WO2025007937A1 - Snrna targeting ush2a pre-mrna pseudo-exon pe40 and use thereof - Google Patents

Abstract

An snRNA targeting USH2A pre-mRNA pseudo-exon PE40 and the use thereof, which belong to the technical field of genetic engineering. The recognition domain of the snRNA is reversely complementary to the pre-mRNA sequence of USH2A gene, and the snRNA binds to the pre-mRNA of USH2A gene, thereby splicing and skipping exon PE40. The snRNA has a relatively high PE40 skipping efficiency, which is higher than that of the strategy of targeted interference of PRE-mRNA splicing by AON.

Description

snRNA targeting USH2A pre-mRNA pseudoexon PE40 and its application

This application claims the priority of Chinese patent application No. 2023108146551 filed on July 4, 2023. This application cites the entire text of the above Chinese patent application.

Technical Field

The present invention relates to the field of genetic engineering technology, and in particular to a snRNA targeting USH2A pre-mRNA pseudoexon PE40 and an application thereof.

Background Art

Usher syndrome is a genetic disease, also known as deafness-retinitis pigmentosa syndrome, which is characterized by varying degrees of congenital sensorineural deafness and progressive vision loss caused by retinitis pigmentosa (RP).

Clinically, Usher syndrome can be divided into three types: 1. Type I Usher syndrome, in which patients will have congenital severe profound sensorineural hearing loss, vestibular response loss, and vision will develop pigmentary retinitis before puberty, which will gradually lead to blindness. The genes associated with this type include MYO7A, CDH23, USH1C, PCHD15, etc.; 2. Type II Usher syndrome, in which patients will have congenital moderate to severe sensorineural hearing loss, normal vestibular response, and vision will develop pigmentary retinitis during puberty, which will gradually lead to blindness. The genes associated with this type include USH2A, GPR98, WHRN, etc.; 3. Type III Usher syndrome, in which patients will have progressive sensorineural hearing loss, normal vestibular response, and vision will develop pigmentary retinitis at the end of puberty, which will gradually lead to blindness. The genes associated with this type include CLRN1, etc.

Among them, type II Usher syndrome accounts for more than 50% of Usher syndrome, and USH2A gene mutation is the most common cause of type II Usher syndrome, covering more than 50% of Usher syndrome patients. At the same time, mutation of the USH2A gene is also one of the important causes of non-syndromic retinitis pigmentosa (NSRP).

USH2A is located on 1q41 (chromosome 1, long arm, region 4, band 1), spanning more than 800kb in the genome, encoding a large transmembrane protein, Usherin, which is anchored on the plasma membrane of retinal photoreceptor cells and inner ear hair cells and is an essential component for the development and maintenance of cilia. In the retina, Usherin is an important part of the USH2 complex and is believed to play a role in stabilizing the outer segments of photoreceptors. USH2A has two subtypes. The main subtype in retinal cells contains 72 Exons and the coding region is approximately 15.6kb in length. The extracellular part of the Usherin protein contains many repeated domains, including 10 Laminin EGF-like (LE) domains and 35 Fibronectin type 3 (FN3) domains.

More than 1,000 pathogenic mutations distributed throughout the USH2A gene have been identified to date. The mutation c.7595-2144A＞G in intron 40 of the USH2A gene forms a splice donor site, resulting in the inclusion of a 152bp abnormal exon PE40 in USH2A mRNA and premature termination of translation, leading to Usher syndrome or retinitis pigmentosa RP.

For the abnormal exon PE40 (also known as pseudoexon) of the USH2A gene, the existing technology uses antisense oligonucleotides (AONs) to target and interfere with pre-mRNA splicing. However, this method promotes PE40 skipping. The reading efficiency is low.

Summary of the invention

Based on this, it is necessary to provide a snRNA targeting the USH2A pre-mRNA pseudoexon PE40 to address the above-mentioned problem of low skipping efficiency, which has a higher PE40 skipping efficiency and is higher than the AON targeted interference strategy for pre-mRNA splicing.

A snRNA targeting exon PE40 pre-mRNA of the USH2A gene, wherein the recognition domain of the snRNA is reverse complementary to the pre-mRNA sequence of the USH2A gene, and the snRNA splices and skips exon PE40 by binding to the pre-mRNA of the USH2A gene.

After investigating and studying USH2A, the inventors found that because the USH2A coding region is about 15.6 kb in length, 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 treat by directly delivering USH2A.

Considering that exon 12 of mouse USH2A is homologous to exon PE40 of human USH2A, both are 642bp in length, the removal of this exon did not cause subsequent frameshift mutations. At the same time, the inventors found that after knocking out exon 12 of mouse USH2A, Usherin can still be correctly positioned and perform normal functions. Therefore, for human USH2A exon PE40 containing pathogenic mutations, a series of means can be used to cause it to skip reading for treatment.

In some of the embodiments, the USH2A gene pre-mRNA sequence is selected from the following regions: USH2A exon PE40 and its flanking sequences or the 40th intron.

In some of the embodiments, the genomic localization region corresponding to the pre-mRNA sequence of the USH2A gene is selected from: Chr1: 215891250-215891281, Chr1: 215891248-215891281, Chr1: 215891246-215891281, Chr1: 215891246-215891281, Chr1: 215891215-215891244, Chr1: 215891174-215891205, Chr1: Chr1: 215891178-215891201, Chr1: 215891345-215891408, Chr1: 215891130-215891173.

In some of the embodiments, the USH2A gene pre-mRNA sequence is selected from the sequence shown in SEQ ID NO:1-SEQ ID NO:9.

In some of the embodiments, the recognition domain of the snRNA is selected from the sequences shown in: SEQ ID NO:10-SEQ ID NO:18, SEQ ID NO:21-SEQ ID NO:40.

In some of the embodiments, the genomic location corresponding to the pre-mRNA sequence of the USH2A gene is selected from: Chr1: 215891258-215891281, Chr1: 215891254-215891277, Chr1: 215891250-215891273, Chr1: 215891246-215891269, Chr1: 215891258-215891277, Chr1: 215891258-215891277, Chr1: 215891293-215891311, Chr1: 215891217-215891240, Chr1: 215891180-215891201.

In some of these embodiments, the recognition domain of the snRNA is selected from the sequences shown in SEQ ID NO:10-SEQ ID NO:18.

In some of the embodiments, the genomic location corresponding to the pre-mRNA sequence of the USH2A gene is selected from: Chr1: 215891258-215891281, Chr1: 215891250-215891273, Chr1: 215891258-215891277.

