WO2023160454A1 - 表达盒组合及其应用 - Google Patents

表达盒组合及其应用 Download PDF

Info

Publication number
WO2023160454A1
WO2023160454A1 PCT/CN2023/076459 CN2023076459W WO2023160454A1 WO 2023160454 A1 WO2023160454 A1 WO 2023160454A1 CN 2023076459 W CN2023076459 W CN 2023076459W WO 2023160454 A1 WO2023160454 A1 WO 2023160454A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression cassette
abca4
combination
protein
terminal truncation
Prior art date
Application number
PCT/CN2023/076459
Other languages
English (en)
French (fr)
Inventor
钟显成
陈邵宏
史天永
Original Assignee
北京中因科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京中因科技有限公司 filed Critical 北京中因科技有限公司
Publication of WO2023160454A1 publication Critical patent/WO2023160454A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

Definitions

  • This application relates to the field of biomedicine, in particular to an expression cassette combination and its application.
  • ABCA4 is a lipid inward flipping enzyme in retinal photoreceptor cells or retinal pigment epithelial cells, which can convert phosphatidylethanolamine (PE) and all-trans retinal (ATR), a lipid derivative—retinyl phosphatidylethanolamine (NRPE), transported from the luminal to the cytoplasmic side of the outer segmental disc. Subsequently, NRPE will be hydrolyzed into ATR and PE, and ATR will be catalyzed by retinal reductase in the cytoplasm to form all-trans retinol (ATRol), thereby returning to the visual cycle.
  • PE phosphatidylethanolamine
  • ATR all-trans retinal
  • NRPE lipid derivative—retinyl phosphatidylethanolamine
  • ATR and NRPE will accumulate excessively in the lumen of the outer segmental disc, and the excessively accumulated ATR and NRPE will further irreversibly form toxic bis-retinyl derivatives, eventually causing a A series of retinal degenerative diseases, including the most common hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa and cone-rod dystrophy.
  • the present application provides an expression cassette combination.
  • the expression cassette combination can co-express the N-terminal truncation (N-terminal domain) of the ABCA4 protein and the C-terminal truncation (N-terminal domain) of the ABCA4 protein expressed by the second expression cassette in the cell.
  • 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 a complete ABCA4 protein between the two (for example, with Consistent cellular localization of intact ABCA4 protein; ATP hydrolase activity; and/or, co-expression in photoreceptor cells, reduced retinal A2E deposition).
  • the present application creatively finds that because the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein can have a complete ABCA4 protein without forming a complete ABCA4 protein between the two Therefore, there is no need to transfect and express the complete ABCA4 protein to achieve therapeutic purposes.
  • the double AAV vector system is usually used to transfect and express the complete ABCA4 protein, for example, the complete ABCA4 expression cassette is split into two parts, and the two Some share a set of promoters, stop codons, poly A, etc.
  • double AAV vectors are injected into the subretinal cavity of mice, each AAV is injected with 3 ⁇ 10 9 vg/eye, and the expression intensity of ABCA4 in mice is about the same as that of endogenous expression 10% (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. ).
  • mice Using the intein protein shearing method, the subretinal cavity of mice was injected with double AAV vectors, each AAV was injected with 5 ⁇ 10 9 vg/eye, and the expression intensity of ABCA4 in the mice was still not higher 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).). Due to the low expression efficiency of the above method, in order to achieve or approach the endogenous expression level, a large amount of AAV virus needs to be injected during the treatment process, so the immunotoxicity is very high, and the expression cassette combination provided by the application solves these difficulties.
  • the application provides an expression cassette combination, which includes a first expression cassette and a second expression cassette, wherein the first expression cassette can express an N-terminal truncation of the ABCA4 protein, and the second expression cassette can express Expressing the C-terminal truncation of the ABCA4 protein, the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can be It has the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein between the two.
  • 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 with each other. It acts to form the complete ABCA4 protein.
  • the protein molar ratio of the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette to the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette About 3:1 ⁇ 1:3.
  • the first expression cassette and/or the second expression cassette do not express a linker sequence capable of truncation of the N-terminus of the ABCA4 protein expressed by the first expression cassette.
  • the short body forms a complete ABCA4 protein with the C-terminal truncation body of the ABCA4 protein expressed by the second expression cassette.
  • the connecting sequence is selected from the group consisting of homologous recombination, mRNA splicing and protein splicing, so that the N-terminal truncated body of the ABCA4 protein expressed by the first expression cassette is combined with The second expression cassette is represented by The achieved C-terminal truncation of the ABCA4 protein forms a complete ABCA4 protein.
  • the linking sequence includes a homology arm sequence.
  • the linking sequence includes a sequence that is complementary to at least part of the sequence encoding the N-terminal truncation of the ABCA4 protein; A sequence that is at least partially complementary to the sequence of the C-terminal truncation.
  • the first expression cassette does not comprise a sequence that is complementary to at least a portion of the sequence encoding the N-terminal truncation of the ABCA4 protein; and/or, the second expression cassette does not comprise a sequence that is compatible with A sequence that encodes at least part of the sequence of the C-terminal truncation of the ABCA4 protein.
  • the junction sequence includes splicing signals for mRNA introns.
  • the first expression cassette does not comprise an intron upstream splice acceptor site (SA); and/or, the second expression cassette does not comprise an intron downstream splice donor site (SD) .
  • SA intron upstream splice acceptor site
  • SD intron downstream splice donor site
  • the linker sequence includes an intein.
  • the linker sequence includes a sequence encoding an intein protein.
  • the first expression cassette does not express the sequence encoding the N-terminal portion of the intein protein; and/or, the second expression cassette does not express the sequence encoding the C-terminal portion of the intein protein.
  • the N-terminal truncation of the ABCA4 protein comprises the N-terminal domains of the ABCA4 protein: transmembrane regions TMD1-TMD6, extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and/or R1, preferably comprises the transmembrane region TMD1-TMD6, the extracellular domain ECD1, IH2, EH1, EH2 and NBD1 and optionally IH1 and/or R1.
  • the N-terminal truncation of the ABCA4 protein comprises amino acids 1 to 1160 (aa 1-1160) (SEQ ID NO:35), amino acids 1 to 1220 (aa 1-1220) of the N-terminal of the ABCA4 protein. ) (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 2253-2273 (aa 1-1280 * , 2253-2273) (SEQ ID NO: 38).
  • the N-terminal truncation of the ABCA4 protein may not include IH1 and/or R1.
  • the N-terminal truncations of the ABCA4 protein include sequentially from the N-terminus: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and/or R1 .
  • the N-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 4.
  • the first expression cassette comprises a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.
  • the nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein is as SEQ ID NO:25 or 27.
  • the first expression cassette comprises a promoter
  • the C-terminal truncation of the ABCA4 protein comprises the domains of the C-terminus of the ABCA4 protein: transmembrane region TMD7-TMD12, extracellular domain ECD2, IH3, IH4, EH3, EH4, NBD2 and/or R2, preferably comprises the transmembrane region TMD7-TMD12, the extracellular domain ECD2, IH4, EH3, EH4 and NBD2 and optionally IH3 and/or R2.
  • the C-terminal truncation of the ABCA4 protein comprises amino acid 1348-2273 (aa 1348-2273), amino acid 1369-2273 (aa 1369-2273), amino acid 1348- 2170 (aa 1348-2170) or amino acids 1-20 and 1369-2273 (aa 1-20, 1369-2273).
  • the C-terminal truncation of the ABCA4 protein may not include IH3 and/or R2.
  • the IH1/3 domains of the ABCA4 protein provide the membrane localization signal and the R1/2 domains provide part of the intracellular domain for interaction, but these two parts of the domain do not appear to be N+C structurally and functionally Required for completeness.
  • the C-terminal truncation of the ABCA4 protein includes sequentially from the N-terminus: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2 and/or R2 .
  • the C-terminal truncation of the ABCA4 protein comprises the amino acid sequence shown in SEQ ID NO: 3 or 5.
  • the second expression cassette comprises a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.
  • nucleotide sequence encoding the C-terminal truncation of the ABCA4 protein is as shown in SEQ ID NO: 26 or 28.
  • the second expression cassette comprises a promoter
  • the first expression cassette expresses a first constant region
  • the second expression cassette expresses a second constant region, wherein the first constant region and the second constant region are capable of The interaction makes the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette interact with the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette to form an N-C isoform of the ABCA4 protein. dimer.
  • the first constant region has a first modification
  • the second constant region has a second modification, wherein the first modification and the second modification are capable of promoting heterodimerization body formation.
  • said first constant region and said second constant region are derived from constant regions of an antibody.
  • the first modification and the second modification comprise knob-into-hole modifications.
  • the first constant region comprises the amino acid sequence shown in SEQ ID NO:6 or 7.
  • the second constant region comprises the amino acid sequence shown in SEQ ID NO: 7 or 6.
  • the first expression cassette comprises 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 from the 5' end. Nucleotide sequence.
  • the first expression cassette consists of a promoter sequence and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein from the 5' end.
  • the first expression cassette is composed of a nucleotide sequence of a promoter, a nucleotide sequence encoding the first constant region, and an N-terminal truncated protein encoding the ABCA4 protein from the 5' end.
  • the nucleotide sequence composition of the short body is composed of a nucleotide sequence of a promoter, a nucleotide sequence encoding the first constant region, and an N-terminal truncated protein encoding the ABCA4 protein from the 5' end.
  • the first expression cassette consists of a promoter nucleotide sequence, a nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein, and encoding the first expression cassette from the 5' end.
  • the nucleotide sequence composition of the constant region is not limited to, but not limited to, but not limited to, but not limited to, but not limited to, but not limited to, a promoter nucleotide sequence, a nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein, and encoding the first expression cassette from the 5' end.
  • the second expression cassette comprises a promoter sequence, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein and/or a core encoding the second constant region from the 5' end. nucleotide sequence.
  • the second expression cassette consists of a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein from the 5' end.
  • the second expression cassette is composed of a nucleotide sequence of a promoter, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein and encoding the second constant from the 5' end.
  • the nucleotide sequence composition of the region is composed of a nucleotide sequence of a promoter, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein and encoding the second constant from the 5' end.
  • the second expression cassette consists of a promoter sequence, a nucleotide sequence encoding the second constant region, and a core encoding a C-terminal truncation of the ABCA4 protein from the 5' end. Nucleotide sequence composition.
  • the first expression cassette comprises the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 8-10, 15-18, 34.
  • the second expression cassette comprises the nucleotide sequence shown in any one of SEQ ID NO:3,5,11-14,19-22.
  • said first expression cassette is present separately from said second expression cassette.
  • the first expression cassette and/or the second expression cassette is present in a form selected from the group consisting of isolated nucleic acid molecules, liposomes and/or exosomes.
  • the first expression cassette and/or the second expression cassette is in the form of a plasmid.
  • the first expression cassette and/or the second expression cassette is a viral vector.
  • said first expression cassette and/or said second expression cassette is AAV.
  • the molar ratio of the protein expressed by the first expression cassette to the second expression cassette is about 1:3-3:1.
  • the expression expressed by the first expression cassette and the second expression cassette is about 1:1.
  • the present application provides a kit comprising the combination of expression cassettes described in the present application.
  • the kit comprises reagents and/or instruments for administering the combination of expression cassettes.
  • the present application provides a use of the expression cassette combination described in the present application, and/or the kit described in the present application, in the preparation of medicines for treating diseases, and the diseases include diseases caused by ABCA4 mutations.
  • the disease comprises an inherited retinal disease.
  • the disease comprises hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and/or cone-rod dystrophy.
  • the present application provides a method for expressing a heterologous ABCA4 gene, which comprises administering the combination of expression cassettes described in the present application, and/or the kit described in the present application to a subject in need.
  • the present application provides a method for alleviating cell death caused by retinyl phosphatidylethanolamine (NRPE), which comprises administering the expression cassette combination described in the present application to a subject in need, and/or the present application The kit described.
  • NRPE retinyl phosphatidylethanolamine
  • the present application provides a method for treating a disease caused by an ABCA4 mutation, which comprises administering the expression cassette combination described in the present application, and/or the kit described in the present application to a subject in need.
  • said administering comprises injection.
  • the disease comprises an inherited retinal disease.
  • the disease comprises hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa, and/or cone-rod dystrophy.
  • Figure 1 shows a schematic diagram of the structure of ABCA4.
  • Figure 2 shows the protein expression intensity of codon-optimized ABCA4.
  • Figure 3 shows the dimerization of the N-terminal truncation (aa 1-1347) of the ABCA4 protein described in the application and the C-terminal truncation (aa 1348-2273) of the ABCA4 protein.
  • Figure 4 shows that the N-terminal truncation (aa 1-1347) of the ABCA4 protein described in the application interacts with the C-terminal truncation (aa 1348-2273) of the ABCA4 protein.
  • Figure 5 shows that there is an interaction between N-terminal truncations of ABCA4 protein with different lengths or containing different domains and C-terminal truncations of ABCA4 protein.
  • Figure 6 shows that the N-terminal truncation (aa 1-1347) of the ABCA4 protein described in the present application and the C-terminal truncation (aa 1348-2273) of the ABCA4 protein have cell colocalization.
  • Figure 7 shows the colocalization form of the N-terminal truncation (aa 1-1347) of the ABCA4 protein and the C-terminal truncation (aa 1348-2273) of the ABCA4 protein described in the application and the full-length wild-type ABCA4 protein unanimous.
  • Figure 8 shows the ATP hydrolase activity of the N-terminal truncated body (aa 1-1347) and/or the C-terminal truncated body (aa 1348-2273) of the ABCA4 protein transfected with the present application.
  • Figure 9 shows that there is an interaction between the modified N-terminal truncation (aa 1-1347) of the ABCA4 protein described in the application and the C-terminal truncation (aa 1348-2273) of the ABCA4 protein.
  • Figure 10 shows that there is an interaction 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 shows the ATP hydrolase activity of the N-terminal truncation (aa 1-1347) of the ABCA4 protein and/or the C-terminal truncation (aa 1348-2273) of the ABCA4 protein transfected through the application .
  • Figure 12 shows that the N-terminal truncation (aa 1-1347) of the ABCA4 protein described in the application is transfected and/or the mitigation situation of ABCA4 protein to the cell death that NRPE causes.
  • Figure 13 shows that co-infection with the complex of the N-terminal truncation of AAV-ABCA4 and the complex of the C-terminal truncation of AAV-ABCA4 can alleviate the cell death caused by NRPE.
  • first expression cassette generally refers to an exogenous DNA sequence operably linked to a promoter or other regulatory sequence sufficient to direct the transcription of a gene of interest.
  • the expression cassette generally refers to a nucleic acid construct which, when introduced into a host cell, leads to the transcription and/or translation of RNA or polypeptide, respectively.
  • the expression cassette may have specific Nucleic acid A set of specialized nucleic acid elements that are transcribed in target cells.
  • the expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus or nucleic acid fragment.
  • the expression cassette may include an untranslated or non-translatable antisense or sense construct.
  • the expression cassette may comprise a polynucleotide construct of a transcription factor operably linked to a promoter, which may be a promoter derived from a gene regulated by the transcription factor.
  • the first expression cassette can express a protein (such as an N-terminal truncation of ABCA4 protein).
  • second expression cassette generally refers to an expression cassette that is not identical to said first expression cassette.
  • the second expression cassette may express a different protein (eg, a C-terminal truncation of the ABCA4 protein) than the protein expressed by the first expression cassette.
  • combination of expression cassettes generally refers to a combination comprising at least one expression cassette.
  • a combination of expression cassettes described herein may comprise said first expression cassette and said second expression cassette.
  • ABC4 gene generally refers to the gene encoding the rim protein or RmP.
  • the ABCA4 gene is also known as the ABCR gene. This gene was cloned and identified for the first time as a gene that causes Stargardt disease, an autosomal recessive disorder that causes macular degeneration.
  • NCBI Entrez Gene accession number of the human ABCA4 gene is 24.
  • ABCA4 protein generally refers to member 4 of the ATP-binding cassette subfamily A with its complete structure.
  • the complete ABCA4 protein may be encoded by the human ABCA4 gene.
  • ABCA4 is a lipid inward flipping enzyme in retinal photoreceptor cells or retinal pigment epithelial cells, which can convert phosphatidylethanolamine (PE) and all-trans retinal (ATR), a lipid derivative—retinyl phosphatidylethanolamine ( NRPE), transported from the luminal to the cytoplasmic side of the outer segmental disc.
  • PE phosphatidylethanolamine
  • ATR all-trans retinal
  • NRPE retinyl phosphatidylethanolamine
  • NRPE will be hydrolyzed into ATR and PE, and ATR will be catalyzed by retinal reductase in the cytoplasm to form all-trans retinol (ATRol), thereby returning to the visual cycle.
  • ATRol all-trans retinol
  • N-terminal truncation of the ABCA4 protein generally refers to an N-terminal truncation of the intact human ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein may comprise amino acids 1-1325, amino acids 1-1160, amino acids 1-1220, and amino acids 1-1280 from the N-terminus of the complete human ABCA4 protein. , or the amino acid sequence shown in amino acid positions 1-1347.
  • the amino acid sequence of the N-terminal truncation of the ABCA4 protein can be as shown in amino acid positions 1-1325 from the N-terminal of the complete human ABCA4 protein.
  • N-terminal truncation of the ABCA4 protein and/or the C-terminal truncation of the ABCA4 protein may correspond to the blue and green in Figure 1 in Nature Communications volume 12, Article number: 3853 (2021), respectively. structure.
