DUCHENNE MUSCULAR DYSTROPHY-RELATED EXONIC SPLICING ENHANCER, sgRNA AND GENE EDITING TOOL, AND APPLICATIONS

Abstract

A duchenne muscular dystrophy-related exonic splicing enhancer, sgRNA and gene editing tool can be applied in the preparation of drugs for treating duchenne muscular dystrophy. The gene editing tool designed on the basis of cytosine deaminase AID mutants and Cas9 mutants can perform site-specific modification on a mammalian genome by using an adeno-associated virus (AAV) as a vector. By optimizing an encoding nucleic acid sequence and an element composition structure of the editing tool, site-specific targeted modification of mammalian genetic material DNA can be efficiently achieved; and by performing targeted genetic manipulation on the nucleic acid sequence carrying disease mutations, a pathogenic mutation cannot be retained in a mature protein amino acid sequence or the pathogenic mutation cannot perform its function, so that the purpose of treating various gene mutation type genetic rare diseases is achieved, and the advantages of high efficiency, safety and stability are achieved.

Claims

1. A sgRNA targeting Duchenne muscular dystrophy-related exon splicing enhancer, which is an exon splicing enhancer element targeting the human DMD gene Exon51, wherein the nucleotide sequence of exon splicing enhancer element comprises: 1) the sequence as shown in SEQ ID NO: 21 and reverse complementary sequence thereof; 2) the sequence as shown in SEQ ID NO: 22 and reverse complementary sequence thereof; or 4) the sequence as shown in SEQ ID NO: 24 and reverse complementary sequence thereof, wherein the sgRNA is selected from the following groups: sgRNA-12 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 19; sgRNA-13 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 20.

2. A sgRNA of claim 1, wherein the sgRNA further comprises the sgRNA selected from the following groups: sgRNA-1 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 8; sgRNA-2 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 9; sgRNA-3 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 10; sgRNA-4 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 11; sgRNA-5 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 12; sgRNA-6 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 13; sgRNA-7 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 14; sgRNA-8 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 15; sgRNA-9 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 16; sgRNA-10 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 17; sgRNA-11 targeting the human DMD gene Exon51 with the nucleotide sequence as shown in SEQ ID NO: 18.

3. (canceled)

4. A Duchenne muscular dystrophy-related gene editing tool, comprising fusion protein of cytosine deaminase and Cas9 mutant, the sgRNA of claim 1 and a vector.

5. The gene editing tool of claim 4, wherein the cytosine deaminase is AID, and the amino acid sequence of the fusion protein of AID and Cas9 mutant is as shown in SEQ ID NO: 1.

6. The gene editing tool of claim 4, wherein the gene editing tool is packaged by an adeno-associated viral (AAV) vector.

7. The gene editing tool of claim 6, wherein the promoter of the adeno-associated viral (AAV) vector is Syn100 promoter or a promoter based on ck8a, mhck7.

8. The gene editing tool of claim 6, wherein the nucleotide sequence of the adeno-associated viral (AAV) vector is shown in SEQ ID NO: 3.

9. (canceled)

10. A method for treating Duchenne muscular dystrophy which comprises administering the sgRNA of claim 1, or gene editing tool comprising the sgRNA to a subject in need.

Description

DESCRIPTION OF FIGURE

[0041] FIG. 1 shows the functional elements including the gene editing tool, wherein A shows a separate packaging virus and B shows a combined packaging virus;

[0042] FIG. 2 shows the treatment flow chart of the novel DMD mouse disease model Dmd-E4, wherein A shows preventive treatment for neonatal mice and B shows restorative treatment for adult mice;

[0043] FIG. 3 shows the partial sequencing results of AAV plasmids; wherein, A shows the sequencing alignment result of the Syn100 promoter; B shows the sequencing alignment result of the AID and Cas9 mutant fusion protein; C shows the sequencing result of the U6 promoter.

