Expression Cassette Combinations and Uses Thereof

Abstract

The present application relates to an expression cassette combination and its use, which comprises a first expression cassette capable of expressing an N-terminal truncation of ABCA4 protein and a second expression cassette capable of expressing a C-terminal truncation of ABCA4 protein, wherein the N-terminal truncation of ABCA4 protein and the C-terminal truncation of ABCA4 protein can both have the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein.

Description

[Technical field]

[0001] This application relates to the biopharmaceutical field, specifically to expression cassette combinations and uses thereof. [Background technology]

[0002] ABCA4 is a lipid inward-turning enzyme in retinal photoreceptor cells or retinal pigment epithelial cells that can transport retinosylphosphatidylethanolamine (NRPE), a lipid derivative formed from phosphatidylethanolamine (PE) and all-trans-retinal (ATR), from the inner cavity of the outer segment membrane to the cytoplasm. NRPE is then hydrolyzed to ATR and PE, of which ATR is catalyzed by retinal reductase in the cytoplasm to form all-trans-retinol (ATRol), which is then transported back to the optic nerve. If the lipid transport function of ABCA4 is impaired, ATR and NRPE will accumulate excessively on the inner cavity of the outer segment membrane, and the excessively accumulated ATR and NRPE will further form irreversibly toxic double retinyl derivatives, ultimately causing a series of retinal degenerative diseases, including the most common hereditary macular degeneration diseases, age-related macular degeneration, retinitis pigmentosa and cone-rod dystrophies.

[0003] The human ABCA4 CDS is 6.7 kb long and encodes 2273 amino acids. A schematic diagram of the structure of ABCA4 is shown in Figure 1 (Xie, T., Z. Zhang, Q. Fang, B. Du and X. Gong (2021). "Structural basis of substrate recognition and translocation by human ABCA4." Nat Commun 12(1): 3853).

[0004] Currently, there are no commercially available medications for retinopathy caused by ABCA4 mutations. Summary of the Invention

[0005] The present application provides an expression cassette combination, which can co-express an N-terminal truncation (N-terminal domain) of an ABCA4 protein and a C-terminal truncation (N-terminal domain) of the ABCA4 protein expressed in a second expression cassette in a cell. The N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein can have the biological functions of the complete ABCA4 protein (e.g., cellular localization, ATP hydrolase activity, and / or co-expression in photoreceptor cells, reduced retinal A2E deposition, consistent with the complete ABCA4 protein) without forming a complete ABCA4 protein between them.

[0006] The present application creatively discovered that the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein can have the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein between them, so there is no need to transfect and express the complete ABCA4 protein to achieve the therapeutic purpose. At present, the complete ABCA4 protein is usually transfected and expressed using a double AAV vector system, for example, the complete ABCA4 expression frame is divided into two parts that share a set of promoter, stop codon, poly A, etc. required for expression, and a complete RNA is obtained through DNA homologous recombination or RNA splicing in the cell, which finally codes for the complete ABCA4 protein or becomes the complete ABCA4 protein through protein splicing. By DNA homologous recombination method, the double AAV vector is injected into the mouse subretinal space, and 1 × 10 per AAV is injected. 10vg / eye, and the expression intensity of ABCA4 protein is approximately 1% of the endogenous expression level (McClements, ME, et al. (2019). “An AAV Dual Vector Strategy Ameliorates the Stargardt Phenotype in Adult Abca4(- / -) Mice.” Hum Gene Ther 30(5): 590-600.). In a hybrid method that combines homologous recombination and RNA splicing, double AAV vectors were injected into the mouse subretinal space, and 3 × 10 9 vg / eye, and the ABCA4 expression intensity in mice is approximately 10% of endogenous expression (Dyka, FM, et al. (2019). “Dual ABCA4-AAV Vector Treatment Reduces Pathogenic Retinal A2E Accumulation in a Mouse Model of Autosomal Recessive Stargardt Disease.” Hum Gene Ther 30(11): 1361-1370.). By the intein protein splicing method, double AAV vectors were injected into the mouse subretinal space, and 5 × 10 9 vg / eye, the ABCA4 expression intensity in mice is still less than 10% of the endogenous expression (Tornabene, P., et al. (2019). “Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.” Sci Transl Med 11(492).). The above methods have low expression efficiency and require injection of large amounts of AAV virus during treatment to reach or approach the endogenous expression level, resulting in significant immunotoxicity. The combination of expression cassettes according to the present application has resolved these difficulties.

[0007] According to one aspect, the present application provides an expression cassette combination including a first expression cassette capable of expressing an N-terminal truncation of ABCA4 protein and a second expression cassette capable of expressing a C-terminal truncation of ABCA4 protein, wherein the N-terminal truncation of ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of ABCA4 protein expressed by the second expression cassette can have the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein.

[0008] In some embodiments, 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 do not substantially interact to form an intact ABCA4 protein.

[0009] In some embodiments, the protein molar ratio between 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 is about 3:1 to 1:3.

[0010] In some embodiments, the first expression cassette and / or the second expression cassette do not express a linking sequence that can cause an N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncation of the ABCA4 protein expressed by the second expression cassette to form a complete ABCA4 protein.

[0011] In some embodiments, the linking sequence allows an N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncation of the ABCA4 protein expressed by the second expression cassette to be formed into an intact ABCA4 protein by a method selected from the group consisting of homologous recombination, mRNA splicing, and protein splicing.

[0012] In some embodiments, the linking sequence comprises a homology arm sequence.

[0013] In some embodiments, the linking sequence comprises a sequence that may be complementary to a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the linking sequence comprises a sequence that may be complementary to a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein.

[0014] In some embodiments, the first expression cassette does not contain a sequence that may be complementary to a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the second expression cassette does not contain a sequence that may be complementary to a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein.

[0015] In some embodiments, the linking sequence comprises a splicing signal of an mRNA intron.

[0016] In some embodiments, the first expression cassette does not contain an intron upstream splice acceptor site (SA) and / or the second expression cassette does not contain an intron downstream splice donor site (SD).

[0017] In some embodiments, the linking sequence comprises an intein.

[0018] In some embodiments, the linking sequence comprises a sequence encoding an intein protein.

[0019] In some embodiments, the first expression cassette does not express a sequence encoding the N-terminal portion of an intein protein, and / or the second expression cassette does not express a sequence encoding the C-terminal portion of an intein protein.

[0020] In some embodiments, the N-terminal truncation of the ABCA4 protein comprises the N-terminal domain of the ABCA4 protein, which comprises transmembrane domains TMD1 to TMD6, extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and / or R1, preferably comprising transmembrane domains TMD1 to TMD6, extracellular domains ECD1, IH2, EH1, EH2 and NBD1, and optionally IH1 and / or R1. In a particularly preferred embodiment, the N-terminal truncation of ABCA4 protein is selected from the group consisting of amino acids 1 to 1160 (aa 1-1160) (SEQ ID NO: 35), amino acids 1 to 1220 (aa 1-1220) (SEQ ID NO: 36), amino acids 1 to 1280 (aa 1-1280) (SEQ ID NO: 37), amino acids 1 to 1347 (aa 1-1347) (SEQ ID NO: 2) or amino acids 1 to 1280 and 2253 to 2273 (aa 1-1280) at the N-terminus of ABCA4 protein. * , 2253-2273) (SEQ ID NO: 38). In some embodiments, the N-terminal truncation of ABCA4 protein may not include IH1 and / or R1.

[0021] In some embodiments, the N-terminal truncation of ABCA4 protein comprises, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and / or R1.

[0022] In some embodiments, the N-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0023] In some embodiments, the first expression cassette comprises a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0024] In some embodiments, the nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein is set forth in SEQ ID NO: 25 or 27.

[0025] In some embodiments, the first expression cassette comprises a promoter.

[0026] In some embodiments, the C-terminal truncation of the ABCA4 protein comprises the C-terminal domain of the ABCA4 protein, which comprises transmembrane domains TMD7 to TMD12, extracellular domains ECD2, IH3, IH4, EH3, EH4, NBD2 and / or R2, preferably comprising transmembrane domains TMD7 to TMD12, extracellular domains ECD2, IH4, EH3, EH4 and NBD2, and optionally IH3 and / or R2. In particularly preferred embodiments, the C-terminal truncation of ABCA4 protein comprises amino acids 1348 to 2273 (aa 1348-2273), amino acids 1369 to 2273 (aa 1369-2273), amino acids 1348 to 2170 (aa 1348-2170) or amino acids 1 to 20 and 1369 to 2273 (aa 1-20, 1369-2273) at the C-terminus of the ABCA4 protein. In some embodiments, the C-terminal truncation of ABCA4 protein may not comprise IH3 and / or R2.

[0027] Without being limited to any theory, it is believed that the IH1 / 3 domain of the ABCA4 protein provides a membrane localization signal and the R1 / 2 domain provides some of the interactions of the intracellular domain, but these two domains do not appear to be necessary for the structural and functional integrity of the N+C.

