WO2023134560A1 - 一种核苷酸及其应用 - Google Patents

一种核苷酸及其应用 Download PDF

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WO2023134560A1
WO2023134560A1 PCT/CN2023/070867 CN2023070867W WO2023134560A1 WO 2023134560 A1 WO2023134560 A1 WO 2023134560A1 CN 2023070867 W CN2023070867 W CN 2023070867W WO 2023134560 A1 WO2023134560 A1 WO 2023134560A1
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nucleic acid
acid molecule
exon
sequence
ush2a
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PCT/CN2023/070867
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English (en)
French (fr)
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梁峻彬
欧家裕
徐辉
林思妙
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广州瑞风生物科技有限公司
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Publication of WO2023134560A1 publication Critical patent/WO2023134560A1/zh

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    • 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
    • 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
    • A61P27/06—Antiglaucoma agents or miotics
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular to a nucleotide and its application.
  • Usher Syndrome is a kind of genetic disease, also known as deafness-retinitis pigmentosa syndrome, which is characterized by different degrees of congenital sensorineural deafness and progressive deafness caused by retinitis pigmentosa (RP). Vision loss.
  • RP retinitis pigmentosa
  • mutations in the USH2A gene are the most common cause of type II Usher syndrome, covering more than 50% of Usher syndrome patients.
  • the mutation of USH2A gene is also one of the important causes of non-syndromic retinitis pigmentosa (NSRP). Mutations in exon 13, exon 50, and intron 40 of the USH2A gene cause Usher syndrome.
  • the present application provides a nucleic acid molecule, the nucleic acid molecule has the ability to specifically bind to the 3' sequence of exon No. 13 of USH2A pre-mRNA or a fragment thereof, and the exon No. 13 of USH2A pre-mRNA
  • the genome location corresponding to the 3' sequence of the gene is Chr1: 216246563-216246753.
  • the application provides a nucleic acid molecule, which has the ability to specifically bind to the sequence shown in SEQ ID NO: 1 or a fragment thereof.
  • the application provides a nucleic acid molecule, which is complementary to 16 or more consecutive nucleotides in the sequence shown in SEQ ID NO:1.
  • the application provides a nucleic acid molecule comprising 16 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 1 or its complementary sequence.
  • the present application provides a gene expression cassette, which comprises or encodes the nucleotide sequence of the nucleic acid molecule described in the present application, and optional expression control elements.
  • the present application provides a vector comprising or encoding the nucleotide sequence of the nucleic acid molecule described in the present application, and/or the nucleotide sequence of the gene expression cassette described in the present application.
  • the present application provides a virus particle, which comprises the nucleic acid molecule described in the present application, the nucleotide sequence of the gene expression cassette described in the present application, and/or the vector described in the present application.
  • the present application provides a cell, which comprises the nucleic acid molecule described in the present application, the nucleotide sequence of the gene expression cassette described in the present application, the vector described in the present application, and/or the nucleic acid molecule described in the present application. the virus particles described above.
  • the application provides a pharmaceutical composition, the pharmaceutical composition comprising the nucleic acid molecule described in the application, the nucleotide sequence of the gene expression cassette described in the application, the carrier described in the application, the The virus particles, and/or the cells described in this application, and optionally a pharmaceutically acceptable carrier.
  • the application provides a kit, the kit comprising the nucleic acid molecule described in the application, the nucleotide sequence of the gene expression cassette described in the application, the vector described in the application, the The virus particles of the application, the cells described in the application, and/or the pharmaceutical composition described in the application.
  • the present application provides a method for preparing the nucleic acid molecule described in the present application, comprising expressing and/or synthesizing the nucleic acid molecule capable of binding to the 3' sequence of exon 13 of USH2A or a fragment thereof, the The genomic mapping of the 3' sequence of exon 13 of USH2A is Chr1: 216246563-216246753.
  • the application provides a method for inhibiting the expression and/or function of Exon 13 of USH2A pre-mRNA, comprising providing the nucleic acid molecule described in the application, the vector described in the application, and the virus described in the application Particles, cells described herein, pharmaceutical compositions described herein and/or kits described herein.
  • the present application provides a method for splicing and skipping exon 13 of USH2A pre-mRNA, comprising providing the nucleic acid molecule described in the present application, the vector described in the present application, the virus particle described in the present application, the The cell described in the application, the pharmaceutical composition described in the application and/or the kit described in the application.
  • the application provides a method for preparing mature USH2A mRNA lacking exon No. 13, comprising providing the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, the The cell, the pharmaceutical composition described in this application and/or the kit described in this application.
  • the application provides a method for reducing the Usherin protein level comprising the No. 13 exon expression product, comprising providing the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, The cells described herein, the pharmaceutical compositions described herein and/or the kits described herein.
  • the application provides a method for preparing Usherin protein that does not contain the expression product of exon No. 13 and/or increasing the amount of Usherin protein that does not contain the expression product of exon No. 13, comprising providing the nucleic acid described in the application Molecules, vectors described herein, viral particles described herein, cells described herein, pharmaceutical compositions described herein, and/or kits described herein.
  • the application provides a method for restoring the function of the mutant Usherin protein, comprising providing the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, the cell described in the application, The pharmaceutical composition described in the application and/or the kit described in the application.
  • the application provides the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, the cell described in the application, the pharmaceutical composition described in the application and/or the The use of the kit in the preparation of medicines for preventing and/or treating diseases caused by USH2A gene mutations.
  • the present application provides a nucleic acid molecule capable of specifically binding to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof.
  • AONs antisense oligonucleotides
  • AONs Antisense oligonucleotides
  • the full length of exon 12 is 196bp, which is not an integer multiple of 3, and the deletion will lead to frameshift mutation and inactivation of USH2A protein.
  • exon 12 of USH2A described in the present application is human USH2A exon 12.
  • exon 13 of USH2A described in the present application is human USH2A exon 13.
  • the oligonucleotides provided by this application can target and interfere with pre-mRNA splicing, and can increase the ratio of single skip reads in exon 13, which can ensure safety while significantly improving efficiency.
  • Figure 1 shows a schematic diagram of the structure and function of the U7-snRNA of the present application.
  • Figure 2 shows that AON at different target sites induces the splicing skipping effect of exon 13 of USH2A pre-mRNA.
  • Figure 3A-3B shows that U7 snRNA with different target sites induces splicing skipping effect of USH2A pre-mRNA exon 13 in reporter cells.
  • Figures 4A-4B show that U7 snRNA combinations at different target sites induce splicing skipping efficiency in exon 13 of USH2A pre-mRNA.
  • Figure 5 shows a schematic diagram of the snRNA vector with hnRNP A1.
  • Figure 6A-6B shows that U7-hnRNP A1-snRNA induces splicing skipping efficiency of exon 13 of USH2A pre-mRNA.
  • Figure 7 shows that the snRNAs of different targets efficiently induce the splicing skipping efficiency of exon 13 of USH2A pre-mRNA.
  • Figure 8A shows the efficiency of chemically synthesized U7 snRNA inducing USH2A pre-mRNA exon 13 splicing skipping in WERI cells.
  • Lane 1 50pmol chemically synthesized and modified U7-snRNA#30-#4;
  • swimming lane 2 50pmol chemically synthesized and modified U7-snRNA#26-#16;
  • swimming lane 3 50pmol AON1;
  • swimming lane 4 50pmol AON2;
  • swimming lane 5 EGFP;
  • Lane 6 GL DNA Marker 2000.
  • Figure 8B shows the histogram of quantitative analysis of RT-PCR electrophoresis bands.
  • ⁇ E12-E13 means the USH2A mRNA that skipped exon 12 and exon 13 by simultaneous splicing, total ⁇ means that exon 13 was skipped by splicing or exon 12 and exon 13 were skipped by simultaneous splicing The sum of the USH2A mRNA of the child.
  • an isolated nucleic acid may refer to a nucleic acid molecule that is separated from other materials present in a natural source.
  • an isolated molecule in the present application may be an artificial molecule, such as an artificially synthesized molecule.
  • expression regulatory element generally refers to a sequence provided for the transcription and translation of a gene and/or for controlling the expression of a protein in vivo, eg in a desired host cell.
  • a viral particle generally refers to a nucleic acid vector.
  • a viral particle can be a viral vector that can be used as a nucleic acid delivery vehicle, comprising a vector genome (eg, DNA and/or RNA) packaged within the viral particle.
  • a vector genome eg, DNA and/or RNA
  • antisense oligonucleotide generally refers to a single-stranded oligonucleotide having a nucleobase sequence that allows hybridization to a corresponding fragment of a target nucleic acid.
  • nucleic acid generally refers to a molecule composed of monomeric nucleotides.
  • Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA), and microRNA (miRNA).
  • microRNA generally refers to a small nucleic acid molecule.
  • small nuclear RNAs can be involved in the processing of RNA in the nucleus of eukaryotes.
  • nuclear small RNAs can be combined with related proteins to form small nuclear ribonucleoproteins (snRNPs, small nuclear ribonucleoproteins), which are involved in the splicing of messenger RNA precursors (pre-mRNA).
  • snRNPs small nuclear ribonucleoproteins
  • pre-mRNA messenger RNA precursors
  • complementarity generally refers to the ability of the nucleic acid base of the antisense oligonucleotide to perform accurate base pairing (i.e. hybridization) with the corresponding nucleic acid base in the target nucleic acid, And it is mediated by the combination of Watson-Crick (Watson-Crick) interaction force between corresponding nucleic acid bases.
  • Watson-Crick Watson-Crick
  • gene expression cassette generally refers to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination.
  • a gene expression cassette comprises a polynucleotide segment encoding a nucleic acid of interest.
  • the term "genomic location” generally refers to chromosomal coordinates describing a DNA region of interest.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • splice skipping generally means that during the processing of an RNA molecule, the spliced skipped sequence is not included in the processed RNA molecule. For example, through splice skipping, a portion of a sequence can be skipped. For example, through splice skipping, a portion of the sequence may not be included in the spliced RNA molecule.
  • modified oligonucleotide generally refers to an oligonucleotide comprising at least one modified internucleoside linkage, modified sugar and/or modified nucleobase.
  • modified oligonucleotide generally refers to an oligonucleotide comprising at least one modified internucleoside linkage, modified sugar and/or modified nucleobase.
  • 2'-O-methoxyethyl also 2'-MOE and 2'-OCH2CH2-OCH3 and MOE
  • 2'-O-methoxyethyl modified sugars are modified sugars.
  • the term "2'-MOE nucleoside” (also 2'-O-methoxyethyl nucleoside) generally refers to a nucleoside comprising a MOE modified sugar moiety.
  • the term “2'-substituted nucleoside” generally refers to a nucleoside comprising a substituent other than H or OH at the 2' position of the furanose ring.
  • 2'-substituted nucleosides include nucleosides with bicyclic sugar modifications.
  • the term "5-methylcytosine” generally refers to cytosine modified with a methyl group attached to the 5' position. 5-methylcytosine is a modified nucleobase.
  • modified phosphate linkage generally refers to a substitution or any change in a modified phosphate linkage relative to that from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).
  • stem-loop domain generally refers to the secondary structure formed by the nucleic acid molecule itself through base pairing.
  • base pairing the double-stranded portion formed by base pairing is the "stem”, and the sequence between paired bases forms the "loop”.
  • the term “contiguous” generally refers to two or more nucleobases that are immediately adjacent to each other.
  • the term “exon” generally refers to a part of a gene that is present in the mature form of the mRNA.
  • vector generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Cas enzyme generally refers to CRISPR-associated nuclease, a DNA endonuclease. For example it can form double-strand breaks at specific DNA sequences.
  • Cas nucleases can usually be complementary to CRISPR sequences, and can use CRISPR sequences as guides to recognize and cut specific DNA strands.
  • pre-mRNA generally refers to precursor mRNA.
  • primary transcripts are produced by DNA
  • the synthetic single-stranded ribonucleic acid product of transcription said transcript can be unmodified.
  • the genomic structure of exons and introns in pre-mRNAs remains unchanged.
  • sm protein generally refers to a protein or a variant thereof capable of binding snRNAs to form the small nuclear ribonucleoprotein complex snRNP.
  • USH2A generally refers to a gene, which encodes Usherin protein.
  • the NCBI gene accession number of USH2A can be 7399.
  • USH2A may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • administering generally refers to providing an agent to an animal, and includes, but is not limited to, administration by a medical professional and self-administration.
  • the present application provides a nucleic acid molecule
  • the nucleic acid molecule may have the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the exon 13 of USH2A pre-mRNA
  • the genome location corresponding to the 3' sequence of the gene is Chr1: 216246563-216246753 (corresponding to the GRch38 version of the NCBI database).
  • the genomic location corresponding to the 3' sequence of the USH2A pre-mRNA exon 13 of the present application is Chr1: 216246563-216246626, Chr1: 216246563-216246621, Chr1: 216246563-216246624, Chr1: 216246563-216 246617, Chr1: 216246563-216246591, Chr1: 216246563-216246596, Chr1: 216246563-216246593, Chr1: 216246563-216246590, and/or Chr1: 216246563-216246586 .
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246626, Chr1: 216246567-216246626, Chr1: 216246570-216246626, Chr1: 216246594-2162 46626, Chr1 : 216246598-216246626, and/or Chr1: 216246603-216246626.
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246621, Chr1: 216246567-216246621, Chr1: 216246570-216246621, Chr1: 216246594-2162 46621, Chr1 : 216246598-216246621, and/or Chr1: 216246603-216246621.
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246624, Chr1: 216246567-216246624, Chr1: 216246570-216246624, Chr1: 216246594-2162 46624, Chr1 : 216246598-216246624, and/or Chr1: 216246603-216246624.
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246617, Chr1: 216246567-216246617, Chr1: 216246570-216246617, Chr1: 216246594-2162 46617, Chr1 : 216246598-216246617, and/or Chr1: 216246603-216246617.
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246593, Chr1: 216246567-216246593, and/or Chr1: 216246570-216246593.
  • the genomic location corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA described in this application is Chr1: 216246563-216246626, and its sequence can be shown in SEQ ID NO: 39.
  • it may be the combination of mutant sequences.
  • the combination may comprise a 1 bp, 2 bp, 3 bp, 4 bp or 5 bp mismatch.
  • Exon 13 of the USH2A gene can contain mutations, including c.2802T>G (p.Cys934Trp, the most frequent mutation in Chinese patients), c.2299delG (p.E767SfsX21, the most frequent mutation in European and American patients), c.2276G> T (amino acid change: p.C759F), C.2522C>A (p.S841Y), c.2242C>T (p.Gln748X), c.2541C>A (C847X), c.2761delC (Leu921fs) and C.
  • the application provides a nucleic acid molecule, which may have the ability to specifically bind to the sequence shown in SEQ ID NO: 1 or a fragment thereof. For example, there may be more than 70%, 80%, 90%, 95%, 98%, or 99% incorporation of base pairs. For example, it may be the combination of mutant sequences. For example, the combination may comprise a 1 bp, 2 bp, 3 bp, 4 bp or 5 bp mismatch.
  • the application provides a nucleic acid molecule, which can be complementary to 16 or more consecutive nucleotides in the sequence shown in SEQ ID NO:1.
  • a nucleic acid molecule which can be complementary to 16 or more consecutive nucleotides in the sequence shown in SEQ ID NO:1.
  • it may be the complement of a mutant sequence.
  • the complementarity may comprise a 1 bp, 2 bp, 3 bp, 4 bp or 5 bp mismatch.
  • the application provides a nucleic acid molecule, which may comprise 16 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 1 or its complementary sequence. For example, there may be more than 70%, 80%, 90%, 95%, 98%, or 99% base paired complementarity. For example, it may be the complement of a mutant sequence.
  • the complementarity may comprise a 1 bp, 2 bp, 3 bp, 4 bp or 5 bp mismatch.
  • the nucleic acid molecule comprises at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof.
  • the nucleic acid molecule comprises 22 to 27 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof.
  • the nucleic acid molecule comprises 15 to 27, 16 to 27, 18 to 27, 20 to 27 complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof , 22 to 27, 25 to 27, 15 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 15 to 22, 16 to 22, 18 to 22, 20 to 22, 15 to 20, 16 to 20, 18 to 20, 15 to 18, or 16 to 18 nucleosides acid.
  • the sequence length of the recognition domain can be more than 16bp, further can be 18bp-40bp, and further can be 20bp-27bp.
  • the length of the recognition domain sequence can be extended along the 5' end or/and 3' end of the target sequence through reverse complementary pairing, preferably a single recognition domain sequence after extension The length is less than or equal to 40bp.
  • the nucleic acid molecule may comprise at least 16 nucleotides.
  • the nucleic acid molecule may comprise 22 to 27 nucleotides.
  • the nucleic acid molecule may comprise 15 to 27, 16 to 27, 18 to 27, 20 to 27, 22 to 27, 25 to 27, 15 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 15 to 22, 16 to 22, 18 to 22, 20 to 22, 15 to 20, 16 to 20, 18 to 20, 15 to 18, or 16 to 18 nucleotides.
