WO2023130959A1 - 靶向USH2A pre-mRNA的snRNA及其应用 - Google Patents

靶向USH2A pre-mRNA的snRNA及其应用 Download PDF

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WO2023130959A1
WO2023130959A1 PCT/CN2022/140599 CN2022140599W WO2023130959A1 WO 2023130959 A1 WO2023130959 A1 WO 2023130959A1 CN 2022140599 W CN2022140599 W CN 2022140599W WO 2023130959 A1 WO2023130959 A1 WO 2023130959A1
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chr1
snrna
sequence
seq
ush2a
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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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Definitions

  • the invention relates to the technical field of genetic engineering, in particular to snRNA targeting USH2A pre-mRNA 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.
  • Usher syndrome can be divided into 3 types: 1. Type I Usher syndrome, in which patients will have congenital severe deep sensorineural deafness in hearing, vestibular response disappearance in vestibular response, and visual impairment in vision. Retinitis pigmentosa occurs before puberty, and then gradually leads to blindness. The genes associated with this type include MYO7A, CDH23, USH1C, PCHD15, etc.; 2.
  • Type II Usher syndrome patients with congenital moderate to severe sensorineural hearing
  • retinitis pigmentosa will appear in adolescence and gradually lead to blindness.
  • the genes associated with this type include USH2A, GPR98, WHRN, etc.; With sensorineural deafness, the vestibular response is normal, and retinitis pigmentosa will appear at the end of puberty in vision, and gradually lead to blindness.
  • the genes associated with this type include CLRN1 and so on.
  • type II Usher syndrome accounts for more than 50% of Usher syndrome
  • USH2A gene mutation is the most common cause of Usher syndrome type II, 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).
  • NSRP non-syndromic retinitis pigmentosa
  • 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 first is to edit the genomic DNA through the CRISPR/Cas system to directly delete the No. 13 exon, or destroy the RNA splicing-related positions. point.
  • fragment deletion such as chromosomal rearrangement, viral integration, reverse reintegration, and 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.
  • the second is to modify the key bases of the above-mentioned splicing-related sites by using a single base editor, which can also promote exon skipping.
  • the present invention provides a snRNA targeting USH2A pre-mRNA, and the snRNA promotes exon 13 skipping more efficiently than AON.
  • the present invention provides a snRNA targeting USH2A pre-mRNA, the recognition domain of the snRNA is reverse complementary to the USH2A pre-mRNA sequence, and the snRNA induces the first Exon 13 splice skipping.
  • snRNAs small nuclear RNAs
  • snRNAs small nuclear RNAs
  • snRNPs small nuclear RNAs
  • Its length is about 100-215 nucleotides in mammals, and it is divided into 7 categories. Because of its rich U content, it is numbered U1-U7. But U7-snRNP is not involved in splicing, but is a key factor in the unique 3' end processing of replication-dependent histone (RDH) pre-mRNA.
  • RDH replication-dependent histone
  • the inventors replaced the non-canonical Sm-binding site of U7-snRNA with a consensus sequence derived from major spliceosome U-snRNPs, and changed the histone-binding sequence of the 5' region of U7-snRNA to be complementary to that of the gene to be modified. Sequences that can induce exon splice skipping by targeting exons.
  • the present inventors found 7 target regions and 21 target sites for U7-snRNP to induce splicing skipping through target screening, which can induce the splicing skipping effect of exon 13, and at the same time designed the recognition domain of U7-smOPT-snRNA and USH2A pre-mRNA exon 13 splicing skipping related site reverse complementation, thereby inducing U7-smOPT-snRNA to combine with USH2A pre-mRNA exon 13 splicing skipping related site, and then induce USH2A pre-mRNA 13
  • the splicing skipping of exon No. 12 and the retention of exon No. 12 significantly reduce the probability of unexpected splicing skipping such as exon No. 12 and 13 double-skipping reads, which ensures safety while significantly improving efficiency.
  • the USH2A pre-mRNA sequence is selected from the following regions: exon 13, intron 12 or intron 13 of USH2A.
  • the USH2A pre-mRNA sequence is selected from exon 13 of USH2A and a region extending 50 bp to both sides.
  • the USH2A pre-mRNA sequence is selected from exon 13 of USH2A and a region extending 20 bp to both sides.
  • the No. 13 exon includes: the No. 13 wild type exon or the No. 13 exon containing mutation.
  • the mutation includes at least one of the following mutation sites: c.2242C>T, c.2276G>T, c.2299delG, c.2522C>A, c.2541C>A, c .2761delC, c.2776C>T, c.2802T>G, 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.
  • the genomic location corresponding to the USH2A pre-mRNA sequence (corresponding to the NCBI database GRch38 version) region is selected from: Chr1: 216247142-216247185, Chr1: 216247130-216247161, Chr1: 216246616-216246649, Chr1: 216247213-216247246, Chr1: 216247204-216247232, Chr1: 216247187-216247220, Chr1: 216247169-216247202.
  • the USH2A pre-mRNA sequence is selected from the following sequences: SEQ ID NO: 1-7 sequence.
  • the genomic location region corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247223-216247246, Chr1: 216247218-216247241, Chr1: 216247213-216247236, Chr1: 216247209-216247232, Chr1: 216247204- 216247227, Chr1: 216247197-216247220, Chr1: 216247191-216247214, Chr1: 216247187-216247210, Chr1: 216247179-216247202, Chr1: 216247174-2 16247197, Chr1: 216247169-216247192, Chr1: 216247162-216247185, Chr1: 216247155-216247178, Chr1: 216247147-216247168, Chr1: 216247147-216247173, Chr1: 216247142-216247165, Chr1: 216247138-216247161, Chr1: 216247130-216247
  • the recognition domain of the snRNA is reverse complementary to at least 16 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 17 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 18 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 19 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 20 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 21 bp of the continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 22 bp of the continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 23 bp of continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to at least 24 bp of the continuous sequence in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to the continuous sequence of 18bp-40bp in the USH2A pre-mRNA sequence.
  • the recognition domain of the snRNA is reverse complementary to the continuous sequence of 20bp-27bp in the USH2A pre-mRNA sequence.
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 8-28.
  • the genomic location corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247213-216247236, Chr1: 216247209-216247232, Chr1: 216247204-216247227, Chr1: 216247197-216247220, Chr1: 216247197-216247220, Chr1: r1: 216247191-216247214 Chr1: 216247187-216247210, Chr1: 216247179-216247202, Chr1: 216247174-216247197, Chr1: 216247169-216247192.
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 10-18.
  • the genome location corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247162-216247185, Chr1: 216247155-216247178, Chr1: 216247147-216247168, Chr1: 216247147-216247173, Chr1: 216247147-216247173, Chr1 r1: 216247142-216247165 , CHR1: 2162471-216247161, CHR1: 216247130-216247153, CHR1: 216246626246649, CHR1: 216246622-216246645, CHR1: 21624661624666666666666666666662 39.
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 19-28.
  • the genomic location corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247213-216247236, Chr1: 216247209-216247232, Chr1: 216247204-216247227, Chr1: 216247197-216247220, Chr1: 216247197-216247220, Chr1: r1: 216247191-216247214 .
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 10-23, SEQ ID NO: 25.
  • the genomic location corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247213-216247236, Chr1: 216247209-216247232, Chr1: 216247204-216247227, Chr1: 216247197-216247220, Chr1: 216247197-216247220, Chr1: r1: 216247191-216247214 .
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 10-19, SEQ ID NO: 21-23, SEQ ID NO: 25.
  • the genomic location corresponding to the USH2A pre-mRNA sequence is selected from: Chr1: 216247218-216247241, Chr1: 216247187-216247210, Chr1: 216247147-216247168, Chr1: 216247147-216247173, Chr1: 216247147-216247173, Chr1 r1: 216247142-216247165 , Chr1: 216247130-216247153.
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 23, SEQ ID NO: 25.
  • the snRNA recognition domain is selected from the following sequences: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23.
  • the snRNA is U1-snRNA or U7-snRNA.
  • the U7-snRNA is chemically synthesized.
  • At least one nucleotide in the chemically synthesized U7-snRNA is chemically modified.
  • the chemical modification includes at least one of 2'-O alkyl modification, 2'-O-methoxy modification or 2'-O-methoxyethyl modification.
  • the 2'-O-alkyl modification is 2'-O-methyl modification.