In some embodiments, the snRNA recognition domain is selected from: SEQ ID NO: 10, SEQ ID NO: 12. The sequence shown in SEQ ID NO:14.

In some of the embodiments, the recognition domain of the snRNA is reverse complementary to a continuous sequence of at least 16 bp in the pre-mRNA sequence of the USH2A gene.

In some embodiments, at least two snRNAs are included.

In some of these embodiments, the recognition domains of at least two of the snRNAs target different pre-mRNA sequence regions of the USH2A gene.

In some embodiments, the recognition domains of at least two of the snRNAs have no overlapping sequences.

In some embodiments, the snRNA is U1-snRNA or U7-snRNA.

There are small nuclear RNAs (snRNAs) in cells. They are the main components of RNA spliceosomes in the post-transcriptional processing of eukaryotic organisms. They participate in the processing of mRNA precursors by binding to snRNP proteins. Their length in mammals is about 100-215 nucleotides, and they are divided into 7 categories. Because they are rich in U, they are numbered U1-U7. However, 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.

Therefore, the inventors replaced the non-canonical Sm binding site of U7-snRNA with a consensus sequence derived from the major spliceosomal U-snRNPs and changed the histone binding sequence in the 5' region of U7-snRNA to the complementary sequence of the gene to be modified, which can induce splicing skipping of exons by targeting exons.

In some embodiments, the snRNA comprises an sm sequence.

In some of the embodiments, the sm sequence is a smOPT sequence, and the smOPT sequence is shown in SEQ ID NO:19.

In some of these embodiments, the snRNA further comprises a motif for recruiting splicing regulatory proteins.

In some embodiments, the splicing regulatory protein includes at least one of hnRNPA1, SRSF1, RBM4, DAZAP1 or SR.

In some embodiments, 3-40 bases on both sides of the chemically synthesized snRNA are modified and connected by special phosphate bonds.

In some embodiments, only 1-10, 6-80 or all nucleotides on both sides of the snRNA are modified and connected with special phosphate bonds, the modification is a combination of one or more modifications, and the special phosphate bond is a combination of one or more phosphate bonds.

In some embodiments, all nucleotides in the chemically synthesized U7 snRNA are interconnected by phosphorothioate bonds and are all 2′-O-methoxy modified.

In some embodiments, only the three nucleotides on either side of the snRNA are linked by phosphorothioate bonds and are 2'-O-methoxy modified.

In some preferred embodiments of chemically synthesized and modified U7 snRNA, the first nucleotide at the 5’ end of the U7 snRNA is preferably adenylic acid (A). If the first nucleotide at the 5’ end of the recognition domain is not adenylic acid (A), adenylic acid (A) is connected to the 5’ end.

In some embodiments, the modified nucleotide comprises a modification selected from the group consisting of 2'-O-alkyl, 2'-O-methoxy, and 2'-O-methoxyethyl.

In some embodiments, the modified nucleotide may comprise a modification selected from the group consisting of 2'-O-methyl, and 2'-O-ethyl.

The invention also discloses a nucleic acid, which comprises a nucleotide sequence encoding the snRNA.

The invention also discloses a gene expression box, which comprises the nucleic acid.

The present invention also discloses a vector, which comprises the snRNA, the nucleic acid, and/or the gene expression box.

The present invention also discloses a virus particle, which comprises the snRNA, the nucleic acid, and/or the vector.

The present invention also discloses a cell, which comprises the snRNA, the nucleic acid, the vector, and/or the virus particle.

The present invention also discloses a pharmaceutical composition, which comprises the snRNA, the nucleic acid, the vector, and/or the virus particle.

The present invention also discloses a method for obtaining Usherin protein with exon PE40 deleted, which comprises contacting the pre-mRNA of USH2A gene with the snRNA, the nucleic acid, the vector, the gene expression cassette, the virus particle, the cell, and/or the composition.

The present invention also discloses the use of the snRNA, the nucleic acid, the gene expression cassette, the vector, the virus particle, the cell, the pharmaceutical composition or the method in preparing drugs for preventing eye diseases and/or ear diseases, or preparing drugs for treating eye diseases and/or ear diseases.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discloses an snRNA targeting the pre-mRNA of exon PE40 of the USH2A gene, selects USH2A exon PE40 and its two side sequences or intron 40 as the targeting region, and induces splicing skipping of exon PE40, thereby treating eye and ear diseases caused by abnormal function of USH2A protein due to missense, frameshift, stop codon, nonsense, synonymous mutations, etc. of USH2A exon PE40.

At the same time, the snRNA targeting USH2A gene exon PE40 pre-mRNA of the present invention is U7-snRNA, which utilizes the key factor that U7-snRNP does not participate in splicing but is the unique 3' end processing of replication-dependent histone (RDH) pre-mRNA, and the modified U7-snRNA replaces the non-canonical Sm binding site of U7-snRNA with a common sequence derived from the major spliceosome U-snRNPs, and changes the histone binding sequence in the 5' region of U7-snRNA into a complementary sequence of the gene to be modified. The unique working mechanism, combined with the snRNA targeting USH2A gene pre-mRNA, achieves the induction of splicing jump of exon PE40 of USH2A gene pre-mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure and function of U7-snRNA;

Figure 2 is a map of the pUC57-U7 snRNA backbone vector;

Figure 3A is a plasmid map of the reporter vector pCMV-EGFP _left -PE40 ^WT -EGFP _right ;

FIG3B is a plasmid map of the reporter vector pCMV-EGFP _left -PE40 ^mut -EGFP _right ;

Figure 4 shows the RT-PCR detection of jumps 96 hours after co-transfection of PE40-EGFP-reporter and pUC57-U7 RNA plasmids Efficiency diagram;

Figure 5 is a schematic diagram of RT-PCR detection of the efficiency of U7 RNA-induced pseudo-exon splicing skipping targeting different regions of PE40;

Figure 6 is a schematic diagram of RT-PCR detection of the efficiency of pseudo-exon splicing skipping induced by U7 RNA targeting PE40 region 5;

FIG7 is a schematic diagram of the splicing skipping effect of USH2A exon PE40 mediated by U7-snRNA;

FIG8 is the result of splicing skipping of USH2A pseudoexon PE40 mediated by U7-snRNA observed under fluorescence microscopy;

FIG9 is the result of detecting the splicing skipping efficiency of USH2A pseudoexon PE40 mediated by U7-snRNA by flow cytometry;

Figure 10 is a plasmid map of the reporter vector pCMV-EGFPleft-Exon13c.2802T>G-EGFPright;

Figure 11 is a graph showing the percentage of cells in which U7-snRNA targeting target region 1 induces splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells;

FIG12 is a graph showing the percentage of cells in which U7-snRNA targeting target region 2 induces splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells;

FIG13 is a graph showing the percentage of cells in which U7-snRNA targeting target region 3 induces splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells;

FIG14 shows the splicing skipping effect of USH2A pseudoexon PE40 mediated by U7-snRNA combinations (in parallel or in series) at different target sites;

Figure 15 is a schematic diagram of the U7 snRNA structure with two recognition domains connected in series.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used herein includes any and all combinations of one or more related listed items.

