A method for inhibiting DUX4 gene expression based on cytosine base editing technology

Abstract

This invention discloses a method for inhibiting DUX4 gene expression based on cytosine base editing technology, which targets the cytosine base editor via sgRNA. LEADER4 Introducing a premature stop codon into the Exon 1 region of the gene suppresses the reporter cell line N2a-i. LEADER4 skeletal muscle in FSHD mouse models LEADER4 The expression of PTCs. This invention successfully introduced PTCs into in vitro experiments using cytosine base editing technology. LEADER4 The Exon 1 target site was effectively inhibited. LEADER4 The expression of the drug; in in vivo experiments, delivery of CBE to FSHD mouse models via LNPs improved muscle strength and endurance and alleviated pathological symptoms in the mouse models.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a method for inhibiting DUX4 gene expression based on cytosine base editing technology. Background Technology

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular genetic disorders, 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 primarily affects the muscles of the face, shoulders, and upper arms, typically presenting as asymmetrical and slowly progressive atrophy. Most patients develop symptoms during puberty, while severe cases may even develop symptoms 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] Based on different pathogenic mechanisms, FSHD can be divided into two types: In FSHD1 patients, the number of D4Z4 tandem repeat sequences on chromosome 4 is reduced (≤10 repeat units), resulting in shortened chromatin domains; in FSHD2 patients, ... SMCHD1 or DNMT3B Mutations in epigenetic regulatory genes lead to hypomethylation of the D4Z4 region. Both of these conditions disrupt chromatin stability, resulting in [the formation of] hypomethylation in skeletal muscle. DUX4 Increased desuppressive expression, thereby causing DUX4The abnormal expression of these genes leads to disease development (Lemmers RJ, van der Vliet PJ, Klooster R, et al. A unifying genetic model for 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 unique and complex pathogenesis of FSHD, there is currently no effective cure, and research on treatment for 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 myosin inhibitors (vander Kooi EL, Kalkman JS, Lindeman E, et al. Effects of training and albuterol 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., all failed to achieve the expected results. Regarding... DUX4The main targeted therapy strategies include small molecule drugs (Mellion ML, Ronco L, Berends CL, et al. Phase 1 clinical trial of losmapimod infacioscapulohumeral dystrophy: Safety, tolerability, pharmacokinetics, and target 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 of a 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 methods are still in the preclinical stage, while losmapimod is undergoing clinical trials. In terms of gene therapy, CRISPR / Cas9 technology has been considered a promising tool due to the genetic characteristics of FSHD. For example, the dCAS9-KRAB system has been used to induce DUX4Epigenetic silencing reduces its transcripts and downregulates the expression of its target genes (Himeda CL, Jones TI, Jones PL. CRISPR / dCas9-mediated Transcriptional Inhibition Ameliorates the Epigenetic Dysregulation at D4Z4 and Represses DUX4-fl in FSH Muscular Dystrophy[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.). Other strategies include targeting SMCHD1 Intron mutations to restore and suppress their expression DUX4 (Goossens R, vanden Boogaard ML, Lemmers R, et al. Intronic SMCHD1 variants in FSHD: testing the 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 The PAS signaling sequence, thereby downregulating PAS 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 research has explored the use of cytosine base editing technology to inhibit DUX4Gene expression. Cytosine base editors (CBEs) are achieved by fusing an inactivated or nicked Cas protein with a single-stranded DNA-specific cytosine deaminase. This technology converts cytosine to uracil and, through a uracil glycosylase inhibitor (UGI), prevents uracil base excision repair, promoting the C•G to U•A conversion. Ultimately, during replication or DNA repair, it is converted to T•A base pairs without the introduction of homology-directed repair (HDR) or double-stranded DNA breaks. Studies have shown that CBEs can effectively convert codons such as CGA(R), CAG(Q), CAA(Q), and TGG(W) into premature termination codons (PTCs) (TGA, TAG, or TAA), effectively silencing genes in eukaryotes. In addition, some studies have demonstrated the significant potential of CBE in the treatment of metabolic liver disease, autosomal recessive deafness, β-thalassemia, and allogeneic CAR-T cell therapy.