  • At least one nucleotide in the chemically synthesized U7-snRNA is linked by a phosphate bond
  • the phosphate bond includes a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphine At least one of ester bond, amidophosphate bond, borane phosphate bond, or chiral link phosphorus.
  • the 6-80 nucleotides on both sides of the chemically synthesized U7-snRNA are all chemically modified and linked by the phosphate bond.
  • the 1-10 nucleotides on both sides of the chemically synthesized U7-snRNA are chemically modified and linked by the phosphate bond.
  • the 3-40 bases on both sides of the chemically synthesized U7-snRNA are chemically modified and linked by the phosphate bonds.
  • all nucleotides of the chemically synthesized U7-snRNA are linked by phosphorothioate bonds, and are all modified with 2′-O-methoxy.
  • the three nucleotides on both sides of the chemically synthesized U7-snRNA are linked by phosphorothioate bonds and modified with 2'-O-methoxy.
  • the first nucleotide at the 5' end of the chemically synthesized U7-snRNA is adenosine.
  • the first nucleotide at the 5' end of the recognition domain of the chemically synthesized U7-snRNA is adenosine.
  • sequence length of the recognition domain of the chemically synthesized U7-snRNA is ⁇ 16 bp.
  • the sequence length of the recognition domain of the chemically synthesized U7-snRNA is 18bp-40bp.
  • the sequence length of the recognition domain of the chemically synthesized U7-snRNA is 20bp-27bp.
  • the chemically synthesized U7-snRNA continues to extend the sequence length of the recognition domain through reverse complementary pairing along the 5' end or/and 3' end of the target sequence.
  • the sequence length of the extended recognition domain is ⁇ 40bp.
  • the snRNA includes sm sequence.
  • the sm sequence is a smOPT sequence, and the smOPT sequence is shown in SEQ ID NO:31.
  • the snRNA comprises a recognition domain, a smOPT sequence, and a U1-snRNA scaffold or U7-snRNA scaffold sequence; preferably, the U7-snRNA scaffold sequence is shown in SEQ ID NO:49.
  • the snRNA further includes a motif for recruiting a splicing regulatory protein.
  • a free tail is introduced at the 5' end of the U7-snRNA, and the sequence of the free tail includes a motif for recruiting splicing regulatory proteins.
  • the splicing regulatory protein includes at least one of hnRNPA1, SRSF1, RBM4, DAZAP1 or SR.
  • the free tail sequence includes at least one hnRNPA1 binding motif.
  • the free tail sequence includes two hnRNPA1 binding motifs.
  • the present invention also provides a nucleotide, which includes the nucleotide sequence encoding the snRNA.
  • the present invention also provides a gene expression cassette, which includes the snRNA, and/or includes the nucleotide.
  • the present invention also provides a carrier, which includes the snRNA, includes the nucleotide, and/or includes the gene expression cassette.
  • the vector comprises a vector backbone and the snRNA
  • the vector backbone comprises a promoter, a smOPT sequence and a snRNA gene-specific 3' box
  • the promoter is passed through a Tpye IIs type restriction endonuclease A recognition site is linked to the smOPT sequence.
  • the vector is selected from: pUC57, pAAV-CMV, lentivirus or transposon.
  • the snRNA gene-specific 3' cassette is a gene fragment extending 28-131 bp at the 3' end of the snRNA gene.
  • the snRNA gene-specific 3' box is a gene fragment with a length of 106 bp at the 3' end of the snRNA gene.
  • the present invention also provides a virus particle, which includes the snRNA, includes the nucleotide, and/or includes the vector.
  • the virus particle is an AAV virus.
  • the capsid protein of the AAV virus is a natural source or a variant based on a natural source capsid protein.
  • the capsid protein of the AAV virus is derived from animals or plants.
  • the capsid protein of the AAV virus is selected from: AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 or AAVrh43.
  • the variant AAV virus based on the capsid protein from natural sources is an AAV virus after directed evolution or rational modification of amino acids/peptides based on the capsid protein from natural sources.
  • the variant AAV virus is selected from AAV2.5, AAV2i8, AAV-TT, AAV9.HR or CAM130.
  • the serotype is consistent with the Rep gene serotype.
  • the present invention also provides a cell comprising the snRNA, the nucleotide, the vector, and/or the virus particle.
  • the present invention also provides a pharmaceutical composition, which comprises the snRNA, the nucleotide, the carrier, and/or the virus particle.
  • the present invention also provides a method for obtaining the Usherin protein whose expression product of the No. 13 exon is deleted, by making the pre-mRNA of the USH2A gene and the snRNA, the nucleotide, the carrier, and the gene The expression cassette, the viral particle, the cell, and/or the pharmaceutical composition are contacted.
  • the method is used in scientific research.
  • the present invention also provides the method for splicing and skipping exon 13 of USH2A pre-mRNA, preparing mature USH2A mRNA lacking exon 13, obtaining Usherin protein with exon 13 deletion, and/or inhibiting USH2A Application in expression and function of exon 13 of pre-mRNA.
  • the present invention also provides the use of the snRNA, the nucleotide, the carrier, the gene expression cassette, the virus particle or the cell in the preparation of a drug for preventing and/or treating eye disease and/or ear disease application.
  • the eye disease is deafness-retinitis pigmentosa syndrome or non-syndromic retinitis pigmentosa.
  • the present invention has the following beneficial effects:
  • the snRNA targeting USH2A pre-mRNA of the present invention selects the No. 13 exon of USH2A as the target region, induces the splicing skipping of the No. 13 exon, thereby treating the missense, frameshift and stop codon of the No. 13 USH2A exon , nonsense, synonymous mutations and other eye and ear diseases that cause abnormal function of USH2A protein.
  • a U7-snRNA targeting USH2A of the present invention utilizes that U7-snRNP does not participate in splicing, but is a key factor in the unique 3' end processing of replication-dependent histone (RDH) pre-mRNA, and the modified U7 -snRNA that changes the histone-binding sequence of the 5' region of U7-snRNA to the complementary sequence of the gene to be modified by replacing the non-canonical Sm-binding site of U7-snRNA with a consensus sequence derived from major spliceosome U-snRNPs
  • the unique working mechanism, combined with snRNA targeting USH2A pre-mRNA can induce the splicing skipping of exon 13 of USH2A pre-mRNA, and retain exon 12, so that exon 12 and exon 13 can double
  • the probability of unexpected splicing jumps such as skipping is significantly reduced, and the safety is guaranteed while significantly improving the single-skip reading efficiency of exon 13.
  • Figure 1 is a schematic diagram of the structure and function of U7-snRNA
  • Figure 2 is a map of the pUC57-U7snRNA backbone vector
  • Figure 3 is the plasmid map of the reporter vector pCMV-EGFP left -Exon13 c.2802T>G -EGFP right ;
  • Figure 4 is the plasmid map of the reporter vector pCMV-EGFP left -Exon13 c.2299delG -EGFP right ;
  • 5 is a schematic diagram of the position of U7-snRNA targeting target region 1 on the genome
  • Figure 6 is a graph showing the proportion of cells in which U7-snRNA targeting target region 1 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter carrier cells;
  • Figure 7 is a histogram of the average FITC intensity of GFP-positive cells induced by U7-snRNA targeting target region 1 (USH2A pre-mRNA No. 13 exon splicing skipping);
  • Figure 8 is a schematic diagram of the position of U7-snRNA targeting target region 2 on the genome
  • Figure 9 is a graph showing the proportion of cells in which U7-snRNA targeting target region 2 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter carrier cells;
  • Figure 10 is a histogram of the average FITC intensity of GFP-positive cells induced by U7-snRNA targeting target region 2 (USH2A pre-mRNA No. 13 exon splice skipping);
  • Figure 11 is a schematic diagram of the position of U7-snRNA targeting target region 3 on the genome
  • Figure 12 is a graph showing the proportion of cells in which U7-snRNA targeting target region 3 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter carrier cells;
  • Figure 13 is a histogram of the average FITC intensity of GFP-positive cells induced by U7-snRNA targeting target region 3 (USH2A pre-mRNA No. 13 exon splicing skipping);
  • Figure 14 is a schematic diagram of the position of U7-snRNA targeting target region 4 on the genome
  • Figure 15 is a graph showing the proportion of cells in which U7-snRNA targeting target region 4 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter cells;
  • Figure 16 is a schematic diagram of the position of U7-snRNA targeting target region 5 on the genome
  • Figure 17 is a graph showing the proportion of cells in which U7-snRNA targeting target region 5 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter cells;
  • Figure 18 is a schematic diagram of the position of U7-snRNA targeting target region 6 on the genome
  • Figure 19 is a graph showing the percentage of cells in which U7-snRNA targeting target region 6 induces splicing skipping of exon 13 of USH2A pre-mRNA in reporter cells;
  • Figure 20 is a schematic diagram of the position of U7-snRNA targeting target region 7 on the genome
  • Figure 21 is a graph showing the results of the cell ratio of U7-snRNA targeting target region 7 inducing USH2A pre-mRNA exon 13 splicing skipping in reporter cells;
  • Figure 22 is a graph showing the average FITC intensity results of U7 snRNA targeting different regions inducing USH2A pre-mRNA exon 13 splicing skipping cells in reporter cells;
  • Figure 23 is a graph showing the experimental results of U7-snRNA-induced USH2A pre-mRNA exon 13 splicing skipping efficiency is significantly better than that of AON1;
  • Figure 24 is a graph showing the results of detecting the splicing skipping efficiency of exon 13 of USH2A pre-mRNA induced by chemically synthesized U7snRNA in WERI cells;
  • Figure 25 is a schematic diagram of the structure of U7-hnRNP A1-snRNA
  • Figure 26 is a graph showing the results of splicing skipping efficiency of USH2A pre-mRNA exon 13 induced by U7-hnRNP A1-snRNA.
