Expression cassette combinations and their use

Abstract

The present invention relates to a combination of expression cassettes and its use. The combination of expression cassettes comprises a first expression cassette and a second expression cassette, wherein the first expression cassette is capable of expressing a fusion protein of the N-terminal cleavage of the ABCA4 protein and the N-terminus of the intein, and the second expression cassette is capable of expressing a fusion protein of the C-terminal cleavage of the ABCA4 protein and the C-terminus of the intein, wherein, when expressed, the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette and the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette can form a complete full-length ABCA4 protein via splicing function of the N-terminus and C-terminus of the intein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to combinations of expression cassettes and their use.

Background Art

[0002] ABCA4 is a lipid flippase in retinal photoreceptor cells or retinal pigment epithelial cells, and transports N-retinyl-phosphatidylethanolamine (NRPE), a lipid derivative formed from phosphatidylethanolamine (PE) and all-trans retinal (ATR), from the luminal side of the outer segment papilla to the cytoplasmic side. Subsequently, NRPE is hydrolyzed into ATR and PE, and ATR therein is catalyzed by retinal reductase in the cytoplasm to become all-trans retinal (ATRol) and return to the visual cycle system. When the lipid transport function of ABCA4 is impaired, ATR and NRPE accumulate excessively on the luminal side of the outer segment papilla, and the excessively accumulated ATR and NRPE further irreversibly form toxic bis-retinyl derivatives, ultimately causing a series of retinal degenerative diseases (including the most common hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and cone-rod dystrophy, etc.). The full length of the human ABCA4 CDS is 6.7 kb and encodes 2273 amino acids.

[0003] Intein is several peptide fragments present inside the peptide chain of the precursor of a specific protein, and is cleaved by a non-enzymatic transpeptidation reaction during the conversion process to a mature protein, while the corresponding one is an exopeptide retained in the mature protein. Intein is widely used in protein purification, protein labeling, and the stitching and expression of large protein fragments.

[0004] CN115074369A relates to a technique for expressing the ABCA4 protein using a dual AAV vector strategy with split intein. The most important aspects of a dual AAV vector strategy with split intein are the intein sequence and cleavage site. These significantly affect the splicing efficiency of the target protein, the tertiary structure and post-translational modifications of the target protein, and furthermore, the protein function. In addition, since dual AAV vectors with split intein express the N-terminus and C-terminus of the ABCA4 protein separately, inefficient stitching results in N-terminal and C-terminal byproducts, which pose a potential safety risk. In CN115074369A, the full-length ABCA4 protein is cleaved at p1150Cys to obtain two fragments, and the intein used is either Rma intein or Npu intein.

[0005] CN115698307A discloses an intein and its use, wherein a process-modified split intein and a combination thereof with a degradation signal (destabilization domain, degradation determinant) are used to reconstruct a large gene. One of the main limitations in the above application of the intein disclosed in CN115698307A is the accumulation of protein intermediates (N-exopeptide-IntN and IntC-C-exopeptide) and cleaved split intein. Those skilled in the art will understand that the accumulation of these unwanted proteins may lead to undesirable side effects (immune reactions). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] To solve the above problems, the inventors conducted extensive research and found that by selecting a specific cleavage site of the ABCA4 protein, they obtained N-terminal and C-terminal cleavages of the ABCA4 protein. Furthermore, by binding these to the N-terminal and C-terminal portions of a specific intein, they obtained a first expression cassette that expresses a fusion protein of the N-terminal cleavage of the ABCA4 protein and the N-terminal portion of the intein, and a second expression cassette that expresses a fusion protein of the C-terminal cleavage of the ABCA4 protein and the C-terminal portion of the intein. The first and second expression cassettes form by efficiently expressing the complete ABCA4 protein in cells, successfully packaging AAV5 and AAV8 viruses, and effectively expressing them in mouse retinal cells, thereby restoring retinal function in ABCA4 KO mice. Co-infection of IPSC-RPE or IPSC-PRC cells derived from Stargardt disease patients with the above-mentioned viruses restores the cells' retinol conversion function and reduces A2E (retinoid dimer (N-retinyl-N-retinolamine)) deposition. Furthermore, using GP41-1 intein has the potential advantage of producing less intein protein by the expression cassette mentioned above, resulting in a lower immune response.

[0007] In particular, the inventors unexpectedly discovered that by selecting GP41-1 intein, not only is the highest complete ABCA4 protein expression efficiency achieved compared to other inteins, but the generation of additional inteins is also reduced, resulting in a decrease in undesirable immunogenicity and side effects when used in vivo.

[0008] Furthermore, the inventors unexpectedly discovered that, when using the same type of intein (e.g., GP41-1), selecting a specific cleavage site of the ABCA4 protein (i.e., the 1224Cys site from the N-terminus) resulted in remarkably efficient expression of the complete ABCA4 protein. [Means for solving the problem]

[0009] Accordingly, in one embodiment, the present invention provides a combination of expression cassettes comprising a first expression cassette and a second expression cassette, wherein the first expression cassette is capable of expressing a fusion protein of the N-terminal cleavage of the ABCA4 protein and the N-terminus of the intein, and the second expression cassette is capable of expressing a fusion protein of the C-terminal cleavage of the ABCA4 protein and the C-terminus of the intein, wherein, upon expression, the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette and the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette form a complete full-length ABCA4 protein via splicing function of the N-terminus and C-terminus of the intein.

[0010] In some embodiments, the intent is selected from the group consisting of P41-1 (SEQ ID NO: 5), DnaE (SEQ ID NO: 4), GP41-8 (SEQ ID NO: 6), NrdJ-1 (SEQ ID NO: 7), IMPDH-1 (SEQ ID NO: 8), SspGyrB (SEQ ID NO: 9), and Rma (SEQ ID NO: 60), and is preferably GP41-1.

[0011] In some embodiments, the complete ABCA4 protein is human ABCA4 protein (SEQ ID NO: 1), and the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette and the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette are two corresponding N-terminal and C-terminal cleavages obtained by cleaving the complete ABCA4 protein from the N-terminus of the ABCA4 protein at the following amino acid (Cys) sites: 1224, 1140, 1150, or 1188, preferably 1188 or 1224.

[0012] In some embodiments, the N-terminal section and the C-terminal section are in the following combinations: The first 1-1223 amino acids and the 1224-2273 amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1), The first 1-1139 amino acids and the first 1140-2273 amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1), The first 1-1149 amino acids and the 1150-2273 amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1), or The amino acids selected are those from the 1st to 1187th and 1188th to 2273rd amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

[0013] In some embodiments, the N-terminus and C-terminus of the intein are as follows: 1)-9): 1) The N-terminus of GP41-1 (SEQ ID NO: 17) and the C-terminus of GP41-1 (SEQ ID NO: 18), 2) The N-terminus of DnaE (SEQ ID NO: 15) and the C-terminus of DnaE (SEQ ID NO: 16), 3) The N-terminus of GP41-8 (SEQ ID NO: 19) and the C-terminus of GP41-8 (SEQ ID NO: 20), 4) The N-terminus of NrdJ-1 (SEQ ID NO: 21) and the C-terminus of NrdJ-1 (SEQ ID NO: 22), 5) The N-terminus of IMPDH-1 (SEQ ID NO: 23) and the C-terminus of IMPDH-1 (SEQ ID NO: 24), 6) The N-terminus of SspGyrB (SEQ ID NO: 25) and the C-terminus of SspGyrB (SEQ ID NO: 26), 7) The N-terminus of Mja-KlbA (SEQ ID NO: 27) and the C-terminus of Mja-KlbA (SEQ ID NO: 28), 8) The N-terminus of NpuSsp (SEQ ID NO: 29) and the C-terminus of NpuSsp (SEQ ID NO: 30), 9) Selected from the group consisting of the N-terminus (SEQ ID NO: 61) and the C-terminus (SEQ ID NO: 62) of Rma.

[0014] In some embodiments, the intein is GP41-1, and the N-terminal and C-terminal cleavages of the ABCA4 protein are two corresponding N-terminal and C-terminal cleavages obtained by cleaving the ABCA4 protein from the N-terminus at the 1224th amino acid (Cys) site, namely, the 1-1223rd and 1224-2273rd amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

[0015] In some embodiments, the intein is GP41-1, and the N-terminal and C-terminal cleavages of the ABCA4 protein are the corresponding N-terminal and C-terminal cleavages obtained by cleaving the ABCA4 protein from the N-terminus at the 1188th amino acid (Cys) site, i.e., the 1-1187th and 1188-2273rd amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

[0016] In some embodiments, the codon expressing the full-length ABCA4 protein is an optimized codon, the sequence of which is shown in Sequence ID No. 3. In some embodiments, the codons expressing the N-terminal cleavage and the C-terminal cleavage of the ABCA4 protein are optimized codons, and the sequence after combining (merging / linking) the two, i.e., the coding sequence of the full-length ABCA4 protein, is shown in Sequence ID No. 3. Those skilled in the art will readily understand that the codon optimization may be an optimization of the sequence of the full-length ABCA4, or an optimization of the codon for the sequences of the N-terminal cleavage and the C-terminal cleavage, respectively.

