Expression cassette combination and application thereof

Abstract

The invention discloses an expression cassette combination and application thereof. The expression cassette combination comprises a first expression cassette and a second expression cassette, the first expression cassette can express a fusion protein of an N-terminal truncation of ABCA4 protein and an intein N terminal, the second expression cassette can express a fusion protein of a C-terminal truncation of ABCA4 protein and an intein C terminal, and when expressed, the fusion protein of the C-terminal truncation of ABCA4 protein and the intein C terminal is separated from the fusion protein of the C-terminal truncation of ABCA4 protein. The N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can form a complete full-length ABCA4 protein through the shearing function of the N terminal and the C terminal of intein.

Description

Expression cassette combinations and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an expression cassette combination and its application. Background Art

[0002] ABCA4 is a lipid inward-flipping enzyme in retinal photoreceptors and retinal pigment epithelial cells. It transports retinylphosphatidylethanolamine (NRPE), a lipid derivative formed from phosphatidylethanolamine (PE) and all-trans-retinal (ATR), from the lumen of the outer segment disc to the cytoplasm. NRPE is then hydrolyzed into ATR and PE. ATR is then converted to all-trans-retinol (ATRol) by cytoplasmic retinal reductase, which then returns to the visual cycle. Impairment of ABCA4's lipid transport function leads to excessive accumulation of ATR and NRPE in the lumen of the outer segment disc. These accumulations irreversibly form toxic di-retinyl derivatives, ultimately causing a range of retinal degenerative diseases, including the most common inherited macular degeneration, age-related macular degeneration, retinitis pigmentosa, and cone-rod dystrophy. The human ABCA4 CDS is 6.7 kb long and encodes 2273 amino acids.

[0003] Inteins are peptides that exist within the peptide chains of certain protein precursors. During the conversion to mature proteins, they are removed through a non-enzymatic transpeptidation reaction, leaving exteins as their counterparts. Inteins have been widely used in protein purification, protein labeling, and the splicing and expression of larger protein fragments.

[0004] CN115074369 A uses an intein dual AAV vector strategy to express the ABCA4 protein. It discloses that the most critical aspects of the split intein dual AAV vector strategy are the intein sequence and the cleavage site, because this will greatly affect the cleavage efficiency of the target protein, as well as the tertiary structure and post-translational modification of the target protein, thereby affecting the protein function. In addition, since the split intein dual AAV vector expresses the N-terminus and C-terminus of the ABCA4 protein respectively, the N-terminal and C-terminal byproducts that are not effectively spliced ​​have potential safety risks. In CN115074369 A, the full-length ABCA4 protein is split with p1150Cys as the cleavage site to obtain two fragments, and the intein used is the Rma intein or the Npu intein.

[0005] CN115698307A discloses inteins and their uses, wherein engineered split inteins and their combination with degradation signals (destabilizing domains, degrons) are used to reconstruct large genes. CN115698307A discloses that one of the main limitations of the above-mentioned applications of inteins is the accumulation of protein intermediates (N-extein - IntN and IntC-C-extein) and the cleaved split intein. Those skilled in the art will understand that the accumulation of these unwanted proteins may lead to adverse side effects (immune responses).

[0006] Summary of the Invention

[0007] To address the above issues, the present inventors conducted in-depth research and discovered that by selecting a specific cleavage site of the ABCA4 protein, N-terminal truncations and C-terminal truncations of the ABCA4 protein were obtained, and these truncations were then linked to the N-terminal and C-terminal portions of a specific intein, respectively, thereby obtaining a first expression cassette expressing a fusion protein of the N-terminal truncations of the ABCA4 protein and the N-terminus of the intein, and a second expression cassette expressing a fusion protein of the C-terminal truncations of the ABCA4 protein and the C-terminus of the intein. The first and second expression cassettes are expressed in cells with high efficiency to form a complete ABCA4 protein; they can successfully package AAV5 and AAV8 viruses, and can be effectively expressed in mouse retinal cells, restoring retinal function in ABCA4 KO mice; the above viruses co-infect IPSC-RPE or IPSC-PRC cells derived from Stargardt disease patients, which can restore the cells' retinol conversion function and reduce A2E (retinoid dimer (N-retinyl-N-retinolamine)) deposition; and when the GP41-1 intein is used, the above expression cassettes produce a low amount of intein protein, which has the potential advantage of low immune response.

[0008] In particular, the present inventors unexpectedly discovered that by selecting the GP41-1 intein, not only can the highest complete ABCA4 protein expression efficiency be achieved compared to other inteins, but also less additional intein is produced, thereby having less unnecessary immunogenicity and side effects when used for in vivo therapy.

[0009] In addition, the present inventors unexpectedly discovered that when using the same type of intein (such as GP41-1), selecting a specific cleavage site of the ABCA4 protein (i.e., the 1224Cys site from the N-terminus) can achieve very excellent expression efficiency of the complete ABCA4 protein.

[0010] Therefore, in one aspect, the present invention provides an expression cassette combination, comprising a first expression cassette and a second expression cassette, wherein the first expression cassette is capable of expressing a fusion protein of an N-terminal truncation of an ABCA4 protein and the N-terminus of an intein, and the second expression cassette is capable of expressing a fusion protein of a C-terminal truncation of an ABCA4 protein and the C-terminus of an intein, wherein when expressed, the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette are capable of forming a complete full-length ABCA4 protein through the cleavage functions of the N-terminus and C-terminus of the intein.

[0011] In certain embodiments, the intein 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), preferably GP41-1.

[0012] In certain embodiments, the complete ABCA4 protein is a human ABCA4 protein (SEQ ID NO: 1), and the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette are the corresponding two N-terminal truncations and C-terminal truncations obtained by truncating 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.

[0013] In certain embodiments, the N-terminal truncation and the C-terminal truncation are selected from the following combinations:

[0014] amino acids 1 to 1223 and 1224 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1);

[0015] amino acids 1 to 1139 and 1140 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1);

[0016] Amino acids 1 to 1149 and amino acids 1150 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1); or

[0017] Amino acids 1 to 1187 and 1188 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1).

[0018] In certain embodiments, the intein N-terminus and the intein C-terminus are selected from the group consisting of:

[0019] 1) N-terminus of GP41-1 (SEQ ID NO: 17) and C-terminus of GP41-1 (SEQ ID NO: 18);

[0020] 2) the N-terminus of DnaE (SEQ ID NO: 15) and the C-terminus of DnaE (SEQ ID NO: 16);

[0021] 3) the N-terminus of GP41-8 (SEQ ID NO: 19) and the C-terminus of GP41-8 (SEQ ID NO: 20);

[0022] 4) N-terminus of NrdJ-1 (SEQ ID NO: 21) and C-terminus of NrdJ-1 (SEQ ID NO: 22);

[0023] 5) N-terminus of IMPDH-1 (SEQ ID NO: 23) and C-terminus of IMPDH-1 (SEQ ID NO: 24);

[0024] 6) N-terminus of SspGyrB (SEQ ID NO: 25) and C-terminus of SspGyrB (SEQ ID NO: 26)

[0025] 7) the N-terminus of Mja-KlbA (SEQ ID NO: 27) and the C-terminus of Mja-KlbA (SEQ ID NO: 28);

[0026] 8) the N-terminus of NpuSsp (SEQ ID NO: 29) and the C-terminus of NpuSsp (SEQ ID NO: 30); and

[0027] 9) N-terminus of Rma (SEQ ID NO: 61) and C-terminus of Rma (SEQ ID NO: 62).

[0028] In certain embodiments, the intein is GP41-1 and the N-terminal truncation and C-terminal truncation of the ABCA4 protein are the corresponding two N-terminal truncation and C-terminal truncation obtained by truncating the ABCA4 protein at the 1224 amino acid (Cys) site from the N-terminus, i.e., amino acids 1-1223 and amino acids 1224-2273 from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

[0029] In certain embodiments, the intein is GP41-1 and the N-terminal truncation and C-terminal truncation of the ABCA4 protein are the corresponding N-terminal truncation and C-terminal truncation obtained by truncating the ABCA4 protein at amino acid position 1188 (Cys) from the N-terminus, i.e., amino acids 1-1187 and amino acids 1188-2273 from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

[0030] In certain embodiments, the codons for expressing the full-length ABCA4 protein are optimized, and the sequence thereof is shown in SEQ ID NO: 3. In certain embodiments, the codons for expressing the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein are optimized, and the combined (merged / concatenated) sequence, i.e., the coding sequence for the full-length ABCA4 protein, is shown in SEQ ID NO: 3. It will be readily understood by those skilled in the art that codon optimization can be performed for the full-length ABCA4 sequence, or for the N-terminal truncation and C-terminal truncation sequences, respectively.