[0028] In some embodiments, the C-terminal truncation of the ABCA4 protein comprises, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and / or R2.

[0029] In some embodiments, the C-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 3 or 5.

[0030] In some embodiments, the second expression cassette comprises a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0031] In some embodiments, the nucleotide sequence encoding the C-terminal truncation of the ABCA4 protein is set forth in SEQ ID NO: 26 or 28.

[0032] In some embodiments, the second expression cassette comprises a promoter.

[0033] In some embodiments, the first expression cassette expresses a first constant region, and / or the second expression cassette expresses a second constant region, and the first constant region and the second constant region can interact such that an N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncation of the ABCA4 protein expressed by the second expression cassette interact to form an NC heterodimer of ABCA4 protein.

[0034] In some embodiments, the first constant region has a first modification and / or the second constant region has a second modification, and the first modification and the second modification can promote the formation of a heterodimer.

[0035] In some embodiments, the first constant region and the second constant region are derived from a constant region of an antibody.

[0036] In some embodiments, the first modification and the second modification comprise a knob-into-hole modification.

[0037] In some embodiments, the first constant region comprises the amino acid sequence set forth in SEQ ID NO:6 or 7.

[0038] In some embodiments, the second constant region comprises the amino acid sequence set forth in SEQ ID NO:7 or 6.

[0039] In some embodiments, the first expression cassette comprises, from the 5' end, a promoter sequence, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and / or a nucleotide sequence encoding the first constant region.

[0040] In some embodiments, the first expression cassette consists of, from the 5' end, a promoter sequence and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0041] In some embodiments, the first expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding the first constant region, and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0042] In some embodiments, the first expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the first constant region.

[0043] In some embodiments, the second expression cassette comprises, from the 5' end, a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein and / or a nucleotide sequence encoding the second constant region.

[0044] In some embodiments, the second expression cassette consists of, from the 5' end, a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0045] In some embodiments, the second expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the second constant region.

[0046] In some embodiments, the second expression cassette is composed of, from the 5' end, a promoter sequence, a nucleotide sequence encoding the second constant region, and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0047] In some embodiments, the first expression cassette comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 2, 4, 8-10, 15-18, 34.

[0048] In some embodiments, the second expression cassette comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 3, 5, 11-14, 19-22.

[0049] In some embodiments, the first expression cassette and the second expression cassette are present alone.

[0050] In some embodiments, 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.

[0051] In some embodiments, the first expression cassette and / or the second expression cassette are present in the form of a plasmid.

[0052] In some embodiments, the first expression cassette and / or the second expression cassette is a viral vector.

[0053] In some embodiments, the first expression cassette and / or the second expression cassette is AAV.

[0054] In some embodiments, in the expression cassette combination, the molar ratio of the protein expressed by the first expression cassette to the protein expressed by the second expression cassette is about 1:3 to 3:1.

[0055] In some embodiments, in the expression cassette combination, the molar ratio of the protein expressed in the first expression cassette to the protein expressed in the second expression cassette is about 1:1.

[0056] According to another aspect, the present application provides a kit comprising an expression cassette combination described herein.

[0057] In some embodiments, the kit comprises reagents and / or equipment for administering the expression cassette combination.

[0058] According to another aspect, the present application provides the use of an expression cassette combination described herein and / or a kit described herein in the manufacture of a medicament for treating a disease, including a disease caused by an ABCA4 mutation.

[0059] In some embodiments, the disease comprises an inherited retinal disease.

[0060] In some embodiments, the disease comprises inherited macular degenerative diseases, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0061] According to another aspect, the present application provides a method for expressing a heterologous ABCA4 gene comprising administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0062] According to another aspect, the present application provides a method for mitigating retinylphosphatidylethanolamine (NRPE)-induced cell death comprising administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0063] According to another aspect, the present application provides a method for treating a disease caused by an ABCA4 mutation, comprising administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0064] In some embodiments, the administration comprises an injection.

[0065] In some embodiments, the disease comprises an inherited retinal disease.

[0066] In some embodiments, the disease comprises inherited macular degenerative diseases, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy.

[0067] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Therefore, the descriptions in the accompanying drawings and the specification of the present application are merely illustrative, not limiting. [Brief description of the drawings]

[0068] Particular features of the present invention are set forth in the appended claims. The features and advantages of the present invention can be better understood with reference to the exemplary embodiments described in detail below and the accompanying drawings, the brief description of which is as follows: [Figure 1] FIG. 1 shows a schematic diagram of the structure of ABCA4. [Diagram 2] FIG. 2 shows the protein expression intensity of codon-optimized ABCA4. [Diagram 3] FIG. 3 shows the dimerization phenomenon between the N-terminal truncated form of the ABCA4 protein (aa 1-1347) and the C-terminal truncated form of the ABCA4 protein (aa 1348-2273) described in the present application. [Figure 4] FIG. 4 shows that an interaction exists between the N-terminal truncated form of the ABCA4 protein (aa 1-1347) and the C-terminal truncated form of the ABCA4 protein (aa 1348-2273) described in the present application. [Diagram 5] FIG. 5 shows that interactions exist between N-terminal truncations of ABCA4 protein and C-terminal truncations of ABCA4 protein containing different lengths or different domains. [Figure 6] FIG. 6 shows that there is cellular colocalization of the N-terminal truncated form of the ABCA4 protein (aa 1-1347) and the C-terminal truncated form of the ABCA4 protein (aa 1348-2273) described in this application. [Figure 7] FIG. 7 shows that the colocalization morphology of the N-terminal truncated ABCA4 protein (aa 1-1347) and the C-terminal truncated ABCA4 protein (aa 1348-2273) described in this application is consistent with that of the full-length wild-type ABCA4 protein. [Figure 8] FIG. 8 shows the ATP hydrolase activity when the N-terminal truncated form (aa 1-1347) of the ABCA4 protein and / or the C-terminal truncated form (aa 1348-2273) of the ABCA4 protein described in the present application are transfected. [Figure 9]FIG. 9 shows that there is an interaction between the modified N-terminal truncated ABCA4 protein (aa 1-1347) and the C-terminal truncated ABCA4 protein (aa 1348-2273) described in this application. [Figure 10] FIG. 10 shows that interactions exist between N-terminal truncations-Fca and Fcb-C-terminal truncations or C-terminal truncations-Fcb of ABCA4 proteins of different lengths or containing different domains. [Figure 11] FIG. 11 shows the ATP hydrolase activity of cells transfected with the modified N-terminal truncated ABCA4 protein (aa 1-1347) and / or the C-terminal truncated ABCA4 protein (aa 1348-2273) described in the present application. [Figure 12] FIG. 12 shows the mitigation of NRPE-induced cell death by transfection of the modified N-terminal truncated form (aa 1-1347) of the ABCA4 protein described in the present application and / or the ABCA4 protein. [Figure 13] FIG. 13 shows that coinfection with a complex of an N-terminal truncation of AAV-ABCA4 and a C-terminal truncation of AAV-ABCA4 alleviates NRPE-induced cell death. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Hereinafter, the embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0070] Term definition

[0071] The term "first expression cassette" typically refers to a foreign DNA sequence operably linked to a promoter or other regulatory sequence sufficient to direct transcription of a gene of interest. In this application, the expression cassette typically refers to a nucleic acid construct that, when introduced into a host cell, causes transcription and / or translation of an RNA or polypeptide, respectively. The expression cassette may have a series of specialized nucleic acid elements that allow transcription of a particular nucleic acid in a target cell. The expression cassette may be incorporated into a plasmid, a chromosome, mitochondrial DNA, a plasmid DNA, a virus, or a nucleic acid fragment. The expression cassette may include an antisense or sense construct that is not translated or cannot be translated. When a transgenic is expressed and an endogenous gene is suppressed (e.g., by antisense, RNAi, or sense suppression), the skilled artisan 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 include a polynucleotide construct of a transcription factor operably linked to a promoter, which may be the promoter of a gene regulated by the transcription factor. In the present application, the first expression cassette is capable of expressing a protein (eg, an N-terminal truncation of the ABCA4 protein).

[0072] The term "second expression cassette" usually refers to an expression cassette that is not completely identical to the first expression cassette. For example, the second expression cassette can express a protein that is different from the protein expressed in the first expression cassette (e.g., a C-terminal truncation of the ABCA4 protein).

[0073] The term "expression cassette combination" generally refers to a combination that includes at least one expression cassette. For example, the expression cassette combination described herein may include the first expression cassette and the second expression cassette.

[0074] The term "ABCA4 gene" generally refers to the gene encoding the rim protein or RmP. The ABCA4 gene is also called the ABCR gene. It was first cloned and identified as the gene causing Stargardt disease, an autosomal recessive genetic disorder that causes macular degeneration. The NCBI Entrez Gene accession number for the human ABCA4 gene is 24.

