Homology-Directed Repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy

Homology-Directed Repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Homology-Directed Repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy Jiali Nie, Yu Han, Zhiyuan Jin, Weijian Hang, Hongyang Shu, Zheng Wen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2199328/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Variants in myosin-binding protein C3 ( MYBPC3 ) gene are a main cause of hypertrophic cardiomyopathy (HCM), accounting for 30–40% of the total number of HCM cases. Gene editing represents a potential permanent cure for HCM. The aim of this study was to investigate whether genome editing of MYBPC3 using the CRISPR/Cas9 system in vivo could rescue the phenotype of rats with HCM. We generated a rat model of HCM (“1098hom”) that carried an Mybpc3 premature termination codon mutation (p.W1098x) discovered in a human HCM pedigree. On postnatal day 3, the CRISPR/Cas9 system was introduced into rat pups by a single dose of AAV9 particles to correct the variant using homology-directed repair (HDR). Analysis was performed 6 months after AAV9 injection. The 1098hom rats didn’t express MYBPC3 protein and developed an HCM phenotype with increased ventricular wall thickness and diminished cardiac function. Importantly, CRISPR HDR genome editing corrected 3.56% of total mutations, restored MYBPC3 protein expression by 2.12%, and normalized the HCM phenotype of 1098hom rats. Our work demonstrates that the HDR strategy is a promising approach for treating HCM associated with MYBPC3 mutation, and that CRISPR technology has great potential for treating hereditary heart disease. Health sciences/Diseases/Cardiovascular diseases Biological sciences/Biological techniques/Genetic engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hypertrophic cardiomyopathy (HCM) is a hereditary disease with a global prevalence of approximately 1 in 500 ( 1 ). It is characterized by left ventricular hypertrophy, myocardial hypercontractility, reduced compliance, myofibrillar disarray, and fibrosis. Symptoms in patients with HCM range from mild chest pain, syncope, and dyspnea to severe sudden cardiac death ( 2 ). HCM is a major cause of sudden cardiac death in young adults, especially young athletes ( 3 ). Current therapy for HCM includes pharmacological intervention represented by 𝛽-blockers or non-dihydropyridine calcium-channel blockers, surgical septal myectomy, alcohol septal ablation, and radiofrequency ablation. These treatments provide some relief, but not a permanent cure. HCM is predominantly caused by mutations in sarcomere protein encoding genes ( 4 ). Up to 1,500 mutations in 11 genes have been reported as potential causes of HCM. The gene coding cardiac myosin-binding protein C, myosin-binding protein C3 ( MYBPC3 ), is a major pathogenic gene, accounting for 30–40% of all HCM mutations ( 5 ). The MYBPC3 gene encodes a 144-kDa protein located in in the C-zone of the A-band of the sarcomere duplex ( 6 ). MYBPC3 protein promotes the structural integrity of the sarcomere by linking myosin, titan and actin. It also regulates cardiac contractility in response to adrenergic stimulation ( 7 ). Variants in MYBPC3 mainly lead to a premature termination codon (PTC), resulting in a defective mRNA unable to express full-length MYBPC3 protein with normal titin and/or the major myosin-binding sites. Patients carrying these variants suffered impaired sarcomere function and developed an HCM phenotype. Studies have shown that correction of MYBPC3 mutations can restore sarcomere function in induced pluripotent stem cell-derived cardiomyocytes. However, in vivo studies of correcting MYBPC3 mutations to treat HCM have not been reported ( 1 ). The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has been proven to be a powerful tool for genetic engineering. To date, the CRISPR/Cas9 system have been implemented as gene therapy for many disease models, such as hearing loss, liver disease and muscle dystrophy( 8 – 10 ). However, its role in correcting MYBPC3 mutations in vivo has not been elucidated. In this study, we generated a novel rat model of HCM (referred to as “1098hom”) that carries the p.W1098x PTC mutation discovered in an HCM pedigree. By employing a CRISPR/Cas9 based