the TPL sequence and the eMLP sequence in a 5” to 3” sequence.

[0021] In some specific embodiments, the expression regulatory element of the present invention further includes an RNA output signal. The RNA output signal is exemplary selected from human hepatitis B virus post-transcriptional element (HPRE) sequences and marmot hepatitis virus post-transcriptional element (WPRE) sequences.

[0022] In some embodiments, a polyadenylation signal (polyA) is further included downstream or lateral to the gene B-promoter B and promoter A-gene A described in this invention. The polyadenylation signal protects mRNA from exonuclease attack and is crucial for transcription termination, mRNA export from the nucleus, and translation. The polyadenylation signal comprises multiple consecutive adenosine monophosphates, typically containing an AAUAAA repeat sequence. The polyadenylation signal described in this invention is located downstream of the coding gene. In some embodiments, the polyadenylation signal includes simian vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or β-globin (RGB).

[0023] In some embodiments, the protein co-expression vector system of the present invention has the following structure: polyadenylation signal B-gene B-intron B-promoter B-enhancer-promoter A-intron A-gene A-polyadenylation signal A.

[0024] In some embodiments, the expression vector of the present invention includes a prokaryotic expression vector, a eukaryotic expression vector, a recombinant bacterium, a cell, or a virus, and the expression vector contains a nucleic acid or DNA sequence with the structure or composition described above in the present invention.

[0025] The prokaryotic expression vector described in this invention is a vector capable of carrying inserted exogenous nucleic acid sequences into prokaryotic cells for expression, and has elements such as a replication origin, selection markers, promoters, and terminators. In some preferred embodiments, the prokaryotic expression vector of this invention is a plasmid or an expression cassette.

[0026] The eukaryotic expression vector described in this invention is a vector for expressing exogenous genes in eukaryotic cells. It typically possesses independent and stable DNA self-replication capability within recipient cells, is easily isolated and purified from host cells, has a single restriction endonuclease site, and a marker gene for easy screening. Common eukaryotic expression vectors include plasmids and viruses.

[0027] In prokaryotic expression vector systems, plasmids are commonly used to carry foreign genes for expression in prokaryotes such as *E. coli*. These plasmids may contain selection markers such as antibiotic resistance genes, facilitating the screening of successfully transformed cells in culture media containing the appropriate antibiotics. Plasmid replication and expression are controlled by prokaryotic genetic mechanisms. In eukaryotic expression systems, plasmids are similarly used to carry foreign genes for expression in eukaryotic cells.

[0028] The recombinant bacteria described in this invention refer to bacteria into which exogenous genes have been introduced through genetic engineering, enabling them to express new traits or functions. Common recombinant bacteria include Escherichia coli and yeast.

[0029] The cell lines described in this invention may be transgenic cell lines or fusion cell lines. The transgenic cell lines may be mammalian cell lines transfected with genes A and B described in this invention, preferably CHO cell lines, or 293 cells and their sublines. The fusion cell lines may be hybridoma cells that secrete proteins or antibodies encoded by genes A and B of this invention.

[0030] Viral vectors are widely used due to their high transduction efficiency and long-term stable gene expression. Commonly used viral vectors include adeno-associated virus (AAV), adenovirus (Ad), lentivirus (LV), and retrovirus (RV). In some preferred embodiments, the viral vector described in this invention is adeno-associated virus (AAV). AAV is a small, single-stranded DNA virus that cannot replicate autonomously. It is commonly used as a vector for gene therapy due to its excellent safety, low immunogenicity, high stability, and ability to express exogenous genes long-term. AAV vectors can be designed with different serotypes to achieve targeting in different tissues.

[0031] In some preferred embodiments, the expression vector of the present invention is a plasmid or adeno-associated virus (AAV), wherein the nucleic acid of the plasmid or virus is flanked by a functional adenoviral inverted terminal repeat (ITR) sequence at the 5' and 3' ends. A functional adenoviral inverted terminal repeat (ITR) sequence refers to an ITR sequence used for integration, replication, and packaging of AAV viral particles. The inverted terminal repeat sequence is a serotype of adeno-associated virus ITR, selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

[0032] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0033] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0034] (b) Polyadenylation signal B;

[0035] (c) Gene B;

[0036] (d) Intron B;

[0037] (e) Promoter B;

[0038] (f) Enhancer;

[0039] (g) Promoter A;

[0040] (h) Intron A;

[0041] (i) Gene A;

[0042] (j) Polyadenylation signal A;

[0043] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0044] The polyadenylation signal A or polyadenylation signal B is selected from simian vacuolating virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or β-globin (RGB); the gene A or gene B is selected from the coding sequences encoding complement factors or complement regulatory proteins, neurotrophic factors, anti-vascular endothelial growth factor (VEGF), endostatin, and anti-angiogenic factors; preferably, the gene A or gene B is selected from the coding sequences encoding complement factors or complement regulatory proteins and neurotrophic factors; the intron A or intron B is selected from the PI intron, VH4 intron, SV40 intron, Chi intron, U12 intron, RHD intron, and MBL intron; the enhancer is selected from the CAG enhancer, CMV enhancer, SV40 enhancer, EF1a enhancer, or UBC enhancer.

[0045] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0046] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0047] (b) Polyadenylation signal B;

[0048] (c) Neurotrophic factor coding sequence;

[0049] (d) Intron B;

[0050] (e) miniCMV promoter;

[0051] (f) CMV enhancer;

[0052] (g)CMV promoter;

[0053] (h) Intron A;

[0054] (i) The coding sequence of the C5 complement inhibitor;

[0055] (j) Polyadenylation signal A;

[0056] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0057] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0058] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0059] (b) Polyadenylation signal B;

[0060] (c) Neurotrophic factor coding sequence;

[0061] (d) Intron B;

[0062] (e) CMV promoter;

[0063] (f) CMV enhancer;

[0064] (g)CB promoter;

[0065] (h) Intron A;

[0066] (i) The coding sequence of the C5 complement inhibitor;

[0067] (j) Polyadenylation signal A;

[0068] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0069] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0070] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0071] (b) Polyadenylation signal B;

[0072] (c) The coding sequence of the C5 complement inhibitor;

[0073] (d) Intron B;

[0074] (e) CMV promoter;

[0075] (f) CMV enhancer;

[0076] (g)CB promoter;

[0077] (h) Intron A;

[0078] (i) Neurotrophic factor coding sequences;

[0079] (j) Polyadenylation signal A;

[0080] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0081] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0082] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0083] (b) RGB polyadenylation signal;

[0084] (c) Neurotrophic factor coding sequence;

[0085] (d) MBL introns;

[0086] (e) miniCMV promoter;

[0087] (f) CMV enhancer;

[0088] (g)CMV promoter;

[0089] (h)PI introns;

[0090] (i) The coding sequence of the C5 complement inhibitor;

[0091] (j) BGH polyadenylation signal;

[0092] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0093] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0094] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0095] (b) RGB polyadenylation signal;

[0096] (c) Neurotrophic factor coding sequence;

[0097] (d) MBL introns;

[0098] (e) CMV promoter;

[0099] (f) CMV enhancer;

[0100] (g)CB promoter;

[0101] (h)PI introns;

[0102] (i) The coding sequence of the C5 complement inhibitor;

[0103] (j) BGH polyadenylation signal;

[0104] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0105] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0106] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0107] (b) RGB polyadenylation signal;

[0108] (c) The coding sequence of the C5 complement inhibitor;

[0109] (d) MBL introns;

[0110] (e) CMV promoter;

[0111] (f) CMV enhancer;

[0112] (g)CB promoter;

[0113] (h)PI introns;

[0114] (i) Neurotrophic factor coding sequences;

[0115] (j) BGH polyadenylation signal;

[0116] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0117] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0118] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0119] (b) RGB polyadenylation signal;

[0120] (c) The coding sequence of the C5 complement inhibitor;

[0121] (d) MBL introns;

[0122] (e) CMV promoter;

[0123] (f) CMV enhancer;

[0124] (g)CB promoter;

[0125] (h)PI introns;

[0126] (i) Neurotrophic factor coding sequences;

[0127] (j)SV40 polyadenylation signal;

[0128] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0129] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0130] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0131] (b) RGB polyadenylation signal;

[0132] (c) The coding sequence of the C5 complement inhibitor;

[0133] (d) MBL introns;

[0134] (e) CMV promoter;

[0135] (f) CMV enhancer;

[0136] (g)CB promoter;

[0137] (h)PI introns;

[0138] (i) Neurotrophic factor coding sequences;

[0139] (j)RGH polyadenylation signal;

[0140] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0141] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0142] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0143] (b) RGB polyadenylation signal;

[0144] (c) The coding sequence of the C5 complement inhibitor;

[0145] (d) PI introns;

[0146] (e) CMV promoter;

[0147] (f) CMV enhancer;

[0148] (g)CB promoter;

[0149] (h)SV40 intron;

[0150] (i) Neurotrophic factor coding sequences;

[0151] (j) BGH polyadenylation signal;

[0152] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0153] In some preferred embodiments, the C5 complement inhibitor is selected from Ravulizumab or Eculizamub and their antigen-binding fragments, preferably Eculizamub and its antigen-binding fragments. In some specific embodiments, the Eculizamub has the heavy chain variable region described in SEQ ID NO:1 and the light chain variable region described in SEQ ID NO:2. In some specific embodiments, the C5 complement inhibitor is the scFv fragment of Eculizamub (abbreviated as Ecu-scFv), which connects the light and heavy chain variable regions through a polypeptide segment (SEQ ID NO:3); in some more specific embodiments, the scFv of Eculizamub has the amino acid sequence described in SEQ ID NO:22. In some specific embodiments, the gene encoding the Eculizamub scFv contains the coding sequence described in SEQ ID NO:4.