[0075] The term "intact ABCA4 protein" refers to a member 4 of ATP-binding cassette subfamily A having a normal, complete structure. The intact ABCA4 protein can be encoded by the human ABCA4 gene. ABCA4 is a lipid inward-turning enzyme in retinal photoreceptor cells or retinal pigment epithelial cells, and can transport retinosylphosphatidylethanolamine (NRPE), a lipid derivative formed from phosphatidylethanolamine (PE) and all-trans-retinal (ATR), from the luminal side of the outer segment membrane disk to the cytoplasmic side. NRPE is then hydrolyzed to ATR and PE, of which ATR is catalyzed by retinal reductase in the cytoplasm to form all-trans-retinol (ATRol), which is then transported back to the optic nerve. The UniProt accession number of the human intact ABCA4 protein is P78363.

[0076] The term "N-terminal truncation of ABCA4 protein" generally refers to an N-terminal truncation of human complete ABCA4 protein. For example, the N-terminal truncation of ABCA4 protein may include an amino acid sequence shown in amino acids 1 to 1325, 1 to 1160, 1 to 1220, 1 to 1280, or 1 to 1347 from the N-terminus of human complete ABCA4 protein. For example, the amino acid sequence of the N-terminal truncation of ABCA4 protein may be shown in amino acids 1 to 1325 from the N-terminus of human complete ABCA4 protein. In the present application, the N-terminal truncation of ABCA4 protein and / or the C-terminal truncation of ABCA4 protein may correspond to the blue and green structures in FIG. 1 in Nature Communications volume 12, Article number: 3853 (2021), respectively.

[0077] The term "C-terminal truncation of ABCA4 protein" generally refers to a C-terminal truncation of the complete human ABCA4 protein. For example, the C-terminal truncation of ABCA4 protein may include the amino acid sequence shown in the amino acids 1326 to 2273, 1348 to 2273, 1369 to 2273, or 1348 to 2170 from the N-terminus of the complete human ABCA4 protein. For example, the amino acid sequence of the C-terminal truncation of ABCA4 protein may be the amino acid sequence shown in the amino acids 1326 to 2273 from the N-terminus of the complete human ABCA4 protein.

[0078] The term "biological function" usually refers to an activity that is native to, or a desired activity of, a biological entity being tested, such as a cell, protein, or analogue. Ideally, an in vitro functional assay can be used to test for the presence of a biological function.

[0079] The term "homologous recombination" generally refers to recombination that occurs as a result of the interaction between homologous or identical fragments of genetic material. For example, the homologous recombination may involve the exchange of DNA fragments between two DNA molecules. The exchanged fragments may be fragments that flank a site where the two DNA molecules have the same nucleic acid sequence (i.e., a "region of homology"). The "region of homology" may include stretches of nucleic acid sequences that are homologous to each other on the nucleic acid fragments involved in homologous recombination. These regions of homology may be at least about 10 bp in length.

[0080] The term "mRNA splicing" refers to a cellular event that typically occurs in the eukaryotic nucleus, in which introns are removed from the previous mRNA form. Generally, the process may require the formation of a splicesome complex that brings the 5' splice donor site into close proximity with the 3' splice acceptor site and removes interfering intron sequences from the transcript.

[0081] The term "protein splicing" usually refers to the process in which an internal region (intein) of a precursor protein is excised and flanking regions (exteins) of the protein are ligated to form a mature protein. An intein unit may contain the essential components required to catalyze protein splicing, usually including an endonuclease domain involved in intein mobility. The resulting protein may be ligated but is not expressed as a single protein. Protein splicing may be performed in trans by spontaneously combining split inteins expressed in separate polypeptides to form a single intein, which then undergoes the protein splicing step and ligates to the isolated protein.

[0082] The term "homologous arm sequence" generally refers to a sequence having sufficient homology to a corresponding target sequence in a cell genome to perform homologous recombination. For example, the homologous arm sequence may comprise a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the target sequence. The homologous arm sequence may comprise an upstream homologous arm sequence and a downstream homologous arm sequence.

[0083] The term "mRNA intron splicing signal" generally refers to a signal for RNA splicing. For example, the RNA splicing may include intramolecular splicing (cis splicing) and intermolecular splicing (trans splicing). In an intron, a 5' splice site, a 3' splice site and a splicing branch point are necessary for splicing. Splicing can be catalyzed by a spliceosome, which is composed of five different small nuclear RNAs (snRNAs) and a large RNP complex consisting of more than 100 proteins called small nuclear ribonucleoproteins (snRNPs). The RNA of the small nuclear ribonucleoproteins hybridizes with the intron and participates in the catalytic reaction of splicing.

[0084] The term "upstream splicing acceptor site (SA)" usually refers to a splicing acceptor, splice acceptor, SA. In some cases, there are regions at the boundaries between the upstream and downstream adjacent exons of an intron, called SD and SA, respectively. In the early mRNA strand formed after transcription, the sequences corresponding to SA and SD contain the spliced ​​signal. At the boundary of the spliced ​​site, the part of the SD sequence related to the determination of the upstream splicing site, which is in the exon part, can be called A1, and the part of the SD sequence related to the determination of the downstream splicing site, which is in the exon part, can be called B1. Similarly, the part of the SA sequence related to the determination of the downstream splicing site, which is in the exon part, can be called A2, and the part of the SA sequence related to the determination of the downstream splicing site, which is in the intron part, can be called B2. Thus, after the target gene is transcribed, the signal RNA finally formed leaves a sequence corresponding to A1A2 at the splicing boundary.

[0085] The term "downstream splicing donor (SD)" generally refers to the splicing donor, SD.

[0086] The term "intein" generally refers to a functional protein that can mediate the self-excision of a protein molecule from a precursor molecule and the binding of extein proteins on both sides of it by peptide bonds at the same time. The intein gene does not have to be an independent gene, but needs to be inserted into an extein gene for replication and transcription, and can be excised from the precursor protein and linked to the exteins on both sides to become a mature protein. The nucleotide sequence corresponding to the intein can be inserted into the nucleic acid sequence corresponding to the host protein, and can be present in the same open reading frame as the host protein gene, and can be transcribed and translated in sync with the host protein gene, and after the protein precursor is formed by translation, the intein can be excised from the host protein to form an active mature protein. Based on the form of existence of intein, it can be divided into integral intein and truncated intein, and the two splicing regions of the integral intein are commonly present on the same polypeptide fragment, while the two splicing regions of the truncated intein are split into two or more fragments, and the two splicing regions are present on different polypeptide fragments, so it can be called a separated intein.

[0087] The term "first constant region" generally refers to a constant region that can be expressed in the first expression cassette described herein. The constant region is constant with respect to the "variable" and refers to a region that can interact with the N-terminal truncation of the ABCA4 protein expressed in the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed in the second expression cassette to form an NC heterodimer of ABCA4 protein. In some cases, the constant region may include a protein encoded by an immunoglobulin constant region gene. The constant region may be selected from any one of five isotypes: α, δ, ε, γ, or μ.

[0088] The term "second constant region" generally refers to a constant region expressed in a second expression cassette as described herein, which may be the same as the first constant region or may be different from the first constant region.

[0089] The term "knob-into-hole modification" typically refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains. In one embodiment, the "knob-into-hole modification" comprises the amino acid substitution T366W and optional amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V and optional Y349C in another antibody heavy chain. The knob-into-hole technique is described, for example, in U.S. Pat. No. 8,216,805.

[0090] The term "liposome" generally refers to a lipid structure formed by amphiphilic vesicular lipids. The liposome may be a closed vesicle consisting of a single or more lipid bilayers with an internal aqueous phase. For example, liposome may refer to a lipid complex particle in a broader sense. The liposome may further include a complex whose aqueous phase is confirmed to be uncertain. The lipid complex includes at least one lipid, and may further include hydrophilic polymers, polysaccharides, amino acids, etc. The lipid complex may refer to a particle formed from these components by covalent or non-covalent bonds.

[0091] The term "exosome" refers to a membrane sac with a membrane structure consisting of a lipid bilayer that is usually present in extracellular secretory cells or cells. The exosome may include a membrane body having an average diameter of about 10 nm to about 2,000 nm. The exosome may include a microvesicle. Microvesicles, also called circulating microvesicles or microparticles, are fragments of plasma membrane with an approximate diameter range of 100 nm to 1000 nm that are shed from almost all cell types. The exosome may also include small intracellularly generated extracellular vesicles, which are formed by the inward budding of the limiting membrane of multivesicular bodies (MVBs), which, upon fusion with the plasma membrane, result in 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 state of the producing cell.

[0092] The term "AAV" is usually an abbreviation for adeno-associated virus and may 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.

[0093] The term "kit" typically refers to a package containing one or more active ingredients in one or more suitable containers.

[0094] The term "ABCA4 mutation-associated disease" generally refers to diseases associated with mutations in the ABCA4 gene, such as rare ophthalmologic diseases.