homology-directed repair (HDR) strategy in diseased pups, we partially restored MYBPC3 protein expression and alleviated cardiac function. Our work demonstrates that the HDR genome editing strategy is a promising approach for treating HCM associated with Mybpc3 mutation, and that this CRISPR-based technique has great potential for treating hereditary heart diseases. Materials And Methods Human subjects The study was approved by the Ethics Review Board of Tongji Hospital and Tongji Medical College. It complied with the principles of the Declaration of Helsinki. Written informed consent was obtained from individual subjects of the HCM pedigree in this study. Animal Experiment All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health (NIH Publication No. 85 − 23, revised 1996). This study was approved by the Institutional Animal Research Committee of Tongji Medical College. The Mybpc3 -p.W1098x SD rats were generated by the Beijing Laboratory Animal Research Center of Chinese Academy of Medical Sciences. Experimental rats were housed at the animal care center of Tongji Medical College at 25°C with 12 h/12 h light/dark cycles. Adequate water and food supply were provided throughout the duration of the study. Genotypes of the rats were confirmed with validation primers ( Supplementary Information 1.1 ). Male homozygous p.W1098x mutant rats and male wild type rats were used in this study. T7e1 Assay The T7E1 detection kit was from New England Biolabs (#M0302). Guide RNA (gRNA) sequences were cloned into the px458 vector (a plasmid expressing spCas9 and gRNA backbone, bought from Addgene, RRID: Addgene_48138). Rat fibroblast cells 208F were transfected with the gRNA-px458 vector for genome editing. Genome DNA was extracted and amplified by PCR. Heteroduplex DNA was obtained by denaturation/renaturation of the following 25 µl mixture: 20 µl purified genomic PCR sample, 2.5 µl of 10X NEB buffer 2 and 2.5 µl ddH2O using the following conditions: 95ºC for 10 min, 95ºC to 85ºC (-1.0ºC/s), 85ºC for 1 min, 85ºC to 75ºC (-0.2ºC/s), 75ºC for 1 min, 75ºC to 65ºC (-0.2ºC/s), 65ºC for 1 min, 65ºC to 55ºC (-0.2ºC/s), 55ºC for 1 min, 55ºC to 45ºC (-0.2ºC/s), 45ºC for 1 min, 45 ºC to 35 ºC (-0.2ºC/s), 35ºC for 1 min, 35ºC to 25ºC (-0.2ºC/s), 25ºC for 5 min, hold at 4ºC. After denaturation/renaturation, 0.5 µl T7E1 enzyme was added to cut the heteroduplex and the mixture was incubated for 1 hour at 37ºC. 1.5% agarose gel electrophoresis was performed for undigested and T7E1 digested PCR products to analyze the mismatch frequency. Aav9 Delivery The spCas9-AAV vector was generated by cloning the spCas9 transgene from the px458 vector into the AAV2/9 backbone. The gRNA-donor-AAV vector was generated by cloning the U6-gRNA-donor sequence into the AAV2/9 backbone. Cloning details and AAV9 production were previously described ( 11 ). Rat pups were injected with AAV9 at postnatal day 3 using a 30-gauge syringe retro-orbitally ( 12 ). The wild type rats were allocated to the WT group; the 1098hom littermates were randomly allocated to receive different AAV9 treatments. Each rat received a total of 5 × 10 11 vg SpCas9-AAV9 plus 5 × 10 11 vg gRNA-donor-AAV9 (or BbsI-donor-AAV9). Immunofluorescence Rat myocardium was fixed with 4% paraformaldehyde, embedded with paraffin, and sliced into heart sections. After dewaxing and tissue autofluorescence quenching, the sections were blocked with 5% donkey serum (Biossci Biotechnology, Wuhan, China)/TBST, followed by incubation with MYBPC3 antibody, ɑ-actinin antibody and GFP antibody in 5% donkey serum/TBST at 4°C overnight. The MYBPC3 antibodies are sc-137237 from Santa Cruz Biotechnology (Dallas, Texas, USA) or bs-9868R-Cy3 from Bioss Antibodies (Beijing, China). ɑ-actinin (11313-2-AP) and GFP (66002-1-Ig) antibodies are from Proteintech (Wuhan, China). The sections were then incubated with secondary antibodies (1:200, biossci biotechnology, Wuhan, China) and for 1 hour. Nuclei were stained with DAPI (AR1176, BOSTER Biological Technology, Wuhan, China). Echocardiography Vevo 1100 Imaging System (Visual Sonics Inc., Toronto, Canada) was used for echocardiography examination. Rats were anesthetized with isoflurane (21% in oxygen). The rats were maintained under a controlled temperature of 22°C and a 12-hours-light- 12-hours-dark