[0031] In certain embodiments, the C-terminus of the first expression cassette is connected to a first tag, and / or the C-terminus of the second expression cassette is connected to a second tag, wherein the first tag and the second tag are different. The first tag and the second tag are independently selected from the group consisting of: a hemagglutinin (HA) tag, a Flag tag, a V5 tag, a Myc tag, a His tag, a 1D4 tag (Rho1D4), and a combination 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).

[0032] In certain embodiments, the N-terminus or C-terminus of the intein is further directly or indirectly linked to a degradation signal via a linker. Preferably, the degradation signal is selected from polypeptides related to 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).

[0033] In certain 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 an enhancer and an intron for enhancing the expression of the fusion protein and operably linked to the fusion protein.

[0034] In certain embodiments, the promoter is a constitutive promoter or an inducible promoter, preferably selected from the group consisting of a CAG-containing promoter (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1 alpha short (EFS) promoter, elongation factor 1 alpha (EF-1 alpha) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, ABCA4 self promoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter or smCAG promoter, more preferably OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48), smCAG promoter (SEQ ID NO: 49), enOPEFS promoter (SEQ ID NO: 50), or smCAG promoter (SEQ ID NO: 51). 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).

[0035] In certain embodiments, the terminator is selected from the group consisting of: SV40 terminator (SEQ ID NO:50), BGH terminator (SEQ ID NO:51), WPRE terminator (such as WPRE-3 terminator (SEQ ID NO:52)), WPRE-SV40 terminator (SEQ ID NO:56), WPRE-BGH terminator, B-globin-ployA terminator (SEQ ID NO:65) or sNRP1 terminator (SEQ ID NO:66).

[0036] In certain embodiments, the enhancer is a CMV enhancer (SEQ ID NO: 53). In certain embodiments, the intron is an intron from rhesus betaherpesvirus-3 (MHV-3) (SEQ ID NO: 54) or an SV40 intron (SEQ ID NO: 55).

[0037] In certain embodiments, the first expression cassette comprises, in order from the 5' end, an optional enhancer, a promoter, an optional intron, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, a nucleotide sequence encoding an N-terminal intein, an optional degradation signal, a nucleotide sequence encoding 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 sequence encoding an C-terminal intein, an optional degradation signal, a nucleotide sequence encoding an C-terminal truncation of the ABCA4 protein, an optional nucleotide sequence encoding a tag, and a terminator. The nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, the nucleotide sequence encoding an N-terminal intein, the nucleotide sequence encoding an C-terminal intein, the nucleotide sequence encoding an C-terminal truncation of the ABCA4 protein, the optional nucleotide sequence encoding a tag, and the optional degradation signal sequence are operably linked to the promoter / enhancer.

[0038] In certain embodiments, the first expression cassette and / or the second expression cassette are present in a form selected from the group consisting of an isolated nucleic acid molecule, a liposome, and / or an exosome.

[0039] In certain embodiments, the first expression cassette and / or the second expression cassette are present in the form of a plasmid.

[0040] In certain embodiments, the first expression cassette and / or the second expression cassette is a viral vector.

[0041] In certain embodiments, the viral vector is an adeno-associated virus (AAV) of the same or different serotype, and the AAV is selected from the group consisting of: 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 / 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.

[0042] In certain embodiments, the expression cassette assembly comprises the following combination of elements:

[0043] 1) The promoter is hGRK1, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is Rma, and the terminator is SV40;

[0044] 2) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is SV40;

[0045] 3) The promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is Rma, and the terminator is B-globin-ployA;

[0046] 4) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is B-globin-ployA;

[0047] 5) the promoter is hGRK1, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is SV40;

[0048] 6) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1150, the intein is GP41-1, and the terminator is SV40; or

[0049] 7) The promoter is enRK-b-globin intron, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is B-globin-ployA.

[0050] In another aspect, the present invention provides a kit comprising the above-described expression cassette combination.

[0051] In another aspect, the present invention provides use of the expression cassette combination or kit of the present invention in the preparation of a medicament for treating a disease, including a disease caused by an ABCA4 mutation.

[0052] In certain embodiments, the disease comprises an inherited retinal disease.

[0053] In certain embodiments, the disease comprises an inherited macular degeneration disease, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0054] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0056] Figure 1 shows the protein expression intensity of codon-optimized ABCA4;

[0057] FIG2 shows a schematic diagram of forming a complete ABCA4 protein according to the intein dual expression cassette strategy of the present invention;

[0058] FIG3 shows the ABCA4 protein expression efficiency of different types of split inteins in the ABCA4 protein expression strategy of the present invention;

[0059] Figure 4 shows the cleavage efficiency of GP41-1 and the Rma intein to form full-length ABCA4;

[0060] FIG5 shows that in the ABCA4 protein expression strategy of the present invention, different truncation positions of the ABCA4 protein lead to different ABCA4 protein expression efficiencies;

[0061] FIG6 shows that the double expression cassette containing the degradation peptide can form the complete ABCA4 protein, and the degradation signal leads to significant degradation of the intein;

[0062] FIG7 shows that the expression cassette constructed in Example 5 can form a complete ABCA4 protein in 293T cells;

[0063] FIG8 shows that the expression cassette constructed in Example 6 can restore the ERG amplitude level in ABCA4 KO mice;

[0064] FIG9 shows the A2E content in the retinal tissue of mice after injection of the expression cassette constructed in Example 6;

[0065] FIG10 shows that the expression cassette constructed in Example 6 can successfully express the complete ABCA4 protein in ABCA4 KO mice;

[0066] Figure 11 shows that different cleavage sites can effectively express full-length ABCA4 protein in vivo;

[0067] Figure 12 shows that different stop codons can effectively express full-length ABCA4 protein in vivo;

[0068] Figure 13 shows that different promoters can effectively express full-length ABCA4 protein in the mouse retina;

[0069] Figure 14 shows that expression cassettes with different element combinations can effectively express the full-length ABCA4 protein in the retina of model mice;

[0070] Figure 15 shows the expression of full-length ABCA4 protein in mouse retina by different serotypes;

[0071] Figure 16 shows that the amount of additional small peptides generated by cleaving the intein GP41-1 is lower; and

[0072] FIG17 shows the ERG, fundus OCT and FP detection results after AAV8-GP41-1 and AAV8-DnaE were injected into the subretinal space of ABCA4 KO mice, respectively. DETAILED DESCRIPTION

[0073] Unless otherwise indicated, the terms used herein have their ordinary technical meanings as understood by those skilled in the art. For definitions and terms in the art, the skilled person is particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0074] The term "first expression cassette" generally comprises an exogenous DNA sequence operably linked to a promoter or other regulatory sequences sufficient to direct transcription of a gene of interest. In this application, the expression cassette generally refers to a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. The expression cassette may comprise a series of specialized nucleic acid elements that allow transcription of a specific nucleic acid in the target cell. The expression cassette may be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. The expression cassette may include untranslatable or non-translatable antisense or sense constructs. In the case of expressing a transgene and suppressing an endogenous gene (e.g., via antisense, RNAi, or sense suppression), those skilled in the art will recognize that the inserted polynucleotide sequence need not be identical but may simply be substantially identical to the sequence of the gene from which it is derived. The expression cassette may comprise a polynucleotide construct of a transcription factor operably linked to a promoter, which may be a promoter from a gene regulated by the transcription factor. In this application, the first expression cassette may express a fusion protein (e.g., a fusion protein of an N-terminal truncation of the ABCA4 protein and the N-terminus of an intein).

[0075] The term "second expression cassette" generally refers to an expression cassette that is not identical to the first expression cassette. For example, the second expression cassette can express a different protein than the protein expressed by the first expression cassette (e.g., a fusion protein of a C-terminal truncation of the ABCA4 protein and the C-terminus of an intein).

[0076] The term "expression cassette combination" generally refers to a combination comprising at least one expression cassette. For example, the expression cassette combination described in the present application may comprise the first expression cassette and the second expression cassette.