  • the snRNA of the present invention is the main component of RNA spliceosome (spliceosome) in the process of eukaryotic post-transcriptional processing, and participates in the processing process of mRNA precursor by combining with snRNP protein.
  • the reagents, materials, and equipment used in this example are all commercially available sources unless otherwise specified; the experimental methods are all conventional experimental methods in the art unless otherwise specified.
  • the wild-type U7-snRNA includes a stem-loop structure (scafford), a U7-specific Sm sequence (AAUUUGUCUAG) and a recognition domain (complementary to histone pre-mRNA).
  • the U7-snRNA of this embodiment is based on the gene sequence (NCBI Reference Sequence: NR_024201.3) of the mouse wild-type U7-snRNA on NCBI, and the U7-specific sm sequence AATTTGTCTAG (SEQ ID NO: 30) is replaced by an optimized The consensus sm sequence, namely smOPT: AATTTTTGGAG (SEQ ID NO: 31), the original recognition domain at the 5' end of the smOPT sequence is replaced with the recognition domain that is reverse complementary to the specific target site of USH2A pre-mRNA, the smOPT sequence
  • the original stem-loop structure sequence of U7 is retained at the 3' end, as shown in Figure 1.
  • Targeted induction of U7-snRNA recognition domain sequence of exon 13 of USH2A pre-mRNA is opposite to the target sequence selected from intron 12-exon 13-intron 13 of USH2A pre-mRNA to complementary pairing.
  • 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 of gtctacaatgaaa (SEQ ID NO: 32), which is involved in the processing of pre-snRNA, preferably connected to U7snRNA
  • the sequence behind the 3' end of the gene is preferably a gene fragment with a sequence length of 28-131bp, and more preferably a sequence length of 106bp.
  • 21 target sites were respectively set for 7 target regions of USH2A pre-mRNA, and the 7 target regions of USH2A pre-mRNA are as follows.
  • Exon 13 region 1 (SEQ ID NO: 1) (Chr1: 216247142-216247185): CGAAGCUUUAAUGAUGUUGGAUGUGAGCCCUGCCAGUGUAACCU;
  • Exon 13 region 2 (SEQ ID NO: 2) (Chr1: 216247130-216247161): GAGCCCUGCCAGUGUAACCUCCAUGGCUCAGU;
  • Exon 13 region 3 (SEQ ID NO: 3) (Chr1: 216246616-216246649): AAUCAGUGGCCAGUGCCUGUGUGUGCCUAAUCGU;
  • Exon 13 region 4 (SEQ ID NO: 4) (Chr1: 216247213-216247246): UAAAUAUAUUUUAUCUUUAGGGCUUAGGUGUGAU;
  • Exon 13 region 5 (SEQ ID NO: 5) (Chr1: 216247204-216247232): CUUUAGGGCUUAGGUGUGAUCAUUGCAAU;
  • Exon 13 region 6 (SEQ ID NO: 6) (Chr1: 216247187-216247220): GGUGUGAUCAUUGCAAUUUUGGAUUUAAAUUUCU;
  • Exon 13 region 7 (SEQ ID NO: 7) (Chr1: 216247169-216247202): UUGGAUUUAAAUUUCUCCGAAGCUUUAAUGAUGU.
  • the 21 target sites are shown in the table below.
  • U7-snRNA can also be directly chemically synthesized to produce RNA containing the recognition domain, smOPT and U7-snRNA scaffold.
  • the U7-snRNA synthesized in vitro can be modified to resist nuclease degradation or 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'methoxyl (2'-OME) and sulfo-modified to increase nuclease resistance.
  • 2'-OME 2'methoxyl
  • snRNA#16 and snRNA#15 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 for reverse complementary pairing with the target sequence domain, italics indicate smOPT sequence):
  • the above-mentioned modified sequence is based on the sequence shown in SEQ ID NO: 33, adding phosphorothioate backbone modification and methoxy modification on the first three bases at the 5' end and the 3' end;
  • the above-mentioned modified sequence is based on the sequence shown in SEQ ID NO: 34, adding phosphorothioation backbone modification and methoxy modification on the first three bases at the 5' end and the 3' end.
  • 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), RGleft indicates the first half of the 5' end of the reporter gene without the reporter function, RGright indicates the reporter gene 3 without the reporter function In the second half of the 'end, the tandem expression of RGleft and RGright 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 represents USH2A exon 13 containing pathogenic mutations, and its upstream and downstream intron sequences (the upstream intron sequence is the tandem sequence 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 tandem gene sequence of 703 bp at the 5' end and 216 bp at the 3' end of intron 13 of human USH2A).
  • the pathogenic mutations in exon 13 of USH2A may include c.2802T>G, c.2299delG, c.2276G>T, c.2522C>A, c.2242C>T, c.2541C>A, c.
  • the pathogenic mutations of exon 13 of USH2A described in the examples of the present invention are c.2802T>G, c.2299delG, that is, the obtained vector structures are pCMV-EGFP left -Exon13 c.2802T>G -EGFP right (report vector map as shown in Figure 3), pCMV-EGFP left -Exon13 c.2299delG -EGFP right (report vector map as shown in Figure 4), wherein the EGFP left sequence is as shown in SEQ As shown in ID NO:35, the sequence of EGFP right is shown in SEQ ID NO:36.
  • Detection method 293T cells are inoculated into a 24-well plate according to a certain amount, so that the cell confluence reaches about 80% after 24 hours.
  • Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFP left -Exon13 c.2802T>G -EGFP right and pUC57-U7-snRNA plasmid targeting USH2A pre-mRNA (vector mass ratio: 100ng:400ng), using separate transfection Reporter plasmids, 293T cells co-transfected with reporter plasmids and pUC57-U7-con were used as two negative controls, and 293T cells not transfected with any plasmids were used as blank controls.
  • 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, USH2A pre-mRNA No. 13 exon is induced to be spliced Proportion of jumping cells) and the average FITC intensity of GFP-positive cells (ie, the average level of splicing skipping of USH2A pre-mRNA exon 13 in GFP cells), the position of U7-snRNA targeting target region 1 on the genome (Fig. Left to right corresponds to genome from 5' end to 3' end) as shown in Figure 5.
  • U7-snRNA groups that is, USH2A pre-mRNA No. 13 exon is induced to be spliced Proportion of jumping cells
  • the average FITC intensity of GFP-positive cells ie, the average level of splicing skipping of USH2A pre-mRNA exon 13 in GFP cells
  • Examples 4, 5, and 6 Based on the analysis of Examples 4, 5, and 6, it is found that although the target regions 1, 2, and 3 are shown in the prior art to be AON-targeted non-sensitive regions, that is, targeting this region cannot/low-efficiency induces splicing skipping of exon 13, However, snRNA targeting this region can significantly induce exon 13 splicing skipping. Therefore, although both snRNA and AON can induce splicing skipping, the mechanism of action of the two is different, and the sensitivity of the target site (applicable target area target site) is also different.