[0044] FIG. 4 shows the results diagram that the disease phenotypes caused by Dystrophin expression defects in Dmd-E4 mice were successfully repaired by AAV treatment. Wherein, A, RNA in the heart of treated Dmd-E4 mice were performed by reverse transcription PCR, and primers were designed for Exon3 and Exon5 to detect skipping of Exon4 carrying mutations. Dmd is the gene encoding Dystrophin protein in mice, and Gapdh is the internal reference of PCR. B, using method of capillary electrophoresis quantification to determine the ratio of the content of nucleic acid contained in the band with Exon4 skipping to the band without skipping (i.e., included); C, Sanger sequencing was performed on the band with Exon4 skipping, and it was confirmed that Exon4 was completely skipped, and Exon3 and Exon5 were spliced together; D, Western blotting was performed to detect proteins in the heart of treated Dmd-E4 mice. WT mice and untreated mice were used as positive and negative controls, and VCL was the internal reference of large molecular weight. E, Quantitative statistics of bands in FIG. D. F, the condition of expression of Dystrophin protein in the heart of Dmd-E4 mice was detected by immunofluorescence staining, including two post-treatment samples. G, the method of small animal heart ultrasound was used to investigate whether the changes in the heart-related physiological structure of Dmd-E4 mice were repaired after AAV treatment. H is the quantification of F, which quantified the proportion of Dystrophin positive expression cells. P-value: * p<0.05, **p<0.01, ***p<0.001.

[0045] FIG. 5 shows AAV treatment successfully restored muscle function and prolonged survival in Dmd-E4 mice. A, the creatine kinase content in serum of treated Dmd-E4 mice was determined, and WT and untreated Dmd-E4 mice samples were used as controls. B, HE staining and Masson staining were used to evaluate the degree of myocardial inflammatory cell infiltration and fibrosis of Dmd-E4 mice after treatment. C, according to the results of Masson staining, the recovery of myocardial fibrosis in Dmd-E4 mice after treatment was quantitatively counted. D, the method of micro-CT was used to detect the degree of spinal curvature in Dmd-E4 mice, and WT mice and untreated mice samples were used as controls. E, the quantitative statistics of the degree of spinal curvature in FIG. D; F, a tension device was used to detect the degradation range of the maximum tension of the whole body muscle of the treated Dmd-E4 mice during the cyclic force process. G, survival statistics of WT mice, and AAV-treated and untreated Dmd-E4 mice; H, the molecular biological evidence of gene editing in cardiomyocytes of Dmd-E4 mice, pre-mRNA of corresponding cells was performed by reverse transcription PCR, and then high-throughput sequencing was performed. It was found that the expected mutation was generated near the location of sgRNA targeting, which is the molecular foundation and basis for the treatment of cardiac disease phenotype in Dmd-E4 mice. * p<0.05, **p<0.01, ***p<0.001.

[0046] FIG. 6 shows gene editing tools can successfully induce the corresponding modification of DMD genes in human cells. A, two sgRNAs were successfully screened in the K562 cell line, which can induce the skipping of Exon51. The figure shows the results of reverse transcription PCR after RNA extraction in edited K562 cells, indicating that the combination of two sgRNAs can effectively induce Exon50-deficient K562 cells to successfully skip Exon51. B, Exon51 of the DMD gene was induced to be skipped in normal human iPS cells and DMDexon50-deficient cells. C, immunofluorescence detection was used to determine that the expression of Dystrophin protein was restored in the edited iPS cells. D, western blot was used to determine that the expression of Dystrophin was restored in the edited iPS cells. E, the quantitative statistics of protein restored expression in FIG. D.