It should be noted that when a sequence is referred to as "selected from" another sequence, it can be directly another sequence or a sequence fragment in another sequence; when a genomic localization region is "selected from" another genomic localization region, its corresponding sequence can be directly the corresponding sequence of another genomic localization region or a sequence fragment in the corresponding sequence of another genomic localization region; the sequence includes DNA or RNA.

definition:

The snRNA described in the present invention is the main component of RNA spliceosome in the post-transcriptional processing of eukaryotic organisms, and participates in the processing of mRNA precursors by binding to snRNP proteins.

Sources:

Helper Free System (AAV5) kit (TAKARA, Code No. 6650)

Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

Example 1

Design of U7-snRNA system and vector construction.

1. Synthesize the U7-snRNA expression vector backbone.

The wild-type U7-snRNA includes a stem-loop structure (scafford), a U7-specific Sm sequence (AAUUUGUCUAG, SEQ ID NO:64) and a recognition domain (complementary to histone pre-mRNA).

The U7-snRNA in this embodiment is based on the gene sequence of mouse wild-type U7-snRNA on NCBI (NCBI Reference Sequence: NR_024201.3), and the U7-specific sm sequence AATTTGTCTAG (SEQ ID NO: 20) is replaced with an optimized common sm sequence, namely smOPT: AATTTTTTGGAG (SEQ ID NO: 19), and the original recognition domain at the 5' end of the smOPT sequence is replaced with a recognition domain that is reverse complementary to the specific target site of the USH2A gene pre-mRNA, and the 3' end of the smOPT sequence retains the original stem-loop structure sequence of U7, as shown in Figure 1.

The U7-snRNA recognition domain sequence that targets and induces USH2A pre-mRNA exon PE40 is reverse-complementarily paired with a target sequence selected from intron 40 of USH2A pre-mRNA (carrying the c.7595-2144A＞G mutation).

The specific operation process is as follows:

First, a pUC57 vector containing a gene sequence, U7-snRNA gene expression cassette skeleton (5'-mouse U7 promoter-smOPT sequence-U7 snRNA scafford-snRNA gene specific 3' cassette-3') was synthesized by full gene synthesis (as shown in Figure 2). Two Type IIs restriction endonuclease recognition sites were added between the U7 promoter and smOPT to facilitate subsequent excision, replacement and insertion of other recognition domain sequences. The snRNA gene specific 3' cassette is a sequence containing gtctacaatgaaa (SEQ ID NO: 32) after the 3' end of the U7-snRNA gene in the mouse genome (GenBank: X54748.1), which participates in the processing of pre-snRNA, preferably a sequence connected to the 3' end of the U7 snRNA gene, preferably a gene fragment with a sequence length of 28-131 bp, and further preferably a sequence length of 106 bp.

2. Construct a U7-snRNA vector targeting USH2A exon PE40 and different nearby sites.

In this embodiment, multiple target sites are set for USH2A pre-mRNA to cause skipping splicing of the pseudo exon PE40, as shown below.

Table 1 The recognition domain sequences of snRNA

Note: The above snRNA6, 6 truncated, 6A, 6B target region 1 (corresponding genomic location Chr1: 215891250-215891281), the above snRNA6, 6 truncated, 6A, 6B, 6D target region 2 (corresponding genomic location Chr1: 215891248-215891281), the above snRNA6, 6 truncated, 6A, 6B, 6D, 6C target region 3 (corresponding genomic location Chr1: 215891246-215891281), the above snRNA 5D targets region 4 (corresponding genomic location Chr1: 215891246-215891281), the above snRNA 7A targets region 5 (corresponding genomic location Chr1: 215891215-215891244), the above snRNA 7B, 7C, 7D target region 6 (corresponding genomic location Chr1: 215891174-215891205), the above snRNA 7B, 7C, 7D target region 7 (corresponding genomic location Chr1: 215891250-215891281), the above snRNA01, 02, 03, 04, 05A, 05B, 05C target region 8 (corresponding genomic location Chr1: 215891345-215891408), the above snRNA7, 8, 9, 10, 11, 12 target region 9 (corresponding genomic location Chr1: 215891130-215891173).

Example 2

Chemical synthesis and modification of U7-snRNA.

Similar to oligonucleotides, U7-snRNA can also be produced by direct chemical synthesis to produce RNA containing a guide sequence, smOPT, and U7-snRNA scafford. U7-snRNA synthesized in vitro can be specifically modified to make it resistant to nuclease degradation or to increase its affinity for the target sequence.

In this example, U7-snRNA was chemically synthesized, and the three bases at the 5' and 3' ends were each 2'-methoxy (2'-OME) modified and thio-modified to increase nuclease resistance. The chemically synthesized snRNA sequence (SEQ ID NO: 65) and the modifications are as follows (* indicates a thiophosphorylated backbone, m indicates a 2'-methoxy modification, the underline indicates a recognition domain that is reverse complementary to the target sequence, and italics indicate a smOPT sequence):

Chemically synthesized and modified U7-snRNA:

Example 3

A reporter vector was constructed for quantitative evaluation of the splicing skipping efficiency of USH2A exon PE40.

The RG _left -USH2A PE40-RG _right sequence (with AgeI and EcoRI restriction sites added to the 5' and 3' ends, respectively) was obtained by total gene synthesis. The synthetic sequence and pX601 plasmid (Addgene, 61591) were digested with restriction endonucleases AgeI and EcoRI, electrophoresed, gel-cut and recovered, and connected. The synthesized sequence was inserted between the AgeI and EcoRI restriction sites of the pX601 vector to replace the SaCas9 gene sequence of the original vector to obtain the reporter vector. The purified reporter vector plasmid was further obtained by transforming E. coli competent cells, picking single clones, PCR and sequencing verification, and stored at -20°C for future use.