[0077] In this application, the term "operably connected" generally refers to placing the regulatory sequence necessary for the expression of the coding sequence in a suitable position relative to the coding sequence so as to achieve expression of the coding sequence. For example, when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence are operably connected. In some embodiments, the arrangement of the coding sequence and the transcription control element in the expression vector can be represented. The control element can include a promoter, an enhancer and a termination element. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably connected to the coding sequence. In some embodiments, "operably connected" can also refer to that the target gene is connected to the vector so that the transcription and translation control sequences in the vector play their expected functions of regulating the transcription and translation of the target gene.

[0078] The term "ABCA4 gene" generally refers to the gene encoding the rim protein, or RmP. The ABCA4 gene is also known as the ABCR gene. This gene was first cloned and identified as the cause of Stargardt disease, an autosomal recessive disorder that leads 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 set forth in SEQ ID NO:2.

[0079] The term "intact (full-length) ABCA4 protein" generally refers to member 4 of the ATP-binding cassette subfamily A with a complete structure. The intact ABCA4 protein can be encoded by the human ABCA4 gene. ABCA4 is a lipid inward-flipping enzyme in retinal photoreceptor cells or retinal pigment epithelial cells, which can transport retinylphosphatidylethanolamine (NRPE), a lipid derivative formed by phosphatidylethanolamine (PE) and all-trans retinal (ATR), from the lumen of the outer segment membrane disc to the cytoplasmic side. Subsequently, NRPE is hydrolyzed into ATR and PE, of which ATR is catalyzed by retinal reductase in the cytoplasm to form all-trans retinol (ATRol), thereby returning to the visual cycle. The UniProt accession number of the human intact ABCA4 protein is P78363. In one embodiment, the sequence of the human intact (full-length) ABCA4 protein is shown in SEQ ID NO: 1.

[0080] The term "N-terminal truncation of an ABCA4 protein" generally refers to an N-terminal truncation of an intact human ABCA4 protein. For example, the N-terminal truncation of an ABCA4 protein may comprise the amino acid sequence of amino acids 1 to 1139, amino acids 1 to 1149, amino acids 1 to 1187, amino acids 1 to 1223, amino acids 1 to 1325, amino acids 1 to 1160, amino acids 1 to 1220, amino acids 1 to 1280, or amino acids 1 to 1347 of an intact human ABCA4 protein from the N-terminus. For example, the amino acid sequence of the N-terminal truncation of an ABCA4 protein may be as shown in amino acids 1 to 1325 of an intact human ABCA4 protein from the N-terminus. In the present application, the N-terminal truncation of the ABCA4 protein and / or the C-terminal truncation of the ABCA4 protein may correspond to the blue and green structures in Figure 1 in Nature Communications volume 12, Article number: 3853 (2021), respectively.

[0081] In the present application, the N-terminal truncation of the ABCA4 protein may comprise the N-terminal domains of the ABCA4 protein: transmembrane regions TMD1 to TMD6, extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and / or R1.

[0082] In the present application, the N-terminal truncation of the ABCA4 protein may include, from the N-terminus, the following: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, and / or R1. For example, the N-terminal truncation of the ABCA4 protein may include, from the N-terminus, the following: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, and / or NBD1.

[0083] In the present application, the first expression cassette may comprise a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0084] The term "C-terminal truncation of an ABCA4 protein" generally refers to a C-terminal truncation of the intact human ABCA4 protein. For example, the C-terminal truncation of the ABCA4 protein may comprise the amino acid sequence of amino acids 1140-2273, 1150-2273, 1188-2273, 1224-2273, 1326-2273, 1348-2273, 1369-2273, or 1348-2170 of the intact human ABCA4 protein from the N-terminus. For example, the amino acid sequence of the C-terminal truncation of the ABCA4 protein may be as shown in amino acids 1326-2273 of the intact human ABCA4 protein from the N-terminus.

[0085] In the present application, the first expression cassette may comprise a promoter. In the present application, the promoter may comprise a constitutive promoter and / or an inducible promoter. For example, the promoter can be selected from the group consisting of a CAG-containing promoter (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1 alpha short (EFS) promoter, elongation factor 1 alpha (EF-1 alpha) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, ABCA4 self promoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter or smCAG promoter, more preferably OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48) or smCAG promoter (SEQ ID NO: 49).

[0086] In the present application, the C-terminal truncation of the ABCA4 protein may comprise the C-terminal domains of the ABCA4 protein: transmembrane regions TMD7 to TMD12, extracellular domains ECD1, IH3, IH4, EH3, EH4, NBD2 and / or R2.

[0087] In the present application, the C-terminal truncation 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 truncation 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.

[0088] In the present application, the second expression cassette may comprise a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0089] In the present application, the second expression cassette may comprise a promoter. In the present application, the promoter may comprise a constitutive promoter and / or an inducible promoter. For example, the promoter is selected from the group consisting of a CAG-containing promoter (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), human β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1 alpha short (EFS) promoter, elongation factor 1 alpha (EF-1 alpha) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, ABCA4 self promoter, RPE65 promoter, OPEFS promoter, hGRK1 promoter or smCAG promoter, more preferably OPEFS promoter (SEQ ID NO: 47), hGRK1 promoter (SEQ ID NO: 48) or smCAG promoter (SEQ ID NO: 49).

[0090] Herein, the N-terminal truncation and C-terminal truncation of the ABCA4 protein can be the corresponding two N-terminal truncation and C-terminal truncation obtained by truncating the complete ABCA4 protein at a specific site (e.g., Cys). In other words, the N-terminal truncation and C-terminal truncation of the ABCA4 protein of the present invention can be spliced ​​from the N-terminus to the C-terminus (e.g., by cleavage of the N-terminus and C-terminus of the intein attached to each other) to form a complete ABCA4 protein, thereby exerting its biological function. Preferably, the truncation site is the following amino acid (Cys) sites from the N-terminus of the ABCA4 protein: 1224, 1140, 1150, or 1188, more preferably 1188 or 1224. For example, when the truncation site is at site 1188, the N-terminal truncation of the ABCA4 protein is amino acids 1-1187 from the N-terminus, while the corresponding C-terminal truncation is amino acids 1188-2273 from the N-terminus. For example, when the truncation position is at position 1224, the N-terminal truncation of the ABCA4 protein is amino acids 1-1223 from the N-terminus, and the corresponding C-terminal truncation is amino acids 1224-2273 from the N-terminus.

[0091] In the present application, the protein molar ratio of the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette to the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette is about 3:1 to 1:3. For example, the protein molar ratio of the N-terminal truncation of the ABCA4 protein to the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette may be about 3:1 to 1:3, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1 to 2:1, about 2:1 to 1:3, about 2:1 to 1:2, about 2:1 to 1:1, about 1:1 to 1:3, about 1:2 to 1:1, or about 1:1.

[0092] The term "biological function" generally refers to an activity of interest that is native to or is a part of the biological entity being tested, such as a native activity of a cell, protein, or the like. Ideally, an in vitro functional assay can be used to test for the presence of a biological function.

[0093] In the present application, the biological functions of the complete ABCA4 protein may include cellular localization function; ATP hydrolase activity; expression in photoreceptor cells, and / or reduction of retinal A2E deposition.

[0094] As used herein, the term "intein" refers to a naturally occurring or artificially constructed polypeptide sequence that catalyzes the protein splicing reaction, where the intein sequence is cleaved from a precursor protein and flanking sequences (N-extein and C-extein) are added via peptide bonds. A list of known inteins is published at http: / / www.inteins.com. Table 1 of CN 114698379 A summarizes hundreds of naturally occurring inteins. In one embodiment, the intein of the present invention is a split intein. In the present invention, the intein can be 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 below in the specification.

[0095] The term "protein splicing" generally refers to the process by which the internal regions of a precursor protein (inteins) are removed and the flanking regions of the protein (exteins) are joined to form the mature protein. The intein unit may contain the necessary components required to catalyze protein splicing and typically includes an endonuclease domain involved in intein mobility. The resulting proteins may be linked together but not expressed as separate proteins. Protein splicing can also occur in trans, with broken inteins expressed on separate polypeptides spontaneously combining to form a single intein, which then undergoes the protein splicing process to combine into separate proteins.

[0096] As used herein, the term "intein N-terminus" refers to any intein comprising an N-terminal amino acid sequence that is functional for the trans-splicing reaction, i.e., is capable of associating with a functional split intein C fragment to form a complete intein, which complete intein sequence is capable of cleaving itself from the host protein, catalyzing the ligation of exteins or flanking sequences via peptide bonds, or, when associated with a split intein C fragment, catalyzing "N-terminal cleavage," i.e., nucleophilic attack on the peptide bond between the extein and the N-terminus of the split intein N fragment, resulting in the cleavage of said peptide bond.