  • Table 5 The average FITC intensity of U7-snRNA-induced (USH2A pre-mRNA exon 13 splicing skipping) targeting regions 1, 2, and 3 GFP-positive cells
  • Detection method the same as in Example 4, the position of the U7-snRNA targeting region 4 on the genome (the picture is from left to right corresponds to the genome from 5' to 3') as shown in Figure 14.
  • Targeting region 2 not only obtained a higher proportion of cells that induced splice skipping (GFP%), but also induced splice skipping mRNA and its protein levels (average FITC intensity) in the same cells.
  • the target site #2 and its adjacent sites #1 and #3 which are more efficient in inducing splice skipping by AON in the prior art, have lower mRNA and protein levels that induce splice skipping in the same cell in the snRNA system.
  • the AON efficiency of the prior art targeting region 3 is higher than that of region 2, but the efficiency of targeting region 2 in the snRNA system is higher than that of region 3. Therefore, although both snRNA and AON can induce splicing skipping, the mechanism of action of the two is different, and the sensitivity of the target site is also different.
  • Detection method Inoculate a certain amount of 293T into a 24-well plate, so that the cell confluence reaches about 80% after 24 hours.
  • the transfected cells were cultured for 72 hours, and then the RNA of each experimental group was extracted, cDNA was obtained by reverse transcription, and RT-PCR experiments were performed with primers AGCCTTTCCGCCAAGGTGATC (SEQ ID NO: 37) and CACAACGTTGCCCAGCAATGG (SEQ ID NO: 38) , to detect whether exon 13 exists in the mature USH2A mRNA, and the electrophoresis results are shown in FIG. 24 .
  • U7-snRNA#14 is a target site close to the AON site with a high probability of double splicing skipping in exon 12 and exon 13 in the prior art. The occurrence rate of exon splicing skipping is very low. It was further proved that U7-snRNA can significantly reduce the double skipping probability of exon 12 and exon 13 while inducing splicing skipping of exon 13.
  • 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 (SEQ ID NO:39), the antisense strand is the target sequence 5' plus AATT and 3' plus (SEQ ID NO: 40).
  • the recognition domain sequence of snRNA#14 is ACACUGGCAGGGCUCACAUCCA (SEQ ID NO: 41)
  • the sense strand of the synthesized Oligo DNA is (SEQ ID NO:42)
  • the antisense strand is (SEQ ID NO:43)
  • the underline indicates the DNA double-stranded sequence corresponding to the recognition domain sequence
  • the bold italic indicates the DNA double-stranded sequence corresponding to the binding motif "UAGGGU" of the hnRNP A1 protein.
  • U7-hnRNP A1-snRNA can also be chemically synthesized and modified according to the methods described in Example 2 and Example 4.
  • 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 the smOPT sequence, bold indicates the hnRNP A1 protein binding motif):
  • FIG. 25 The schematic diagram of the structure of U7-hnRNP A1-snRNA is shown in FIG. 25 .
  • the above-mentioned modified sequence is based on the sequence shown in SEQ ID NO: 44, adding phosphorothioation backbone modification and methoxyl modification on the first three bases at the 5' end and the 3' end.
  • Detection method Inoculate a certain amount of 293T into a 24-well plate, so that the cell confluence reaches about 80% after 24 hours.
  • 293T cells transfected with reporter plasmid alone, co-transfected with reporter plasmid and pUC57-U7Scramble were used as two negative controls, 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 using trypsin, and then flow cytometry was used to detect the splicing skipping efficiency induced by different s
  • This embodiment introduces a free tail at the 5' end of U7-snRNA, the free tail sequence includes the binding motif "UAGGGU" of the hnRNP A1 protein, and the free tail sequence is preferably (SEQ ID NO:45), 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, and will not affect its targeting specificity Sex, will not cause or increase off-target effects.
  • 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 to construct the pAAV-U7-snRNA vector, and AAV packaging plasmid: serotype pRC plasmid (containing the Rep gene of AAV2 and the respective Cap gene of each serotype), pHelper plasmid (the vector plasmid containing the E2A, E4 and VA genes of adenovirus) co-transfected host cells, and obtained by packaging AAV-U7-snRNA virus targeting USH2A pre-mRNA exon 13 splice skipping.
  • AAV packaging plasmid serotype pRC plasmid (containing the Rep gene of AAV2 and the respective Cap gene of each serotype), pHelper plasmid (the vector plasmid containing the E2A, E4 and VA genes of
  • Example 1 According to the method described in Example 1 and Example 6, according to the pre-transcriptional DNA sequence corresponding to the snRNA recognition domain sequence in Table 1, the corresponding Oligo DNA sense strand and antisense strand were respectively synthesized, and a type similar to Type IIs was added at both ends. Sticky ends after restriction endonuclease recognition site cleavage.
  • T4 ligase ligated into the linearized pAAV-U7-snRNA backbone plasmid recovered after digestion with the corresponding Type IIs restriction endonuclease to form USH2A-targeting pre-mRNA
  • the pAAV-U7-snRNA plasmid that induces splicing skipping at the specific site of exon 13 is named according to the snRNA number corresponding to the recognition domain sequence, such as pAAV-U7-snRNA#3, pAAV-U7-snRNA#14, pAAV-U7-snRNA#16 et al.
  • the target gene (U7-snRNA gene expression cassette that induces splicing skipping of exon 13 of USH2A pre-mRNA) and replacing the gene sequence between the AAV2-ITR domains of the pAAV-CMV plasmid, pAAV-U7- snRNA plasmid vector.
  • the AAV-U7-snRNA virus that targets and induces the splicing skipping of exon 13 of USH2A pre-mRNA was packaged and obtained according to the instructions and standard cell operation procedures.
  • 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 system in the table below, 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
  • the AAV2-ITR domain of the pAAV-U7-snRNA plasmid and the inserted target gene fragment should be less than 4.7kb, it is possible to insert multiple U7-snRNA gene expression cassettes (5'-mouse U7 promoter-recognition structure Domain-smOPT sequence, 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 -
  • the snRNA plasmid carries 1-10 U7-snRNA gene expression cassettes, and multiple U7-snRNA gene expression cassettes in the pAAV-U7-snRNA plasmid may have the same recognition domain.