The reporter vector structure is: pCMV-RG _left -USH2A PE40-RG _right , RG represents a reporter gene, RGleft represents the first half of the 5' end of the reporter gene without a reporter function, RGright represents the second half of the 3' end of the reporter gene without a reporter function, and the tandem expression of RGleft and RGright can normally exercise the complete reporter gene function. In the embodiment, the reporter gene is the green fluorescent gene EGFP, then the reporter vector structure is pCMV-EGFP _left -PE40 ^mut -EGFP _right (the reporter vector map is shown in Figure 3B), PE40 ^mut represents the USH2A No. 40 intron partial sequence containing the pathogenic mutation c.7595-2144A＞G; the positive control vector structure is pCMV-EGFP _left -PE40 ^WT -EGFP _right (the reporter vector map is shown in Figure 3A), PE40 ^WT represents the USH2A No. 40 intron partial sequence that does not contain the pathogenic mutation.

The EGFP _left sequence is as follows:

The EGFP _right sequence is as follows:

The PE40 ^mut sequence in the vector is as follows:

The PE40 ^WT sequence in the vector is as follows:

Example 4

Detect the splicing skipping effect of USH2A exon PE40 pre-mRNA mediated by targeting U7-snRNA.

1. Targeting area 1

1. Detection method: 293T cells were seeded into 24-well plates so that the cell confluence reached about 80% after 24 hours. Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFP _left -PE40 ^mut -EGFP _right and pUC57-U7-snRNA plasmid targeting USH2A pre-mRNA (the vector mass ratio was 100ng:400ng), and 293T cells transfected with plasmid CMV-GFP and plasmid pCMV-EGFP _left -PE40 ^WT -EGFP _right were used as two positive controls, 293T cells transfected with plasmid pCMV-EGFP _left -PE40 ^mut -EGFP _right were used as negative controls, and 293T cells not transfected with any plasmid were used as blank controls. The transfected cells were cultured for 48-72 hours, and then RNA was extracted from the cells in each experimental group and reverse transcribed to obtain cDNA. RT-PCR experiments were performed using primers GACGTAAACGGCCACAAGTT (SEQ ID NO: 45) and CCTCCTTGAAGTCGATGCCC (SEQ ID NO: 46) to detect whether GFP gene transcription existed. The electrophoresis results are shown in FIG4 .

2. Experimental results: As shown in Figure 4, the positive control PCR product is 337 bp, and the negative control contains a pseudo The exon PCR product is 489 bp, and the meanings of the numbers are as follows: 1: positive control CMV-EGFP; 2: positive control PE40-WT; 3: negative control point mutation c7595-2144A>G; 4: PE40-ANO1; 5: PE40-ANO2; 6: RF-06B; 7: RF-06A; 8: RF-06; 9: RF-05C; 10: RF-05B; 11: RF-05A; 12: RF-04; 13: RF-03; 14: RF-02; 15: RF-01.

The plasmid pCMV-EGFP _left -PE40 ^WT -EGFP _right is a single complete GFP band, proving that the PE40 ^WT sequence in the vector does not affect the splicing of the GFP gene, while pCMV-EGFP _left -PE40 ^mut -EGFP _right is a single invalid GFP band with the PE40 exon introduced, and there is no spontaneous splicing jump.

The results also showed that all U7-snRNAs RF-01, RF-02, RF-03, RF-04, RF-05A, RF-05B, and RF-05C targeting the 5' region near the pseudoexon PE40 (region 8) could not induce splicing jumps of USH2A exon PE40 in reporter gene cells. However, U7-snRNAs RF-06, RF-06A, and RF-06B targeting region 1 could significantly induce splicing jumps of pseudoexon PE40. Conventional technology comparison experiments found that AONs targeting this region could not induce splicing jumps of exon PE40, but snRNAs targeting region 1 could efficiently induce splicing jumps of exon PE40, and were superior to conventional technology AON1 and AON2 target sites.

2. Targeting area 2

1. Detection method: same as above.

2. Experimental results: As shown in Figure 5, U7-snRNA RF-06, RF-06A, and RF-06D targeting region 2 can significantly induce splicing skipping of pseudoexon PE40. However, target sites RF-5E and AON2, which differ by 1 base and 3 bases from RF-06 at the 5' end and RF-06D at the 3' end of targeting region 2, respectively, have almost no effect on splicing skipping of pseudoexon PE40.

RF-5D targeting region 4, RF-7A targeting region 5, and RF-7B targeting region 6 can also induce splicing skipping of the pseudoexon PE40.

The AON targeting RF-7B found in previous research and experiments has a significant effect, but the effect is not high in snRNA. It can be inferred that AON and snRNA have different sensitivities to different sites due to differences in their mechanisms of action. We also found through experiments that the target sites RF-7D and RF-7C, which are more effective than RF-7B in AON, are weaker than RF-7B in snRNA. It can be seen that snRNA targeting region 7 can induce splicing jump of the USH2A pre-mRNA pseudoexon PE40.

3. Targeting area 3

Detection method: Same as above.

Experimental results: As shown in Figure 7, U7-snRNA RF-06 truncated (20bp), RF-06, RF-06B, RF-06A, RF-06D, and RF-06C targeting target region 3 can all induce splicing skipping of USH2A exon PE40 in reporter gene cells.

Example 5

The splicing skipping effect of USH2A pseudoexon PE40 mediated by U7-snRNA was detected by fluorescence microscopy and flow cytometry.

1. Detection method: 293T cells were seeded into 24-well plates so that the cell confluence reached about 80% after 24 hours. Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFP _left -PE40 ^mut -EGFP _right and pUC57-U7-snRNA plasmid targeting USH2A pre-mRNA (the vector mass ratio was 100ng:400ng), and 293T cells transfected with plasmid pCMV-EGFP _left -PE40 ^mut -EGFP _right alone were used as negative controls, and 293T cells not transfected with any plasmid were used as blank controls. The transfected cells were cultured for 96 hours, and the expression of GFP fluorescent protein in the cells was observed under a fluorescence microscope; thereafter, trypsin was used to digest the cells into single cells, and then flow cytometry was used to detect the GFP positive rate of different snRNA groups (i.e., the proportion of cells in which the USH2A pre-mRNA pseudoexon PE40 was induced to splice jump).

2. Experimental results: As shown in Figures 8-9, the efficiency of U7-snRNA RF-06A, RF-06D, and RF-06C targeting target region 3 in inducing PE40 pseudo-exon splicing skipping is more than 5 times that of the prior art control group AON2 (the proportion of PE40 pseudo-exon splicing skipping cells to total cells is 8.18%), while the proportion of cells induced by splicing skipping by RF-6D is 56.86%, which is better than RF-6A, followed by RF-6C.

Example 6

A reporter vector was constructed for quantitative evaluation of USH2A exon 13 splicing skipping efficiency.