[0154] In some preferred embodiments, the neurotrophic factor is selected from PEDF, CNDF, CNTF or RdCVF, with CNTF being preferred.

[0155] In some specific embodiments, the CNTF has the amino acid sequence described in SEQ ID NO: 5. In some specific embodiments, the gene encoding the CNTF comprises the coding sequence described in SEQ ID NO: 6.

[0156] In some specific embodiments, the PEDF has the amino acid sequence described in SEQ ID NO: 23.

[0157] In some specific embodiments, the RdCVF has the amino acid sequence described in SEQ ID NO: 24.

[0158] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0159] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0160] (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7;

[0161] (c) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6;

[0162] (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8;

[0163] (e) a miniCMV promoter comprising the nucleotide sequence described in SEQ ID NO:9;

[0164] (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10;

[0165] (g) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11;

[0166] (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12;

[0167] (i) The Eculizamub scFv coding sequence, which comprises the nucleotide sequence described in SEQ ID NO:4;

[0168] (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13;

[0169] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0170] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises the nucleotide sequence described in SEQ ID NO:14 from the 5' to 3' direction.

[0171] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0172] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0173] (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7;

[0174] (c) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6;

[0175] (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8;

[0176] (e) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11;

[0177] (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10;

[0178] (g) A CB promoter comprising the nucleotide sequence described in SEQ ID NO:15;

[0179] (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12;

[0180] (i) The Eculizamub scFv coding sequence, which comprises the nucleotide sequence described in SEQ ID NO:4;

[0181] (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13;

[0182] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0183] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises the nucleotide sequence described in SEQ ID NO:16 from the 5' to 3' direction.

[0184] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises, from the 5' to the 3' direction:

[0185] (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;

[0186] (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7;

[0187] (c) The coding sequence of Eculizamub scFv, which comprises the nucleotide sequence described in SEQ ID NO:4;

[0188] (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8;

[0189] (e) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11;

[0190] (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10;

[0191] (g) A CB promoter comprising the nucleotide sequence described in SEQ ID NO:15;

[0192] (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12;

[0193] (i) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6;

[0194] (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13;

[0195] (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0196] In some specific embodiments, the nucleic acid of the expression vector of the present invention comprises the nucleotide sequence described in SEQ ID NO:17 from the 5' to 3' direction.

[0197] The nucleotides or amino acids described in this invention also include nucleotide or amino acid sequences having at least 85%, such as 90%, 95%, 99%, or 100%, homology with them. "Homology" refers to the degree of similarity in amino acid sequences or base sequences when two or more nucleotide sequences or amino acid sequences are compared, expressed as a percentage, such as 85%, 90%, 95%, 99%, or 100%.

[0198] In some embodiments, the present invention provides a plasmid comprising the nucleic acid described above.

[0199] In some embodiments, the present invention provides a host cell containing the aforementioned nucleic acids or plasmids; preferably, the host cell is a mammalian cell, yeast cell, bacterial cell, or insect cell.

[0200] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising a capsid protein and the aforementioned nucleic acid of the present invention.

[0201] In some preferred embodiments, the capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10 serotypes or mutants thereof. In some preferred embodiments, the capsid protein is the AAV2 serotype capsid protein or a mutant thereof. When present in an AAV viral vector, the mutant exhibits increased infectivity to target tissues or cells (e.g., retina, muscle, or joint cavity) compared to an AAV viral vector containing the unmutated parental AAV capsid protein.

[0202] In some embodiments, the capsid protein mutant includes the capsid protein mutant described above in this invention.

[0203] In other embodiments, the capsid protein mutant is inserted relative to the wild-type AAV capsid protein into a heterologous polypeptide comprising about 7 to 8 amino acids, the heterologous polypeptide comprising an amino acid sequence selected from IADSSRT (SEQ ID NO: 18), IGETRT (SEQ ID NO: 20), or IADNTRP (SEQ ID NO: 21), and the insertion site is located between amino acid positions 587 and 588 of the wild-type AAV2 capsid protein or at the corresponding position of other serotype capsid proteins.

[0204] In some specific embodiments, the capsid protein mutant, relative to the wild-type AAV2 capsid protein, further includes amino acid substitutions of I240T, V708I, Y444F, Y500F, and / or Y730F, or corresponding amino acid substitutions of other serotype capsid proteins. In some specific embodiments, the capsid protein mutant, relative to the wild-type AAV2 capsid protein, further includes amino acid substitutions of I240T and V708I. In some specific embodiments, the capsid protein mutant, relative to the wild-type AAV2 capsid protein, further includes amino acid substitutions of I240T, V708I, Y444F, Y500F, and Y730F.

[0205] In some embodiments, the present invention provides a pharmaceutical composition characterized by the aforementioned nucleic acid, plasmid, or recombinant adeno-associated virus and a pharmaceutically acceptable vector. In some preferred embodiments, the pharmaceutical composition is an intravitreal injection, subretinal injection, choroidal injection, intravenous injection, intratumoral injection, or intramuscular injection formulation.

[0206] In some embodiments, the present invention provides the use of the aforementioned nucleic acids, plasmids, or recombinant adeno-associated viruses in the preparation of medicaments for treating fundus diseases. In some preferred embodiments, the fundus disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoid choroidal vascular disease, wet age-related macular degeneration with extremely low vision, and choroidal neovascularization secondary to pathological myopia. In some embodiments, the eye disease is dry age-related macular degeneration. In some embodiments, the fundus disease is geographic atrophy (GA).

[0207] To enhance infectivity to retinal cells, in one aspect, the present invention provides an adeno-associated virus (AAV) capsid protein mutant and a viral vector (such as an AAV viral vector) containing the capsid protein mutant to desired cells or tissues. Specifically, the present invention comprises a modified capsid protein having one or more modifications (such as substitution, insertion, or mutation) in the amino acid sequence relative to the wild-type AAV capsid protein, which, when present in an AAV viral vector, exhibits increased infectivity to target tissues or cells (e.g., retina, muscle, or joint cavity) compared to an AAV viral vector containing the unmutated wild-type AAV capsid protein.

[0208] In another aspect, this invention provides an adeno-associated virus (AAV) capsid protein mutant comprising an insertion of a heterologous polypeptide comprising approximately 7-8 amino acids relative to the wild-type AAV capsid protein. The heterologous polypeptide comprises an amino acid sequence selected from IADSSRTs (SEQ ID NO: 18), IGETRTs (SEQ ID NO: 20), or IADNTRP (SEQ ID NO: 21), and the insertion site is located between amino acid positions 587 and 588 of the wild-type AAV2 capsid protein or at a corresponding position in other serotype capsid proteins. Compared to AAV viruses containing the corresponding wild-type AAV capsid protein, AAV viruses containing the capsid protein mutant exhibit enhanced retinal cell infectivity.

[0209] In some specific embodiments, the heterologous polypeptide includes an amino acid sequence selected from IADSSRTS (SEQ ID NO:18), IGETSRTS (SEQ ID NO:20), or IADNTRP (SEQ ID NO:21).

[0210] In some preferred embodiments, the heterologous polypeptide comprises an amino acid sequence selected from IADSSRTS (SEQ ID NO:18) or IGETRTS (SEQ ID NO:20).

[0211] In some preferred embodiments, the amino acid sequence of the heterologous polypeptide is IADSSRTS (SEQ ID NO: 18).