  • the nucleic acid molecule is an AON molecule.
  • the nucleic acid molecule may comprise at least 60 nucleotides.
  • the nucleic acid molecule can comprise at least 65, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or 500 nucleotides.
  • the nucleic acid molecule is an snRNA molecule.
  • snRNA molecules can contain many more elements and can be of theoretically infinite length.
  • the total length of the chemically synthesized snRNA sequence is greater than or equal to 96bp.
  • the 3-40 bases flanking the chemically synthesized snRNA are modified and linked by specific phosphate bonds.
  • the nucleic acid molecule may comprise a sequence complementary to a genome location selected from the following group: Chrl: 216246603-216246626, Chrl: 216246598-216246621, Chrl: 216246598-216246624, Chrl: 216246594-216246617, Chrl: 216 246570-216246593, Chr1: 216246570-216246591, Chr1: 216246570-216246596, Chr1: 216246570-216246593, Chr1: 216246570-216246596, Chr1: 216246567-216246590, and Chr1 r1: 216246563-216246586.
  • the nucleic acid molecule may comprise a sequence complementary to any one of SEQ ID NOs: 10-21.
  • it may be 70%, 80%, 90%, 95%, 98%, or 99% or more base-paired complementary.
  • it may be the complement of a mutant sequence.
  • the complementarity may comprise a 1 bp, 2 bp, 3 bp, 4 bp or 5 bp mismatch.
  • there may be 0-5 mismatched nucleotides in the reverse complementary pairing between the recognition domain and the target site, for example, it may be 0-1.
  • the nucleic acid molecule can comprise the sequence shown in any one of SEQ ID NO: 10-21.
  • the nucleic acid molecule may comprise modified nucleotides.
  • the modified nucleotide may comprise a modification selected from the group consisting of 2'-O-alkyl, 2'-O-methoxy and/or 2'-O-methoxyethyl.
  • the modified nucleotide may comprise a modification selected from the group consisting of 2'-O-methyl and/or 2'-O-ethyl.
  • the nucleic acid molecule may comprise at least one modified nucleotide.
  • only 1-10, 6-80 or all nucleotides on both sides of the snRNA are modified and connected with special phosphate bonds, and the modification is one or a combination of more than two modifications,
  • the special phosphate bond is one or a combination of two or more phosphate bonds.
  • all nucleotides in the chemically synthesized U7 snRNA are connected to each other through phosphorothioate bonds, and are all modified with 2′-O-methoxy.
  • only 3 nucleotides flanking the snRNA are linked by phosphorothioate linkages and are 2'-O-methoxy modified.
  • the first nucleotide at the 5' end of U7 snRNA is preferably adenosine (A), if the first nucleotide at the 5' end of the recognition domain is not adenine (A), then connect adenosine (A) at the 5' end.
  • the 5' end of the nucleic acid molecule may comprise at least one modified nucleotide.
  • the 5' end of the nucleic acid molecule may contain 1 to 3 modified nucleotides.
  • the 5' end of the nucleic acid molecule may comprise 1, 2, 3, 4 or 5 modified nucleotides.
  • the 3' end of the nucleic acid molecule may comprise at least one modified nucleotide.
  • the 3' end of the nucleic acid molecule may contain 1 to 3 modified nucleotides.
  • the 3' end of the nucleic acid molecule may comprise 1, 2, 3, 4 or 5 modified nucleotides.
  • the nucleotide molecule in the nucleic acid molecule may comprise a group selected from the group consisting of: 6'-modified bicyclic nucleosides, 5'-modified bicyclic nucleosides, 6'-disubstituted bicyclic nucleosides Glycosides, tetrahydropyranucleoside analogs and/or 2'-deoxy2'-fluoro- ⁇ -D-arabinonucleotides (2'-FANA modified nucleotides).
  • the nucleic acid molecule has a modified phosphate bond.
  • the nucleotide molecules are linked via a phosphate bond selected from the group consisting of phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages, alkylphosphonate linkages, aminoalkylphosphotriester linkages , alkylene phosphonate linkage, phosphinate linkage, phosphoramidate linkage and aminoalkyl phosphoramidate linkage, phosphorothioamidate linkage, thiocarbonylalkylphosphonate linkage, thiocarbonylalkylphosphonate linkage Ester linkages, phosphorothioate linkages, phosphoroselenolate linkages and/or borylphosphate linkages.
  • a phosphate bond selected from the group consisting of phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages, alkylphosphonate linkages, aminoalkylphosphotriester linkages , alkylene phosphonate linkage,
  • the modified phosphate linkage may comprise a phosphodiester linkage, a phosphotriester linkage, a phosphorothioate linkage (5'OP(S)O-3O-, 5'S-P(O)O -3'-O- and 5'OP(O)O-3'S-), phosphorodithioate linkage, Rp-phosphorothioate linkage, Sp-phosphorothioate linkage, borane phosphate linkage, methylene group bond (methylimino), amide bond (3'-CH 2 -CO-NH-5' and 3'-CH 2 -NH-CO-5'), methyl phosphonate bond, 3'-thio Formal linkage, (3'S- CH2- O5'), amide linkage ( 3'CH2 -C(O)NH-5'), and/or phosphoramidate group.
  • the modified phosphate linkage may comprise a group selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl phosphate, amide phosphate and/or borane phosphate.
  • the modified phosphate linkage may comprise a phosphodiester linkage, a phosphotriester linkage, a phosphorothioate linkage (5'OP(S)O-3O-, 5'S-P(O)O -3'-O- and 5'OP(O)O-3'S-), phosphorodithioate linkage, Rp-phosphorothioate linkage, Sp-phosphorothioate linkage, borane phosphate linkage, methylene radical bond (methylimino), amide bond (3'-CH 2 -CO-NH-5' and 3'-CH 2 -NH-CO-5'), alkyl phosphate bond, amide phosphate bond, Methylphosphonate linkage, 3'-thioformal linkage, (3'S-CH 2 -O5'), amide linkage (3'CH 2 -C(O)NH-5') and/or phosphoramidate group.
  • a phosphodiester linkage a phosphotriester linkage,
  • the nucleic acid molecule may comprise at least one modified phosphate bond.
  • the 5' end of the nucleic acid molecule may comprise at least one modified phosphate bond.
  • the 5' end of the nucleic acid molecule may contain 1 to 3 modified phosphate bonds.
  • the 5' end of the nucleic acid molecule may contain 1, 2, 3, 4 or 5 modified phosphate linkages.
  • the 3' end of the nucleic acid molecule may comprise at least one modified phosphate bond.
  • the 3' end of the nucleic acid molecule may contain 1 to 3 modified phosphate bonds.
  • the 5' end of the nucleic acid molecule may contain 1, 2, 3, 4 or 5 modified phosphate linkages.
  • the nucleic acid molecule may comprise DNA and/or RNA.
  • the nucleic acid molecule is single-stranded or double-stranded.
  • the nucleic acid molecule may comprise antisense oligonucleotides, shRNA, siRNA, miRNA and/or aptamers.
  • the nucleic acid molecule may comprise small nuclear RNA (Small nuclear RNA).
  • the small nucleoRNA may comprise a U1 microRNA and/or a U7 microRNA.
  • the small nucleoRNA can comprise U1, U2, U3, U4, U5, U6 and/or U7 small nucleoRNA.
  • the small nuclear RNA can comprise a U1 small nuclear RNA.
  • the small nuclear RNA can comprise a U7 small nuclear RNA.
  • the nucleic acid molecule does not bind the Cas enzyme.
  • the nucleic acid molecule may not comprise a structure capable of binding a Cas enzyme.
  • the Cas enzyme is Cas13 enzyme.
  • the nucleic acid molecule may comprise a stem-loop domain or a derivative thereof.
  • the stem-loop domain may comprise the sequence shown in SEQ ID NO:6.
  • the nucleic acid molecule may comprise a domain capable of binding an sm protein or a derivative thereof.
  • the domain capable of binding to the sm protein may comprise the sequence shown in SEQ ID NO:5.
  • the domain capable of binding to sm protein may comprise an optimized smOPT sequence.
  • the sm protein-binding domain may comprise human, murine or porcine origin.
  • the nucleic acid molecule can comprise a domain capable of recruiting a splicing regulatory protein.
  • the nucleic acid molecule may comprise a domain capable of binding histones selected from: SRSF1 (Serine And Arginine Rich Splicing Factor 1), RBM4 (RNA Binding Motif Protein 4), DAZAP1 (DAZ Associated Protein 1), and SR ( Serine And Arginine-Rich Protein).
  • the nucleic acid molecule can comprise a domain capable of binding the hnRNP A1 protein.
  • the domain capable of binding hnRNP A1 protein may comprise the sequence shown in SEQ ID NO:30.
  • the nucleic acid molecule of the present application has the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or its fragment, and the genome corresponding to the 3' sequence of exon 13 of USH2A pre-mRNA Positioned as Chr1: 216246563-216246753, the nucleic acid molecule contains at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the nucleic acid molecule may contain at least 16 Nucleotides.
  • the nucleic acid molecules of the present application have the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or its fragments, and the genome location corresponding to the 3' sequence of exon 13 of the USH2A pre-mRNA Chr1: 216246563-216246753, the nucleic acid molecule includes at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the nucleic acid molecule may include at least 60 nuclei glycosides.
  • the nucleic acid molecules of the present application have the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or its fragments, and the genome location corresponding to the 3' sequence of exon 13 of the USH2A pre-mRNA Chr1: 216246563-216246753, the nucleic acid molecule includes at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the nucleic acid molecule may include at least 60 nuclei Nucleotides, the 3-40 bases on both sides of the chemically synthesized snRNA are modified and linked by special phosphate bonds.
  • the nucleic acid molecules of the present application have the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or its fragments, and the genome location corresponding to the 3' sequence of exon 13 of the USH2A pre-mRNA Chr1: 216246563-216246753, the nucleic acid molecule includes at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the nucleic acid molecule may include at least 60 nuclei Nucleic acid, the 3-40 bases on both sides of the chemically synthesized snRNA are 2'-methoxy modified and linked by phosphorothioate bonds.
  • the nucleic acid molecules of the present application have the ability to specifically bind to the 3' sequence of exon 13 of USH2A pre-mRNA or its fragments, and the genome location corresponding to the 3' sequence of exon 13 of the USH2A pre-mRNA Chr1: 216246563-216246753, the nucleic acid molecule includes at least 16 nucleotides that may be complementary to the 3' sequence of exon 13 of USH2A pre-mRNA or a fragment thereof, and the nucleic acid molecule may include at least 60 nuclei Nucleic acid, the three bases on both sides of the chemically synthesized snRNA are 2'-methoxy modified and connected by phosphorothioate bonds, and the nucleic acid molecule may contain a domain capable of binding to the sm protein or its derivative structure .
  • the nucleic acid molecule may comprise a domain capable of binding the hnRNP A1 protein.
  • the present application provides a gene expression cassette, which may comprise or encode the nucleotide sequence of the nucleic acid molecule described in the present application, and optional expression control elements.
  • the present application provides a vector, which may contain or encode the nucleotide sequence of the nucleic acid molecule described in the present application, and/or the nucleotide sequence of the gene expression cassette described in the present application.
  • the present application provides a virus particle, which may comprise the nucleic acid molecule described in the present application, the nucleotide sequence of the gene expression cassette described in the present application, and/or the vector described in the present application.
  • the carrier of the present application can be pUC57, and can also be selected from pAAV-CMV (TAKARA Company, Code No.6650), lentivirus, transposon and nucleic acid vectors known in the art.
  • the present application can induce USH2A pre-mRNA No. 13 splicing skipping by delivering U7 snRNA through AAV, and the capsid protein of the AAV can be a natural source, or a variant based on a natural source capsid protein, or Carry out directed evolution, or carry out rational modification of amino acids/peptides (codon optimization, chimera functional peptides of different serotypes, etc.), etc., to improve the characteristics of tissues and organs such as tropism, immunogenicity, and transfection efficiency, such as AAV2. 5.
  • the AAV capsid protein of natural origin can be derived from animal body, also can be derived from plant, and the AAV capsid protein derived from animal body can be derived from human body (such as AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8 and AAV9, etc.), can also be derived from non-human primates (such as AAVrh.8, AAVrh.10 and AAVrh.43), or from vertebrates such as mice and pigs , can also be derived from insects.
  • the AAV ITR serotype should be consistent with the Rep gene serotype, and may be inconsistent with the Cap gene serotype.
  • the present application provides a cell, which may comprise the nucleic acid molecule described in the present application, the nucleotide sequence of the gene expression cassette described in the present application, the vector described in the present application, and/or the the virus particles.
  • the application provides a pharmaceutical composition, which may comprise the nucleic acid molecule described in the application, the nucleotide sequence of the gene expression cassette described in the application, the vector described in the application, the The virus particle described in the application, and/or the cell described in the application, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can include a first carrier and a second carrier, the first carrier can include the nucleic acid molecule described in the application, and the second carrier can include a protein that can specifically bind USH2A pre-mRNA.
  • the second carrier may comprise a nucleic acid molecule having the ability to specifically bind to the group selected from intron No. 12-exon No. 13-intron No. 13 of USH2A pre-mRNA, e.g. The child and the adjacent target area on both sides.
  • it can be selected from the pre-mRNA region corresponding to chr1:216246563-216247246.
  • it may be used in combination of two or more U7-snRNAs whose target sequences are not exactly the same.
  • the regions on both sides of exon 13 of the USH2A pre-mRNA may include intron 12 and/or intron 13 of USH2A pre-mRNA.
  • the application provides a kit, which may include the nucleic acid molecule described in the application, the nucleotide sequence of the gene expression cassette described in the application, the vector described in the application, the The virus particle described in the application, the cell described in the application, and/or the pharmaceutical composition described in the application.
  • the present application provides a method for preparing the nucleic acid molecule described in the present application, which may comprise expressing and/or synthesizing the nucleic acid molecule capable of binding to the 3' sequence of exon 13 of USH2A or a fragment thereof, so The genomic mapping of the 3' sequence of exon 13 of USH2A is Chr1: 216246563-216246753.
  • the application provides a method for inhibiting the expression and/or function of exon 13 of USH2A pre-mRNA, which may include providing the nucleic acid molecule described in the application, the carrier described in the application, the vector described in the application Virus particles, cells described herein, pharmaceutical compositions described herein and/or kits described herein.
  • inhibiting the expression and/or function of exon 13 of USH2A pre-mRNA may refer to making the mature USH2A mRNA not include the region of exon 13 expression product.
  • the present application provides a method for splicing and skipping exon 13 of USH2A pre-mRNA, which may include providing the nucleic acid molecule described in the present application, the vector described in the present application, the virus particle described in the present application, The cells described herein, the pharmaceutical compositions described herein and/or the kits described herein.
  • the application provides a method for preparing mature USH2A mRNA lacking exon 13, which may include providing the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, the The cell described in the application, the pharmaceutical composition described in the application and/or the kit described in the application.
  • the application provides a method for reducing the Usherin protein level that may comprise the No. 13 exon expression product, which may include providing the nucleic acid molecule described in the application, the carrier described in the application, the virus described in the application Particles, cells described herein, pharmaceutical compositions described herein and/or kits described herein.
  • the application provides a method for preparing the Usherin protein that may not contain the No. 13 exon expression product and/or increasing the Usherin protein quantity that does not contain the No. 13 exon expression product, which may include providing the Usherin protein described in this application.
  • the Usherin protein prepared in the present application does not contain the polypeptide expressed in exon 13 or its mutants.
  • the application provides a method for restoring the function of the mutant Usherin protein, which may include providing the nucleic acid molecule described in the application, the carrier described in the application, the virus particle described in the application, the cell described in the application , the pharmaceutical composition described in the application and/or the kit described in the application.
  • the Usherin protein prepared by the present application maintains or basically maintains the function of the wild-type Usherin protein in a healthy person.
  • the symptoms of retinitis pigmentosa, congenital degenerative neurological deafness, vestibular dysfunction and other diseases in subjects expressing mutant Usherin protein were alleviated.
  • the application provides the nucleic acid molecule described in the application, the carrier described in the application, the cell described in the application, the pharmaceutical composition described in the application and/or the test kit described in the application
  • the medicine is used for preventing and/or treating diseases caused by mutation of USH2A gene.
  • the disease may comprise eye disease and/or ear disease.
  • the disease may comprise Usher's syndrome.
  • the disease may comprise Usher Syndrome Type II.
  • the present application provides a method for preventing and/or treating diseases caused by USH2A gene mutations, which may include administering the nucleic acid molecules described in the present application, the vectors described in the present application, the cells described in the present application, the The pharmaceutical composition and/or the kit described in this application.
  • the disease may comprise eye disease and/or ear disease.
  • the disease may comprise Usher's syndrome.
  • the disease may comprise Usher Syndrome Type II.