photoperiod. Echocardiographic parameters were obtained with a 30 MHz transducer. Hemodynamics After anesthetization, a pressure–volume catheter (Millar 1.4Fr, SPR 835, Millar Instruments, USA) was inserted into the left ventricle through the right carotid artery. After stabilization, the readout signals were analyzed using the PVAN software (Millar Instruments, Houston, Texas, USA). Sgrna Design And Plasmid Cloning SgRNAs are designed using the CCTop tool ( https://crispr.cos.uni-heidelberg.de/)(13, 14) . For in vitro studies, an sgRNA expressing backbone plasmid, BK23264, was constructed by cloning the sgRNA expressing scaffold into the backbone of pEGFP-C1 plasmid. For in vivo study, sgRNA backbone and Cas9 were cloned into two separate AAV plasmids and processed to AAV9 virus packaging. Western Blot Rat myocardium were homogenized using Protein or IP lysate buffer (Beyotime Technology, Shanghai, China) containing 1:100 protease inhibitor and 1:100 phosphatase inhibitor. Protein concentrations were determined using the Bicinchoninic Acid Assay Kit (Boster, Wuhan, China). After denaturation, tissue lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and blocked with 5% BSA in TBST. After incubation with primary antibody for 12-16h at 4℃ and incubation with secondary antibody for 2h at room temperature, the membrane was developed with ECL system (Advansta, California, USA). The antibodies are MYBPC3 (sc-137237, Santa Cruz Biotechnology, Dallas, Texas, USA) and GAPDH (AC002, ABclonal Technology, Wuhan, China). Cell Culture 208F and H9c2 cells were cultured in MEM supplemented with 10% FBS. Transfection was performed with 3µl Lipofectamine 2000 (Life Technologies, Carlsbad, CA) and 2µg of total plasmid each well of a 12-well culture plate. Rna Extraction And Qrt-pcr Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Reverse-transcription was performed using MultiScribe system (ABI, Waltham, MA) for complementary DNA (cDNA). 7900HT Fast Real‐Time PCR System was used for real‐time PCR. The primer sequences are listed in Supplementary XX. Isolation Of Genomic Dna Genome DNA were isolated from the cultured cell or myocardium using TIANamp Genomic DNA Kit (DP304, TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s protocols. Briefly, the myocardium tissue was cut into pieces and digested with Proteinase K in buffer GA overnight at 56℃. Genomic DNA was sedimented and retained in a spin column. Elute the column with buffer TE and centrifuge. Genomic DNA was thus obtained. Deep Sequencing The primer details for deep sequencing were listed in Supplementary Information 4 . Genomic DNA was extracted and was PCR amplified by locus-specific primers targeting on- or off-target sites using AlleleID version 6 (PREMIER Biosoft). A pair of 8 base unique barcodes were added to 5’ end of the forward and reverse primers of each sample. The PCR products were purified using Universal DNA Purification Kit (DP214, TIANGEN BIOTECH, Beijing, China). All barcoded amplicons in an equal molar ratio were pooled together and processed on the Illumina MiSeq platform by 2×250 paired-end sequencing. Statistics Data were expressed as mean ± standard error of the mean. Comparisons among groups were performed by unpaired Student’s t-test or one-way analysis of variance using SPSS version 20 (IBM) or GraphPad Prism version 5. Biostatistics significance was accepted at p < 0.05. Results Generation of HCM rat model and designation of gene editing strategy The p.W1098x variant in MYBPC3 was discovered in a Chinese Han HCM pedigree (Fig. 1 a). This three-generation pedigree consists of two family members who presented with HCM. The proband (II:2) is a 33-year-old man diagnosed with HCM eighteen years ago. He suffered from chest pain and syncope. Echocardiography revealed a ventricular septum thickness of 16 mm. High-throughput sequencing identified a c.3293G > A mutation causing the p.W1098x variant in the proband. Subsequent investigation revealed that the proband’s mother (I:2) is also an HCM patient carrying the same variant. The p.W1098x variant is located within the 30th exon and converts the tryptophan 1098 (Trp, W) codon to a premature termination codon (Fig. 1 b). While there have been a few sporadic reports of HCM cases with this variant ( 7 ), there is no functional