Method for inhibiting DUX4 gene expression based on cytosine base editing technology

Abstract

The invention discloses a method for inhibiting DUX4 gene expression based on a cytosine base editing technology, which comprises the following steps: targeting a cytosine base editor to an Exon 1 region of a DUX4 gene through sgRNA, introducing a premature termination codon, and inhibiting expression of DUX4 in skeletal muscles of a report cell line N2a-iDUX4 and an FSHD mouse model. According to the invention, PTCs is successfully introduced into an Exon 1 target site of DUX4 in an in-vitro experiment through a cytosine base editing technology, and the expression of DUX4 is effectively inhibited; in an in vivo experiment, CBE is delivered to an FSHD mouse model through LNPs, muscle strength and endurance of the mouse model are improved, and pathological symptoms are relieved.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing technology, and specifically relates to a method for inhibiting DUX4 gene expression based on cytosine base editing technology. Background Art

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common genetic neuromuscular diseases, with an incidence of approximately 1 / 15,000 to 1 / 20,000 (Hamel J, Johnson N, Tawil R, et al. Patient-Reported Symptoms in Facioscapulohumeral Muscular Dystrophy (PRISM-FSHD) [J]. Neurology, 2019, 93(12): e1180-e1192.). FSHD mainly affects the muscles of the face, shoulders, and upper arms, usually showing asymmetry and slowly progressive atrophy. Most patients develop symptoms during adolescence, and severe cases may even develop the disease before the age of 10 (Giardina E, Camaño P, Burton Jones S, et al. Best practice guidelines on genetic diagnostics of facioscapulohumeralmuscular dystrophy: Update of the 2012 guidelines[J]. Clinical Genetics, 2024, 106(1): 13-26.).

[0003] FSHD can be divided into two types based on the different pathogenic mechanisms: In FSHD1 patients, the number of D4Z4 tandem repeats on chromosome 4 is reduced (≤10 repeat units), resulting in a shortened chromatin domain; in FSHD2 patients, SMCHD1 or DNMT3B Mutations in epigenetic regulatory genes such as β-actin and β-actin lead to hypomethylation of the D4Z4 region. Both of these conditions disrupt chromatin stability, leading to DUX4 Increased disinhibitory expression of DUX4Abnormal expression of FSHD leads to the development of the disease (Lemmers RJ, van der Vliet PJ, Klooster R, et al. A unifying genetic modelfor facioscapulohumeral muscular dystrophy[J]. Science, 2010, 329(5999):1650-1653. Sacconi S, Briand-Suleau A, Gros M, et al. FSHD1 and FSHD2 form adisease continuum[J]. Neurology, 2019, 92(19): e2273-e2285.). Due to the particularity and complexity of the pathogenic mechanism of FSHD, there is currently no effective cure, and research on the treatment of this disease is still in the exploratory stage.