[0095] The term "inherited retinal diseases" generally refers to inherited retinal dystrophies or inherited retinal diseases (IRDs), which belong to rare ophthalmic diseases in which genetic defects cause the patient to lose retinal function and / or progressive degeneration. The inherited retinal diseases often cause reversible or irreversible visual impairment in childhood or adolescence. IRDs are intractable ophthalmic diseases, and most of them are expressed as metabolic disorders at the protein level, and the therapeutic goal can be achieved by regulating genes, transcription and translation during the genetic information transmission process. The hereditary retinal disease may include autosomal recessive retinitis pigmentosa (RP, MERTK mutation), achoroidopathy (CHM mutation), juvenile macular degeneration (ABCA4 mutation), Usher syndrome 1B subtype (Myo7a mutation), X-linked congenital retinal detachment (RS1 mutation), mitochondrial-associated Leber hereditary optic neuropathy (ND4 mutation), total achromatopsia (CNGA3 mutation and CNGB3 mutation), sex-linked hereditary RP (RPGR mutation), and the like.

[0096] The term "hereditary macular degeneration disease" refers to a common, rare familial genetic disease. The hereditary macular degeneration disease may include Stargardt disease. Stargardt disease is an autosomal recessive genetic disease that occurs primarily in the retinal pigment epithelium layer, and sporadic cases are relatively rare, with many occurring in children of consanguineous marriages. Patients suffer from macular atrophy damage combined with retinal macular deposits. There is currently no approved treatment, and Stargardt disease may cause thousands of vision losses worldwide each year. The hereditary macular degeneration disease may further include Best disease, in which the lesions mainly affect the macula of both eyes and generally do not spread to the surrounding retina. The hereditary macular degeneration disease may occur at any age.

[0097] The term "age-related macular degeneration" usually refers to age-related macular degeneration (AMD), which is one of the important causes of blindness in the population over 50 years of age. AMD can be described as a progressive, irreversible loss of central vision, mainly affecting the macula, retinal pigment epithelium, choroid, and other areas. Clinically, early AMD is mainly characterized by the deposition of subretinal vitreous membrane warts, while end-stage AMD is divided into two types: dry and wet. Dry AMD, also known as atrophic AMD, is characterized by the geographic atrophy and detachment of the RPE layer, and the vision of the affected eye is slow and gradual loss, while wet AMD is also known as neovascular or exudative AMD, and its main pathological changes are choroidal neovascularization (CNV) and secondary fluid accumulation and hemorrhage. The pathogenesis of AMD is still unclear and may involve RPE damage, mitochondrial dysfunction, oxidative stress, inflammation, complement pathway activation, etc. It is now generally believed that the development of AMD is the result of the combined effects of genetic and environmental factors.

[0098] The term "retinitis pigmentosa" usually refers to Retinitis Pigmentosa, RP, which refers to a group of hereditary eye diseases characterized by progressive deterioration of the retinal photoreceptors. The retinitis pigmentosa may include progressive degenerative disease of the retina (the transparent photosensitive membrane on the inner surface of the back of the eye), which may eventually cause moderate to severe vision loss. Retinitis pigmentosa is often hereditary. The clinical manifestations of retinitis pigmentosa may include early night blindness, concentric reduction of the visual field, and eventually tubular vision, leading to binocular blindness or impending blindness, which accounts for a significant proportion of blinding eye diseases. The retinitis pigmentosa may belong to hereditary rod-cone dystrophic diseases.

[0099] The term "cone-rod dystrophy" generally refers to cone-rod dystrophy (CRD), which belongs to hereditary macular degeneration diseases. The cone-rod dystrophy disease may include clinical and genetic inheritance syndromes such as progressive visual impairment, photophobia, ocular tremor, etc., mainly affecting cone cells, and in severe cases, also affecting rod cells, and the inheritance mode is mainly autosomal dominant, recessive, X-linked inheritance.

[0100] The term "heterologous", when referring to a polynucleotide, gene, or polypeptide, generally refers to a polynucleotide, gene, or polypeptide not normally found in the host organism. Said heterologous may include native coding regions, or parts thereof, i.e., reintroduced into the source organism in a form different from the corresponding native gene, e.g., not in its native location in the genome of said organism.

[0101] The term "retinosylphosphatidylethanolamine (NRPE)" refers to the physiological lipid substrate of ABCA4, which is normally sandwiched between the two TMDs in the luminal leaflet and further stabilized by an extension ring from extracellular domain 1.

[0102] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0103] According to one aspect, the present application provides an expression cassette combination including a first expression cassette capable of expressing an N-terminal truncation of ABCA4 protein and a second expression cassette capable of expressing a C-terminal truncation of ABCA4 protein, wherein the N-terminal truncation of ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of ABCA4 protein expressed by the second expression cassette can have the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein.

[0104] In the present application, the biological function of the intact ABCA4 protein may include cellular localization function, ATP hydrolase activity, expression in photoreceptor cells, and / or reduction of retinal A2E deposition.

[0105] In the present application, the N-terminal truncated form of the ABCA4 protein may comprise an amino acid sequence represented by the amino acids 1 to 1325, 1 to 1160, 1 to 1220, 1 to 1280, or 1 to 1347 from the N-terminus of the human complete ABCA4 protein. For example, the amino acid sequence of the N-terminal truncated form of the ABCA4 protein may comprise an amino acid sequence represented by the amino acids 1 to 1325 from the N-terminus of the human complete ABCA4 protein. In the present application, the C-terminal truncated form of the ABCA4 protein may comprise an amino acid sequence represented by the amino acids 1326 to 2273, 1348 to 2273, 1369 to 2273, or 1348 to 2170 from the N-terminus of the human complete ABCA4 protein. For example, the amino acid sequence of the N-terminal truncated form of the ABCA4 protein may comprise an amino acid sequence represented by the amino acids 1326 to 2273 from the N-terminus of the human complete ABCA4 protein. In the present application, the N-terminal truncation of ABCA4 protein and / or the C-terminal truncation of ABCA4 protein may correspond to the blue and green structures in Figure 1 in Nature Communications volume 12, Article number: 3853 (2021), respectively.

[0106] In the present application, the complete ABCA4 protein may be composed of an N-terminal truncation of the ABCA4 protein and a C-terminal truncation of the ABCA4 protein. The N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein are structurally similar to each other, and therefore may interact in a spatial conformation (e.g., form an NC heterodimer) and may interact to form the complete ABCA4 protein. At the same time, the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein may exist alone and separately without forming the complete ABCA4 protein.

[0107] In the present application, the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette may not substantially interact (e.g., at least about 50% molar ratio, at least about 60% molar ratio, at least about 70% molar ratio, at least about 80% molar ratio, at least about 90% molar ratio, at least about 95% molar ratio or higher) to form the complete ABCA4 protein.

[0108] In the present application, the protein molar ratio of the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette to the C-terminal truncated form 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 truncated form of the ABCA4 protein to the C-terminal truncated form 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.

[0109] In the present application, the first expression cassette and / or the second expression cassette do not express a linking sequence that can form 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 into a complete ABCA4 protein.

[0110] In the present application, the linking sequence allows 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 to be formed into the complete ABCA4 protein by a method selected from the group consisting of homologous recombination, mRNA splicing, and protein splicing.

[0111] The N-terminal truncated form of the ABCA4 protein and the C-terminal truncated form of the ABCA4 protein can interact with each other (e.g., by homologous recombination, mRNA splicing, and / or protein splicing) via the linking sequence to form a complete ABCA4 protein. In the present application, the first expression cassette and / or the second expression cassette do not express the linking sequence, and the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette may not substantially interact with each other (e.g., at least about 50% molar ratio, at least about 60% molar ratio, at least about 70% molar ratio, at least about 80% molar ratio, at least about 90% molar ratio, at least about 95% molar ratio, or higher) to form a complete ABCA4 protein.

[0112] In the present application, the linking sequence may comprise a homology arm sequence.

[0113] In the present application, the linking sequence may comprise a sequence that may be complementary to a sequence that encodes at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the linking sequence may comprise a sequence that may be complementary to a sequence that encodes at least a portion of a C-terminal truncation of the ABCA4 protein. For example, the homology arm sequence may comprise an upstream homology arm sequence and / or a downstream homology arm sequence. The upstream homology arm sequence may comprise a sequence that may be complementary to a sequence that encodes at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the downstream homology arm sequence may comprise a sequence that may be complementary to a sequence that encodes at least a portion of a C-terminal truncation of the ABCA4 protein.

[0114] In the present application, the first expression cassette should not include a sequence that may be complementary to a sequence encoding at least a part of the N-terminal truncation of the ABCA4 protein, and / or the second expression cassette should not include a sequence that may be complementary to a sequence encoding at least a part of the C-terminal truncation of the ABCA4 protein. The first expression cassette and / or the second expression cassette cannot allow the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein that they express to interact with each other through homologous recombination to form the complete ABCA4 protein.

[0115] In the present application, the linking sequence may include a splicing signal of an mRNA intron.