[0006] Although cytosine base editors are powerful gene-editing tools, there is currently a lack of applications for them. DUX4 Gene expression suppression. Summary of the Invention

[0007] Technical Problem Solved: To address the aforementioned technical problem, this invention provides a method for inhibiting [certain activities] based on cytosine base editing technology. DUX4 Gene expression methods, 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 genes lead to more lasting therapeutic effects.

[0008] Technical Solution: A method for inhibiting DUX4 gene expression based on cytosine base editing technology, which targets the cytosine base editor (CBE) via sgRNA. DUX4 Introducing premature termination codons (PTCs) into the Exon 1 region of the gene suppresses the reporter cell line N2a-i. DUX4 skeletal muscle in FSHD mouse models DUX4 The expression.

[0009] Preferably, the sgRNA targets the cytosine base editor. DUX4The 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: SEQ ID NO. 1: CTGAAACCAAATCTGGACCC.

[0010] Preferably, the reporter cell line N2a-i DUX4 It utilizes the PiggyBac (PB) transposable system to transport complete human bodies. DUX4 - fl The gene structure transposed into N2a cells.

[0011] Furthermore, the reporter cell line N2a-i DUX4 Induction with doxycycline (DOX) DUX4 Express.

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

[0013] Furthermore, the induction time of the doxycycline is 24 to 72 hours, preferably not exceeding 48 hours.

[0014] Preferably, the FSHD mouse model is derived from... Myf6 - CreERT2 Heterozygous mice and FLExDUX4 Double transgenic heterozygous mice obtained by breeding heterozygous mice, i.e. 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 FSHD mouse models no more than 6 weeks old, preferably no more than 4 weeks old. DUX4 The administration frequency is once a week via intraperitoneal injection.

[0017] Furthermore, the induced 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 into the skeletal muscle of an FSHD mouse model.

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

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

[0021] Beneficial effects: This invention successfully introduced PTCs into in vitro experiments using cytosine base editing technology. DUX4 The Exon 1 target site was effectively inhibited. DUX4 The expression of the drug; in in vivo experiments, delivery of CBE to FSHD mouse models via LNPs improved muscle strength and endurance and alleviated pathological symptoms in the mouse models. Attached Figure Description