The RG _left -USH2A EXON13 ^mut -RG _right sequence (with AgeI and EcoRI restriction sites added to the 5' and 3' ends, respectively) was obtained by total gene synthesis. The synthetic sequence and pX601 plasmid (Addgene, 61591) were digested with restriction endonucleases AgeI and EcoRI, electrophoresed, gel-cut and recovered, and connected. The synthesized sequence was inserted between the AgeI and EcoRI restriction sites of the pX601 vector to replace the SaCas9 gene sequence of the original vector to obtain the reporter vector. The purified reporter vector plasmid was further obtained by transforming E. coli competent cells, picking single clones, PCR and sequencing verification, and stored at -20°C for future use.

The reporter vector structure is: pCMV-RG _left -USH2A EXON13 ^mut -RG _right , RG represents a reporter gene, RGleft represents the first half of the 5' end of the reporter gene without a reporter function, RGright represents the second half of the 3' end of the reporter gene without a reporter function, and the tandem expression of RGleft and RGright can normally exercise the complete reporter gene function. In this embodiment, the reporter gene is the green fluorescent gene EGFP, and the vector structure is pCMV-EGFP _left -Exon13 ^mut -EGFP _right (the reporter vector map is shown in Figure 10). EXON13 ^mut represents USH2A exon 13 containing pathogenic mutations, and its upstream and downstream intron sequences (the upstream intron sequence is the gene sequence of the 5' end 204bp and 3' end 490bp of the intron 12 of the human USH2A gene; the downstream intron sequence is the gene sequence of the 5' end 703bp and 3' end 216bp of the intron 13 of the human USH2A gene).

Example 7

Detect the effect of USH2A exon 13 splicing skipping mediated by targeted U7-snRNA.

In the previous research, the inventors conducted a splicing skipping experiment on exon 13 (see CN 202210003448.3). This example compares the differences in editing efficiency between AON technology and U7-snRNA technology.

1. Detection method: 293T cells were seeded into 24-well plates, and the cell confluence reached 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 (the vector mass ratio was 100ng:400ng). 293T cells transfected with reporter plasmid alone, co-transfected with reporter plasmid and pUC57-U7-Scramble were used as two negative control cells. The 293T cells without any plasmid transfection were used as blank control. The transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then flow cytometry was used to detect the GFP positive rate (i.e., the proportion of cells in which USH2A pre-mRNA exon 13 was induced to splice jump) and the average FITC intensity of GFP-positive cells (i.e., the average level of USH2A pre-mRNA exon 13 splicing jump in GFP cells) of different U7-snRNA groups.

2. Results of targeting target area A:

The recognition domain sequence of the snRNA targeting target region A is shown in the following table.

Table 2 The recognition domain sequences of snRNA

The experimental results are shown in Figure 11. All U7-snRNAs targeting target region A can induce splicing skipping of USH2A exon 13 in reporter gene cells. Conventional AONs targeting region A cannot induce splicing skipping of exon 13, but snRNAs targeting region A can efficiently induce splicing skipping of exon 13.

3. Results of targeting target area B:

The recognition domain sequence of the snRNA targeting target region B is shown in the following table.

Table 3. The recognition domain sequences of snRNA

The experimental results are shown in Figure 12. All U7-snRNAs targeting target region B can induce splicing skipping of USH2A exon 13 in reporter gene cells. The AONs targeting region 2 in the prior art are less effective in inducing splicing skipping of exon 13, but snRNAs targeting region B can efficiently induce splicing skipping of exon 13.

4. Targeted area C results:

The recognition domain sequence of the snRNA targeting target region C is shown in the following table.

Table 4 snRNA recognition domain sequences

Experimental results: As shown in Figure 13 and the table below, all U7-snRNAs targeting target region C can induce splicing skipping of USH2A exon 13 in reporter gene cells. The AONs targeting region C in the prior art are less effective in inducing splicing skipping of exon 13, but snRNAs targeting region C can efficiently induce splicing skipping of exon 13.

Experimental analysis found that although the prior art shows that target areas A, B, and C are AON-targeted non-sensitive areas, i.e., target This region is unable/low-efficiency to induce splicing skipping of exon 13, but snRNA targeting this region can significantly induce splicing skipping of exon 13. Therefore, although both snRNA and AON can induce splicing skipping, their mechanisms of action are different and their target site sensitivity (target region and target site to which they are applicable) is also different.

Example 8

Chemically synthesized U7-snRNA induces USH2A exon splicing skipping in WERI cells.

Human host cells were seeded into 24-well plates at 6×10 ⁵ /well. The human retinal neural cells selected in this example were WERI-Rb-1 cells (retinal neural cell line). WERI cells were transfected with 100 pmol U7-snRNA synthesized in vitro using Lipofectamine 2000, and 1 μg EGFP plasmid was transfected as a negative control. WERI cells without any plasmid were used as blank controls. The transfected cells were cultured for 72 hours, and then RNA was extracted from cells in each experimental group, and cDNA was obtained by reverse transcription. The mature USH2A mRNA was tested for exon splicing skipping by RT-PCR.

The results showed that chemically synthesized and modified U7-snRNA could efficiently induce splicing skipping of USH2A pseudoexon PE40 in WERI cells.

Example 9

Effects of splicing skipping of USH2A pseudoexon PE40 mediated by U7-snRNA combinations with different target sites

Construct a U7-snRNA multi-target combination vector. According to the Golden Gate Assembly technology, different U7 snRNA plasmids were used as templates to PCR amplify the U7 snRNA cassette (expression box). At the same time, additional 5' flanking bases and BsaI restriction sites in the correct direction were introduced at both ends of the amplicon through primers, so that the adjacent different U7 snRNA cassettes were digested by BsaI to produce specific complementary sticky ends, and the first and last U7 snRNA cassettes were digested by BsaI to produce the same sticky ends as the linearized backbone vector digested by HindIII+NotI. Finally, use Golden Gate Assembly Kit( v2) (NEB#E1601) Assemble the above PCR products and the pUC57-U7 snRNA Backbone recovered by HindIII+NotI digestion. The assembly method is as follows: 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; T4 DNA Ligase Buffer (10X), 2μl; NEB Golden Gate Assembly Mix, 1μl; Reaction process: (37℃, 5min→16℃, 5min)×20→60℃, 5min.

The Golden Gate Assembly product was further transformed into E. coli competent cells, single clones were selected, PCR and sequencing were verified to obtain the U7 snRNA multi-target combination vector for inducing USH2A exon splicing skipping. The plasmid was purified and stored at -20°C for future use. The constructed vector was exemplarily named pUC57-U7 snRNA#A+U7 snRNA#B+U7 snRNA#C.