  • C-terminal truncation of ABCA4 protein generally refers to a C-terminal truncation of the intact human ABCA4 protein.
  • the C-terminal truncation of the ABCA4 protein may comprise the 1326-2273rd amino acid from the N-terminus of the complete human ABCA4 protein. amino acid, amino acid 1348-2273, amino acid 1369-2273 or amino acid 1348-2170.
  • the amino acid sequence of the C-terminal truncated product of the ABCA4 protein may be as shown in amino acid positions 1326-2273 from the N-terminal of the complete human ABCA4 protein.
  • biological function generally refers to an activity that is native to, or is an activity of interest of, the biological entity being tested, such as a native activity of a cell, protein or the like. Ideally, in vitro functional assays can be used to test for the presence of biological function.
  • homologous recombination generally refers to recombination occurring as a result of interactions between homologous or identical segments of genetic material.
  • the homologous recombination may involve the exchange of DNA segments between two DNA molecules. Segments that are exchanged may be those flanking the site at which the two DNA molecules have identical nucleic acid sequences (ie, "regions of homology").
  • the “homologous region” may include a range of nucleic acid sequences that are homologous to each other on nucleic acid fragments that participate in homologous recombination. These regions of homology may be at least about 10 bp in length.
  • mRNA splicing generally refers to the cellular event that occurs in the eukaryotic nucleus in which introns are removed from pre-mRNA types. Generally, this process may require the formation of a spliceosome complex, in which a 5' splice donor site is brought adjacent to a 3' splice acceptor site, while interfering intron sequences are removed from the transcript.
  • protein splicing generally refers to the process by which an internal region (intein) of a precursor protein is excised and flanking regions of the protein (extein) are joined to form a mature protein.
  • the intein unit can contain the essential components required to catalyze protein splicing and often contains an endonuclease domain involved in intein mobility.
  • the resulting proteins may be linked together, but are not expressed as separate proteins.
  • Protein splicing can also occur in trans by spontaneous association with split inteins expressed on separate polypeptides to form a single intein, which then undergoes a process of protein splicing to combine into separate proteins.
  • the term "homology arm sequence” generally refers to a sequence having sufficient homology to a corresponding target sequence within the genome of a cell to undergo homologous recombination.
  • the homology arm sequence may comprise 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%, Polynucleotide sequences that are at least 97%, at least 98%, at least 99%, or 100% identical.
  • the homology arm sequence may comprise an upstream homology arm sequence and a downstream homology arm sequence.
  • RNA intron splicing signal generally refers to a signal for RNA splicing.
  • the RNA splicing may include intramolecular (cis splicing) and intermolecular (intermolecular) splicing (trans splicing).
  • intramolecular cis splicing
  • intermolecular intermolecular splicing
  • a 5' splice site, a 3' splice site, and a splice branch point may be required for splicing.
  • Splicing can be catalyzed by the spliceosome, which is a large ribonucleic acid protein complex composed of five different small nuclear ribonucleic acids (snRNAs) and no less than one hundred proteins, called small nuclear ribonucleic acid proteins (snRNPs) ) composed of. Small riboprotein RNA hybridizes to introns (hybridization), and participate in the catalytic reaction of splicing.
  • snRNAs small nuclear ribonucleic acids
  • snRNPs small nuclear ribonucleic acid proteins
  • upstream splice acceptor site generally refers to a splice acceptor, SA.
  • upstream and downstream of an intron there are regions called SD and SA, respectively, at the junction with an adjacent exon.
  • the sequences corresponding to SA and SD contain the splicing signal.
  • the part of the SD sequence that is related to the determination of the upstream splicing site, located in the exon part, is called A1, and the part located in the intron part is called B1;
  • the part related to the determination of the downstream splicing site, located in the exon part is called A2
  • the part located in the intron part is called B2; in this way, after the target gene is transcribed, it will eventually form The signal RNA, at the cut junction, leaves the sequence corresponding to A1A2.
  • downstream splicing donor site generally refers to the splicing donor, SD.
  • intein generally refers to a functional protein that can mediate the self-cleavage of protein molecules from precursor molecules, and at the same time connect the extein proteins on both sides through peptide bonds.
  • the intein gene may not be an independent gene, it needs to be inserted into the extein gene to copy and transcribe, and it can be excised from the precursor protein and connected with the exteins on both sides to become a mature protein.
  • the nucleotide sequence corresponding to the intein can be chimeric in the nucleic acid sequence corresponding to the host protein, exists in the same open reading frame as the host protein gene, and performs synchronous transcription and translation with the host protein gene.
  • inteins can be excised from host proteins to form mature, active proteins. According to the form of intein, it can be divided into integral intein and fragmented intein. The two splicing regions of the integral intein co-exist on the same polypeptide fragment, and the two splicing regions of the fragmented intein are split. Inteins can be divided into two or more fragments, and the two splicing regions exist on different polypeptide fragments, so they can also be called isolated inteins.
  • first constant region generally refers to a constant region that can be expressed by the first expression cassette described herein.
  • the constant region may refer to the opposite of the "variable", and the constant region can interact to make the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette interact with the second expression cassette.
  • the constant region may comprise a protein encoded by an immunoglobulin constant region gene.
  • the constant region may be selected from any of the following 5 isotypes: alpha, delta, epsilon, gamma or mu.
  • second constant region generally refers to a constant region that can be expressed by the second expression cassette described herein.
  • the second constant region may be the same as the first constant region, or may be different from the first constant region.
  • knob-into-hole modification generally refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains.
  • the "knob-into-hole modification” comprises amino acid substitution T366W and optional amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and any amino acid substitution in the other antibody heavy chain. The chosen Y349C.
  • the knob-into-hole technique is described, for example, in US Patent No. 8,216,805.
  • liposome generally refers to lipid structures formed from amphiphilic vesicle-forming lipids.
  • the liposomes may be closed vesicles consisting of a single or multilamellar lipid bilayer with an inner aqueous phase.
  • liposome can refer to a lipid complex particle in a broader sense.
  • the liposomes may even include complexes whose aqueous phases are not definitively identified.
  • the lipoplex contains at least one type of lipid, and may additionally contain hydrophilic polymers, polysaccharides, amino acids, and the like. Lipoplexes may refer to particles formed from these components via covalent or non-covalent bonds.
  • exosome generally refers to an extracellular secretory cell or a membrane vesicle having a membrane structure composed of a lipid bilayer present in the cell.
  • the exosomes may comprise membranous bodies having an average diameter of from about 10 nm to about 2,000 nm.
  • the exosomes may comprise microvesicles.
  • Microvesicles are also known as circulating microvesicles or microparticles and are fragments of the plasma membrane with an approximate diameter ranging from 100 nm to 1000 nm that are shed from almost all cell types.
  • the exosomes may also include smaller intracellularly generated extracellular vesicles formed by inward budding of the limiting membrane of multivesicular bodies (MVBs), which upon fusion with the plasma membrane cause their secretion and deposition in body fluids (e.g. blood, urine).
  • MVBs multivesicular bodies
  • Exosomes can contain complex mixtures of microRNAs (miRs), mRNAs, and proteins that reflect the transcriptional and translational state of the producing cell.
  • AAV is commonly used as 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 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.
  • kit generally refers to a package comprising one or more active ingredients in one or more suitable containers.
  • a disease caused by an ABCA4 mutation generally refers to a disease associated with an ABCA4 gene mutation, such as a rare eye disease.
  • inherited retinal diseases generally refers to inherited retinal dystrophies or inherited retinal diseases, IRDs, which are rare ophthalmic diseases that cause loss of retinal function and/or progressive degeneration in patients due to genetic defects.
  • the genetic retinal diseases often present reversible or irreversible visual impairment in early childhood or adolescence.
  • IRDs are refractory ophthalmic diseases, mostly manifested as abnormal protein metabolism, and can be cured by regulating genes, transcription and translation in the process of genetic information transmission.
  • the inherited retinal diseases may include autosomal recessive retinitis pigmentosa (RP, MERTK mutation), choroideremia (CHM mutation), juvenile macular degeneration (ABCA4 mutation), Usher syndrome subtype 1B (Myo7a mutation) , X-linked congenital retinoschisis (RS1 mutation), mitochondria-related Leber hereditary optic neuropathy (ND4 mutation), achromatopsia (CNGA3 mutation and CNGB3 mutation), sex-linked genetic RP (RPGR mutation), etc.
  • RP autosomal recessive retinitis pigmentosa
  • CHM mutation choroideremia
  • ABCA4 mutation juvenile macular degeneration
  • Usher syndrome subtype 1B Myo7a mutation
  • RS1 mutation X-linked congenital retinoschisis
  • ND4 mutation mitochondria-related Leber hereditary optic neuropathy
  • CNGA3 mutation and CNGB3 mutation sex-linked genetic RP
  • the term "hereditary macular degeneration" generally refers to a less common genetic disorder that runs in families.
  • the hereditary macula Degenerative diseases can include Stargardt's disease.
  • Stargardt disease is an autosomal recessive genetic disease that originates primarily in the retinal pigment epithelium. Sporadic cases are relatively rare, and more often occur in the children of consanguineous marriages. The patient had macular atrophy and retinal yellow spots. There are currently no approved treatments, and Stargardt disease causes vision loss in tens of thousands of people worldwide each year.
  • the hereditary macular degeneration disease may also include Best's disease, the lesion mainly involves the macular area of both eyes, and generally does not spread to the surrounding omentum.
  • the inherited macular degeneration disease can occur at any age.
  • age-related macular degeneration generally refers to age-related macular degeneration, AMD, which is one of the leading causes of blindness in people over the age of 50.
  • AMD can manifest as progressive and irreversible loss of central vision, mainly involving the macula, retinal pigment epithelium, and choroid.
  • early AMD is characterized by subretinal drusen deposits, while late AMD is divided into dry and wet types.
  • Dry AMD is also called atrophic AMD, and its main pathological changes are geographic atrophy and detachment of the RPE layer, with slow and progressive loss of vision in the affected eye; wet AMD is also called neovascular or exudative AMD, and its main pathological changes are Choroidal neovascularization (CNV) with subsequent fluid accumulation and hemorrhage.
  • CNV Choroidal neovascularization
  • retinitis pigmentosa generally refers to Retinitis Pigmentosa, RP, a group of inherited eye diseases characterized by progressive degeneration of retinal photoreceptors.
  • the retinitis pigmentosa can involve progressive degenerative disease of the retina (the clear, light-sensitive membrane lining the inner surface of the back of the eye) that can eventually lead to moderate to severe vision loss.
  • Retinitis pigmentosa is mostly hereditary.
  • the clinical features of retinitis pigmentosa may include early night blindness, concentric narrowing of visual field, and finally tubular visual field, binocular blindness or near blindness, which account for a considerable proportion of blind eye diseases.
  • the retinitis pigmentosa may belong to hereditary rod and cone dystrophy diseases.
  • cone-rod dystrophy generally refers to cone-rod dystrophy, or CRD, which is a type of inherited macular degeneration.
  • the cone-rod dystrophy may include a group of clinical and genetic syndromes such as progressive vision loss, photophobia, nystagmus, etc., mainly involving cone cells, and may be combined to involve rod cells in severe cases, and the inheritance mode is mainly autosomal Dominant, recessive, X-linked inheritance.
  • heterologous generally refers to a polynucleotide, gene, or polypeptide that is not normally found in the host organism when used in reference to a polynucleotide, gene, or polypeptide.
  • the heterologous may also include native coding regions, or parts thereof, reintroduced into the source organism in a form different from the corresponding native gene, eg, may not be at a native location in the organism's genome.
  • retinylphosphatidylethanolamine generally refers to the physiological lipid substrate of ABCA4, which is sandwiched between two TMDs in the luminal leaflet and further stabilized by an extended loop from extracellular domain 1 .
  • the application provides an expression cassette combination, which includes a first expression cassette and a second expression cassette, wherein the first expression cassette can express an N-terminal truncation of the ABCA4 protein, and the second expression cassette can express Expressing the C-terminal truncation of the ABCA4 protein, the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can be It has the biological function of the complete ABCA4 protein without forming the complete ABCA4 protein.
  • the biological function of the complete ABCA4 protein may include cell localization function; ATP hydrolase activity; expression in photoreceptor cells, and/or, reducing retinal A2E deposition.
  • the N-terminal truncation of the ABCA4 protein may comprise amino acids 1-1325, amino acids 1-1160, amino acids 1-1220, amino acids 1- The 1280th amino acid, or the amino acid sequence shown by the 1st-1347th amino acid.
  • the amino acid sequence of the N-terminal truncation of the ABCA4 protein can be as shown in amino acid positions 1-1325 from the N-terminal of the complete human ABCA4 protein.
  • the C-terminal truncation of the ABCA4 protein may comprise amino acids 1326-2273, amino acids 1348-2273, amino acids 1369-2273 or amino acids 1348-2273 from the N-terminus of the complete human ABCA4 protein.
  • the amino acid sequence shown at amino acid position 2170 can be as shown in amino acid positions 1326-2273 from the N-terminal of the complete human ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein and/or the C-terminal truncation of the ABCA4 protein may correspond to the blue and green in Figure 1 in Nature Communications volume 12, Article number: 3853 (2021), respectively. structure.
  • the complete ABCA4 protein may consist of an N-terminal truncation of the ABCA4 protein and a C-terminal truncation of the ABCA4 protein.
  • the N-terminal truncated body of the ABCA4 protein and the C-terminal truncated body of the ABCA4 protein can interact in spatial conformation (for example, form an N-C heterodimer) due to their similar structures, and there is an interaction to form a complete possibility of the ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein can also exist separately and separately, without forming the complete ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can be substantially (for example, can be at least about 50% molar, at least about 60% molar, at least about 70% molar, at least about 80% molar, at least about 90% molar, at least about 95% molar, or higher ratio) do not interact to form the complete ABCA4 protein.
  • the protein molar ratio of the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette to the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette is about 3:1 ⁇ 1:3.
  • the ABCA4 The protein molar ratio of the N-terminal truncation of the protein to the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can be about 3:1-1:3, about 3:1-1: 2. About 3:1 ⁇ 1:1, about 3:1 ⁇ 2:1, about 2:1 ⁇ 1:3, about 2:1 ⁇ 1:2, about 2:1 ⁇ 1: 1. About 1:1 ⁇ 1:3, about 1:2 ⁇ 1:1, about 1:1.
  • the first expression cassette and/or the second expression cassette do not express a linking sequence, and the linking sequence can make the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette forming a complete ABCA4 protein with the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette.
  • the connecting sequence is selected from the following methods: homologous recombination, mRNA splicing and protein splicing, so that the N-terminal truncated body of the ABCA4 protein expressed by the first expression cassette and the The C-terminal truncation of the ABCA4 protein expressed by the second expression cassette forms a complete ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein and the C-terminal truncation of the ABCA4 protein can interact (eg, homologous recombination, mRNA splicing and/or protein splicing) through the linker sequence to form a complete ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette is compatible with the
  • the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can be substantially (for example, at a molar ratio of at least about 50%, at a molar ratio of at least about 60%, at a molar ratio of at least about 70%, A molar ratio of at least about 80%, a molar ratio of at least about 90%, a molar ratio of at least about 95% or higher) does not interact to form an intact ABCA4 protein.
  • the connecting sequence may include a homology arm sequence.
  • the linking sequence may include a sequence complementary to at least part of the sequence encoding the N-terminal truncation of the ABCA4 protein; and/or, the linking sequence may include a sequence capable of encoding the ABCA4 protein.
  • 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 complementary to at least part of the sequence encoding the N-terminal truncation of the ABCA4 protein; and/or the downstream homology arm sequence may comprise a sequence capable of encoding the ABCA4 protein A sequence complementary to at least part of the sequence of the C-terminal truncation of the protein.
  • the first expression cassette may not contain a sequence that is complementary to at least a portion of the sequence encoding the N-terminal truncation of the ABCA4 protein; and/or, the second expression cassette may not contain a sequence that is compatible with A sequence that encodes at least part of the sequence of the C-terminal truncation of the ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and/or the second expression cassette cannot interact with the C-terminal truncation of the ABCA4 protein through homologous recombination to form a complete ABCA4 protein.
  • the connecting sequence may include splicing signals of mRNA introns.
  • the splicing signal of the mRNA intron may include an upstream splice acceptor site (SA) of the intron and/or a downstream Splice Donor Site (SD).
  • SA upstream splice acceptor site
  • SD downstream Splice Donor Site
  • the intron upstream splice acceptor site (SA) may be located downstream of the sequence encoding at least part of the N-terminal truncation of the ABCA4 protein
  • the downstream splice donor site may be located downstream of a sequence capable of interacting with upstream of the sequence encoding at least part of the C-terminal truncation of the ABCA4 protein.
  • the first expression cassette may not comprise an intron upstream splice acceptor site (SA); and/or, the second expression cassette may not comprise an intron downstream splice donor site (SD) .
  • SA intron upstream splice acceptor site
  • SD intron downstream splice donor site
  • the linker sequence may include an intein.
  • the connecting sequence may include a sequence encoding an intein protein.
  • the sequence encoding the intein protein may include a sequence encoding the N-terminal part of the intein protein; and/or, a sequence encoding the C-terminal part of the intein protein.
  • the sequence encoding the N-terminal portion of the intein protein may be located downstream of the sequence encoding at least part of the N-terminal truncation of the ABCA4 protein; and/or the sequence encoding the C-terminal portion of the intein protein may be located at the upstream of the sequence encoding at least part of the C-terminal truncation of the ABCA4 protein.