[0022] Figure 1 To introduce PTCs after converting cytosine (C) to thymine (T) via CBE. DUX4 A schematic diagram of the Exon1 process; Figure 2 For N2a-i DUX4 The cell line was constructed and its DOX dose-dependent DUX4 protein expression was verified by Western Blot. Figure 3 The Sanger sequencing results are shown using the W66 locus as an example. Figure 4 To quantitatively analyze the editing efficiency of BE4max and TadCBEa at five target sites with high editing efficiency using NGS; Figure 5 The heatmap shows the target base editing and flanking editing effects at the W66 and R145 sites, which have the highest editing efficiency. The data are plotted based on the mean of three biological replicates. Figure 6 For N2a-i DUX4 In cells (induced by 250 ng / mL DOX for 48 hours), the expression of DUX4 protein after editing five TadCBEa high-efficiency editing target sites; Figure 7 The heatmap shows the on-target editing efficiency of W66, R145, Q222 and W309 & W310 sites after editing with TadCBEa in HEK293T cells, as well as the editing efficiency at each of the seven predicted potential off-target sites. The data are expressed as the mean of three biological replicates. Figure 8Schematic diagram of LNPs or PBS injection into the gastrocnemius muscle and TMX intraperitoneal injection protocol; Figure 9 The results of limb grip strength tests for each mouse group (mice were 9 weeks old) have been standardized according to their respective body weights. Figure 10 For the inverted grid experiment results, each point represents the longest suspension time for each mouse in three trials (mice were 9 weeks old). Figure 11 The weight of the gastrocnemius muscle to the body weight ratio of each group of mice at 9 weeks of age; Figure 12 H&E stained images of frozen sections of gastrocnemius muscle, scale bar 20 μm; Figure 13 Quantitative analysis of the proportion of central nucleus muscle fibers (using a double-blind method, counting 1000 muscle fibers per mouse). Figure 14 Sirius red stained image of frozen sections of gastrocnemius muscle, scale bar 20 μm; Figure 15 To use Fiji to quantitatively analyze the area of ​​fibrosis; Figure 16 The heatmap shows the results after NGS analysis. M6D4 / + (sgRNA) group and M6D4 In the / + (sgRNA & CBE) group, the average editing efficiency of target bases in gDNA of each tissue is expressed as the mean of five biological replicates; Figure 17 The heatmap shows the results after NGS analysis. M6D4 / + (sgRNA) group and M6D4 In the / + (sgRNA & CBE) group, the average conversion rate of cDNA target bases in each tissue is expressed as the mean of five biological replicates. Figure 18 For those obtained by qRT-PCR DUX4 Relative expression in the gastrocnemius muscle of mice in each group; Figure 19 For those obtained by qRT-PCR DUX4 target genes Wfdc3 Relative expression in the gastrocnemius muscle of mice in each group; Figure 20 For those obtained by qRT-PCR DUX4 target genes Agtr2 Relative expression in the gastrocnemius muscle of mice in each group; Figure 21 For those obtained by qRT-PCR DUX4 target genes Serpinb6cRelative expression in the gastrocnemius muscle of mice in each group; The data in the above figures are expressed as mean ± standard deviation. ± s) indicates that the differences among multiple groups were analyzed using one-way ANOVA with Bonferroni correction. ns indicates no statistically significant difference. * P < 0.05,** P < 0.01, *** P <0.001, **** P < 0.0001. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments: Example 1: Screening of Editing Sites in In Vitro First, such as Figure 1 As shown: Induction using doxycycline (DOX) DUX4 The reporter cell line expressing (N2a-i) DUX4 The editing efficiency (i.e., reducing the effect of DUX4 protein) at multiple sites was validated. N2a-i DUX4 Methods for constructing cell lines: [The text abruptly shifts to a seemingly unrelated topic about cell line construction.] DUX4 The gene (Jones T, Jones PL, Asakura A. A cre-inducible DUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy[J]. PloS one, 2018, 13(2): e192657. It includes a 5' untranslated region, all three exons, two introns, endogenous PAS, and distal accessory elements) was inserted into the PiggyBac (PB) plasmid vector and co-transfected into N2a cells with the transposase plasmid and rtTA plasmid. After drug screening based on the resistance gene carried by the plasmid, stable transgenic cell lines were retained. The expression of DOX dose-dependent DUX4 protein in the cell line was verified by Western Blot, such as Figure 2 As shown, the expression level of DUX4 protein in this cell line after DOX induction showed a DOX dose-dependent range of 0-500 ng / mL.

[0024] Design a single-guide RNA (sgRNA) targeting the protospacer adjacent motif (PAM) of spCas9 (D10A) to guide CBE to act on... DUX4The target sites in Exon 1 are 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. N2a-i was then co-transfected with paired sgRNAs and two CBEs (BE4max and TadCBEa). DUX4 Cells, with nucleotide changes assessed using Sanger sequencing, taking W66 as an example, such as... Figure 3 As shown: PTCs were successfully introduced at the target site after editing BE4max and TadCBEa.