[0004] Currently, the treatment of FSHD mainly focuses on supportive care (Aguirre AS, Astudillo MO, Mosquera J, et al. Treatment of Facioscapulohumeral Muscular Dystrophy(FSHD): A Systematic Review[J]. Cureus, 2023, 15(6): e39903.). Early treatment attempts included the use of steroids (Tawil R, McDermott MP, Pandya S, et al. A pilot trial of prednisone in facioscapulohumeral muscular dystrophy. FSH-DY Group[J]. Neurology, 1997, 48(1): 46-49.), β2-adrenergic receptor agonists, and myostatin inhibitors (vander Kooi EL, Kalkman JS, Lindeman E, et al. Effects of training andalbuterol on pain and fatigue in facioscapulohumeral muscular dystrophy[J]. JNeurol, 2007, 254(7): 931-940. Kissel JT, McDermott MP, Mendell JR, et al. Randomized, double-blind, placebo-controlled trial of albuterol in facioscapulohumeral dystrophy[J]. Neurology, 2001, 57(8): 1434-1440.), but failed to achieve the expected results. DUX4The main targeted treatment strategies include small molecule drugs (Mellion ML, Ronco L,Berends CL, et al. Phase 1 clinical trial of losmapimod infacioscapulohumeral dystrophy: Safety, tolerability, pharmacokinetics, andtarget engagement[J]. Br J Clin Pharmacol, 2021, 87(12): 4658-4669.), oligonucleotide therapy (Bouwman LF, den Hamer B, van den Heuvel A, et al. Systemic delivery ofa DUX4-targeting antisense oligonucleotide to treat facioscapulohumeralmuscular dystrophy[J]. Molecular Therapy - Nucleic Acids, 2021, 26: 813-827.Lim K, Bittel A, Maruyama R, et al. DUX4 Transcript Knockdown with Antisense2'-O-Methoxyethyl Gapmers for the Treatment of Facioscapulohumeral Muscular Dystrophy[J]. Mol Ther, 2021, 29(2): 848-858.) and gene therapy. Most of these approaches are still in the preclinical stage, while losmapimod is undergoing clinical trials. In terms of gene therapy, CRISPR / Cas9 technology has been considered a tool with great potential due to the genetic characteristics of FSHD. For example, the dCAS9-KRAB system has been used to induce DUX4Other strategies include targeting the epigenetic silencing of D4Z4, reducing its transcriptional products and downregulating the expression of its target genes (Himeda CL, Jones TI, JonesP L. CRISPR / dCas9-mediated Transcriptional Inhibition Ameliorates theEpigenetic Dysregulation at D4Z4 and Represses DUX4-fl in FSH MuscularDystrophy[J]. Mol Ther, 2016, 24(3): 527-535.Himeda CL, Jones TI, Jones PL. Targeted epigenetic repression by CRISPR / dSaCas9 suppresses pathogenicDUX4-fl expression in FSHD[J]. Mol Ther Methods Clin Dev, 2021, 20: 298-311.). SMCHD1 Intronic mutation to restore its expression and suppress DUX4 (Goossens R, vanden Boogaard ML, Lemmers R, et al. Intronic SMCHD1 variants in FSHD: testingthe potential for CRISPR-Cas9 genome editing[J]. J Med Genet, 2019, 56(12):828-837.), and using adenine base editing technology to interfere with 4qA DUX4 PAS signaling sequence, thereby downregulating the expression of cytokines in FSHD skeletal muscle cells. DUX4 Transcript (Sikrova D, Cadar VA, Ariyurek Y, et al. Adenine baseediting of the DUX4 polyadenylation signal for targeted genetic therapy infacioscapulohumeral muscular dystrophy[J]. Mol Ther Nucleic Acids, 2021, 25:342-354.).

[0005] To date, no studies have explored the use of cytosine base editing technology to inhibit DUX4Gene expression. Cytosine base editors (CBEs) are created by fusing inactivated or nicking Cas proteins with single-stranded DNA-specific cytosine deaminases. This technology converts cytosine to uracil and blocks uracil base excision repair with a uracil glycosylase inhibitor (UGI), promoting the conversion of C•G to U•A, ultimately converting the base pair to a T•A during replication or DNA repair without the need for homology-directed repair (HDR) or the introduction of double-stranded DNA breaks. Previous studies have demonstrated that CBEs can efficiently convert codons such as CGA (R), CAG (Q), CAA (Q), and TGG (W) to premature termination codons (PTCs) (TGA, TAG, or TAA), effectively silencing genes in eukaryotes. In addition, several studies have demonstrated the significant potential of CBE in the treatment of metabolic liver diseases, autosomal recessive deafness, β-thalassemia, and allogeneic CAR-T cell therapy.

[0006] Although cytosine base editors are powerful gene editing tools, their application is currently lacking. DUX4 Gene expression inhibition. Summary of the Invention

[0007] Technical problems to be solved: In response to the above technical problems, the present invention provides a method for inhibiting DUX4 Gene expression method, using CBE to introduce PTCs into DUX4 The Exon1 region achieves inhibition DUX4 The effect of expression, CBE through precise editing DUX4 The exons of the gene bring about a more lasting therapeutic effect.

[0008] Technical solution: A method based on cytosine base editing technology to inhibit DUX4 gene expression, targeting the cytosine base editor (CBE) through sgRNA DUX4 Introducing premature termination codons (PTCs) into the Exon 1 region of the gene inhibited the reporter cell line N2a-i DUX4 and skeletal muscle in FSHD mouse models DUX4 expression.

[0009] Preferably, the sgRNA targets the cytosine base editor DUX4The target site of Exon 1 of the gene is W66, and the nucleotide sequence of the target site W66 is shown in SEQ ID NO.1: SEQ ID NO. 1: CTGAAACCAAATCTGGACCC.

[0010] Preferably, the reporter cell line N2a-i DUX4 The PiggyBac (PB) transposition system is used to transform the complete human DUX4 - fl The gene construct was transposed into N2a cells.

[0011] Furthermore, the reporter cell line N2a-i DUX4 Doxycycline (DOX) was used to induce DUX4 Express.