[0017] In some embodiments, a first tag is fused to the C-terminus of the first expression cassette and / or a second tag is fused to the C-terminus of the second expression cassette, where the first tag and the second tag are different. The first tag and the second tag are independently selected from the group consisting of hemagglutinin (HA) tags, Flag tags, V5 tags, Myc tags, His tags, 1D4 tags (Rho1D4) and combinations thereof, preferably the first tag is a hemagglutinin (HA) tag (SEQ ID NO: 12) and / or the second tag is a Flag tag (SEQ ID NO: 13).

[0018] In some embodiments, the N-terminus or C-terminus of the intein is further linked directly or indirectly via a linker to a degradation signal, preferably selected from polypeptides associated with DHFR (dehydrofolate reductase), FKBP (FK506-binding protein), FRB (FKBP-rapamycin-binding protein), and PDE5 (phosphodiesterase type 5), and degradation signals of the endoplasmic reticulum-associated degradation (ERAD) pathway, more preferably the degradation signal is ecDHFR (SEQ ID NO: 14).

[0019] In some embodiments, the first expression cassette and / or the second expression cassette comprises a promoter and a terminator for expressing the fusion protein, and optionally comprises enhancers and introns operably linked to the fusion protein to enhance the expression of the fusion protein.

[0020] In some embodiments, the promoter is a constitutive promoter or an inductive promoter, preferably a CAG promoter (also referred to as hybrid CMV initial enhancer / chicken β-actin promoter, CAGGS promoter, CB promoter or CBA promoter), a human β-actin promoter, a small CBA (smCBA) promoter, a CBS promoter or CBh promoter, an elongation factor 1α short (EFS) promoter, an elongation factor 1α (EF-1α) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a VMD2( The promoter is selected from the group consisting of the BEST1 promoter, ABCA4 self-promoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter, or smCAG promoter, and more preferably the OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48), smCAG promoter (SEQ ID NO: 49), enOPEFS promoter (SEQ ID NO: 67), enhGRK1 (enRK) promoter (SEQ ID NO: 68), hGRK1-b-globin-intron promoter (SEQ ID NO: 69), or enhGRK1-b-globin-intron promoter (SEQ ID NO: 70).

[0021] In some embodiments, the terminator is selected from the group consisting of SV40 terminator (SEQ ID NO: 50), BGH terminator (SEQ ID NO: 51), WPRE terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), WPRE-SV40 terminator (SEQ ID NO: 56), WPRE-BGH terminator, B-globin-polyA terminator (SEQ ID NO: 65), or sNRP1 terminator (SEQ ID NO: 66).

[0022] In some embodiments, the enhancer is a CMV enhancer (SEQ ID NO: 53). In some embodiments, the intron is an intron derived from rhesus macaque beta-herpesvirus-3 (MHV-3) (SEQ ID NO: 54) or an SV40 intron (SEQ ID NO: 55).

[0023] In some embodiments, the first expression cassette includes, in order from the 5'-end, an optional enhancer, a promoter, an optional intron, a nucleotide coding sequence of an N-terminal truncation of the ABCA4 protein, a nucleotide coding sequence of the N-terminal of an intein, an optional degradation signal, a nucleotide coding sequence of an optional tag, and a terminator, and / or the second expression cassette includes, in order from the 5'-end, an optional enhancer, a promoter, an optional intron, a nucleotide coding sequence of the C-terminal of an intein, an optional degradation signal, a nucleotide coding sequence of a C-terminal truncation of the ABCA4 protein, a nucleotide coding sequence of an optional tag, and a terminator. The nucleotide coding sequence of the N-terminal truncation of the ABCA4 protein, the nucleotide coding sequence of the N-terminal of the intein, the nucleotide coding sequence of the C-terminal of the intein, the nucleotide coding sequence of the C-terminal truncation of the ABCA4 protein, the nucleotide coding sequence of an optional tag, and an optional degradation signal sequence are operably linked to the promoter / enhancer.

[0024] In some embodiments, the first expression cassette and / or the second expression cassette exist in a form selected from the group consisting of an isolated nucleic acid molecule, a liposome, and / or an exosome.

[0025] In some embodiments, the first expression cassette and / or the second expression cassette exist in the form of a plasmid.

[0026] In some embodiments, the first expression cassette and / or the second expression cassette is a viral vector.

[0027] In some embodiments, the viral vector is an adeno-associated virus (AAV) of the same or different serotypes, where the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, Selected from the group consisting of AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, AAV2 / 10, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV8-Y447, 733F, or AAVtYF, preferably AAV5, AAV8, AAV8-Y447, 733F, AAVtYF, or AAV2.7m8.

[0028] In some embodiments, the expression cassette combination is a combination of the following elements: 1) The promoter is hGRK1, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is Rma, and the terminator is SV40. 2) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is SV40. 3) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is Rma, and the terminator is B-globin-polyA. 4) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is B-globin-polyA. 5) The promoter is hGRK1, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is SV40. 6) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1150, the intent is GP41-1, the terminator is SV40, or 7) The promoter is enRK-b-globin intron, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intein is GP41-1, and the terminator is B-globin-polyA.

[0029] On the other hand, the present invention provides a kit that includes the above-mentioned combination of expression cassettes.

[0030] On the other hand, the present invention provides the use of a combination or kit of the expression cassettes of the present invention in the manufacture of pharmaceuticals for treating diseases including diseases caused by ABCA4 mutations.

[0031] In some embodiments, the disease includes hereditary retinal diseases.

[0032] In some embodiments, the disease includes hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0033] Those skilled in the art will readily understand other aspects and advantages of the present invention from the following detailed description. The detailed description below shows and describes only exemplary embodiments of the present invention. As those skilled in the art will understand, the content of the present invention allows those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Accordingly, the drawings and description of the present invention are illustrative and not limiting. [Brief explanation of the drawing]

[0034] The above features and advantages of the present invention will become more apparent from the following detailed description, which combines the attached drawings. [Figure 1] This shows the expression intensity of the ABCA4 protein with optimized codons. [Figure 2] This is a schematic diagram illustrating the formation of the complete ABCA4 protein by the dual expression cassette strategy of the intein according to the present invention. [Figure 3]The ABCA4 protein expression strategy according to the present invention demonstrates the expression efficiency of ABCA4 proteins with different types of split-intane proteins. [Figure 4] This shows the splicing efficiency of GP41-1 and Rma Intein forming the overall length ABCA4. [Figure 5] The present invention demonstrates that different cleavage sites of the ABCA4 protein result in different ABCA4 protein expression efficiencies. [Figure 6] This study demonstrates that a dual expression cassette containing degradation peptides can form a complete ABCA4 protein, and that the degradation signal leads to significant degradation of the intein. [Figure 7] This demonstrates that the expression cassette constructed in Example 5 can form a complete ABCA4 protein in 293T cells. [Figure 8] This example demonstrates that the expression cassette constructed in Example 6 can restore ERG amplitude levels in ABCA4 KO mice. [Figure 9] This shows the A2E content in the retinal tissue of mouse eyes after injection of the expression cassette constructed in Example 6. [Figure 10] This example demonstrates that the expression cassette constructed in Example 6 successfully expressed the complete ABCA4 protein in ABCA4 KO mice. [Figure 11] This indicates that all different cleavage sites effectively expressed the full-length ABCA4 protein in the body. [Figure 12] This indicates that all different stop codons effectively expressed the full-length ABCA4 protein in the body. [Figure 13] This shows that all different promoters effectively expressed the full-length ABCA4 protein in the mouse retina. [Figure 14] This shows that expression cassettes with different combinations of elements all effectively expressed the full-length ABCA4 protein in the model mouse retina. [Figure 15]This shows that different serotypes expressed the full-length ABCA4 protein in the mouse retina. [Figure 16] This indicates a lower content of the additional small peptides produced by GP41-1 in split-intine. [Figure 17] The results of ERG, fundus OCT, and FP detection after subretinal injection of AAV8-GP41-1 and AAV8-DnaE, ​​respectively, in ABCA4 KO mice are shown. [Modes for carrying out the invention]

[0035] Unless otherwise stated, terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are especially encouraged to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0036] The term “first expression cassette” generally includes an exogenous DNA sequence operably ligated to a promoter or other regulatory sequence sufficient to induce transcription of a target gene. In the present invention, the expression cassette generally refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and / or translation of RNA or polypeptide, respectively. The expression cassette may have a set of specialized nucleic acid elements that enable transcription of a particular nucleic acid in the target cell. The expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. The expression cassette may include an untranslatable or nontranslatable antisense or sense construct. When expressing a transgene and repressing an endogenous gene (e.g., repression via antisense, RNAi, or sense), those skilled in the art will understand that the inserted polynucleotide sequence does not need to be identical, but only needs to be substantially identical to the sequence of its origin gene. The expression cassette may include a polynucleotide construct of a transcription factor operably ligated to a promoter, the promoter may be a promoter derived from the gene controlled by the transcription factor. In the present invention, the first expression cassette can express a fusion protein (for example, a fusion protein of the N-terminal cleavage of the ABCA4 protein and the N-terminus of the intein).

[0037] The term "second expression cassette" generally refers to an expression cassette that is not exactly identical to the first expression cassette. For example, the second expression cassette may express a protein different from the protein expressed by the first expression cassette (e.g., a fusion protein of the C-terminal cleavage of the ABCA4 protein and the C-terminus of the intein).

[0038] The term "expression cassette combination" generally refers to a combination of at least one expression cassette. For example, the expression cassette combination according to the present invention may include the first expression cassette and the second expression cassette.