  • the first expression cassette does not express the sequence encoding the N-terminal part of the intein protein; and/or, the second expression cassette does not express the sequence encoding the C-terminal part of the intein protein.
  • the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and/or the second expression cassette cannot interact with the C-terminal truncation of the ABCA4 protein through protein splicing to form a complete ABCA4 protein.
  • the N-terminal truncation of the ABCA4 protein may comprise the N-terminal domains of the ABCA4 protein: transmembrane regions TMD1-TMD6, extracellular domains ECD1, IH1, IH2, EH1, EH2, NBD1 and/or R1 .
  • the N-terminal truncation of the ABCA4 protein may include: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and/or R1 in sequence from the N-terminus.
  • the N-terminal truncation of the ABCA4 protein may include: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6 and/or NBD1 in order from the N-terminus.
  • the N-terminal truncated body of the ABCA4 protein may comprise the amino acid sequence shown in SEQ ID NO: 2 or 4.
  • the first expression cassette may comprise a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein.
  • nucleotide sequence encoding the N-terminal truncation of the ABCA4 protein can be as shown in SEQ ID NO: 25 or 27.
  • said first expression cassette may comprise a promoter.
  • the promoter may include the composition promoters and/or inducible promoters.
  • the promoter can include a CMV promoter.
  • the C-terminal truncation of the ABCA4 protein may comprise the C-terminal domains of the ABCA4 protein: transmembrane region TMD7-TMD12, extracellular domain ECD1, IH3, IH4, EH3, EH4, NBD2 and/or R2 .
  • the C-terminal truncation of the ABCA4 protein may include: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2 and/or R2 in sequence from the N-terminus.
  • the C-terminal truncation of the ABCA4 protein may include: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12 and/or NBD2 in order from the N-terminus.
  • the C-terminal truncated body of the ABCA4 protein may comprise the amino acid sequence shown in SEQ ID NO: 3 or 5.
  • the second expression cassette may comprise a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein.
  • nucleotide sequence encoding the C-terminal truncation of the ABCA4 protein can be as shown in SEQ ID NO: 26 or 28.
  • said second expression cassette may comprise a promoter
  • the first expression cassette may express a first constant region
  • the second expression cassette may express a second constant region, wherein the first constant region and the second constant region are capable of The interaction makes the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette interact with the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette to form an N-C isoform of the ABCA4 protein. dimer.
  • the first constant region may have a first modification
  • the second constant region may have a second modification, wherein the first modification and the second modification can promote heterodimerization body formation.
  • the first constant region and the second constant region may be derived from constant regions of antibodies.
  • it can be derived from an IgG constant region of an antibody.
  • it can be derived from the IgG1 constant region of an antibody.
  • it can be derived from the human IgG constant region of an antibody.
  • 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.
  • the first modification can promote heterodimeric formation with the second modification through charge attraction.
  • the first modification and the second modification may include knob-into-hole modifications.
  • 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.
  • the first constant region may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the second constant region may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the first constant region may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the second constant region may comprise the amino acid sequence shown in SEQ ID NO:6.
  • R1 in the N-terminal truncation of the ABCA4 protein can interact with R2 in the C-terminal truncation of the ABCA4 protein to facilitate the interaction between the N-terminal truncation of the ABCA4 protein and
  • the C-terminal truncation of the ABCA4 protein forms an N-C heterodimer of the ABCA4 protein.
  • R1 in the N-terminal truncation of the ABCA4 protein can be replaced by the first constant region
  • R2 in the C-terminal truncation of the ABCA4 protein can be replaced by the The second constant region is replaced, so that the first constant region and the second constant region interact to make the N-terminal truncation of the ABCA4 protein expressed by the first expression cassette interact with the second
  • the C-terminal truncated body of the ABCA4 protein expressed by the two expression cassettes interacts to form an N-C heterodimer of the ABCA4 protein.
  • downstream of R1 in the N-terminal truncation of the ABCA4 protein may be further connected to the first constant region, and/or, the downstream of R2 in the C-terminal truncation of the ABCA4 protein
  • the second constant region can be further connected, thereby further promoting 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 Interacts to form N-C heterodimers of ABCA4 proteins.
  • the first expression cassette may comprise a promoter sequence, a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein and/or a core encoding the first constant region from the 5' end. nucleotide sequence.
  • the first expression cassette may comprise a promoter sequence, a nucleotide sequence encoding the first constant region and/or a core encoding an N-terminal truncation of the ABCA4 protein from the 5' end. nucleotide sequence.
  • the first expression cassette may consist of a promoter sequence and a nucleotide sequence encoding an N-terminal truncation of the ABCA4 protein from the 5' end.
  • the first expression cassette may be truncated from the 5' end by the nucleotide sequence of the promoter, the nucleotide sequence encoding the first constant region, and the N-terminal encoding the ABCA4 protein The body's nucleotide sequence composition.
  • the first expression cassette may be composed of the nucleotide sequence of the promoter, the nucleotide sequence of the N-terminal truncation of the ABCA4 protein and the first constant region from the 5' end. nucleotide sequence composition.
  • the second expression cassette may comprise a promoter sequence, a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein and/or a nucleoside encoding the second constant region from the 5' end acid sequence.
  • the second expression cassette may comprise a promoter sequence, a nucleotide sequence encoding the second constant region and/or a nucleoside encoding a C-terminal truncation of the ABCA4 protein from the 5' end acid sequence.
  • the second expression cassette may consist of a promoter sequence and a nucleotide sequence encoding a C-terminal truncation of the ABCA4 protein from the 5' end.
  • the second expression cassette may be composed of the nucleotide sequence of the promoter, the nucleotide sequence of the C-terminal truncation of the ABCA4 protein and the second constant region from the 5' end. nucleotide sequence composition.
  • the second expression cassette may be composed of a promoter sequence from the 5' end, encoding the second constant region, and the nucleotide sequence encoding the C-terminal truncation of the ABCA4 protein.
  • the protein expressed by the first expression cassette may include: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and/or R1, and the first constant region.
  • the protein expressed by the first expression cassette may sequentially include: the first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 from the N-terminus and R1.
  • the protein expressed by the first expression cassette may be: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1, R1 and the first constant region in sequence from the N-terminus.
  • the protein expressed by the first expression cassette can be: IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and the first constant region in sequence from the N-terminus.
  • the protein expressed by the first expression cassette may be: the first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6, NBD1 and R1 in sequence from the N-terminus.
  • the protein expressed by the first expression cassette may be: the first constant region, IH1, TMD1, ECD1, TMD2, IH2, TMD3, TMD4, TMD5, EH1, EH2, TMD6 and NBD1 in sequence from the N-terminus.
  • the protein expressed by the second expression cassette may include: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2 and/or R2, and a second constant region.
  • the protein expressed by the second expression cassette may include: the second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, and NBD2 and/or R2.
  • the protein expressed by the second expression cassette may be: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, R2 and the second constant region in sequence from the N-terminus.
  • the protein expressed by the second expression cassette may be: IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2, and the second constant region in sequence from the N-terminus.
  • the protein expressed by the second expression cassette may be: the second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12, NBD2 and R2 in sequence from the N-terminus.
  • the protein expressed by the second expression cassette may be: the second constant region, IH3, TMD7, ECD2, TMD8, IH4, TMD9, TMD10, TMD11, EH3, EH4, TMD12 and NBD2 in sequence from the N-terminus.
  • the first expression cassette may comprise the nucleotide sequence shown in any one of SEQ ID NO: 2, 4, 8-10, 15-18, 34.
  • the second expression cassette may comprise the nucleotide sequence shown in any one of SEQ ID NO:3,5,11-14,19-22.
  • the first expression cassette may exist separately from the second expression cassette.
  • the first expression cassette and/or the second expression cassette may exist in a form selected from the group consisting of isolated nucleic acid molecules, liposomes and/or exosomes.
  • the first expression cassette and/or the second expression cassette may exist in the form of a plasmid.
  • the first expression cassette and/or the second expression cassette may be a viral vector.
  • the first expression cassette and/or the second expression cassette may be AAV.
  • the molar ratio of the protein expressed by the first expression cassette and the second expression cassette may be about 1:3-3:1.
  • it can be about 3:1 ⁇ 1:3, about 3:1 ⁇ 1:2, about 3:1 ⁇ 1:1, about 3:1 ⁇ 2:1, about 2:1 ⁇ 1 :3, about 2:1 ⁇ 1:2, about 2:1 ⁇ 1:1, about 1:1 ⁇ 1:3, about 1:2 ⁇ 1:1, about 1:1.
  • the molar ratio of the protein expressed by the first expression cassette and the second expression cassette may be about 1:1.
  • the present application provides a kit comprising the combination of expression cassettes described in the present application.
  • the kit may comprise reagents and/or instruments for administering the combination of expression cassettes.
  • the kit may comprise reagents and/or equipment for transfection of AAV comprising said first expression cassette and/or said second expression cassette.
  • said kit may comprise reagents and/or instruments for injecting AAV comprising said first expression cassette and/or said second expression cassette.
  • the injection may include local injection.
  • the present application provides a use of the expression cassette combination described in the present application, and/or the kit described in the present application, in the preparation of medicines for treating diseases, and the diseases include diseases caused by ABCA4 mutations.
  • the diseases may include hereditary retinal diseases.
  • the disease may include hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa and/or cone-rod dystrophy.
  • the heterologous N-terminal truncation of the ABCA4 protein and the heterologous C-terminal truncation of the ABCA4 protein can be expressed in the subject in need, because the ABCA4
  • the N-terminal truncation of the protein and the C-terminal truncation of the heterologous ABCA4 protein can exert the biological function of the complete ABCA4 protein, and thus can be used to alleviate/treat/improve diseases caused by ABCA4 mutations.
  • the present application provides a method for expressing heterologous ABCA4 gene, which may comprise administering the combination of expression cassettes described in the present application, and/or the kit described in the present application to a subject in need.
  • the present application provides a method for alleviating cell death caused by retinyl phosphatidylethanolamine (NRPE), It may comprise administering an expression cassette combination described herein, and/or a kit described herein to a subject in need thereof.
  • NRPE retinyl phosphatidylethanolamine
  • the present application provides a method for treating a disease caused by an ABCA4 mutation, which may comprise administering the expression cassette combination described in the present application, and/or the kit described in the present application to a subject in need.
  • said administering may comprise injection.
  • the diseases may include hereditary retinal diseases.
  • the disease may include hereditary macular degeneration, age-related macular degeneration, retinitis pigmentosa and/or cone-rod dystrophy.
  • HEK293 cells (provided by Peking University Health Science Center) were transfected with wild-type ABCA4 plasmid fused with FLAG tag (ABCA4-WT) or codon-optimized ABCA4 plasmid fused with FLAG tag (ABCA4-CO). After 48 hours, the cells were lysed, and the expression intensity of ABCA4 protein in the cells was detected using the endogenous antibody of ABCA4 (NBP1-30032, NovusBio) or the flag tag antibody (F1804, Sigma), respectively.
  • the nucleotide sequence of wild-type ABCA4 is shown in SEQ ID NO.23; the nucleotide sequence of codon-optimized ABCA4 is shown in SEQ ID NO.24.
  • 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; the amino acid sequence of the C-terminal truncation of ABCA4 is shown in SEQ ID NO .3 shown.
  • HA hemagglutinin
  • the N-terminal truncation of ABCA4 (aa 1-1347), the C-terminal truncation (aa 1348-2273) or the N+C co-transfection (that is, the co-transfection of the N-terminal truncation (aa 1-1347) and C-terminal truncation (aa 1348-2273)).
  • the backbone vector for the transfection plasmid was purchased from Addgene.
  • the cells were lysed, and the C-terminal truncations containing the flag tag in the cells were enriched using flag antibody (F1804, Sigma) coupled to magnetic beads, and then detected by HA antibody (AE036, ABclonal) western blot. N-terminal truncations of short-body interactions.
  • Example 4 There is an interaction between the N-terminal truncations of ABCA4 of different lengths or containing different domains and the C-terminal truncations of ABCA4
  • N-terminal truncations and C-terminal truncations of different lengths or containing different domains wherein the N-terminal truncations include: 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 * , 2253-2273) (SEQ ID NO:38); C-terminal truncations include: C(aa 1348-2273) (SEQ ID NO:3), C * (aa 1-20 , 1369-2273) (SEQ ID NO: 39).
  • the HA tag is connected to the C-terminal of the N-terminal truncated body, and the C-terminal of the C-terminal truncated body is
  • N1347 (aa 1-1347), N1160 (aa 1-1160), N1220(aa 1-1220), N1280(aa 1-1280), N1280 * (aa 1-1280 * , 2253-2273) can all detect the interaction with C(aa 1348-2273); N1347(aa 1-1347), N1220 (aa 1-1220), N1280 * (aa 1-1280 * , 2253-2273) and detectable interactions with C * (aa 1-20, 1369-2273).
  • N1347(aa 1-1347) had the strongest interaction with C or C * .
  • Example 5 The N-terminal truncation of ABCA4 and the C-terminal truncation exist in cell colocalization, and the localization position is consistent with the full-length wild-type ABCA4
  • the full-length ABCA4 (aa 1-2273), the N-terminal truncation of ABCA4 (aa 1-1347) and the C-terminal truncation of ABCA4 (aa 1348-2273) were transfected into HEK293 cells (wherein the N-terminal truncated The C-terminal of the C-terminal truncated body is connected to the HA tag, and the C-terminal of the C-terminal truncated body is connected to the Flag tag), and then the corresponding tag is labeled with the corresponding antibody (flag antibody (F1804, Sigma), HA antibody (K200003M, Solarbio)), and ABCA4 can be observed
  • the N-terminal truncation (aa 1-1347) and the C-terminal truncation (aa 1348-2273) co-localize in cells (see Figure 6), and the localization position is consistent with the full-length wild-type ABCA4, both of which exist in the cell in the inner
  • Example 6 The N-terminal truncation and C-terminal truncation complex of ABCA4 has ATP hydrolase activity
  • the full-length ABCA4 (aa 1-2273), the N-terminal truncation of ABCA4 (aa 1-1347) and the C-terminal truncation of ABCA4 (aa 1348-2273) or N+C co-transfection were respectively transfected
  • cells were lysed, and flag-tagged full-length ABCA4 or C-terminal truncations, or N+C complexes (that is, N-terminal truncations and C-terminal truncations) were enriched in cells using flag antibody-coupled magnetic beads. short complex), and then use the ATPase detection kit (Promega) to detect the ATP hydrolase activity of the protein enriched on the magnetic beads.
  • Example 7 There is an interaction between N(aa 1-1347)-Fca of ABCA4 and Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb
  • Fca and Fcb were used to increase the proportion of heterodimers and reduce the proportion of homodimers.
  • amino acid sequence of Fca is shown in SEQ ID NO.6; the amino acid sequence of Fcb is shown in SEQ ID NO.7.
  • Fca and Fcb are adjacent to each other in the amino acid secondary structure, that is, the C-terminus of the N-terminal truncated body (aa 1-1347) of ABCA4 is connected to Fca (adjacent to R1) and constructed as N (aa 1-1347) -Fca (wherein the amino acid sequence of N(aa 1-1347)-Fca is shown in SEQ ID NO.8), the N-terminus of the C-terminal truncated body (aa 1348-2273) of ABCA4 is connected to Fcb (adjacent to IH3 ) was constructed as Fcb-C (aa 1348-2273) (wherein, the amino acid sequence of Fcb-C (aa 1348-2273) is shown in SEQ ID NO.14).
  • the second is that Fca and Fcb are adjacent in the tertiary structure of the protein, that is, the C-terminus of the N-terminal truncation (aa 1-1347) of ABCA4 is connected to Fca (adjacent to R1) to construct N (aa 1-1347) -Fca (wherein, the amino acid sequence of N(aa 1-1347)-Fca is shown in SEQ ID NO.8), the C-terminal (aa 1348-2273) of the C-terminal truncated body of ABCA4 is connected to Fcb (adjacent to R2 ) was constructed as C(aa 1348-2273)-Fcb (wherein, the amino acid sequence of C(aa 1348-2273)-Fcb is shown in SEQ ID NO.13).
  • HEK293 cells were single-transfected or co-transfected with N(aa 1-1347)-Fca, Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb respectively.
  • Protein expression (Input) IgG coupled magnetic beads can be enriched to N(aa 1-1347)-Fca in cell lysis, and Fcb-C(aa 1348) that interacts with N(aa 1-1347)-Fca -2273) or C(aa 1348-2273)-Fcb was also enriched.
  • N-Fc means transfection of N(aa 1-1347)-Fca alone
  • C-Fc means transfection of C(aa 1348-2273)-Fcb alone
  • Fc-C means transfection of Fcb-C (aa 1348-2273)
  • N(aa 1-1347)-Fc+C-Fc means co-transfection of N(aa 1-1347)-Fca and C(aa 1348-2273)-Fcb
  • N(aa 1-1347)- Fc+Fc-C means co-transfected N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273)
  • NTD-Fc means enriched N(aa 1-1347)-Fca
  • CTD-Fc /Fc-CTD indicates Fcb-C(aa 1348-2273) or C(aa 1348-2273)-Fcb interacting with N(aa 1-1347)-Fca.
  • Example 8 There is an interaction between N-terminal truncations of ABCA4 with different lengths or different domains-Fca and Fcb-C-terminal truncations or C-terminal truncations-Fcb
  • N-terminal truncations-Fca and Fcb-C-terminal truncations or C-terminal truncations-Fcb of ABCA4 with different lengths or containing different domains wherein the N-terminal truncations include: N(aa 1-1347) -Fca, N(aa 1-1160)-Fca; C-terminal truncations include: C(aa 1348-2273)-Fcb, C(aa 1348-2170)-Fcb, Fcb-C(aa 1348-2273).
  • Example 9 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 complex has ATP hydrolysis Enzyme activity
  • the results are shown in Figure 11, the results show that neither the blank magnetic beads nor the independent N(aa 1-1347)-Fca has ATP hydrolase activity, and the N+C complex (ie N-terminal truncated body and C-terminal truncated
  • the complex of the body can be obtained by co-transfecting N(aa 1-1347)-Fca and Fcb-C(aa 1348-2273), or co-transfecting N(aa 1-1347)-Fca and C(aa 1348- 2273)-Fcb) is similar to the full-length ABCA4 enriched by Flag-coupled magnetic beads, and has ATP hydrolase activity.
  • Example 10 The N-terminal truncation body and C-terminal truncation body complex of ABCA4 can alleviate the cell death caused by NRPE
  • Example 11 Co-infection of the N-terminal truncation of AAV-ABCA4 and the complex of the C-terminal truncation of AAV-ABCA4 can alleviate the cell death caused by NRPE
  • NRPE retinyl phosphatidylethanolamine
  • the ATR1 conversion ability in the cells infected with the control virus AAV-EV decreased, and the cell activity decreased; while the N-terminal truncated body (aa 1-1347) and the C-terminal truncated body of AAV-ABCA4 (aa 1348-2273), the transformation ability of ATR1 is improved, and the cell activity is relatively good.