[0025] Table 1 Target sites and their corresponding sequences

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

[0027] The side-editing effect of the base editor was further analyzed, such as... Figure 5 As shown, at the W66 site, compared to BE4max, TadCBEa exhibited higher editing efficiency on the target bases (C7 and C8), while significantly reducing flanking edits (C1 and C13). At the R145 site, TadCBEa not only showed higher editing efficiency on the target base (C5), but also induced more flanking edits (C12 and C13) compared to BE4max. Based on TadCBEa's high editing efficiency and low flanking edit effect at the target sites 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 The decrease in DUX4 protein in the cell line was verified by Western blot, and the results are as follows: Figure 6 As shown, after 48 hours of DOX induction, the expression of DUX4 protein decreased significantly after editing at all five highly efficient editing sites.

[0029] To assess potential off-target effects, CRISPR RGEN was used to identify genomic sites with sequence homology to the sgRNAs corresponding to the four sites (W66, R145, Q222, and W309 & W310) where protein reduction was most pronounced. Selected off-target sites were chosen to meet the following criteria: 1) a maximum of three mismatched bases allowed; 2) identical PAM sequences; 3) the C site was identical to the target site, or at least one C was present within the TadCBEa editing window. Based on these criteria, in this example, the corresponding sgRNA plasmid and the TadCBEa plasmid were co-transfected into HEK293T cells. Figure 7 As shown: Post-NGS analysis revealed that the off-target site chr20 (chr20:29,411,397-29,411,419) of W66 contains a site identical to the target sequence, with editing efficiency comparable to the target site (average editing efficiency of approximately 10%). This site is located at... DUX4 Similar gene 34 (pseudogene); chr12 site 1 (chr12:34,209,474-34,209,496) contains a mismatched base, located at DUX4 Similar to gene 27 (pseudogene), its maximum average editing efficiency was 0.374%; the remaining 5 off-target sites did not show significant editing activity. However, the off-target effects and flanking editing of sites R145, Q222, and W309 & W310 were more pronounced.

[0030] The above process selected W66 as the optimal editing site. After CBE editing, this site significantly reduced the DUX4 protein, and had good on-target editing efficiency, good off-target effect, and low flanking editing.

[0031] Example 2: In vivo experiments to verify 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 times. They were divided into 3 groups based on their genotype and the specific medication injected into their bilateral gastrocnemius muscles: Myf6 - CreERT2 / + mice injected with PBS (marked as control group) M6D4 / + mice were injected with LNPs containing only the W66 site sgRNA (labeled as M6D4 / +(sgRNA) group), M6D4 / + mice were injected with LNPs containing W66 site sgRNA and TadCBEa mRNA ( M6D4 (sgRNA & CBE group), with 5 mice in each group. Figure 8As shown: Each injection of 50 μL of PBS or LNPs into the gastrocnemius muscle was administered every 72 hours for a total of 3 injections; phenotypic evaluation was performed on all mice at 9 weeks of age.

[0032] (1) Mouse limb grip strength test The grip strength measurement system used was the Shanghai Xinruan Information Technology Co., Ltd. XR501. Mice were allowed 30 minutes to acclimatize to the test area before testing. During the test, each mouse was placed in the center of the grid to ensure all four paws gripped the support. 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 strength achieved. Each mouse underwent three tests, with a 10-minute rest period between tests to reduce the effect of fatigue. The average maximum grip strength for each mouse was calculated. This value was then normalized to the mouse's body weight to obtain the limb strength / body weight ratio (gf / g). Figure 9 As shown: M6D4 The ratio of limb grip strength to body weight in the / + (sgRNA & CBE) group was significantly higher than that in the sgRNA & CBE group. M6D4 The group with +(sgRNA) was significantly lower than the control group.