[0039] In the present invention, the term “operably linked” generally refers to locating regulatory sequences necessary for the expression of a coding sequence in appropriate positions relative to the coding sequence to achieve the expression of the coding sequence. For example, if a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. In some embodiments, this can represent the arrangement of coding sequences and transcriptional regulatory elements in an expression vector. The regulatory elements may include promoters, enhancers, and stop elements. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. In some embodiments, “operably linked” can also refer to linking a target gene to a vector such that transcriptional and translational regulatory sequences in the vector perform the intended function of controlling the transcription and translation of the target gene.

[0040] The term "ABCA4 gene" generally refers to the gene encoding the rim protein, or RmP. The ABCA4 gene is also called the ABCR gene. This gene was first cloned and identified as the causative gene for Stargardt disease, an autosomal recessive genetic disorder leading to macular degeneration. The NCBI Entrez Gene accession number for the human ABCA4 gene is 24. In one embodiment, the nucleotide sequence of the ABCA4 gene is shown in Sequence ID No. 2.

[0041] The term “complete (full-length) ABCA4 protein” generally refers to component 4 of the ATP-binding cassette subfamily A, which has a complete structure. The complete ABCA4 protein can be encoded by the human ABCA4 gene. ABCA4 is a lipid inversion enzyme in retinal photoreceptor cells or retinal pigment epithelial cells that transports N-retinyl-phosphatidylethanolamine (NRPE), a lipid derivative formed from phosphatidylethanolamine (PE) and all-trans retinal (ATR), from the luminal side to the cytoplasmic side of the external papilla. Subsequently, NRPE is hydrolyzed into ATR and PE, where ATR is catalyzed by cytoplasmic retinal reductase to all-trans retinal (ATRol), which returns to the visual circulation. The UniProt accession number for the complete human ABCA4 protein is P78363. In one embodiment, the sequence of the complete human (full-length) ABCA4 protein is shown in SEQ ID NO: 1.

[0042] The term “N-terminal cleavage of ABCA4 protein” generally refers to the N-terminal cleavage of the complete human ABCA4 protein. For example, the N-terminal cleavage of ABCA4 protein may contain amino acid sequences represented by amino acids 1-1139, 1-1149, 1-1187, 1-1223, 1-1325, 1-1160, 1-1220, 1-1280, or 1-1347 from the N-terminus of the complete human ABCA4 protein. For example, the amino acid sequence of the N-terminal cleavage of ABCA4 protein appears to be represented by amino acids 1-1325 from the N-terminus of the complete human ABCA4 protein. In the present invention, the N-terminal cleavage of ABCA4 protein and / or the C-terminal cleavage of ABCA4 protein can correspond to the blue and green structures shown in Figure 1 of Nature Communications volume 12, Article number: 3853 (2021), respectively.

[0043] In the present invention, the N-terminal cleavage of the ABCA4 protein may include the transmembrane domains TMD1-TMD6, which are the N-terminal domains of the ABCA4 protein, as well as the extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1, and / or R1.

[0044] In the present invention, the N-terminal cleavage of the ABCA4 protein may include, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and / or R1. For example, the N-terminal cleavage of the ABCA4 protein may include, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6 and / or NBD1.

[0045] In the present invention, the first expression cassette may contain a nucleotide sequence encoding the N-terminal cleavage of the ABCA4 protein.

[0046] The term "C-terminal cleavage of ABCA4 protein" generally refers to the C-terminal cleavage of the complete human ABCA4 protein. For example, the C-terminal cleavage of ABCA4 protein may contain amino acid sequences represented by the complete human ABCA4 protein from the N-terminus at positions 1140-2273, 1150-2273, 1188-2273, 1224-2273, 1326-2273, 1348-2273, 1369-2273, or 1348-2170. For example, the amino acid sequence of the C-terminal cleavage of ABCA4 protein appears to be represented by the amino acid sequence from the N-terminus at positions 1326-2273 of the complete human ABCA4 protein.

[0047] In the present invention, the first expression cassette may include a promoter. In the present invention, the promoter may include a constitutive promoter and / or an inducible promoter. For example, the promoter is selected from the group consisting of the CAG promoter (also referred to as the hybrid CMV initial enhancer / chicken β-actin promoter, CAGGS promoter, CB promoter or CBA promoter), human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1α short (EFS) promoter, elongation factor 1α (EF-1α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also referred to as BEST1) promoter, ABCA4 autopromoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter or smCAG promoter, and more preferably the OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48) or smCAG promoter (SEQ ID NO: 49).

[0048] In the present invention, the C-terminal cleavage of the ABCA4 protein may include the transmembrane domains TMD7-TMD12, which are the C-terminal domains of the ABCA4 protein, the extracellular domains ECD1, IH3, IH4, EH3, EH4, NBD2 and / or R2.

[0049] In the present invention, the C-terminal cleavage of the ABCA4 protein may include, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and / or R2. For example, the C-terminal cleavage of the ABCA4 protein may include, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, and / or NBD2.

[0050] In the present invention, the second expression cassette may include a nucleotide sequence encoding the C-terminal cleavage of the ABCA4 protein.

[0051] In the present invention, the second expression cassette may include a promoter. In the present invention, the promoter may include a constitutive promoter and / or an inducible promoter. For example, the promoter is selected from the group consisting of the CAG promoter (also referred to as the hybrid CMV initial enhancer / chicken β-actin promoter, CAGGS promoter, CB promoter or CBA promoter), human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1α short (EFS) promoter, elongation factor 1α (EF-1α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also referred to as BEST1) promoter, ABCA4 autopromoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter or smCAG promoter, and more preferably the OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48) or smCAG promoter (SEQ ID NO: 49).

[0052] In this specification, the N-terminal and C-terminal cleavage of the ABCA4 protein may be two corresponding N-terminal and C-terminal cleavage obtained by cleaving the complete ABCA4 protein at a specific site (e.g., Cys). In other words, both the N-terminal and C-terminal cleavage of the ABCA4 protein of the present invention (e.g., via the splicing function of the N-terminus and C-terminus of the respective bound inteins) can form the complete ABCA4 protein after splicing in the N-terminal to C-terminal direction, thereby enabling them to exert their biological function. Preferred cleavage sites are the following amino acid (Cys) sites starting from the N-terminus of the ABCA4 protein: 1224, 1140, 1150, or 1188, more preferably 1188 or 1224. For example, if the cleavage site is 1188, the N-terminal cleavage of the ABCA4 protein consists of amino acids 1-1187 from the N-terminus, and the corresponding C-terminal cleavage consists of amino acids 1188-2273 from the N-terminus. For example, if the cleavage site is 1224, the N-terminal cleavage of the ABCA4 protein consists of amino acids 1-1223 from the N-terminus, and the corresponding C-terminal cleavage consists of amino acids 1224-2273 from the N-terminus.

[0053] In the present invention, the protein molar ratio of the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette to the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette is approximately 3:1 to 1:3. For example, the protein molar ratio of the N-terminal cleavage of the ABCA4 protein to the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette may be approximately 3:1 to 1:3, approximately 3:1 to 1:2, approximately 3:1 to 1:1, approximately 3:1 to 2:1, approximately 2:1 to 1:3, approximately 2:1 to 1:2, approximately 2:1 to 1:1, approximately 1:1 to 1:3, approximately 1:2 to 1:1, or approximately 1:1.

[0054] The term "biological function" generally refers to the natural activity of the biological entity under test, or the activity intended for the biological entity, such as the innate activity of a cell, protein, or analogue. Ideally, the presence of a biological function can be tested using in vitro functional assays.

[0055] In the present invention, the biological functions of the complete ABCA4 protein may include cell localization function, ATP hydrolase activity, expression in photoreceptor cells, and / or reduction of retinal A2E deposition.

[0056] In this specification, the term "intine" refers to a naturally occurring or artificially constructed polypeptide sequence that can catalyze a protein splicing reaction that cleaves the intein sequence from a precursor protein and adds flanking sequences (N-exopeptides and C-exopeptides) via peptide bonds. A list of known inteins is published at http: / / www.inteins.com. Table 1 of CN114698379A summarizes several hundred naturally occurring inteins. In one embodiment, the intein of the present invention is a split intein. In the present invention, the intein is selected from, but is not limited to, the group consisting of GP41-1, DnaE, ​​GP41-8, NrdJ-1, IMPDH-1, SspGyrB, Mja-KlbA, NpuSsp, and Rma. In one embodiment, the sequences of these inteins are shown in Table 1 of the following specification.

[0057] The term "protein splicing" generally refers to the process by which the internal region (intane) of a precursor protein is cleaved and its adjacent region (exopeptide) is joined to form a mature protein. The intein unit may contain the components necessary for catalysis of protein splicing and generally includes an endonuclease domain involved in intein movement. The resulting proteins may bind to each other but are not expressed as individual proteins. Protein splicing can also be performed in a trans manner, as a split intein expressed in another polypeptide can spontaneously bind to a single intein, which can then undergo the protein splicing process to bind to an isolated protein.

[0058] In this specification, the term “N-terminus of intein” refers to such intein: comprising an N-terminal amino acid sequence that is functional for trans-splicing reactions, i.e., binding to a functional split intein C fragment to form a complete intein, the complete intein sequence either cleaving itself from the host protein to catalyze binding to an exopeptide or adjacent sequence via a peptide bond, or, upon binding to the split intein C fragment, “N-terminal cleavage,” i.e., catalyzing a nucleophilic attack on the peptide bond between the exopeptide and the N-terminus of the split intein N fragment, thereby cleaving the peptide bond.