[0012] Furthermore, the induction concentration of doxycycline is 5 ng / mL to 500 ng / mL, preferably 250 ng / mL.

[0013] Furthermore, the induction time of doxycycline is 24 hours to 72 hours, preferably no more than 48 hours.

[0014] Preferably, the FSHD mouse model is Myf6 - CreERT2 Heterozygous mice and FLExDUX4 The double transgenic heterozygous mice obtained by breeding heterozygous mice are Myf6 - CreERT2-FLExDUX4 / +, named M6D4 / +.

[0015] Preferably, the skeletal muscle of the FSHD mouse model is induced by tamoxifen (TMX). DUX4 Express.

[0016] Furthermore, the tamoxifen induces FSHD in mice no older than 6 weeks, preferably no older than 4 weeks. DUX4 The frequency of intraperitoneal injection was once a week.

[0017] Furthermore, the induction concentration of tamoxifen is 5-10 mg / kg, preferably 10 mg / kg.

[0018] Preferably, lipid nanoparticles (LNPs) are used to deliver cytosine base editor mRNA and sgRNA to the skeletal muscle of FSHD mouse models.

[0019] Furthermore, the mass ratio of sgRNA to cytosine base editor mRNA encapsulated by the lipid nanoparticles is 1:2, and the total RNA concentration is 1 μg / 100 μL~8 μg / 100 μL, preferably 8 μg / 100 μL.

[0020] Furthermore, the lipid nanoparticles are injected before the mice are 6 weeks old, preferably before 4 weeks old, with a frequency of once every 24 to 72 hours, preferably once every 72 hours.

[0021] Beneficial effects: The present invention successfully introduced PTCs into the cell line through cytosine base editing technology in vitro. DUX4 Exon 1 target site and effectively inhibited DUX4 In vivo experiments, CBE was delivered to FSHD mouse models via LNPs, which improved the muscle strength and endurance of the mouse models and alleviated the pathological symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To convert cytosine (C) into thymine (T) by CBE and then introduce PTCs into DUX4 Schematic diagram of the process of Exon1; Figure 2 N2a-i DUX4 The cell construction method and the verification of DOX-dose-dependent DUX4 protein expression in the cell line by Western Blot; Figure 3 This is the Sanger sequencing result of the W66 site as an example; Figure 4 To quantitatively analyze the editing efficiency of BE4max and TadCBEa at five target sites with high editing efficiency by NGS; Figure 5 The heat map shows the on-target base editing and flanking editing effects at the W66 and R145 sites with the highest editing efficiency. The data are plotted based on the mean of three biological replicates. Figure 6 N2a-i DUX4 DUX4 protein expression in cells (250 ng / mL DOX induction for 48 hours) after editing at five highly efficient TadCBEa target sites. Figure 7 The heat map shows the on-target editing efficiency of W66, R145, Q222, and W309&W310 sites after TadCBEa editing in HEK293T cells, as well as the editing efficiency at the seven predicted potential off-target sites. The data are expressed as the mean of three biological replicates. Figure 8Schematic diagram of the injection of LNPs or PBS into the gastrocnemius muscle and the intraperitoneal injection of TMX; Figure 9 The results of the limb grasping strength test of each mouse group (mice were 9 weeks old) were normalized according to their body weight; Figure 10 The results of the inverted grid experiment are shown. Each point represents the longest hanging time of each mouse in three trials (mice were 9 weeks old). Figure 11 is the ratio of gastrocnemius muscle weight to body weight of mice in each group at 9 weeks of age; Figure 12 H&E staining images of frozen sections of gastrocnemius muscle, scale bar is 20 μm; Figure 13 Quantitative analysis of the proportion of central nuclear muscle fibers (using a double-blind method, counting 1000 muscle fibers per mouse); Figure 14 The image is Sirius red stained image of gastrocnemius frozen section, the scale bar is 20 μm; Figure 15 To quantitatively analyze the fibrosis area using Fiji; Figure 16 The heat map shows the M6D4 / + (sgRNA) group and M6D4 In the / + (sgRNA & CBE) group, the average editing efficiency of target bases in gDNA of each tissue was expressed as the mean of five biological replicates; Figure 17 The heat map shows the M6D4 / + (sgRNA) group and M6D4 In the / + (sgRNA & CBE) group, the average conversion rate of cDNA target bases in each tissue, the data are expressed as the mean of five biological replicates; Figure 18 was obtained by qRT-PCR DUX4 The relative expression in the gastrocnemius muscle of each group of mice; Figure 19 was obtained by qRT-PCR DUX4 target genes Wfdc3 The relative expression in the gastrocnemius muscle of each group of mice; Figure 20 was obtained by qRT-PCR DUX4 target genes Agtr2 The relative expression in the gastrocnemius muscle of each group of mice; Figure 21 was obtained by qRT-PCR DUX4 target genes Serpinb6cThe relative expression in the gastrocnemius muscle of each group of mice; The above figures are expressed as mean ± standard deviation ( ± s) were displayed. One-way ANOVA combined with Bonferroni correction was used for comparison among multiple groups. ns indicates no statistically significant difference. * P < 0.05, ** P < 0.01,*** P <0.001, **** P < 0.0001. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments: Example 1: In vitro screening of editing sites First, if Figure 1 Doxycycline (DOX)-induced DUX4 Reporter cell lines expressing DUX4 ) to verify the editing efficiency of multiple sites (i.e., reduce the effect of DUX4 protein). N2a-i DUX4 Cell line construction method: full-length synthetic DUX4 The gene (Jones T, Jones PL, Asakura A. A cre-inducibleDUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy[J]. PloS one, 2018, 13(2): e192657. It includes the 5' untranslated region, all 3 exons, 2 introns, the endogenous PAS and distal auxiliary elements) was inserted into the PiggyBac (PB) plasmid vector and co-transfected with the transposase plasmid and rtTA plasmid into N2a cells. Stable transgenic cell lines were retained after drug screening based on the resistance gene carried by the plasmid. The DOX dose-dependent expression of DUX4 protein in the cell line was verified by Western Blot, as shown in Figure 5. Figure 2 As shown: After DOX induction, the expression level of DUX4 protein in this cell line showed DOX dose dependence within the range of 0-500 ng / mL.