DETAILED DESCRIPTION

[0047] Taking the pathogenic mutations carried by DMD mouse models and pathogenic mutations carried by human DMD patients as examples, the present invention achieved the modification of pathogenic mutations by designing and constructing gene editing tools. The present invention is further described below combining specific examples and accompanying drawings:

Example 1 AAV Virus Carrying Gene Editing Tools

[0048] The gene editing tool designed according to the present invention is shown in FIG. 1. Taking AID as an example, we cloned the corresponding sequence into the AAV plasmid, including the following steps:

[0049] First, pAAV2 backbone vectors (purchased from addgene, but not limited to it) were double-digested based on the digestion sites of XhoI and NotI. At the same time, the amino acid sequence of AID and Cas9 fusion protein in gene editing tools was designed. The amino acid sequence and nucleic acid sequence of AID and Cas9 fusion proteins are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. After codon optimization, double-stranded DNA fragment was directly synthesized, and it was connected with a Syn100 promoter, a tail signaling element and other elements to AAV backbone vector to obtain an AAV vector plasmid expressing AID-Cas9 mutant fusion protein, and the sequence of that is as shown in SEQ ID NO: 3.

[0050] In addition, by primer synthesis and PCR, the sequences of the U6 promoter, H1 promoter and 7SK promoter can be connected with sgRNA that identifies the splicing site of the pathogenic mutation exon, and the Syn100 promoter and tail signaling element were used to express green fluorescent protein and related components to increase protein expression tags and help improving gene editing efficiency. In addition, gene editing tool can be constructed using an AAV plasmid with combined packaging virus. On the basis of AID and Cas9 fusion protein expression elements, the U6 promoter was connected with sgRNA targeting the pathogenic mutant exon splicing site to construct an AAV plasmid vector with a 4.9kbp insertion sequence. Partial results for related plasmid cloning are shown in FIG. 3 below.

[0051] After constructing the completed AAV vector plasmid, according to previous literature [Grieger, J., Choi, V. & Samulski, R. Production and characterization of adeno-associated viral vectors. Nat Protoc 1, 1412-1428 (2006).], AAV virus of a serotype AAV9 was packaged and purified with a titer of 1 × 10.sup.13 v.g./mL. The separate packaging virus was mixed proportionally when used, and the combined packaging virus can be directly used for in vivo treatment.

Example 2: In Vivo Treatment of DMD Model Mice Using AAV Carrying Gene Editing Tools

[0052] A new DMD mouse disease model Dmd-E4 with abnormal cardiac function was selected in the present example. The model can be purchased from Jiangsu Jicui Pharma Biotechnology Co., Ltd., but not limited to it. Dmd-E4 showed cardiac hypertrophy, fibrosis and other phenotypes in the heart at 6-8 weeks, and showed severe cardiac degeneration at about 8 months. This process well mimiced the cardiac pathological process of DMD patients. For this model, cytosine deaminase and Cas9 were used to design gene editing tools to target exons carrying pathogenic mutations, and mutations near the 5′ splicing sites of that were induced to make them skip, and maximize the preservation of Dystrophin protein expression and restore its biological functions without affecting the open reading frame of the protein.

[0053] Specifically, the method of Example 1 was used to construct a gene editing tool, wherein the sgRNA sequence designed for the Dmd-E4 mouse mutation site is as shown in SEQ ID NO: 4, and the AAV vector plasmid that expressing the sgRNA targeting the Dmd-E4 mouse mutation site was obtained, and its sequence is shown in SEQ ID NO: 5. The corresponding sequence containing the AID-Cas9 fusion protein and the sgRNA targeting Dmd-E4 mice in the same AAV vector plasmid is shown in SEQ ID NO: 6.

[0054] Serotype AAV9 was selected for viral synthesis and purification, and Dmd-E4 mice were treated according to the two regimens of preventive treatment for neonatal mice and restorative treatment for adult mice, as shown in FIG. 2.

(A) Gene Therapy for Neonatal Mice

[0055] Grouping: Homozygous KO male and female Dmd-E4 mice were mated. After the female mice were pregnant, the male and female mice were caged, and the pregnant female mice were observed every two days to confirm whether they gave birth. After the birth of the newborn Dmd-E4 mice, the sex was observed, then 3-5 male mice were selected as the experimental group, and the other 3-5 male mice were as the negative control group.

[0056] Administration: 50-75 .Math.L of adeno-associated virus (AAV) carrying the gene editing tool (with a titer of 10.sup.13 v.g./mL) was administered by intraperitoneal injection or facial intravenous injection, and control mice were given an equal volume of sterile PBS at the same time, and then they were housed normally with female mice.