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Abstract

提供了一种靶向USH2A pre-mRNA的snRNA，该snRNA的识别结构域与USH2A pre-mRNA序列反向互补，所述snRNA通过与USH2A pre-mRNA结合从而剪接跳跃第13号外显子。该snRNA促进第13号外显子跳读的效率比AON更高。

Description

靶向USH2A pre-mRNA的snRNA及其应用 技术领域
本发明涉及基因工程技术领域，特别是涉及靶向USH2A pre-mRNA的snRNA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。在临床上Usher综合征可分为3种类型：1、I型Usher综合征，患者的听力方面会出现先天性重深度感音神经性耳聋，前庭反应方面会出现前庭反应消失，视力方面会在***前出现色素性视网膜炎，然后逐渐致盲，与该类型关联的基因有MYO7A、CDH23、USH1C、PCHD15等；2、II型Usher综合征，患者的听力方面会出现先天性中重度感音神经性耳聋，前庭反应正常，视力方面会在***出现色素性视网膜炎，逐渐致盲，与该类型关联的基因有USH2A、GPR98、WHRN等；3、III型Usher综合征，患者的听力方面会出现进行性感音神经性耳聋，前庭反应正常，视力方面会在***末出现色素性视网膜炎，逐渐致盲，与该类型关联的基因有CLRN1等。其中，II型Usher综合征占Usher综合症的比例超过50％，而USH2A基因突变是Usher综合征II型的最常见原因，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.Glu767Serfs*21，欧美患者频率最高突变)、c.2276G>T(p.cys759phe，导致非综合征性RP中最常见的突变位点)、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、c.2339G>T(PMID:31904091)。
针对USH2A基因的第13号外显子，现有技术中常见手段一般为3种，第一种是通过CRISPR/Cas***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。但使用片段删除存在风险，如染色体重排、病毒整合、反向重新整合，以及长时间表达CAS***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高。第二种是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。第三种是通过反义寡核苷酸(AONs，Antisense oligonucleotides)靶向干扰pre-mRNA剪接，促进外显子跳读的效率较高。然而，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处理导致的全是双跳读，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，剪接双跳后的USH2A蛋白失活，且AON效果持续性不长，用药频率高；诱导剪接跳跃效率不高。
发明内容
针对上述问题，本发明提供了一种靶向USH2A pre-mRNA的snRNA，该snRNA促进第13号外显子跳读的效率比AON更高。
为了达到上述目的，本发明提供了一种靶向USH2A pre-mRNA的snRNA，所述snRNA的识别结构域与USH2A pre-mRNA序列反向互补，所述snRNA通过与USH2A pre-mRNA结合从而诱导第13号外显子剪接跳跃。
本发明人对USH2A进行调查研究后发现，因为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，所以难以通过直接递送USH2A进行治疗。而小鼠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。同时，本发明人发现在敲除了小鼠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。因此，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。
同时，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。但U7-snRNP不参与剪接，而是复制依赖型组蛋白(RDH)pre-mRNA独特3'末端加工的关键因素。
因此，本发明人将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。
本发明人通过靶点筛选发现了U7-snRNP诱导剪接跳跃的7个靶区域，21个靶位点，可 诱导第13号外显子剪接跳跃效果，同时设计U7-smOPT-snRNA的识别结构域与USH2A pre-mRNA第13号外显子剪接跳跃相关位点反向互补，从而诱导U7-smOPT-snRNA结合USH2A pre-mRNA第13号外显子剪接跳跃相关位点，进而诱导USH2A pre-mRNA的第13号外显子的剪接跳跃，并且保留第12号外显子，使第12号外显子和13双跳读等非预期剪接跳跃的概率显著降低，在显著提升效率的同时，保证了安全性。
在其中一个实施例中，所述USH2A pre-mRNA序列选自以下区域：USH2A第13号外显子、第12号内含子或第13号内含子。
在其中一个实施例中，所述USH2A pre-mRNA序列选自USH2A第13号外显子及向两侧延伸50bp的区域。
在其中一个实施例中，所述USH2A pre-mRNA序列选自USH2A第13号外显子及及向两侧延伸20bp的区域。
在其中一个实施例中，所述第13号外显子包括：野生型的第13号外显子或含有突变的第13号外显子。
在其中一个实施例中，所述突变包括以下突变位点中的至少1个：c.2242C>T、c.2276G>T、c.2299delG、c.2522C>A、c.2541C>A、c.2761delC、c.2776C>T、c.2802T>G、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。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位(对应于NCBI数据库GRch38版本)区域选自：Chr1：216247142-216247185、Chr1：216247130-216247161、Chr1：216246616-216246649、Chr1：216247213-216247246、Chr1：216247204-216247232、Chr1：216247187-216247220、Chr1：216247169-216247202。
在其中一个实施例中，所述USH2A pre-mRNA序列选自以下序列：SEQ ID NO：1-7序列。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位区域选自：Chr1：216247223-216247246、Chr1：216247218-216247241、Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247138-216247161、Chr1：216247130-216247153、Chr1：216246626-216246649、Chr1：216246622-216246645、Chr1：216246616-216246639。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少16bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少17bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少 18bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少19bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少20bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少21bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少22bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少23bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少24bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中18bp-40bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中20bp-27bp的连续序列反向互补。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：8-28。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位选自：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：10-18。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位选自：Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247138-216247161、Chr1：216247130-216247153、Chr1：216246626-216246649、Chr1：216246622-216246645、Chr1：216246616-216246639。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：19-28。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位选自：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：10-23、 SEQ ID NO：25。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位选自：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：10-19、SEQ ID NO：21-23、SEQ ID NO：25。
在其中一个实施例中，所述USH2A pre-mRNA序列对应的基因组定位选自：Chr1：216247218-216247241、Chr1：216247187-216247210、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：9、SEQ ID NO：15、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25。
在其中一个实施例中，所述snRNA的识别结构域选自以下序列：SEQ ID NO：15、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23。
在其中一个实施例中，所述snRNA为U1-snRNA或U7-snRNA。
在其中一个实施例中，所述U7-snRNA为化学合成。
在其中一个实施例中，所述化学合成的U7-snRNA中至少1个核苷酸进行了化学修饰。
在其中一个实施例中，所述化学修饰包括2′-O烷基修饰、2′-O-甲氧基修饰或2′-O-甲氧基乙基修饰中的至少1种。
在其中一个实施例中，所述2′-O烷基修饰为2′-O-甲基修饰。
在其中一个实施例中，所述化学合成的U7-snRNA中至少1个核苷酸通过磷酸酯键连接，所述磷酸酯键包括硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、酰胺磷酸酯键、硼烷磷酸酯键或手性连接磷中的至少1种。
在其中一个实施例中，所述化学合成的U7-snRNA两侧的6-80个核苷酸均进行了所述化学修饰并通过所述磷酸酯键连接。
在其中一个实施例中，所述化学合成的U7-snRNA两侧的1-10个核苷酸均进行了所述化学修饰并通过所述磷酸酯键连接。
在其中一个实施例中，所述化学合成的U7-snRNA两侧的3-40个碱基均进行了所述化学修饰并通过所述磷酸酯键连接。
在其中一个实施例中，所述化学合成的U7-snRNA的所有核苷酸均通过硫代磷酸酯键连接，且均进行了2′-O-甲氧基修饰。
在其中一个实施例中，所述化学合成的U7-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。
在其中一个实施例中，所述化学合成的U7-snRNA的5’端的首个核苷酸为腺苷酸。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域5’端的首个核苷酸为腺苷酸。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域与靶位点反向互补配对中有0-5个错配核苷酸。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域与靶位点反向互补配对中有0-1个错配核苷酸。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域的序列长度≥16bp。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域的序列长度为18bp-40bp。