[0097] As used herein, the term "intein C-terminus" refers to any intein comprising a C-terminal amino acid sequence that is functional for the trans-splicing reaction, i.e., capable of associating with a functional split intein N-fragment upon association to form a complete intein, wherein the complete intein sequence is capable of cleaving itself from the host protein, catalyzing the joining of exteins or flanking sequences via peptide bonds, or, when associated with a split N-intein, catalyzing "C-terminal cleavage," i.e., nucleophilic attack on the peptide bond between the extein and the C-terminus of the split intein C-fragment, thereby resulting in the cleavage of said peptide bond.

[0098] The N-terminus of the intein and the C-terminus of the intein can be fused to the N-terminal truncations of the ABCA4 protein and the C-terminal truncations of the ABCA4 protein, respectively, to connect the N-terminal truncations of the ABCA4 protein and the C-terminal truncations of the ABCA4 protein. For example, in some embodiments, the N-terminus of the intein is fused to the C-terminus of the N-terminal portion of the fragmented ABCA4 protein, i.e., forming a structure of N-[N-terminal truncations of the ABCA4 protein]-[N-terminus of the intein]--C. In some embodiments, the C-terminus of the intein is fused to the N-terminus of the C-terminal truncations of the ABCA4 protein, i.e., forming a structure of N-[C-terminus of the intein]--[C-terminal truncations of the ABCA4 protein]-C. The mechanisms for intein-mediated protein splicing that join proteins to which inteins are fused (e.g., split ABCA4 protein) are known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference.

[0099] In one embodiment, the "intein N-terminus" and "intein C-terminus" in the present invention are a homologous pair, i.e., derived from the same intein (see Shah et al. Journal of the American Chemical Society 2012, https: / / doi.org / 10.1021 / ja303226x). It should be understood that regardless of whether the "intein N-terminus" and "intein C-terminus" in the present invention are derived from the same intein, they should be able to associate or reconstruct into a single fragment, thereby causing trans-splicing of the intein. In the present invention, preferably, the "intein N-terminus" and "intein C-terminus" are the N-terminus and C-terminus combinations of the following inteins: the N-terminus and C-terminus of GP41-1; the N-terminus and C-terminus of DnaE; the N-terminus and C-terminus of GP41-8; the N-terminus and C-terminus of NrdJ-1; the N-terminus and C-terminus of NrdJ-1; the N-terminus and C-terminus of IMPDH-1; the N-terminus and C-terminus of SspGyrB; the N-terminus and C-terminus of Mja-KlbA; the N-terminus and C-terminus of NpuSsp; or the N-terminus and C-terminus of Rma. In one embodiment, the sequences of the N-terminus and C-terminus of the above-mentioned inteins according to the present invention are shown in Table 1 below.

[0100] As used herein, the term "degradation signal" refers to a polypeptide that, when fused directly or indirectly via a linker to the N-terminus or C-terminus of an intein of the present invention, can induce degradation of the intein N-terminus or C-terminus. Non-limiting examples of degradation signals include polypeptides associated with DHFR (dehydrofolate reductase), FKBP (FK506 binding protein), FRB (FKBP-rapamycin binding protein), and PDE5 (phosphodiesterase type 5), as well as degradation signals of the endoplasmic reticulum-associated degradation (ERAD) pathway.

[0101] In one embodiment, the N-terminus of the intein or the C-terminal fragment of the intein in the first expression cassette can be linked to a degradation signal at its C-terminus, either directly or indirectly via a linker, to promote degradation of the intein formed after the ABCA4 protein is released by intein splicing. It should be noted that when a degradation signal is used, it does not adversely affect the splicing yield or negatively impact the expression of the intact ABCA4 protein.

[0102] In a preferred embodiment, the first expression cassette and the second expression cassette respectively comprise, starting from the 5' end:

[0103] 1) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0104] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0105] 2) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of DnaE (SEQ ID NO: 15)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0106] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of DnaE (SEQ ID NO: 16)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0107] 3) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (the N-terminus of GP41-8 (SEQ ID NO: 19)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0108] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-8 (SEQ ID NO: 20)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0109] 4) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (N-terminus of NrdJ-1 (SEQ ID NO: 21)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0110] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of NrdJ-1 (SEQ ID NO: 22)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0111] 5) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of IMPDH-1 (SEQ ID NO: 23)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0112] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of IMPDH-1 (SEQ ID NO: 24)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0113] 6) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (the N-terminus of SspGyrB (SEQ ID NO: 25)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0114] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of SspGyrB (SEQ ID NO: 26)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0115] 7) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of Mja-KlbA (SEQ ID NO: 27)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0116] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of Mja-KlbA (SEQ ID NO: 28)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0117] 8) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of NpuSsp (SEQ ID NO: 29)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0118] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of NpuSsp (SEQ ID NO: 30)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0119] 9) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0120] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0121] 10) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0122] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0123] 11) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0124] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0125] 12) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of DnaE (SEQ ID NO: 15)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0126] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of DnaE (SEQ ID NO: 16)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0127] 13) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of DnaE (SEQ ID NO: 15)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0128] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of DnaE (SEQ ID NO: 16)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0129] 14) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of DnaE (SEQ ID NO: 15)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0130] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of DnaE (SEQ ID NO: 16)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0131] 15) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of DnaE (SEQ ID NO: 15)), a degradation signal (e.g., ecDHFR (SEQ ID NO: 14)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0132] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of DnaE (SEQ ID NO: 16)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0133] 16) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), and a terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and

[0134] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., SV40 terminator (SEQ ID NO: 50));

[0135] 17) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), and a terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56)), and

[0136] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56));

[0137] 18) a promoter (e.g., the OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (the N-terminus of GP41-1 (SEQ ID NO: 17)), and a terminator (e.g., the WPRE-3 terminator (SEQ ID NO: 52)), and

[0138] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52));

[0139] 19) a promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)), and

[0140] a promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), a nucleotide coding sequence for the C-terminus of an intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for a C-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)); or

[0141] 20) a promoter (e.g., an smCAG promoter (SEQ ID NO: 49)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (the N-terminus of GP41-1 (SEQ ID NO: 17)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)), and

[0142] promoter (e.g., smCAG promoter (SEQ ID NO: 49)), a nucleotide coding sequence for the C-terminus of the intein (the C-terminus of GP41-1 (SEQ ID NO: 18)), an (optimized) nucleotide coding sequence for the C-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., the SV40 terminator (SEQ ID NO: 50)).

[0143] 21) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0144] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0145] 22) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1139 from the N-terminus, i.e., amino acids 1-1139 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0146] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1140 from the N-terminus, i.e., amino acids 1140-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0147] 23) a promoter (e.g., an OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1-1149 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0148] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1150 from the N-terminus, i.e., amino acids 1150-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0149] 24) a promoter (e.g., an OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1-1187 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51)), and

[0150] a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1188 from the N-terminus, i.e., amino acids 1188-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., a BGH terminator (SEQ ID NO: 51));

[0151] 25) a promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), and a terminator (e.g., SV40 terminator (SEQ ID NO: 50)), and

[0152] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., SV40 terminator (SEQ ID NO: 50));

[0153] 26) a promoter (e.g., the OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the intein N-terminus (the N-terminus of RMA (SEQ ID NO: 61)), and a terminator (e.g., the WPRE-SV40 terminator (SEQ ID NO: 56)), and

[0154] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence of the C-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., WPRE-SV40 terminator (SEQ ID NO: 56));

[0155] 27) promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of an ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1 to 1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of an intein N-terminus (N-terminus of RMA (SEQ ID NO: 61)), and a terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52)), and

[0156] A promoter (e.g., OPEFS promoter (SEQ ID NO: 47)), a nucleotide coding sequence of the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence of the C-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), and a terminator (e.g., WPRE-3 terminator (SEQ ID NO: 52));

[0157] 28) promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)), and

[0158] a promoter (e.g., hGRK1 promoter (SEQ ID NO: 48)), a nucleotide coding sequence for the C-terminus of an intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for a C-terminal truncation of an ABCA4 protein (NTD) (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)); or

[0159] 29) promoter (e.g., smCAG promoter (SEQ ID NO: 49)), an (optimized) nucleotide coding sequence of an N-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1-1223 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence of the N-terminus of the intein (N-terminus of RMA (SEQ ID NO: 61)), a nucleotide coding sequence of a first tag (e.g., an HA tag (SEQ ID NO: 12)), and a terminator (e.g., an SV40 terminator (SEQ ID NO: 50)), and

[0160] promoter (e.g., smCAG promoter (SEQ ID NO: 49)), a nucleotide coding sequence for the C-terminus of the intein (C-terminus of RMA (SEQ ID NO: 62)), an (optimized) nucleotide coding sequence for the C-terminal truncation (NTD) of the ABCA4 protein (truncation site: amino acid 1224 from the N-terminus, i.e., amino acids 1224-2273 from the N-terminus) (the corresponding nucleotide coding sequence in SEQ ID NO: 3), a nucleotide coding sequence for a second tag (e.g., a Flag tag (SEQ ID NO: 13)), and a terminator (e.g., the SV40 terminator (SEQ ID NO: 50)).