[0024] A single guide RNA (sgRNA) targeting the protospacer adjacent motif (PAM) of spCas9 (D10A) was designed to guide CBE to act on DUX4The target sites in Exon 1 of α-glucan were W66, W141, R145, W177, W186, Q222, Q231, Q259, Q273, Q305, W309, W310, Q334, Q346, Q348, Q357 and Q360, and the specific target site sequences are shown in Table 1. The paired sgRNA was co-transfected with two CBEs (i.e., BE4max and TadCBEa) into N2a-i DUX4 Cells were sequenced by Sanger sequencing to assess nucleotide changes. Taking W66 as an example, Figure 3 Shown: PTCs were successfully introduced into the target site after editing with BE4max and TadCBEa.

[0025] Table 1 Target sites and their corresponding sequences

[0026] The editing efficiency of these target sites was quantitatively evaluated using NGS. The results showed that the two editors exhibited different editing efficiencies at different sites. Except for the extremely low efficiencies at Q273 and Q360 (the highest average editing efficiency was only 3.24%), most sites showed significant average editing efficiencies, such as Figure 4 As shown: The editing efficiency of these two sites, W66 and R145, is the highest.

[0027] The side editing effects of base editors were further analyzed, such as Figure 5 As shown: At the W66 site, TadCBEa exhibited higher editing efficiency at the target base (C7 and C8) compared to BE4max, while significantly reducing the amount of side edits (C1 and C13); at the R145 site, TadCBEa not only had a higher editing efficiency at the target base (C5), but also triggered more side edits (C12 and C13) than BE4max. Based on the high editing efficiency and low side editing effect of TadCBEa at the target site with the highest editing efficiency, as well as its smaller protein size compared to BE4max, this editor was selected for further research.

[0028] In N2a-i DUX4 Western Blot was used to verify the decrease of DUX4 protein in the cell line. The results are as follows Figure 6 As shown: After 48 hours of DOX induction, the expression of DUX4 protein was significantly decreased after editing at the five efficient editing sites.

[0029] In order to evaluate potential off-target effects, the CRISPR RGEN tool was used to identify genomic sites with sequence homology to the sgRNA corresponding to the four sites with the most obvious protein reduction (W66, R145, Q222, and W309&W310). The selected off-target sites should meet the following criteria as much as possible: 1) a maximum of three mismatched bases are allowed; 2) the same PAM sequence; 3) the C site is the same as the target site, or at least one C is within the editing window of TadCBEa. Based on these criteria, in this example, the corresponding sgRNA plasmid was co-transfected with the TadCBEa plasmid into HEK293T cells. Figure 7 As shown: NGS analysis showed that the off-target site chr20 (chr20:29,411,397-29,411,419) of W66 has a site that is exactly the same as the target sequence, and its editing efficiency is comparable to that of the target site (the average editing efficiency is about 10%). DUX4 Similar gene 34 (pseudogene); chr12 site 1 (chr12:34,209,474-34,209,496) has a mismatched base located at DUX4 Similar gene 27 (pseudogene) had a maximum average editing efficiency of 0.374%, while the remaining five off-target sites showed no significant editing activity. However, the off-target effects and flanking editing at sites R145, Q222, and W309 & W310 were more pronounced.