Abstract

本申请涉及一种表达盒组合及其应用。所述表达盒组合包括第一表达盒和第二表达盒，其中所述第一表达盒能够表达ABCA4蛋白的N端截短体，所述第二表达盒能够表达ABCA4蛋白的C端截短体，所述ABCA4蛋白的N端截短体与所述ABCA4蛋白的C端截短体能够在两者不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能。

Description

表达盒组合及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种表达盒组合及其应用。
背景技术
ABCA4是视网膜感光细胞或视网膜色素上皮细胞中的脂质内向翻转酶，可以将磷脂酰乙醇胺(PE)和全反式视黄醛(ATR)形成的脂质衍生物—视黄基磷脂酰乙醇胺(NRPE)，从外节膜盘的腔内侧转运到细胞质侧。随后，NRPE会水解为ATR和PE，其中的ATR会被细胞质内的视黄醛还原酶催化形成全反式视黄醇(ATRol)，从而重新回到视循环。如果ABCA4的脂质转运功能受损，会使得ATR和NRPE在外节膜盘的腔内侧发生过量积累，过量积累的ATR和NRPE会进一步不可逆的形成具有毒性的双视黄基衍生物，最终引起一系列的视网膜退行性疾病，包括最为常见的遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和锥杆营养不良等。
人ABCA4 CDS全长6.7kb，编码2273个氨基酸。ABCA4的结构示意图如图1所示(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)。
目前尚无针对ABCA4突变引起的视网膜病的上市药物。
发明内容
本申请提供了一种表达盒组合。该表达盒组合可以在细胞内共表达ABCA4蛋白的N端截短体(N端结构域)与第二表达盒所表达的所述ABCA4蛋白的C端截短体(N端结构域)。其中，所述ABCA4蛋白的N端截短体与所述ABCA4蛋白的C端截短体能够在两者之间不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能(例如与完整的ABCA4蛋白一致的细胞定位；ATP水解酶活性；和/或，在感光细胞中共表达，减少视网膜A2E沉积)。
本申请创造性地发现，由于所述ABCA4蛋白的N端截短体与所述ABCA4蛋白的C端截短体能够在两者之间不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能，因此就无需转染、表达完整的ABCA4蛋白来实现治疗目的。目前通常使用双AAV载体系统转染、表达完整的ABCA4蛋白，例如，将完整的ABCA4表达框拆成两部分，两 部分共用一套表达所需的启动子、终止密码子、poly A等，利用细胞里的DNA同源重组或RNA剪切，获得一条完整的RNA，从而最终编码为完整的ABCA4蛋白；或利用蛋白剪切成为完整的ABCA4蛋白。利用DNA同源重组方法，小鼠视网膜下腔注射双AAV载体，每种AAV各注射1×1010vg/眼，ABCA4蛋白的表达强度约为内源表达量的1％(McClements,M.E.,et al.(2019)."An AAV Dual Vector Strategy Ameliorates the Stargardt Phenotype in Adult Abca4(-/-)Mice."Hum Gene Ther 30(5):590-600.)。即便将同源重组和RNA剪切结合起来的hybrid方法，小鼠视网膜下腔注射双AAV载体，每种AAV各注射3×109vg/眼，小鼠中的ABCA4表达强度约为内源表达的10％(Dyka,F.M.,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.)。利用内含肽蛋白剪切方法，小鼠视网膜下腔注射双AAV载体，每种AAV各注射5×109vg/眼，小鼠中的ABCA4表达强度仍然不高于内源表达的10％(Tornabene,P.,et al.(2019)."Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina."Sci Transl Med 11(492).)。由于上述方法表达效率较低，为了达到或接近内源表达水平，在治疗过程中需要注射大量的AAV病毒，因此免疫毒性很大，而本申请提供的表达盒组合则解决了这些困难。
一方面，本申请提供了一种表达盒组合，其包括第一表达盒和第二表达盒，其中所述第一表达盒能够表达ABCA4蛋白的N端截短体，所述第二表达盒能够表达ABCA4蛋白的C端截短体，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体能够在两者之间不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能。
在某些实施方式中，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体基本上不相互作用形成完整的ABCA4蛋白。
在某些实施方式中，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体的蛋白摩尔比例约为3:1～1:3。
在某些实施方式中，所述第一表达盒和/或所述第二表达盒不表达连接序列，所述连接序列能够使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
在某些实施方式中，所述连接序列通过选自下组的方法：同源重组、mRNA剪接和蛋白剪接，使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表 达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
在某些实施方式中，所述连接序列包括同源臂序列。
在某些实施方式中，所述连接序列包括能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述连接序列包括能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。
在某些实施方式中，所述第一表达盒不包含能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述第二表达盒不包含能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。
在某些实施方式中，所述连接序列包括mRNA内含子的剪切信号。
在某些实施方式中，所述第一表达盒不包含内含子上游剪接受体位点(SA)；和/或，所述第二表达盒不包含内含子下游剪接供体位点(SD)。
在某些实施方式中，所述连接序列包括内含肽。
在某些实施方式中，所述连接序列包括编码intein蛋白的序列。
在某些实施方式中，所述第一表达盒不表达编码intein蛋白的N端部分的序列；和/或，所述第二表达盒不表达编码intein蛋白的C端部分的序列。
在某些实施方式中，所述ABCA4蛋白的N端截短体包含所述ABCA4蛋白N端的结构域：跨膜区TMD1～TMD6、胞外域ECD1、IH1、IH2、EH1、EH2、NBD1和/或R1，优选包含跨膜区TMD1～TMD6、胞外域ECD1、IH2、EH1、EH2和NBD1以及任选地IH1和/或R1。在特别优选的实施方式中，所述ABCA4蛋白的N端截短体包含ABCA4蛋白N端的氨基酸1至1160(aa 1-1160)(SEQ ID NO:35)、氨基酸1至1220(aa 1-1220)(SEQ ID NO:36)、氨基酸1至1280(aa 1-1280)(SEQ ID NO:37)、氨基酸1至1347(aa 1-1347)(SEQ ID NO:2)或氨基酸1至1280和2253-2273(aa 1-1280*，2253-2273)(SEQ ID NO:38)。在某些实施方式中，所述ABCA4蛋白的N端截短体可以不包含IH1和/或R1。
在某些实施方式中，所述ABCA4蛋白的N端截短体自N端依次包括：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和/或R1。
在某些实施方式中，所述ABCA4蛋白的N端截短体包含SEQ ID NO:2或4所示的氨基酸序列。
在某些实施方式中，所述第一表达盒包含编码所述ABCA4蛋白的N端截短体的核苷酸序列。
在某些实施方式中，编码所述ABCA4蛋白的N端截短体的核苷酸序列如SEQ ID NO:25 或27所示。
在某些实施方式中，所述第一表达盒包含启动子。
在某些实施方式中，所述ABCA4蛋白的C端截短体包含所述ABCA4蛋白C端的结构域：跨膜区TMD7～TMD12、胞外域ECD2、IH3、IH4、EH3、EH4、NBD2和/或R2，优选包含跨膜区TMD7～TMD12、胞外域ECD2、IH4、EH3、EH4和NBD2以及任选地IH3和/或R2。在特别优选的实施方式中，所述ABCA4蛋白的C端截短体包含所述ABCA4蛋白C端的氨基酸1348-2273(aa 1348-2273)、氨基酸1369-2273(aa 1369-2273)、氨基酸1348-2170(aa 1348-2170)或氨基酸1-20和1369-2273(aa 1-20，1369-2273)。在某些实施方式中，所述ABCA4蛋白的C端截短体可以不包含IH3和/或R2。
不限于任何理论，据信ABCA4蛋白的IH1/3结构域提供了膜定位信号，R1/2结构域提供了胞内区的一部分相互作用，但是这两部分结构域似乎不是N+C结构和功能完整性所必需的。
在某些实施方式中，所述ABCA4蛋白的C端截短体自N端依次包括：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2和/或R2。
在某些实施方式中，所述ABCA4蛋白的C端截短体包含SEQ ID NO:3或5所示的氨基酸序列。
在某些实施方式中，所述第二表达盒包含编码所述ABCA4蛋白的C端截短体的核苷酸序列。
在某些实施方式中，编码所述ABCA4蛋白的C端截短体的核苷酸序列如SEQ ID NO:26或28所示。
在某些实施方式中，所述第二表达盒包含启动子。
在某些实施方式中，所述第一表达盒表达第一恒定区，和/或，所述第二表达盒表达第二恒定区，其中所述第一恒定区与所述第二恒定区能够相互作用使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体。
在某些实施方式中，所述第一恒定区具有第一修饰，和/或，所述第二恒定区具有第二修饰，其中所述第一修饰与所述第二修饰能够促进异二聚体的形成。
在某些实施方式中，所述第一恒定区与所述第二恒定区源自抗体的恒定区。
在某些实施方式中，所述第一修饰与所述第二修饰包括knob-into-hole修饰。
在某些实施方式中，所述第一恒定区包含SEQ ID NO:6或7所示的氨基酸序列。
在某些实施方式中，所述第二恒定区包含SEQ ID NO:7或6所示的氨基酸序列。
在某些实施方式中，所述第一表达盒自5’端起包含启动子序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列和/或编码所述第一恒定区的核苷酸序列。
在某些实施方式中，所述第一表达盒自5’端起由启动子序列和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。
在某些实施方式中，所述第一表达盒自5’端起由启动子的核苷酸序列、编码所述第一恒定区的核苷酸序列、和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。
在某些实施方式中，所述第一表达盒自5’端起由启动子的核苷酸序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列、和编码所述第一恒定区的核苷酸序列组成。
在某些实施方式中，所述第二表达盒自5’端包含启动子序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和/或编码所述第二恒定区的核苷酸序列。
在某些实施方式中，所述第二表达盒自5’端起由启动子序列和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。
在某些实施方式中，所述第二表达盒自5’端起由启动子的核苷酸序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和编码所述第二恒定区的核苷酸序列组成。
在某些实施方式中，所述第二表达盒自5’端起由启动子序列、编码所述第二恒定区的核苷酸序列、和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。
在某些实施方式中，所述第一表达盒包含SEQ ID NO:2，4，8-10，15-18，34中任一项所示的核苷酸序列。
在某些实施方式中，所述第二表达盒包含SEQ ID NO:3，5，11-14，19-22中任一项所示的核苷酸序列。
在某些实施方式中，所述第一表达盒与所述第二表达盒单独存在。
在某些实施方式中，所述第一表达盒和/或所述第二表达盒以选自下组的形式存在：分离的核酸分子、脂质体和/或外泌体。
在某些实施方式中，所述第一表达盒和/或所述第二表达盒以质粒的形式存在。
在某些实施方式中，所述第一表达盒和/或所述第二表达盒为病毒载体。
在某些实施方式中，所述第一表达盒和/或所述第二表达盒为AAV。
在某些实施方式中，在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的蛋白的摩尔比为约1:3～3:1。
在某些实施方式中，在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的 蛋白的摩尔比为约1:1。
另一方面，本申请提供一种试剂盒，其包含本申请所述的表达盒组合。
在某些实施方式中，所述的试剂盒包含用于施用所述表达盒组合的试剂和/或仪器。
另一方面，本申请提供一种本申请所述的表达盒组合，和/或本申请所述的试剂盒，在制备治疗疾病的药物中的用途，所述疾病包括ABCA4突变引起的疾病。
在某些实施方式中，所述疾病包括遗传性视网膜疾病。
在某些实施方式中，所述疾病包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
另一方面，本申请提供一种表达异源ABCA4基因的方法，其包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
另一方面，本申请提供一种缓解视黄基磷脂酰乙醇胺(NRPE)导致的细胞死亡的方法，其包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
另一方面，本申请提供一种治疗ABCA4突变引起的疾病的方法，其包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
在某些实施方式中，所述施用包括注射。
在某些实施方式中，所述疾病包括遗传性视网膜疾病。
在某些实施方式中，所述疾病包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是ABCA4的结构示意图。
图2显示的是密码子优化的ABCA4的蛋白表达强度。
图3显示的是本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和ABCA4蛋白的C端截短体(aa 1348-2273)的二聚化现象。
图4显示的是本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和ABCA4蛋白的C端截短体(aa 1348-2273)存在相互作用。
图5显示的是不同长度或包含不同结构域的ABCA4蛋白的N端截短体与ABCA4蛋白的C端截短体之间存在相互作用。
图6显示的是本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和ABCA4蛋白的C端截短体(aa 1348-2273)存在细胞共定位。
图7显示的是本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和ABCA4蛋白的C端截短体(aa 1348-2273)的共定位形式与全长野生型的ABCA4蛋白一致。
图8显示的是转染本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和/或ABCA4蛋白的C端截短体(aa 1348-2273)的ATP水解酶活性。
图9显示的是经修饰的本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和ABCA4蛋白的C端截短体(aa 1348-2273)之间存在相互作用。
图10显示的是不同长度或包含不同结构域的ABCA4蛋白的N端截短体-Fca与Fcb-C端截短体或C端截短体-Fcb之间存在相互作用。
图11显示的是转染经修饰的本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和/或ABCA4蛋白的C端截短体(aa 1348-2273)的ATP水解酶活性。
图12显示的是转染经修饰的本申请所述ABCA4蛋白的N端截短体(aa 1-1347)和/或ABCA4蛋白对NRPE导致的细胞死亡的缓解情况。
图13显示的是共感染AAV-ABCA4的N端截短体与AAV-ABCA4的C端截短体复合物可缓解NRPE导致的细胞死亡。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
术语“第一表达盒”通常是指可操作地连接至启动子或足以指导目的基因的转录的其他调控序列的外源DNA序列。在本申请中，所述表达盒通常是指这样的核酸构建体：当被引入宿主细胞中时，其分别导致RNA或多肽的转录和/或翻译。所述表达盒可以具有允许特定 核酸在靶细胞中转录的一系列专门核酸元件。所述表达盒可以掺入质粒、染色体、线粒体DNA、质粒DNA、病毒或核酸片段内。所述表达盒可以包括未翻译或不可翻译的反义或有义构建体。在表达转基因和抑制内源基因(例如，通过反义、RNAi或有义抑制)的情况下，本领域技术人员会认识到，插入的多核苷酸序列不需要是相同的，但是可以仅仅与它的来源基因的序列基本上相同。所述表达盒可以包含与启动子可操作地连接的转录因子的多核苷酸构建体，所述启动子可以是得自由所述转录因子调节的基因的启动子。在本申请中，所述第一表达盒可以表达一种蛋白质(例如ABCA4蛋白的N端截短体)。
术语“第二表达盒”通常是指与所述第一表达盒不完全相同的一种表达盒。例如，所述第二表达盒可以表达与所述第一表达盒所表达的蛋白质不同的蛋白质(例如ABCA4蛋白的C端截短体)。
术语“表达盒组合”通常是指包含至少一个表达盒的组合。例如，本申请所述的表达盒组合可以包含所述第一表达盒和所述第二表达盒。
术语“ABCA4基因”通常是指编码rim蛋白或RmP的基因。ABCA4基因也称作ABCR基因。该基因首次克隆并被鉴定为一种可引起Stargardt病的基因，这是一种常染色体隐性遗传疾病，可导致黄斑变性。人ABCA4基因的NCBI Entrez Gene登录号为24。
术语“完整的ABCA4蛋白”通常是指具有完整结构的ATP结合盒亚家族A的成员4。所述完整的ABCA4蛋白可以由人ABCA4基因编码。ABCA4是视网膜感光细胞或视网膜色素上皮细胞中的脂质内向翻转酶，可以将磷脂酰乙醇胺(PE)和全反式视黄醛(ATR)形成的脂质衍生物—视黄基磷脂酰乙醇胺(NRPE)，从外节膜盘的腔内侧转运到细胞质侧。随后，NRPE会水解为ATR和PE，其中的ATR会被细胞质内的视黄醛还原酶催化形成全反式视黄醇(ATRol)，从而重新回到视循环。人完整的ABCA4蛋白的UniProt登录号为P78363。
术语“ABCA4蛋白的N端截短体”通常是指人完整的ABCA4蛋白的N端截短体。例如，所述ABCA4蛋白的N端截短体可以包含人完整的ABCA4蛋白自N端起第1-1325位氨基酸、第1-1160位氨基酸、第1-1220位氨基酸、第1-1280位氨基酸、或第1-1347位氨基酸所示的氨基酸序列。例如，所述ABCA4蛋白的N端截短体的氨基酸序列可以如人完整的ABCA4蛋白自N端起第1-1325位氨基酸所示。在本申请中，所述ABCA4蛋白的N端截短体和/或ABCA4蛋白的C端截短体可以分别对应于Nature Communications volume 12,Article number:3853(2021)中图1蓝色和绿色的结构。
术语“ABCA4蛋白的C端截短体”通常是指人完整的ABCA4蛋白的C端截短体。例如，所述ABCA4蛋白的C端截短体可以包含人完整的ABCA4蛋白自N端起第1326-2273位氨 基酸、第1348-2273位氨基酸、第1369-2273位氨基酸或第1348-2170位氨基酸所示的氨基酸序列。例如，所述ABCA4蛋白的C端截短体的氨基酸序列可以如人完整的ABCA4蛋白自N端起第1326-2273位氨基酸所示。
术语“生物学功能”通常是指对于被测试的生物实体是天然的或作为所述生物实体的目的活性，例如细胞、蛋白质或类似物的天然活性。理想地，可使用体外功能测定来测试生物学功能的存在。
术语“同源重组”通常是指重组以同源或同一的遗传物质片段之间相互作用的结果的形式发生。例如，所述同源重组可以包括两个DNA分子之间DNA片段的交换。交换的片段可以是两个DNA分子具有相同核酸序列的位点(即，“同源区”)侧翼的片段。所述“同源区”可以包括在参与同源重组的核酸片段上彼此之间具有同源性的核酸序列范围。在这些同源区的长度可以为至少约10bp。
术语“mRNA剪接”通常是指发生在真核细胞核内的细胞事件，其中内含子被从前-mRNA类型中去除。一般地，该过程可能需要剪接酶体复合物的形成，其中5’剪接供体位点被带到3’剪接受体位点附近，同时从所述转录本中去除干扰内含子序列。
术语“蛋白剪接”通常是指前体蛋白的内部区域(内含肽)被切除并且蛋白质的侧翼区域(外显肽)被连接形成成熟蛋白质的过程。内含肽单元可以包含催化蛋白剪接所需的必要组分，并且通常包含参与内含肽移动性的核酸内切酶结构域。产生的蛋白质可以是连接在一起的，但是并不以单独的蛋白质表达。蛋白剪接也可以与在分开的多肽上表达的分裂内含肽自发结合形成单个内含肽，然后进行蛋白剪接过程以结合成分离的蛋白质，从而以反式方式进行。