在其中一个实施例中，所述化学合成的U7-snRNA的识别结构域的序列长度为20bp-27bp。
在其中一个实施例中，所述化学合成的U7-snRNA沿着靶序列的5’端或/和3’端继续通过反向互补配对延申识别结构域序列长度。
在其中一个实施例中，所述延申识别结构域序列长度≤40bp。
在其中一个实施例中，所述snRNA包括sm序列。
在其中一个实施例中，所述sm序列为smOPT序列，所述smOPT序列如SEQ ID NO:31所示。
在其中一个实施例中，所述snRNA包含识别结构域、smOPT序列以及U1-snRNA scafford或U7-snRNA scafford序列；优选的，所述U7-snRNA scafford序列如SEQ ID NO:49所示。
在其中一个实施例中，所述snRNA还包括募集剪接调控蛋白的基序。
在其中一个实施例中，在所述U7-snRNA的5’端引入游离尾部，所述游离尾部序列包括募集剪接调控蛋白的基序。
在其中一个实施例中，所述剪接调控蛋白包括hnRNPA1、SRSF1、RBM4、DAZAP1或SR中的至少1种。
在其中一个实施例中，所述游离尾部序列包括至少1个hnRNPA1结合基序。
在其中一个实施例中，所述游离尾部序列包括2个hnRNPA1结合基序。
本发明还提供了一种核苷酸，该核苷酸包括编码所述snRNA的核苷酸序列。
本发明还提供了一种基因表达盒，该基因表达盒包括所述snRNA，和/或包括所述核苷酸。
本发明还提供了一种载体，该载体包括所述snRNA，包括所述核苷酸，和/或包括所述基因表达盒。
在其中一个实施例中，所述载体包括载体骨架和所述snRNA，所述载体骨架包括启动子、smOPT序列和snRNA基因特异性3’盒，所述启动子通过Tpye IIs型限制性内切酶识别位点连接所述smOPT序列。
在其中一个实施例中，所述载体选自：pUC57、pAAV-CMV、慢病毒或转座子。
在其中一个实施例中，所述snRNA基因特异性3’盒为snRNA基因3’端外延28-131bp的基因片段。
在其中一个实施例中，所述snRNA基因特异性3’盒为snRNA基因3’端长度为106bp的基因片段。
本发明还提供了一种病毒颗粒，该病毒颗粒包括所述snRNA，包括所述核苷酸，和/或包括所述载体。
在其中一个实施例中，所述病毒颗粒为AAV病毒。
在其中一个实施例中，所述AAV病毒的衣壳蛋白为天然来源、基于天然来源衣壳蛋白的变体。
在其中一个实施例中，所述AAV病毒的衣壳蛋白来源于动物或植物。
在其中一个实施例中，所述AAV病毒的衣壳蛋白选自：AAV1、AAV2、AAV3、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh8、AAVrh10或AAVrh43。
在其中一个实施例中，所述基于天然来源衣壳蛋白的变体AAV病毒，为基于天然来源衣壳蛋白进行定向进化或进行氨基酸/肽段合理改造后的AAV病毒。
在其中一个实施例中，所述变体AAV病毒选自AAV2.5、AAV2i8、AAV-TT、AAV9.HR或CAM130。
在其中一个实施例中，所述AAV病毒为AAV ITR时，血清型与Rep基因血清型一致。
本发明还提供了一种细胞，该细胞包括所述snRNA，包括所述核苷酸，包括所述载体，和/或所述病毒颗粒。
本发明还提供了一种药物组合物，该药物组合物包括所述snRNA，包括所述核苷酸，包括所述载体，和/或包括所述病毒颗粒。
本发明还提供了一种获得第13号外显子表达产物缺失的Usherin蛋白的方法，该方法通过使USH2A 基因的pre-mRNA与所述snRNA，所述核苷酸，所述载体，所述基因表达盒，所述病毒颗粒，所述细胞，和/或所述药物组合物接触。
在其中一个实施例中，所述方法用于科学研究。
本发明还提供了所述方法在使USH2A pre-mRNA第13号外显子剪接跳跃，制备缺失第13号外显子的成熟USH2A mRNA，获得第13号外显子缺失的Usherin蛋白，和/或抑制USH2A pre-mRNA第13号外显子表达和功能中的应用。
本发明还提供了所述snRNA、所述核苷酸、所述载体、所述基因表达盒、所述病毒颗粒或所述细胞在制备预防和/或治疗眼病和/或耳病的药物中的应用。
在其中一个实施例中，所述眼病为耳聋-色素性视网膜炎综合征或非综合征性视网膜色素变性疾病。
与现有技术相比，本发明具有以下有益效果：
本发明的靶向USH2A pre-mRNA的snRNA，选择USH2A第13号外显子作为靶向区域，诱导第13号外显子剪接跳跃，从而治疗USH2A第13号外显子的错义、移码、终止密码、无义、同义突变等引起USH2A蛋白功能异常的眼部和耳部疾病。同时，本发明的一种靶向USH2A的U7-snRNA，利用U7-snRNP不参与剪接，而是复制依赖型组蛋白(RDH)pre-mRNA独特3'末端加工的关键因素，且修饰后的U7-snRNA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的特有工作机制，结合靶向靶向USH2A pre-mRNA的snRNA，实现诱导USH2A pre-mRNA的第13号外显子的剪接跳跃，并且保留第12号外显子，使第12号外显子和13双跳读等非预期剪接跳跃的概率显著降低，在显著提升第13号外显子单跳读效率的同时，保证了安全性。
附图说明
图1为U7-snRNA的结构和作用示意图；
图2为pUC57-U7snRNA骨架载体图谱；
图3为报告载体pCMV-EGFP left-Exon13 c.2802T>G-EGFP right质粒图谱；
图4为报告载体pCMV-EGFP left-Exon13 c.2299delG-EGFP right质粒图谱；
图5为靶向靶区域1的U7-snRNA在基因组上的位置示意图；
图6为靶向靶区域1的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图7为靶向靶区域1的U7-snRNA诱导的(USH2A pre-mRNA第13号外显子剪接跳跃)GFP阳性细胞的平均FITC强度柱状图；
图8为靶向靶区域2的U7-snRNA在基因组上的位置示意图；
图9为靶向靶区域2的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图10为靶向靶区域2的U7-snRNA诱导的(USH2A pre-mRNA第13号外显子剪接跳跃)GFP阳性细胞的平均FITC强度柱状图；
图11为靶向靶区域3的U7-snRNA在基因组上的位置示意图；
图12为靶向靶区域3的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图13为靶向靶区域3的U7-snRNA诱导的(USH2A pre-mRNA第13号外显子剪接跳跃)GFP阳性细胞的平均FITC强度柱状图；
图14为靶向靶区域4的U7-snRNA在基因组上的位置示意图；
图15为靶向靶区域4的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图16为靶向靶区域5的U7-snRNA在基因组上的位置示意图；
图17为靶向靶区域5的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图18为靶向靶区域6的U7-snRNA在基因组上的位置示意图；
图19为靶向靶区域6的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图20为靶向靶区域7的U7-snRNA在基因组上的位置示意图；
图21为靶向靶区域7的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA第13号外显子剪接跳跃的细胞比例结果图；
图22为靶向不同区域的U7snRNA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的平均FITC强度结果图；
图23为U7-snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃效率显著优于AON1的实验结果图；
图24为在WERI细胞中检测化学合成的U7snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃效率的结果图；
图25为U7-hnRNP A1-snRNA的结构示意图；
图26为U7-hnRNP A1-snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃效率结果图。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。需要说明的是，当一段序列被称为“选自”另一段序列，它可以直接是另一段序列或者另一段序列中的序列片段；当一个基因组定位区域“选自”另一个基因组定位区域，则它对应序列可以直接是另一个基因定位区域对应序列或者另一个基因定位区域对应序列中的序列片段；所述序列包括DNA或RNA。
定义：
本发明所述的snRNA：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。
材料来源：
Figure PCTCN2022140599-appb-000001
Helper Free System(AAV5)试剂盒(TAKARA公司，Code No.6650)
本实施例所用试剂、材料、设备如无特殊说明，均为市售来源；实验方法如无特殊说明，均为本领域的常规实验方法。
实施例1
U7-snRNA体系的设计和载体构建。
1、合成U7-snRNA表达载体骨架。
野生型U7-snRNA包括茎环结构(scafford)、U7特异性的Sm序列(AAUUUGUCUAG)和识别结构域(与组蛋白pre-mRNA互补)。
本实施例U7-snRNA是在NCBI上小鼠野生型U7-snRNA的基因序列(NCBI Reference Sequence:NR_024201.3)的基础上，将U7特异性sm序列AATTTGTCTAG(SEQ ID NO:30)替换为优化的共有sm序列，即smOPT：AATTTTTGGAG(SEQ ID NO:31)，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，如图1所示。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。
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-U7snRNA scafford-snRNA基因特异性3’盒-3’)的pUC57载体(如图2所示)。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，以方便后续切除、替换以及***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:32)的序列，参与pre-snRNA的加工，优选连接在U7snRNA基因3’端后的序列，优选序列长度为28-131bp的基因片段，进一步优选序列长度为106bp。
2、构建靶向USH2A 第13号外显子及其附近不同位点的U7-snRNA载体。
本实施例分别针对USH2A pre-mRNA的7个靶区域设置了21个靶位点，USH2A Pre-mRNA的7个靶区域如下所示。
第13号外显子区域1(SEQ ID NO:1)(Chr1：216247142-216247185)：CGAAGCUUUAAUGAUGUUGGAUGUGAGCCCUGCCAGUGUAACCU；
第13号外显子区域2(SEQ ID NO:2)(Chr1：216247130-216247161)：GAGCCCUGCCAGUGUAACCUCCAUGGCUCAGU；
第13号外显子区域3(SEQ ID NO:3)(Chr1：216246616-216246649)：AAUCAGUGGCCAGUGCCUGUGUGUGCCUAAUCGU；
第13号外显子区域4(SEQ ID NO:4)(Chr1：216247213-216247246)：UAAAUAUAUUUUAUCUUUAGGGCUUAGGUGUGAU；
第13号外显子区域5(SEQ ID NO:5)(Chr1：216247204-216247232)：CUUUAGGGCUUAGGUGUGAUCAUUGCAAU；
第13号外显子区域6(SEQ ID NO:6)(Chr1：216247187-216247220)：GGUGUGAUCAUUGCAAUUUUGGAUUUAAAUUUCU；
第13号外显子区域7(SEQ ID NO:7)(Chr1：216247169-216247202)：UUGGAUUUAAAUUUCUCCGAAGCUUUAAUGAUGU。
所述21个靶位点如下表所示。
表1 snRNA的识别结构域序列
Figure PCTCN2022140599-appb-000002
Figure PCTCN2022140599-appb-000003
Figure PCTCN2022140599-appb-000004
实施例2
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，产生包含识别结构域、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#16和snRNA#15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#16：