[0030] Through the above process, W66 was screened as the optimal editing site. After CBE editing, this site has a significant effect on reducing DUX4 protein, and has good on-target editing efficiency, good off-target effect and low side editing.

[0031] Example 2: In vivo experiments verifying the therapeutic effect of CBE All mice were intraperitoneally injected with tamoxifen at a dose of 10 mg / kg, once a week for a total of 4 injections. According to the genotype of the mice and the injection of bilateral gastrocnemius muscles, they were divided into 3 groups: Myf6 - CreERT2 / + mice were injected with PBS (marked as control group), M6D4 / + mice were injected with LNPs containing only W66 site sgRNA (marked as M6D4 / + (sgRNA) group), M6D4 / + mice were injected with LNPs containing W66 site sgRNA and TadCBEa mRNA ( M6D4 / + (sgRNA & CBE) group), each group had 5 mice. Figure 8As shown: PBS or LNPs were injected into each gastrocnemius muscle at a volume of 50 μL each time, once every 72 hours for a total of 3 injections; all mice were evaluated for phenotype at 9 weeks of age.

[0032] (1) Mouse limb grip strength test The grip strength measurement system used was XR501 from Shanghai Xinruan Information Technology Co., Ltd. The mice were adapted to the test area for 30 minutes before the test. During the test, each mouse was placed in the center of the grid to ensure that all four paws of the mice grasped the bracket. The tester then gently pulled the mouse's tail into a straight line until the mouse released the grid, and the system recorded the maximum grip force achieved. Each mouse underwent 3 tests, with a 10-minute rest period between tests to reduce the effects of fatigue. The average maximum grip strength of each mouse was calculated. This value was then normalized with the mouse's body weight to obtain the limb strength / body weight ratio (gf / g). Figure 9 As shown: M6D4 The ratio of limb grasping strength to body weight in the / + (sgRNA & CBE) group was significantly higher than that in the M6D4 / + (sgRNA) group, but still significantly lower than the control group.

[0033] (2) Mouse inverted grid experiment The device used was homemade: a transparent acrylic plate was used to make a lidless container (length × width × height = 15 cm × 15 cm × 35 cm), and a wire mesh with a thickness of 0.6 mm and a side length of 0.6 cm per hole was used to make a grid cover. All groups of mice were adapted to the test area for 30 minutes before testing at the age of 9 weeks. First, the mice were placed upright in the center of the wire mesh so that their limbs could grasp the mesh. Then, the mesh was inverted and the tail of the mouse was released at the same time, and the hanging time of each mouse on the mesh was recorded with a timer. Each mouse underwent 3 trials, with a rest period of 10 minutes between the two trials. The longest hanging time of each mouse in the 3 trials was used for comparison. Figure 10 As shown: M6D4 The average longest hanging time of the / + (sgRNA & CBE) group was M6D4 The expression of sgRNA in the HBeAg / IgG4 T cells was significantly increased compared with the / + (sgRNA) group, but still significantly lower than that in the control group (P < 0.0001).

[0034] (3) Mouse gastrocnemius muscle / body weight Weigh the gastrocnemius muscle of each mouse and divide it by the corresponding mouse body weight to obtain the gastrocnemius muscle to body weight ratio (mg / g). Figure 11 As shown: M6D4 The ratio of gastrocnemius muscle to body weight in the / + (sgRNA & CBE) group was significantly higher than that in the M6D4 / + (sgRNA) group, but still significantly lower than the control group.