Usherin high-expressing 293T cells were inoculated into 24-well plates in a certain amount so that the cell confluence reached about 80% after 24 hours. Lipofectamine2000 was used to transfect 293T cells with pUC57-U7-snRNA combination plasmids targeting USH2A pre-mRNA exons (the vector mass ratio was 100ng:400ng). 293T cells transfected with single-target snRNA plasmids were used as the control group, 293T cells transfected with pUC57-U7-Scramble plasmids were used as negative controls, and 293T cells not transfected with any plasmids were used as blank controls. The transfected cells were cultured for 48-72 hours, and the cells were collected to extract RNA. RT-PCR and qRT-PCR were used to detect the splicing skipping of USH2A exons induced by snRNA combination.

As shown in Figure 14A and Figure 14B, the efficiency of snRNA#6 and snRNA#7A in parallel (RF-6-RF-7A group in the figure) in inducing USH2A exon splicing skipping was higher than that of snRNA#6 (RF-6 group in the figure) or snRNA#7A (RF-7A group in the figure) alone.

The efficiency of snRNA#6C in inducing USH2A exon splicing skipping in parallel with snRNA#5D (RF-5D-RF-6C group in the figure) was higher than that of snRNA#5D (RF-5D group in the figure) or snRNA#6C (RF-6C group in the figure) alone.

In this embodiment, by combining U7-snRNA targeting different target sites, it is found that the efficiency of inducing splicing jump of USH2A pseudoexon PE40 in Usherin high-expressing 293T cells can be improved. The splicing jump efficiency induced by the combination of different targets in this embodiment can be higher than the effect of a single target. In the combined application of U7-snRNA at different target sites, U7-snRNA can not only be expressed by vector delivery, but also can be a combination of chemically synthesized and modified U7-snRNA molecules for administration.

Example 10

Recognition domain-containing tandem U7 snRNA induces USH2A exon splicing skipping

1. Preparation of recognition domain tandem U7 snRNA

The U7 snRNA with tandem recognition domains means a U7 snRNA stem-loop structure and a smOPT sequence connected to two or more recognition domains, and its structure is 5′-recognition domain B-recognition domain A-smOPT sequence-stem-loop structure-3′, as shown in Figure 15. The recognition domains A and B of the tandem U7 snRNA recognize RNA sequences at different target sites.

According to the pre-transcriptional DNA sequence corresponding to the snRNA recognition domain sequence, the corresponding oligo DNA is synthesized. The sense chain of the oligo DNA is the DNA corresponding to the recognition domain sequence, and CCGCA is added to the 5′, and the antisense chain is the antisense complementary sequence of the recognition domain sequence with AATT added to the 5′ and T added to the 3′. The synthesized sense and antisense chains of the oligo DNA are mixed according to the annealing reaction system (total reaction volume 20μl: Oligo-F (100μM) 2μl + Oligo-R (100μM) 2μl + 10×NEB Cutter smart buffer 2μl + deionized water 16μl), incubated at 95℃ for 5 minutes, and then placed on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, take 1μl and mix with 10ng BsaI-digested and recovered linearized pUC57- The U7 snRNA backbone plasmid was ligated with T4 ligase. The ligation product was further verified by transformation of E. coli competent cells, single cloning, PCR and sequencing to obtain the U7 snRNA vector for inducing USH2A exon splicing skipping. The plasmid was purified and stored at -20°C for future use. The constructed vector was named pUC57-U7 snRNA#B-#A, where A and B represent the recognition domain numbers, corresponding to different recognition domain sequence columns. For example, pUC57-snRNA#RF-06-#RF-07B.

The recognition domain-tandem U7 snRNA can also be chemically synthesized and modified. In some embodiments, the total length of the chemically synthesized snRNA sequence is preferably greater than or equal to 96 bp.

2. Recognition domain tandem U7 snRNA induces USH2A exon splicing skipping in Usherin-overexpressing 293T cells

Usherin high-expressing 293T cells were inoculated into 24-well plates in a certain amount so that the cell confluence reached about 80% after 24 hours. Lipofectamine2000 was used to transfect 293T cells with pUC57-U7-recognition domain tandem snRNA plasmid targeting USH2A pre-mRNA exon (the vector mass ratio was 100ng:400ng). 293T cells transfected with single recognition domain U7 snRNA plasmid were used as the control group, 293T cells transfected with pUC57-U7-Scramble were used as negative control, and 293T cells not transfected with any plasmid were used as blank control. The transfected cells were cultured for 48-72 hours, and the cells were collected to extract RNA. RT-PCR and qRT-PCR were used to detect the splicing skipping of USH2A exons induced by snRNA combination.

The results are shown in Figures 14A and 14B. The efficiency of snRNA#6 and snRNA#7A in tandem (RF-7A-6 group in the figure) in inducing USH2A exon splicing skipping is higher than that of snRNA#6 (RF-6 group in the figure) or snRNA#7A (RF-7A group in the figure) alone.

The efficiency of snRNA#6C and snRNA#5D in tandem (RF-6C-5D group in the figure) in inducing USH2A exon splicing skipping was higher than that of snRNA#5D (RF-5D group in the figure) or snRNA#6C (RF-6C group in the figure) alone.

In addition, the inventors constructed AONs with different target sites in series (ASO-RF-6+7A group, ASO-RF-5D+6C group) and tried to induce USH2A pre-mRNA exon splicing skipping, but found that compared with single AONs (ASO-RF-6, ASO-RF-7A, ASO-RF-5D, ASO-RF-6C), the splicing skipping efficiency did not increase or even decreased significantly.

Embodiment 11

Detection of the splicing skipping effect of U7-snRNA linked to the hnRNP A1 binding motif.

1. Construction of U7-snRNA linked to the hnRNP A1 binding motif.

According to the pre-transcription DNA sequence corresponding to the gRNA sequence in the table, the corresponding oligo DNA is synthesized. The positive strand of the oligo DNA is the reverse complementary sequence of the target sequence (the DNA sequence corresponding to the recognition domain sequence), and the 5' CCGCAATATGATAGGGACTTAGGGTG (SEQ ID NO: 56), the antisense strand is the target sequence 5' plus AATT and 3' plus CACCCTAAGTCCCTATCATATT (SEQ ID NO: 57). For example, the recognition domain sequence of snRNA #14 is ACACUGGCAGGGCUCACAUCCA (SEQ ID NO: 58), then the synthetic oligo DNA positive strand is TGGATGTGAGCCCTGCCAGTGT (SEQ ID NO: 59), the antisense strand is AATT ACACTGGCAGGGCTCACATCCA (SEQ ID NO:60), the underline indicates the double-stranded DNA sequence corresponding to the recognition domain sequence, and the bold italics indicate the double-stranded DNA sequence corresponding to the binding motif "UAGGGU" of the hnRNP A1 protein.