Figure PCTCN2022140599-appb-000005
上述修饰的序列为在SEQ ID NO:33所示序列的基础上，在5’端及3’端的前三个碱基上增加硫代磷酸化骨架修饰和甲氧基修饰；
化学合成和修饰的U7-snRNA#15：
Figure PCTCN2022140599-appb-000006
上述修饰的序列为在SEQ ID NO:34所示序列的基础上，在5’端及3’端的前三个碱基上增加硫代磷酸化骨架修饰和甲氧基修饰。
实施例3
构建用于定量评价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***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)，RGleft表示没有报告功能的报告基因5’端前半部分，RGright表示没有报告功能的报告基因3’端后半部分，RGleft和RGright串联表达可正常行使完整报告基因功能。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串联的基因序列；下游内含子序列为人USH2A第13号内含子的5’端703bp和3’端216bp串联的基因序列)。所述USH2A 第13号外显子的致病突变可以包括c.2802T>G、c.2299delG、c.2276G>T、c.2522C>A、c.2242C>T、c.2541C>A、c.2761delC或c.2776C>T等，在本发明实施例中所述的USH2A 第13号外显子的致病突变为c.2802T>G、c.2299delG，即获得的载体结构分别为pCMV-EGFP left-Exon13 c.2802T>G-EGFP right(报告载体图谱如图3所示)，pCMV-EGFP left-Exon13 c.2299delG-EGFP right(报告载体图谱如图4所示)，其中EGFP left序列如SEQ ID NO:35所示，EGFP right序列如SEQ ID NO:36所示。
实施例4
检测靶向靶区域1的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：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 c.2802T>G-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、共转染报告质粒和pUC57-U7-con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 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)，靶向靶区域1的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图5所示。
实验结果：如图6和下表所示，靶向靶区域1的所有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。现有技术靶向该区域1的AON不能诱导第13号外显子剪接跳跃，但靶向该区域1的snRNA却可高效诱导第13号外显子剪接跳跃。
表2 靶向区域1的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 PCTCN2022140599-appb-000007
Figure PCTCN2022140599-appb-000008
实施例5
检测靶向靶区域2的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向靶区域2的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图8所示。
实验结果：如图9和下表所示，靶向靶区域2的所有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。现有技术靶向该区域2的AON诱导第13号外显子剪接跳跃的效果较低，但靶向该区域2的snRNA却可高效诱导第13号外显子剪接跳跃。
表3 靶向区域2的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 PCTCN2022140599-appb-000009
实施例6
检测靶向靶区域3的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向靶区域3的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图11所示。
实验结果：如图12和下表所示，靶向靶区域3的所有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。现有技术靶向该区域3的AON诱导第13号外显子剪接跳跃的效果较低，但靶向该区域3的snRNA却可高效诱导第13号外显子剪接跳跃。
表4 靶向区域3的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 PCTCN2022140599-appb-000010
结合实施例4、5、6分析发现，尽管现有技术中显示靶区域1、2、3是AON靶向非敏感区，即靶向该区域无法/低效率诱导第13号外显子剪接跳跃，但是靶向该区域的snRNA却可显著诱导第13号外显子剪接跳跃。因此，尽管snRNA与AON都可以诱导剪接跳跃，但两者的作用机制不同，靶位点敏感性(所适用的靶区域靶位点)也不相同。
同时，如图7、10、13和下表所示，尽管靶向同一区域的诱导剪接跳跃的细胞比例(GFP％)接近，但是靶向同一区域不同靶点的snRNA在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)有所区别。且靶区域1、靶区域2在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平均优于靶区域3。
表5 靶向区域1、2、3的U7-snRNA诱导的(USH2A pre-mRNA第13号外显子剪接跳跃)GFP阳性细胞的平均FITC强度
Figure PCTCN2022140599-appb-000011
实施例7
检测靶向靶区域4的U7-snRNA和snRNA#24介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向区域4的U7-snRNA在基因组上的位置(图片从左到 右对应于基因组从5’端到3’端)如图14所示。
实验结果：如图15和下表所示，靶向靶区域4的所有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。
表6 靶向区域4的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 PCTCN2022140599-appb-000012
实施例8
检测靶向靶区域5的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向靶区域5的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图16所示。
实验结果：如图17和下表所示，靶向靶区域5的所有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。
表7 靶向区域5的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 PCTCN2022140599-appb-000013
实施例9
检测靶向靶区域6的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向靶区域6的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图18示。
实验结果：如图19和下表所示，靶向靶区域6的所有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。
表8 靶向区域6的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 PCTCN2022140599-appb-000014
实施例10
检测靶向靶区域7的U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
检测方法：和实施例4相同，靶向靶区域7的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5’端到3’端)如图20示。
实验结果：如图21和下表所示，靶向靶区域7的所有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。
表9 靶向区域7的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 PCTCN2022140599-appb-000015
实施例11
检测U7-snRNA介导的USH2A第13号外显子剪接跳跃效果。
验证方法：和实施例4相同。
实验结果：不同区域的U7-snRNA诱导的GFP阳性细胞的平均FITC强度如图22和下表所示。
表10 靶向不同区域的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的平均FITC强度
Figure PCTCN2022140599-appb-000016
Figure PCTCN2022140599-appb-000017
尽管靶向同一区域的(诱导剪接跳跃的细胞比例)GFP％接近，但是靶向同一区域的不同靶点snRNA在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)不同；
靶向区域2不仅获得较高的诱导剪接跳跃的细胞比例(GFP％)，而且在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)较高。
而现有技术AON诱导剪接跳跃效率较高的靶位点#2以及其临近位点#1和#3，在snRNA体系中在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平较低。现有技术靶向区域3的AON效率比区域2更高，但是在snRNA体系靶向区域2的效率比区域3更高。因此，尽管snRNA与AON都可以诱导剪接跳跃，但两者的作用机制不同，靶位点敏感性也不相同。且结合实施例12结果分析，由于snRNA#24的效率与snRNA#2、AON1的效率近似，则可推知snRNA#3-#11均效果均优于snRNA#2、snRNA#24，优于AON1。
实施例12
验证U7-snRNA介导的USH2A第13号外显子剪接跳跃效率显著优于AON。
检测方法：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 c.2802T>G-EGFP right分别和不同靶点的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、共转染报告质粒的和pUC57-U7Scramble的293T细胞作为两种阴性对照，共转染报告质粒的和10pmol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)的293T细胞作为阳性 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的GFP阳性率和平均FITC强度。
实验结果：如图23和下表所示，本发明筛选出来的snRNA#8和snRNA#18均优于AON1，snRNA#2与AON1效率相近。
表11 U7-snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃效率显著优于AON1
Figure PCTCN2022140599-appb-000018
实施例13
化学合成的U7-snRNA在WERI细胞中诱导USH2A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#1、snRNA#4、snRNA#8、snRNA#10、snRNA#14分别转染WERI细胞，转染相同剂量(100pmol)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:37)和CACAACGTTGCCCAGCAATGG(SEQ ID NO:38)进行RT-PCR实验，检测成熟的USH2A mRNA是否存在第13号外显子，电泳结果如图24所示。
实验结果：在内源性表达Usherin蛋白的WERI细胞中，将化学合成的U7-snRNA诱导USH2A pre-mRNA第13号外显子剪接跳跃的效果与AON技术方案进行比较，由RT-PCR试验数据和分析结果显示，AON单跳条带相对snRNA弱，且AON单跳条带下存在更为明显的双跳条带，由此可知，化学合成的U7-snRNA实验组1和U7-snRNA实验组2诱导第13号外显子单剪接跳跃的效果显著优于AON1和AON2技术方案。且进一步通过ImageJ软件对rt-PCR电泳条带进行定量分析，并第12号外显子和第13号外显子双跳读的USH2A mRNA 占总跳读USH2A mRNA(第13号外显子单跳读、第12号外显子和第13号外显子双跳读的USH2A mRNA总和)的比例进行分析，结果显示U7-snRNA诱导第12号外显子和第13号外显子双剪接跳跃的比例非常低，且低于AON1、AON2的双跳比例。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，U7-snRNA#14为靶向位点临近于现有技术第12号外显子和第13号外显子双剪接跳跃概率极高的AON位点，然而，U7-snRNA#14处理后双外显子剪接跳跃的发生概率却非常低。进一步证明，U7-snRNA在诱导第13号外显子剪接跳跃的同时，可显著降低第12号外显子和第13号外显子的双跳概率。