  • the present application provides the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the pharmaceutical composition described in the present application and/or the test kit described in the present application, it is used It is used to prevent and/or treat diseases caused by mutations in the USH2A gene.
  • the disease may comprise eye disease and/or ear disease.
  • the disease may comprise Usher's syndrome.
  • the disease may comprise Usher Syndrome Type II.
  • USH2A generally refers to the gene encoding Usherin.
  • USH2A is located at 1q41, which spans more than 800kb in the genome and encodes a large transmembrane protein, Usherin, which anchors on the plasma membrane of retinal photoreceptor cells and inner ear hair cells, and is an essential component for cilia development and maintenance. In the retina, Usherin is an important part of the USH2 complex and is thought to function in stabilizing the outer segments of photoreceptors.
  • USH2A has two subtypes, the main subtype contains 72 Exons in retinal cells, and the length of the coding region is about 15.6kb.
  • the extracellular part of Usherin protein contains many repeated domains, including 10 Laminin EGF-like (LE) domains and 35 Fibronectin type 3 (FN3) domains.
  • Exon 13 of human USH2A is 642bp in length, encoding amino acids 723-936, which are 4 of the 10 LE domains in Usherin protein.
  • the length of the USH2A coding region is about 15.6kb.
  • Conventional gene therapy delivery methods (such as recombinant lentivirus, recombinant adeno-associated virus, etc.) are difficult to package such a large coding sequence, so it is difficult to directly deliver USH2A for treatment.
  • Exon 12 of mouse USH2A is homologous to exon 13 of human USH2A, both of which are 642 bp in length. Removal of this exon did not cause subsequent frameshift mutations. Studies have shown that after knocking out exon 12 of mouse USH2A, Usherin can still be correctly positioned and perform normal functions. For exon 13 of human USH2A containing pathogenic mutations, a series of means can be used to make them skip for treatment.
  • Editing of genomic DNA through the CRISPR/Cas system directly deletes exon 13, or destroys sites related to RNA splicing. There may be risks in the use of fragment deletions, such as chromosomal rearrangement, viral integration, reverse reintegration, and the possibility of high off-target probability of expressing the CAS system for a long time or performing two gRNA-induced double cuts based on a relatively large genomic background. Exon skipping can also be promoted by using a single base editor to modify key bases at the aforementioned splicing-related sites. However, existing single base editors may not be loaded by a single AAV vector, and limited by PAM, editing window, and base conversion type, there may be no suitable gRNA near the splicing-related site.
  • Antisense oligonucleotides are used to target and interfere with pre-mRNA splicing to promote exon skipping with high efficiency. While AON promotes the skipping of exon 13, it may also promote the co-reading of exon 12 and exon 13. Some AON treatment may lead to double skipping. However, the full length of exon 12 is 196bp, which is not an integer multiple of 3. The deletion will lead to frameshift mutation and inactivation of USH2A protein. Targeted interference with pre-mRNA splicing by U7 snRNA promotes exon skipping more efficiently than AON, and increases the proportion of single skipping of exon 13, which not only improves efficiency significantly, but also ensures safety.
  • snRNA small nuclear RNA
  • snRNA small nuclear RNA
  • U7 snRNA is by replacing the non-canonical Sm binding site of U7 snRNA with the consensus sequence derived from the major spliceosome U snRNPs, changing the histone binding sequence of the 5' region of U7 snRNA to the complementary sequence of the gene to be modified, Splice skipping of exons can be induced by targeting exons.
  • the wild-type U7 snRNA includes a stem-loop structure (scafford), a U7-specific Sm sequence (AAUUUGUCUAG, SEQ ID NO: 2) and a recognition domain (complementary to histone pre-mRNA).
  • the U7 snRNA of the present application can be based on the gene sequence (NCBI Reference Sequence: NR_024201.3) of the mouse wild-type U7 snRNA on NCBI, wherein the U7-specific Sm binding site (AATTTGTCTAG, SEQ ID NO: 3) is replaced
  • SmOPT AATTTTTGGAG, SEQ ID NO: 4; or AAUUUUUGGAG, SEQ ID NO: 5
  • the original recognition domain at the 5' end of the SmOPT sequence was replaced with a USH2A pre-mRNA specific target site.
  • the 3' end of the SmOPT sequence retains the U7 original stem-loop structure sequence (CAGGUUUUCUGACUUCGGUCGGAAAACCCCU, SEQ ID NO: 6).
  • Figure 1 shows a schematic diagram of the structure and function of the U7-snRNA of the present application.
  • the sequence of the U7 snRNA recognition domain of target-induced USH2A pre-mRNA exon 13 is reversed from the target sequence selected from USH2A pre-mRNA intron 12-exon 13-intron 13 Complementary pairing, the target sequence can be selected from the 3' sequence target region of exon 13 of USH2A pre-mRNA.
  • the snRNA recognition domain sequence preferably has a length of 16 bp or more, more preferably 18 bp-40 bp, and still more preferably 20 bp-27 bp.
  • the snRNA gene-specific 3' box is after the 3' end of the U7 snRNA gene in the mouse genome (GenBank: X54748.1), including the sequence "GTCTACAATGAAA (SEQ ID NO: 7)", which is involved in the processing of pre-snRNA, preferably U7 snRNA A gene fragment with a sequence length of 28-131 bp behind the 3' end of the gene, and a further preferred sequence length of 106 bp.
  • the corresponding Oligo DNA was synthesized respectively.
  • the sense strand of Oligo DNA is the DNA sequence corresponding to the recognition domain sequence, and 5' plus CCGCA
  • the antisense strand is the antisense complementary sequence of the recognition domain sequence 5' plus AATT and 3' plus T.
  • the recognition domain sequence is 5'-NNN-3'
  • the sense strand of the synthesized Oligo DNA is 5'-CCGCA NNN- 3'
  • the antisense strand is 5'-AATT NNN T-3'.
  • the ligation product was further verified by transforming Escherichia coli competent cells, picking a single clone, PCR and sequencing to obtain the U7 snRNA vector for inducing splicing skipping of exon 13 of USH2A. Plasmids were purified and stored at -20°C for future use.
  • the sequence of the antisense oligonucleotide AON that can be combined with this region can be designed and synthesized.
  • U7 snRNA can also be directly chemically synthesized to generate RNA containing guide sequence, smOPT and U7 snRNA scaffold.
  • U7 snRNA synthesized in vitro can be specifically modified to make it resistant to nuclease degradation, or to increase the affinity for the target sequence.
  • U7 snRNA was chemically synthesized, and the 3 bases at the 5' and 3' ends were respectively modified with 2'methoxy (2'-OME) and sulfo-modified to increase nuclease resistance.
  • 2'-OME 2'methoxy
  • snRNA#25 and snRNA#26 the chemically synthesized snRNA sequences and modifications are as follows (* indicates the phosphorothioated backbone, m indicates the 2'-methoxy modification, and the underline indicates the recognition structure that is reversely complementary to the target sequence domain, italics indicate smOPT sequence):
  • nucleotides in the U7 snRNA of this application have carried out one or both of 2'-O alkyl, 2'-O-methoxy, 2'-O-methoxyethyl
  • the 2'-O alkyl group is preferably 2'-O-methyl modification.
  • the phosphate bond linked by the nucleotide of the snRNA can be connected by a special phosphate bond, and the special phosphate bond is a phosphorothioate bond, a phosphorodithioate bond, an alkyl phosphonate bond, an amide phosphate bond ( Phosphoroamidate), boranophosphate (boranophosphate) bond, chiral linkage phosphorus (chiral linkage phosphor) or two or more.
  • the special phosphate bond is a phosphorothioate bond, a phosphorodithioate bond, an alkyl phosphonate bond, an amide phosphate bond ( Phosphoroamidate), boranophosphate (boranophosphate) bond, chiral linkage phosphorus (chiral linkage phosphor) or two or more.
  • nucleotides on both sides of the snRNA are modified and connected with special phosphate bonds, and the modification is one or a combination of more than two modifications,
  • the special phosphate bond is one or a combination of two or more phosphate bonds.
  • all nucleotides in the chemically synthesized U7 snRNA are connected to each other through phosphorothioate bonds, and are all modified with 2′-O-methoxy.
  • only 3 nucleotides flanking the snRNA are linked by phosphorothioate linkages and are 2'-O-methoxy modified.
  • the first nucleotide at the 5' end of U7 snRNA is preferably adenosine (A), if the first nucleotide at the 5' end of the recognition domain is not adenine (A), then connect adenosine (A) at the 5' end.
  • the sequence length of the recognition domain is preferably 16 bp or more, more preferably 18 bp-40 bp, and still more preferably 20 bp-27 bp.
  • the length of the recognition domain sequence can be extended along the 5' end or/and 3' end of the target sequence through reverse complementary pairing, preferably a single recognition domain sequence after extension The length is less than or equal to 40bp. In some embodiments, preferably, the total length of the chemically synthesized snRNA sequence is greater than or equal to 96 bp. In some embodiments, preferably the 3-40 bases flanking the chemically synthesized snRNA are modified and linked by specific phosphate bonds.
  • the RG left -USH2A EXON13 mut -RG right sequence (AgeI and EcoRI restriction sites were added to the 5' end and 3' end respectively) was obtained by whole gene synthesis, and the synthetic sequence and pX601 plasmid (Addgene, 61591) Restriction endonuclease AgeI and EcoRI digestion, electrophoresis, gel recovery and ligation, insert the synthesized sequence between the AgeI and EcoRI restriction sites of the pX601 vector, replace the SaCas9 gene sequence of the original vector, and obtain the reporter vector. Further, through transformation of Escherichia coli competent cells, selection of single clones, PCR and sequencing verification, the purified reporter vector plasmid was obtained and stored at -20°C for later use.
  • the structure of the reporter vector is: pCMV-RG left -USH2A EXON13 mut -RG right , RG indicates the reporter gene (reporter gene), RG left indicates the first half of the 5' end of the reporter gene without the reporter function, RG right indicates the report without the reporter function In the second half of the 3' end of the gene, the tandem expression of RG left and RG right can normally function as a complete reporter gene.
  • the reporter gene is the green fluorescent gene EGFP
  • the vector structure is pCMV-EGFP left -Exon13 mut -EGFP right .
  • EXON13 mut means USH2A exon 13 containing pathogenic mutations, and its upstream and downstream intron sequences (the upstream intron sequence is a combination of 204bp at the 5' end and 490bp at the 3' end of intron 12 of the human USH2A gene Gene sequence; the downstream intron sequence is the gene sequence of human USH2A No. 13 intron 703bp at the 5' end and 216bp at the 3' end).
  • the pathogenic mutation of exon 13 of USH2A described in the examples of the present invention can be c.2299delG or c.2802T>G or any mutation, and the obtained vector structures are respectively pCMV-EGFP left -Exon13 c .
  • the mutations in some embodiments can also be or include c.2276G>T, C.2522C>A, c.2242C>T, c.2541C>A, c.2761delC and C.2776C>T, etc.
  • RG left for example, the sequence of EGFP left is:
  • RG right for example, the sequence of EGFP right is:
  • 293T cells were inoculated into a 24-well plate in a certain amount so that the confluence of the cells reached about 80% after 24 hours.
  • Use Lipofectamine2000 to synthesize 100pmol antisense oligonucleotides AON#20, AON#24, AON#25, AON#31 all nucleoside monomers of AON synthesized are all carried out 2′-O-methoxy modification and thiolation Phosphorylation modification), were co-transfected with reporter plasmid pCMV-EGFP left -Exon13 mut -EGFP right 293T cells.
  • the transfected cells continued to be cultured for 48-72 hours, digested into single cells using trypsin, and then used flow cytometry to detect the GFP positive rate of different AON groups (that is, the USH2A pre-mRNA exon 13 was induced to splice skipping cell ratio) and the average FITC intensity of GFP-positive cells (i.e. the average level of splicing skipping in exon 13 of USH2A pre-mRNA in GFP cells), the following Table 2 and Figure 2 show that AON induction targeting two regions Splicing skipping effect of USH2A pre-mRNA exon 13. The experimental results show that the effect of AON targeting the 3' end region is significantly better than that of AON targeting other regions, such as #20 region.
  • 293T cells were inoculated into a 24-well plate in a certain amount so that the confluence of the cells reached about 80% after 24 hours.
  • Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFP left -Exon13 mut -EGFP right and pUC57-U7 snRNA plasmid targeting USH2A pre-mRNA (the vector mass ratio was 100ng:400ng), and a separate transfection reporter plasmid (Report, Reporter group), 293T cells co-transfected with reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls respectively, and 293T cells not transfected with any plasmid were used as blank control.
  • the transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then used flow cytometry to detect the GFP positive rate of different U7 snRNA groups (that is, the proportion of cells with USH2A exon 13 induced splicing skipping ).
  • the average FITC intensity of different experimental groups that is, the average FITC fluorescence intensity of GFP-positive cells, the GFP-positive rate, and the expression level of GFP protein in positive cells were detected.
  • Human host cells were inoculated into 24-well plates at 6 ⁇ 10 5 /well, and the human retinal nerve cells used in this example were WERI-Rb-1 cells (retinal nerve cell line).
  • 100 pmol U7-snRNA #24, #25, #26, #27, #28, #29, #30, #33, #34 synthesized in vitro were transfected into WERI cells with Lipofectamine2000.
  • the transfected cells continued to culture for 72 hours, and then the RNA of each experimental group was extracted, reverse-transcribed to obtain cDNA, and RT-PCR experiments were performed with primers AGCCTTTCCGCCAAGGTGATC (SEQ ID NO: 34) and CACAACGTTGCCCAGCAATGG (SEQ ID NO: 35) , to detect whether there is exon splicing skipping in the mature USH2A mRNA, and the electrophoresis results are shown in FIG. 7 .
  • U7-snRNA multi-target combination vector Construction of U7-snRNA multi-target combination vector.
  • PCR amplifies the U7 snRNA cassette (expression cassette) and introduces additional 5' flanking bases and BsaI restriction sites in the correct direction at both ends of the amplicon through primers point, so that the adjacent different U7 snRNA cassettes are digested with BsaI to produce specific complementary cohesive ends, and the first and last U7 snRNA cassettes are digested with BsaI to produce the same cohesive ends as the HindIII+NotI digested linearized backbone vector.
  • the Golden Gate Assembly assembly product was further transformed into Escherichia coli competent cells, single clones were selected, PCR and sequencing verification, and a U7 snRNA multi-target combination vector for inducing USH2A exon 13 splicing skipping was obtained. Plasmids were purified and stored at -20°C for future use.
  • the constructed vector is exemplarily named as pUC57-U7 snRNA#A+U7 snRNA#B+U7 snRNA#C.
  • U7-snRNA combinations with different target sites more efficiently induce USH2A pre-mRNA exon 13 splicing skipping.
  • 293T cells were inoculated into a 24-well plate in a certain amount so that the confluence of the cells reached about 80% after 24 hours.
  • Lipofectamine2000 to co-transfect 293T cells with pCMV-EGFP left -Exon13 mut -EGFP right respectively and U7 snRNA combination plasmids or single-target plasmids of different targets (vector mass ratio is 100ng:400ng), using a separate transfection reporter plasmid ( Report, reporter group), 293T cells co-transfected with reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells not transfected with any plasmid were used as blank control.
  • transfected cells were cultured for 48-72 hours, digested into single cells by trypsin, and then detected the GFP positive rate of different snRNA groups by flow cytometry.
  • Table 4 below and Figures 4A-4B show that U7-snRNA combinations at different target sites induce splicing skipping efficiencies in exon 13 of USH2A pre-mRNA.
  • one of the snRNAs can target the 3' sequence or its fragment of exon 13 of USH2A pre-mRNA of this application, and the other or multiple snRNAs can target USH2A Pre-mRNA No. 12 intron-No. 13 exon-No. 13 intron sequence or its fragment, such as U7-snRNA with its recognition domain sequence and USH2A pre-mRNA No. 12 intron, Exon 13 or intron 13 target sequence reverse complementary pairing, preferably selected from exon 13 and adjacent target regions on both sides (pre-mRNA region corresponding to chr1:216246563-216247246).
  • the U7-snRNA combination of different target sites is the combination of two or more U7-snRNAs whose target sequences are not exactly the same.
  • snRNA combination 1 (U7-snRNA#30 and U7-snRNA#4) and combination 2 (U7-snRNA#26 and U7-snRNA#16) synthesized in vitro were transfected into WERI cells with Lipofectamine2000 respectively, and the same dose ( 50pmol) antisense oligonucleotide AON1 (5′-MA*MG*MC*MU*MU*MC*MG*MG*MA*MG*MA*MA*MA*MU*MU*MU*MA*MA* MA*MU*MC*-3′, “M” indicates 2′-O-methoxy modification, “*” indicates phosphorothioation) and AON2 (5′-MU*MG*MA*MU*MC*MA *MC*MA*MC*MC*MU*MA*MA*MG*MC*MC*MU*MA*MA*-3′
  • the transfected cells continued to culture for 72 hours, and then the RNA of each experimental group was extracted, reverse-transcribed to obtain cDNA, and RT-PCR experiments were performed with primers AGCCTTTCCGCCAAGGTGATC (SEQ ID NO: 34) and CACAACGTTGCCCAGCAATGG (SEQ ID NO: 35) , to detect whether there is exon splicing skipping in the mature USH2A mRNA, and the electrophoresis results are shown in FIG. 8A .