[0161] The term "liposome" generally refers to a lipid structure formed from amphiphilic vesicle-forming lipids. The liposome can be a closed vesicle composed of a single or multiple lipid bilayers with an internal aqueous phase. For example, liposomes can be more broadly referred to as lipid complex particles. Liposomes can even include complexes whose aqueous phase is not definitively identified. Lipoplexes contain at least one type of lipid and may additionally contain hydrophilic polymers, polysaccharides, amino acids, etc. Lipoplexes can also refer to particles formed from these components via covalent or non-covalent bonds.

[0162] The term "exosome" generally refers to a membrane vesicle secreted outside a cell or having a membrane structure composed of a lipid bilayer present in a cell. The exosome may include membrane bodies with an average diameter ranging from about 10 nm to about 2,000 nm. The exosome may include microvesicles. Microvesicles, also known as circulating microvesicles or microparticles, are fragments of the plasma membrane with an approximate diameter ranging from 100 nm to 1000 nm that are shed from almost all cell types. The exosome may also include smaller intracellularly produced extracellular vesicles, which are formed by inward budding of the limiting membrane of multivesicular bodies (MVBs). Upon fusion with the plasma membrane, the MVBs lead to their secretion and deposition in body fluids (e.g., blood, urine). Exosomes may contain a complex mixture of microRNAs (miRs), mRNAs, and proteins, which reflect the transcriptional and translational status of the producing cell.

[0163] The term "AAV" is generally an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The AAV may include AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 (AAV-3 or AAV3), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, among others.

[0164] In a preferred embodiment, the first expression cassette and the second expression cassette of the present invention are both AAV viral packaging plasmids, wherein the first and second expression cassettes include an AAV 5' inverted terminal repeat (5'-ITR) sequence at the 5' end of the promoter sequence and an AAV 3' inverted terminal repeat (3'-ITR) sequence at the 3' end of the terminator.

[0165] The term "kit" generally refers to a package comprising one or more active ingredients in one or more suitable containers.

[0166] The term "diseases caused by ABCA4 mutations" generally refers to diseases associated with ABCA4 gene mutations, such as rare eye diseases.

[0167] The term "inherited retinal disease" generally refers to inherited retinal dystrophies or inherited retinal diseases, IRDs, which are rare ophthalmic diseases that cause loss of retinal function and / or progressive degeneration in patients due to genetic defects. The inherited retinal diseases often cause reversible or irreversible visual impairment in infancy or adolescence. IRDs are intractable ophthalmic diseases that are often manifested as abnormal protein metabolism. The purpose of treatment can be achieved by regulating genes, transcription and translation in the process of genetic information transmission. The inherited retinal diseases may include autosomal recessive retinitis pigmentosa (RP, MERTK mutation), choroideremia (CHM mutation), juvenile macular degeneration (ABCA4 mutation), Usher syndrome 1B subtype (Myo7a mutation), X-linked congenital retinoschisis (RS1 mutation), mitochondrial-related Leber hereditary optic neuropathy (ND4 mutation), achromatopsia (CNGA3 mutation and CNGB3 mutation), sex-linked inherited RP (RPGR mutation), etc.

[0168] The term "hereditary macular degeneration" generally refers to a less common familial genetic disease. The hereditary macular degeneration may include Stargardt disease. Stargardt disease is an autosomal recessive genetic disease originating in the retinal pigment epithelium. Sporadic cases are relatively rare and occur more often in children of consanguineous marriages. Patients have macular atrophy damage combined with retinal yellow spot deposits. There is currently no approved treatment option, and Stargardt disease can cause vision loss in tens of thousands of people worldwide each year. The hereditary macular degeneration may also include Best disease, in which the lesions mainly affect the macular area of ​​both eyes and generally do not spread to the surrounding retina. The hereditary macular degeneration may occur at any age.

[0169] The term "age-related macular degeneration" generally refers to age-related macular degeneration (AMD), a major cause of blindness in people over 50 years old. AMD can manifest as progressive, irreversible loss of central vision, primarily affecting the macula, retinal pigment epithelium, and choroid. Clinically, early-stage AMD is characterized by subretinal drusen deposits, while late-stage AMD is divided into two types: dry and wet. Dry AMD, also known as atrophic AMD, primarily manifests as geographic atrophy and detachment of the RPE layer, with slow, progressive vision loss in the affected eye. Wet AMD, also known as neovascular or exudative AMD, primarily manifests as choroidal neovascularization (CNV) and subsequent fluid accumulation and hemorrhage. The pathogenesis of AMD remains unclear, but may involve RPE damage, mitochondrial dysfunction, oxidative stress, inflammation, and complement pathway activation. It is generally believed that the development of AMD results from a combination of genetic and environmental factors.

[0170] The term "retinitis pigmentosa" usually refers to Retinitis Pigmentosa, RP, which is a group of hereditary eye diseases characterized by progressive degeneration of retinal photoreceptors. The retinitis pigmentosa may include a progressive retinal (a transparent photosensitive membrane on the inner surface of the back of the eyeball) degenerative disease, which may eventually lead to moderate to severe vision loss. Retinal pigmentary lesions are mostly hereditary. The clinical characteristics of the retinitis pigmentosa may include early night blindness, centripetal narrowing of the visual field, and eventually tubular vision, binocular blindness or near-blindness, which accounts for a considerable proportion of blind eye diseases. The retinitis pigmentosa may belong to a hereditary rod and cone dystrophy disease.

[0171] The term "cone-rod dystrophy" generally refers to cone-rod dystrophy (CRD), a hereditary macular degeneration disorder. CRD encompasses a group of clinical and genetic syndromes characterized by progressive visual loss, photophobia, and nystagmus. These disorders primarily affect cones and, in severe cases, may also affect rods. They are inherited in autosomal dominant, recessive, or X-linked patterns.

[0172] The term “retinylphosphatidylethanolamine (NRPE)” generally refers to the physiological lipid substrate of ABCA4, which is sandwiched between two TMDs in the luminal leaflet and further stabilized by an extended loop from the extracellular domain 1.

[0173] In the present application, the first expression cassette and the second expression cassette may exist separately.

[0174] In the present application, the first expression cassette and / or the second expression cassette may be present in a form selected from the group consisting of: an isolated nucleic acid molecule, a liposome and / or an exosome.

[0175] In the present application, the first expression cassette and / or the second expression cassette may exist in the form of a plasmid.

[0176] In the present application, the first expression cassette and / or the second expression cassette may be a viral vector.

[0177] In the present application, the first expression cassette and / or the second expression cassette may be AAV.

[0178] In another aspect, the present application provides a kit comprising the expression cassette combination described in the present application.

[0179] In the present application, the kit may comprise reagents and / or instruments for administering the expression cassette combination. For example, the kit may comprise reagents and / or instruments for transfecting an AAV comprising the first expression cassette and / or the second expression cassette. For example, the kit may comprise reagents and / or instruments for injecting an AAV comprising the first expression cassette and / or the second expression cassette. In the present application, the injection may comprise a local injection.

[0180] On the other hand, the present application provides a use of the expression cassette combination described in the present application, and / or the kit described in the present application, in the preparation of a drug for treating a disease, including a disease caused by an ABCA4 mutation.

[0181] In the present application, the disease may include inherited retinal diseases.

[0182] In the present application, the disease may include hereditary macular degeneration diseases, age-related macular degeneration, retinitis pigmentosa and / or cone-rod dystrophy.