[0116] For example, the splicing signal of the mRNA intron may comprise an intron upstream splicing acceptor site (SA) and / or a downstream splicing donor site (SD). For example, the intron upstream splicing acceptor site (SA) may be located downstream of a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the downstream splicing donor site (SD) may be located upstream of a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein.

[0117] In the present application, the first expression cassette must not contain an intron upstream splicing acceptor site (SA) and / or the second expression cassette must not contain an intron downstream splicing donor site (SD). The first expression cassette and / or the second expression cassette cannot allow the N-terminal truncated form of the ABCA4 protein and the C-terminal truncated form of the ABCA4 protein expressed by them to interact with each other through mRNA splicing to form the complete ABCA4 protein.

[0118] In the present application, the linking sequence may include an intein.

[0119] In the present application, the linking sequence may include a sequence encoding an intein protein. For example, the sequence encoding an intein protein may include a sequence encoding an N-terminal portion of an intein protein and / or a sequence encoding a C-terminal portion of an intein protein. For example, the sequence encoding the N-terminal portion of an intein protein may be located downstream of a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the sequence encoding the C-terminal portion of an intein protein may be located upstream of a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein.

[0120] In the present application, the first expression cassette does not express a sequence encoding the N-terminal portion of an intein protein, and / or the second expression cassette does not express a sequence encoding the C-terminal portion of an intein protein. The first expression cassette and / or the second expression cassette do not allow the N-terminal truncated form of the ABCA4 protein and the C-terminal truncated form of the ABCA4 protein, which they express, to interact with each other through protein splicing to form the complete ABCA4 protein.

[0121] In the present application, the N-terminal truncation of the ABCA4 protein comprises the N-terminal domain of the ABCA4 protein, and the N-terminal domain of the ABCA4 protein may comprise the transmembrane domains TMD1 to TMD6, and the extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and / or R1.

[0122] In the present application, the N-terminal truncation of the ABCA4 protein may include, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, and / or R1. For example, the N-terminal truncation of the ABCA4 protein may include, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, and / or NBD1.

[0123] In the present application, the N-terminal truncation of the ABCA4 protein may comprise the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0124] In the present application, the first expression cassette may comprise a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0125] In the present application, the nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein may be shown in SEQ ID NO: 25 or 27.

[0126] 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 may comprise a CMV promoter.

[0127] In the present application, the C-terminal truncation of the ABCA4 protein comprises the C-terminal domain of the ABCA4 protein, and the C-terminal domain of the ABCA4 protein may comprise the transmembrane domains TMD7 to TMD12, and the extracellular domains ECD1, IH3, IH4, EH3, EH4, NBD2 and / or R2.

[0128] 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.

[0129] In the present application, the C-terminal truncation of the ABCA4 protein may comprise the amino acid sequence shown in SEQ ID NO: 3 or 5.

[0130] In the present application, the second expression cassette may comprise a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0131] In the present application, the nucleotide sequence encoding the C-terminal truncation of the ABCA4 protein may be shown in SEQ ID NO: 26 or 28.

[0132] In the present application, the second expression cassette may comprise a promoter.

[0133] In the present application, the first expression cassette can express a first constant region, and / or the second expression cassette can express a second constant region, and the first constant region and the second constant region can interact such that an N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncation of the ABCA4 protein expressed by the second expression cassette interact with each other to form an NC heterodimer of the ABCA4 protein.

[0134] In the present application, the first constant region may have a first modification and / or the second constant region may have a second modification, and the first modification and the second modification may promote the formation of a heterodimer.

[0135] In the present application, the first constant region and the second constant region can be derived from the constant region of an antibody. For example, they can be derived from the IgG constant region of an antibody. For example, they can be derived from the IgG1 constant region of an antibody. For example, they can be derived from the human IgG constant region of an antibody.

[0136] In the present application, the first modification may be the same as the second modification. In the present application, the first modification may be different from the second modification. For example, the first modification and the second modification may promote the formation of a heterodimer by charge attraction. For example, the first modification and the second modification may include a knob-into-hole modification. For example, the first modification may have a knob modification and the second modification may have a hole modification, and / or the first modification may have a hole modification and the second modification may have a knob modification.

[0137] In the present application, the first constant region may comprise the amino acid sequence shown in SEQ ID NO: 6. In the present application, the second constant region may comprise the amino acid sequence shown in SEQ ID NO: 7.

[0138] In the present application, the first constant region may comprise the amino acid sequence shown in SEQ ID NO: 7. In the present application, the second constant region may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0139] In the present application, R1 in the N-terminal truncated form of the ABCA4 protein can interact with R2 in the C-terminal truncated form of the ABCA4 protein to help the N-terminal truncated form of the ABCA4 protein and the C-terminal truncated form of the ABCA4 protein to form an NC heterodimer of the ABCA4 protein. In some cases, R1 in the N-terminal truncated form of the ABCA4 protein can be replaced with the first constant region, and / or R2 in the C-terminal truncated form of the ABCA4 protein can be replaced with the second constant region, so that the N-terminal truncated form of the ABCA4 protein expressed in the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed in the second expression cassette interact with each other to form an NC heterodimer of the ABCA4 protein through the interaction between the first constant region and the second constant region. In some cases, the downstream of R1 in the N-terminal truncated form of the ABCA4 protein can be further linked to the first constant region, and / or the downstream of R2 in the C-terminal truncated form of the ABCA4 protein can be further linked to a second constant region, thereby further promoting the interaction between the N-terminal truncated form of the ABCA4 protein expressed in the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed in the second expression cassette to form an NC heterodimer of the ABCA4 protein.

[0140] In the present application, the first expression cassette may include, from the 5' end, a promoter sequence, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and / or a nucleotide sequence encoding the first constant region. In the present application, the first expression cassette may include, from the 5' end, a promoter sequence, a nucleotide sequence encoding the first constant region, and / or a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.

[0141] In the present application, the first expression cassette may be composed of, from the 5' end, a promoter sequence and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein. In the present application, the first expression cassette may be composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding the first constant region, and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein. In the present application, the first expression cassette may be composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the first constant region.

[0142] In the present application, the second expression cassette may include, from the 5' end, a promoter sequence, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein, and / or a nucleotide sequence encoding the second constant region. In the present application, the second expression cassette may include, from the 5' end, a promoter sequence, a nucleotide sequence encoding the second constant region, and / or a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0143] In the present application, the second expression cassette may be composed of, from the 5' end, a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein. In the present application, the second expression cassette may be composed of, from the 5' end, a promoter nucleotide sequence, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the second constant region. In the present application, the second expression cassette may be composed of, from the 5' end, a promoter sequence, a nucleotide sequence encoding the second constant region, and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.

[0144] In the present application, the protein expressed by the first expression cassette may include, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and / or R1, and a first constant region.

[0145] In the present application, the protein expressed by the first expression cassette may include, in order from the N-terminus, a first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and R1.

[0146] For example, the protein expressed by the first expression cassette may be, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, R1, and the first constant region. For example, the protein expressed by the first expression cassette may be, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, and the first constant region. For example, the protein expressed by the first expression cassette may be, in order from the N-terminus, the first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, and R1. For example, the protein expressed by the first expression cassette may be, in order from the N-terminus, a first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6 and NBD1.

[0147] In the present application, the protein expressed by the second expression cassette may include, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2 and / or R2, and a second constant region.

[0148] In the present application, the protein expressed by the second expression cassette may include, in order from the N-terminus, a second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, and NBD2 and / or R2.

[0149] For example, the protein expressed by the second expression cassette may be, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, R2, and a second constant region. For example, the protein expressed by the second expression cassette may be, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and a second constant region. For example, the protein expressed by the second expression cassette may be, in order from the N-terminus, a second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and R2. For example, the protein expressed by the second expression cassette may be, in order from the N-terminus, a second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12 and NBD2.

[0150] In the present application, the first expression cassette may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 2, 4, 8-10, 15-18, and 34.

[0151] In the present application, the second expression cassette may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 3, 5, 11-14, 19-22.

[0152] In the present application, the first expression cassette can exist independently of the second expression cassette.

[0153] 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.

[0154] In the present application, the first expression cassette and / or the second expression cassette may be present in the form of a plasmid.

[0155] In the present application, the first expression cassette and / or the second expression cassette may be a viral vector.

[0156] In the present application, the first expression cassette and / or the second expression cassette may be AAV.

[0157] In the present application, in the expression cassette combination, the molar ratio of the protein expressed by the first expression cassette to the protein expressed by the second expression cassette may be about 1:3 to 3:1. For example, it 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.

[0158] In the present application, in the expression cassette combination, the molar ratio of the protein expressed by the first expression cassette to the protein expressed by the second expression cassette may be about 1:1.

[0159] According to another aspect, the present application provides a kit comprising an expression cassette combination described herein.

[0160] In the present application, the kit may include a reagent and / or equipment for administering the expression cassette combination. For example, the kit may include a reagent and / or equipment for transfecting an AAV comprising the first expression cassette and / or the second expression cassette. For example, the kit may include a reagent and / or equipment for injecting an AAV comprising the first expression cassette and / or the second expression cassette. In the present application, the injection may include a local injection.