术语“同源臂序列”通常是指与在细胞基因组内的对应靶序列具有足以经历同源重组的同源性的序列。例如，所述同源臂序列可以包括与靶序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的多核苷酸序列。所述同源臂序列可以包括上游同源臂序列和下游同源臂序列。
术语“mRNA内含子的剪切信号”通常是指用于RNA剪接的信号。例如，所述RNA剪接可以包括分子内(intramolecular)剪接(cis splicing)、及分子间(intermolecular)剪接(trans splicing)。在内含子里，可以需要有5'剪接位点(5'splice site)、3'剪接位点(3'splice site)及剪接分枝位点(branch point)来进行剪接。剪接可以由剪接体(Spliceosome)来催化，它是以五个不同的小核核糖核酸(snRNAs)以及不下于一百个蛋白质所组成的大型核糖核酸蛋白质复合物，称为小核核糖蛋白(snRNP)所组成。小核核糖蛋白的RNA会与内含子行杂交作用 (hybridization)，并且参与剪接的催化反应。
术语“上游剪接受体位点(SA)”通常是指剪接受体，splice acceptor，SA。在某些情况下，一个内含子的上游和下游的与邻近外显子的交界处，分别有一段被称为SD以及SA的区域。在转录后形成的早期mRNA链上，与SA和SD对应的序列，则包含了剪切的信号。以剪切的位点为界，可以将SD序列中，与决定上游剪切位点相关的部分，位于外显子部分的，称为A1，位于内含子部分的，称为B1；与之类似，可以将SA序列中，与决定下游剪切位点相关的部分，位于外显子部分的，称为A2，位于内含子部分的，称为B2；这样，目标基因转录后，最终形成的信号RNA，在剪切的交界处，就留下与A1A2相对应的序列。
术语“下游剪接供体位点(SD)”通常是指剪接供体splicing donor，SD。
术语“内含肽”通常是指能够介导蛋白质分子从前体分子中自我切除，同时将其双侧的外显肽蛋白通过肽键连接起来的功能性蛋白质。内含肽基因可以不是一个独立的基因，需要插入于外显肽基因才能复制转录，可从前体蛋白中切除并将两侧外显肽连接起来成为成熟蛋白质。内含肽对应的核苷酸序列可以嵌合在宿主蛋白对应的核酸序列之中，与宿主蛋白基因存在于同一开放阅读框架内，并与宿主蛋白质基因进行同步转录和翻译，当翻译形成蛋白质前体后，内含肽可以从宿主蛋白质中切除，从而形成成熟的具有活性的蛋白。根据内含肽的存在形式，可以分为整体内含肽和断裂型内含肽，整体内含肽的两个剪接区域共同存在于同一多肽片段上，断裂型内含肽的两个剪接区域分裂成两份或者更多片段，两个剪接区域存在于不同的多肽片段上，所以又可以称为分离内含肽。
术语“第一恒定区”通常是指可以通过本申请所述第一表达盒表达的恒定区。所述恒定区可以是指与所述“可变”相对的，所述恒定区能够相互作用使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体的区域。在某些情况下，所述恒定区可以包括免疫球蛋白的恒定区基因所编码的蛋白质。所述恒定区可以选自以下5个同种型中的任一个：α、δ、ε、γ或μ。
术语“第二恒定区”通常是指可以通过本申请所述第二表达盒表达的恒定区。在本申请中，所述第二恒定区可以与所述第一恒定区相同，或者，可以与所述第一恒定区不同。
术语“knob-into-hole修饰”通常是指两个免疫球蛋白重链的CH3结构域之间的界面内的修饰。在一种实施方案中，“knob-into-hole修饰”包含一条抗体重链中的氨基酸取代T366W和任选的氨基酸取代S354C，以及另一条抗体重链中的氨基酸取代T366S、L368A、Y407V和任选的Y349C。knob-into-hole技术在例如美国专利第8216805号中描述。
术语“脂质体”通常是指由两亲成囊泡脂质形成的脂质结构。所述脂质体可以为由具有内部水相的单层或更多层脂双层构成的封闭囊泡。例如，脂质体在更广意义上可以是指脂质复合粒子。所述脂质体甚至可以包括其水相未确定性证实的复合物。该脂质复合物含有至少一种类型的脂质，并可另外含有亲水聚合物、多糖、氨基酸等。脂质复合物可以是指由这些成分经共价或非共价键形成的粒子。
术语“外泌体”通常是指细胞外分泌细胞或具有由存在于细胞中的脂质双层构成的膜结构的膜囊。所述外泌体可以包括具有从约10nm至约2,000nm的平均直径的膜体。所述外泌体可以包括微囊泡。微囊泡又称为循环的微囊泡或微颗粒，并且是从几乎所有细胞类型中脱落的近似直径范围为从100nm至1000nm的质膜的片段。所述外泌体还可以包括较小的细胞内生成的细胞外囊泡，其通过多囊泡体(MVB)的界膜的向内芽殖形成，在与质膜融合时所述MVB导致它们的分泌以及在体液(例如血液、尿液)里沉积。外泌体可以含有microRNA(miR)、mRNA以及蛋白质的复杂混合物，其反映了生产细胞的转录和翻译的状态。
术语“AAV”通常是指腺伴随病毒的缩写，并且可以用来指代病毒本身或其衍生物。所述AAV可以包括AAV 1型(AAV-1或AAV1)、AAV 2型(AAV-2或AAV2)、AAV 3型(AAV-3或AAV3)、AAV 4型(AAV-4或AAV4)、AAV 5型(AAV-5或AAV5)、AAV 6型(AAV-6或AAV6)、AAV 7型(AAV-7或AAV7)、AAV 8型(AAV-8或AAV8)、AAV 9型(AAV-9或AAV9)、鸟AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV、以及羊AAV。
术语“试剂盒”通常是指在一个或多个合适的容器中包含一种或多种活性成分的包装。
术语“ABCA4突变引起的疾病”通常是指与ABCA4基因突变相关的疾病，例如眼科罕见病。
术语“遗传性视网膜疾病”通常是指inherited retinal dystrophies or inherited retinal diseases，IRDs，属于由于遗传缺陷而导致患者视网膜功能丧失和/或进行性退化的眼科罕见病。所述遗传性视网膜疾病常在幼儿或青少年时期即出现可逆性或不可逆性视力损害。IRDs是难治性眼科疾病，多表现为蛋白质水平代谢异常，可通过调节遗传信息传递过程中的基因、转录和翻译而实现治疗的目的。所述遗传性视网膜疾病可以包括常染色体隐性遗传的视网膜色素变性(RP，MERTK突变)、无脉络膜症(CHM突变)、青少年黄斑变性(ABCA4突变)、Usher综合征1B亚型(Myo7a突变)、X性连锁先天性视网膜劈裂症(RS1突变)、线粒体相关Leber遗传性视神经病变(ND4突变)、全色盲(CNGA3突变和CNGB3突变)、性连锁遗传RP(RPGR突变)等。
术语“遗传性黄斑变性疾病”通常是指一种较少见的家族性遗传性疾病。所述遗传性黄斑 变性疾病可以包括Stargardt病。Stargardt病是一种原发于视网膜色素上皮层的常染色体隐性遗传病，散发性者较为少见，较多发生于近亲婚配的子女中。患者黄斑萎缩性损害合并视网膜黄色斑点沉着。目前尚无批准的治疗方案，Stargardt病每年可以在世界范围内造成成千上万人视力丧失。所述遗传性黄斑变性疾病还可以包括Best病，病变主要累及双眼黄斑区，一般不向周围网膜扩散。所述遗传性黄斑变性疾病可以在任何年龄发生。
术语“年龄相关的黄斑变性”通常是指age-related macular degeneration，AMD，是50岁以上人群致盲的重要要原因之一。AMD可以表现为中心视力进行性、不可逆性丧失，主要累及黄斑、视网膜色素上皮、脉络膜等部位。临床上早期AMD以视网膜下玻璃膜疣沉积为主要特征，而晚期AMD分为干性及湿性2种类型。干性AMD又称为萎缩型AMD，其主要病理改变为RPE层地图状萎缩和脱离，患眼视力缓慢、渐进性丧失；湿性AMD又称为新生血管型或渗出型AMD，其主要病理改变为脉络膜新生血管(CNV)以及继发的液体积聚及出血。AMD的发病机制尚不明确，可能涉及RPE损伤、线粒体功能障碍、氧化应激、炎症、补体途径激活等。目前普遍认为AMD的发生是遗传和环境因素共同作用的结果。
术语“色素性视网膜炎”通常是指Retinitis Pigmentosa，RP，是一组以视网膜光感受器进行性退化为特征的遗传性眼病。所述色素性视网膜炎可以包括进行性视网膜(眼球后部内表面的一层透明感光膜)退行性疾病，最终可导致中重度的视力缺失。视网膜色素病变多具有遗传性。所述色素性视网膜炎的临床特点表现可以包括早期夜盲、视野向心性缩小，最终呈管状视野，双目失明或频临失明，在失明眼病中占有相当大的比例。所述色素性视网膜炎可以属于遗传性视杆、视锥细胞营养不良性疾病。
术语“锥杆营养不良”通常是指cone-rod dystrophy，CRD，属于一种遗传性黄斑变性疾病。所述锥杆营养不良可以包括一组由进行性视力减退、畏光、眼球震颤等临床及基因遗传综合征，主要累及视锥细胞，严重时可合并累及视杆细胞，遗传方式主要是常染色体显性、隐性、X染色体连锁遗传。
术语“异源”通常是指在用于提及多核苷酸、基因、或多肽时，指在宿主生物体中没有正常地发现的多核苷酸、基因、或多肽。所述异源也可以包括原生的编码区、或它们的一部分，即以不同于对应的原生基因的形式重新导入源生物体，例如，可以不在所述生物体基因组中的原生位置上。
术语“视黄基磷脂酰乙醇胺(NRPE)”通常是指ABCA4的生理脂质底物，其夹在管腔小叶中的两个TMD之间，并通过来自细胞外结构域1的延伸环进一步稳定。
发明详述
一方面，本申请提供了一种表达盒组合，其包括第一表达盒和第二表达盒，其中所述第一表达盒能够表达ABCA4蛋白的N端截短体，所述第二表达盒能够表达ABCA4蛋白的C端截短体，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体能够在不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能。
在本申请中，所述完整的ABCA4蛋白的生物学功能可以包括细胞定位功能；ATP水解酶活性；表达于感光细胞，和/或，减少视网膜A2E沉积。
在本申请中，所述ABCA4蛋白的N端截短体可以包含人完整的ABCA4蛋白自N端起第1-1325位氨基酸、第1-1160位氨基酸、第1-1220位氨基酸、第1-1280位氨基酸、或第1-1347位氨基酸所示的氨基酸序列。例如，所述ABCA4蛋白的N端截短体的氨基酸序列可以如人完整的ABCA4蛋白自N端起第1-1325位氨基酸所示。在本申请中，所述ABCA4蛋白的C端截短体可以包含人完整的ABCA4蛋白自N端起第1326-2273位氨基酸、第1348-2273位氨基酸、第1369-2273位氨基酸或第1348-2170位氨基酸所示的氨基酸序列。例如，所述ABCA4蛋白的N端截短体的氨基酸序列可以如人完整的ABCA4蛋白自N端起第1326-2273位氨基酸所示。在本申请中，所述ABCA4蛋白的N端截短体和/或ABCA4蛋白的C端截短体可以分别对应于Nature Communications volume 12,Article number:3853(2021)中图1蓝色和绿色的结构。
在本申请中，所述完整的ABCA4蛋白可以由所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体组成。其中，所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体由于结构相近，可以在空间构象上相互作用(例如形成N-C异二聚体)，存在相互作用而形成完整的ABCA4蛋白的可能性。同时，所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体也可以单独的、分离地存在，而不形成所述完整的ABCA4蛋白。
在本申请中，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体可以基本上(例如，可以以至少约50％的摩尔比例、至少约60％的摩尔比例、至少约70％的摩尔比例、至少约80％的摩尔比例、至少约90％的摩尔比例、至少约95％的摩尔比例或更高比例)不相互作用形成完整的ABCA4蛋白。
在本申请中，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体的蛋白摩尔比例约为3:1～1:3。例如，所述ABCA4 蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体的蛋白摩尔比例可以约为3:1～1:3、约为3:1～1:2、约为3:1～1:1、约为3:1～2:1、约为2:1～1:3、约为2:1～1:2、约为2:1～1:1、约为1:1～1:3、约为1:2～1:1、约为1:1。
在本申请中，所述第一表达盒和/或所述第二表达盒不表达连接序列，所述连接序列能够使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
在本申请中，所述连接序列通过选自下组的方法：同源重组、mRNA剪接和蛋白剪接，使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体可以通过所述连接序列而相互作用(例如同源重组、mRNA剪接和/或蛋白剪接)形成完整的ABCA4蛋白。在本申请中，当所述第一表达盒和/或所述第二表达盒不表达所述连接序列，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体就可以基本上(例如，可以以至少约50％的摩尔比例、至少约60％的摩尔比例、至少约70％的摩尔比例、至少约80％的摩尔比例、至少约90％的摩尔比例、至少约95％的摩尔比例或更高比例)不相互作用形成完整的ABCA4蛋白。
在本申请中，所述连接序列可以包括同源臂序列。
在本申请中，所述连接序列可以包括能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述连接序列可以包括能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。例如，所述同源臂序列可以包括上游同源臂序列和/或下游同源臂序列。其中，所述上游同源臂序列可以包含能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或所述下游同源臂序列可以包含能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。
在本申请中，所述第一表达盒不可以包含能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述第二表达盒不可以包含能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。所述第一表达盒和/或所述第二表达盒无法使其表达的所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体通过同源重组而相互作用形成完整的ABCA4蛋白。
在本申请中，所述连接序列可以包括mRNA内含子的剪切信号。
例如，所述mRNA内含子的剪切信号可以包括内含子上游剪接受体位点(SA)和/或下游 剪接供体位点(SD)。例如，所述内含子上游剪接受体位点(SA)可以位于编码所述ABCA4蛋白的N端截短体至少部分的序列的下游；和/或下游剪接供体位点(SD)可以位于能够与编码所述ABCA4蛋白的C端截短体至少部分的序列的上游。
在本申请中，所述第一表达盒不可以包含内含子上游剪接受体位点(SA)；和/或，所述第二表达盒不可以包含内含子下游剪接供体位点(SD)。所述第一表达盒和/或所述第二表达盒无法使其表达的所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体通过mRNA剪接而相互作用形成完整的ABCA4蛋白。
在本申请中，所述连接序列可以包括内含肽。
在本申请中，所述连接序列可以包括编码intein蛋白的序列。例如，所述编码intein蛋白的序列可以包括编码intein蛋白的N端部分的序列；和/或，编码intein蛋白的C端部分的序列。例如，所述编码intein蛋白的N端部分的序列可以位于编码所述ABCA4蛋白的N端截短体至少部分的序列的下游；和/或所述编码intein蛋白的C端部分的序列可以位于能够与编码所述ABCA4蛋白的C端截短体至少部分的序列的上游。
在本申请中，所述第一表达盒不表达编码intein蛋白的N端部分的序列；和/或，所述第二表达盒不表达编码intein蛋白的C端部分的序列。所述第一表达盒和/或所述第二表达盒无法使其表达的所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体通过蛋白剪接而相互作用形成完整的ABCA4蛋白。
在本申请中，所述ABCA4蛋白的N端截短体可以包含所述ABCA4蛋白N端的结构域：跨膜区TMD1～TMD6、胞外域ECD1、IH1、IH2、EH1、EH2、NBD1和/或R1。
在本申请中，所述ABCA4蛋白的N端截短体自N端依次可以包括：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和/或R1。例如，所述ABCA4蛋白的N端截短体自N端依次可以包括：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6和/或NBD1。
在本申请中，所述ABCA4蛋白的N端截短体可以包含SEQ ID NO:2或4所示的氨基酸序列。
在本申请中，所述第一表达盒可以包含编码所述ABCA4蛋白的N端截短体的核苷酸序列。
在本申请中，编码所述ABCA4蛋白的N端截短体的核苷酸序列可以如SEQ ID NO:25或27所示。
在本申请中，所述第一表达盒可以包含启动子。在本申请中，所述启动子可以包括组成 型启动子和/或诱导型启动子。例如，所述启动子可以包括CMV启动子。
在本申请中，所述ABCA4蛋白的C端截短体可以包含所述ABCA4蛋白C端的结构域：跨膜区TMD7～TMD12、胞外域ECD1、IH3、IH4、EH3、EH4、NBD2和/或R2。
在本申请中，所述ABCA4蛋白的C端截短体自N端依次可以包括：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2和/或R2。例如，所述ABCA4蛋白的C端截短体自N端依次可以包括：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12和/或NBD2。
在本申请中，所述ABCA4蛋白的C端截短体可以包含SEQ ID NO:3或5所示的氨基酸序列。
在本申请中，所述第二表达盒可以包含编码所述ABCA4蛋白的C端截短体的核苷酸序列。
在本申请中，编码所述ABCA4蛋白的C端截短体的核苷酸序列可以如SEQ ID NO:26或28所示。
在本申请中，所述第二表达盒可以包含启动子。
在本申请中，所述第一表达盒可以表达第一恒定区，和/或，所述第二表达盒可以表达第二恒定区，其中所述第一恒定区与所述第二恒定区能够相互作用使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体。
在本申请中，所述第一恒定区可以具有第一修饰，和/或，所述第二恒定区可以具有第二修饰，其中所述第一修饰与所述第二修饰能够促进异二聚体的形成。
在本申请中，所述第一恒定区与所述第二恒定区可以源自抗体的恒定区。例如，可以源自抗体的IgG恒定区。例如，可以源自抗体的IgG1恒定区。例如，可以源自抗体的人IgG恒定区。
在本申请中，所述第一修饰可以与所述第二修饰相同。在本申请中，所述第一修饰可以与所述第二修饰不同。例如，所述第一修饰可以与所述第二修饰通过电荷的相吸作用而促进异二聚体的形式。例如，所述第一修饰与所述第二修饰可以包括knob-into-hole修饰。例如，所述第一修饰可以具有knob修饰且所述第二修饰可以具有hole修饰；和/或，所述第一修饰可以具有hole修饰且所述第二修饰可以具有knob修饰。
在本申请中，所述第一恒定区可以包含SEQ ID NO:6所示的氨基酸序列。在本申请中，所述第二恒定区可以包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述第一恒定区可以包含SEQ ID NO:7所示的氨基酸序列。在本申请中，所述第二恒定区可以包含SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述ABCA4蛋白的N端截短体中的R1可以与所述ABCA4蛋白的C端截短体中的R2相互作用而有助于所述ABCA4蛋白的N端截短体和所述ABCA4蛋白的C端截短体形成ABCA4蛋白的N-C异二聚体。在某些情况下，所述ABCA4蛋白的N端截短体中的R1可以被所述第一恒定区所替代，和/或，所述ABCA4蛋白的C端截短体中的R2可以被所述第二恒定区所替代，从而使所述第一恒定区与所述第二恒定区通过相互作用使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体。在某些情况下，所述ABCA4蛋白的N端截短体中的R1的下游可以进一步连接所述第一恒定区，和/或，所述ABCA4蛋白的C端截短体中的R2的下游可以进一步连接第二恒定区，从而进一步促进所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体。
在本申请中，所述第一表达盒自5’端起可以包含启动子序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列和/或编码所述第一恒定区的核苷酸序列。在本申请中，所述第一表达盒自5’端起可以包含启动子序列、编码所述第一恒定区的核苷酸序列和/或编码所述ABCA4蛋白的N端截短体的核苷酸序列。
在本申请中，所述第一表达盒自5’端起可以由启动子序列和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。在本申请中，所述第一表达盒自5’端起可以由启动子的核苷酸序列、编码所述第一恒定区的核苷酸序列、和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。在本申请中，所述第一表达盒自5’端起可以由启动子的核苷酸序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列和编码所述第一恒定区的核苷酸序列组成。
在本申请中，所述第二表达盒自5’端可以包含启动子序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和/或编码所述第二恒定区的核苷酸序列。在本申请中，所述第二表达盒自5’端可以包含启动子序列、编码所述第二恒定区的核苷酸序列和/或编码所述ABCA4蛋白的C端截短体的核苷酸序列。