[0033] (2) Mouse inverted grid test The apparatus used was homemade: an open container (length × width × height = 15 cm × 15 cm × 35 cm) was made from a transparent acrylic sheet, and a mesh cover was made from 0.6 mm thick wire mesh with each hole edge length of 0.6 cm. All groups of mice were acclimatized to the test area for 30 minutes before the test at 9 weeks of age. The mice were first placed upright in the center of the wire mesh, allowing them to grip the mesh with their limbs. Then, the mesh was inverted while the mice's tails were released, and a timer was used to record the suspension time of each mouse on the mesh. Each mouse underwent three trials, with a 10-minute rest period between trials. The longest suspension time for each mouse across the three trials was used for comparison. Figure 10 As shown: M6D4 The mean longest suspension time in the / + (sgRNA & CBE) group and M6D4 The level of sgRNA was significantly increased compared to the + group, but still significantly lower than that of 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 mass to body weight was significantly higher in the / + (sgRNA & CBE) group than in the other group. M6D4 The group with +(sgRNA) was significantly lower than the control group.

[0035] (4) Pathological changes in mouse skeletal muscle Fresh gastrocnemius muscle, coated with OCT embedding agent, was rapidly frozen in pre-cooled isopentane and then transferred to liquid nitrogen. Frozen sections with a thickness of 10 μm were prepared using a cryostat. 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 below. Figure 12 As shown: M6D4 / + (sgRNA) group and M6D4 The proportion of central nucleus myofibrils in the / + (sgRNA & CBE) group was significantly higher than that in the control group. M6D4 / + (sgRNA) group compared to M6D4 Mononuclear cell infiltration was observed in the / + (sgRNA & CBE) group. Quantitative analysis was performed as follows: Figure 13 As shown: M6D4 The proportion of central nucleus muscle fibers in the / + (sgRNA & CBE group compared to M6D4 The / + (sgRNA & CBE) group showed a significant decrease. For example... Figure 14 and Figure 15 As shown: M6D4 The fibrosis area in the / + (sgRNA & CBE group was smaller than that in the sgRNA & CBE group. M6D4 The level of sgRNA in the + group was significantly lower, but still significantly higher than that in the control group.

[0036] (5) Base editing efficiency in various mouse tissues NGS analysis was used to analyze the editing efficiency of target bases in gDNA c and cDNA in the gastrocnemius, tibialis anterior, quadriceps femoris, liver, heart, and brain of mice. The results are as follows: Figure 16 and Figure 17 As shown, base editing is concentrated in the injected gastrocnemius muscle, and the base conversion rate in cDNA is 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 femoral muscle using RNA Isolater Total RNA Extraction Reagent (Novizan, R401-01) according to the manufacturer's instructions. Subsequent steps included removal of genomic DNA and synthesis of cDNA using a HiScriptIII RT SuperMix qPCR kit. Quantitative Real-time PCR was performed using a QuantStudio™ 7 Flex real-time quantitative PCR system and a ChamQ SYBR qPCR Master Mix (all reagents were purchased from Novizan). The amplified gene was... DUX4 , Wfdc3 , Agtr2 and Serpinb6c ,by Gapdh The primer sequences used as internal reference genes are shown in Table 2: Table 2 qRT-PCR primer sequences

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

[0039] This invention screened the optimal editing sites in in vitro studies and verified the effects of CBE-edited skeletal muscle in FSHD mouse models in in vivo experiments. 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 within the protection scope of the present invention.

Claims

1. A cytosine base editing composition, characterized in that: The composition comprises sgRNA and a cytosine base editor, and the composition targets the cytosine base editor via sgRNA. DUX4 An early stop codon is introduced into the Exon 1 region of the gene; The sgRNA targets the cytosine base editor. DUX4 The target site of the gene's Exon 1 is W66, and the nucleotide sequence of the target site W66 is shown in SEQ ID NO.

1.

2. The composition according to claim 1, characterized in that: The composition utilizes lipid nanoparticles to deliver cytosine base editor mRNA and sgRNA.

3. The composition according to claim 2, characterized in that: The lipid nanoparticles encapsulate sgRNA to cytosine base editor mRNA in a mass ratio of 1:2, with a total RNA concentration of 1 μg / 100 μL to 8 μg / 100 μL.

4. The composition according to claim 3, characterized in that: The total RNA concentration was 8 μg / 100 μL.

Images