[0161] According to another aspect, the present application provides the use of an expression cassette combination described herein and / or a kit described herein in the manufacture of a medicament for treating a disease, including a disease caused by an ABCA4 mutation.

[0162] In the present application, the disease may include an inherited retinal disease.

[0163] In the present application, the disease may include inherited macular degenerative diseases, age-related macular degeneration, retinitis pigmentosa and / or cone-rod dystrophies.

[0164] The methods described herein allow for the expression in vivo of the heterologous N-terminal truncated ABCA4 protein and the heterologous C-terminal truncated ABCA4 protein in a subject in need thereof, and the N-terminal truncated ABCA4 protein and the heterologous C-terminal truncated ABCA4 protein can exert the biological functions of the intact ABCA4 protein and can therefore be used to alleviate / treat / ameliorate diseases caused by ABCA4 mutations.

[0165] According to another aspect, the present application provides a method of expressing a heterologous ABCA4 gene, which may comprise administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0166] According to another aspect, the present application provides a method for mitigating retinylphosphatidylethanolamine (NRPE) induced cell death, which may comprise administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0167] According to another aspect, the present application provides a method for treating a disease caused by an ABCA4 mutation, which may comprise administering to a subject in need thereof an expression cassette combination described herein and / or a kit described herein.

[0168] In the present application, the administration may include injection.

[0169] In the present application, the disease may include an inherited retinal disease.

[0170] In the present application, the disease may include inherited macular degenerative diseases, age-related macular degeneration, retinitis pigmentosa and / or cone-rod dystrophies.

[0171] Without wishing to be limited by any theory, the following examples are merely illustrative of the expression cassettes, preparation methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0172] Working Example

[0173] Example 1: Codon-optimized ABCA4 protein expression intensity was improved

[0174] HEK293 cells (provided by Peking University School of Medicine) were transfected with wild-type ABCA4 plasmid fused with FLAG tag (ABCA4-WT) or codon-optimized ABCA4 plasmid fused with FLAG tag (ABCA4-CO), respectively. After 48 hours, cells were lysed and the expression intensity of ABCA4 protein in cells was examined using endogenous ABCA4 antibody (NBP1-30032, NovusBio) or flag tag antibody (F1804, Sigma), respectively. The nucleotide sequence of wild-type ABCA4 is shown in SEQ ID NO. 23, and the nucleotide sequence of codon-optimized ABCA4 is shown in SEQ ID NO. 24.

[0175] According to the Western results (see Figure 2 ), the expression intensity of codon-optimized ABCA4 (ABCA4-CO) was significantly higher than that of wild-type ABCA4 (ABCA4-WT).

[0176] Example 2 Homodimerization occurred in N-terminal and C-terminal truncations of ABCA4

[0177] Using the codon-optimized ABCA4 full-length CDS sequence (aa 1-2273), the ABCA4 protein was divided based on its structural and functional domains (Scortecci, JF, et al. (2021). Nat Commun 12(1): 5902.; Xie, T., et al. (2021). Nat Commun 12(1): 3853.), and two truncations of ABCA4 were constructed, an N-terminal truncation (aa 1-1347) and a C-terminal truncation (aa 1348-2273), respectively. The full-length amino acid sequence of ABCA4 is shown in SEQ ID NO. 1, the amino acid sequence of the N-terminal truncation of ABCA4 is shown in SEQ ID NO. 2, and the amino acid sequence of the C-terminal truncation of ABCA4 is shown in SEQ ID NO. 3.

[0178] To facilitate antibody testing, a hemagglutinin (HA) tag was ligated to the C-terminus of the N-terminal truncation, and a Flag tag was ligated to the C-terminus of the C-terminal truncation.

[0179] Expression plasmids for full-length ABCA4, N-terminal truncations, or C-terminal truncations were transfected into HEK293 cells, and then the corresponding tags were labeled with the corresponding antibodies.

[0180] According to the Western results (see Figure 3), homodimerization occurred in the N- and C-terminal truncations of ABCA4, and cotransfection of the N- and C-terminal truncations could reduce the rate of homodimerization.

[0181] Example 3: Interactions were observed between N-terminal truncations and C-terminal truncations of ABCA4

[0182] In HEK293 cells, ABCA4 N-terminal truncations (aa 1-1347), C-terminal truncations (aa 1348-2273) were transfected, or N+C cotransfected (i.e., N-terminal truncations (aa 1-1347) and C-terminal truncations (aa 1348-2273) were cotransfected). The backbone vectors of the transfection plasmids were purchased from Addgene.

[0183] After 48 hours, cells were lysed, and C-terminal truncations containing the flag tag were enriched in the cells using flag antibody (F1804, Sigma)-coupled magnetic beads, and the presence or absence of N-terminal truncations interacting with the C-terminal truncations was detected using HA antibody (AE036, ABclonal) western blot.

[0184] The results are shown in Figure 4. HEK293 cells were transfected with N-terminal truncation, C-terminal truncation, or N+C co-transfected, and the corresponding protein expression (Input) was observed. The flag antibody-coupled magnetic beads were able to enrich the C-terminal truncation containing the flag tag in the cell lysate, and also enriched the N-terminal truncation interacting with the C-terminal truncation. Therefore, there was an interaction between the N-terminal truncation (aa 1-1347) and the C-terminal truncation (aa 1348-2273) of ABCA4.

[0185] Example 4 There was an interaction between N-terminal truncations of ABCA4 containing different lengths or different domains and C-terminal truncations of ABCA4

[0186] According to the codon-optimized full-length CDS sequence of ABCA4, N- and C-terminal truncations of different lengths or containing different domains were designed. The N-terminal truncations were N1347 (aa 1-1347) (SEQ ID NO: 2), N1160 (aa 1-1160) (SEQ ID NO: 35), N1220 (aa 1-1220) (SEQ ID NO: 36), N1280 (aa 1-1280) (SEQ ID NO: 37), N1280 (aa 1-1280) (SEQ ID NO: 38), N1280 (aa 1-1280) (SEQ ID NO: 39), N1280 (aa 1-1280) (SEQ ID NO: 40), N1280 (aa 1-1280) (SEQ ID NO: 41), N1280 (aa 1-1280) (SEQ ID NO: 42), N1280 (aa 1-1280) (SEQ ID NO: 43), N1280 (aa 1-1280) (SEQ ID NO: 44), N1280 (aa 1-1280) (SEQ ID NO: 45), N1280 (aa 1-1280) (SEQ ID NO: 46), N1280 (aa 1-1280) (SEQ ID NO: 47), N1280 (aa 1-1280) (SEQ ID NO: 48), N1280 (aa 1-1280) (SEQ ID NO: 49), N1280 (aa 1-1280) (SEQ ID NO: 50), N1280 (aa 1-1280) (SEQ ID NO * (aa 1~1280 * , 2253-2273) (SEQ ID NO: 38), and C-terminal truncations include C(aa 1348-2273) (SEQ ID NO: 3), C * (aa 1-20, 1369-2273) (SEQ ID NO: 39), in which an HA tag was linked to the C-terminus of the N-terminal truncation product, and a Flag tag was linked to the C-terminus of the C-terminal truncation product.

[0187] The above two plasmids, N-terminal truncated and C-terminal truncated, were combined and co-transfected into HEK293 cells. After 48 hours, the cells were lysed, and the C-terminal truncated forms containing the flag tag were enriched in the cells using flag antibody-coupled magnetic beads. Western blotting using HA antibody was then used to detect whether or not there were N-terminal truncated forms that interacted with the C-terminal truncated forms.

[0188] The results are shown in FIG. 5, which shows that there is an interaction between N-terminal and C-terminal truncations of ABCA4 containing different lengths or different domains, and N1347 (aa 1-1347), N1160 (aa 1-1160), N1220 (aa 1-1220), N1280 (aa 1-128 ... * (aa 1~1280 * , 2253-2273) were found to have interactions with C (aa 1348-2273), and N1347 (aa 1-1347), N1220 (aa 1-1220), and N1280 (aa 1-1220) were found to have interactions with C (aa 1-1220). * (aa 1~1280 * , 2253~2273) is C * (aa 1-20, 1369-2273). Here, N1347 (aa 1-1347) is C or C * The strongest interaction was between

[0189] Example 5: N- and C-terminal truncations of ABCA4 were colocalized in cells, and the localization positions were consistent with those of full-length wild-type ABCA4

[0190] Full-length ABCA4 (aa 1-2273), N-terminal truncated ABCA4 (aa 1-1347), and C-terminal truncated ABCA4 (aa 1348-2273) were transfected into HEK293 cells (an HA tag was linked to the C-terminus of the N-terminal truncated ABCA4, and a Flag tag was linked to the C-terminus of the C-terminal truncated ABCA4). The corresponding tags were then labeled with corresponding antibodies (flag antibody (F1804, Sigma) and HA antibody (K200003M, Solarbio)). As a result, it was observed that the N-terminal truncated ABCA4 (aa 1-1347) and C-terminal truncated ABCA4 (aa 1348-2273) were colocalized in cells (see Figure 6), and the localization positions were consistent with those of full-length wild-type ABCA4, and both were present in intracellular blebs (see Figure 7).