[0212] In some specific embodiments, the AAV described in this invention can be derived from any serotype of AAV, for example, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh10. In a preferred embodiment, the AAV described in this invention is selected from AAV2.

[0213] In some specific embodiments, the capsid protein mutant further includes, relative to the wild-type AAV2 capsid protein, amino acid substitutions at corresponding positions of I240T, V708I, Y444F, Y500F and / or Y730F, or other serotype capsid proteins.

[0214] In some specific implementations, the capsid protein mutant, relative to the wild-type AAV2 capsid protein, includes amino acid substitutions I240T and V708I.

[0215] In some specific embodiments, the capsid protein mutant, relative to the wild-type AAV2 capsid protein, includes amino acid substitutions of I240T, V708I, Y444F, Y500F, and Y730F.

[0216] Another aspect of the present invention provides a recombinant adeno-associated virus (rAAV), comprising:

[0217] i. The adeno-associated virus capsid protein mutant provided in the first aspect of the present invention;

[0218] ii. Heterologous nucleic acids encoding gene products.

[0219] In some specific implementations, the gene product is a VEGF antagonist, pigment epithelial-derived factor, endostatin, angiopoietin, complement inhibitor, complement regulatory protein, and / or neurotrophic factor, etc.

[0220] In some specific implementation schemes, the VEGF antagonist is selected from aflibercept, conbercept, ranibizumab, and broxolizumab.

[0221] In some specific implementations, the complement inhibitor or complement regulatory protein is selected from complement factor I, complement factor H, complement factor C3, complement factor C5, complement factor B, complement factor D inhibitors, or complement regulatory protein CD59, etc.

[0222] In some more specific embodiments, the C5 complement inhibitor is selected from Ravulizumab or Eculizamub and their antigen-binding fragments, preferably Eculizamub and its antigen-binding fragments.

[0223] In some specific implementations, the neurotrophic factor is selected from PEDF, CNDF, CNTF, or RdCVF, with CNTF being preferred.

[0224] In some more specific embodiments, the gene product is a C5 complement inhibitor and a neurotrophic factor.

[0225] In some specific implementations, the complement regulatory protein is human soluble CD59 (sCD59).

[0226] In some specific embodiments, the complement factor is selected from human soluble CD59 (sCD59). The protein structure of human soluble CD59 (sCD59) includes a single cysteine-rich domain, a hydrophobic core with three loops, and a small fourth helical loop. sCD59 includes 26 amino acids at the C-terminus, which designate a signal sequence for linking a glycosylphosphatidylinositol anchor (GPI anchor) at the asparagine amino acid at position 77. In some specific embodiments, human soluble CD59 (sCD59) has the amino acid sequence as described in SEQ ID NO:25. Specifically, human soluble CD59 (sCD59) has the amino acid sequence as described in SEQ ID NO:25.

[0227] Another aspect of the present invention provides a pharmaceutical composition comprising:

[0228] a) The recombinant adeno-associated virus provided by the present invention;

[0229] b) Pharmaceutically acceptable excipients.

[0230] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition of the present invention is administered via intravitreal, retinal, choroidal injection, intravenous, subcutaneous, intramuscular, or intra-articular injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition of the present invention is administered via intravitreal, retinal, or suprachoroidal injection.

[0231] In another aspect, the present invention provides the use of the recombinant adeno-associated virus in the preparation of medicaments for the prevention or treatment of eye diseases.

[0232] Another aspect of the present invention provides a method for preventing or treating eye diseases, the method comprising administering an effective amount of the recombinant adeno-associated virus or pharmaceutical composition according to the present invention to an individual in need.

[0233] In some specific embodiments, the ocular diseases described in this invention are selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascularization, non-infectious uveitis, or glaucoma. In some specific embodiments, the ocular diseases described in this invention are selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular edema caused by branch retinal vein occlusion, diabetic retinopathy, diabetic proliferative retinopathy, diabetic retinal ischemia, polypoid choroidal angiopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity.

[0234] In some specific implementations, the eye disease described in this invention is dry age-related macular degeneration.

[0235] In another aspect, this invention also discovered that the C5 complement inhibitor protein, in combination with neurotrophic factors, has a synergistic effect, which can significantly improve the transfection capacity of retinal cells, and is expected to provide a new treatment strategy for the treatment of fundus diseases.

[0236] Therefore, in some embodiments, the present invention provides the use of C5 complement inhibitor protein combined with neurotrophic factor drugs in the preparation of medicaments for treating fundus diseases.

[0237] In some embodiments, the present invention also provides the use of C5 complement inhibitors and neurotrophic factors in the preparation of viral vector drugs for the treatment of fundus diseases.

[0238] In some specific implementations, the viral vector simultaneously encodes a C5 complement inhibitor and a neurotrophic factor.

[0239] In some specific implementations, the viral vector includes adeno-associated virus (AAV), adenovirus (Ad), lentivirus (LV), retrovirus (RV), etc., with adeno-associated virus being preferred.

[0240] In some specific implementations, exemplary C5 complement inhibitor proteins are selected from Ravulizumab or Eculizamub and their antigen-binding fragments, preferably Eculizamub and its antigen-binding fragments (such as Fab, sc-Fv, Fab', F(ab')2, etc.).

[0241] In some specific implementations, exemplary neurotrophic factors are selected from PEDF, CNDF, CNTF, or RdCVF, with CNTF being preferred.

[0242] In some specific implementations, the fundus disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoid choroidal vascular disease, wet age-related macular degeneration with extremely low vision, and choroidal neovascularization secondary to pathological myopia.

[0243] In some more specific implementations, the fundus disease is dry age-related macular degeneration.

[0244] In some more specific implementations, the fundus disease is geographic atrophy (GA). Attached Figure Description

[0245] Figure 1 shows the infection status of ARPE19 cells in Example 2.

[0246] Figure 2 shows the infection status of ARPE19 cells in Example 3.

[0247] Figure 3 shows the transduction of mice via intravitreal injection in Example 4.

[0248] Figure 4 shows the expression of mice via intravitreal injection in Example 6.

[0249] Figure 5A shows the fluorescence staining in the in vitro immunofluorescence experiment of inhibiting the MAC complex after transduction of ARPE cells with protein drugs in Example 7.

[0250] Figures 5B-5C show the fluorescence statistics in the in vitro MAC complex immunoassay after transduction of ARPE cells with protein drugs in Example 7.

[0251] Figures 6A-6D show schematic diagrams of the insulator carrier and bidirectional starter carrier in Example 8.

[0252] Figure 7A Ecu-scFv protein expression in the cell infection experiment of ARPE19 Example 9

[0253] Figure 7B CNTF protein expression in the cell infection experiment of ARPE19 Example 9

[0254] Figure 8A. Expression of Ecu-scFv protein in cell infection experiment in HEK293 Example 10.

[0255] Figure 8B: Expression of CNTF protein in cell infection experiment of HEK293 in Example 10.

[0256] Figure 9A. Ecu-scFv protein expression in the cell infection experiment of ARPE19 Example 11.

[0257] Figure 9B CNTF protein expression in the cell infection experiment of ARPE19 Example 11.

[0258] Figure 10A shows the fluorescence staining in the in vitro MAC complex immunofluorescence experiment in Example 12.

[0259] Figure 10B Fluorescence statistics of the in vitro MAC complex immunofluorescence experiment in Example 12.

[0260] Figure 11. Staining in the β-galactosidase staining experiment of cell senescence in Example 13.

[0261] Figure 12A shows the fluorescence staining in the apoptosis experiment in Example 14.

[0262] Figure 12B Fluorescence statistics in the apoptosis experiment of Example 14

[0263] Figure 13. Slice results from the sodium iodate-induced dry AMD model experiment in Example 15.

[0264] Figure 14 shows the results of the Mianyang erythrocyte hemolysis experiment in Example 16. Detailed Implementation

[0265] The present invention will be further described below with reference to embodiments, but these embodiments do not constitute any limitation on the present invention.

[0266] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0267] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0268] Example 1: Construction of Mutants

[0269] AAV2 mutants were generated using site-directed mutagenesis and recombinant DNA techniques. These mutants contained a polypeptide substitution between amino acid positions 587 and A588 of the wild-type AAV2 capsid protein, or further contained amino acid substitutions of I240T, V708I, Y444F, Y500F, and Y730F relative to the wild-type AAV2 capsid protein, as shown in Table 1.