[0059] In this specification, the term “C-terminus of intein” refers to such intein: having function for trans-splicing reactions, i.e., comprising a C-terminal amino acid sequence that, upon binding, binds to the N-fragment of a functional split intein to form a complete intein, the complete intein sequence either cleaving itself from the host protein to catalyze binding to an exopeptide or adjacent sequence via a peptide bond, or, upon binding to the N-fragment of the split intein, “C-terminal cleavage,” i.e., catalyzing a nucleophilic attack on the peptide bond between the exopeptide (extein) and the C-terminus of the split intein C-fragment, thereby cleaving the peptide bond.

[0060] The N-terminus and C-terminus of intein can be fused to the N-terminal and C-terminal cleavage of the ABCA4 protein, respectively, to link the N-terminal and C-terminal cleavage of the ABCA4 protein. For example, in some embodiments, the N-terminus of intein is fused to the C-terminus of the N-terminal portion of the split ABCA4 protein, i.e., forming an N-[N-terminal cleavage of ABCA4 protein]-[N-terminus of intein]--C structure. In some embodiments, the C-terminus of intein is fused to the N-terminus of the C-terminal cleavage of the ABCA4 protein, i.e., forming an N-[C-terminus of intein]--[C-terminal cleavage of ABCA4 protein]-C structure. Mechanisms of intein-mediated protein splicing used to link intein to a fused protein (e.g., split ABCA4 protein) are known in the art. For example, see Shah et al., Chem Sci. 2014, 5(1):446-461, which are incorporated herein by reference.

[0061] In one embodiment, the "N-terminus" and "C-terminus" of the present invention are homologous pairs, i.e., derived from the same intein (Shah et al. Journal of the American Chemical Society 2012, https: / / doi.org / 10.1021 / ja303226x). Regardless of whether the "N-terminus" and "C-terminus" of the present invention originate from the same intein, it can be understood that they can be reconstituted into a bond or a single fragment to carry out trans-splicing reactions of intein. In the present invention, preferably, the "N-terminus of intein" and the "C-terminus of intein" are the following combinations of intein N-terminus and C-terminus, respectively: N-terminus of GP41-1, C-terminus of GP41-1, N-terminus of DnaE, ​​C-terminus of DnaE, ​​N-terminus of GP41-8, C-terminus of GP41-8, N-terminus of NrdJ-1, C-terminus of NrdJ-1, N-terminus of IMPDH-1, C-terminus of IMPDH-1, N-terminus of SspGyrB, C-terminus of SspGyrB, N-terminus of Mja-KlbA, C-terminus of Mja-KlbA, N-terminus of NpuSsp, C-terminus of NpuSsp, or N-terminus of Rma, C-terminus of Rma. In one embodiment, the sequences of the intein N-terminus and C-terminus according to the present invention are shown in Table 1 below.

[0062] In this specification, the term “degradation signal” refers to such polypeptides: when the N-terminus or C-terminus of the intente of the present invention is fused directly or indirectly via a linker to a degradation signal, the degradation of the N-terminus or C-terminus fragment of the intente can be induced. Non-limiting realizations of degradation signals include polypeptides associated with DHFR (dehydrofolate reductase), FKBP (FK506-binding protein), FRB (FKBP-rapamycin-binding protein), and PDE5 (phosphodiesterase type 5), and degradation signals of the endoplasmic reticulum-associated degradation (ERAD) pathway.

[0063] In one embodiment, a fragment of the N-terminus or C-terminus of the first expression cassette can directly or indirectly via a linker bind a degradation signal to its C-terminus to promote the degradation of the intein formed after the degradation of the ABCA4 protein by intein splicing. When using a degradation signal, it should be noted that it does not adversely affect the splicing yield or the complete ABCA4 protein expressed.

[0064] In a preferred embodiment, the first expression cassette and the second expression cassette each include, in order from the 5' end: 1) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)), and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 2) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of DnaE (SEQ ID NO: 15)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of DnaE (SEQ ID NO: 16)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 3) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-8 (SEQ ID NO: 19)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-8 (SEQ ID NO: 20)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 4) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of NrdJ-1 (SEQ ID NO: 21)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of NrdJ-1 (SEQ ID NO: 22)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 5) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of IMPDH-1 (SEQ ID NO: 23)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of IMPDH-1 (SEQ ID NO: 24)), the (optimized) nucleotide coding sequence of the C-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)) and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 6) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of SspGyrB (SEQ ID NO: 25)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of SspGyrB (SEQ ID NO: 26)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 7) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of Mja-KlbA (SEQ ID NO: 27)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of Mja-KlbA (SEQ ID NO: 28)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 8) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of NpuSsp (SEQ ID NO: 29)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of NpuSsp (SEQ ID NO: 30)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)) and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 9) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 10) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 11) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 12) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of DnaE (SEQ ID NO: 15)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of DnaE (SEQ ID NO: 16)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 13) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of DnaE (SEQ ID NO: 15)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of DnaE (SEQ ID NO: 16)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 14) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of DnaE (SEQ ID NO: 15)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of DnaE (SEQ ID NO: 16)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 15) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of DnaE (SEQ ID NO: 15)), degradation signal (e.g., ecDHFR (SEQ ID NO: 14)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of DnaE (SEQ ID NO: 16)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 16) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)), 17) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)) and terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56)), 18) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)) and terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), 19) Promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)) and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)), or 20) Promoter (e.g., smCAG promoter (SEQ ID NO: 49)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of GP41-1 (SEQ ID NO: 17)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., smCAG promoter (SEQ ID NO: 49)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of GP41-1 (SEQ ID NO: 18)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)) and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)), 21) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of the RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 22) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 23) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of the RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 24) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., BGH terminator (SEQ ID NO: 51)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., BGH terminator (SEQ ID NO: 51)), 25) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)), 26) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)) and terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56)), 27) Promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)) and terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), and The promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), and the terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), 28) Promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)) and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)), or 29) Promoter (e.g., smCAG promoter (SEQ ID NO: 49)), (optimized) nucleotide coding sequence of the N-terminal cleavage (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), nucleotide coding sequence of the N-terminus of the intein (N-terminus of the RMA (SEQ ID NO: 61)), nucleotide coding sequence of the first tag (e.g., HA tag (SEQ ID NO: 12)) and terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and The promoter (e.g., smCAG promoter (SEQ ID NO: 49)), the nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), the (optimized) nucleotide coding sequence of the C-terminal cleavage product (NTD) of the ABCA4 protein (cleavage site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (corresponding nucleotide coding sequence of SEQ ID NO: 3), the nucleotide coding sequence of the second tag (e.g., Flag tag (SEQ ID NO: 13)), and the terminator (e.g., SV40 terminator (SEQ ID NO: 50)).

[0065] The term "liposome" generally refers to a lipid structure formed from amphiphilic vesicle lipids. The liposome may be a closed vesicle consisting of a single layer or multiple lipid bilayers with an aqueous phase inside. More broadly, the term "liposome" may also refer to lipid complex particles. The liposome may also include complexes where the aqueous phase is not clearly identified. The lipid complex may contain at least one type of lipid and may further contain hydrophilic polymers, polysaccharides, amino acids, etc. The lipid complex may refer to particles formed by covalent or non-covalent bonds between these components.

[0066] The term "exosome" generally refers to a membrane vesicle with a lipid bilayer membrane structure present in extracellular secretory cells or cells. The exosome may contain membrane bodies with an average diameter of approximately 10 nm to 2000 nm. The exosome may also contain microvesicles. Microvesicles, also called circulating microvesicles or microparticles, are fragments of the cell membrane with an approximate diameter range of 100 nm to 1000 nm that detach from almost all types of cells. The exosome may also contain extracellular vesicles produced within smaller cells, which are formed by the inward budding of the boundary membranes of multiple polyendoplasmic reticulum (MVBs). Upon fusion with the cell membrane, the MVBs cause secretion and deposition in bodily fluids (e.g., blood, urine). The exosome may contain a complex mixture of microRNA (miR), mRNA, and proteins, reflecting the transcriptional and translational state of the producing cell.

[0067] The term "AAV" is generally an abbreviation for adeno-associated virus and may refer to the virus itself or its derivatives. The AAV may include AAV1 (AAV-1 or AAV1), AAV2 (AAV-2 or AAV2), AAV3 (AAV-3 or AAV3), AAV4 (AAV-4 or AAV4), AAV5 (AAV-5 or AAV5), AAV6 (AAV-6 or AAV6), AAV7 (AAV-7 or AAV7), AAV8 (AAV-8 or AAV8), AAV9 (AAV-9 or AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, among others.

[0068] In preferred embodiments, the first and second expression cassettes of the present invention are all AAV virus packaging plasmids, where the first and second expression cassettes include an AAV 5' reverse end repeat (5'-ITR) sequence at the 5' end of the promoter sequence and an AAV 3' reverse end repeat (3'-ITR) sequence at the 3' end of the terminator.

[0069] The term "kit" generally refers to packaging that contains one or more active ingredients in one or more suitable containers.

[0070] The term "diseases caused by ABCA4 mutations" generally refers to diseases associated with ABCA4 gene mutations, such as rare eye diseases.