evidence for this variation in ClinVar database. To explore the efficacy of genetic engineering for the treatment of a MYBPC3- associated HCM missense variant, we generated the p.W1098x rat model (Fig. 1 c). Different from human, heterozygous rats with the p.W1098x allele and the wild-type allele do not exhibit an HCM phenotype ( Table S1 ). However, homozygous rats carrying both mutant p.W1098x alleles developed cardiac hypertrophy and reduced cardiac function, reproducing the phenotype of the HCM patients (Fig. 1 d,e,f,g). Therefore, homozygous p.W1098x mutant rats (1098hom) were used as the HCM model in this study. An HDR genome editing strategy was designed to correct the stop codon back to the original TGG (Trp, W) codon. To achieve efficient genome editing, we designed 3 guide RNAs (gRNA) to target exon 30 (Fig. 2 a). These gRNAs were cloned into plasmid with a SpCas9 backbone and transfected into 208F rat fibroblast. Genomic DNA was extracted from 208F cells, and target PCR products were generated for the T7E1 assay and Sanger sequencing. T7E1 endonuclease detected mismatched double-stranded DNA and cut it into fragments. As shown in Fig. 2 b and Figure S1 , gRNA1 showed the highest editing activity and was selected for in vivo gene editing studies. As shown in Fig. 2 c, a dual AAV9 strategy were employed, with one virus carrying the gRNA1 plus donor sequence, and the other virus carrying the SpCas9 sequence. The unedited BbsI clone site was used as a negative control sequence. Five synonymous mutations were introduced into the donor sequence at the gRNA target site to avoid repeated editing (Fig. 2 d). Detailed information of gRNA targeting and donor sequence were provided in Supplemental Methods . Alleviation Of Cardiac Hypertrophy And Cardiac Function By Hdr Treatment Rat pups were divided into 3 groups: wild type (WT), 1098hom rats receiving control treatment (1098hom + Con), and 1098hom receiving HDR treatment (1098hom + HDR). AAV9 viruses were injected retro-orbitally at postnatal day 3. The rats were equally fed and raised for 6 months without any other interventions. Six months later, echocardiographic, hemodynamic, biochemical and heart histochemical analysis were performed for all the rats. As shown in Fig. 3 , control 1098hom rats had enlarged hearts and thickened ventricular wall, as assessed by heart/body weight ratio and left anterior ventricular wall thickness. HDR treatment alleviated the cardiac hypertrophy of 1098hom rats. We next assessed cardiac function by echocardiography and in vivo hemodynamics. As shown in Fig. 4 , cardiac function was impaired in control 1098hom rats. Ejection fraction (EF) was 61% in control 1098hom rats compared with 78% in the WT group. Fractional shortening (FS), max dP/dt and min dP/dt were also decreased in control 1098hom rats. HDR treatment partially restored the impaired cardiac function (Fig. 4 a,b,c,d and Table S2 ). In addition, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), biomarkers of heart failure that were elevated in control 1098hom rat hearts, were normalized by HDR editing (Fig. 4 e,f). Sirius red staining of heart section revealed increased cardiac fibrosis in control 1098hom rat, which was attenuated by HDR treatment. There was no significant difference in cardiac hypertrophy and cardiac function between 1098hom rats with and without control AAV treatment ( Table S3 ). Together, these observations revealed that HDR treatment partially improved the HCM phenotype in 1098hom rats. Hdr Treatment Partially Corrected P.w1098x Mutation And Restored Mybpc3 Protein Expression The functional and histological studies described above revealed therapeutic effects of the HDR treatment. To verify the efficacy of genome editing by HDR strategy, we performed Sanger sequencing on rat heart tissue samples, as shown in Fig. 5 a. A considerable number of alleles was edited according to the donor template, as indicated by the changes in peak pattern. Of interest to us, the p.W1098x mutation was also partially corrected by A > G conversion. To further evaluate the precise editing efficiency, we performed deep sequencing, as shown in Fig. 5 b and 5 c. Figure 5 b shows the proportion of base substitution of 5 synonymous mutations, and the p.W1098x position were about 25%. However, the