  • the rt-PCR electrophoresis bands were further quantitatively analyzed by ImageJ software, and statistics and analysis were performed on the proportion of mature USH2A mRNA that skipped exon 13 or spliced skipped exons 12 and 13, as shown in Figure 8B .
  • snRNA combination 2 targets the AON site adjacent to exon 12 and exon 13 of the prior art with a high probability of double splicing skipping, however, the occurrence probability of double exon splicing skipping of snRNA combination 2 is very low.
  • the sense strand of Oligo DNA is the reverse complementary sequence of the target sequence (the DNA sequence corresponding to the recognition domain sequence), and the 5' plus The antisense strand is the target sequence 5' plus AATT and 3' plus
  • the sequence of the recognition domain is NNN (the length of the recognition domain is preferably greater than 16 nucleotides)
  • the synthetic Oligo DNA sense strand is Antisense strand is (The underline indicates the DNA double-stranded sequence corresponding to the recognition domain sequence , and the bold italic indicates the DNA double-stranded sequence corresponding to the binding motif "UAGGGU (SEQ ID NO: 30)" of the hnRNP A1 protein).
  • U7-hnRNP A1-snRNA can also be chemically synthesized and modified according to the methods described in the examples of this application.
  • the chemically synthesized U7-hnRNP A1-snRNA sequence and modifications are as follows (* indicates the phosphorothioated backbone, m indicates the 2'-methoxy modification, and the underline indicates the recognition of reverse complementary pairing with the target sequence domain , italics indicate smOPT sequence, bold indicates hnRNP A1 protein binding motif):
  • U7 snRNA linked to hnRNP A1-binding motif induces splice skipping of USH2A exon 13 in reporter cells.
  • 293T was inoculated into a 24-well plate in a certain amount so that the cell confluency reached about 80% after 24 hours.
  • Lipofectamine2000 to co-transfect 293T cells with pCMV-EGFP left -Exon13 mut -EGFP right respectively with pUC57-U7-hnRNP A1-snRNA plasmid and pUC57-U7 snRNA plasmid (vector mass ratio is 100ng:400ng), using a separate transfection reporter Plasmid (reporter group), 293T cells co-transfected with reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells not transfected with any plasmid were used as blank control.
  • transfected cells were cultured for 48-72 hours, digested into single cells using trypsin, and then flow cytometry was used to detect the splicing skipping efficiency induced by different snRNA groups.
  • Table 5 below and Figures 6A-6B show the splicing skipping efficiency of exon 13 of U7-hnRNP A1-snRNAUSH2A pre-mRNA.
  • the free tail sequence includes the binding motif "UAGGGU" of the hnRNP A1 protein, and the free tail sequence can contain 1, 2 or more than 2 hnRNP A1 Protein binding motifs, preferably 2, the free tail sequence is preferably It can recruit hnRNP A1 protein, promote the splicing skipping of exon 13 of USH2A, and will not increase the double skipping of exon 12 and exon 13, which may not affect its targeting specificity, may not cause or increase off-target effects.
  • the No. 12 exon of USH2A described in this application is the No. 12 exon of human USH2A.
  • the No. 13 exon of USH2A described in this application is the No. 13 exon of human USH2A.
  • the 5' end of the U7 snRNA introduces a free tail as a motif that can recruit splicing regulatory proteins
  • the splicing regulatory proteins are hnRNP A1 (Heterogeneous Nuclear Ribonucleoprotein A1), SRSF1 (Serine And Arginine Rich Splicing Factor 1), RBM4 (RNA Binding Motif Protein 4), DAZAP1 (DAZ Associated Protein 1), SR (Serine And Arginine-Rich Protein), etc.
  • the U7 snRNA gene that targets the splicing skipping of exon 13 of USH2A pre-mRNA is inserted and replaced with the intermediate gene sequence of the two ITR domains in the pAAV-CMV vector, and the pAAV-U7 snRNA vector is constructed and packaged with AAV Plasmid: serotype pRC plasmid (containing Rep gene of AAV2 and Cap gene of each serotype), pHelper plasmid (carrier plasmid containing E2A, E4 and VA genes of adenovirus) co-transfect host cells, packaged to obtain targeted USH2A pre-mRNA exon 13 splice skipping AAV-U7 snRNA virus.
  • the specific operation process is as follows:
  • the corresponding Oligo DNA sense strand and antisense strand were synthesized respectively, and the two ends were added with similar Sticky ends cleaved by Type IIs restriction endonuclease recognition sites.
  • the pAAV-U7 snRNA plasmid that induces splicing skipping at the specific site of exon 13 of USH2A pre-mRNA is named according to the snRNA number corresponding to the recognition domain sequence, such as pAAV-U7 snRNA#25, etc.
  • the pAAV-U7snRNA plasmid was obtained by inserting the target gene (the U7-snRNA gene expression box targeting the splicing skipping of exon 13 of USH2A pre-mRNA) and replacing the gene sequence between the AAV2-ITR domains of the pAAV-CMV plasmid carrier.
  • kit instructions and standard cell operation procedures are packaged to obtain the AAV-U7 snRNA virus that targets and induces the splicing skipping of exon 13 of USH2A pre-mRNA.
  • HEK293/293T cells were inoculated into 100 mm cell culture dishes, the medium was DMEM medium with 10% FBS, and transfected when the confluence reached 80%-90%. 3 hours before transfection, discard the old medium and replace with fresh medium.
  • transfection simultaneously prepare pAAV-U7 snRNA plasmid, pRC plasmid, pHelper plasmid and PEI (polyethyleneimine) transfection reagent according to the following system, and add them dropwise to the culture dish. After adding the PEI transfection mixture, gently shake the culture dish to distribute the transfection reagent evenly, and place the medium in a 37°C, 5% CO 2 incubator for cultivation.
  • PEI polyethyleneimine
  • PEI transfection system pAAV plasmid (1 ⁇ g/ ⁇ l), 6 ⁇ L; pRC1/2/5/6 plasmid (1 ⁇ g/ ⁇ l); (pRC plasmid capsid gene determines serotype), 6 ⁇ L; pHelper plasmid (1 ⁇ g/ ⁇ l), 6 ⁇ L; serum-free DMEM medium, 500 ⁇ L; PEI (1 mg/mL), 110 ⁇ L
  • Treatment method vortex mixing several times, incubate at room temperature for 5 min.
  • the target gene fragment inserted between the pAAV-U7 snRNA plasmid AAV2-ITR domain should be less than 2.5kb, it can be inserted into multiple U7-snRNA gene expression cassettes (5'-mouse U7 promoter-smOPT sequence, U7 snRNA scaffold-snRNA gene-specific 3'box-3'), so as to ensure the expression of U7 snRNA under the same amount of AAV virus particles, the gene sequence length is about 450bp, then preferably pAAV-U7 snRNA plasmid Carrying 1-5 U7-snRNA gene expression cassettes in the pAAV-U7 snRNA plasmid, multiple U7-snRNA gene expression cassettes in the pAAV-U7 snRNA plasmid may have the same recognition domain or combination of recognition domains, or may have different or Combinations of recognition domains that are not identical.
  • the capsid protein of the AAV can be from natural sources, or can be a variant based on natural source capsid proteins, or undergo directed evolution , or carry out reasonable modification of amino acids/peptides (codon optimization, chimeric functional peptides of different serotypes, etc.), etc., to improve the characteristics of tissues and organs such as tropism, immunogenicity, and transfection efficiency, such as AAV2.5, AAV2i8 , AAV-TT, AAV9.HR, CAM130, etc.
  • the AAV capsid protein of natural origin can be derived from animal body, also can be derived from plant, and the AAV capsid protein derived from animal body can be derived from human body (such as AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8 and AAV9, etc.), can also be derived from non-human primates (such as AAVrh.8, AAVrh.10 and AAVrh.43), or from vertebrates such as mice and pigs , can also be derived from insects.
  • the AAV ITR serotype should be consistent with the Rep gene serotype, and may not be consistent with the Cap gene serotype.

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Abstract

提供了一种核苷酸及其应用，具体提供了一种特异性结合USH2A pre-mRNA第13号外显子的核酸分子，还提供了所述核酸分子的制备方法和用途。核酸分子具有特异性结合USH2A pre-mRNA第13号外显子的3'段序列或其片段的能力，可以靶向干扰pre-mRNA 剪接，并且可以增加USH2A pre-mRNA第13号外显子单跳读的比例，在显著提升效率的同时，可以保证安全性。

Description

一种核苷酸及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种核苷酸及其应用。
背景技术
Usher综合征(Usher Syndrome)是一类遗传性疾病，又称耳聋-色素性视网膜炎综合征，其特征是不同程度的先天性感音神经性耳聋，以及色素性视网膜炎(RP)引起的进行性视力丧失。其中，USH2A基因突变是II型Usher综合征的最常见原因，涵盖超过50％的Usher综合征患者。同时，USH2A基因的突变也是导致非综合征性视网膜色素变性(NSRP)的重要原因之一。USH2A基因的第13号外显子、第50号外显子和第40号内含子的突变会引发Usher综合征。迄今为止已经鉴定出超过1000个分布在整个USH2A基因中的致病性突变，其中的第13号外显子是USH2A基因中突变最频繁的外显子，约占35％。USH2A编码区长度约为15.6kb，目前的常规基因治疗递送方法(如重组慢病毒、重组腺相关病毒等)难以包装如此庞大的编码序列，导致难以通过直接递送USH2A进行治疗。
因此，本领域急需一种能够提高外显子跳读的效率，保证治疗安全性的USH2A基因突变相关疾病的治疗方法。
发明内容
一方面，本申请提供了一种核酸分子，所述核酸分子具有特异性结合USH2A pre-mRNA第13号外显子的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753。
一方面，本申请提供了一种核酸分子，所述核酸分子具有特异性结合SEQ ID NO：1所示序列或其片段的能力。