[0183] The method described in the present application can be used to express a heterologous N-terminal truncation of the ABCA4 protein and a heterologous C-terminal truncation of the ABCA4 protein in a subject in need thereof. Since the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the heterologous ABCA4 protein form a complete ABCA4 protein after splicing by the intein, they can exert the biological function of the complete ABCA4 protein and can therefore be used to alleviate / treat / amend diseases caused by ABCA4 mutations.

[0184] In another aspect, the present application provides a method for expressing a heterologous ABCA4 gene, which may comprise administering the expression cassette combination described herein and / or the kit described herein to a subject in need thereof.

[0185] On the other hand, the present application provides a method for alleviating cell death caused by retinyl phosphatidylethanolamine (NRPE), which may include administering the expression cassette combination described herein and / or the kit described herein to a subject in need thereof.

[0186] In another aspect, the present application provides a method for treating a disease caused by an ABCA4 mutation, which may comprise administering the expression cassette combination described herein and / or the kit described herein to a subject in need thereof.

[0187] In the present application, the administration may include injection.

[0188] In the present application, the disease may include inherited retinal diseases.

[0189] In the present application, the disease may include hereditary macular degeneration diseases, age-related macular degeneration, retinitis pigmentosa and / or cone-rod dystrophy.

[0190] The present invention is further described in the following examples which are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0191] Example 1 Codon Optimization of ABCA4 Protein

[0192] The present inventors codon-optimized the CDS region of the wild-type human ABCA4 protein (SEQ ID NO: 1), and the optimized codons are shown in SEQ ID NO: 3 in Table 1. The CDS region of the wild-type human ABCA4 protein and the optimized CDS region were respectively inserted into the EF1A promoter of the pFG12-PURO vector (the pFG12-PURO vector was modified from pFG12 (Addgene #14884), and the EGFP CDS region of the pFG12 vector was replaced with the PURO CDS (amino acid sequence shown in SEQ ID NO: 57, encoding nucleotide sequence shown in SEQ ID NO: 58), and the EF1A promoter (sequence shown in SEQ ID NO: 59) was inserted in the opposite direction of the UBC promoter. NO:59), pFG12-PURO-ABCA4-WT-Flag and pFG12-PURO-ABCA4-CO-Flag were obtained respectively. PEI (23966-1, Polyscience) was used for transfection, and pFG12-PURO-ABCA4-WT-Flag and pFG12-PURO-ABCA4-CO-Flag were transfected into human HEK293A (Peking University), human retinal pigment epithelial cells ARPE19 (CRL-2302 , ATCC) and mouse retinal photoreceptor cell lines (Peking University). After 48 hours, cells were lysed with RIPA lysis buffer (C1053-100, Priligen), and ABCA4 protein expression was detected by Western blot. The optimization results (see Figure 1) showed that compared with the unoptimized wild-type ABCA4 (WT), the optimized ABCA4 coding sequence (CO) significantly increased ABCA4 protein expression in human HEK293A and ARPE19 cells (detected using an endogenous ABCA4 antibody (clone 5B4, MABN2440, Merck)), but no enhanced expression was observed in mouse 661W cells. The endogenous control protein ACTB (β-actin, detected by antibody: AC026, ABclonal) was used.

[0193] Example 2 Different types of split inteins lead to different ABCA4 protein expression efficiencies

[0194] The strategy of using an intein double expression cassette to form a complete ABCA4 protein according to the present invention is shown in Figure 2, which is similar to the method of Tornabene, P., et al. (2019). "Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina." Sci Transl Med 11(492), with the main difference being the truncation position of ABCA4 and / or the intein used.

[0195] We truncated the ABCA4 protein (SEQ ID NO: 1) at position 1224 and combined the N-terminal truncation (NTD) and C-terminal truncation (CTD) of ABCA4 with different types of 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: 12). NO:60), and the N-terminus and C-terminus of these inteins (for specific sequences, see Table 1 below) were respectively inserted into the 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 includes the nucleotide coding sequence of the N-terminal truncation of the ABCA4 protein; CTD indicates that the plasmid includes the nucleotide coding sequence of the C-terminal truncation of the ABCA4 protein; intN indicates that the plasmid includes the nucleotide coding sequence of the N-terminus of the intein; intC indicates that the plasmid includes the nucleotide coding sequence of the C-terminus of the intein; HA indicates that the plasmid includes the nucleotide coding sequence of the HA tag (SEQ ID NO:12); Flag indicates that the plasmid includes the nucleotide coding sequence of the Flag tag (SEQ ID NO:13)). HEK293A cells (Peking University) were transfected with the corresponding plasmid combinations using PEI, with CO serving as a positive control for the transfection plasmid pFG12-PURO-ABCA4-CO-Flag. The results, as shown in Figure 3, indicate that the inteins DnaE, ​​GP41-1, GP41-8, NrdJ-1, IMPDH-1, and SspGyrB produced intact ABCA4 protein of the correct molecular weight. DnaE, ​​GP41-1, and NrdJ showed the highest efficiency in reconstructing intact ABCA4 protein. The order of reconstructing efficiency among the different inteins was GP41-1 > DnaE > NrdJ > GP41-8 ~ IMPDH-1 ~ SspGyrB. Mja-KlbA and NpuSsp failed to produce intact ABCA4 protein of the correct molecular weight. Antibodies used in Western blot: anti-Flag, F1804, Sigma; anti-HA, 3724S, CST; anti-ACTB, AC026, ABclonal.

[0196] Furthermore, we further compared the cleavage efficiency of the GP41-1 and Rma inteins. Western blot results, shown in Figure 4, show that both GP41-1 and Rma inteins effectively reconstituted the intact ABCA4 protein, with comparable reconstitution efficiencies. Antibodies used in the Western blot analysis were: anti-ABCA4, clone 5B4; anti-ABCA4, clone 3F4; and anti-ACTB, clone AC026, AB.

[0197] Example 3 Different truncation positions of ABCA4 protein lead to different ABCA4 protein expression efficiencies

[0198] Similar to the method described in Example 2, we truncated ABCA4 at the following amino acid positions, starting from the N-terminus: 1140, 1150, 1188, and 1224. These N-terminal truncations (NTD) and C-terminal truncations (CTD) of ABCA4 were combined with either DnaE or GP41-1 splicing peptides, respectively. HEK293A cells (Peking University) were transfected with the corresponding plasmid combinations, with CO serving as a positive control for the transfection plasmid pFG12-PURO-ABCA4-CO-Flag. Results showed that truncation at amino acid position 1224 was most efficient. The efficiency ranking of different truncation positions was 1224 > 1188 > 1150 > 1140. Furthermore, we found that GP41-1 produced a lower level of additional small peptide intein, potentially offering a safer alternative for gene therapy (see Figure 5). Antibodies used in Western blot: anti-Flag, F1804, Sigma; anti-HA, 3724S, CST; anti-ACTB, AC026, ABclonal.

[0199] Example 4 Dual expression cassettes containing degradation peptides can form complete ABCA4 protein

[0200] Similar to the above example, the N-terminal truncation (NTD) and C-terminal truncation (CTD) of ABCA4 were obtained by truncating ABCA4 at amino acid position 1150 from the N-terminus. These truncation fragments were combined with the N-terminus of the intein DnaE (SEQ ID NO: 15) and the C-terminus of DnaE (SEQ ID NO: 16), respectively, and ligated with respective tags to generate two corresponding expression cassette constructs (pAV-OPEFS-NTD(1150)-int(DnaE)N-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag). A nucleotide sequence encoding the degradation signal (degradation peptide) ecDHFR (SEQ ID NO: 14) was inserted into pAV-OPEFS-NTD(1150)-int(DnaE)N-HA to generate the pAV-OPEFS-NTD(1150)-int(DnaE)N-ecDHFR-HA vector. pAV-OPEFS-NTD(1150)-int(DnaE)N-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag (without degradation signal, represented as -ecDHFR in FIG6 ), or pAV-OPEFS-NTD(1150)-int(DnaE)N-ecDHFR-HA and pAV-OPEFS-int(DnaE)C-CTD(1150)-Flag (with degradation signal, represented as +ecDHFR in FIG6 ) were co-transfected into HEK293A cells with PEI. After 48 hours, the expression of the complete ABCA4 protein was detected by Western blotting (as shown in FIG6 ), where CO is a positive control of the transfected plasmid pFG12-PURO-ABCA4-CO-Flag. The results showed that the dual expression cassette containing the degradation peptide was able to form the complete ABCA4 protein, and fewer inteins were detected, indicating that the inteins were partially degraded by the degradation signal. Antibodies used in Western blot: anti-Flag, F1804, Sigma; anti-HA, 3724S, CST.