[0057] Sampling and detection: When the mice grew to about 2 months, in addition to the experimental group and the control group, 3-5 WT male mice of the same age were taken, and the following treatment was performed at the same time: after anesthetizing the mouse, the function test of the tibial anterior muscle, echocardiogram detection and the like was first performed, and then cardiac arteriovenous blood was collected to sacrifice the mouse. The serum was separated by centrifugation and stored at -80° C., while myocardium, skeletal muscle, tibial anterior muscle, back muscle, liver, brain, kidney and other tissues were collected, and proteins, RNA, genomic DNA of that were extracted. Enough tissues were retained for immunofluorescence staining, hematoxylin eosin staining, etc.

[0058] As shown in FIG. 4A, RNA in the hearts of treated Dmd-E4 mice was performed reverse transcription PCR, and primers were designed for Exon3 and Exon5 to detect skipping of Exon4 carrying mutations. At the same time, using method of capillary electrophoresis quantification to determine the ratio of the content of nucleic acid contained in the band with Exon4 skipping to the band without skipping (i.e., included). The results are shown in FIG. 4B. Further, Sanger sequencing was performed on the band with Exon4 skipping, and as shown in FIG. 4C, Exon4 was completely skipped, and Exon3 and Exon5 were spliced together. FIGS. 3D-F shows that western blotting was performed to detect proteins in the heart of mice, wherein FIG. 4D is the band plot, FIG. 3 panel E is the quantitative statistics of the band in the FIG. 3D. FIG. 4F shows the condition of expression of Dystrophin protein, the results show that the treated Dmd-E4 significantly restored the expression of Dystrophin protein. In addition, the method of small animal heart ultrasound detection was used to investigate whether the changes in the heart-related physiological structure of Dmd-E4 mice were repaired after AAV treatment. Results are shown in FIG. 4G, which shows that the heart-related physiological structure of Dmd-E4 mice was basically repaired after treatment.

[0059] Furthermore, the muscle function and survival of Dmd-E4 mice were verified to recover and prolong or not. FIG. 5A shows the results of the determination of creatine kinase content in the serum of mice, from which it can be seen that the creatine kinase content of treated Dmd-E4 mice was significantly reduced compared to WT and untreated Dmd-E4 mice samples. The method of HE staining and Masson staining was used to evaluate the degree of myocardial inflammatory cell infiltration and fibrosis of Dmd-E4 mice after treatment, and according to the results of Masson staining, the recovery of myocardial fibrosis in treated Dmd-E4 mice was quantitatively counted. As the results shown in FIGS. 5B-5C, the degree of myocardial fibrosis of treated Dmd-E4 mice was significantly improved. In addition, the micro-CT method was also used to detect the degree of spinal curvature in Dmd-E4 mice (FIGS. 5D-5E), and a pulling device was used to detect the degradation range of the maximum tension of the whole body muscle of the treated Dmd-E4 mice during the cyclic force process (FIG. 5F). The results show that the the spine curvature of Dmd-E4 mice was relieved after treatment, and the whole body muscle tension of the mice was enhanced, and the survival of Dmd-E4 mice was greatly prolonged (FIG. 5G). FIG. 5H shows the molecular biological evidence of gene editing in cardiomyocytes of Dmd-E4 mice. The pre-mRNA of the corresponding cells was performed by reverse-transcribed PCR, followed by high-throughput sequencing, and it was found that the expected mutation was generated near the location of the sgRNA targeting, which is the molecular foundation and basis for the treatment of the cardiac disease phenotype of Dmd-E4 mice.

[0060] The above results show that the gene editing tool of the present invention can effectively treat and prevent neonatal Dmd-E4 mice.

(B) Gene Therapy in Adult Mice

[0061] Grouping: 3-5 homozygous KO Dmd-E4 male mice aged 4-6 weeks were taken as the experimental group to give gene therapy, and 3-5 homozygous KO Dmd-E4 male mice were taken as the control group to give the same amount of PBS.