一方面，本申请提供了一种核酸分子，所述核酸分子与SEQ ID NO：1所示序列中的16个或更多的连续核苷酸互补。
一方面，本申请提供了一种核酸分子，所述核酸分子包含SEQ ID NO：1所示序列、或其互补序列中的16个或更多的连续核苷酸。
一方面，本申请提供了一种基因表达盒，所述基因表达盒包含或编码如本申请所述的核酸分子的核苷酸序列，以及任选的表达调控元件。
一方面，本申请提供了一种载体，所述载体包含或编码本申请所述的核酸分子的核苷酸序列，和/或本申请所述的基因表达盒的核苷酸序列。
一方面，本申请提供了一种病毒颗粒，所述病毒颗粒包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，和/或本申请所述的载体。
一方面，本申请提供了一种细胞，所述细胞包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，和/或本申请所述的病毒颗粒。
一方面，本申请提供了一种药物组合物，所述药物组合物包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，本申请所述的病毒颗粒，和/或本申请所述的细胞，以及任选的药学可接受的载剂。
一方面，本申请提供了一种试剂盒，所述试剂盒包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，本申请所述的病毒颗粒，本申请所述的细胞，和/或本申请所述的药物组合物。
一方面，本申请提供了一种制备本申请所述的核酸分子的方法，包含表达和/或合成能够结合USH2A第13号外显子的3’段序列或其片段的所述核酸分子，所述USH2A第13号外显子的3’段序列的基因组定位为Chr1：216246563-216246753。
一方面，本申请提供了一种抑制USH2A pre-mRNA第13号外显子表达和/或功能的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种使USH2A pre-mRNA第13号外显子剪接跳跃的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种制备缺失第13号外显子的成熟USH2A mRNA的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种降低包含第13号外显子表达产物的Usherin蛋白水平的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种制备不包含第13号外显子表达产物的Usherin蛋白和/或增加不包含第13号外显子表达产物的Usherin蛋白数量的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物 和/或本申请所述的试剂盒。
一方面，本申请提供了一种恢复突变Usherin蛋白的功能的方法，包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗USH2A基因突变引发的疾病。
本申请提供了一种具有特异性结合USH2A pre-mRNA第13号外显子的3’段序列或其片段的能力的核酸分子。目前有针对外显子跳读的反义寡核苷酸(AONs，Antisense oligonucleotides)的技术。然而，现有的AON在促进第13号外显子跳读的同时，也促进第12号外显子与第13号外显子共同跳读，甚至有的AON处理导致的全是第12号外显子与第13号外显子双跳读，而没有单跳读。而第12号外显子全长196bp，非3整数倍，缺失会导致移码突变以及USH2A蛋白失活。例如，本申请所述的USH2A的外显子12为人的USH2A外显子12。例如，本申请所述的USH2A的外显子13为人的USH2A外显子13。本申请提供的寡核苷酸可以靶向干扰pre-mRNA剪接，并且可以增加第13号外显子单跳读的比例，在显著提升效率的同时，可以保证安全性。
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。对附图简要说明如下：
图1显示的是，本申请U7-snRNA的结构和作用示意图。
图2显示的是，不同靶位点的AON诱导USH2A pre-mRNA第13号外显子剪接跳跃效果。
图3A-3B显示的是，不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃效果。
图4A-4B显示的是，不同靶位点的U7 snRNA组合诱导USH2A pre-mRNA第13号外显子剪接跳跃效率。
图5显示的是，带有hnRNP A1的snRNA载体示意图。
图6A-6B显示的是，U7-hnRNP A1-snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃效率。
图7显示的是，不同靶点的snRNA高效诱导USH2A pre-mRNA第13号外显子单独剪接跳跃效率。
图8A显示的是，在WERI细胞中检测化学合成的U7 snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃的效率。泳道1：50pmol化学合成和修饰的U7-snRNA#30-#4；泳道2：50pmol化学合成和修饰的U7-snRNA#26-#16；泳道3：50pmol AON1；泳道4：50pmol AON2；泳道5：EGFP；泳道6：GL DNA Marker 2000。图8B显示的是，RT-PCR电泳条带定量分析柱状图。▲E12-E13表示同时剪接跳过第12号外显子和第13号外显子的USH2A mRNA，总▲表示剪接跳过第13号外显子或同时剪接跳过第12号外显子和第13号外显子的USH2A mRNA总和。
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
在本申请中，术语“分离”通常是指包括从天然来源中存在的其他物质中分离的分子。例如分离的核酸可以是指从天然来源中存在的其他物质中分离的核酸分子。例如，本申请中分离的分子可以是人工的分子，例如人工合成的分子。
在本申请中，术语“表达调控元件”通常是指针对基因的转录和翻译和/或控制蛋白质在例如期望的宿主细胞中体内表达而提供的序列。
在本申请中，术语“病毒颗粒”通常是指一种核酸载体。例如，病毒颗粒可以是病毒载体，可以用于充当核酸递送媒介物，包含包装在病毒颗粒内的载体基因组(例如DNA和/或RNA)。
在本申请中，术语“反义寡核苷酸”通常是指具有允许与靶核酸的对应片段杂交的核碱基序列的单链寡核苷酸。
在本申请中，术语“核酸”通常是指单体核苷酸组成的分子。核酸包括但不限于核糖核酸(RNA)、脱氧核糖核酸(DNA)、单链核酸、双链核酸、小干扰核糖核酸(siRNA)和微RNA(miRNA)。
在本申请中，术语“核小RNA”通常是指一种小的核酸分子。例如，核小RNA可以参与真核生物细胞核中RNA的加工。例如，核小RNA可以与相关蛋白结合在一起成为小胞核核糖核蛋白(snRNPs即small nuclear ribonucleoproteins)，参与信使RNA前体(pre-mRNA)的剪接。
在本申请中，术语“互补”或“碱基互补性”通常是指反义寡核苷酸的核酸碱基与靶核酸中的相应核酸碱基进行准确碱基配对(即杂交)的能力，且由相应核酸碱基之间的沃森-克里克(Watson-Crick)相互作用力结合介导。
在本申请中，术语“基因表达盒”通常是指可在特定限制性位点或通过同源重组插入核酸或多核苷酸中的DNA区段。例如，基因表达盒包含编码目标核酸的多核苷酸片段。
在本申请中，术语“基因组定位”通常是指描述目的DNA区域的染色体坐标。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
在本申请中，术语“剪接跳跃”通常是指RNA分子在加工过程中，被剪接跳跃的序列不被包含在加工后的RNA分子中。例如，通过剪接跳跃，一部分序列可以被跳读。例如，通过剪接跳跃，一部分序列可以不被包含在剪接后的RNA分子中。
在本申请中，术语“经修饰的寡核苷酸”通常是指包含至少一个修饰的核苷间键、修饰的糖和/或修饰的核碱基的寡核苷酸。在本申请中，术语“2'-O-甲氧基乙基”(又为2’-MOE和2’-OCH2CH2-OCH3以及MOE)通常是指呋喃糖环的2'位的O-甲氧基-乙基修饰。2’-O-甲氧基乙基修饰的糖是修饰的糖。在本申请中，术语“2'-MOE核苷”(又为2'-O-甲氧基乙基核苷)通常是指包含MOE修饰的糖部分的核苷。在本申请中，术语“2'-取代的核苷”通常是指在呋喃糖环的2'位处包含除H或OH以外的取代基的核苷。在某些实施方案中，2'-取代的核苷包括具有双环糖修饰的核苷。在本申请中，术语“5-甲基胞嘧啶”通常是指使用连接至5’位置的甲基修饰的胞嘧啶。5-甲基胞嘧啶是修饰的核碱基。
在本申请中，术语“经修饰的磷酸键”通常是指相对于来自天然存在的核苷间键(即，磷酸二酯核苷间键)，经修饰的磷酸键存在的取代或任何变化。
在本申请中，术语“茎环结构域”通常是指核酸分子自身通过碱基配对而形成二级结构。例如，通过碱基配对形成的双链部分为“茎部”，配对碱基之间的序列则形成“环部”。
在本申请中，术语“连续”通常是指彼此紧邻的两个或更多个核碱基。
在本申请中，术语“外显子”通常是指存在于mRNA成熟形式中的基因的一部分。
在本申请中，术语“载体”通常是指能够转运与它连接的另一核酸的核酸分子。
在本申请中，术语“Cas酶”通常是指CRISPR相关核酸酶，一种DNA核酸内切酶。例如其可在特定的DNA序列处形成双链断裂。Cas核酸酶通常可以与CRISPR序列互补，能够使用CRISPR序列作为指导(guide)，从而识别和切割特定的DNA链。
在本申请中，术语“pre-mRNA”通常是指前体mRNA。例如，初级转录物是通过DNA
转录合成的单链核糖核酸产物，所述转录物可以未经修饰。例如，前体mRNA中外显子和内含子的基因组结构维持不变。
在本申请中，术语“sm蛋白”通常是指能够结合snRNAs形成小核核糖核蛋白复合物snRNP的蛋白或其变体。
在本申请中，术语“USH2A”通常是指一种基因，其编码Usherin蛋白。例如，USH2A(Usher Syndrome Type-2A)的NCBI基因登录号可以是7399。本申请中，USH2A可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
在本申请中，术语“施用”通常是指向动物提供药剂，且包括但不限于由医学专业人员施用及自行施用。
发明详述
一方面，本申请提供了一种核酸分子，所述核酸分子可以具有特异性结合USH2A pre-mRNA外显子13的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753(对应于NCBI数据库GRch38版本)。例如，本申请的所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246626、Chr1：216246563-216246621、Chr1：216246563-216246624、Chr1：216246563-216246617、Chr1：216246563-216246591、Chr1：216246563-216246596、Chr1：216246563-216246593、Chr1：216246563-216246590、和/或Chr1：216246563-216246586。例如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246626、Chr1：216246567-216246626、Chr1：216246570-216246626、Chr1：216246594-216246626、Chr1：216246598-216246626、和/或Chr1：216246603-216246626。例如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246621、Chr1：216246567-216246621、Chr1：216246570-216246621、Chr1：216246594-216246621、Chr1：216246598-216246621、和/或Chr1：216246603-216246621。例 如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246624、Chr1：216246567-216246624、Chr1：216246570-216246624、Chr1：216246594-216246624、Chr1：216246598-216246624、和/或Chr1：216246603-216246624。例如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246617、Chr1：216246567-216246617、Chr1：216246570-216246617、Chr1：216246594-216246617、Chr1：216246598-216246617、和/或Chr1：216246603-216246617。例如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246593、Chr1：216246567-216246593、和/或Chr1：216246570-216246593。例如，本申请所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246626，其序列可以如SEQ ID NO：39所示。例如，可以是70％、80％、90％、95％、98％或99％以上的碱基配对的结合。例如，可以是对突变序列的结合。例如，所述结合可以包含1bp、2bp、3bp、4bp或5bp错配。
USH2A基因第13号外显子可以包含突变，突变可以包括c.2802T＞G(p.Cys934Trp，中国患者频率最高突变)、c.2299delG(p.E767SfsX21，欧美患者频率最高突变)、c.2276G＞T(氨基酸变化:p.C759F)、C.2522C＞A(p.S841Y)、c.2242C＞T(p.Gln748X)、c.2541C＞A(C847X)、c.2761delC(Leu921fs)和C.2776C＞T(p.R926C)、C.2209C＞T、C.2310delA、c.2391_2392deITG、c.2431A＞T、C.2431_2432delAA、c.2440C＞T、c.2525dup、C.2610C＞A、C.2755C＞T、C.2176T＞C、C.2236C＞G、c.2296T＞C、和/或C.2332G＞T。
一方面，本申请提供了一种核酸分子，所述核酸分子可以具有特异性结合SEQ ID NO：1所示序列或其片段的能力。例如，可以是70％、80％、90％、95％、98％或99％以上的碱基配对的结合。例如，可以是对突变序列的结合。例如，所述结合可以包含1bp、2bp、3bp、4bp或5bp错配。
一方面，本申请提供了一种核酸分子，所述核酸分子可以与SEQ ID NO：1所示序列中的16个或更多的连续核苷酸互补。例如，可以是70％、80％、90％、95％、98％或99％以上碱基配对的互补。例如，可以是对突变序列的互补。例如，所述互补可以包含1bp、2bp、3bp、4bp或5bp错配。
一方面，本申请提供了一种核酸分子，所述核酸分子可以包含SEQ ID NO：1所示序列、或其互补序列中的16个或更多的连续核苷酸。例如，可以是70％、80％、90％、95％、98％或99％以上碱基配对的互补。例如，可以是对突变序列的互补。例如，所述互补可以包含1bp、2bp、3bp、4bp或5bp错配。
例如，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸。例如，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的22个至27个核苷酸。例如，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的15个至27个、16个至27个、18个至27个、20个至27个、22个至27个、25个至27个、15个至25个、16个至25个、18个至25个、20个至25个、22个至25个、15个至22个、16个至22个、18个至22个、20个至22个、15个至20个、16个至20个、18个至20个、15个至18个、或16个至18个核苷酸。例如，识别结构域序列长度可以为16bp以上，进一步可以为18bp-40bp，再进一步可以为20bp-27bp。在一些化学合成和修饰的U7 snRNA实施例中，可沿着靶序列的5’端或/和3’端继续通过反向互补配对延伸识别结构域序列长度，优选延伸后的单个识别结构域序列长度小于或等于40bp。
例如，所述核酸分子可以包含至少16个核苷酸。例如，所述核酸分子可以包含22至27个核苷酸。例如，所述核酸分子可以包含15个至27个、16个至27个、18个至27个、20个至27个、22个至27个、25个至27个、15个至25个、16个至25个、18个至25个、20个至25个、22个至25个、15个至22个、16个至22个、18个至22个、20个至22个、15个至20个、16个至20个、18个至20个、15个至18个、或16个至18个核苷酸。例如所述核酸分子是AON分子。
例如，所述核酸分子可以包含至少60个核苷酸。例如，所述核酸分子可以包含至少65个、至少70个、至少80个、至少90个、至少100个、至少150个、至少200个、或500个核苷酸。例如所述核酸分子是snRNA分子。例如，snRNA分子可以包含更多的元件，而可以具有理论上无限长的长度。在一些实施例中，化学合成的snRNA序列总长度大于等于96bp。在一些实施例中，化学合成的snRNA两侧的3-40个碱基为经过修饰并通过特殊磷酸酯键连接的。
例如，所述核酸分子可以包含互补于选自以下组基因组定位对应的序列：Chr1：216246603-216246626、Chr1：216246598-216246621、Chr1：216246598-216246624、Chr1：216246594-216246617、Chr1：216246570-216246593、Chr1：216246570-216246591、Chr1：216246570-216246596、Chr1：216246570-216246593、Chr1：216246570-216246596、Chr1：216246567-216246590、和Chr1：216246563-216246586。
例如，所述核酸分子可以包含互补于SEQ ID NO:10-21中任一项所示的序列。例如，可以是70％、80％、90％、95％、98％或99％以上碱基配对的的互补。例如，可以是对突变序列 的互补。例如，所述互补可以包含1bp、2bp、3bp、4bp或5bp错配。在一些化学合成和修饰的U7 snRNA实施例中，识别结构域可以与靶位点反向互补配对中可存在0-5个错配核苷酸，例如可以为0-1个。
例如，所述核酸分子可以包含SEQ ID NO:10-21中任一项所示的序列。
例如，所述核酸分子可以包含经修饰的核苷酸。
例如，所述经修饰的核苷酸可以包含选自以下组的修饰：2’-O-烷基、2’-O-甲氧基和/或2’-O-甲氧基乙基。
例如，所述经修饰的核苷酸可以包含选自以下组的修饰：2’-O-甲基和/或2’-O-乙基。
例如，所述核酸分子可以包含至少一个经修饰的核苷酸。在一些实施例中，仅snRNA两侧的1-10、6-80个或者全部的核苷酸都进行了修饰和特殊磷酸酯键连接，所述修饰是一种或两种以上的修饰组合，所述特殊磷酸酯键是一种或者两种以上磷酸酯键的组合。在一些实施例中，所述化学合成的U7 snRNA中所有的核苷酸均通过硫代磷酸酯键相互连接，且均进行了2′-O-甲氧基修饰。在一些实施例中，仅snRNA两侧的3个核苷酸通过硫代磷酸酯键连接，并进行了2′-O-甲氧基修饰。在一些化学合成和修饰的U7 snRNA优选实施例中，U7 snRNA的5’端首个核苷酸优选为腺苷酸(A)，若识别结构域5’端的首个核苷酸不是腺苷酸(A)，则在5’端连接腺苷酸(A)。
例如，所述核酸分子的5’端可以包含至少一个经修饰的核苷酸。例如，所述核酸分子的5’端可以包含1个至3个经修饰的核苷酸。例如，所述核酸分子的5’端可以包含1个、2个、3个、4个或5个经修饰的核苷酸。
例如，所述核酸分子的3’端可以包含至少一个经修饰的核苷酸。例如，所述核酸分子的3’端可以包含1个至3个经修饰的核苷酸。例如，所述核酸分子的3’端可以包含1个、2个、3个、4个或5个经修饰的核苷酸。