[0270] Table 1

[0271] The AAV2 mutant virus packaging was generated through cell co-transfection with three plasmids: a first plasmid (pAAV-CBA-EGFP or pAAV-CMV-sCD59) containing an expression cassette (EGFP reporter gene or sCD59) flanked by an ITR; a second plasmid (the mutant plasmid encoding the Rep / Cap gene constructed in Example 1); and a third plasmid containing an adenovirus helper gene. The target gene plasmid / capsid protein plasmid / helper plasmid were mixed at a 1:2:1 ratio, filtered, and added to the packaging reagent. After mixing and incubation at room temperature for 30 minutes, the mixture was added to suspended HEK293 cells. Cells were cultured at 120 rpm, 37°C, and 5% CO2 for 72 hours, then centrifuged. Cells were incubated overnight at -80°C, allowed to return to room temperature, and then lysed with acid. The pH was adjusted to neutral, filtered through a 0.22 μm filter, and purified using an AAVX purification column to obtain the virus. The viral titer was determined by PCR and used for further processing.

[0272] Example 2 ARPE19 cell infection experiment

[0273] ARPE19 cells were seeded in 24-well plates with F12 medium containing 10% fetal bovine serum. ARPE19 cells were then seeded at a rate of 2e5 cells / well and cultured at 37°C for 16 h. Wild-type AAV2 cells or mutants SJ48, SJ18, SJ58, SJ20, SJM1, and SJM4 (carrying the EGFP reporter gene) were added to the culture dish at a dose of 2e8 vg. After transduction for 48 h, the fluorescence signal of EGFP was captured using 488 nm excitation light to observe the expression of the fluorescent protein. The infection status of ARPE19 cells after 48 hours is shown in Figure 1. The results showed that the mutants SJ48, SJM1, and SJM4 had stronger fluorescent protein signals than the wild-type AAV2.

[0274] Example 3 ARPE19 cell infection experiment

[0275] ARPE19 cells were seeded in 24-well plates with F12 medium containing 10% fetal bovine serum. ARPE19 cells were then seeded at a rate of 2e5 cells / well and cultured at 37°C for 16 h. Wild-type AAV2 or mutants SJM1, SJM4, SJM1-3YF, and SJM4-3YF (carrying the EGFP reporter gene) were added to the culture dish at a dose of 2e8 vg. After transduction for 48 h, the EGFP fluorescence signal was captured using 488 nm excitation light to observe the expression of the fluorescent protein. The infection status of ARPE19 cells after 48 h is shown in Figure 2. The results showed that mutants SJM1, SJM4, SJM1-3YF, and SJM4-3YF had stronger fluorescent protein signals and significantly improved infectivity compared to wild-type AAV2.

[0276] Example 4: Intravitreal Injection Transduction Experiment in Mice

[0277] Male C57BL / 6 mice, aged 6–8 weeks, were purchased and acclimatized for 5 days. Then, 2 μL of various AAV variants (carrying the EGFP reporter gene) were injected intravitreally (totaling 2.5 E9 vg); 3 mice per group. After 13 days of continued feeding, eyeballs from each group were harvested, fixed overnight in FAS eye fixative, and then paraffin-embedded. EGFP fluorescence signals were captured from each section under 488 nm excitation light, as shown in Figure 3. SJM1 and SJM4 primarily transduced photoreceptor cells and RPE cells, with superior transduction efficiency compared to AAV2.

[0278] Example 5: Intravitreal Injection Transduction Experiment in Rhesus Monkeys

[0279] The AAV variant (carrying the EGFP reporter gene) was injected intravitreally into the eyes of rhesus monkeys (animal numbers 2M003 and 2M004) at a dose of 1e11vg / eye. The right eye (OD) was injected with either the SJM1 or SJM4 variant, with 50 μL injected into each eye. Twenty-one days later, the rhesus monkeys were sacrificed, and retinal tissue was isolated. EGFP protein expression levels were measured by ELISA, and the results are shown in Table 2.

[0280] Table 2

[0281] Example 6: Mouse intravitreal injection expression experiment

[0282] Male C57BL / 6 mice, aged 6–8 weeks, were purchased and acclimatized for 5 days. Then, 2 μL of various AAV variants (carrying the sCD59 gene) were injected intravitreally (totaling 2.5 E9 vg). Four mice were used per group, resulting in a total of eight eyes. After 14 days of continued acclimatization, the retina and choroid were separated from the eight eyes in each group, and sCD59 protein expression levels were detected by ELISA. The results are shown in Figure 4.

[0283] Example 7: Immunofluorescence assay of inhibition of MAC complex in vitro after transduction of ARPE cells with protein drugs.

[0284] Experimental Methods: ARPE19 cells were seeded in 3E4 wells of 96-well plates and cultured overnight at 37°C with 5% CO2. Protein drugs Izervay (avacincaptad pegol, commercially available), Ecu-scFv (SEQ ID NO: 3), PEDF (SEQ ID NO: 23), CNTF (SEQ ID NO: 5), RdcvF (SEQ ID NO: 24), and Ecu-scFv combined with PEDF, CNTF, and RdcvF at a 1:1 ratio were administered to cells at different MOI gradients (0, 2.3, 4.6, 9.1, 18.2, 36.3, 72.5, 125 nM, with the NC group receiving no subsequent treatment) were used to infect cells. After 6 hours, the medium was replaced with DMEM / F-12 medium + 10% FBS + 1% penicillin-dextrose antibody. After 48 hours, 7.5% non-inactivated human serum was added to a final concentration, and the cells were incubated at 37°C for 30 minutes. The supernatant was discarded, and the cells were washed twice with 1X PBS. Discard the supernatant, add 100 μL / well of 4% paraformaldehyde, and fix at 4°C for 10 min. Wash cells three times with 1X PBS, and block with 300 μL / well of 3% BSA for 20 min. Dilute Anti-C5b-9 antibody (abcam, ab55811) 1:500 with 3% BSA, add 100 μL / well to cells, and incubate at 37°C for 1.5 h. Wash cells three times with 1X PBS, and discard the supernatant. Goat anti-rabbit IgG H&L (Alexa) 488)(abcam, ab150077) was diluted 1:500 with 3% BSA, and 100 μL / well was added to each cell. Cells were incubated at 37°C for 1 hour. Cells were washed three times with 1X PBS, and the supernatant was discarded. Hochest staining was performed, and images were taken using a high-content cell imaging system.

[0285] Experimental results: Complement activation ultimately leads to the formation of the membrane attack complex (MAC), which damages cells. In the in vitro immunofluorescence assay, green fluorescence represents MAC complex staining; stronger green fluorescence indicates the formation of more MAC complexes and greater cell damage. The fluorescence staining results are shown in Figure 5A, and the statistical results are shown in Figures 5B and 5C.

[0286] Figures 5A-5C show that the combined use of Ecu-scFv with PEDF, CNTF, and RdcvF protein drugs has a stronger effect on inhibiting MAC complex formation after transducing ARPE cells compared to single drug use, and also has a certain synergistic effect.

[0287] Example 8 Construction of capsid mutants and preparation of rAAV

[0288] A mutant of the capsid protein of AAV2 (abbreviated as SJM1-3YF or M1-3YF) was generated using site-directed mutagenesis and recombinant DNA technology. The mutant contains a polypeptide IADSSRTS (SEQ ID NO:18) inserted between amino acids 587 to A588 of wild-type AAV2, and amino acid substitutions at I240T, V708I, Y444F, Y500F and Y730F of wild-type AAV2 capsid protein.

[0289] A multinucleotide expression cassette containing regulatory elements and the coding sequence of the target gene was constructed using standard recombinant DNA cloning techniques or general molecular biology techniques. In this embodiment, target gene A encodes Ecu-scFv, which contains the heavy chain variable region described in SEQ ID NO:1 and the light chain variable region described in SEQ ID NO:2, linked by a linker peptide (SEQ ID NO:3). The complete amino acid sequence includes the amino acid sequence described in SEQ ID NO:22, and Ecu-scFv has the coding nucleic acid described in SEQ ID NO:4. Target gene B encodes ciliary neurotrophic factor-CNTF, which contains the amino acid sequence described in SEQ ID NO:5 and has the coding nucleic acid described in SEQ ID NO:6.

[0290] As shown in Figure 6A, this embodiment exemplarily illustrates the main constituent elements of the multi-expression cassettes KHP-T-561, KHP-T-581, KHP-T-582, and KHP-T-583 containing target gene A and target gene B, sequentially from 5'ITR to 3'ITR. KHP-T-561 is an insulator polynucleotide expression cassette vector, which sequentially contains 5'ITR, RGB polyadenylation signal, gene B, CMV promoter, insulator, U1a promoter, PI intron, gene A, BGH polyadenylation signal, and 3'ITR.