The synthesized Oligo DNA sense and antisense strands were mixed according to the annealing reaction system (total reaction volume 20μl: Oligo-F (100μM) 2μl + Oligo-R (100μM) 2μl + 10×NEB Cutter smart buffer 2μl + deionized water 16μl), incubated at 95℃ for 5 minutes, and then placed on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, 1μl was taken and connected with 10ng BsaI digested and recovered linearized pUC57-U7-snRNA backbone plasmid. Further transformation of Escherichia coli competent cells, single clone selection, PCR and sequencing verification were performed to obtain the U7-snRNA vector containing the hnRNP A1 binding motif for inducing USH2A pre-mRNA exon PE40 splicing skipping, and the vector was named pUC57-U7-hnRNP A1-snRNA# number. The purified plasmid was stored at -20°C for future use.

U7-hnRNP A1-snRNA can also be chemically synthesized and modified according to the method described in the above examples. Taking snRNA 6 as an example, the sequence and modification of the chemically synthesized U7-hnRNP A1-snRNA are as follows (* indicates phosphorothioate backbone, m indicates 2'-methoxy modification, underline indicates recognition domain that is reverse complementary to the target sequence, italics indicate smOPT sequence, and bold indicates hnRNP A1 protein binding motif):

The above-mentioned modified sequence is based on the sequence shown in SEQ ID NO:61, with thiophosphate backbone modification and methoxy modification added to the first three bases at the 5’ end and the 3’ end.

2. To detect the efficiency of U7-snRNA linked to the hnRNP A1 binding motif in inducing splicing skipping of USH2A exon PE40 in reporter gene cells.

Detection method: 293T cells were seeded into 24-well plates, so that the cell confluence reached about 80% after 24 hours. Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFP _left -PE40 ^mut -EGFP _right and pUC57-U7-hnRNP A1-snRNA plasmid and pUC57-U7-snRNA plasmid (the vector mass ratio was 100ng:400ng), and 293T cells transfected with reporter plasmid alone, co-transfected with reporter plasmid and pUC57-U7 Scramble were used as two negative controls. 293T cells without any plasmid transfection were used as blank control. The transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then flow cytometry was used to detect the splicing skipping efficiency induced by different snRNA groups.

Experimental results: As shown in the table below, the introduction of the hnRNP A1 binding motif at the 5' end of U7-snRNA can significantly enhance the effect of inducing splicing skipping of USH2A pre-mRNA exon PE40, which not only increases the proportion of cells with exon splicing skipping (GFP+), but also increases the mRNA level of splicing skipped exons in each cell (average FITC intensity).

In this embodiment, a free tail is introduced at the 5' end of U7-snRNA, and the free tail sequence includes the binding motif "UAGGGU" of hnRNP A1 protein. The free tail sequence is preferably "UAUGA UAGGGA CU UAGGGU G" (SEQ ID NO: 62), which can recruit hnRNP A1 protein and promote splicing skipping of USH2A exon PE40 without affecting its targeting specificity and causing or increasing off-target effects.

Example 12

Construction and viral packaging of AAV-U7-snRNA related plasmid vector targeting and inducing splicing skipping of USH2A pre-mRNA pseudoexon PE40.

In this example, the U7-snRNA gene that targets and induces splicing skipping of the USH2A pre-mRNA pseudoexon PE40 is inserted and replaced with the middle gene sequence of the two ITR domains in the pAAV-CMV vector to construct the pAAV-U7-snRNA vector, and the AAV packaging plasmids: serotype pRC plasmid (containing the Rep gene of AAV2 and the Cap gene of each serotype) and pHelper plasmid (a vector plasmid containing the E2A, E4 and VA genes of adenovirus) are co-transfected into the host cells to package and obtain the AAV-U7-snRNA virus that targets splicing skipping of the USH2A pre-mRNA pseudoexon PE40. The specific operation process is as follows:

First, the gene sequence was synthesized by whole gene synthesis - U7-snRNA gene expression box skeleton (excluding recognition domain): 5'-mouse U7 promoter-smOPT sequence-U7-snRNA scafford-snRNA gene specific 3' box-3'. Two Type IIs restriction endonuclease recognition sites were added between the U7 promoter and smOPT to facilitate subsequent excision, replacement and insertion of other recognition domain sequences. The whole gene synthesis sequence was inserted and replaced the gene sequence between the two AAV2-ITR domains of the pAAV-CMV plasmid to obtain the pAAV-U7-snRNA backbone vector.

U7-snRNA gene expression cassette skeleton (excluding the recognition domain) (SEQ ID NO: 63):

According to the method described in Example 1, the corresponding oligo DNA positive strand and antisense strand were synthesized according to the pre-transcription DNA sequence corresponding to the snRNA recognition domain sequence in Table 1, and a restriction endonuclease similar to Type IIs was added to both ends. The sticky ends after the enzyme recognition site is cut. Annealing forms a double-stranded DNA with a recognition domain with sticky ends, which is then ligated with T4 ligase into the linearized pAAV-U7-snRNA backbone plasmid that has been digested and recovered by the corresponding Type IIs restriction endonuclease to form a pAAV-U7-snRNA plasmid that targets a specific site of the USH2A pre-mRNA exon to induce splicing skipping, and is named according to the snRNA number corresponding to the recognition domain sequence.

After inserting the target gene (U7-snRNA gene expression box that targets and induces USH2A pre-mRNA exon splicing skipping) and replacing the gene sequence between the AAV-ITR domains of the pAAV-CMV plasmid, the pAAV-U7-snRNA plasmid vector was obtained. The AAV-U7-snRNA virus that targets and induces USH2A pre-mRNA exon splicing skipping was packaged according to the instructions and standard cell operation procedures.

24 hours before transfection, HEK293/293T cells were inoculated into 100mm cell culture dishes with 10% FBS DMEM culture medium. Transfection was performed when the confluence reached 80%-90%. 3 hours before transfection, the old culture medium was discarded and replaced with fresh culture medium. During transfection, pAAV-U7-snRNA plasmid, pRC plasmid, pHelper plasmid and PEI (polyethyleneimine) transfection reagent were prepared according to the system in the table below and added dropwise to the culture dish. After the PEI transfection mixture was added, the culture dish was gently shaken to evenly distribute the transfection reagent, and the culture medium was placed in a 37°C, 5% CO ₂ incubator for culture.