在本申请中，所述第二表达盒自5’端起可以由启动子序列和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。在本申请中，所述第二表达盒自5’端起可以由启动子的核苷酸序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和编码所述第二恒定区的核苷酸序列组成。在本申请中，所述第二表达盒自5’端起可以由启动子序列、编码所述第二恒定 区的核苷酸序列、和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。
在本申请中，所述第一表达盒所表达的蛋白自N端依次可以包括：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和/或R1、以及第一恒定区。
在本申请中，所述第一表达盒所表达的蛋白自N端依次可以包括：第一恒定区、IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和R1。
例如，所述第一表达盒所表达的蛋白自N端依次可以为：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1、R1和第一恒定区。例如，所述第一表达盒所表达的蛋白自N端依次可以为：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和第一恒定区。例如，所述第一表达盒所表达的蛋白自N端依次可以为：第一恒定区、IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和R1。例如，所述第一表达盒所表达的蛋白自N端依次可以为：第一恒定区、IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6和NBD1。
在本申请中，所述第二表达盒所表达的蛋白自N端依次可以包括：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2和/或R2、以及第二恒定区。
在本申请中，所述第二表达盒所表达的蛋白自N端依次可以包括：第二恒定区、IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、以及NBD2和/或R2。
例如，所述第二表达盒所表达的蛋白自N端依次可以为：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2、R2和第二恒定区。例如，所述第二表达盒所表达的蛋白自N端依次可以为：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2、第二恒定区。例如，所述第二表达盒所表达的蛋白自N端依次可以为：第二恒定区、IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2和R2。例如，所述第二表达盒所表达的蛋白自N端依次可以为：第二恒定区、IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12和NBD2。
在本申请中，所述第一表达盒可以包含SEQ ID NO:2，4，8-10，15-18，34中任一项所示的核苷酸序列。
在本申请中，所述第二表达盒可以包含SEQ ID NO:3，5，11-14，19-22中任一项所示的核苷酸序列。
在本申请中，所述第一表达盒可以与所述第二表达盒单独存在。
在本申请中，所述第一表达盒和/或所述第二表达盒可以以选自下组的形式存在：分离的核酸分子、脂质体和/或外泌体。
在本申请中，所述第一表达盒和/或所述第二表达盒可以以质粒的形式存在。
在本申请中，所述第一表达盒和/或所述第二表达盒可以为病毒载体。
在本申请中，所述第一表达盒和/或所述第二表达盒可以为AAV。
在本申请中，在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的蛋白的摩尔比可以为约1:3～3:1。例如，可以约为3:1～1:3、约为3:1～1:2、约为3:1～1:1、约为3:1～2:1、约为2:1～1:3、约为2:1～1:2、约为2:1～1:1、约为1:1～1:3、约为1:2～1:1、约为1:1。
在本申请中，在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的蛋白的摩尔比可以为约1:1。
另一方面，本申请提供一种试剂盒，其包含本申请所述的表达盒组合。
在本申请中，所述的试剂盒可以包含用于施用所述表达盒组合的试剂和/或仪器。例如，所述试剂盒可以包含用于转染包含所述第一表达盒和/或所述第二表达盒的AAV的试剂和/或仪器。例如，所述试剂盒可以包含用于注射包含所述第一表达盒和/或所述第二表达盒的AAV的试剂和/或仪器。在本申请中，所述注射可以包括局部注射。
另一方面，本申请提供一种本申请所述的表达盒组合，和/或本申请所述的试剂盒，在制备治疗疾病的药物中的用途，所述疾病包括ABCA4突变引起的疾病。
在本申请中，所述疾病可以包括遗传性视网膜疾病。
在本申请中，所述疾病可以包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
通过本申请所述的方法，可以使有需要的受试者体内表达异源的所述ABCA4蛋白的N端截短体和异源的所述ABCA4蛋白的C端截短体，由于所述ABCA4蛋白的N端截短体和异源的所述ABCA4蛋白的C端截短体能够发挥完整的ABCA4蛋白的生物学功能，因此能够用于缓解/治疗/改善ABCA4突变引起的疾病。
另一方面，本申请提供一种表达异源ABCA4基因的方法，其可以包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
另一方面，本申请提供一种缓解视黄基磷脂酰乙醇胺(NRPE)导致的细胞死亡的方法， 其可以包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
另一方面，本申请提供一种治疗ABCA4突变引起的疾病的方法，其可以包括向有需要的受试者施用本申请所述的表达盒组合，和/或本申请所述的试剂盒。
在本申请中，所述施用可以包括注射。
在本申请中，所述疾病可以包括遗传性视网膜疾病。
在本申请中，所述疾病可以包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的表达盒、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1密码子优化的ABCA4的蛋白表达强度提高
在HEK293细胞(北京大学医学部提供)中分别转染融合FLAG标签的野生型的ABCA4质粒(ABCA4-WT)，或者融合FLAG标签的密码子优化的ABCA4质粒(ABCA4-CO)。48小时后裂解细胞，分别使用ABCA4的内源抗体(NBP1-30032,NovusBio)或flag标签抗体(F1804，Sigma)检测细胞内的ABCA4蛋白表达强度。其中，野生型ABCA4的核苷酸序列如SEQ ID NO.23所示；密码子优化的ABCA4的核苷酸序列如SEQ ID NO.24所示。
Western结果显示(参见图2)，密码子优化的ABCA4(ABCA4-CO)表达强度显著高于野生型的ABCA4(ABCA4-WT)。
实施例2 ABCA4的N端截短体与C端截短体存在同源二聚化
利用密码子优化的ABCA4全长CDS序列(aa 1-2273)，并依据ABCA4蛋白结构功能域划分(Scortecci,J.F.,et al.(2021).Nat Commun 12(1):5902.；Xie,T.,et al.(2021).Nat Commun 12(1):3853.)，分别构建了2种ABCA4的截短体，分别为N端截短体(aa 1-1347)以及C端截短体(aa 1348-2273)。其中，ABCA4的全长氨基酸序列如SEQ ID NO.1所示；ABCA4的N端截短体的氨基酸序列如SEQ ID NO.2所示；ABCA4的C端截短体的氨基酸序列如SEQ ID NO.3所示。
为方便抗体检测，在N端截短体的C端连接血凝素(HA)标签，在C端截短体的C端连接Flag标签。
将全长ABCA4、N端截短体或C端截短体的表达质粒转染HEK293细胞，而后用相应 抗体标记对应标签。
Western结果显示(参见图3)，ABCA4的N端截短体与C端截短体存在同源二聚化，而共转染N端截短体与C端截短体，可以减少同源二聚化的比例。
实施例3 ABCA4的N端截短体与C端截短体存在相互作用
在HEK293细胞中分别转染ABCA4的N端截短体(aa 1-1347)，C端截短体(aa 1348-2273)或者将N+C共转染(即共转染N端截短体(aa 1-1347)以及C端截短体(aa 1348-2273))。转染质粒的骨架载体为购自Addgene。
48小时后裂解细胞，使用flag抗体(F1804，Sigma)偶连磁珠富集细胞中含有flag标签的C端截短体，然后使用HA抗体(AE036，ABclonal)western blot检测是否存在与C端截短体相互作用的N端截短体。
结果如图4所示，分别转染N端截短体，C端截短体或N+C共转染的HEK293细胞中分别有相应的蛋白表达(Input)，flag抗体偶连磁珠可富集到细胞裂解中含有flag标签的C端截短体，同时与C端截短体相互作用的N端截短体也被富集下来。因此，ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)存在相互作用。
实施例4不同长度或包含不同结构域的ABCA4的N端截短体与ABCA4的C端截短体之间存在相互作用
利用密码子优化的ABCA4全长CDS序列，设计不同长度或包含不同结构域的N端截短体和C端截短体，其中N端截短体包括：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*，2253-2273)(SEQ ID NO:38)；C端截短体包括：C(aa 1348-2273)(SEQ ID NO:3)、C*(aa 1-20，1369-2273)(SEQ ID NO:39)。其中在N端截短体的C端连接HA标签，C端截短体的C端连接Flag标签。
将以上质粒N端截短体和C端截短体两两组合后共转染HEK293细胞，48小时后裂解细胞，使用flag抗体偶连磁珠富集细胞中含有flag标签的C端截短体，然后使用HA抗体western blot检测是否存在与C端截短体相互作用的N端截短体。
结果如图5所示，不同长度或包含不同结构域的ABCA4的N端截短体与C端截短体之间存在相互作用，N1347(aa 1-1347)、N1160(aa 1-1160)、N1220(aa 1-1220)、N1280(aa 1-1280)、N1280*(aa 1-1280*，2253-2273)均可检测到与C(aa 1348-2273)之间存在相互作用；N1347(aa  1-1347)、N1220(aa 1-1220)、N1280*(aa 1-1280*，2253-2273)与可检测到与C*(aa 1-20，1369-2273)之间存在相互作用。其中N1347(aa 1-1347)与C或C*之间相互作用最强。
实施例5 ABCA4的N端截短体与C端截短体存在细胞共定位，且定位位置与全长的野生型ABCA4一致
分别将全长ABCA4(aa 1-2273)，ABCA4的N端截短体(aa 1-1347)以及ABCA4的C端截短体(aa 1348-2273)转染HEK293细胞(其中在N端截短体的C端连接HA标签，C端截短体的C端连接Flag标签)，而后用相应抗体(flag抗体(F1804，Sigma)，HA抗体(K200003M，Solarbio))标记对应标签，可以观察到ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)存在细胞共定位(参见图6)，且定位位置与全长的野生型ABCA4一致，均存在于胞内小泡中(参见图7)。
实施例6 ABCA4的N端截短体与C端截短体复合物具有ATP水解酶活性
在HEK293细胞中分别转染全长ABCA4(aa 1-2273)，ABCA4的N端截短体(aa 1-1347)以及ABCA4的C端截短体(aa 1348-2273)或N+C共转染，48小时后裂解细胞，使用flag抗体偶连磁珠富集细胞中含有flag标签的全长ABCA4或C端截短体，或N+C复合物(即N端截短体以及C端截短体的复合物)，然后使用ATPase检测试剂盒(Promega)对磁珠上富集的蛋白的ATP水解酶活性进行检测。
结果如图8所示，空白磁珠与单独的C端截短体(aa 1348-2273)均不具有ATP水解酶活性，而N+C复合物与全长的ABCA4类似，具有ATP水解酶活性。
实施例7 ABCA4的N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb之间存在相互作用
由于ABCA4的N端截短体与C端截短体之间的高度相似性，为二者之间形成异二聚体提供了结构基础，但也有形成同源二聚体的可能性。
为了增加异二聚体比例，减少同源二聚体比例，利用“knob-into-hole”原理，采用Fca和Fcb增加异二聚体比例，减少同源二聚体比例。
其中，Fca的氨基酸序列如SEQ ID NO.6所示；Fcb的氨基酸序列如SEQ ID NO.7所示。
Fc与ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)相连采用两种策略：
一是Fca与Fcb在氨基酸二级结构上相邻，即分别在ABCA4的N端截短体(aa 1-1347)的C端连接Fca(与R1相邻)构建为N(aa 1-1347)-Fca(其中，N(aa 1-1347)-Fca的氨基酸序列如SEQ ID NO.8所示)，ABCA4的C端截短体(aa 1348-2273)的N端连接Fcb(与IH3相邻)构建为Fcb-C(aa 1348-2273)(其中，Fcb-C(aa 1348-2273)的氨基酸序列如SEQ ID NO.14所示)。
二是Fca与Fcb在蛋白三级结构上相邻，即分别在ABCA4的N端截短体(aa 1-1347)的C端连接Fca(与R1相邻)构建为N(aa 1-1347)-Fca(其中，N(aa 1-1347)-Fca的氨基酸序列如SEQ ID NO.8所示)，ABCA4的C端截短体的C端(aa 1348-2273)连接Fcb(与R2相邻)构建为C(aa 1348-2273)-Fcb(其中，C(aa 1348-2273)-Fcb的氨基酸序列如SEQ ID NO.13所示)。
在HEK293细胞中分别共转染N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)，或者共转染N(aa1-1347)-Fca与C(aa 1348-2273)-Fcb，48小时后裂解细胞，使用IgG偶连磁珠富集细胞中含有Fca标签的N(aa 1-1347)-Fca，然后使用抗人抗体(AS002，ABclonal)western blot检测是否存在与N(aa 1-1347)-Fca相互作用的Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb。
结果如图9所示，分别单转或共转染N(aa 1-1347)-Fca，Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb的HEK293细胞中分别有相应的蛋白表达(Input)，IgG偶连磁珠可富集到细胞裂解中的N(aa 1-1347)-Fca，同时与N(aa 1-1347)-Fca相互作用的Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb也被富集下来。图9中，N-Fc表示单独转染N(aa 1-1347)-Fca，C-Fc表示单独转染C(aa 1348-2273)-Fcb，Fc-C表示单独转染Fcb-C(aa 1348-2273)，N(aa 1-1347)-Fc+C-Fc表示共转染N(aa 1-1347)-Fca和C(aa 1348-2273)-Fcb；N(aa 1-1347)-Fc+Fc-C表示共转染N(aa 1-1347)-Fca和Fcb-C(aa 1348-2273)；NTD-Fc表示富集获得的N(aa 1-1347)-Fca；CTD-Fc/Fc-CTD表示与N(aa 1-1347)-Fca相互作用的Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb。
因此，ABCA4的N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)或C(aa 1348-2273)-Fcb之间均存在相互作用。
实施例8不同长度或包含不同结构域的ABCA4的N端截短体-Fca与Fcb-C端截短体或C端截短体-Fcb之间存在相互作用
构建不同长度或包含不同结构域的ABCA4的N端截短体-Fca与Fcb-C端截短体或C端截短体-Fcb，其中N端截短体包括：N(aa 1-1347)-Fca，N(aa 1-1160)-Fca；C端截短体包括：C(aa 1348-2273)-Fcb，C(aa 1348-2170)-Fcb，Fcb-C(aa 1348-2273)。
在HEK293细胞中分别共转染上述ABCA4的N端截短体和C端截短体，48小时后裂 解细胞，使用IgG偶连磁珠富集细胞中含有Fca标签的N端截短体，然后使用抗人抗体(AS002，ABclonal)western blot检测是否存在与N端截短体相互作用的C端截短体。
结果如图10所示，ABCA4的N(aa 1-1347)-Fca或N(aa 1-1160)-Fca与C(aa 1348-2273)-Fcb、C(aa 1348-2170)-Fcb或Fcb-C(aa 1348-2273)之间均存在较强的相互作用。
实施例9 ABCA4的N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)，或N(aa 1-1347)-Fca与C(aa 1348-2273)-Fcb复合物具有ATP水解酶活性
在HEK293细胞中分别共转染N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)，或者共转染N(aa1-1347)-Fca与C(aa 1348-2273)-Fcb，48小时后裂解细胞，使用IgG偶连磁珠富集细胞中含有Fca标签的N(aa 1-1347)-Fca(IgG偶连磁珠不能结合Fcb，参见图11)，然后使用ATPase检测试剂盒(Promega)对beads上富集的蛋白的ATP水解酶活性进行检测。
结果如图11所示，结果显示空白磁珠与单独的N(aa 1-1347)-Fca均不具有ATP水解酶活性，而N+C复合物(即N端截短体以及C端截短体的复合物，其可以由共转染N(aa 1-1347)-Fca与Fcb-C(aa 1348-2273)，或者共转染N(aa 1-1347)-Fca与C(aa 1348-2273)-Fcb所获得)与经Flag偶连磁珠富集后的全长ABCA4类似，具有ATP水解酶活性。
实施例10 ABCA4的N端截短体与C端截短体复合物可缓解NRPE导致的细胞死亡
在661W细胞(北京大学医学部提供)中分别转染阴性对照质粒RHO(金唯智合成)，全长质粒ABCA4，或共转ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)。48小时后，细胞加入30μM～50μM的全反式视黄醛(ATR)或者视黄基磷脂酰乙醇胺(NRPE)(aladdin/阿拉丁)刺激，24小时后使用CellTiter-Glo(Promega)检测细胞活性。
结果如图12所示，由于高浓度的ATR或NRPE具有细胞毒性，使用30μM～50μM的ATR或NRPE刺激24小时，在阴性对照转染RHO细胞组，可见细胞活性显著下降(50μM时细胞活性为约3％)。
转染全长ABCA4或共转ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)，30μM～50μM的ATR刺激24小时，细胞活性下降情况与RHO组类似(50μM时细胞活性为约3％)；而30μM～50μM的NRPE刺激24小时，两组的细胞活性都得到了一定程度的恢复(50μM时细胞活性为约10％)。
说明表达全长ABCA4或共表达ABCA4的N端截短体(aa 1-1347)与C端截短体(aa 1348-2273)，均可以促进其底物NRPE的转运或转化，而减少NRPE的细胞毒性。
实施例11共感染AAV-ABCA4的N端截短体与AAV-ABCA4的C端截短体复合物可缓解NRPE导致的细胞死亡
在661W细胞中感染不同MOI(MOI＝1E5或5E5)的对照病毒AAV-EV，或共感染AAV-ABCA4的N端截短体(aa 1-1347)与AAV-ABCA4的C端截短体(aa 1348-2273)。48小时后，细胞加入0μM～75μM的全反式视黄醛(ATR)或者视黄基磷脂酰乙醇胺(NRPE)刺激，24小时后使用酶标仪测量细胞中转化产生的全反式视黄醇(ATRol)，并通过CellTiter-Glo(Promega)检测细胞活性。
结果如图13所示，使用高浓度的ATR刺激细胞后，细胞中ATRol转化能力下降，且细胞活性降低，感染对照病毒AAV-EV，或共感染AAV-ABCA4的N端截短体(aa 1-1347)与AAV-ABCA4的C端截短体(aa 1348-2273)无差异。使用高浓度的NRPE刺激细胞后，感染对照病毒AAV-EV的细胞中ATRol转化能力下降，且细胞活性降低；而共感染高感染复数(MOI＝5E5)AAV-ABCA4的N端截短体(aa 1-1347)与AAV-ABCA4的C端截短体(aa 1348-2273)的细胞，ATRol转化能力提高，同时细胞活性相对较好。
说明在661W细胞中共感染AAV-ABCA4的N端截短体(aa 1-1347)与AAV-ABCA4的C端截短体(aa 1348-2273)，可以促进其底物NRPE的转运并转化为ATRol，而减少NRPE的细胞毒性，提高细胞活性。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (59)