[0291] The construction of the insulator plasmid vector included: designing homologous arm primers for PCR amplification of the insulator gene; performing DNA gel electrophoresis on the PCR product; and recovering the target band by gel extraction. The backbone vector was digested with enzymes to obtain the digested vector. Digestion conditions: 37℃, >3 hours. The PCR fragment and the digested vector were ligated using a homologous recombinase at 50℃ for 10 minutes. 10 μL of the ligation product was added to stbl3 competent cells, incubated on ice for 20 minutes, heat-shocked at 42℃ for 60 seconds, and then quickly placed on ice for 2-3 minutes. 2XYT medium was added, and the cells were incubated at 37℃ and 220 rpm for 1 hour. The cells were then plated on KANA plates and incubated overnight at 37℃. The next day, clones were selected for sequencing. KHP-T-561 contained the nucleotide sequence described in SEQ ID NO:19. Successfully sequenced clones were subjected to plasmid extraction to obtain the target plasmid.

[0292] KHP-T-581, KHP-T-582, and KHP-T-583 are bidirectional promoter polynucleotide expression cassette vectors. The KHP-T-581 polynucleotide expression cassette, from 5' ITR to 3' ITR, contains the following sequence: 5' ITR, RGB polyadenylation signal, gene B, MBL intron, miniCMV promoter, CMV enhancer, CMV promoter, PI intron, gene A, BGH polyadenylation signal, and 3' ITR. The KHP-T-582 polynucleotide expression cassette, from 5' ITR to 3' ITR, contains the following sequence: 5' ITR, RGB polyadenylation signal, gene B, MBL intron, CMV promoter, CMV enhancer, CB promoter, PI intron, gene A, BGH polyadenylation signal, and 3' ITR. The KHP-T-583 polynucleotide expression cassette, from 5' ITR to 3' ITR, contains the following components in sequence: 5' ITR, RGB polyadenylation signal, gene A, MBL intron, CMV promoter, CMV enhancer, CB promoter, PI intron, gene B, BGH polyadenylation signal, and 3' ITR. Figures 6B-6D illustrate the specific component composition of the KHP-T-581, KHP-T-582, and KHP-T-583 polynucleotide expression cassettes.

[0293] Construction of bidirectional promoter plasmid vector: The bidirectional promoter gene and backbone vector were double-digested with enzymes at 37°C for >3 hours. The digested fragments were ligated to the digested vector using ligase at 22°C for 30 minutes. 10 μL of the ligation product was added to stbl3 competent cells, incubated on ice for 20 minutes, heat-shocked at 42°C for 60 seconds, and then quickly placed on ice for 2-3 minutes. 2XYT medium was added, and the cells were incubated at 37°C and 220 rpm for 1 hour. The cells were then plated on KANA plates and incubated overnight at 37°C. Clones were selected the following day for sequencing. KHP-T-581, KHP-T-582, and KHP-T-583 contained the nucleotide sequences described in SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:17 from 5' ITR to 3' ITR, respectively. Correctly sequenced clones were subjected to plasmid extraction to obtain the target plasmid.

[0294] This embodiment also constructs polynucleotide expression cassettes containing only target gene A, target gene B, or reporter gene (mCherry). For example, the KHP-T-536 polynucleotide expression cassette contains, in the order from 5'ITR to 3'ITR, 5'ITR, CMV enhancer, CMV promoter, MBL intron, gene A, BGH polyadenylation signal, and 3'ITR; the KHP-T-547 polynucleotide expression cassette contains, in the order from 5'ITR to 3'ITR, 5'ITR, CMV enhancer, CMV promoter, MBL intron, gene B, BGH polyadenylation signal, and 3'ITR; and the KHP-T-mCherry polynucleotide expression cassette contains, in the order from 5'ITR to 3'ITR, 5'ITR, CMV enhancer, CMV promoter, MBL intron, mCherry, BGH polyadenylation signal, and 3'ITR.

[0295] The AAV2 mutant virus packaging was generated through cell co-transfection with three plasmids: a first plasmid consisting of an expression cassette flanked by the target gene on the ITR; a second plasmid encoding the Rep / Cap gene; and a third plasmid containing an adenovirus helper gene. Specifically, the target gene plasmid / capsid mutant plasmid / phelper plasmid were mixed in a 1:2:1 ratio, filtered, and added to the packaging reagent. After mixing and incubation at room temperature for 30 minutes, the mixture was added to suspended HEK293 cells. Cells were cultured at 120 rpm, 37°C, and 5% CO2 for 72 hours, then centrifuged. The cells were incubated overnight at -80°C, allowed to return to room temperature, and then subjected to acid lysis. The pH was adjusted to neutral, and the cells were filtered through a 0.22 μm filter and purified using an AAVX purification column to obtain the virus. The viral titer was determined by PCR and used for further processing.

[0296] The main components of the exemplary polynucleotide expression cassette vector of the present invention are shown in Table 3.

[0297] Table 3

[0298] Example 9 ARPE19 cell infection experiment

[0299] Experimental methods

[0300] After digestion, ARPE19 cells were seeded at 5E5 cells / well in 6-well plates and cultured overnight at 37°C with 5% CO2 in DMEM / F-12 medium + 10% FBS + 1% antibiotics. The cells were then replaced with Opti-MEM medium and cultured at 37°C with 5% CO2. 2.5 μg each of plasmids (T536, T561, T547, T581, T582, T583) were added to 250 μL of Opti-MEM medium, and 5 μL each of Lipofectamine 2000 transfection reagent were added to the same medium. After mixing, the plasmids and Lipofectamine 2000 transfection reagent were mixed and incubated at room temperature for 5 min. The mixture was then incubated at room temperature for 20 min. The mixed sample was added to the cells after the medium change and cultured at 37°C with 5% CO2 for 48 hours. The cell supernatant was collected, centrifuged at 2000 rpm for 5 min, and then used for ELISA detection.

[0301] Detection methods

[0302] Ecu-scFv protein detection method: Human C5 antigen is coated onto the ELISA plate using 1X PBS at a concentration of 4 μg / mL (100 μL / well). Coating is carried out overnight at 4°C. The supernatant is discarded, and the ELISA plate is blotted dry. The plate is washed three times with 1X PBST (PBS + 0.05% Tween 20) (0.05%), 300 μL each time. The ELISA plate is blotted dry, and 300 μL / well is added to blocking buffer (1% BSA + 0.05% Tween 20). Blocking is carried out at 37°C for 1 hour. The supernatant is discarded, and the ELISA plate is blotted dry. The plate is washed three times with 1X PBST (0.05%), 300 μL each time. The ELISA plate is blotted dry, and 100 μL / well is added to diluted cell supernatant containing the protein drug. The sample is diluted with the coating buffer and incubated at 37°C for 1 hour. Discard the supernatant, blot dry the ELISA plate, and wash three times with 300 μL of 1X PBST (0.05%). Blot dry the ELISA plate after each wash. MonoRab TM Rabbit Anti-scFv Cocktail (HRP) was diluted 1:5000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1X PBST (0.05%) each time. The ELISA plate was blotted dry, and 100 μL / well of TMB chromogenic buffer was added. The plate was incubated at 37°C for 5-10 minutes. The reaction was stopped by adding 50 μL / well of 2N H2SO4, and the plate was read using an OD450 microplate reader.

[0303] CNTF protein detection method: The human CNTF ELISA kit (abcam, ab264608, 1*96 tests) was used for detection, and the plate was read by an OD450 microplate reader.

[0304] The expression of Ecu-scFv protein is shown in Figure 7A. The results show that the expression level of Ecu-scFv protein is 2279.93 ng / ml in the single-target plasmid T536, 889.39 ng / ml in the insulator plasmid T561, and it is also expressed in plasmids T581, T582, and T583, with expression levels of 1667.36 ng / ml, 686.94 ng / ml, and 1769.75 ng / ml, respectively.

[0305] The expression of CNTF protein is shown in Figure 7B. The results show that the expression level of CNTF protein in the single-target plasmid T547 is 20541.55 pg / ml, and it is also expressed in plasmids T581, T582, and T583, with expression levels of 12270.98 pg / ml, 20375.00 pg / ml, and 14185.51 pg / ml, respectively. However, CNTF protein expression was not detected in the insulator plasmid T561.