[0071] The term "hereditary retinal disease" generally refers to inherited retinal dystrophies or inherited retinal diseases (IRDs), which are rare eye diseases caused by genetic defects that result in retinal loss and / or progressive degeneration in patients. These hereditary retinal diseases often present with reversible or irreversible visual impairment in childhood or adolescence. IRDs are intractable ophthalmic diseases that often manifest as abnormalities in protein metabolism, and therapeutic goals can be achieved by controlling gene, transcription, and translation processes in genetic information transmission. The aforementioned hereditary retinal diseases may include autosomal recessive genetic disorders such as retinitis pigmentosa (RP, MERTK mutation), choroidal aplasia (CHM mutation), juvenile macular degeneration (ABCA4 mutation), Usher syndrome subtype 1B (Myo7a mutation), X-linked retinoschisis (RS1 mutation), mitochondrial-associated Leber optic neuropathy (ND4 mutation), total color blindness (CNGA3 and CNGB3 mutations), and sex-linked RP (RPGR mutation).

[0072] The term "hereditary macular degeneration" generally refers to rare familial genetic disorders. These hereditary macular degenerations may include Stargardt disease. Stargardt disease is an autosomal recessive genetic disorder caused by the retinal pigment epithelium, with relatively few sporadic cases and a higher incidence in children of consanguineous parents. Patients present with macular atrophy and macular deposition in the retina's macula. Currently, there is no approved treatment, and Stargardt disease causes tens of thousands of vision losses worldwide each year. These hereditary macular degenerations may also include Best's disease, in which the lesions primarily affect the macula of both eyes and usually do not spread to the peripheral retina. These hereditary macular degenerations can develop at any age.

[0073] The term "age-related macular degeneration" generally refers to age-related macular degeneration (AMD), which is the leading cause of blindness in people over 50 years of age. AMD presents as progressive and irreversible central vision loss, primarily affecting the macula, retinal pigment epithelium (RPE), and choroid. Clinically, early AMD is characterized by subretinal drusen deposition, while advanced AMD is classified into two types: dry and wet. Dry AMD (also called atrophic AMD) is characterized by geographical atrophy and detachment of the RPE layer and a slow progression of vision loss in the affected eye. Wet AMD (also called neovascular AMD or exudative AMD) is characterized by choroidal neovascularization (CNV) and secondary fluid retention and hemorrhage. The pathogenesis of AMD is not fully understood, but RPE damage, mitochondrial dysfunction, oxidative stress, inflammation, and complement pathway activation may be involved. Currently, AMD is generally believed to be caused by a combination of genetic and environmental factors.

[0074] The term "retinitis pigmentosa" generally refers to Retinitis Pigmentosa (RP). It is a group of hereditary eye diseases characterized by progressive degeneration of retinal photoreceptors. Retinitis pigmentosa can include progressive degenerative diseases of the retina (the transparent photosensitive membrane on the inner surface of the posterior part of the eyeball), which ultimately lead to moderate to severe vision loss. Retinitis pigmentosa is often hereditary. Clinical features of retinitis pigmentosa include early night blindness, concentric visual field constriction, and ultimately tubular visual field, bilateral blindness or near-blindness, accounting for a significant proportion of eye diseases that lead to blindness. Retinitis pigmentosa is also classified as hereditary rod cell and cone cell dystrophy.

[0075] The term "cone-rod dystrophy" generally refers to cone-rod dystrophy (CRD), a hereditary macular degeneration. This cone-rod dystrophy may encompass a range of clinical and genetic syndromes characterized by progressive vision loss, photophobia, and nystagmus, primarily affecting cone cells. In severe cases, rod cells may also be affected, and the mode of inheritance is mainly autochromatic dominant, recessive, and X-linked.

[0076] The term "N-retinylidene-phosphatidylethanolamine (NRPE)" generally refers to the physiological lipid substrate of ABCA4, flanked by two intracellular TMDs within the luminal lobule and further stabilized by an elongation loop from extracellular domain 1.

[0077] In the present invention, the first expression cassette may exist independently of the second expression cassette.

[0078] In the present invention, the first expression cassette and / or the second expression cassette may exist in a form selected from the group consisting of isolated nucleic acid molecules, liposomes and / or exosomes.

[0079] In the present invention, the first expression cassette and / or the second expression cassette may exist in the form of a plasmid.

[0080] In the present invention, the first expression cassette and / or the second expression cassette may be a viral vector.

[0081] In the present invention, the first expression cassette and / or the second expression cassette may be AAV.

[0082] On the other hand, the present invention provides a kit that includes a combination of expression cassettes according to the present invention.

[0083] In the present invention, the kit may include reagents and / or equipment for administering the combination of expression cassettes. For example, the kit may include reagents and / or equipment for transfecting an AAV containing the first expression cassette and / or the second expression cassette. For example, the kit may include reagents and / or equipment for injecting an AAV containing the first expression cassette and / or the second expression cassette. In the present invention, the injection may include a local injection.

[0084] On the other hand, the present invention provides the use of a combination of expression cassettes according to the present invention and / or a kit according to the present invention in the manufacture of pharmaceuticals for treating diseases including diseases caused by ABCA4 mutations.

[0085] In the present invention, the disease may include hereditary retinal diseases.

[0086] In the present invention, the disease may include hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0087] The method according to the present invention makes it possible to express heterologous N-terminal cuts and heterologous C-terminal cuts of the ABCA4 protein in the body of the subject of interest, and since the N-terminal cuts and heterologous C-terminal cuts of the ABCA4 protein form a complete ABCA4 protein after intein splicing, they can exert the biological function of the complete ABCA4 protein and can therefore be used to alleviate / treat / improve diseases caused by ABCA4 mutations.

[0088] On the other hand, the present invention provides a method for expressing heterologous ABCA4 genes, the method may include the step of administering a combination of expression cassettes and / or a kit according to the present invention to a subject requiring such a combination.

[0089] On the other hand, the present invention provides a method for mitigating N-retinyl-phosphatidylethanolamine (NRPE)-induced cell death, the method which may include the step of administering a combination of expression cassettes and / or a kit according to the present invention to a subject requiring such a combination.

[0090] On the other hand, the present invention provides a method for treating a disease caused by an ABCA4 mutation, the method may include the step of administering a combination of expression cassettes and / or a kit according to the present invention to a subject requiring such a combination.

[0091] In the present invention, the administration may include injection.

[0092] In the present invention, the disease may include hereditary retinal diseases.

[0093] In the present invention, the disease may include hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0094] The present invention will be described in more detail below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0095] Example 1: Optimization of codons in the ABCA4 protein The inventors performed codon optimization on the CDS region of the wild-type human-derived ABCA4 protein (SEQ ID NO: 1), and the optimized codon is shown in SEQ ID NO: 3 in Table 1. After inserting the CDS region of wild-type human ABCA4 protein and an optimized CDS region into the EF1A promoter of the pFG12-PURO vector (the pFG12-PURO vector is modified from pFG12 (Addgene #14884), and the EGFP CDS region of the pFG12 vector is replaced with PURO CDS (amino acid sequence is shown in SEQ ID NO: 57, coding nucleotide sequence is shown in SEQ ID NO: 58) and simultaneously inserting the EF1A promoter in the reverse direction of the UBC promoter (sequence is shown in SEQ ID NO: 59), we obtained pFG12-PURO-ABCA4-WT-Flag and pFG12-PURO-ABCA4-CO-Flag, respectively. Using PEI (23966-1, Polyscience) transfect, we obtained pFG12-PURO-ABCA4-WT-Flag and pFG12-PURO-ABCA4-CO-Flag from human HEK293A (Beijing The ABCA4 coding sequence (CO) was transfected into human retinal pigment epithelial cells ARPE19 (CRL-2302, ATCC) and mouse retinal photoreceptor cells 661W (Beijing University). After 48 hours, the cells were lysed with RIPA lysis buffer (C1053-100, Polyscience), and ABCA4 protein expression was detected by Western blot assay. The optimized results (see Figure 1) showed that the optimized ABCA4 coding sequence (CO) significantly increased ABCA4 protein expression (detected using an endogenous antibody for ABCA4 (clone 5B4, MABN2440, Merck)) in human HEK293A and ARPE19 cells compared to unoptimized wild-type ABCA4 (WT), but no increase in expression was observed in mouse-derived 661W cells. The internal control protein was ACTB (β-actin, detection antibody: AC026, ABclonal).

[0096] Example 2 Expression efficiency of different ABCA4 proteins using different types of split inteins A strategy for forming the complete ABCA4 protein using the intein dual expression cassette according to the present invention is shown in Figure 2. This is similar to the method in Tornabene, P., et al. (2019). "Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina." Sci Transl Med 11(492), but the main difference is the cleavage site of ABCA4 and / or the intein used.