[0201] Example 5 Expression cassette is able to form complete ABCA4 protein in 293T cells

[0202] Similar to the above example, the N-terminal truncation (NTD) and C-terminal truncation (CTD) of ABCA4 were obtained by truncating ABCA4 at amino acid position 1150 from the N-terminus, and they were combined with the N-terminus and C-terminus of the intein GP41-1 (GP41-1 N-terminus (SEQ ID NO: 17) and GP41-1 C-terminus (SEQ ID NO: 18)), respectively, and the respective tags were connected to obtain two corresponding expression cassette constructs (pAV-OPEFS-NTD (1150) -int (GP41-1) N-HA, pAV-OPEFS-int (GP41-1) C-CTD (1150) -Flag). 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 the expression cassettes of the NTD and CTD truncated at the 1150 site described in Example 3 and GP41-1, respectively. 293T cells (3216, ATCC) were co-infected at multiplicity of infection (MOI) of 0, 2E4, and 2E5, respectively. Complete ABCA4 protein was formed 72 hours after infection (see Figure 7 for results). CO is a positive control for the transfection plasmid pFG12-PURO-ABCA4-CO-Flag. Antibodies used in Western blot: anti-Flag, F1804, Sigma; anti-HA, 3724S, CST.

[0203] Example 6 The expression cassette can successfully express the complete ABCA4 protein in ABCA4 KO mice

[0204] The AAV8 vectors pAV-OPEFS-NTD(1150)-int(GP41-1)N-HA and pAV-OPEFS-int(GP41-1)C-CTD(1150)-Flag constructed as described in the above examples were used to package the expression cassettes (daul-AAV(GP41-1-1150)) of the NTD and CTD truncated at the 1150 site in combination with GP41-1 as described in Example 3, respectively. The cassettes were injected subretinally into 1-2 month old ABCA4 KO mice (S-KO-00815, C57BL / 6J, Saiye Bio). Five weeks later, the mice were anesthetized, the pupils were dilated, and the mice were stimulated with 20,000 lux, 430 nm blue light for 30 minutes. ERG was measured after one week of recovery. The results showed that blue light stimulation could significantly reduce the ERG amplitude level of mice, and dual-AAV injection could restore the ERG amplitude level of ABCA4 KO mice (see Figure 8, wherein 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 (wherein AAV-EV virus represents empty AAV virus, i.e., AAV virus that does not contain the target genome (i.e., ABCA4 expression cassette)); KO+B+dualAAV represents ABCA4 KO mice injected with AAV virus expressing the dual expression cassette of the present invention after blue light stimulation).

[0205] After 6 weeks, the animals were euthanized and the retinal tissues were isolated. A portion of the retinal tissue was used for phenol-chloroform-methanol extraction to extract A2E. The A2E content in the samples was determined using an ACQUITY UPLC H-Class ultra-high performance liquid chromatography with an Atlantic dC18 column using acetonitrile and water containing 0.1% TFA 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).

[0206] Table A

[0207] The remaining mouse retinal tissue was used for Western blot analysis. The results (Figure 10) showed that 9 of the 12 mouse retinal samples expressed intact ABCA4 protein, with the highest expression intensity being approximately 20% of the endogenous expression intensity in wild-type mice. Antibodies used in Western blot analysis were: anti-ABCA4, clone 5B4, MABN2440, Merck; anti-ACTB, AC026, ABclonal.

[0208] Example 7: Expression cassettes containing different cleavage sites of ABCA4 protein can successfully express complete ABCA4 protein in ABCA4 KO mice

[0209] According to the method of the above embodiment, the following AAVs were packaged using AAV8 vectors: an expression cassette of NTD and CTD truncated at site 1150 combined with GP41-1 (daul-AAV (GP41-1-1150)), an expression cassette of NTD and CTD truncated at site 1150 combined with DnaE (daul-AAV (DnaE-1150)), an expression cassette of NTD and CTD truncated at site 1188 combined with GP41-1 (daul-AAV (GP41-1-1188)), and an expression cassette of NTD and CTD truncated at site 1224 combined with GP41-1 (daul-AAV (GP41-1-1224)). Each double AAV combination was injected into the subretinal space of ABCA4 KO mice. The animals were euthanized after 6 weeks, and the retinal tissue of the eyeballs was isolated for Western BLOT detection. The experimental results are shown in Figure 11. Sites 1150, 1188, and 1224 can all effectively express full-length ABCA4 protein in the mouse retina. A comparison found that the cleavage efficiency of sites 1188 and 1224 was higher than that of site 1150.

[0210] Example 8 Expression cassettes containing different regulatory elements can successfully express complete ABCA4 protein in ABCA4 KO mice

[0211] To further screen the effects of regulatory elements on the in vivo expression efficiency of ABCA4, based on the above examples, the Flag tag and HA tag were deleted from the dual AAV vectors with 1224 or 1188 cleavage sites, and the original BGH terminator was replaced with the SV40 terminator or WPRE-SV40 terminator. In addition, the dual AAV terminator was further replaced with the B-globin-ployA terminator (SEQ ID NO: 65) or the sNRP1 terminator (SEQ ID NO: 66) at the 1188 cleavage site.

[0212] The above vectors were packaged using AAV8 vectors. Each dual-AAV combination was injected subretinally into ABCA4 KO mice. Six weeks later, the animals were euthanized, and retinal tissue was isolated for Western blotting. The experimental results are shown in Figure 12. Each terminator can effectively express the full-length ABCA4 protein, and WPRE can promote the expression of the complete ABCA4 protein.

[0213] Example 9 Expression cassettes containing different promoters can successfully express the complete ABCA4 protein in ABCA4 KO mice

[0214] The vectors pAV-OPEFS-NTD(1224)-int(GP41-1)N-SV40 and pAV-OPEFS-int(GP41-1)C-CTD(1224)-SV40 constructed as described in the above examples were used. The original promoter sequence EFS was replaced with the hGRK1 promoter or the smCAG promoter. In addition, 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). At the same time, a partial sequence of the hGRK1 promoter was further 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 respectively packaged using AAV8 vectors. Each dual-AAV combination was injected subretinally into ABCA4 KO mice. Six weeks later, the animals were euthanized, and retinal tissue was isolated for Western blotting. The results, shown in Figure 13, demonstrate that each promoter effectively expressed the full-length ABCA4 protein in the mouse retina.

[0215] Example 10: Reducing the length of the expression cassette allows for successful expression of the complete ABCA4 protein in ABCA4 KO mice

[0216] The ABCA4 1224 cleavage site encodes an excessively long amino acid sequence, which can cause the ABCA4-NTD expression cassette to exceed the AAV vector packaging capacity, making efficient production difficult. Based on the methods described in the previous examples, we screened expression cassettes based on the 1188 and 1150 cleavage sites and further validated these genome-shortening expression cassette combinations in mice.

[0217] The carrier element combination includes:

[0218] The NTD and CTD expressed by the dual expression cassettes described above were packaged into AAV8. Each dual AAV combination was injected subretinally into ABCA4 KO mice. Eight weeks later, the animals were euthanized, and retinal tissue was isolated for Western blotting. The results, shown in Figure 14, A, demonstrate that all combinations effectively expressed the full-length ABCA4 protein in the retina of the model mice. Furthermore, as shown in Figure 14, B, the A2E assay results show that subretinal administration of the expression cassette vectors in each group significantly reduced A2E deposition in the ABCA4 KO mouse model, demonstrating the potential therapeutic efficacy of these expression cassettes.

[0219] Example 11 Expression cassettes packaged in different vectors can successfully express complete ABCA4 protein in ABCA4 KO mice

[0220] According to the method of the above embodiment, AAV8, AAV8-Y447,733F vector, double AAVtYF or AAV2.7m8 were used to package the expression cassette of the NTD and CTD truncated at the 1224 site combined with GP41-1 (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 was packaged by Guangzhou Paizhen Biotechnology Co., Ltd.), ABCA4 KO mice were injected into the subretinal space, and the animals were euthanized after 6 weeks. The retinal tissue of the eyeball was isolated for Western Blot. BLOT assay results, as shown in Figure 15, show that AAV8, the AAV8-Y447,733F variant, and AAV2.7m8 serotypes can all effectively express full-length ABCA4 protein in the mouse retina, while the AAV2-tYF serotype has lower expression efficiency. Compared to AAV8, the AAV7m8 serotype exhibits stronger expression.