[0306] Example 10 HEK293 cell infection experiment

[0307] Experimental methods

[0308] After digestion, HEK293 cells were seeded at 5E5 cells / well in 6-well plates and cultured overnight at 37°C with 5% CO2 in DMEM medium + 10% FBS + 1% antibiotics. The cells were then replaced with Opti-MEM medium and cultured at 37°C with 5% CO2. 2.5 μg each of plasmids (T536, T561, T547, T581, T582, T583) were added to 250 μL of Opti-MEM medium, and 5 μL each of Lipofectamine 2000 transfection reagent were added to the same medium. The mixture was incubated at room temperature for 5 min, then the plasmids and Lipofectamine 2000 transfection reagent were mixed and incubated at room temperature for 20 min. The mixed sample was then added to the cells after the medium change and cultured at 37°C with 5% CO2 for 48 hours. The cell supernatant was collected, centrifuged at 2000 rpm for 5 min, and used for ELISA detection.

[0309] Detection methods

[0310] Ecu-scFv protein detection method: Human C5 antigen is coated onto the ELISA plate using 1X PBS at a concentration of 4 μg / mL (100 μL / well). The plate is incubated overnight at 4°C. The supernatant is discarded, the plate is blotted dry, and the plate is washed three times with 1X PBST (0.05%), 300 μL each time. The plate is blotted dry, and 300 μL / well is added to blocking buffer (1% BSA + 0.05% Tween 20). Blocking is performed at 37°C for 1 hour. The supernatant is discarded, the plate is blotted dry, and the plate is washed three times with 1X PBST (0.05%), 300 μL each time. The plate is blotted dry, and 100 μL / well is added to diluted sample (diluted with the coating buffer). The plate is incubated at 37°C for 1 hour. The supernatant is discarded, the plate is blotted dry, and the plate is washed three times with 1X PBST (0.05%), 300 μL each time. The plate is then blotted dry. MonoRab TM Rabbit Anti-scFv Cocktail (HRP) was diluted 1:5000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1X PBST (0.05%) each time. The ELISA plate was blotted dry, and 100 μL / well of TMB chromogenic buffer was added. The plate was incubated at 37°C for 5-10 minutes. The reaction was stopped by adding 50 μL / well of 2N H2SO4, and the plate was read using an OD450 microplate reader.

[0311] CNTF protein detection method: The human CNTF ELISA kit (abcam, ab264608, 1*96 tests) was used for detection, and the plate was read by an OD450 microplate reader.

[0312] The expression of Ecu-scFv protein is shown in Figure 8A. The results show that the expression level of Ecu-scFv protein is 3527.42 ng / ml in the single-target plasmid T536, 1809.87 ng / ml in the insulator plasmid T561, and also in plasmids T581, T582, and T583, with expression levels of 1806.75 ng / ml, 811.62 ng / ml, and 1919.47 ng / ml, respectively.

[0313] The expression of CNTF protein is shown in Figure 8B. The results show that CNTF is expressed at a level of 17078.59 pg / ml in the single-target plasmid T547, and is also expressed in plasmids T581, T582, and T583, with expression levels of 7222.02 pg / ml, 14131.86 pg / ml, and 6366.93 pg / ml, respectively. However, CNTF protein expression was not detected in the insulator plasmid T561.

[0314] Example 11 ARPE19 cell infection experiment

[0315] After digestion, ARPE19 cells were seeded at 5E4 cells / well in 48-well plates and cultured overnight at 37°C with 5% CO2 in DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. Viruses (rAAV-T583, rAAV-T855, rAAV-T860, rAAV-T861) were added to the cells at an MOI of 1E3. After 6 hours, the supernatant was removed, and the medium was replaced with DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. The cells were then cultured at 37°C with 5% CO2 for 72 hours. The cell supernatant was collected, centrifuged at 2000 rpm for 5 min, and then used for ELISA detection.

[0316] Detection methods

[0317] Ecu-scFv protein detection method: Human C5 antigen is coated onto the ELISA plate using 1X PBS at a concentration of 4 μg / mL (100 μL / well). The plate is incubated overnight at 4°C. The supernatant is discarded, and the plate is blotted dry. The plate is washed three times with 1X PBST (PBS + 0.05% Tween 20) (0.05%), 300 μL each time. The plate is blotted dry, and 300 μL / well is added to blocking buffer (1% BSA + 0.05% Tween 20). Blocking is performed at 37°C for 1 hour. The supernatant is discarded, and the plate is blotted dry. The plate is washed three times with 1X PBST (0.05%), 300 μL each time. The plate is blotted dry, and 100 μL / well is added to diluted supernatant sample. The sample is diluted with the coating buffer and incubated at 37°C for 1 hour. The supernatant is discarded, and the plate is blotted dry. The plate is washed three times with 1X PBST (0.05%), 300 μL each time. The plate is then blotted dry. MonoRab TM Rabbit Anti-scFv Cocktail (HRP) was diluted 1:5000 with blocking buffer, and 100 μL / well was added to the plate and incubated at 37°C for 1 hour. The supernatant was discarded, the ELISA plate was blotted dry, and the plate was washed three times with 300 μL of 1X PBST (0.05%) each time. The ELISA plate was blotted dry, and 100 μL / well of TMB chromogenic buffer was added. The reaction was incubated at 37°C for 5–10 min, and the reaction was stopped by adding 50 μL / well of 2N H₂SO₄. The plate was read using an OD450 microplate reader. The expression of Ecu-scFv protein is shown in Figure 9A.

[0318] CNTF protein detection method: The human CNTF ELISA kit (abcam, ab264608, 1*96 tests) was used for detection, and the plate was read by an OD450 microplate reader. The expression of CNTF protein is shown in Figure 9B.

[0319] Example 12 In vitro MAC complex immunofluorescence assay

[0320] Experimental Methods: ARPE19 cells were seeded in 96-well plates at 2E4 wells each, cultured in DMEM / F-12 medium with 10% FBS and 1% penicillin antibody, and incubated overnight at 37°C with 5% CO2. rAAV viruses (rAAV-T536, rAAV-T547, rAAV-T583) were infected with different MOI gradients (0, 2E3, 3E8, 4E4, 2E5, with the NC group receiving no subsequent treatment). After 6 hours, the virus-added medium was removed and replaced with DMEM / F-12 medium with 10% FBS and 1% penicillin antibody. After 48 hours, 3.5% non-inactivated human serum was added to the medium, and the cells were incubated at 37°C for 30 minutes. The supernatant was discarded, and the cells were washed twice with 1X PBS. The supernatant was discarded again, and 100 μL / well of 4% paraformaldehyde was added for fixation at 4°C for 10 minutes. Wash cells three times with 1X PBS, then block with 300 μL / well of 3% BSA for 20 min. Add 100 μL / well of Anti-C5b-9 antibody (abcam, ab55811) diluted 1:500 with 3% BSA and incubate at 37°C for 1.5 h. Wash cells three times with 1X PBS and discard the supernatant. Goat anti-rabbit IgG H&L (Alexa) 488)(abcam, ab150077) was diluted 1:500 with 3% BSA, and 100 μL / well was added to each cell. Cells were incubated at 37°C for 1 hour. Cells were washed three times with 1X PBS, and the supernatant was discarded. Hochest staining was performed, and images were taken using a high-content cell imaging system.

[0321] Experimental Results: Complement activation ultimately leads to the formation of the membrane attack complex (MAC), which damages cells. In the in vitro immunofluorescence assay, green fluorescence represents MAC complex staining; stronger green fluorescence indicates greater MAC complex formation and stronger cell damage. The fluorescence staining results are shown in Figure 10A, and the statistical results are shown in Figure 10B. Figures 10A and 10B show that both single-target groups (rAAV-T536 and rAAV-T547) and one dual-target group (rAAV-T583) significantly inhibited MAC complex formation and protected cells compared to the positive control (MOI concentration of 0). The dual-target group (rAAV-T583) showed significantly better inhibitory effects than the two single-target groups (rAAV-T536 and rAAV-T547) at viral MOI concentrations of 2E3 and above.

[0322] Example 13 Cell senescence β-galactosidase staining experiment

[0323] Experimental Methods: ARPE19 cells were seeded in 2E3 wells of 96-well plates and cultured overnight at 37°C with 5% CO2 in DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. ARPE19 cells were infected with viruses (rAAV-mCherry, rAAV-T536, rAAV-T547, rAAV-T583) at an MOI of 1E5. After 24 hours, the virus-added medium was removed and replaced with DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. Detection was performed on day 6 post-infection using a β-galactosidase staining kit (Solepro, G1580).