[0062] Administration: About 50 .Math.L of adeno-associated virus (AAV) carrying the gene-editing tool (with a titer of 10.sup.13 v.g./mL) was administered by tail vein injection or skeletal muscle in situ injection, and control mice were given an equal volume of sterile PBS at the same time;

[0063] Sampling and detection: When the mice were treated for about 2 months, in addition to the experimental group and the control group, 3-5 WT male mice of the same age were taken, and the following treatment was carried out at the same time: after anesthetizing the mouse, the function test of the tibial anterior muscle, echocardiogram detection and the like was first performed, and then cardiac arteriovenous blood was collected to sacrifice the mouse. The serum was separated by centrifugation and stored at -80° C., while myocardium, skeletal muscle, tibial anterior muscle, back muscle, liver, brain, kidney and other tissues were collected, and proteins, RNA, genomic DNA of that were extracted. Enough tissue was retained for immunofluorescence staining, hematoxylin eosin staining, etc.

[0064] The results show that AAV can be used as a carrier for gene editing tools to achieve efficient gene repair of mutant exons. In the treated Dmd-E4 mice, the pathogenic exon skipping could be observed in the myocardium and multiple muscle tissues, and the expression of Dystrophin protein was restored, and the phenotype of myocardial injury was also significantly repaired, so that adult Dmd-E4 mice were treated.

Example 3 Gene Editing of the DMD Model of Human Induced Pluripotent Stem Cells (iPSCs) Successfully Restored the Expression of Dystrophin Protein

[0065] Gene editing therapy of human cells has also been successfully implemented in the present invention. Firstly, we constructed induced pluripotent stem cells (iPSCs) from normal human peripheral blood mononuclear cells. Then CRISPR-cas9 was used to specifically delete Exon 50 in the Dystrophin coding gene DMD, resulting in a frame shift mutation in the coding sequence of the Dystrophin protein, thereby a mutation type mimicking DMD patients was constructed, which became a good DMD disease model cell. For this cell, we designed the sequence of AID and Cas9 fusion protein and the corresponding sgRNA, and a series of potential regulatory exon splicing elements targeting Exon51 of the DMD gene. The sgRNAs used in this example were sgRNA-12 as shown in SEQ ID NO: 19 and sgRNA-13 as shown in SEQ ID NO: 20, wherein sgRNA-12 mainly targeted exon splicing enhancer as shown in SEQ ID NO: 21 and SEQ ID NO: 22, and sgRNA-13 mainly targeted exon splicing enhancer as shown in SEQ ID NO: 24. The above two sgRNA-12 were screened in human K562 cell lines and could induce the skipping of Exon51. As shown in FIG. 6A, the results of reverse transcription PCR after RNA extraction of edited K562 cells show that both sgRNAs can induce mutations, and the combination of that can effectively induce Exon50-deficient K562 cells to successfully skip Exon51. By inducing the skipping of Exon5 1, the open reading frame of Dystrophin protein in Exon50-deficient K562 cells can be restored, and the expression of Dystrophin protein can be reconstructed at the same time. The specific implementation plan is as follows:

3.1 Induction of Differentiation of iPS Cells Into Cardiomyocytes

[0066] Human iPS cells cultured on matrix gel were digested with Accutase at 37° C. for 6 min. The reaction was terminated with DMEM medium, and the cells were collected, centrifuged at 1500 rpm for 3 min, and were counted under a microscope.

[0067] iPS cells were placed in 12-well plates pre-coated with matrix glue, and the cell density was adjusted to 10,000-20,000 cells /cm.sup.2. iPS cells were cultured with mTeSR1 medium for 4 days and 10 .Math.M ROCK inhibitor (Y-27632) was added, and the fresh medium was changed every day. ROCK inhibitors are not required when changing the medium.

[0068] After 4 days of cell culture, mTeSR1 medium was changed to RPMI/B27-insulin medium containing 6 uM CHIR99021 for 2 days of culture.