Table 5 PEI transfection system

24 hours after transfection, replace the DMEM medium with fresh 2% FBS. 48-72 hours after transfection, collect the cells containing AAV virus, wash and centrifuge, collect the cell pellet, and vortex to loosen the cell pellet. Then, according to the instructions of the kit, add 0.5mL of AAV Extraction Solution A to the cell pellet and vortex for 15 seconds to fully suspend the cell pellet. After standing at room temperature for 5 minutes, vortex for another 15 seconds. Centrifuge at 4℃, 2000-14000g for 10 minutes to remove cell debris. Collect the supernatant into a new sterile centrifuge tube, add 50μL AAV Extraction Solution B, use a pipette to mix, and obtain AAV-U7-snRNA virus solutions with different recognition domains. Take a portion to detect the virus titer by qPCR and store at 80℃ for later use.

Since the AAV-ITR domain inserted into the pAAV-U7-snRNA plasmid and the target gene fragment inserted therein should be less than 4.7kb, multiple U7-snRNA gene expression cassettes (5'-mouse U7 promoter-recognition domain-smOPT sequence, snRNA gene-specific 3'cassette-3') can be inserted to ensure that the expression amount of U7-snRNA is increased under the same number of AAV virus particles. The gene sequence length is about 450bp, so preferably the pAAV-U7-snRNA plasmid carries 1-10 U7-snRNA gene expression cassettes, and the multiple U7-snRNA gene expression cassettes in the pAAV-U7-snRNA plasmid may have the same recognition domain.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (26)

A snRNA targeting USH2A gene exon PE40 pre-mRNA, characterized in that the recognition domain of the snRNA is reverse complementary to the USH2A gene pre-mRNA sequence, and the snRNA splices and skips exon PE40 by binding to the USH2A gene pre-mRNA. The snRNA according to claim 1, characterized in that the USH2A gene pre-mRNA sequence is selected from the following regions: USH2A exon PE40 and its flanking sequences or intron 40. The snRNA according to claim 2, characterized in that the genomic localization region corresponding to the pre-mRNA sequence of the USH2A gene is selected from: Chr1: 215891250-215891281, Chr1: 215891248-215891281, Chr1: 215891246-215891281, Chr1: 21589124 6-215891281, Chr1: 215891215-215891244, Chr1: 215891174-215891205, Chr1: Chr1 : 215891178-215891201, Chr1: 215891345-215891408, Chr1: 215891130-215891173. The snRNA according to claim 3 is characterized in that the USH2A gene pre-mRNA sequence is selected from: the sequences shown in SEQ ID NO: 1-SEQ ID NO: 9. The snRNA according to claim 4 is characterized in that the recognition domain of the snRNA is selected from: the sequences shown in SEQ ID NO:10-SEQ ID NO:18, SEQ ID NO:21-SEQ ID NO:40. The snRNA according to claim 3 is characterized in that the genomic location corresponding to the pre-mRNA sequence of the USH2A gene is selected from: Chr1: 215891258-215891281, Chr1: 215891254-215891277, Chr1: 215891250-215891273, Chr1: 215891246-215891269, Chr1: 215891258-215891277, Chr1: 215891258-215891277, Chr1: 215891293-215891311, Chr1: 215891217-215891240, Chr1: 215891180-215891201. The snRNA according to claim 6 is characterized in that the recognition domain of the snRNA is selected from the sequence shown in SEQ ID NO:10-SEQ ID NO:18. The snRNA according to claim 2, characterized in that the genomic location corresponding to the USH2A gene pre-mRNA sequence is selected from: Chr1: 215891258-215891281, Chr1: 215891250-215891273, Chr1: 215891258-215891277. The snRNA according to claim 8 is characterized in that the recognition domain of the snRNA is selected from the sequences shown in SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. The snRNA according to claim 1, characterized in that the recognition domain of the snRNA is reverse complementary to a continuous sequence of at least 16 bp in the pre-mRNA sequence of the USH2A gene. The snRNA according to claim 1, characterized in that it comprises at least two snRNAs. The snRNA according to claim 11, characterized in that the recognition domains of at least two of the snRNAs target different USH2A gene pre-mRNA sequence regions. The snRNA according to claim 12, characterized in that the recognition domains of at least two of the snRNAs have no overlapping sequences. The snRNA according to claims 1-13, characterized in that the snRNA is U1-snRNA or U7- snRNA. The snRNA according to claim 14, characterized in that the snRNA comprises an sm sequence. The snRNA according to claim 15 is characterized in that the sm sequence is a smOPT sequence, and the smOPT sequence is shown in SEQ ID NO:19. The snRNA according to claim 15, characterized in that the snRNA also includes a motif for recruiting splicing regulatory proteins. The snRNA according to claim 17, characterized in that the splicing regulatory protein includes at least one of hnRNPA1, SRSF1, RBM4, DAZAP1 or SR. A nucleic acid, characterized in that it comprises a nucleotide sequence encoding the snRNA according to any one of claims 1 to 18. A gene expression cassette, characterized in that the gene expression cassette comprises the snRNA according to any one of claims 1 to 18, and/or comprises the nucleic acid according to claim 19. A vector, characterized in that the vector comprises the snRNA according to any one of claims 1 to 18, the nucleic acid according to claim 19, and/or the gene expression cassette according to claim 20. A virus particle, characterized in that the virus particle comprises the snRNA according to any one of claims 1 to 18, the nucleic acid according to claim 19, and/or the vector according to claim 21. A cell, characterized in that the cell comprises the snRNA according to any one of claims 1 to 18, the nucleic acid according to claim 19, the vector according to claim 21, and/or the virus particle according to claim 22. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the snRNA according to any one of claims 1 to 18, the nucleic acid according to claim 19, the vector according to claim 21, and/or the virus particle according to claim 22. A method for obtaining Usherin protein with exon PE40 deleted, characterized in that the method is carried out by contacting the pre-mRNA of USH2A gene with the snRNA according to any one of claims 1 to 18, the nucleic acid according to claim 19, the vector according to claim 21, the gene expression cassette according to claim 20, the virus particle according to claim 22, the cell according to claim 23, and/or the composition according to claim 24. Use of the snRNA of any one of claims 1 to 18, the nucleic acid of claim 19, the gene expression cassette of claim 20, the vector of claim 21, the viral particle of claim 22, the cell of claim 23, the pharmaceutical composition of claim 24 or the method of claim 25 in the preparation of a medicament for preventing eye diseases and/or ear diseases, or in the preparation of a medicament for treating eye diseases and/or ear diseases.