[0097] The ABCA4 protein (SEQ ID NO: 1) was cleaved at position 1224, and the N-terminal cleavage (NTD) and C-terminal cleavage (CTD) of ABCA4 were combined with different types of inteins (splicing peptides), including GP41-1 (SEQ ID NO: 5), DnaE (SEQ ID NO: 4), GP41-8 (SEQ ID NO: 6), NrdJ-1 (SEQ ID NO: 7), IMPDH-1 (SEQ ID NO: 8), SspGyrB (SEQ ID NO: 9), Mja-KlbA (SEQ ID NO: 10), NpuSsp (SEQ ID NO: 11), and Rma (SEQ ID NO: 60), respectively. The N-terminus and C-terminus of these inteins (see Table 1 below for specific sequences) were used to insert into a pAV vector (pX601, Addgene) to construct the corresponding plasmids pAV-OPEFS-NTD-intN-HA and pAV-OPEFS-intC-CTD-Flag. (Note: The plasmid promoter is OPEFS (SEQ ID NO: 47), NTD indicates that the plasmid contains the nucleotide coding sequence of the N-terminal cleavage of the ABCA4 protein, CTD indicates that the plasmid contains the nucleotide coding sequence of the C-terminal cleavage of the ABCA4 protein, intN indicates that the plasmid contains the nucleotide coding sequence of the N-terminus of the intein, intC indicates that the plasmid contains the nucleotide coding sequence of the C-terminus of the intein, HA indicates that the plasmid contains the HA-tag (SEQ ID NO: 12) nucleotide coding sequence, and Flag indicates that the plasmid contains the Flag-tag (SEQ ID NO: 13) nucleotide coding sequence.) The corresponding plasmid combinations were transfected into HEK293A cells (Beijing University) using PEI, where CO is a positive control for transfecting the plasmid pFG12-PURO-ABCA4-CO-Flag. Referring to Figure 3, the results confirmed that complete ABCA4 proteins with the correct molecular weight could be generated when the inteins were DnaE, ​​GP41-1, GP41-8, NrdJ-1, IMPDH-1, and SspGyrB. Here, DnaE, ​​GP41-1, and NrdJ showed the highest efficiency in recombination to complete ABCA4 proteins.The recombination efficiency of each intein is in the order of GP41-1 > DnaE > NrdJ > GP41-8 ~ IMPDH-1 ~ SspGyrB. Mja-KlbA and NpuSsp do not produce complete ABCA4 proteins with the correct molecular weight. Antibodies used in the Western study: anti-Flag, F1804, Sigma, anti-HA, 3724S, CST, anti-ACTB, AC026, ABclonal.

[0098] Furthermore, comparing the splicing efficiency of GP41-1 and Rma intein, the Western Blot experimental results are shown in Figure 4. Both GP41-1 and Rma intein can be effectively recombined into the complete ABCA4 protein. The recombination efficiencies of the two inteins are similar. Antibodies used in the Western Blot: anti-ABCA4, clone 5B4, anti-ABCA4, clone 3F4, anti-ACTB, AC026, ABclonal.

[0099] Example 3 Expression efficiency of different ABCA4 proteins at different cleavage sites of the ABCA4 protein Similar to the method described in Example 2, ABCA4 was cleaved from the N-terminus at amino acid positions 1140, 1150, 1188, and 1224, and the N-terminal cleavage (NTD) and C-terminal cleavage (CTD) of ABCA4 were combined with DnaE or GP41-1 intein (splicing peptides), respectively. The corresponding plasmid combinations were transfected into HEK293A cells (Beijing University), where CO is a positive control for transfecting with the plasmid pFG12-PURO-ABCA4-CO-Flag. The results confirmed that efficiency was highest when the cleavage site was 1224 amino acid. The ranking of efficiency for different cleavage sites was 1224 > 1188 > 1150 > 1140. Furthermore, it was confirmed that the content of additional small peptide intein generated by GP41-1 was lower, which has potentially higher safety in gene therapy (see Figure 5 for results). Antibodies used in the Western group: anti-Flag, F1804, Sigma, anti-HA, 3724S, CST, anti-ACTB, AC026, ABclonal.

[0100] Example 4: A dual expression cassette containing a degraded peptide can form a complete ABCA4 protein. Similar to the above example, 1150 amino acids were cleaved from the N-terminus of ABCA4 to obtain the N-terminal cleavage (NTD) and C-terminal cleavage (CTD) of ABCA4. These were then combined with the N-terminus (SEQ ID NO: 15) and C-terminus (SEQ ID NO: 16) of the intein DnaE, ​​respectively, and tags were attached to each to obtain two corresponding expression cassette constructs (pAV-OPEFS-NTD(1150)-int(DnaE)N-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag). The coding nucleotide sequence expressing the degradation signal (degradation peptide) ecDHFR (SEQ ID NO: 14) was inserted into pAV-OPEFS-NTD(1150)-int(DnaE)N-HA to obtain the pAV-OPEFS-NTD(1150)-int(DnaE)N-ecDHFR-HA vector. Both pAV-OPEFS-NTD(1150)-int(DnaE)N-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag (no degradation signal, indicated by -ecDHFR in Figure 6), or both pAV-OPEFS-NTD(1150)-int(DnaE)N-ecDHFR-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag (with degradation signal, indicated by +ecDHFR in Figure 6), were jointly transfected into HEK293A cells using PEI, and complete ABCA4 protein expression (shown in Figure 6) was detected by Western after 48 hours. Here, CO is a positive control for transfecting with plasmid pFG12-PURO-ABCA4-CO-Flag. The results showed that the dual expression cassette containing the degraded peptide was able to form a complete ABCA4 protein, while simultaneously producing less intein, which means that the intein was partially degraded by the degradation signal. Antibodies used in the Western region: anti-Flag, F1804, Sigma, anti-HA, 3724S, CST.

[0101] Example 5: The expression cassette can form a complete ABCA4 protein in 293T cells. Similar to the above example, 1150 amino acids were cleaved from the N-terminus of ABCA4 to obtain the N-terminal cleavage (NTD) and C-terminal cleavage (CTD) of ABCA4. These were then combined with the N-terminus and C-terminus of the intein GP41-1 (N-terminus of GP41-1 (SEQ ID NO: 17) and C-terminus of GP41-1 (SEQ ID NO: 18)), respectively, and tags were attached to each to obtain two corresponding expression cassette constructs (pAV-OPEFS-NTD(1150)-int(GP41-1)N-HA and pAV-OPEFS-int(GP41-1)C-CTD(1150)-Flag). Using the constructed AAV8 vectors pAV-OPEFS-NTD(1150)-int(GP41-1)N-HA and pAV-OPEFS-int(GP41-1)C-CTD(1150)-Flag, NTD and CTD cleaved at the 1150 site as described in Example 3 were packaged into GP41-1 expression cassettes, respectively, and 293 T cells (3216, ATCC) were co-infected. The multiplicity of infection (MOI) was 0, 2E4, and 2E5, respectively. After 72 hours of infection, complete ABCA4 protein was formed (see Figure 7 for results). Here, CO is a positive control for transfecting with plasmid pFG12-PURO-ABCA4-CO-Flag. Antibodies used in Western: anti-Flag, F1804, Sigma; anti-HA, 3724S, CST.

[0102] Example 6: The expression cassette can successfully express the complete ABCA4 protein in ABCA4 KO mice. As described in the above examples, the constructed AAV8 vectors pAV-OPEFS-NTD(1150)-int(GP41-1)N-HA and pAV-OPEFS-int(GP41-1)C-CTD(1150)-Flag were used to package expression cassettes (daul-AAV(GP41-1-1150)) combining NTD and CTD cleaved at the 1150 site with GP41-1, as described in Example 3, and administered by subretinal injection to 1-2 month old ABCA4 KO mice (S-KO-00815, C57BL / 6J, Cyagen Biosciences). Five weeks later, the mice were anesthetized and pupil-dilated, and stimulated with 20000 lux, 430 nm blue light for 30 minutes. ERG was detected one week after recovery. The results showed that blue light stimulation significantly reduced mouse ERG amplitude levels, and that ERG amplitude levels recovered in BCA4 KO mice after dual-AAV injection (see Figure 8, where WT represents wild-type mice, WT+B represents wild-type mice after blue light stimulation, KO represents ABCA4 KO mice, KO+B represents uninjected ABCA4 KO mice after blue light stimulation, KO+B+EV represents ABCA4 KO mice injected with AAV-EV virus after blue light stimulation (where AAV-EV virus represents empty-shell AAV virus, i.e., an AAV virus that does not contain the target genome (i.e., the ABCA4 expression cassette)), and KO+B+dualAAV represents ABCA4 KO mice injected with AAV virus expressing the dual expression cassette of the present invention after blue light stimulation).

[0103] After 6 weeks, once detection was complete, the animals were euthanized. Retinal tissue from the eyeballs was isolated, and a portion of it was used for phenol-chloroform-methanol extraction to extract A2E. A2E content in the samples was detected using ACQUITY UPLC H-Class ultrahigh performance liquid chromatography with an Atlantic dC18 column, using acetonitrile containing 0.1% TFA and water as the mobile phase (Radu, RA, et al. (2008). "Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for ABCA4-mediated retinal dystrophies following Vitamin A supplementation." Invest Ophthalmol Vis Sci 49(9):3821-3829.) (See Figure 9 and Table A).

[0104] Table A TIFF2026519810000001.tif116170

[0105] The remaining mouse retinal tissue was used for a Western blot assay. The results (Figure 10) showed that 9 out of 12 mouse retinal samples expressed the complete ABCA4 protein, with the highest expression level being approximately 20% of the endogenous expression level in wild-type mice. Antibodies used in the Western blot assay: anti-ABCA4, clone 5B4, MABN2440, Merck, anti-ACTB, AC026, ABclonal.