precise genome editing efficiency was much lower. As shown in Fig. 5 c, though insertions/deletions were found in 20.6% of all reads, the proportion of precise HDR editing was 3.56%. Our data was consistent with previous studies, indicating the high efficiency of NHEJ and low efficiency of precise editing by HDR strategy ( 15 ). In addition, we assessed the frequency of off-target edits. We predicted the potential off-target sites of gRNA1 using the CCTop tool ( https://crispr.cos.uni-heidelberg.de/ ) and amplified the sequence of top 10 potential off-target sites. Deep sequence revealed that no obvious off-target sites were detected ( Table S4, S5, and Figure S2 ). We also performed immunostaining of the myocardium to detect MYBPC3 protein expression. As shown in Fig. 6 a and 6 b, control 1098hom rats did not express detectable levels of MYBPC3, while in rats with HDR treatment, 6.6% of the cardiac myocytes expressed MYBPC3 protein. Immunoblotting revealed MYBPC3 protein in the myocardium of HDR treated 1098hom rats (Fig. 6 c and 6 d). Together, these data indicate that the HDR strategy partially corrected the p.W1098x mutation and restored MYBPC3 protein expression. Discussion In this study, we first demonstrated that the p.W1098x variant in MYBPC3 causes HCM by establishing a 1098hom rat model. Next, we used a CRSIPR/Cas9 strategy to correct the p.W1098x variant using HDR, which partially restored MYBPC3 protein expression levels. Furthermore, HDR treatment also attenuated cardiac hypertrophy and cardiac function in 1098hom rats. Our study suggests that CRISPR/Cas9 may be a potential treatment for HCM. This is the first study in vivo to reveal the therapeutic potential of HDR in treating inherited heart disease. The HDR pathway is an ideal way of precise gene engineering. With efficient HDR, it is theoretically possible to program the genome with any desired change. Prior studies have shown that HDR has great potential for the treatment of genetic diseases such as hereditary tyrosinemia ( 16 ), OTC deficiency( 9 ), and inherited blindness( 17 ). For myopathy treatment, nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA template corrected 5.4% of the dystrophin gene and improved animal strength in the mdx mouse model of Duchenne’s muscular dystrophy( 18 ). Collectively, these studies demonstrated the potential of an HDR strategy for disease treatment. The biggest obstacle to the application of HDR is its low efficiency in postmitotic cell types. Since most somatic cells are nondividing, the efficiency of HDR repair is naturally low. Adult mammalian heart, which is thought to be postmitotic, has only 1% of its cardiomyocyte renewed at the age of 20 and 0.3% at the age of 70, as measured by the integration tests of 14 C ( 19 ). The mammalian heart, on the other hand, retains its ability to regenerate for some time after birth. Mouse cardiomyocytes exit the cell cycle after about a week of birth, while human heart cardiomyocyte regeneration continues for years( 20 ). Retention of cardiomyocyte regeneration provides a time window for therapeutic gene editing of postnatal hearts. In this study, the CRISPR/Cas9 HDR strategy was used to restore the expression of MYBPC3 protein by correcting 3.6% of the p.W1098x variant as evaluated by deep sequence. It is interesting that such a small portion of corrected cardiomyocytes would improve cardiac function, which is consistent with previous studies. For instance, expression of 1.1 ± 1.1% and 3.2 ± 2.4% of dystrophin by cardiomyocytes improved cardiac symptoms in a mouse model of muscular dystrophy( 21 ). Low levels of dystrophin (3%-15%) are sufficient to delay the onset of cardiomyopathy ( 22 ). Disruption of about 2–6% of mutant alleles restores heart morphology and function in PRKAG2 transgenic mice with glycogen-storage cardiomyopathy ( 23 ). These findings suggest that relatively low editing efficiencies are sufficient to produce therapeutic effects. The major cause of HCM are variants in genes encoding sarcomere components, such as the MYBPC3 and MYH7 genes ( 24 ). Individuals carrying homozygous, double or triple mutations have more severe disease progression ( 25 ). At present, there is no effective treatment for HCM caused by truncating variants in MYBPC3 . Our study has established an