[0324] Experimental Results: Senescent cells possess highly active β-galactosidase. In situ staining with X-Gal as a substrate, a deep blue product is generated under the catalysis of β-galactosidase, which can be observed under an optical microscope. The results are shown in Figure 11. Cells containing deep blue material represent senescent cells. The results show that the dual-target virus rAAV-T583 produced relatively less deep blue substrate, indicating that the dual-target virus rAAV-T583 has a certain protective effect against ARPE19 cell senescence.

[0325] Example 14 Apoptosis Experiment

[0326] Experimental Methods: ARPE19 cells were seeded in 2E4 wells of 96-well plates and cultured overnight at 37°C with 5% CO2 in DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. ARPE19 cells were infected with viruses (rAAV-T536, rAAV-T547, rAAV-T583) at different MOI gradients (0, 5E3, 1E4, 5E4, 1E5, 5E5, with the NC group receiving no further treatment). After 6 hours, the virus-added medium was removed and replaced with DMEM / F-12 medium + 10% FBS + 1% penicillin antibody. After 48 hours, 5% non-inactivated human serum was added for 2 hours, and the cells were cultured at 37°C with 5% CO2. Apoptosis was detected using the Annexin V-FITC apoptosis detection kit (Beyotime, C1062S).

[0327] Experimental Results: The fluorescence staining results of AEPE19 cells are shown in Figure 12A, and the fluorescence intensity statistics are shown in Figure 12B. Phosphatidylserine is mainly distributed on the inner side of the cell membrane. In the early stages of apoptosis, different types of cells evert phosphatidylserine to the cell surface. Annexin V, labeled with the FITC fluorescent probe carrying green fluorescence, directly detected this important characteristic of apoptosis—the eversion of phosphatidylserine. Propidium iodide staining solution: Propidium iodide can stain necrotic cells or cells that have lost their cell membrane integrity in the late stage of apoptosis, showing red fluorescence. When it is only green, it indicates apoptosis; the more apoptotic cells there are, the greener it is. When it shows yellow (co-localization of green and red), it indicates cell necrosis; the more necrotic cells there are, the yellower it is. The fluorescence results in Figures 12A and 12B show that the green color of the dual-target virus group (rAAV-T583) is significantly less than that of the single-target virus groups (rAAV-T536 and rAAV-T547), indicating that both single-target groups and one dual-target group showed an inhibitory effect on apoptosis compared to the untreated group. Among them, the rAAV-T583 group showed better apoptosis inhibition than the two single-target groups at viral MOI 5E4 and above.

[0328] Example 15: Sodium iodate-induced dry AMD model experiment in mice

[0329] Experimental methods: Six-week-old C57BL / 6 male mice (N=6) were injected intravitreally with viral drugs (rAAV-T536, rAAV-T547, rAAV-T583) at a dose of 2E9 vg / eye. Two weeks later, a dry AMD model was induced by intravenous injection of NaIO3 (sodium iodate) at a dose of 35 mg / kg. The negative control group received PBS; the NaIO3 model group received no drugs. One week after NaIO3 injection to induce the model, the mice were anesthetized, and their eyeballs were removed, paraffin sections were prepared, stained with hematoxylin and eosin (HE), and photographed.

[0330] Experimental Results: The results are shown in Figure 13. In the NaIO3 model group, the ONL layer was severely disordered and thinned, and the RPE layer was severely damaged. In contrast, the ONL layer in the single-target group (rAAV-T536, rAAV-T547) was less disordered and thicker than that in the model group, indicating that the single-target drug did not have a significant protective effect on the ONL and RPE layers. In the dual-target group (rAAV-T583), the ONL and RPE layers were comparable to those in the control group (PBS), indicating a good protective effect, and the protective effect on the RPE layer was significantly better than that of the single-target group.

[0331] Example 16: Hemolysis test of red blood cells in Mianyang

[0332] Experimental Methods: ARPE19 cells were seeded in 2E4 wells of 96-well plates and cultured overnight at 37°C with 5% CO2 in DMEM / F-12 medium + 10% FBS + 1% penicillin-dip antibiotics. Different viruses (rAAV-T536, rAAV-T547, rAAV-T583) were introduced into the cells at an MOI of 2E5. After 6 hours, the virus-added medium was removed and replaced with DMEM / F-12 medium + 10% FBS + 1% penicillin-dip antibiotics. After 7-12 hours, the cell supernatant was collected, and the concentrations of expressed Ecu-scFv and CNTF proteins were measured. The concentrations of rAAV-expressed Ecu scFv and CNTF proteins were then normalized to 1600 nmol and diluted using a 2-fold dilution method, resulting in concentrations of 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 nmol. Each protein was then mixed with 2% human serum and set aside for later use. Take 5 mL of 2% Mianyang red blood cells (Beijing Bosch Co., Ltd., P2791905), centrifuge at 400×g for 5 min, resuspend in 5 mL of GVB++ buffer and wash repeatedly until no blood cells are present in the supernatant, add 2.5 μL of hemolysin (Beijing Bosch Co., Ltd., BM351Y) and mix well with the protein-serum complex, aspirate 100 μL / well and add to a 96-well plate, incubate at 37℃ for 30 min, centrifuge at 400×g for 10 min, collect the supernatant and add to a new 96-well plate to the microplate reader to read the OD415 value, and calculate the inhibition rate and IC50.

[0333] Experimental Results: The results are shown in Figure 14 and Table 4. The IC50 values ​​of Ecu scFv protein expressed by rAAV-T536 and CNTF protein expressed by rAAV-T547 were both high, and Ecu scFv showed better protection against hemolysis of Mianyang erythrocytes than CNTF. However, the IC50 value of Ecu scFv+CNTF protein expressed by rAAV-T583 was significantly lower than that of Ecu scFv protein expressed by rAAV-T536 or CNTF protein expressed by rAAV-T547, and superior to the control C5 aptamer-Lzervay. These results indicate that rAAV expression of a dual-target protein provides better protection against hemolysis of Mianyang erythrocytes than rAAV expression of a single-target protein.

[0334] Table 4

Claims

1. The application of the combination of complement inhibitors and neurotrophic factors in the preparation of drugs for the treatment of eye diseases.

2. The application of complement inhibitors and neurotrophic factors in the preparation of viral vector drugs for the treatment of eye diseases.

3. Use according to claim 1 or 2, characterized in that, The viral vector includes adeno-associated virus (AAV), adenovirus (Ad), lentivirus (LV), and retrovirus (RV), with adeno-associated virus being preferred.

4. Use according to claim 1 or 2, characterized in that, The complement inhibitor is a C5 complement inhibitor; preferably, the C5 complement inhibitor is selected from Ravulizumab or Eculizamub and their antigen-binding fragments; the neurotrophic factor is selected from PEDF, CNDF, CNTF or RdCVF, preferably CNTF.

5. Use according to claim 1 or 2, characterized in that, The eye disease is a fundus disease; preferably, the fundus disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoid choroidal vascular disease, wet age-related macular degeneration with extremely low vision, and choroidal neovascularization secondary to pathological myopia; more preferably, the fundus disease is dry age-related macular degeneration; even more preferably, the fundus disease is geographic atrophy (GA).

6. A nucleic acid, characterized in that, The nucleic acid contains two target genes and a bidirectional promoter, and the nucleic acid has the following structure: gene B - promoter B - enhancer - promoter A - gene A; wherein promoter A and promoter B are located on both sides of the promoter and the transcription directions are opposite.

7. The nucleic acid of claim 6, wherein The gene A or gene B encodes a complement inhibitor or complement regulatory protein, neurotrophic factor, anti-vascular endothelial growth factor, endostatin, or anti-angiogenic factor, and the gene A and gene B may be the same or different; preferably, the gene A and gene B encode a complement inhibitor and a neurotrophic factor, respectively.

8. The nucleic acid of claim 6, wherein The promoter A and promoter B are selected from: CB, CMV, miniCMV promoter, U1a, EF1α, MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, artificial splicing promoter, pMNTC promoter, and the promoter A and promoter B may be the same or different; preferably, the promoter is selected from CB promoter, CMV promoter, CAG promoter or miniCMV promoter.

9. The nucleic acid of claim 6, wherein The enhancer is selected from CAG enhancer, CMV enhancer or UBC enhancer.

10. The nucleic acid of claim 6, wherein The promoter B-enhancer-promoter A is selected from: miniCMV promoter-CMV enhancer-CMV promoter, CMV promoter-CMV enhancer-miniCMV promoter, CMV promoter-CMV enhancer-CB promoter, CB promoter-CMV enhancer-CMV promoter, CAG promoter-CMV enhancer-CMV promoter, or CMV promoter-CMV enhancer-CAG promoter.