[0106] Example 7: By including different cleavage sites of the ABCA4 protein in the expression cassette, the complete ABCA4 protein can be successfully expressed in ABCA4 KO mice. Following the method of the above example, the following AAVs were packaged using the AAV8 vector: an expression cassette combining NTDs and CTDs cleaved at 1150 sites with GP41-1 (daul-AAV(GP41-1-1150)), an expression cassette combining NTDs and CTDs cleaved at 1150 sites with DnaE (daul-AAV(DnaE-1150)), an expression cassette combining NTDs and CTDs cleaved at 1188 sites with GP41-1 (daul-AAV(GP41-1-1188)), and an expression cassette combining NTDs and CTDs cleaved at 1224 sites with GP41-1 (daul-AAV(GP41-1-1224)). Each dual AAV combination was administered by subretinal injection to ABCA4 KO mice. After six weeks, the animals were euthanized, and retinal tissue from the eyeballs was isolated and subjected to a Western blot assay. As shown in Figure 11, the experimental results indicated that sites 1150, 1188, and 1224 all effectively expressed full-length ABCA4 protein in the mouse retina, and furthermore, the cleavage efficiency at sites 1188 and 1224 was higher than that at site 1150.

[0107] Example 8 Expression cassettes containing different regulatory elements can successfully express the complete ABCA4 protein in ABCA4 KO mice. To further screen the effects of regulatory factors on the in vivo expression efficiency of ABCA4, based on the above examples, the Flag tag and HA tag were removed from dual AAV vectors targeting the 1224 or 1188 cleavage sites, and the original BGH terminator was replaced with an SV40 terminator or a WPRE-SV40 terminator. Furthermore, the dual AAV terminator at the 1188 cleavage site was replaced with a B-globin-polyA terminator (SEQ ID NO: 65) or an sNRP1 terminator (SEQ ID NO: 66).

[0108] The above vectors were packaged using the AAV8 vector. Each of the dual AAV combinations was administered by subretinal injection to ABCA4 KO mice. After 6 weeks, the animals were euthanized, and retinal tissue from the eyeballs was isolated and a Western blot assay was performed. As shown in Figure 12, the experimental results indicate that each terminator can effectively express the full-length ABCA4 protein, and WPRE can promote the expression of the complete ABCA4 protein.

[0109] Example 9 Expression cassettes containing different promoters can successfully express the complete ABCA4 protein in ABCA4 KO mice. As described in the above examples, the constructed vectors pAV-OPEFS-NTD(1224)-int(GP41-1)N-SV40 and pAV-OPEFS-int(GP41-1)C-CTD(1224)-SV40 were used to replace the original promoter sequence EFS with the hGRK1 promoter or the smCAG promoter. Furthermore, a CMV enhancer was added before the EFS promoter and the hGRK1 promoter to obtain the corresponding expression cassette vectors (enOPEFS promoter, SEQ ID NO: 67; enhGRK1(enRK) promoter, SEQ ID NO: 68). Simultaneously, a portion of the hGRK1 promoter sequence was additionally replaced with a b-globin-intron sequence to form a new hGRK1-b-globin-intron promoter (SEQ ID NO: 69), and a CMV enhancer was added to obtain the enhGRK1-b-globin-intron promoter (SEQ ID NO: 70). The above vectors were packaged using the AAV8 vector. Each of the dual AAV combinations was administered via subretinal injection to ABCA4 knockout mice. After 6 weeks, the animals were euthanized, and retinal tissue from the eyeballs was isolated and subjected to Western blot assays. As shown in Figure 13, the experimental results indicate that each promoter can effectively express the full-length ABCA4 protein in the mouse retina.

[0110] Example 10: A shortened expression cassette can successfully express the complete ABCA4 protein in ABCA4 KO mice. The ABCA4 1224 cleavage site is too long to encode an amino acid sequence, so the length of the ABCA4-NTD expression cassette is greater than the AAV vector packaging capacity, which can inhibit efficient production. Based on the methods of the above examples, expression cassettes were screened based on the 1188 and 1150 cleavage sites, and combinations of genome-reduced expression cassettes were further validated in mice.

[0111] The combinations of vector elements include the following: TIFF2026519810000002.tif88170

[0112] NTD and CTD expressed by the dual expression cassette described above were packaged into AAV8, and the dual AAV combinations were administered by subretinal injection to ABCA4 KO mice. After 8 weeks, the animals were euthanized, and retinal tissue from the eyeballs was isolated and subjected to Western blot assay. As shown in Figure 14A, the experimental results indicate that all combinations can effectively express full-length ABCA4 protein in the model mouse retina. Furthermore, the A2E detection results are shown in Figure 14B. Subretinal administration of each group's expression cassette vector significantly reduced A2E deposition in the ABCA4 KO mouse model, indicating that all of the expression cassettes have potential therapeutic effects.

[0113] Example 11 Expression cassettes packaged in different vectors can successfully express the complete ABCA4 protein in ABCA4 KO mice. Expression cassettes were packaged in combination with GP41-1, containing NTDs and CTDs cleaved at 1224 sites using AAV8, AAV8-Y447, 733F vector, dual AAVtYF, or AAV2.7m8 (daul-AAV8(GP41-1-1224), daul-AAV8-Y447, 733F(GP41-1-1224), daul-AAVtYF(GP41-1-1224), daul-AAV2.7m8(GP41-1-1224), each virus packaged by Guangzhou PackGene Biotechnology Co.,Ltd.). Subretinal injection was administered to ABCA4 KO mice. After 6 weeks, the animals were euthanized, retinal tissue from the eyeballs was isolated, and a Western blot assay was performed. As shown in Figure 15, experimental results indicated that AAV8, AAV8-Y447, 733F variants, and AAV2.7m8 serotypes all effectively expressed full-length ABCA4 protein in the mouse retina, but the expression efficiency of the AAV2-tYF serotype was low. Compared to AAV8, the AAV7m8 serotype showed stronger expression intensity.

[0114] Example 12: Lower content of additional small peptides produced by GP41-1 in split-intine. To detect whether additional small peptides generated after splicing of the dual AAV vector split peptides are retinotoxic, separate GP41-1 split peptides and DnaE split peptides were constructed in plasmids pAV-OPEFS-int(GP41-1)-HA and pAV-OPEFS-int(DnaE)-HA(pX601, Addgene), respectively, and packaged in AAV8 virus. The expression status 48 hours after plasmid transfection to HEK293A and 72 hours after infection of 293T cells with AAV is shown below (Figure 16).

[0115] AAV8-GP41-1 and AAV8-DnaE were administered via subretinal injection to ABCA4 KO mice, respectively. After 6 weeks, ERG (electroretinography) (see Figure 17), fundus OCT (optical coherence tomography), and FP (fundus photography) detection were performed. Compared to controls, the content of the additional small peptide intein produced by GP41-1 was lower, and no retinal toxicity was observed in either case.

[0116] Those skilled in the art will understand that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Without departing from the spirit of the invention, those skilled in the art can make appropriate modifications or improvements based on the methods and technical solutions taught herein, and all equivalent implementations obtained therefrom are within the scope of the present invention.

[0117] Table 1 List of sequences used in the specification and examples TIFF2026519810000003.tif247170TIFF2026519810000004.tif247170TIFF2026519810000005.tif247170TIFF2026519810000006.tif247170TIFF2026519810000007.tif247170TIFF2026519810000008.tif247170TIFF2026519810000009.tif247170TIFF2026519810000010.tif247170TIFF2026519810000011.tif247170TIFF2026519810000012.tif247170TIFF2026519810000013.tif247170TIFF2026519810000014.tif247170TIFF2026519810000015.tif247170TIFF2026519810000016.tif247170TIFF2026519810000017.tif247170TIFF2026519810000018.tif247170TIFF2026519810000019.tif247170TIFF2026519810000020.tif247170TIFF2026519810000021.tif247170TIFF2026519810000022.tif247170TIFF2026519810000023.tif247170TIFF2026519810000024.tif247170TIFF2026519810000025.tif247170TIFF2026519810000026.tif247170TIFF2026519810000027.tif247170TIFF2026519810000028.tif247170TIFF2026519810000029.tif247170TIFF2026519810000030.tif247170TIFF2026519810000031.tif247170TIFF2026519810000032.tif247170TIFF2026519810000033.tif247170TIFF2026519810000034.tif247170TIFF2026519810000035.tif247170TIFF2026519810000036.tif247170TIFF2026519810000037.tif247170TIFF2026519810000038.tif247170TIFF2026519810000039.tif247170TIFF2026519810000040.tif247170TIFF2026519810000041.tif247170TIFF2026519810000042.tif247170TIFF2026519810000043.tif247170TIFF2026519810000044.tif247170TIFF2026519810000045.tif247170TIFF2026519810000046.tif247170TIFF2026519810000047.tif247170TIFF2026519810000048.tif247170.

Claims

1. A combination of expression cassettes, It includes a first expression cassette and a second expression cassette, The first expression cassette is capable of expressing a fusion protein of the N-terminal cleavage of the ABCA4 protein and the N-terminus of the intein, and the second expression cassette is capable of expressing a fusion protein of the C-terminal cleavage of the ABCA4 protein and the C-terminus of the intein, wherein, upon expression, the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette and the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette form a complete full-length ABCA4 protein via splicing of the N-terminus and C-terminus. A combination of expression cassettes characterized by the above.