[0221] Example 12: The content of additional small peptides generated by cleaving the intein GP41-1 is lower

[0222] To test whether the additional small peptides generated by splicing the split peptides in the dual AAV vectors are retinotoxic, we constructed separate GP41-1 and DnaE split peptides into plasmids pAV-OPEFS-int(GP41-1)-HA and pAV-OPEFS-int(DnaE)-HA (pX601, Addgene), and packaged them into AAV8 viruses. The following are the expression levels 48 hours after plasmid transfection into HEK293A cells and 72 hours after AAV infection in 293T cells (Figure 16).

[0223] AAV8-GP41-1 and AAV8-DnaE were injected into the subretinal space of ABCA4 KO mice, respectively. Six weeks later, ERG (electroretinogram) (see Figure 17), fundus OCT (optical coherence tomography) and FP (fundus color photography) tests were performed. Compared with the control group, it was found that the content of additional small peptide intein produced by GP41-1 was less, and no retinal toxicity was found.

[0224] Those skilled in the art will appreciate 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. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are within the scope of the present invention.

Claims

1. An expression cassette combination, comprising a first expression cassette and a second expression cassette, wherein the first expression cassette is capable of expressing a fusion protein of an N-terminal truncation of an ABCA4 protein and an N-terminal intein, and the second expression cassette is capable of expressing a fusion protein of a C-terminal truncation of an ABCA4 protein and an C-terminal intein, wherein when expressed, the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette are capable of forming a complete full-length ABCA4 protein through the cleavage function of the N-terminus and the C-terminus of the intein.

2. The expression cassette combination according to claim 1, wherein the intein 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), preferably GP41-1.

3. The expression cassette combination according to any one of claims 1 to 2, wherein the complete ABCA4 protein is a human ABCA4 protein (SEQ ID NO: 1), and the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette are the corresponding N-terminal truncation and C-terminal truncation obtained by truncating 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, that is, the N-terminal truncation and the C-terminal truncation are selected from the following combination: Amino acids 1 to 1223 and 1224 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1); Amino acids 1 to 1139 and 1140 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1); Amino acids 1 to 1149 and 1150 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1); or Amino acids 1 to 1187 and 1188 to 2273 from the N-terminus of human ABCA4 protein (SEQ ID NO: 1).

4. The expression cassette combination according to claim 2, wherein the intein N-terminus and the intein C-terminus are selected from the group consisting of: 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) N-terminus of Rma (SEQ ID NO:61) and C-terminus of Rma (SEQ ID NO:62).

5. The expression cassette combination according to any one of claims 1 to 4, wherein the intein is GP41-1 and the N-terminal truncation and C-terminal truncation of the ABCA4 protein are the corresponding N-terminal truncation and C-terminal truncation obtained by truncating the ABCA4 protein at the 1224 amino acid (Cys) site from the N-terminus, i.e., amino acids 1 to 1223 and amino acids 1224 to 2273 from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

6. The expression cassette combination according to any one of claims 1 to 4, wherein the intein is GP41-1 and the N-terminal truncation and C-terminal truncation of the ABCA4 protein are the corresponding N-terminal truncation and C-terminal truncation obtained by truncating the ABCA4 protein at the 1188 amino acid (Cys) site from the N-terminus, i.e., amino acids 1 to 1187 and amino acids 1188 to 2273 from the N-terminus of the human ABCA4 protein (SEQ ID NO: 1).

7. The expression cassette combination according to any one of claims 1 to 6, wherein the codons for expressing the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein are optimized codons, and the sequence after the combination of the two, i.e., the coding sequence of the full-length ABCA4 protein, is as shown in SEQ ID NO:

3.

8. The expression cassette combination according to any one of claims 1-7, wherein the C-terminus of the fusion protein expressed by the first expression cassette is connected to a first tag, and / or the C-terminus of the fusion protein expressed by the second expression cassette is connected to a second tag, wherein the first tag and the second tag are different, and the first tag and the second tag are independently selected from the group consisting of: hemagglutinin (HA) tag, Flag tag, V5 tag, Myc tag, His tag, 1D4 tag (Rho1D4) and a combination 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).

9. The expression cassette combination according to any one of claims 1 to 8, wherein the N-terminus or C-terminus of the intein is additionally directly or indirectly linked to a degradation signal via a linker, preferably the degradation signal is 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).

10. The expression cassette combination according to any one of claims 1 to 9, wherein the first expression cassette and / or the second expression cassette comprises a promoter and a terminator for expressing the fusion protein, and optionally comprises an enhancer and an intron for enhancing the expression of the fusion protein and operably linked to the fusion protein.

11. The expression cassette combination according to claim 10, wherein the promoter is a constitutive promoter or an inducible promoter, preferably selected from the group consisting of: a CAG-containing promoter (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), a human β-actin promoter, a small CBA (smCBA) promoter, a CBS promoter or a CBh promoter, an elongation factor 1 alpha short (EFS) promoter, an elongation factor 1 alpha (EF-1 alpha) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a VMD2 (also known as BEST1) promoter, an ABCA4 promoter, an RPE65 promoter, an OPEFS promoter, a hGRK1 promoter or a smCAG promoter, more preferably an OPEFS promoter (SEQ ID NO: 47), an hGRK1 promoter (SEQ ID NO: 48), a smCAG promoter (SEQ ID NO: 49), a 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).

12. The expression cassette combination according to claim 10 or 11, wherein the terminator is selected from the group consisting of: SV40 terminator (SEQ ID NO: 50), BGH terminator (SEQ ID NO: 51), WPRE terminator (such as WPRE-3 terminator (SEQ ID NO: 52)), WPRE-SV40 terminator (SEQ ID NO: 56), WPRE-BGH terminator, B-globin-ployA terminator (SEQ ID NO: 65) or sNRP1 terminator (SEQ ID NO: 66).

13. The expression cassette combination according to any one of claims 10 to 12, wherein the first expression cassette comprises, starting from the 5' end: an optional enhancer, a promoter, an optional intron, a nucleotide coding sequence for an N-terminal truncation of the ABCA4 protein, a nucleotide coding sequence for an intein N-terminus, an optional degradation signal, a nucleotide coding sequence for 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 for an intein C-terminus, an optional degradation signal, a nucleotide coding sequence for a C-terminal truncation of the ABCA4 protein, an optional nucleotide coding sequence for a tag, and a terminator.

14. The expression cassette combination according to any one of claims 1 to 13, wherein the first expression cassette and / or the second expression cassette is present in a form selected from the group consisting of an isolated nucleic acid molecule, a liposome and / or an exosome.

15. The expression cassette combination according to any one of claims 1 to 14, wherein the first expression cassette and / or the second expression cassette is present in the form of a plasmid.

16. The expression cassette combination according to any one of claims 1 to 15, wherein the first expression cassette and / or the second expression cassette is a viral vector.

17. The expression cassette combination according to claim 16, wherein the viral vector is an adeno-associated virus (AAV) of the same or different serotype, and the AAV is selected from the group consisting of: 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 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, AAV2 / 10, AAV-D J. AAV-DJ / 8, AAV-DJ / 9, AAV8-Y447,733F or AAVtYF, preferably AAV5, AAV8, AAV8-Y447,733F, AAVtYF or AAV2.7m8.

18. The expression cassette combination according to any one of claims 1 to 17, wherein: 1) The promoter is hGRK1, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is Rma, and the terminator is SV40; 2) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is SV40; 3) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is Rma, and the terminator is B-globin-ployA; 4) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is B-globin-ployA; 5) the promoter is hGRK1, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is SV40; 6) the promoter is enRK, the truncation site of the N-terminal truncation and the C-terminal truncation is 1150, the intein is GP41-1, and the terminator is SV40; or 7) The promoter is enRK-b-globin intron, the truncation site of the N-terminal truncation and the C-terminal truncation is 1188, the intein is GP41-1, and the terminator is B-globin-ployA.

19. A kit comprising the expression cassette combination according to any one of claims 1 to 18.

20. Use of the expression cassette combination according to any one of claims 1 to 18 or the kit according to claim 19 in the preparation of a drug for treating a disease, wherein the disease includes a disease caused by an ABCA4 mutation.

21. The use according to claim 20, wherein the disease comprises an inherited retinal disease.

22. The use according to any one of claims 20-21, wherein the disease comprises an inherited macular degeneration disease, age-related macular degeneration, retinitis pigmentosa and / or cone-rod dystrophy.

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