本申请所述的核酸分子，所述核酸分子中的核苷酸分子可以包含选自以下组：6’-修饰的双环核苷、5’-修饰的双环核苷、6’-双取代双环核苷、四氢吡喃核苷类似物和/或2'-脱氧2'-氟-β-D-阿拉伯糖核苷酸(2’-FANA修饰的核苷酸)。
例如，所述核酸分子经修饰的磷酸键。
例如，所述核苷酸分子经过选自以下组的磷酸键连接：硫代磷酸酯连接、二硫代磷酸酯连接、磷酸三酯连接、烷基膦酸酯连接、氨基烷基磷酸三酯连接、亚烷基膦酸酯连接、次膦酸酯连接、氨基磷酸酯连接和氨基烷基氨基磷酸酯连接、硫代氨基磷酸酯连接、硫羰基烷基膦酸酯连接、硫羰基烷基磷酸三酯连接、硫代磷酸酯连接、硒代磷酸酯连接和/或硼烷基磷酸 酯连接。
例如，所述经修饰的磷酸键可以包含选自以下组：磷酸二酯键、磷酸三酯键、硫代磷酸酯键(5'O-P(S)O-3O-、5'S-P(O)O-3'-O-和5'O-P(O)O-3'S-)、二硫代磷酸酯键、Rp-硫代磷酸酯键、Sp-硫代磷酸酯键、硼烷磷酸酯键、亚甲基键(甲基亚氨基)、酰胺键(3'-CH 2-CO-NH-5'和3'-CH 2-NH-CO-5')、甲基膦酸酯键、3'-硫代甲缩醛键、(3'S-CH 2-O5')、酰胺键(3'CH 2-C(O)NH-5')、和/或氨基磷酸酯基团。
例如，所述经修饰的磷酸键可以包含选自以下组：硫代磷酸酯键、二硫代磷酸酯键、烷基磷酸酯键、酰胺磷酸酯键和/或硼烷磷酸酯键。
例如，所述经修饰的磷酸键可以包含选自以下组：磷酸二酯键、磷酸三酯键、硫代磷酸酯键(5'O-P(S)O-3O-、5'S-P(O)O-3'-O-和5'O-P(O)O-3'S-)、二硫代磷酸酯键、Rp-硫代磷酸酯键、Sp-硫代磷酸酯键、硼烷磷酸酯键、亚甲基键(甲基亚氨基)、酰胺键(3'-CH 2-CO-NH-5'和3'-CH 2-NH-CO-5')、烷基磷酸酯键、酰胺磷酸酯键、甲基膦酸酯键、3'-硫代甲缩醛键、(3'S-CH 2-O5')、酰胺键(3'CH 2-C(O)NH-5')和/或氨基磷酸酯基团。
例如，所述核酸分子可以包含至少一个经修饰的磷酸键。
例如，所述核酸分子的5’端可以包含至少一个经修饰的磷酸键。例如，所述核酸分子的5’端可以包含1个至3个经修饰的磷酸键。例如，所述核酸分子的5’端可以包含1个、2个、3个、4个或5个经修饰的磷酸键。
例如，所述核酸分子的3’端可以包含至少一个经修饰的磷酸键。例如，所述核酸分子的3’端可以包含1个至3个经修饰的磷酸键。例如，所述核酸分子的5’端可以包含1个、2个、3个、4个或5个经修饰的磷酸键。
例如，所述核酸分子可以包含DNA和/或RNA。
例如，所述核酸分子为单链的或双链的。
例如，所述核酸分子可以包含反义寡核苷酸(antisense oligonucleotide)、shRNA、siRNA、miRNA和/或适体(aptamer)。
例如，所述核酸分子可以包含核小RNA(Small nuclear RNA)。
例如，所述核小RNA可以包含U1核小RNA和/或U7核小RNA。例如，所述核小RNA可以包含U1、U2、U3、U4、U5、U6和/或U7核小RNA。例如，所述核小RNA可以包含U1核小RNA。例如，所述核小RNA可以包含U7核小RNA。
例如，所述核酸分子不结合Cas酶。例如，所述核酸分子可以不包含能够结合Cas酶的结构。例如，所述Cas酶为Cas13酶。
例如，所述核酸分子可以包含茎环结构域或其衍生结构。例如，所述茎环结构域可以包含SEQ ID NO：6所示的序列。
例如，所述核酸分子可以包含能够结合sm蛋白的结构域或其衍生结构。例如，所述能够结合sm蛋白的结构域可以包含SEQ ID NO：5所示的序列。例如，所述能够结合sm蛋白的结构域可以包含优化后的smOPT序列。例如，所述结合sm蛋白的结构域可以包含人源的、鼠源的或猪源的。
例如，所述核酸分子可以包含能够募集剪接调控蛋白的结构域。例如，所述核酸分子可以包含能够结合选自以下组蛋白的结构域：SRSF1(Serine And Arginine Rich Splicing Factor 1)、RBM4(RNA Binding Motif Protein 4)、DAZAP1(DAZ Associated Protein 1)、和SR(Serine And Arginine-Rich Protein)。
例如，所述核酸分子可以包含能够结合hnRNP A1蛋白的结构域。例如，所述能够结合hnRNP A1蛋白的结构域可以包含SEQ ID NO：30所示的序列。
例如，本申请的核酸分子有特异性结合USH2A pre-mRNA第13号外显子的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸，所述核酸分子可以包含至少16个核苷酸。
例如，本申请的核酸分子有特异性结合USH2A pre-mRNA外显子13的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸，所述核酸分子可以包含至少60个核苷酸。
例如，本申请的核酸分子有特异性结合USH2A pre-mRNA外显子13的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸，所述核酸分子可以包含至少60个核苷酸，化学合成的snRNA两侧的3-40个碱基为经过修饰并通过特殊磷酸酯键连接的。
例如，本申请的核酸分子有特异性结合USH2A pre-mRNA外显子13的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸，所述核酸分子可以包含至少60个核苷酸，化学合成的snRNA两侧的3-40个碱基为经过2’-甲氧基修饰并通过硫代磷酸酯键连接的。
例如，本申请的核酸分子有特异性结合USH2A pre-mRNA外显子13的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753，所述核酸分子包含可以与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸，所述核酸分子可以包含至少60个核苷酸，化学合成的snRNA两侧的3个碱基为经过2’-甲氧基修饰并通过硫代磷酸酯键连接的，所述核酸分子可以包含能够结合sm蛋白的结构域或其衍生结构。任选地，所述核酸分子可以包含能够结合hnRNP A1蛋白的结构域。
一方面，本申请提供了一种基因表达盒，所述基因表达盒可以包含或编码本申请所述的核酸分子的核苷酸序列，以及任选的表达调控元件。
一方面，本申请提供了一种载体，所述载体可以包含或编码本申请所述的核酸分子的核苷酸序列，和/或本申请所述的基因表达盒的核苷酸序列。一方面，本申请提供了一种病毒颗粒，所述病毒颗粒可以包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，和/或本申请所述的载体。例如本申请的载体可以是pUC57，还可以选自pAAV-CMV(TAKARA公司，Code No.6650)、慢病毒、转座子和本领域已知的核酸载体。例如，本申请可以通过AAV递送U7 snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃，所述AAV的衣壳蛋白可以是天然来源的，也可以是基于天然来源衣壳蛋白的变体、或进行定向进化、或进行氨基酸/肽段合理改造(密码子优化、嵌合不同血清型功能肽段等)等，提升组织器官亲嗜性、免疫原性、提升转染效率等特性，如AAV2.5、AAV2i8、AAV-TT、AAV9.HR、CAM130等。所述天然来源的AAV衣壳蛋白可以是来源于动物体的，也可以是来源于植物的，所述来源于动物体的AAV衣壳蛋白可以是来源于人体的(如AAV1、AAV2、AAV3、AAV5、AAV6、AAV7、AAV8和AAV9等)，也可以是来源于非人灵长类动物(如AAVrh.8、AAVrh.10和AAVrh.43)，也可以是来源于小鼠、猪等脊椎动物，也可以是来源于昆虫。本发明AAV质粒体系中，例如，AAV ITR血清型应于Rep基因血清型一致，可以与Cap基因血清型可不一致。
一方面，本申请提供了一种细胞，所述细胞可以包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，和/或本申请所述的病毒颗粒。
一方面，本申请提供了一种药物组合物，所述药物组合物可以包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，本申请所述的病毒颗粒，和/或本申请所述的细胞，以及任选的药学可接受的载剂。
例如，所述药物组合物可以包含第一载体和第二载体，所述第一载体可以包含本申请所述的核酸分子，所述第二载体可以包含可以具有特异性结合USH2A pre-mRNA第13号外显 子及其两侧区域或其片段的能力的核酸分子。例如，第二载体可以包含具有特异性结合选自USH2A pre-mRNA第12号内含子-第13号外显子-第13号内含子的能力的核酸分子，例如可以选自第13号外显子及两侧临近靶区域。例如可以选自chr1:216246563-216247246对应的pre-mRNA区域。例如，可以是靶点序列不完全一样的两个或两个以上的U7-snRNA的组合使用。
例如，所述USH2A pre-mRNA第13号外显子的两侧区域可以包含USH2A pre-mRNA第12号内含子和/或第13号内含子。
一方面，本申请提供了一种试剂盒，所述试剂盒可以包含本申请所述的核酸分子，本申请所述的基因表达盒的核苷酸序列，本申请所述的载体，本申请所述的病毒颗粒，本申请所述的细胞，和/或本申请所述的药物组合物。
一方面，本申请提供了一种制备本申请所述的核酸分子的方法，可以包含表达和/或合成能够结合USH2A第13号外显子的3’段序列或其片段的所述核酸分子，所述USH2A第13号外显子的3’段序列的基因组定位为Chr1：216246563-216246753。
一方面，本申请提供了一种抑制USH2A pre-mRNA第13号外显子表达和/或功能的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。例如，抑制USH2A pre-mRNA第13号外显子表达和/或功能可以是指使得成熟的USH2A mRNA中可以不包含第13号外显子表达产物的区域。
一方面，本申请提供了一种使USH2A pre-mRNA第13号外显子剪接跳跃的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种制备缺失第13号外显子的成熟USH2A mRNA的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种降低可以包含第13号外显子表达产物的Usherin蛋白水平的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。
一方面，本申请提供了一种制备可以不包含第13号外显子表达产物的Usherin蛋白和/或增加不包含第13号外显子表达产物的Usherin蛋白数量的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的 药物组合物和/或本申请所述的试剂盒。例如，本申请制备的Usherin蛋白中不包含第13号外显子表达的多肽或其突变体。
一方面，本申请提供了一种恢复突变Usherin蛋白的功能的方法，可以包含提供本申请所述的核酸分子、本申请所述的载体、本申请所述的病毒颗粒、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。例如，通过剪接跳跃第13号外显子，本申请制备的Usherin蛋白保持或基本保持与健康者体内野生型Usherin蛋白的功能。例如，表达突变Usherin蛋白的受试者的视网膜色素变性的症状、先天性退行性神经性耳聋、前庭功能障碍等疾病得到缓解。
一方面，本申请提供了本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗USH2A基因突变引发的疾病。例如，所述疾病可以包含眼病和/或耳病。例如，所述疾病可以包含Usher综合症。例如，所述疾病可以包含Ⅱ型Usher综合症。
一方面，本申请提供了一种预防和/或治疗USH2A基因突变引发的疾病的方法，可以包含施用本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒。例如，所述疾病可以包含眼病和/或耳病。例如，所述疾病可以包含Usher综合症。例如，所述疾病可以包含Ⅱ型Usher综合症。
一方面，本申请提供了本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物和/或本申请所述的试剂盒，其用于预防和/或治疗USH2A基因突变引发的疾病。例如，所述疾病可以包含眼病和/或耳病。例如，所述疾病可以包含Usher综合症。例如，所述疾病可以包含Ⅱ型Usher综合症。
USH2A通常是指编码Usherin的基因。USH2A定位于1q41，其在基因组中的跨度超过800kb，编码一个大型跨膜蛋白Usherin，其锚定在视网膜感光细胞和内耳毛细胞的质膜上，是纤毛发育和维持必不可少的组分。在视网膜中，Usherin是USH2复合物的重要部分，被认为在稳定光感受器的外节段发挥作用。USH2A具有2个亚型，在视网膜细胞中主要的亚型含有72个Exon，编码区长度约为15.6kb。Usherin蛋白的胞外部分包含许多重复的结构域，包括10个Laminin EGF-like(LE)结构域和35个Fibronectin type 3(FN3)结构域。人USH2A第13号外显子长度为642bp，编码着第723～936位氨基酸，为Usherin蛋白中10个LE结构域中的4个。
USH2A基因的第13号外显子、第50号外显子和第40号内含子的突变会引发Usher综合征。迄今为止已经鉴定出超过1000个分布在整个USH2A基因中的致病性突变，其中的第 13号外显子是USH2A基因中突变最频繁的外显子，约占35％。USH2A基因第13号外显子的突变，包括c.2802T＞G(p.Cys934Trp，中国患者频率最高突变)、c.2299delG(p.E767SfsX21，欧美患者频率最高突变)、c.2276G＞T(氨基酸变化:p.C759F)、C.2522C＞A(p.S841Y)、c.2242C＞T(p.Gln748X)、c.2541C＞A(C847X)、c.2761delC(Leu921fs)和C.2776C＞T(p.R926C)、C.2209C＞T、C.2310delA、c.2391_2392deITG、c.2431A＞T、C.2431_2432delAA、c.2440C＞T、c.2525dup、C.2610C＞A、C.2755C＞T、C.2176T＞C、C.2236C＞G、c.2296T＞C、C.2332G＞T。
USH2A编码区长度约为15.6kb，常规的基因治疗递送方法(如重组慢病毒、重组腺相关病毒等)难以包装如此庞大的编码序列，因此难以通过直接递送USH2A进行治疗。小鼠USH2A的第12号外显子与人USH2A第13号外显子同源，长度均为642bp，移除该外显子并没有造成后续的移码突变。有研究显示，在敲除了小鼠USH2A的第12号外显子后，Usherin依然能够正确定位并且行使正常的功能。对于包含致病性突变的人USH2A第13号外显子，可以利用一系列手段使其发生跳读进行治疗。
通过CRISPR/Cas系统进行基因组DNA的编辑直接删除第13号外显子，或者破坏RNA剪接相关的位点。使用片段删除可能存在风险，如染色体重排、病毒整合、反向重新整合，以及长时间表达CAS系统或者基于相对庞大的基因组背景进行两个gRNA诱导的双切的脱靶概率可能较高。通过使用单碱基编辑器修改上述剪接相关位点的关键碱基，亦可促进外显子跳读。但是现有的单碱基编辑器可能无法通过单个AAV载体装载，并且受PAM、编辑窗口以及碱基转换类型的限制，可能在剪接相关位点附近没有合适的gRNA。通过反义寡核苷酸(AONs，Antisense oligonucleotides)靶向干扰pre-mRNA剪接，促进外显子跳读的效率较高。AON在促进第13号外显子跳读的同时，也可能促进第12号外显子与第13号外显子共同跳读，有的AON处理可能导致的全是双跳读。而第12号外显子全长196bp，非3整数倍，缺失会导致移码突变，USH2A蛋白失活。通过U7 snRNA靶向干扰pre-mRNA剪接，促进外显子跳读的效率比AON更高，而且增加第13号外显子单跳读的比例，在显著提升效率的同时，保证了安全性。本申请发现的效果，即靶向本申请区域的snRNA分子促进外显子跳读效率，高于同靶点的AON分子，该效果在已知的现有技术中是非显而易见的。例如，本领域靶向Chr1：216247218-216247241的AON分子，其跳读促进外显子效率效果显著优于靶向该区域的snRNA分子。细胞内有核小RNA(small nuclearRNA，snRNA)，它是真核生物转录后加工过程中RNA剪接体(spliceosome)的主要成分，通过与snRNP蛋白结合参与mRNA前体的加工过程。其长度在哺乳动物中约为100-215个核苷酸，共分为7类，由于含U丰富，故编号为U1～U7。修饰后的U7SnRNA是通过将U7 snRNA的非规范Sm结合位点替换为衍生自 主要剪接体U snRNPs的共有序列，将U7 snRNA的5'区的组蛋白结合序列改变为待修饰基因的互补序列，可以通过靶向外显子来诱导外显子的剪接跳跃。
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核苷酸、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
合成snRNA
snRNA的骨架合成。野生型U7 snRNA包括茎环结构(scafford)、U7特异性的Sm序列(AAUUUGUCUAG，SEQ ID NO：2)和识别结构域(与组蛋白pre-mRNA互补)。本申请的U7 snRNA可以在NCBI上小鼠野生型U7 snRNA的基因序列(NCBI Reference Sequence:NR_024201.3)的基础上，其中U7特异性Sm结合位点(AATTTGTCTAG，SEQ ID NO：3)被替换为优化的共有Sm序列，即SmOPT(AATTTTTGGAG，SEQ ID NO：4；或AAUUUUUGGAG，SEQ ID NO：5)，SmOPT序列的5’端的原识别结构域更换为与USH2A pre-mRNA特定靶位点反向互补配对的识别结构域，SmOPT序列的3’端保留U7原茎环结构序列(CAGGUUUUCUGACUUCGGUCGGAAAACCCCU，SEQ ID NO：6)。
图1显示的是，本申请U7-snRNA的结构和作用示意图。靶向诱导USH2A pre-mRNA第13号外显子的U7 snRNA识别结构域序列与选自USH2A pre-mRNA第12号内含子-第13号外显子-第13号内含子的靶序列反向互补配对，靶序列可以选自USH2A pre-mRNA第13号外显子的3’段序列靶区域。snRNA的识别结构域序列优选长度为16bp以上，进一步优选为18bp-40bp，再进一步优选为20bp-27bp。
具体操作过程：首先，通过全基因合成的方式，合成包含基因序列——U7-snRNA基因表达盒骨架(5’-小鼠U7启动子-smOPT序列-U7 snRNA scafford-snRNA基因特异性3’盒-3’)的pUC57载体。其中U7启动子与smOPT之间加入2个Tpye IIs型限制性内切酶识别位点(如BsaI、AarI、BsmBI等)，以方便后续切除、替换以及插入其他识别结构域序列。snRNA基因特异性3’盒为小鼠基因组(GenBank:X54748.1)U7 snRNA基因3’端后，包含“GTCTACAATGAAA (SEQ ID NO：7)”的序列，参与pre-snRNA的加工，优选U7 snRNA基因3’端后序列长度为28-131bp的基因片段，进一步优选序列长度为106bp。
构建靶向靶区域的U7 snRNA载体
根据表1A中的snRNA识别结构域序列对应的转录前DNA序列，分别合成对应的Oligo  DNA。Oligo DNA正义链为识别结构域序列对应的DNA序列，并且5’加CCGCA，反义链为识别结构域序列的反义互补序列5’加AATT并且3’加T。例如，识别结构域序列为5’-NNN-3’，则合成的Oligo DNA正义链为5’-CCGCA NNN-3’，反义链为5’-AATT NNNT-3’。
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl：Oligo-F(100μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合，95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切回收的线性化pUC57-U7 snRNA骨架质粒进行T4连接酶连接。连接产物进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证，获得用于诱导USH2A第13号外显子剪接跳跃的U7 snRNA载体。提纯质粒，保存于-20℃备用。