[0191] Example 6: Complexes of N-terminal and C-terminal truncations of ABCA4 had ATP hydrolase activity

[0192] Full-length ABCA4 (aa 1-2273), N-terminal truncations of ABCA4 (aa 1-1347), and C-terminal truncations of ABCA4 (aa 1348-2273) were transfected into HEK293 cells, or N+C was cotransfected. After 48 hours, the cells were lysed, and full-length ABCA4 or C-terminal truncations containing flag tags, or N+C complexes (i.e., complexes of N-terminal truncations and C-terminal truncations) were enriched in the cells using flag antibody-coupled magnetic beads, and the ATPase activity of the proteins enriched on the magnetic beads was tested using an ATPase test kit (Promega).

[0193] The results are shown in Figure 8. Neither the blank magnetic beads nor the C-terminal truncation alone (aa 1348-2273) had ATP hydrolase activity, whereas the N+C complex had ATP hydrolase activity similar to that of full-length ABCA4.

[0194] Example 7: Interaction existed between N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb of ABCA4

[0195] The high degree of similarity between the N- and C-terminal truncations of ABCA4 provides a structural basis for the formation of heterodimers between the two, although homodimer formation is also possible.

[0196] In order to increase the ratio of heterodimers and decrease the ratio of homodimers, Fca and Fcb were employed by the "knob-into-hole" principle to increase the ratio of heterodimers and decrease the ratio of homodimers.

[0197] The amino acid sequence of Fca is shown in SEQ ID NO. 6, and the amino acid sequence of Fcb is shown in SEQ ID NO. 7.

[0198] Two policies were adopted to link the N-terminal truncations (aa 1–1347) of Fc and ABCA4 to the C-terminal truncations (aa 1348–2273):

[0199] In one, Fca and Fcb are adjacent to each other in the secondary amino acid structure, i.e., Fca (adjacent to R1) is linked to the C-terminus of an N-terminal truncation of ABCA4 (aa 1-1347) to form N(aa 1-1347)-Fca (wherein the amino acid sequence of N(aa 1-1347)-Fca is shown in SEQ ID NO. 8), and Fcb (adjacent to IH3) is linked to the N-terminus of a C-terminal truncation of ABCA4 (aa 1348-2273) to form Fcb-C(aa 1348-2273) (wherein the amino acid sequence of Fcb-C(aa 1348-2273) is shown in SEQ ID NO. 14).

[0200] In the two, Fca and Fcb are adjacent in the tertiary protein structure, i.e., Fca (adjacent to R1) is linked to the C-terminus of an N-terminal truncation (aa 1-1347) of ABCA4 to form N(aa 1-1347)-Fca (wherein the amino acid sequence of N(aa 1-1347)-Fca is shown in SEQ ID NO. 8), and Fcb (adjacent to R2) is linked to the C-terminus (aa 1348-2273) of a C-terminal truncation of ABCA4 to form C(aa 1348-2273)-Fcb (wherein the amino acid sequence of C(aa 1348-2273)-Fcb is shown in SEQ ID NO. 13).

[0201] In HEK293 cells, N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273) were co-transfected, or N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb were co-transfected, and after 48 hours, the cells were lysed, and N(aa 1-1347)-Fca containing Fca tag was enriched in the cells using IgG-coupled magnetic beads, and anti-human antibody (AS002, ABclonal) western blot was used to detect whether Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb interacting with N(aa 1-1347)-Fca was present.

[0202] The results are shown in FIG. 9, in which N(aa 1-1347)-Fca, Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb were transfected or co-transfected with HEK293 cells, respectively, with corresponding protein expression (Input). IgG-coupled magnetic beads could enrich N(aa 1-1347)-Fca in cell lysis, and at the same time, Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb interacting with N(aa 1-1347)-Fca was also enriched. In FIG. 9, N-Fc indicates the transfection of N(aa 1-1347)-Fca alone, C-Fc indicates the transfection of C(aa 1348-2273)-Fcb alone, Fc-C indicates the transfection of Fcb-C(aa 1348-2273) alone, N(aa 1-1347)-Fc+C-Fc indicates the transfection of N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb together, N(aa 1-1347)-Fc+Fc-C indicates the transfection of N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273) together, and NTD-Fc indicates the transfection of N(aa CTD-Fc / Fc-CTD showed Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb interacting with N(aa 1-1347)-Fca.

[0203] Therefore, there was an interaction between N(aa 1-1347)-Fca of ABCA4 and either Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb.

[0204] Example 8: There was an interaction between N-terminal truncations of ABCA4-Fca and C-terminal truncations of Fcb or C-terminal truncations of Fcb containing different lengths or different domains

[0205] N-terminal truncations-Fca and Fcb-C-terminal truncations or C-terminal truncations-Fcb of ABCA4 containing different lengths or different domains were constructed. The N-terminal truncations included N(aa 1-1347)-Fca and N(aa 1-1160)-Fca, and the C-terminal truncations included C(aa 1348-2273)-Fcb, C(aa 1348-2170)-Fcb, and Fcb-C(aa 1348-2273).

[0206] The above-mentioned N-terminal truncations and C-terminal truncations of ABCA4 were co-transfected into HEK293 cells, and the cells were lysed 48 hours later. The N-terminal truncations containing the Fca tag were enriched in the cells using IgG-coupled magnetic beads, and the presence of C-terminal truncations interacting with the N-terminal truncations was detected using western blotting with an anti-human antibody (AS002, ABclonal).

[0207] The results are shown in FIG. 10 , and show that there was a relatively high interaction between N(aa 1-1347)-Fca or N(aa 1-1160)-Fca of ABCA4 and C(aa 1348-2273)-Fcb, C(aa 1348-2170)-Fcb, or Fcb-C(aa 1348-2273).

[0208] Example 9: The complexes of N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273) of ABCA4, or N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb, had ATP hydrolase activity

[0209] HEK293 cells were co-transfected with N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273), or with N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb, respectively, and 48 hours later, the cells were lysed, and N(aa 1-1347)-Fca containing the Fca tag was enriched in the cells using IgG-coupled magnetic beads (IgG-coupled magnetic beads cannot bind to Fcb, see Figure 11), and the ATPase activity of the proteins enriched in the beads was tested using an ATPase test kit (Promega).

[0210] The results are shown in FIG. 11. According to the results, neither blank magnetic beads nor N(aa 1-1347)-Fca alone had ATP hydrolase activity, but the N+C complex (i.e., the complex of N-terminal truncation and C-terminal truncation obtained by cotransfection of N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273) or cotransfection of N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb) had ATP hydrolase activity similar to that of full-length ABCA4 enriched with Flag-coupled magnetic beads.

[0211] Example 10: Complexes of N- and C-terminal truncations of ABCA4 were able to alleviate NRPE-induced cell death

[0212] 661W cells (provided by Peking University School of Medicine) were transfected with the negative control plasmid RHO (synthesized by GENEWIZ), the full-length plasmid ABCA4, or co-transfected with the N-terminal truncation (aa 1-1347) and C-terminal truncation (aa 1348-2273) of ABCA4. After 48 hours, the cells were stimulated with 30 μM to 50 μM all-trans retinal (ATR) or retinyl phosphatidylethanolamine (NRPE) (aladdin), and the cell activity was examined after 24 hours using CellTiter-Glo (Promega).

[0213] The results are shown in Figure 12. Because high concentrations of ATR or NRPE are cytotoxic, stimulation with 30 μM to 50 μM ATR or NRPE for 24 hours resulted in a significant decrease in cell activity in the negative control transfected RHO cell group (cell activity was approximately 3% at 50 μM).

[0214] Full-length ABCA4 was transfected or the N-terminal truncated form (aa1-1347) and C-terminal truncated form (aa1348-2273) of ABCA4 were co-transfected and stimulated with 30 μM to 50 μM ATR for 24 hours. The decrease in cell activity was similar to that of the RHO group (cell activity was approximately 3% at 50 μM). On the other hand, when stimulated with 30 μM to 50 μM NRPE for 24 hours, the cell activity of both groups was restored to some extent (cell activity was approximately 10% at 50 μM).

[0215] This illustrates that expression of full-length ABCA4 or co-expression of N-terminal truncations (aa 1–1347) and C-terminal truncations (aa 1348–2273) of ABCA4 can both promote the translocation or transformation of its substrate NRPE and reduce the cytotoxicity of NRPE.

[0216] Example 11 Co-infection with a complex of an N-terminal truncation of AAV-ABCA4 and a C-terminal truncation of AAV-ABCA4 could alleviate cell death caused by NRPE.