HCM rat model of the homozygous p.W1098x variants in Mybpc3 . The heart function of these 1098hom rats was restored by CRISPR/Cas9 genome editing, indicating that CRISPR/Cas9 is a good tool for the treatment of HCM. A limitation of this study is that the inability of gRNA to distinguish between the mutant and wild-type alleles. In patients who carry a pathogenic sequence that is heterozygotes, it is important to target the mutant allele while maintaining the integrity of the wild-type allele. This might be more like the real world, where disease-causing allele could not be specifically targeted by current Cas9 variants. For homozygous mutations, HDR strategy would be a better choice. In conclusion, we applied the CRSIPR/Cas9 HDR strategy to correct a truncating Mybpc3 variant, restored MYBPC3 protein expression, and improve cardiac function of HCM rats. Our work demonstrates that HDR strategy is a promising therapy for the treatment of HCM associated with MYBPC3 mutations, and that CRISPR technology has great potential for treating hereditary heart diseases. Declarations Data Availability The datasets analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study. Author Contributions D. W. Wang, L. Ni and J. Nie designed the study. J. Nie and Y. Han conducted the experiments, analyzed data and completed the manuscript. Z. Jin, W. Hang and H. Shu conducted the animal experiments. Z. Wen performed the echocardiography examination of the animals. Sources of Funding This work was supported by National Natural Science Foundation of China [No. 82100401, 82070354, 81470519, 81630010] and Huazhong University of Science and Technology Academic Frontier Youth Team (No. 2019QYTD08). Ethical Approval The study was approved by the Ethics Review Board of Tongji Hospital and Tongji Medical College. It complied with the principles of the Declaration of Helsinki. Written informed consent was obtained from individual subjects of the HCM pedigree in this study. All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996). This study was approved by the Institutional Animal Research Committee of Tongji Medical College. Disclosures The authors claimed no conflict of interest. References Maron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I, Maron MS. 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Supplementary Files SupplementalMaterial.docx Supplemental Material Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 06 Dec, 2022 Review # 3 received at journal 05 Dec, 2022 Review # 2 received at journal 26 Nov, 2022 Review # 1 received at journal 24 Nov, 2022 Reviewer # 3 agreed at journal 22 Nov, 2022 Reviewer # 2 agreed at journal 15 Nov, 2022 Reviewer # 1 agreed at journal 12 Nov, 2022 Reviewers invited by journal 12 Nov, 2022 Editor assigned by journal 11 Nov, 2022 Submission checks completed at journal 11 Nov, 2022 First submitted to journal 24 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2199328","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":151614925,"identity":"c355d883-1046-453e-a83b-da889fdc23ed","order_by":0,"name":"Jiali Nie","email":"","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Nie","suffix":""},{"id":151614926,"identity":"78cf5a32-940d-4909-8ba3-d51d03aa8d19","order_by":1,"name":"Yu Han","email":"","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Han","suffix":""},{"id":151614927,"identity":"a66010c7-da45-4ff3-95ee-71b3ecaca731","order_by":2,"name":"Zhiyuan Jin","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital Xiamen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Jin","suffix":""},{"id":151614928,"identity":"00a87150-9e75-41c0-bdd7-3f1918ebc314","order_by":3,"name":"Weijian Hang","email":"","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weijian","middleName":"","lastName":"Hang","suffix":""},{"id":151614929,"identity":"24e93f4e-b8cb-4ff8-b814-25fddea4d8b8","order_by":4,"name":"Hongyang Shu","email":"","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongyang","middleName":"","lastName":"Shu","suffix":""},{"id":151614930,"identity":"7a3bde1b-ab87-4fa0-8f27-f98a2be5b6b2","order_by":5,"name":"Zheng Wen","email":"","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Wen","suffix":""},{"id":151614931,"identity":"8b492030-8210-4dcb-9d2d-339f74f740f9","order_by":6,"name":"Li