[0069] CHIR99021 stimulation was removed, and the medium was changed to RPMI/B27-insulin medium for 1 day of culture.

[0070] The medium was changed to RPMI/B27-insulin medium containing 5 .Math.m IWR1 for 2 days of culture.

[0071] IWR1 stimulation was removed, and the medium was changed to RPMI/B27-insulin medium for 2 days of culture.

[0072] The cell culture medium was changed to RPMI/B27 medium, and then the cells were cultured with this medium. The medium was changed every two days to obtain human pluripotent stem cells differentiated into cardiomyocytes.

3.2 Transfection of Gene Editing Tools in Human Pluripotent Stem Cells That Induced Differentiation Into Cardiomyocytes

[0073] On the day before transfection, iPS cells that induced differentiation into cardiomyocytes were digested with Accutase, and were seed in a 6-well plate with 4×10.sup.5 cells per well.

[0074] After about 24 hours, when the density of iPS cells that induced differentiation into cardiomyocytes reached about 60%, the cell culture medium was changed to antibiotic-free medium.

[0075] 2.5 .Math.g plasmids expressing AID and Cas9 mutant fusion protein (e.g., Lenti-V2-AIDx-nSaCas9 (KKH)-Ugi plasmid), 500 ng plasmids expressing UGI (e.g., pCDNA3.1-Ugi) and 1.5 .Math.g sgRNA plasmids were mixed in 150 .Math.l opti-MEM, and 2.5 .Math.l PLUS™ reagent was added and gently mixed.

[0076] 12.Math.l Lipofectamine LTX and 150 .Math.l opti-MEM medium were mixed and added into the plasmids of step (3). They were gently mixed, and incubated at room temperature for 15 min. The reaction product was added into the iPS cells differentiated into cardiomyocytes of step (2);

[0077] After 48 h of transfection, 2 .Math.g/ml puromycin was added into the transfected cells. The cells were screened for 3 days and then the drug was withdrawn. After 7 days of transfection, cells were collected for analysis.

3.3 The Detection of the Relevant Indicators of the Edited iPSC

[0078] The genomic DNA of iPSC before and after editing was extracted to detect whether the corresponding Exon51 mutation occurred.

[0079] The iPSC RNA was extracted before and after editing, and reverse transcription PCR was performed to detect whether Exon51 had been skipped at the RNA level. The results are shown in FIG. 6B. Exon51 of the DMD gene was induced to skip in normal human iPS cells and DMD exon50-deficient cells.

[0080] The expression of Dystrophin protein was investigated at the protein level of iPSCs before and after editing, and experimental methods included Western Blot, immunofluorescence staining, etc. FIG. 6C shows that immunofluorescence detection was used to determine that the expression of Dystrophin protein was restored in the edited iPS cells. FIG. 6D shows that western blot was used to determine that the expression of Dystrophin was restored in the edited iPS cells. FIG. 6E is the quantitative statistics of protein restored expression in FIG. D.

[0081] The above results show that in the K562 cell line, a gene editing method that can induce skipping of human DMD gene Exon51 has been successfully constructed, and a series of sequence elements that potentially regulate exon skipping have been identified. This gene editing scheme can be further used to successfully carry out therapeutic modification of DMD disease model cells iPSC and restore the expression of Dystrophin protein.

[0082] In addition, the corresponding exon splicing enhancer of the present invention such as SEQ ID NO: 21- SEQ ID NO: 24 are all targeted by the rest of the sgRNA-1 - sgRNA-11 as shown in SEQ ID NO: 7- SEQ ID NO: 18. When it is constructed into a gene editing tool, it can efficiently induce skipping of Exon51, thereby realizing the treatment of human DMD.

[0083] Obviously, the above embodiments are only examples for clarity, and do not qualify the embodiment. For those of ordinary skill in the art, other different forms of change or variation can be made on the basis of the above description. It is unnecessary and impossible to enumerate all embodiments here. The obvious change or variation derived therefrom remains within the scope of protection of the present invention.