[0217] 661W cells were infected with the control virus AAV-EV at different MOIs (MOI = 1E5 or 5E5) or co-infected with the N-terminal truncation of AAV-ABCA4 (aa 1-1347) and the C-terminal truncation of AAV-ABCA4 (aa 1348-2273). After 48 h, the cells were stimulated with 0 μM to 75 μM all-trans-retinal (ATR) or retinyl phosphatidylethanolamine (NRPE). After 24 h, the total trans-retinol (ATRol) converted in the cells was measured using a microplate reader, and the cellular activity was examined by CellTiter-Glo (Promega).

[0218] The results are shown in Figure 13. After stimulating cells with a high concentration of ATR, the ATRol conversion ability in the cells was reduced and the cellular activity was reduced, while there was no difference between infection with the control virus AAV-EV or co-infection with the N-terminal truncated AAV-ABCA4 (aa 1-1347) and the C-terminal truncated AAV-ABCA4 (aa 1348-2273). After stimulating cells with a high concentration of NRPE, the cells infected with the control virus AAV-EV had a reduced ATRol conversion ability and a reduced cellular activity, while the cells co-infected with the N-terminal truncated AAV-ABCA4 (aa 1-1347) and the C-terminal truncated AAV-ABCA4 (aa 1348-2273) at a high multiplicity of infection (MOI=5E5) had an improved ATRol conversion ability and relatively high cellular activity.

[0219] This explains that coinfection of AAV-ABCA4 N-terminal truncation (aa 1–1347) and AAV-ABCA4 C-terminal truncation (aa 1348–2273) in 661W cells can promote the translocation of its substrate NRPE into ATRol, reduce the cytotoxicity of NRPE, and improve the cellular activity.

[0220] All documents mentioned in this application are incorporated herein by reference as if each document were incorporated by reference alone. It should also be understood that after reading the above teachings of this application, one skilled in the art may make various changes or modifications to the present invention, and that equivalents thereof are within the scope defined by the appended claims of this application.

Claims

1. An expression cassette combination comprising a first expression cassette capable of expressing an N-terminal truncated form of an ABCA4 protein and a second expression cassette capable of expressing a C-terminal truncated form of an ABCA4 protein, wherein the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette can have the biological function of a complete ABCA4 protein without forming the complete ABCA4 protein.

2. The following features: 1) the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette do not substantially interact with each other to form the complete ABCA4 protein; 2) the protein molar ratio of the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette to the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette is about 3:1 to 1:3; 3) the first expression cassette and the second expression cassette exist singly; 4) 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, a plasmid, and a viral vector; 5) the first expression cassette and / or the second expression cassette is AAV; 6) In the expression cassette combination, the molar ratio of the protein expressed by the first expression cassette to the protein expressed by the second expression cassette is about 1:3 to 3:1; and 7) In the expression cassette combination, the molar ratio of the protein expressed by the first expression cassette to the protein expressed by the second expression cassette is about 1:1; The expression cassette combination of claim 1, comprising any one of:

3. The expression cassette combination of claim 1, wherein the first expression cassette and / or the second expression cassette do not express a linking sequence that can form an N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncation of the ABCA4 protein expressed by the second expression cassette into a complete ABCA4 protein.

4. The following features: a) the linking sequence allows 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 to form an intact ABCA4 protein by a method selected from the group consisting of homologous recombination, mRNA splicing, and protein splicing; b) the linking sequence comprises a homology arm sequence; c) the linking sequence comprises a sequence that may be complementary to a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, and / or the linking sequence comprises a sequence that may be complementary to a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein; d) the linking sequence comprises a splicing signal of an mRNA intron; e) the linking sequence comprises an intein; and f) the linking sequence comprises a sequence encoding an intein protein; The expression cassette combination according to claim 3, comprising any one of:

5. The following features: 1) the first expression cassette does not contain a sequence that may be complementary to a sequence encoding at least a portion of an N-terminal truncation of the ABCA4 protein, does not contain an intron upstream splicing acceptor site (SA), or does not express a sequence encoding the N-terminal portion of an intein protein; 2) The N-terminal truncation of the ABCA4 protein comprises the N-terminal domain of the ABCA4 protein, and the N-terminal domain of the ABCA4 protein comprises transmembrane domains TMD1 to TMD6, extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and / or R1, preferably comprising transmembrane domains TMD1 to TMD6, extracellular domains ECD1, IH2, EH1, EH2 and NBD1, and optionally IH1 and / or R1; 3) The N-terminal truncation product of the ABCA4 protein contains, in order from the N-terminus, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and / or R1; 4) The N-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 4; 5) the first expression cassette comprises a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein; 6) the first expression cassette comprises the nucleotide sequence shown in SEQ ID NO: 25 or 27; 7) the first expression cassette comprises a promoter; 8) the first expression cassette is composed of, from the 5' end, a promoter sequence and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein; and 9) The first expression cassette comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 2, 4, 8-10, 15-18, and 34; The expression cassette combination of claim 1, comprising any one of:

6. The following features: 1) the second expression cassette does not contain a sequence that may be complementary to a sequence encoding at least a portion of a C-terminal truncation of the ABCA4 protein, does not contain an intron downstream splice donor (SD), or does not express a sequence encoding the C-terminal portion of an intein protein; 2) The C-terminal truncation of the ABCA4 protein comprises the C-terminal domain of the ABCA4 protein, which comprises transmembrane domains TMD7 to TMD12, extracellular domains ECD2, IH3, IH4, EH3, EH4, NBD2 and / or R2, preferably comprising transmembrane domains TMD7 to TMD12, extracellular domains ECD2, IH4, EH3, EH4 and NBD2, and optionally IH3 and / or R2; 3) the C-terminal truncation of the ABCA4 protein comprises, in order from the N-terminus, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and / or R2; 4) The C-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 3 or 5; 5) the second expression cassette comprises a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein; 6) the second expression cassette comprises the nucleotide sequence shown in SEQ ID NO: 26 or 28; 7) the second expression cassette comprises a promoter; 8) the second expression cassette is composed of, from the 5' end, a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein; and 9) The second expression cassette comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 3, 5, 11-14, 19-22; The expression cassette combination of claim 1, comprising any one of:

7. The expression cassette combination according to claim 1, wherein the first expression cassette expresses a first constant region, and / or the second expression cassette expresses a second constant region, and the first constant region and the second constant region can interact such that an N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and a C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette interact to form an N-C heterodimer of the ABCA4 protein.

8. The following features: a) the first constant region comprises a first modification and / or the second constant region comprises a second modification, and the first modification and the second modification are capable of promoting heterodimer formation; b) the first constant region and the second constant region are derived from constant regions of an antibody; c) the first constant region has a first modification and / or the second constant region has a second modification, wherein the first modification and the second modification can promote heterodimer formation, and the first modification and the second modification comprise a knob-into-hole modification; d) the first constant region comprises the amino acid sequence set forth in SEQ ID NO: 6 or 7; e) the second constant region comprises the amino acid sequence set forth in SEQ ID NO: 6 or 7; f) the first expression cassette comprises, from the 5' end, a promoter sequence, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and / or a nucleotide sequence encoding the first constant region; g) the first expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding the first constant region, and a nucleotide sequence encoding an N-terminal truncated form of the ABCA4 protein; h) the first expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the first constant region; i) the second expression cassette comprises, from the 5′ end, a promoter sequence, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein, and / or a nucleotide sequence encoding the second constant region; j) the second expression cassette is composed of, from the 5' end, a nucleotide sequence of a promoter, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein, and a nucleotide sequence encoding the second constant region; and k) the second expression cassette is composed of, from the 5' end, a promoter sequence, a nucleotide sequence encoding the second constant region, and a nucleotide sequence encoding a C-terminal truncated form of the ABCA4 protein; The expression cassette combination according to claim 7, comprising any one of:

9. A kit comprising the expression cassette combination of any one of claims 1 to 8, which may optionally further comprise reagents and / or equipment for administering said expression cassette combination.

10. Use of an expression cassette combination according to any one of claims 1 to 8 and / or a kit comprising said expression cassette combination in the manufacture of a medicament for treating a disease, including a disease caused by an ABCA4 mutation.

11. The following features: 1) the disease includes an inherited retinal disease, and 2) The disease includes hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy; The use according to claim 10, wherein

12. A method for expressing a heterologous ABCA4 gene, comprising administering to a subject in need thereof an expression cassette combination according to any one of claims 1 to 8, and / or a kit comprising said expression cassette combination.

13. An expression cassette combination described in any one of claims 1 to 8, and / or a kit comprising said expression cassette combination, for alleviating cell death caused by retinylphosphatidylethanolamine (NRPE).

14. An expression cassette combination according to any one of claims 1 to 8, and / or a kit comprising said expression cassette combination, for treating a disease caused by an ABCA4 mutation by administration to a subject in need thereof.

15. The following features: 1) the administration comprises injection; 2) the disease includes an inherited retinal disease, and 3) The disease includes hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and / or cone-rod dystrophy; 15. The expression cassette combination and / or kit according to claim 14, comprising any one of:

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