  1. 表达盒组合，其包括第一表达盒和第二表达盒，其中所述第一表达盒能够表达ABCA4蛋白的N端截短体，所述第二表达盒能够表达ABCA4蛋白的C端截短体，所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体能够在不形成完整的ABCA4蛋白的情况下具备完整的ABCA4蛋白的生物学功能。
  2. 根据权利要求1所述的表达盒组合，其中所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体基本上不相互作用形成完整的ABCA4蛋白。
  3. 根据权利要求1-2中任一项所述的表达盒组合，其中所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体的蛋白摩尔比例约为3:1～1:3。
  4. 根据权利要求1-3中任一项所述的表达盒组合，其中所述第一表达盒和/或所述第二表达盒不表达连接序列，所述连接序列能够使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
  5. 根据权利要求4所述的表达盒组合，其中所述连接序列通过选自下组的方法：同源重组、mRNA剪接和蛋白剪接，使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体形成完整的ABCA4蛋白。
  6. 根据权利要求4-5中任一项所述的表达盒组合，其中所述连接序列包括同源臂序列。
  7. 根据权利要求4-6中任一项所述的表达盒组合，其中所述连接序列包括能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述连接序列包括能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。
  8. 根据权利要求1-7中任一项所述的表达盒组合，其中所述第一表达盒不包含能够与编码所述ABCA4蛋白的N端截短体至少部分的序列互补的序列；和/或，所述第二表达盒不包含能够与编码所述ABCA4蛋白的C端截短体至少部分的序列互补的序列。
  9. 根据权利要求4-8中任一项所述的表达盒组合，其中所述连接序列包括mRNA内含子的剪切信号。
  10. 根据权利要求1-9中任一项所述的表达盒组合，其中所述第一表达盒不包含内含子上游 剪接受体位点(SA)；和/或，所述第二表达盒不包含内含子下游剪接供体位点(SD)。
  11. 根据权利要求4-10中任一项所述的表达盒组合，其中所述连接序列包括内含肽。
  12. 根据权利要求4-11中任一项所述的表达盒组合，其中所述连接序列包括编码intein蛋白的序列。
  13. 根据权利要求1-12中任一项所述的表达盒组合，其中所述第一表达盒不表达编码intein蛋白的N端部分的序列；和/或，所述第二表达盒不表达编码intein蛋白的C端部分的序列。
  14. 根据权利要求1-13中任一项所述的表达盒组合，其中所述ABCA4蛋白的N端截短体包含所述ABCA4蛋白N端的结构域：跨膜区TMD1～TMD6、胞外域ECD1、IH1、IH2、EH1、EH2、NBD1和/或R1，优选包含跨膜区TMD1～TMD6、胞外域ECD1、IH2、EH1、EH2和NBD1以及任选地IH1和/或R1。
  15. 根据权利要求1-14中任一项所述的表达盒组合，其中所述ABCA4蛋白的N端截短体自N端依次包括：IH1、TMD1、ECD1、TMD2、IH2、TMD3、TMD4、TMD5、EH1、EH2、TMD6、NBD1和/或R1。
  16. 根据权利要求1-15中任一项所述的表达盒组合，其中所述ABCA4蛋白的N端截短体包含SEQ ID NO:2或4所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的表达盒组合，其中所述第一表达盒包含编码所述ABCA4蛋白的N端截短体的核苷酸序列。
  18. 根据权利要求17所述的表达盒组合，其中编码所述ABCA4蛋白的N端截短体的核苷酸序列如SEQ ID NO:25或27所示。
  19. 根据权利要求1-18中任一项所述的表达盒组合，其中所述第一表达盒包含启动子。
  20. 根据权利要求1-19中任一项所述的表达盒组合，其中所述ABCA4蛋白的C端截短体包含所述ABCA4蛋白C端的结构域：跨膜区TMD7～TMD12、胞外域ECD2、IH3、IH4、EH3、EH4、NBD2和/或R2，优选包含跨膜区TMD7～TMD12、胞外域ECD2、IH4、EH3、EH4和NBD2以及任选地IH3和/或R2。
  21. 根据权利要求1-20中任一项所述的表达盒组合，其中所述ABCA4蛋白的C端截短体自N端依次包括：IH3、TMD7、ECD2、TMD8、IH4、TMD9、TMD10、TMD11、EH3、EH4、TMD12、NBD2和/或R2。
  22. 根据权利要求1-21中任一项所述的表达盒组合，其中所述ABCA4蛋白的C端截短体包含SEQ ID NO:3或5所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的表达盒组合，其中所述第二表达盒包含编码所述ABCA4蛋白的C端截短体的核苷酸序列。
  24. 根据权利要求23所述的表达盒组合，其中编码所述ABCA4蛋白的C端截短体的核苷酸序列如SEQ ID NO:26或28所示。
  25. 根据权利要求1-24中任一项所述的表达盒组合，其中所述第二表达盒包含启动子。
  26. 根据权利要求1-25中任一项所述的表达盒组合，其中所述第一表达盒表达第一恒定区，和/或，所述第二表达盒表达第二恒定区，其中所述第一恒定区与所述第二恒定区能够相互作用使所述第一表达盒所表达的所述ABCA4蛋白的N端截短体与所述第二表达盒所表达的所述ABCA4蛋白的C端截短体相互作用形成ABCA4蛋白的N-C异二聚体。
  27. 根据权利要求26所述的表达盒组合，其中所述第一恒定区具有第一修饰，和/或，所述第二恒定区具有第二修饰，其中所述第一修饰与所述第二修饰能够促进异二聚体的形成。
  28. 根据权利要求26-27中任一项所述的表达盒组合，其中所述第一恒定区与所述第二恒定区源自抗体的恒定区。
  29. 根据权利要求27-28中任一项所述的表达盒组合，其中所述第一修饰与所述第二修饰包括knob-into-hole修饰。
  30. 根据权利要求26-29中任一项所述的表达盒组合，其中所述第一恒定区包含SEQ ID NO:6或7所示的氨基酸序列。
  31. 根据权利要求26-30中任一项所述的表达盒组合，其中所述第二恒定区包含SEQ ID NO:6或7所示的氨基酸序列。
  32. 根据权利要求26-31中任一项所述的表达盒组合，其中所述第一表达盒自5’端起包含启动子序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列和/或编码所述第一恒定区的核苷酸序列。
  33. 根据权利要求19-32中任一项所述的表达盒组合，其中所述第一表达盒自5’端起由启动子序列和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。
  34. 根据权利要求26-33中任一项所述的表达盒组合，其中所述第一表达盒自5’端起由启动子的核苷酸序列、编码所述第一恒定区的核苷酸序列、和编码所述ABCA4蛋白的N端截短体的核苷酸序列组成。
  35. 根据权利要求26-34中任一项所述的表达盒组合，其中所述第一表达盒自5’端起由启动子的核苷酸序列、编码所述ABCA4蛋白的N端截短体的核苷酸序列、和编码所述第一恒定区的核苷酸序列组成。
  36. 根据权利要求26-35中任一项所述的表达盒组合，其中所述第二表达盒自5’端包含启动子序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和/或编码所述第二恒定区的核苷酸序列。
  37. 根据权利要求25-36中任一项所述的表达盒组合，其中所述第二表达盒自5’端起由启动子序列和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。
  38. 根据权利要求26-37中任一项所述的表达盒组合，其中所述第二表达盒自5’端起由启动子的核苷酸序列、编码所述ABCA4蛋白的C端截短体的核苷酸序列和编码所述第二恒定区的核苷酸序列组成。
  39. 根据权利要求26-38中任一项所述的表达盒组合，其中所述第二表达盒自5’端起由启动子序列、编码所述第二恒定区的核苷酸序列、和编码所述ABCA4蛋白的C端截短体的核苷酸序列组成。
  40. 根据权利要求1-39中任一项所述的表达盒组合，其中所述第一表达盒包含SEQ ID NO:2，4，8-10，15-18，34中任一项所示的核苷酸序列。
  41. 根据权利要求1-40中任一项所述的表达盒组合，其中所述第二表达盒包含SEQ ID NO:3，5，11-14，19-22中任一项所示的核苷酸序列。
  42. 根据权利要求1-41中任一项所述的表达盒组合，其中所述第一表达盒与所述第二表达盒单独存在。
  43. 根据权利要求1-42中任一项所述的表达盒组合，其中所述第一表达盒和/或所述第二表达盒以选自下组的形式存在：分离的核酸分子、脂质体和/或外泌体。
  44. 根据权利要求1-43中任一项所述的表达盒组合，其中所述第一表达盒和/或所述第二表达盒以质粒的形式存在。
  45. 根据权利要求1-44中任一项所述的表达盒组合，其中所述第一表达盒和/或所述第二表达盒为病毒载体。
  46. 根据权利要求1-45中任一项所述的表达盒组合，其中所述第一表达盒和/或所述第二表达盒为AAV。
  47. 根据权利要求1-46中任一项所述的表达盒组合，其中在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的蛋白的摩尔比为约1:3～3:1。
  48. 根据权利要求1-47中任一项所述的表达盒组合，其中在所述表达盒组合中，所述第一表达盒和所述第二表达盒所表达的蛋白的摩尔比为约1:1。
  49. 试剂盒，其包含权利要求1-48中任一项所述的表达盒组合。
  50. 根据权利要求49所述的试剂盒，其包含用于施用所述表达盒组合的试剂和/或仪器。
  51. 权利要求1-48中任一项所述的表达盒组合，和/或权利要求49-50中任一项所述的试剂盒，在制备治疗疾病的药物中的用途，所述疾病包括ABCA4突变引起的疾病。
  52. 根据权利要求51所述的用途，其中所述疾病包括遗传性视网膜疾病。
  53. 根据权利要求51-52中任一项所述的用途，其中所述疾病包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
  54. 表达异源ABCA4基因的方法，其包括向有需要的受试者施用权利要求1-48中任一项所述的表达盒组合，和/或权利要求49-50中任一项所述的试剂盒。
  55. 缓解视黄基磷脂酰乙醇胺(NRPE)导致的细胞死亡的方法，其包括向有需要的受试者施用权利要求1-48中任一项所述的表达盒组合，和/或权利要求49-50中任一项所述的试剂盒。
  56. 治疗ABCA4突变引起的疾病的方法，其包括向有需要的受试者施用权利要求1-48中任一项所述的表达盒组合，和/或权利要求49-50中任一项所述的试剂盒。
  57. 根据权利要求54-56中任一项所述的方法，其中所述施用包括注射。
  58. 根据权利要求54-57中任一项所述的方法，其中所述疾病包括遗传性视网膜疾病。
  59. 根据权利要求54-58中任一项所述的方法，其中所述疾病包括遗传性黄斑变性疾病、年龄相关的黄斑变性、色素性视网膜炎和/或锥杆营养不良。
PCT/CN2023/076459 2022-02-25 2023-02-16 表达盒组合及其应用 WO2023160454A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210181036 2022-02-25
CN202210181036.9 2022-02-25