合成AON
可以根据表1B中的USH2A pre-mRNA第13号外显子的靶向区域，设计和合成可以与该区域的反义寡核苷酸AON的序列。
表1A snRNA的识别结构域序列
Figure PCTCN2023070867-appb-000001
Figure PCTCN2023070867-appb-000002
表1B AON的识别序列
Figure PCTCN2023070867-appb-000003
U7-snRNA的化学合成和修饰
与寡核苷酸类似，U7 snRNA也可以通过直接化学合成的方式，产生包含引导序列、smOPT和U7 snRNA scafford的RNA。体外合成的U7 snRNA可以通过特定修饰使其耐受核酸酶降解，或者增加对靶序列的亲和力。
本实施例通过化学合成了U7 snRNA，其5‘和3'末端的3个碱基各进行2'甲氧基(2’-OME)修饰和硫代修饰，以增加核酸酶抗性。以snRNA#25、snRNA#26为例，化学合成的snRNA序列和修饰如下(*表示硫代磷酸化骨架，m表示2’-甲氧基修饰，下划线表示与靶序列反向互补配对的识别结构域，斜体表示smOPT序列)：
化学合成和修饰的U7-snRNA#25：
Figure PCTCN2023070867-appb-000004
Figure PCTCN2023070867-appb-000005
化学合成和修饰的U7-snRNA#25双向延长：
Figure PCTCN2023070867-appb-000006
化学合成和修饰的U7-snRNA#26：
Figure PCTCN2023070867-appb-000007
化学合成和修饰的U7-snRNA#26单向延长：
Figure PCTCN2023070867-appb-000008
本申请U7 snRNA中的一个或两个以上的核苷酸进行了2′-O烷基、2′-O-甲氧基、2′-O-甲氧基乙基中的一种或者两种以上修饰，所述2′-O烷基优选为2′-O-甲基修饰。snRNA的核苷酸连接的磷酸键可以是通过特殊磷酸酯键连接，所述特殊磷酸酯键为硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、酰胺磷酸酯键(phosphoroamidate)、硼烷磷酸酯(boranophosphate)键、手性连接磷(chiral linkage phosphorus)中的一种或者两种以上组成。在一些实施例中，仅snRNA两侧的1-10、6-80个或者全部的核苷酸都进行了修饰和特殊磷酸酯键连接，所述修饰是一种或两种以上的修饰组合，所述特殊磷酸酯键是一种或者两种以上磷酸酯键的组合。在一些实施例中，所述化学合成的U7 snRNA中所有的核苷酸均通过硫代磷酸酯键相互连接，且均进行了2′-O-甲氧基修饰。在一些实施例中，仅snRNA两侧的3个核苷酸通过硫代磷酸酯键连接，并进行了2′-O-甲氧基修饰。在一些化学合成和修饰的U7 snRNA优选实施例中，U7 snRNA的5’端首个核苷酸优选为腺苷酸(A)，若识别结构域5’端的首个核苷酸不是腺苷酸(A)，则在5’端连接腺苷酸(A)。在一些化学合成和修饰的U7 snRNA实施例中，识别结构域与靶位点反向互补配对中可存在0-5个错配核苷酸，优选为0-1个。识别结构域序列长度优选长度为16bp以上，进一步优选为18bp-40bp，再进一步优选为20bp-27bp。在一些化学合成和修饰的U7 snRNA实施例中，可沿着靶序列的5’端或/和3’端继续通过反向互补配对延伸识别结构域序列长度，优选延伸后的单个识别结构域序列长度小于或等于40bp。在一些实施例中，优选化学合成的snRNA序列总长度大于等于96bp。在一些实施例中，优选化学合成的snRNA 两侧的3-40个碱基为经过修饰并通过特殊磷酸酯键连接的。
实施例2
用于定量评价USH2A第13号外显子剪接跳跃效率的报告载体
将RG left-USH2A EXON13 mut-RG right序列(5’端和3’端分别加入AgeI和EcoRI酶切位点)通过全基因合成的方式获取，通过对合成序列、pX601质粒(Addgene，61591)进行限制性内切酶AgeI和EcoRI酶切、电泳、切胶回收和连接，将合成的序列插入pX601载体的AgeI和EcoRI酶切位点之间，替换原载体的SaCas9基因序列，获得报告载体。进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证，获得提纯报告载体质粒，保存于-20℃备用。
报告载体结构为：pCMV-RG left-USH2A EXON13 mut-RG right，RG表示报告功能基因(reporter gene)，RG left表示没有报告功能的报告基因5’端前半部分，RG right表示没有报告功能的报告基因3’端后半部分，RG left和RG right串联表达可正常行使完整报告基因功能。本发明实施例中报告基因为绿色荧光基因EGFP，则载体结构为pCMV-EGFP left-Exon13 mut-EGFP right。EXON13 mut表示包含致病突变的USH2A第13号外显子，及其上下游内含子序列(上游内含子序列为人USH2A基因第12号内含子的5’端204bp和3’端490bp组合的基因序列；下游内含子序列为人USH2A第13号内含子的5’端703bp和3’端216bp组合的基因序列)。本发明实施例中所述的USH2A第13号外显子的致病突变可以为c.2299delG或c.2802T＞G或任意突变，则获得的载体结构分别为pCMV-EGFP left-Exon13 c . 2299delG-EGFP right、pCMV-EGFP left-Exon13 c . 2802T＞G-EGFP right。一些实施例中的突变还可以是或包括c.2276G＞T、C.2522C＞A、c.2242C＞T、c.2541C＞A、c.2761delC和C.2776C＞T等。
RG left，例如EGFP left序列为：
Figure PCTCN2023070867-appb-000009
RG right，例如EGFP right序列为：
Figure PCTCN2023070867-appb-000010
实施例3
293T细胞按一定量接种至24孔板，使得24小时后细胞汇合度达到约80％。使用Lipofectamine2000将合成的100pmol反义寡核苷酸AON#20、AON#24、AON#25、AON#31(合成的AON所有核苷单体都进行2′-O-甲氧基修饰和硫代磷酸化修饰)，分别与报告质粒pCMV-EGFP left-Exon13 mut-EGFP right共转染293T细胞。转染后的细胞继续培养48-72小时，使用胰酶消化成单细胞，随后使用流式细胞仪检测不同AON组的GFP阳性率(即USH2A pre-mRNA第13号外显子被诱导剪接跳跃的细胞比例)以及GFP阳性细胞的平均FITC强度(即GFP细胞中USH2A pre-mRNA第13号外显子剪接跳跃的平均水平)，下表2和图2显示的是，靶向两个区域的AON诱导USH2A pre-mRNA第13号外显子剪接跳跃效果。从实验结果表明，靶向3’端区域的AON效果显著优于靶向其它区域，例如#20区域的AON。
表2靶向两个区域的AON诱导USH2A pre-mRNA第13号外显子剪接跳跃效果
Figure PCTCN2023070867-appb-000011
实施例4
293T细胞按一定量接种至24孔板，使得24小时后细胞汇合度达到约80％。选用Lipofectamine2000将pCMV-EGFP left-Exon13 mut-EGFP right和靶向USH2A pre-mRNA的pUC57-U7 snRNA质粒共转染293T细胞(载体质量比例为100ng:400ng)，使用单独转染报告质粒(Report，报告组)、共转染报告质粒和pUC57-U7Scramble(SC组)的293T细胞分别作为两种阴性对照，不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时，使用胰酶消化成单细胞，随后使用流式细胞仪检测不同U7 snRNA组的GFP阳性率(即USH2A第13号外显子被诱导剪接跳跃的细胞比例)。本实施例检测了不同实验组的平均FITC强度，即GFP阳性细胞FITC荧光强度的平均值，以及GFP阳性率，阳性细胞的GFP蛋白表达量。
本实施例比对了不同靶位点的U7-snRNA诱导剪接跳跃的效率。下表3和图3A-3B显示的是，不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃效果。结果显示所有靶向USH2A pre-mRNA第13号外显子的3’段序列的U7-snRNA均能在报告基因细胞中诱导USH2A第13号外显子的剪接跳跃，USH2A诱导剪接跳跃的效率 高。
表3不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃细胞比例
Figure PCTCN2023070867-appb-000012
实施例5
化学合成的snRNA高效诱导USH2A pre-mRNA第13号外显子单独剪接跳跃
人源宿主细胞按6×10 5/孔接种至24孔板，本实施例选用的人源视网膜神经细胞为WERI-Rb-1细胞(视网膜神经细胞系)。用Lipofectamine2000将体外合成的100pmol U7-snRNA#24、#25、#26、#27、#28、#29、#30、#33、#34转染WERI细胞。转染后的细胞继续培养72小时，随后提取每个实验组细胞的RNA，反转录获得cDNA，通过引物AGCCTTTCCGCCAAGGTGATC(SEQ ID NO：34)和CACAACGTTGCCCAGCAATGG(SEQ ID NO：35)进行RT-PCR实验，检测成熟的USH2A mRNA是否存在外显子的剪接跳跃，电泳结果如图7所示。结果显示，U7-snRNA#24-34均能高效诱导第13号外显子的剪接跳跃，且几乎未见的第13号外显子和第12号外显子共同剪接跳跃，可知靶向3’端区域的U7 snRNA可高效诱导USH2A pre-mRNA第13号外显子的单独剪接跳跃，具有较高的安全性。
实施例6
不同靶位点的U7-snRNA组合介导的USH2A第13号外显子剪接跳跃效果
构建U7-snRNA多靶点组合载体。根据Golden Gate Assembly技术，以不同U7 snRNA质粒为模板，PCR扩增U7 snRNA cassette(表达盒)同时通过引物在扩增子的两端引入额外的5’侧翼碱基和正确方向的BsaI酶切位点，使得相邻的不同U7 snRNA cassette通过BsaI酶切后产生特异的互补粘性末端，首尾U7 snRNA cassette则通过BsaI酶切后产生与HindIII+NotI 酶切线性化骨架载体相同的粘性末端。最后使用
Figure PCTCN2023070867-appb-000013
Golden Gate Assembly Kit
Figure PCTCN2023070867-appb-000014
(NEB#E1601)将上述PCR产物以及HindIII+NotI酶切回收的pUC57-U7 snRNA Backbone组装。组装方法如下所示：pUC57-U7 snRNA Backbone-HindIII+NotI、80ng；U7 snRNA#A cassette PCR product、20ng；U7 snRNA#B cassette PCR product、20ng；U7 snRNA#C cassette PCR product、20ng；T4DNA Ligase Buffer(10X)、2μl；NEB Golden Gate Assembly Mix、1μl；反应过程：(37℃,5min→16℃,5min)×20→60℃,5min。
Golden Gate Assembly组装产物进一步通过转化大肠杆菌感受态细胞、挑选单克隆、PCR和测序验证，获得用于诱导USH2A第13号外显子剪接跳跃的U7 snRNA多靶点组合载体。提纯质粒，保存于-20℃备用。构建的载体示例性命名为pUC57-U7 snRNA#A+U7 snRNA#B+U7 snRNA#C。
不同靶位点的U7-snRNA组合更高效地诱导USH2A pre-mRNA第13号外显子剪接跳跃。293T细胞按一定量接种至24孔板，使得24小时后细胞汇合度达到约80％。使用Lipofectamine2000将pCMV-EGFP left-Exon13 mut-EGFP right分别和不同靶点的U7 snRNA组合质粒或单靶点质粒共转染293T细胞(载体质量比例为100ng：400ng)，使用单独转染报告质粒(Report，报告组)、共转染报告质粒的和pUC57-U7Scramble(SC组)的293T细胞作为两种阴性对照，不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时，使用胰酶消化成单细胞，随后使用流式细胞仪检测不同snRNA组的GFP阳性率。下表4和图4A-4B显示的是，不同靶位点的U7-snRNA组合诱导USH2A pre-mRNA第13号外显子剪接跳跃效率。
本实施例通过将靶向不同靶位点的U7-snRNA组合应用，发现可以在报告基因细胞中提升诱导USH2A第13号外显子的剪接跳跃的效率。本实施例中的不同靶点组合的诱导的剪接跳跃效率可以高于单个靶点的效果，而且可以优于已知的USH2A第13号外显子的剪接跳跃技术。
表4不同靶位点的U7-snRNA组合诱导USH2A pre-mRNA第13号外显子剪接跳跃效率
Figure PCTCN2023070867-appb-000015
Figure PCTCN2023070867-appb-000016
本申请不同靶位点的U7-snRNA组合中，其中一个snRNA可以靶向本申请的USH2A pre-mRNA第13号外显子的3’段序列或其片段，另一个或多个snRNA可以靶向USH2A pre-mRNA第12号内含子-第13号外显子-第13号内含子的序列或其片段，例如U7-snRNA以其识别结构域序列与USH2A pre-mRNA第12号内含子、第13号外显子或第13号内含子靶点序列反向互补配对，优选地选自第13号外显子及两侧临近靶区域(chr1:216246563-216247246对应的pre-mRNA区域)。不同靶位点的U7-snRNA组合则是靶点序列不完全一样的两个或两个以上的U7-snRNA的组合使用。
实施例7
化学合成U7 snRNA组合在WERI细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃
人源宿主细胞按6×10 5/孔接种至24孔板，本实施例选用的是WERI-Rb-1细胞系。用Lipofectamine2000将体外合成的50pmol snRNA组合1(U7-snRNA#30和U7-snRNA#4)、组合2(U7-snRNA#26和U7-snRNA#16)分别转染WERI细胞，转染相同剂量(50pmol)的反义寡核苷酸AON1(5′-MA*MG*MC*MU*MU*MC*MG*MG*MA*MG*MA*MA*MA*MU*MU*MU*MA*MA*MA*MU*MC*-3′，“M”表示2′-O-甲氧基修饰，“*”表示硫代磷酸化)和AON2(5′-MU*MG*MA*MU*MC*MA*MC*MA*MC*MC*MU*MA*MA*MG*MC*MC*MC*MU*MA*MA*MA*-3′，“M”表示2′-O-甲氧基修饰，“*”表示硫代磷酸化)作为对照组，转染1μg EGFP质粒作为阴性对照，不转染任何质粒的WERI细胞作为空白对照。转染后的细胞继续培养72小时，随后提取每个实验组细胞的RNA，反转录获得cDNA，通过引物AGCCTTTCCGCCAAGGTGATC(SEQ ID NO：34)和CACAACGTTGCCCAGCAATGG(SEQ ID NO：35)进行RT-PCR实验，检测成熟的USH2A mRNA是否存在外显子剪接跳跃，电泳结果如图8A所示。进一步通过ImageJ软件对rt-PCR电泳条带进行定量分析，并针对剪接跳过第13号外显子或剪接跳过第12号外显子和13的成熟USH2A mRNA的比例进行统计和分析，如图8B。
在内源性表达Usherin蛋白的WERI细胞中，将不同靶位点U7 snRNA组合诱导USH2A  pre-mRNA第13号外显子剪接跳跃的效果与现有技术优选AON技术方案进行比较，由RT-PCR试验数据和分析结果可知，snRNA组合1和snRNA组合2诱导第13号外显子单剪接跳跃的效果显著优于现有技术最优技术方案AON1和AON2，且snRNA组合1和snRNA组合2诱导第12号外显子和13双剪接跳跃mRNA占总剪接跳跃mRNA的比例却比AON1、AON2低。因此，可明确U7 snRNA在确保较低的双跳USH2A mRNA副产品的同时，显著提升第13号外显子单剪接跳跃的效率。
此外，snRNA组合2是靶向临近于现有技术第12号外显子和13双剪接跳跃概率极高的AON位点，然而，snRNA组合2的双外显子剪接跳跃的发生概率却非常低。
实施例8
带有可募集剪接调控蛋白的基序的U7 snRNA的剪接跳跃效果
连接hnRNP A1结合基序的U7 snRNA的构建。根据表中的序列对应的转录前DNA序列，分别合成对应的Oligo DNA。Oligo DNA正义链为靶序列的反向互补序列(识别结构域序列对应的DNA序列)，并且5’加
Figure PCTCN2023070867-appb-000017
Figure PCTCN2023070867-appb-000018
反义链为靶序列5’加AATT并且3’加
Figure PCTCN2023070867-appb-000019
Figure PCTCN2023070867-appb-000020
例如，识别结构域序列为NNN(识别结构域长度优选大于16个核苷酸)，则合成的Oligo DNA正义链为
Figure PCTCN2023070867-appb-000021
反义链为
Figure PCTCN2023070867-appb-000022
(下划线表示 识别结构域序列对应的DNA双链 序列，粗斜体表示hnRNP A1蛋白的结合基序“UAGGGU(SEQ ID NO：30)”对应的DNA双链序列)。
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl：Oligo-F(100μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合，95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切、回收的线性化pUC57-U7 snRNA backbone质粒连接。进一步通过转化大肠杆菌感受态细胞、挑选单克隆、PCR和测序验证，获得含有hnRNP A1结合基序的用于诱导USH2A第13号外显子剪接跳跃的U7 snRNA载体，载体命名为pUC57-U7-hnRNP A1-snRNA#A。提纯质粒，保存于-20℃备用。图5显示的是，带有hnRNP A1的snRNA载体示意图。
U7-hnRNP A1-snRNA还可以依据本申请实施例所述的方法进行化学合成和修饰。以snRNA#25为例，化学合成的U7-hnRNP A1-snRNA序列和修饰如下(*表示硫代磷酸化骨架，m表示2’-甲氧基修饰，下划线表示 与靶序列反向互补配对的识别结构域，斜体表示smOPT序列，粗体表示hnRNP A1蛋白结合基序)：
Figure PCTCN2023070867-appb-000023
连接hnRNP A1结合基序的U7 snRNA在报告基因细胞中诱导USH2A第13号外显子剪接跳跃。293T按一定量接种至24孔板，使得24小时后细胞汇合度达到约80％。使用Lipofectamine2000将pCMV-EGFP left-Exon13 mut-EGFP right分别和pUC57-U7-hnRNP A1-snRNA质粒、pUC57-U7 snRNA质粒共转染293T细胞(载体质量比例为100ng:400ng)，使用单独转染报告质粒(报告组)、共转染报告质粒的和pUC57-U7Scramble(SC组)的293T细胞作为两种阴性对照，不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时，使用胰酶消化成单细胞，随后使用流式细胞仪检测不同snRNA组诱导的剪接跳跃效率。下表5和图6A-6B显示的是，U7-hnRNP A1-snRNAUSH2A pre-mRNA第13号外显子剪接跳跃效率。