11. The nucleic acid of claim 6, wherein The relationship between gene B and promoter B or promoter A and gene A further includes introns; preferably, the introns are selected from PI introns, VH4 introns, SV40 introns, Chi introns, U12 introns, RHD introns, and MBL introns.

12. The nucleic acid of claim 6, wherein The downstream of gene B-promoter B or promoter A-gene A further includes a polyadenylation signal; preferably, the polyadenylation signal includes SV40, HGH, BGH or β-RGB.

13. The nucleic acid of claim 6, wherein The nucleic acid comprises the following structure: polyadenylation signal B-gene B-intron B-promoter B-enhancer-promoter A-intron A-gene A-polyadenylation signal A.

14. An expression vector comprising the nucleic acid of any one of claims 6-13; preferably, the expression vector is a prokaryotic expression vector, a eukaryotic expression vector, a recombinant bacterium, a cell, or a virus; preferably, the expression vector is a plasmid or recombinant adeno-associated virus (rAAV).

15. The expression vector of claim 14, wherein, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) Polyadenylation signal B; (c) Gene B; (d) Intron B; (e) Promoter B; (f) Enhancer; (g) Promoter A; (h) Intron A; (i) Gene A; (j) Polyadenylation signal A; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

16. The expression vector of claim 15, wherein, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) Polyadenylation signal B; (c) Neurotrophic factor coding sequence; (d) Intron B; (e) miniCMV promoter; (f) CMV enhancer; (g)CMV promoter; (h) Intron A; (i) The coding sequence of the C5 complement inhibitor; (j) Polyadenylation signal A; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) Polyadenylation signal B; (c) Neurotrophic factor coding sequence; (d) Intron B; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h) Intron A; (i) The coding sequence of the C5 complement inhibitor; (j) Polyadenylation signal A; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) Polyadenylation signal B; (c) The coding sequence of the C5 complement inhibitor; (d) Intron B; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h) Intron A; (i) Neurotrophic factor coding sequences; (j) Polyadenylation signal A; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

17. The expression vector of claim 16, wherein, The intron A or intron B is selected from PI intron, VH4 intron, SV40 intron, Chi intron, U12 intron, RHD intron or MBL intron.

18. The expression vector of claim 16, wherein, The polyadenylation signal A or polyadenylation signal B is selected from SV40, HGH, BGH or β-RGB.

19. The expression vector of claim 16, wherein, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) Neurotrophic factor coding sequence; (d) MBL introns; (e) miniCMV promoter; (f) CMV enhancer; (g)CMV promoter; (h)PI introns; (i) The coding sequence of the C5 complement inhibitor; (j) BGH polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) Neurotrophic factor coding sequence; (d) MBL introns; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h)PI introns; (i) The coding sequence of the C5 complement inhibitor; (j) BGH polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) The coding sequence of the C5 complement inhibitor; (d) MBL introns; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h)PI introns; (i) Neurotrophic factor coding sequences; (j) BGH polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) The coding sequence of the C5 complement inhibitor; (d) MBL introns; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h)PI introns; (i) Neurotrophic factor coding sequences; (j)SV40 polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) The coding sequence of the C5 complement inhibitor; (d) MBL introns; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h)PI introns; (i) Neurotrophic factor coding sequences; (j)RGH polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal; (c) The coding sequence of the C5 complement inhibitor; (d) PI introns; (e) CMV promoter; (f) CMV enhancer; (g)CB promoter; (h)SV40 intron; (i) Neurotrophic factor coding sequences; (j) BGH polyadenylation signal; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

20. The expression vector of any one of claims 14-19, wherein, The C5 complement inhibitor is selected from Ravulizumab, Eculizamub, or their antigen-binding fragments, preferably Eculizamub or its antigen-binding fragments, and more preferably Eculizamub scFv; the neurotrophic factor is selected from PETF, CNDF, CNTF, or RdcvF, preferably CNTF.

21. The expression vector of any one of claims 14-20, wherein, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7; (c) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6; (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8; (e) a miniCMV promoter comprising the nucleotide sequence described in SEQ ID NO:9; (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10; (g) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11; (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12; (i) The Eculizamub scFv coding sequence, which comprises the nucleotide sequence described in SEQ ID NO:4; (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7; (c) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6; (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8; (e) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11; (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10; (g) A CB promoter comprising the nucleotide sequence described in SEQ ID NO:15; (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12; (i) The Eculizamub scFv coding sequence, which comprises the nucleotide sequence described in SEQ ID NO:4; (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; or, The nucleic acid of the expression vector comprises, from the 5' to the 3' direction: (a) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence; (b) RGB polyadenylation signal, wherein the RGB contains the nucleotide sequence described in SEQ ID NO:7; (c) The coding sequence of Eculizamub scFv, which comprises the nucleotide sequence described in SEQ ID NO:4; (d) MBL introns, wherein the MBL contains the nucleotide sequence described in SEQ ID NO:8; (e) A CMV promoter comprising the nucleotide sequence described in SEQ ID NO:11; (f) A CMV enhancer comprising the nucleotide sequence described in SEQ ID NO:10; (g) A CB promoter comprising the nucleotide sequence described in SEQ ID NO:15; (h) PI introns, wherein the PI introns comprise the nucleotide sequence described in SEQ ID NO:12; (i) A CNTF coding sequence comprising the nucleotide sequence described in SEQ ID NO:6; (j) BGH polyadenylation signal, wherein the BGH comprises the nucleotide sequence described in SEQ ID NO:13; (k) Adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

22. The expression vector of claim 21, wherein, The nucleic acid of the expression vector contains, from 5' to 3', the nucleotide sequences described in SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:17 or nucleotide sequences having at least 85% homology with them.

23. The expression vector of any one of claims 14-22, wherein, The capsid protein of the recombinant adeno-associated virus is selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10 or their mutants; preferably, the capsid protein is the AAV2 serotype capsid protein or its mutant.

24. An adeno-associated virus capsid protein mutant, wherein, The capsid protein is an insertion of a heterologous polypeptide comprising about 7 to 8 amino acids relative to the wild-type AAV capsid protein. The heterologous polypeptide includes an amino acid sequence selected from IADSRTS (SEQ ID NO:18), IGETSRTS (SEQ ID NO:20), or IADNTRP (SEQ ID NO:21), and the insertion site is located between amino acid positions 587 and 588 of the wild-type AAV2 capsid protein or at the corresponding position of other serum-type capsid proteins.

25. The mutant of claim 24, wherein The other serum capsid proteins are selected from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh10.

26. The mutant of claim 24, wherein The mutant, relative to the wild-type AAV2 capsid protein, further includes amino acid substitutions at corresponding positions of I240T, V708I, Y444F, Y500F and / or Y730F, or other serum-type capsid proteins.

27. The mutant of claim 26, wherein The mutant, relative to the wild-type AAV2 capsid protein, includes amino acid substitutions of I240T and V708I. Preferably, the mutant, relative to the wild-type AAV2 capsid protein, includes amino acid substitutions of I240T, V708I, Y444F, Y500F, and Y730F.

28. The expression vector of claim 23, wherein, The capsid protein is an AAV2 capsid protein mutant, and the mutant is selected from the capsid protein mutants of any one of claims 24-27; preferably, the mutant comprises the insertion of a heterologous polypeptide at amino acid positions 587 and 588 of wild-type AAV2 and the substitution of amino acids I240T, V708I, Y444F, Y500F and Y730F, and the heterologous polypeptide is IADSSRT (SEQ ID NO:18), IGETRT (SEQ ID NO:20) or IADNTRP (SEQ ID NO:21).

29. A pharmaceutical composition comprising The pharmaceutical composition includes a nucleic acid according to any one of claims 6-13 or an expression vector and a pharmaceutically acceptable vector according to any one of claims 14-23 or 28; preferably, the pharmaceutical composition is an intravitreal injection, a subretinal injection, a choroidal injection, an intravenous injection, an intratumoral injection, or an intramuscular injection.

30. Use of the nucleic acid of any one of claims 6-13 or the expression vector of any one of claims 14-23 or 28 in the manufacture of a medicament for the treatment of an ocular disease; preferably, the ocular fundus disease is an ocular disease; more preferably, the ocular fundus disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with very low visual acuity, choroidal neovascularization secondary to pathologic myopia; more preferably, the ocular disease is dry age-related macular degeneration; more preferably, the ocular disease is geographic atrophy (GA).

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