Publications (1)

Publication Number Publication Date
WO2023160454A1 true WO2023160454A1 (zh) 2023-08-31

Family

ID=87764680

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/076459 WO2023160454A1 (zh) 2022-02-25 2023-02-16 表达盒组合及其应用

Country Status (1)

Country Link
WO (1) WO2023160454A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101952307A (zh) * 2008-02-21 2011-01-19 电视募捐基金会 通过基因疗法治疗眼疾病的方法
WO2017106370A1 (en) * 2015-12-14 2017-06-22 Cold Spring Harbor Laboratory Compositions and methods for treatment of eye diseases
CN109642242A (zh) * 2016-06-15 2019-04-16 牛津大学创新有限公司 用于表达abc4a的双重重叠腺伴随病毒载体系统
WO2021222654A1 (en) * 2020-04-29 2021-11-04 Saliogen Therapeutics, Inc. Compositions and methods for treatment of inherited macular degeneration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101952307A (zh) * 2008-02-21 2011-01-19 电视募捐基金会 通过基因疗法治疗眼疾病的方法
WO2017106370A1 (en) * 2015-12-14 2017-06-22 Cold Spring Harbor Laboratory Compositions and methods for treatment of eye diseases
CN109642242A (zh) * 2016-06-15 2019-04-16 牛津大学创新有限公司 用于表达abc4a的双重重叠腺伴随病毒载体系统
WO2021222654A1 (en) * 2020-04-29 2021-11-04 Saliogen Therapeutics, Inc. Compositions and methods for treatment of inherited macular degeneration

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAN HANDONG, XING YIQIAO: "ABCA4 gene related retinal degenerative diseases", JOURNAL OF CHINESE PRACTICAL DIAGNOSIS AND THERAPY., vol. 34, no. 3, 10 March 2020 (2020-03-10), pages 315 - 317, XP093085725, DOI: 10.13507/j.issn.1674-3474.2020.03.026 *
LENIS TAMARA L., HU JANE, NG SZE YIN, JIANG ZHICHUN, SARFARE SHANTA, LLOYD MARCIA B., ESPOSITO NICHOLAS J., SAMUEL WILLIAM, JAWORS: "Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 115, no. 47, 20 November 2018 (2018-11-20), pages E11120 - E11127, XP055981234, ISSN: 0027-8424, DOI: 10.1073/pnas.1802519115 *

Similar Documents

Publication Publication Date Title
AU2017202320B2 (en) Promoters, expression cassettes, vectors, kits, and methods for the treatment of achromatopsia and other diseases
JP2022190081A (ja) 修飾型閉端ｄｎａ（ｃｅｄｎａ）
JP6875856B2 (ja) 対象の網膜色素上皮において目的のポリヌクレオチドを発現させるための方法および医薬組成物
WO2001029243A1 (en) Method and vector for producing and transferring trans-spliced peptides
RU2762747C2 (ru) Генная терапия офтальмологических нарушений
EP3393522B1 (en) Improved hybrid dual recombinant aav vector systems for gene therapy
US20220042035A1 (en) Non-viral dna vectors and uses thereof for antibody and fusion protein production
CN112779255A (zh) 用于治疗色盲和其它疾病的启动子、表达盒、载体、药盒和方法
Palfi et al. Efficacy of codelivery of dual AAV2/5 vectors in the murine retina and hippocampus
JP2019523648A (ja) Ａｂｃ４ａを発現させるためのデュアル重複アデノ随伴ウイルスベクターシステム
US20220117996A1 (en) Trans-splicing ribozyme targeting rhodopsin transcript and uses thereof
WO2023160454A1 (zh) 表达盒组合及其应用
WO2022262756A1 (zh) Prpf31变体及其用途
EP4071247A1 (en) Trans-splicing ribozyme that is specific to rhodopsin transcriptome, and use thereof
WO2023133561A1 (en) Vector constructs for delivery of nucleic acids encoding therapeutic anti-igf-1r antibodies and methods of using the same
AU2019250266A1 (en) Melatonin agonist treatment
CN117980489A (zh) Retgc基因疗法
CN112063625A (zh) 编码arl2bp的核酸及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23759079

Country of ref document: EP

Kind code of ref document: A1