[0035] (4) Pathological changes in skeletal muscle of mice Fresh gastrocnemius muscles coated with OCT embedding medium were quickly frozen in pre-cooled isopentane and then transferred to liquid nitrogen. 10 μm thick frozen sections were prepared using a freezing microtome. The prepared sections were used for subsequent tissue staining experiments, including H&E staining and Sirius red staining. The H&E staining results are shown in Figure 2. Figure 12 As shown: M6D4 / + (sgRNA) group and M6D4 The proportion of central nucleus muscle fibers in the / + (sgRNA&CBE) group was significantly higher than that in the control group. M6D4 / + (sgRNA) group compared with M6D4 The / + (sgRNA & CBE) group showed mononuclear cell infiltration. Quantitative analysis was as follows Figure 13 As shown: M6D4 The ratio of central nucleus muscle fibers in the / + (sgRNA & CBE) group was compared with M6D4 The / + (sgRNA & CBE) group was significantly reduced. Figure 14 and Figure 15 As shown: M6D4 The fibrosis area in the / + (sgRNA&CBE) group was significantly higher than that in the M6D4 The expression of sgRNA in the sgRNA / / + group was significantly decreased, but still significantly higher than that in the control group.

[0036] (5) Base editing efficiency in various mouse tissues NGS was used to analyze the editing efficiency of target bases of gDNAc and cDNA in mouse gastrocnemius muscle, tibialis anterior muscle, quadriceps femoris, liver, heart and brain. Figure 16 and Figure 17 As shown: Base editing occurred concentratedly in the injected gastrocnemius muscle, and the base conversion rate in cDNA was significantly higher than that in gDNA.

[0037] (6) Expression of DUX4 and its target genes in mouse skeletal muscle Total RNA was extracted from fresh mouse entrails using RNA Isolater Total RNA Extraction Reagent (Norvozymes, R401-01) according to the manufacturer's instructions. Subsequent steps included the removal of genomic DNA and the synthesis of cDNA using the HiScript III RT SuperMix qPCR Kit. Quantitative Real-time PCR was performed using the QuantStudio™ 7 Flex Real-Time Fluorescence Quantitative PCR System and ChamQ SYBR qPCR Master Mix (all reagents purchased from Norvozymes). The amplified gene was DUX4 、 Wfdc3 、 Agtr2 and Serpinb6c ,by Gapdh As the internal reference gene, the primer sequences used are shown in Table 2: Table 2 qRT-PCR primer sequences

[0038] DUX4 、 Wfdc3 、 Agtr2 and Serpinb6c Gene expression such as Figures 18-21 As shown: DUX4 The relative expression levels of IL-6 and its target genes were significantly downregulated in gastrocnemius muscle.

[0039] The present invention screened the optimal editing site in vitro and verified in vivo that the skeletal muscle of FSHD mouse model after CBE editing DUX4 Gene expression is suppressed, and the phenotype is improved.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for inhibiting DUX4 gene expression based on cytosine base editing technology, characterized in that: Targeting of cytosine base editors by sgRNA DUX4 Introducing a premature stop codon in the Exon 1 region of the gene suppressed the reporter cell line N2a-i DUX4 and skeletal muscle in FSHD mouse models DUX4 expression.

2. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 1, characterized in that: The sgRNA targets the cytosine base editor DUX4 The Exon 1 target site of the gene is W66, and the nucleotide sequence of the target site W66 is shown in SEQ ID NO.

1.

3. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 1, characterized in that: The reporter cell line N2a-i DUX4 The PiggyBac transposition system is used to transfer complete human DUX4 - fl The gene construct was transposed into N2a cells.

4. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 3, characterized in that: The reporter cell line N2a-i DUX4 Doxycycline induction DUX4 Express.

5. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 4, characterized in that: The induction concentration of doxycycline is 5 ng / mL to 500 ng / mL, preferably 250 ng / mL; the induction time is 24 hours to 72 hours, preferably no more than 48 hours.

6. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 1, characterized in that: The skeletal muscle of the FSHD mouse model was induced by tamoxifen. DUX4 Express.

7. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 6, characterized in that: The tamoxifen induces FSHD in mice aged no more than 6 weeks, preferably no more than 4 weeks. DUX4 The induction concentration of tamoxifen is 5-10 mg / kg, preferably 10 mg / kg.

8. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 1, characterized in that: Lipid nanoparticles were used to deliver cytosine base editor mRNA and sgRNA to skeletal muscle in a FSHD mouse model.

9. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 8, characterized in that: The mass ratio of sgRNA to cytosine base editor mRNA encapsulated by the lipid nanoparticles is 1:2, and the total RNA concentration is 1 μg / 100 μL~8 μg / 100 μL, preferably 8 μg / 100 μL.

10. The method for inhibiting DUX4 gene expression based on cytosine base editing technology according to claim 8, characterized in that: The lipid nanoparticles are injected before the mouse is 6 weeks old, preferably before 4 weeks old, with a frequency of once every 24 to 72 hours, preferably once every 72 hours.

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