实施例14
检测连接hnRNP A1结合基序的U7-snRNA的剪接跳跃效果。
1、连接hnRNP A1结合基序的U7-snRNA的构建。
根据表中的gRNA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 PCTCN2022140599-appb-000019
(SEQ ID NO:39)，反义链为靶序列5’加AATT并且3’加
Figure PCTCN2022140599-appb-000020
(SEQ ID NO:40)。例如，snRNA#14的识别结构域序列为ACACUGGCAGGGCUCACAUCCA(SEQ ID NO:41)，则合成的Oligo DNA正义链为
Figure PCTCN2022140599-appb-000021
(SEQ ID NO:42)，反义链为
Figure PCTCN2022140599-appb-000022
(SEQ ID NO:43)，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”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#编号。提纯质粒，保存于-20℃备用。
U7-hnRNP A1-snRNA还可以依据实施例2、实施例4所述的方法进行化学合成和修饰。以snRNA#14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 PCTCN2022140599-appb-000023
U7-hnRNP A1-snRNA的结构示意图如图25所示。
上述修饰的序列为在SEQ ID NO:44所示序列的基础上，在5’端及3’端的前三个碱基 上增加硫代磷酸化骨架修饰和甲氧基修饰。
2、检测连接hnRNP A1结合基序的U7-snRNA在报告基因细胞中诱导USH2A第13号外显子剪接跳跃的效率。
检测方法：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 c.2802T>G-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、共转染报告质粒的和pUC57-U7Scramble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。
实验结果：如图26和下表所示，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)。
表12 U7-hnRNP A1-snRNAUSH2A pre-mRNA第13号外显子剪接跳跃效率
Figure PCTCN2022140599-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”，游离尾部序列优选为
Figure PCTCN2022140599-appb-000025
(SEQ ID NO:45)，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，不影响其靶向特异性，不会造成或者增加脱靶效应。
实施例15
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***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，以方便后续切除、 替换以及***其他识别结构域序列。将全基因合成的序列***并替换pAAV-CMV质粒两个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:46)：
Figure PCTCN2022140599-appb-000026
依照实施例1和实施例6所述方法，根据表1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#3、pAAV-U7-snRNA#14、pAAV-U7-snRNA#16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)***并替换pAAV-CMV质粒AAV2-ITR结构域之间的基因序列后，获得pAAV-U7-snRNA质粒载体。依据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)转染试剂按照下表的体系配置好，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。
表13 PEI转染体系
Figure PCTCN2022140599-appb-000027
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***pAAV-U7-snRNA质粒AAV2-ITR结构域及其***的目的基因片段应小于4.7kb，因此，可通过***多个U7-snRNA基因表达盒子(5’-小鼠U7启动子-识别结构域-smOPT序列、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1-10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。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (18)

  1. 一种靶向USH2A pre-mRNA的snRNA，其特征在于，所述snRNA的识别结构域与USH2Apre-mRNA序列反向互补，所述snRNA通过与USH2A pre-mRNA结合从而剪接跳跃第13号外显子。
  2. 根据权利要求1所述的snRNA，其特征在于，所述USH2A pre-mRNA序列选自以下区域：USH2A第13号外显子、第12号内含子或第13号内含子。
  3. 根据权利要求2所述的snRNA，其特征在于，所述USH2A pre-mRNA序列对应的基因组定位区域选自：Chr1：216247142-216247185、Chr1：216247130-216247161、Chr1：216246616-216246649、Chr1：216247213-216247246、Chr1：216247204-216247232、Chr1：216247187-216247220、Chr1：216247169-216247202。
  4. 根据权利要求3所述的snRNA，其特征在于，所述USH2A pre-mRNA序列选自以下序列：SEQ ID NO：1-7。
  5. 根据权利要求1-4中任一项所述的snRNA，其特征在于，所述snRNA的识别结构域与所述USH2A pre-mRNA序列中至少16bp的连续序列反向互补，优选至少20bp的连续序列反向互补，更优选20bp-27bp的连续序列反向互补。
  6. 根据权利要求1所述的snRNA，所述USH2A pre-mRNA序列对应的基因组定位区域选自：Chr1：216247223-216247246、Chr1：216247218-216247241、Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247138-216247161、Chr1：216247130-216247153、Chr1：216246626-216246649、Chr1：216246622-216246645、Chr1：216246616-216246639；
    优选：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192；或者
    优选：Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247138-216247161、Chr1：216247130-216247153、Chr1：216246626-216246649、Chr1：216246622-216246645、Chr1：216246616-216246639；
    更优选：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247155-216247178、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153；
    进一步优选：Chr1：216247213-216247236、Chr1：216247209-216247232、Chr1：216247204-216247227、Chr1：216247197-216247220、Chr1：216247191-216247214、Chr1：216247187-216247210、Chr1：216247179-216247202、Chr1：216247174-216247197、Chr1：216247169-216247192、Chr1：216247162-216247185、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153；
    或者
    所述USH2A pre-mRNA序列对应的基因组定位区域选自：Chr1：216247218-216247241、Chr1：216247187-216247210、Chr1：216247147-216247168、Chr1：216247147-216247173、Chr1：216247142-216247165、Chr1：216247130-216247153。
  7. 根据权利要求1所述的snRNA，其特征在于，所述snRNA的识别结构域选自以下序列：SEQ ID NO：8-28，优选SEQ ID NO：10-18或SEQ ID NO：19-28；更优选SEQ ID NO：10-23、SEQ ID NO：25；进一步优选SEQ ID NO：10-19、SEQ ID NO：21-23、SEQ ID NO：25；
    或者
    所述snRNA的识别结构域选自以下序列：SEQ ID NO：9、SEQ ID NO：15、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25；优选SEQ ID NO：15、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23。
  8. 根据权利要求1-7任一项所述的snRNA，其特征在于，所述snRNA为U1-snRNA或U7-snRNA。
  9. 根据权利要求8所述的snRNA，其特征在于，所述snRNA包括sm序列，所述sm序列优选为smOPT序列，所述smOPT序列更优选如SEQ ID NO:31所示。
  10. 根据权利要求9所述的snRNA，其特征在于，所述snRNA包含识别结构域、smOPT序列以及U1-snRNA scafford或U7-snRNA scafford序列；优选的，所述U7-snRNA scafford序列如SEQ ID NO:49所示。
  11. 根据权利要求10所述的snRNA，其特征在于，所述snRNA还包括募集剪接调控蛋白 的基序，所述剪接调控蛋白优选包括hnRNPA1、SRSF1、RBM4、DAZAP1或SR中的至少1种。
  12. 一种核苷酸，其特征在于，该核苷酸包括编码权利要求1-11中任一项所述snRNA的核苷酸序列。
  13. 一种载体，其特征在于，该载体包括权利要求1-10中任一项所述的snRNA和/或包括权利要求11所述的核苷酸。
  14. 一种病毒颗粒，其特征在于，该病毒颗粒包括权利要求1-11中任一项所述的snRNA，包括权利要求12所述的核苷酸，和/或包括权利要求13所述的载体。
  15. 一种细胞，其特征在于，该细胞包括权利要求1-11中任一项所述的snRNA，包括权利要求12所述的核苷酸，包括权利要求13所述的载体，和/或权利要求14所述的病毒颗粒。
  16. 一种药物组合物，其特征在于，该药物组合物包括权利要求1-11中任一项所述的snRNA，包括权利要求12所述的核苷酸，包括权利要求13所述的载体，包括权利要求14所述的病毒颗粒和/或包括权利要求15所述的细胞。
  17. 一种获得第13号外显子表达产物缺失的Usherin蛋白的方法，其特征在于，该方法通过使USH2A基因的pre-mRNA与权利要求1-11中任一项所述的snRNA，权利要求12所述的核苷酸，权利要求13所述的载体，权利要求14所述的病毒颗粒，权利要求15所述的细胞，和/或权利要求16所述的药物组合物接触。
  18. 权利要求1-11中任一项所述snRNA、权利要求12所述核苷酸、权利要求13所述载体、权利要求14所述病毒颗粒、权利要求15所述细胞、权利要求16所述药物组合物或权利要求17所述方法，在制备预防和/或治疗眼病和/或耳病的药物中的应用。
PCT/CN2022/140599 2022-01-04 2022-12-21 靶向USH2A pre-mRNA的snRNA及其应用 WO2023130959A1 (zh)

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