数据显示，在U7 snRNA的5’端引入hnRNP A1结合基序可显著提升诱导USH2A pre-mRNA第13号外显子剪接跳跃的效果，不仅提升了第13号外显子剪接跳跃的细胞(GFP+)的比例，而且提升了每个细胞中剪接跳过外显子的mRNA水平(平均FITC强度)。同时，在U7 snRNA#25中引入hnRNP A1结合基序平均FITC强度增加了3.25倍，诱导的剪接跳跃效率显著提高。
表5 U7-hnRNP A1-snRNAUSH2A pre-mRNA第13号外显子剪接跳跃效率
Figure PCTCN2023070867-appb-000024
本实施例在U7 snRNA的5’端引入游离尾部，所述游离尾部序列包括hnRNP A1蛋白的结合基序“UAGGGU”，所述游离尾部序列可以含有1个、2个或者2个以上的hnRNP A1蛋白的结合基序，优选为2个，游离尾部序列优选为
Figure PCTCN2023070867-appb-000025
Figure PCTCN2023070867-appb-000026
可募集hnRNP A1蛋白，促进USH2A第13号外显子的剪接跳跃，且不会增加第12号外显子和第13号外显子的双跳，可以不影响其靶向特异性，可以不造成或者增加脱靶效应。例如，本申请所述的USH2A的第12号外显子为人的USH2A第12号外显子。例 如，本申请所述的USH2A的第13号外显子为人的USH2A第13号外显子。
在一些实施例中，所述U7 snRNA的5’端引入游离尾部为可以募集剪接调控蛋白的基序，所述剪接调控蛋白为hnRNP A1(Heterogeneous Nuclear Ribonucleoprotein A1)、SRSF1(Serine And Arginine Rich Splicing Factor 1)、RBM4(RNA Binding Motif Protein 4)、DAZAP1(DAZ Associated Protein 1)、SR(Serine And Arginine-Rich Protein)等。
实施例9
靶向诱导USH2A pre-mRNA第13号外显子剪接跳跃的AAV-U7 snRNA相关质粒载体构建和病毒包装
本实施例将靶向诱导USH2A pre-mRNA第13号外显子剪接跳跃的U7 snRNA基因插入并替换pAAV-CMV载体中两个ITR结构域的中间基因序列，构建pAAV-U7 snRNA载体，与AAV包装质粒：血清型pRC质粒(包含AAV2的Rep基因和每个血清型各自的Cap基因)、pHelper质粒(包含腺病毒的E2A、E4和VA基因的载体质粒)共转染宿主细胞，包装获得靶向USH2A pre-mRNA第13号外显子剪接跳跃的AAV-U7 snRNA病毒。具体操作过程如下：
首先，通过全基因合成的方式，合成基因序列——U7-snRNA基因表达盒骨架(未包含识别结构域)：5’-小鼠U7启动子-smOPT序列-U7 snRNA scafford-snRNA基因特异性3’盒-3’。其中U7启动子与smOPT之间加入2个Tpye IIs型限制性内切酶识别位点(如BsaI、AarI、BsmBI等)，以方便后续切除、替换以及插入其他识别结构域序列。将全基因合成的序列插入并替换pAAV-CMV质粒(
Figure PCTCN2023070867-appb-000027
Helper Free System(AAV5)试剂盒，TAKARA公司，Code No.6650)两个AAV2-ITR结构域之间的基因序列，获得pAAV-U7 snRNA骨架载体。
U7-snRNA基因表达盒骨架(未包含识别结构域)(SEQ ID NO：33)：
Figure PCTCN2023070867-appb-000028
依照上述实施例所述方法，根据本申请的snRNA识别结构域序列或识别结构域序列的串 联组合对应的转录前DNA序列，分别合成对应的Oligo DNA正义链和反义链，两端加入类似于Tpye IIs型限制性内切酶识别位点切割后的粘性末端。退火形成带粘性末端的识别结构域(单独/串联)双链DNA，T4连接酶连接入经过对应Tpye IIs型限制性内切酶酶切回收的线性化pAAV-U7 snRNA骨架质粒中，形成靶向USH2A pre-mRNA第13号外显子特定位点诱导剪接跳跃的pAAV-U7 snRNA质粒，依据识别结构域序列对应的snRNA编号对其进行命名，如pAAV-U7 snRNA#25等。
将目的基因(靶向诱导USH2A pre-mRNA第13号外显子剪接跳跃的U7-snRNA基因表达盒子)插入并替换pAAV-CMV质粒AAV2-ITR结构域之间的基因序列后，获得pAAV-U7snRNA质粒载体。依据
Figure PCTCN2023070867-appb-000029
Helper Free System(AAV5)试剂盒说明书和标准的细胞操作流程包装获取靶向诱导USH2A pre-mRNA第13号外显子剪接跳跃的AAV-U7 snRNA病毒。
在转染之前24小时，将HEK293/293T细胞接种到100mm细胞培养皿，培养基为10％FBS的DMEM培养基，汇合度达到80％-90％时转染。转染前3小时，弃去旧培养基，更换新鲜培养基。转染时，同时将pAAV-U7 snRNA质粒、pRC质粒、pHelper质粒和PEI(聚乙烯亚胺)转染试剂按照以下的体系配置好，逐滴加入培养皿中。PEI转染混合物添加完毕后，轻轻晃动培养皿使转染试剂分布均匀，将培养基放置于37℃，5％CO 2培养箱中培养。
PEI转染体系：pAAV质粒(1μg/μl)、6μL；pRC1/2/5/6质粒(1μg/μl)；(pRC质粒衣壳基因决定血清型)、6μL；pHelper质粒(1μg/μl)、6μL；无血清DMEM培养基、500μL；PEI(1mg/mL)、110μL处理方式：涡旋混合数次，室温孵育5min。
转染后24小时，更换新鲜2％FBS的DMEM培养基。转染48-72小时后，收集含AAV病毒的细胞，清洗、离心，收集细胞沉淀，涡旋振荡使细胞沉淀松散。随后，依照
Figure PCTCN2023070867-appb-000030
Helper Free System(AAV5)试剂盒说明书，在细胞沉淀中加入0.5mL的AAV Extraction Solution A，涡旋振荡15秒使细胞沉淀充分悬浮。室温静置5分钟后，再涡旋振荡15秒。4℃，2000-14000g离心10分钟，去除细胞碎片。收集上清液到新的无菌离心管中，加入50μL AAV Extraction Solution B，使用移液枪吸打混匀，获得不同识别结构域的AAV-U7 snRNA病毒溶液，取部分以qPCR法检测病毒滴度，保存于80℃备用。
由于插入pAAV-U7 snRNA质粒AAV2-ITR结构域之间插入的目的基因片段应小于2.5kb，因此，可通过插入多个U7-snRNA基因表达盒子(5’-小鼠U7启动子-smOPT序列、U7 snRNA scafford-snRNA基因特异性3’盒-3’)，从而确保在相同AAV病毒颗粒数量的情况下，提升U7 snRNA的表达量，基因序列长度约为450bp，则优选地pAAV-U7 snRNA质粒中携带1-5 个U7-snRNA基因表达盒子，所述pAAV-U7 snRNA质粒中的多个U7-snRNA基因表达盒子可以是具有相同的识别结构域或识别结构域组合，也可以是具有不同或者不完全相同的识别结构域组合。
本申请通过AAV递送U7 snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃，所述AAV的衣壳蛋白可以是天然来源的，也可以是基于天然来源衣壳蛋白的变体、或进行定向进化、或进行氨基酸/肽段合理改造(密码子优化、嵌合不同血清型功能肽段等)等，提升组织器官亲嗜性、免疫原性、提升转染效率等特性，如AAV2.5、AAV2i8、AAV-TT、AAV9.HR、CAM130等。所述天然来源的AAV衣壳蛋白可以是来源于动物体的，也可以是来源于植物的，所述来源于动物体的AAV衣壳蛋白可以是来源于人体的(如AAV1、AAV2、AAV3、AAV5、AAV6、AAV7、AAV8和AAV9等)，也可以是来源于非人灵长类动物(如AAVrh.8、AAVrh.10和AAVrh.43)，也可以是来源于小鼠、猪等脊椎动物，也可以是来源于昆虫。本发明AAV质粒体系中，例如，AAV ITR血清型应于Rep基因血清型一致，与Cap基因血清型可不一致。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (62)

  1. 一种核酸分子，所述核酸分子具有特异性结合USH2A pre-mRNA第13号外显子的3’段序列或其片段的能力，所述USH2A pre-mRNA第13号外显子的3’段序列对应的基因组定位为Chr1：216246563-216246753。
  2. 一种核酸分子，所述核酸分子具有特异性结合SEQ ID NO：1所示序列或其片段的能力。
  3. 一种核酸分子，所述核酸分子与SEQ ID NO：1所示序列中的16个或更多的连续核苷酸互补。
  4. 一种核酸分子，所述核酸分子包含SEQ ID NO：1所示序列、或其互补序列中的16个或更多的连续核苷酸。
  5. 如权利要求1-4中任一项所述的核酸分子，所述核酸分子包含与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的至少16个核苷酸。
  6. 如权利要求1-5中任一项所述的核酸分子，所述核酸分子包含与USH2A pre-mRNA第13号外显子的3’段序列或其片段互补的22个至27个核苷酸。
  7. 如权利要求1-6中任一项所述的核酸分子，所述核酸分子包含至少16个核苷酸。
  8. 如权利要求1-7中任一项所述的核酸分子，所述核酸分子包含22个至27个核苷酸。
  9. 如权利要求1-8中任一项所述的核酸分子，所述核酸分子包含至少60个核苷酸。
  10. 如权利要求1-9中任一项所述的核酸分子，所述核酸分子包含互补于选自以下组基因组定位对应的pre-mRNA序列：Chr1：216246603-216246626、Chr1：216246598-216246621、Chr1：216246598-216246624、Chr1：216246594-216246617、Chr1：216246570-216246593、Chr1：216246570-216246591、Chr1：216246570-216246596、Chr1：216246570-216246593、Chr1：216246570-216246596、Chr1：216246567-216246590、和Chr1：216246563-216246586。
  11. 如权利要求1-10中任一项所述的核酸分子，所述核酸分子包含互补于SEQ ID NO:10-21中任一项所示的序列。
  12. 如权利要求1-11中任一项所述的核酸分子，所述核酸分子包含SEQ ID NO:10-21中任一项所示的序列。
  13. 如权利要求1-12中任一项所述的核酸分子，所述核酸分子包含经修饰的核苷酸。
  14. 如权利要求13所述的核酸分子，所述经修饰的核苷酸包含选自以下组的修饰：2’-O-烷基、2’-O-甲氧基和/或2’-O-甲氧基乙基。
  15. 如权利要求13-14中任一项所述的核酸分子，所述经修饰的核苷酸包含选自以下组的修饰：2’-O-甲基和/或2’-O-乙基。
  16. 如权利要求13-15中任一项所述的核酸分子，所述核酸分子包含至少一个经修饰的核苷酸。
  17. 如权利要求13-16中任一项所述的核酸分子，所述核酸分子的5’端包含至少一个经修饰的核苷酸。
  18. 如权利要求13-17中任一项所述的核酸分子，所述核酸分子的5’端包含1个至3个经修饰的核苷酸。
  19. 如权利要求13-18中任一项所述的核酸分子，所述核酸分子的3’端包含至少一个经修饰的核苷酸。
  20. 如权利要求13-19中任一项所述的核酸分子，所述核酸分子的3’端包含1个至3个经修饰的核苷酸。
  21. 如权利要求1-20中任一项所述的核酸分子，所述核酸分子中包含选自以下组的核苷酸或核苷酸类似物单体：6’-修饰的双环核苷、5’-修饰的双环核苷、6’-双取代双环核苷、四氢吡喃核苷类似物和/或2'-脱氧2'-氟-β-D-阿拉伯糖核苷酸(2’-FANA修饰的核苷酸)。
  22. 如权利要求1-21中任一项所述的核酸分子，所述核酸分子包含经修饰的磷酸键。
  23. 如权利要求1-22中任一项所述的核酸分子，所述核苷酸分子经过选自以下组的化学键连接：硫代磷酸酯、二硫代磷酸酯、磷酸三酯、烷基膦酸酯、氨基烷基磷酸三酯、亚烷基膦酸酯、次膦酸酯、氨基磷酸酯、氨基烷基氨基磷酸酯、硫代氨基磷酸酯、硫羰基烷基膦酸酯、硫羰基烷基磷酸三酯、硫代磷酸酯、硒代磷酸酯、硼烷基磷酸酯、磷酸二酯键、Rp-硫代磷酸酯键、Sp-硫代磷酸酯键、硼烷磷酸酯键、亚甲基键(甲基亚氨基)、酰胺键(3'-CH 2-CO-NH-5'和3'-CH 2-NH-CO-5')、甲基膦酸酯键和/或3'-硫代甲缩醛键(3'S-CH 2-O5')。
  24. 如权利要求22-23中任一项所述的核酸分子，所述经修饰的磷酸键包含选自以下组：硫代磷酸酯键、二硫代磷酸酯键、烷基磷酸酯键、酰胺磷酸酯键和/或硼烷磷酸酯键。
  25. 如权利要求22-24中任一项所述的核酸分子，所述核酸分子包含至少一个经修饰的磷酸键。
  26. 如权利要求22-25中任一项所述的核酸分子，所述核酸分子的5’端包含至少一个经修饰的磷酸键。
  27. 如权利要求22-26中任一项所述的核酸分子，所述核酸分子的5’端包含1个至3个经修饰的磷酸键。
  28. 如权利要求22-27中任一项所述的核酸分子，所述核酸分子的3’端包含至少一个经修饰的磷酸键。
  29. 如权利要求22-28中任一项所述的核酸分子，所述核酸分子的3’端包含1个至3个经修饰的磷酸键。
  30. 如权利要求1-29中任一项所述的核酸分子，所述核酸分子包含DNA和/或RNA。
  31. 如权利要求1-30中任一项所述的核酸分子，所述核酸分子为单链的或双链的。
  32. 如权利要求1-31中任一项所述的核酸分子，所述核酸分子包含反义寡核苷酸(antisense oligonucleotide)、shRNA、siRNA、miRNA和/或适体(aptamer)。
  33. 如权利要求1-32中任一项所述的核酸分子，所述核酸分子包含核小RNA(Small nuclear RNA)。
  34. 如权利要求33所述的核酸分子，所述核小RNA包含U1核小RNA和/或U7核小RNA。
  35. 如权利要求1-34中任一项所述的核酸分子，所述核酸分子不结合Cas酶。
  36. 如权利要求1-35中任一项所述的核酸分子，所述核酸分子不包含能够结合Cas酶的结构。
  37. 如权利要求35-36中任一项所述的核酸分子，所述Cas酶为Cas13酶。
  38. 如权利要求1-37中任一项所述的核酸分子，所述核酸分子包含茎环结构域或其衍生结构。
  39. 如权利要求38所述的核酸分子，所述茎环结构域包含SEQ ID NO：6所示的序列。
  40. 如权利要求1-39中任一项所述的核酸分子，所述核酸分子包含能够结合sm蛋白的结构域或其衍生结构。
  41. 如权利要求40所述的核酸分子，所述能够结合sm蛋白的结构域包含SEQ ID NO：5所示的序列。
  42. 如权利要求1-41中任一项所述的核酸分子，所述核酸分子包含能够结合剪接调控蛋白的结构域。
  43. 如权利要求1-42中任一项所述的核酸分子，所述核酸分子包含能够结合选自以下组的蛋白的结构域：SRSF1(Serine And Arginine Rich Splicing Factor 1)、RBM4(RNA Binding Motif Protein 4)、DAZAP1(DAZ Associated Protein 1)、和SR(Serine And Arginine-Rich Protein)。
  44. 如权利要求1-43中任一项所述的核酸分子，所述核酸分子包含能够结合hnRNP A1蛋白的结构域。
  45. 如权利要求44所述的核酸分子，所述能够结合hnRNP A1蛋白的结构域包含SEQ ID NO：30所示的序列。
  46. 一种基因表达盒，所述基因表达盒包含或编码如权利要求1-45中任一项所述的核酸分子的核苷酸序列，以及任选的表达调控元件。
  47. 一种载体，所述载体包含或编码如权利要求1-45中任一项所述的核酸分子的核苷酸序列，和/或如权利要求46所述的基因表达盒的核苷酸序列。
  48. 一种病毒颗粒，所述病毒颗粒包含如权利要求1-45中任一项所述的核酸分子，如权利要求46所述的基因表达盒的核苷酸序列，和/或如权利要求47所述的载体。
  49. 一种细胞，所述细胞包含如权利要求1-45中任一项所述的核酸分子，如权利要求46所述的基因表达盒的核苷酸序列，如权利要求47所述的载体，和/或如权利要求48所述的病毒颗粒。
  50. 一种药物组合物，所述药物组合物包含如权利要求1-45中任一项所述的核酸分子，如权利要求46所述的基因表达盒的核苷酸序列，如权利要求47所述的载体，如权利要求48所述的病毒颗粒，和/或如权利要求49所述的细胞，以及任选的药学可接受的载剂。
  51. 如权利要求50所述的药物组合物，所述药物组合物包含第一载体和第二载体，所述第一载体包含如权利要求1-45中任一项所述的核酸分子，所述第二载体包含具有特异性结合USH2A pre-mRNA第13号外显子及其两侧区域或其片段的能力的核酸分子。
  52. 如权利要求50-51中任一项所述的药物组合物，所述USH2A pre-mRNA第13号外显子的两侧区域包含USH2A pre-mRNA第12号内含子和/或第13号内含子。
  53. 一种试剂盒，所述试剂盒包含如权利要求1-45中任一项所述的核酸分子，如权利要求46所述的基因表达盒的核苷酸序列，如权利要求47所述的载体，如权利要求48所述的病毒颗粒，如权利要求49所述的细胞，和/或如权利要求50-52中任一项所述的药物组合物。
  54. 一种制备如权利要求1-45中任一项所述的核酸分子的方法，包含表达和/或合成能够结合USH2A第13号外显子的3’段序列或其片段的所述核酸分子，所述USH2A第13号外显子的3’段序列的基因组定位为Chr1：216246563-216246753。
  55. 一种抑制USH2A pre-mRNA第13号外显子表达和/或功能的方法，包含提供如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  56. 一种使USH2A pre-mRNA第13号外显子剪接跳跃的方法，包含提供如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  57. 一种制备缺失第13号外显子的成熟USH2A mRNA的方法，包含提供如权利要求1-45中 任一项所述的核酸分子、如权利要求47所述的载体、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  58. 一种降低包含第13号外显子表达产物的Usherin蛋白水平的方法，包含提供如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  59. 一种制备不包含第13号外显子表达产物的Usherin蛋白和/或增加不包含第13号外显子表达产物的Usherin蛋白数量的方法，包含提供如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求48所述的病毒颗粒、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  60. 一种恢复突变Usherin蛋白的功能的方法，包含提供如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求48所述的病毒颗粒、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒。
  61. 如权利要求1-45中任一项所述的核酸分子、如权利要求47所述的载体、如权利要求48所述的病毒颗粒、如权利要求49所述的细胞、如权利要求50-52中任一项所述的药物组合物和/或如权利要求53所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗USH2A基因突变引发的疾病。
  62. 如权利要求61所述的用途，所述疾病包含眼病和/或耳病。
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