2. The aforementioned intent is selected from the group consisting of GP41-1 (SEQ ID NO: 5), DnaE (SEQ ID NO: 4), GP41-8 (SEQ ID NO: 6), NrdJ-1 (SEQ ID NO: 7), IMPDH-1 (SEQ ID NO: 8), SspGyrB (SEQ ID NO: 9), and Rma (SEQ ID NO: 60), and is preferably GP41-1. A combination of expression cassettes according to claim 1, characterized in that

3. The complete ABCA4 protein is human ABCA4 protein (SEQ ID NO: 1), and the N-terminal cleavage of the ABCA4 protein expressed by the first expression cassette and the C-terminal cleavage of the ABCA4 protein expressed by the second expression cassette are the corresponding N-terminal and C-terminal cleavage obtained by cleaving the complete ABCA4 protein from the N-terminus at the following amino acid (Cys) sites: 1224, 1140, 1150, or 1188, preferably 1188 or 1224, i.e., the N-terminal and C-terminal cleavage are in the following combinations: The first 1-1223 amino acids and the 1224-2273 amino acids from the N-terminus of human ABCA4 protein (SEQ ID NO: 1), The first 1-1139 amino acids and the 1140-2273 amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1), The first 1-1149 amino acids and the 1150-2273 amino acids from the N-terminus of human ABCA4 protein (SEQ ID NO: 1), or The first 1-1187 amino acids and the 1188-2273 amino acids from the N-terminus of human ABCA4 protein (SEQ ID NO: 1), Selected from A combination of expression cassettes according to claim 1 or 2, characterized in that

4. The N-terminus and C-terminus of the aforementioned intein are as follows: 1)-7): 1) The N-terminus of GP41-1 (SEQ ID NO: 17) and the C-terminus of GP41-1 (SEQ ID NO: 18), 2) The N-terminus of DNAE (SEQ ID NO: 15) and the C-terminus of DNAE (SEQ ID NO: 16), 3) The N-terminus of GP41-8 (SEQ ID NO: 19) and the C-terminus of GP41-8 (SEQ ID NO: 20), 4) The N-terminus of NrdJ-1 (SEQ ID NO: 21) and the C-terminus of NrdJ-1 (SEQ ID NO: 22), 5) The N-terminus of IMPDH-1 (SEQ ID NO: 23) and the C-terminus of IMPDH-1 (SEQ ID NO: 24), 6) The N-terminus of SspGyrB (SEQ ID NO: 25) and the C-terminus of SspGyrB (SEQ ID NO: 26), and 7) The N-terminus of Rma (SEQ ID NO: 61) and the C-terminus of Rma (SEQ ID NO: 62), Selected from the group consisting of A combination of expression cassettes according to claim 2, characterized in that

5. The aforementioned intein is GP41-1, and the N-terminal and C-terminal cleavage of the ABCA4 protein are the corresponding N-terminal and C-terminal cleavage obtained by cleaving the ABCA4 protein from the N-terminus at the 1224th amino acid (Cys) site, i.e., the 1st-1223rd amino acids and 1224th-2273rd amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1). A combination of expression cassettes according to any one of claims 1 to 4, characterized in that

6. The aforementioned intein is GP41-1, and the N-terminal and C-terminal cleavage of the ABCA4 protein are the corresponding N-terminal and C-terminal cleavage obtained by cleaving the ABCA4 protein from the N-terminus at the 1188th amino acid (Cys) site, i.e., the 1-1187th amino acids and 1188-2273rd amino acids from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1). A combination of expression cassettes according to any one of claims 1 to 4, characterized in that

7. The codons of the N-terminal and C-terminal cleavage of the ABCA4 protein are optimized codons, and the combined sequence of these, i.e., the coding sequence of the full-length ABCA4 protein, is shown in Sequence ID No.

3. A combination of expression cassettes according to any one of claims 1 to 6, characterized in that

8. A first tag is bound to the C-terminus of the fusion protein expressed by the first expression cassette, and / or a second tag is bound to the C-terminus of the fusion protein expressed by the second expression cassette, wherein the first and second tags are different, and the first and second tags are independently selected from the group consisting of hemagglutinin (HA) tag, Flag tag, V5 tag, Myc tag, His tag, 1D4 tag (Rho1D4), and combinations thereof, preferably the first tag is a hemagglutinin (HA) tag (SEQ ID NO: 12), and / or the second tag is a Flag tag (SEQ ID NO: 13). A combination of expression cassettes according to any one of claims 1 to 7, characterized in that

9. The N-terminus or C-terminus of the intein is further linked directly or indirectly via a linker to a degradation signal, preferably selected from DHFR (dehydrofolate reductase), FKB (FK506-binding protein), FRB (FKB-rapamycin-binding protein), and polypeptides associated with PDE5 (phosphodiesterase type 5), and degradation signals of the endoplasmic reticulum-associated degradation (ERAD) pathway, and more preferably the degradation signal is ecDHFR (SEQ ID NO: 14). A combination of expression cassettes according to any one of claims 1 to 8, characterized in that

10. The first expression cassette and / or the second expression cassette comprises a promoter and a terminator for expressing the fusion protein, and selectively includes enhancers and introns operably linked to the fusion protein to enhance the expression of the fusion protein. A combination of expression cassettes according to any one of claims 1 to 9, characterized in that

11. The promoter is a constitutive promoter or an inductive promoter, preferably a CAG promoter (also referred to as a hybrid CMV initial enhancer / chicken β-actin promoter, CAGGS promoter, CB promoter or CBA promoter), a human β-actin promoter, a small CBA (smCBA) promoter, a CBS promoter or CBh promoter, an elongation factor 1α short (EFS) promoter, an elongation factor 1α (EF-1α) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, or a VMD2 (BEST1) promoter. The promoter is selected from the group consisting of the promoter (also known as ABCA4 self-promoter), RPE65 promoter, OPEFS promoter, hGRK1 promoter, or smCAG promoter, and more preferably the OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48), smCAG promoter (SEQ ID NO: 49), enOPEFS promoter (SEQ ID NO: 67), enhGRK1 (enRK) promoter (SEQ ID NO: 68), hGRK1-b-globin-intron promoter (SEQ ID NO: 69), or enhGRK1-b-globin-intron promoter (SEQ ID NO: 70). A combination of expression cassettes according to claim 10, characterized in that

12. The terminator is selected from the group consisting of SV40 terminator (SEQ ID NO: 50), BGH terminator (SEQ ID NO: 51), WPRE terminator (WPRE-3 terminator (SEQ ID NO: 52), etc.), WPRE-SV40 terminator (SEQ ID NO: 56), WPRE-BGH terminator, B-globin-polyA terminator (SEQ ID NO: 65), or sNRP1 terminator (SEQ ID NO: 66). A combination of expression cassettes according to claim 10 or 11, characterized in that

13. The first expression cassette comprises, in order from the 5' end, an optional enhancer, a promoter, an optional intron, a nucleotide coding sequence of the N-terminal cleavage of the ABCA4 protein, a nucleotide coding sequence of the N-terminal intein, an optional degradation signal, a nucleotide coding sequence of an optional tag, and a terminator, and / or the second expression cassette comprises, in order from the 5' end, an optional enhancer, a promoter, an optional intron, a nucleotide coding sequence of the C-terminal intein, an optional degradation signal, a nucleotide coding sequence of the C-terminal cleavage of the ABCA4 protein, a nucleotide coding sequence of an optional tag, and a terminator. A combination of expression cassettes according to any one of claims 10 to 12, characterized in that

14. The first expression cassette and / or the second expression cassette exist in a form selected from the group consisting of isolated nucleic acid molecules, liposomes, and / or exosomes. A combination of expression cassettes according to any one of claims 10 to 13, characterized in that

15. The first expression cassette and / or the second expression cassette exist in the form of a plasmid. A combination of expression cassettes according to any one of claims 1 to 14, characterized in that

16. The first expression cassette and / or the second expression cassette are viral vectors. A combination of expression cassettes according to any one of claims 1 to 15, characterized in that

17. The viral vector is an adeno-associated virus (AAV) of the same or different serotypes, and the AAVs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 A selection from the group consisting of AAV-6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, AAV2 / 10, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV8-Y447, 733F, or AAVtYF, preferably AAV5, AAV8, AAV8-Y447, 733F, AAVtYF, or AAV2.7m8. A combination of expression cassettes according to claim 16, characterized in that

18. 1) The promoter is hGRK1, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is Rma, and the terminator is SV40. 2) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is SV40. 3) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is Rma, and the terminator is B-globin-polyA. 4) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is B-globin-polyA. 5) The promoter is hGRK1, the cleavage site of the N-terminal and C-terminal cleavage sites is 1188, the intent is GP41-1, and the terminator is SV40. 6) The promoter is enRK, the cleavage site of the N-terminal and C-terminal cleavage sites is 1150, the intent is GP41-1, the terminator is SV40, or 7) The promoter is enRK-b-globin intron, the cleavage sites of the N-terminal and C-terminal cleavage sites are 1188, the intein is GP41-1, and the terminator is B-globin-polyA. A combination of expression cassettes according to any one of claims 1 to 17, characterized in that

19. A kit comprising a combination of expression cassettes according to any one of claims 1 to 18.

20. Use of a combination of expression cassettes according to any one of claims 1 to 18 or a kit according to claim 19 in the manufacture of a pharmaceutical product for treating a disease including a disease caused by an ABCA4 mutation.

21. The aforementioned diseases include hereditary retinal diseases. The use according to claim 20, characterized by the features described herein.

22. The aforementioned diseases include hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy. The use according to claim 20 or 21, characterized in that it is the same as the use according to claim 20 or 21.

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