Ni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACZuaGAx8Y2BKATBBmYGwgrIWx4eCMBKAWNqK1ANUw8yQwgLQwEKfFnJ2x8bDtD748BvmGp5t5GGxkNxxgfvYAnxbLZsaGwzkJbMVAW9Ju8zCkGW84wGZugE+LwWGIlsQGiJbDiRsO8LBJENRigdDyn0gtDAgtB4jTcrAnjS2xjS0h7eYcg2TjmYfZzPBrOX/48IcfNscS+5nPpN14U2En23e8+RleLVBwDBhgwNhhAAUVMxHqgaAGiNkPEKd2FIyCUTAKRhwAABDCSPqnYjaeAAAAAElFTkSuQmCC","orcid":"","institution":"Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Ni","suffix":""},{"id":151614932,"identity":"57721c34-8eb5-413f-a797-cfcf12956c69","order_by":7,"name":"Dao Wen Wang","email":"","orcid":"https://orcid.org/0000-0002-9774-3980","institution":"Tongji Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dao","middleName":"Wen","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-10-24 15:41:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2199328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2199328/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29090946,"identity":"c38cc080-7d17-4058-b34a-de9c0129c38a","added_by":"auto","created_at":"2022-11-15 15:52:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7249052,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of the 1098hom rat model of HCM. \u003cstrong\u003e(a)\u003c/strong\u003e Pedigree of human HCM patients carrying the p.W1098x variant. \u003cstrong\u003e(b) \u003c/strong\u003eThe G to A mutation causing the p.W1098x variant leading to a Tryptophan codon converting to a premature stop codon. \u003cstrong\u003e(c) \u003c/strong\u003eSanger sequencing validating this variant in homozygous 1098hom rats. \u003cstrong\u003e(d) \u003c/strong\u003eNo detectable MYBPC3 protein in 1098hom rats. \u003cstrong\u003e(e) \u003c/strong\u003eCardiac M-mode\u003cstrong\u003e \u003c/strong\u003eechocardiography tracings of a 1098hom rats and wild-type littermate at 6-month of age. \u003cstrong\u003e(f) \u003c/strong\u003eEjection fraction was reduced in 1098hom rats. \u003cstrong\u003e(g) \u003c/strong\u003e1098hom rats had thickened ventricular wall. * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Data were expressed as mean ± standard error of the mean.\u003c/p\u003e","description":"","filename":"figure101.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/c200132302ae331846609044.jpg"},{"id":29090943,"identity":"c10c1e98-91c1-4812-b2c0-24e4f06f100e","added_by":"auto","created_at":"2022-11-15 15:52:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4237543,"visible":true,"origin":"","legend":"\u003cp\u003eDesignation of CRISPR/Cas9 HDR strategy to correct the p.W1098x mutation. \u003cstrong\u003e(a)\u003c/strong\u003eDesign of the single guide RNA (sgRNA) using the CCTop tool (\u003ca href=\"https://crispr.cos.uni-heidelberg.de/\"\u003ehttps://crispr.cos.uni-heidelberg.de/\u003c/a\u003e). The top 3 gRNAs with highest predicted on-target activity and low off-target probability are shown. \u003cstrong\u003eb) \u003c/strong\u003eEfficacy screening of the 3 gRNAs. T7E1 recognizes and cleaves the heteroduplex DNA caused by CRISPR/Cas9-mediated genome editing. The arrowheads indicate cleaved bands of T7E1. gRNA1 showed the highest editing efficacy. \u003cstrong\u003e(c)\u003c/strong\u003e Schematic of the dual AAV9 constructs. The Control (Con) treatment included the combination of BbsI-donor-AAV9 and spCas9-AAV9, whereas the HDR treatment included the combination of gRNA1-donor-AAV9 and spCas9-AAV9. \u003cstrong\u003e(d)\u003c/strong\u003e Comparison between the p.W1098x sequence and the donor sequence. The blue letters indicated the 5 synonymous mutations and the target W1098 codon.\u003c/p\u003e","description":"","filename":"figure201.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/b6b616cc08c44600b989e80f.jpg"},{"id":29092049,"identity":"ef11445c-d0a1-4155-aa7e-066a3d731b06","added_by":"auto","created_at":"2022-11-15 16:00:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5299120,"visible":true,"origin":"","legend":"\u003cp\u003eHDR treatment alleviated cardiac hypertrophy in 1098hom rats. \u003cstrong\u003e(a) \u003c/strong\u003eTime schedule of AAV9 injection and subsequent analysis. Images of whole mount hearts \u003cstrong\u003e(b)\u003c/strong\u003e, H\u0026amp;E staining \u003cstrong\u003e(c), \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eM-modeechocardiography recordings \u003cstrong\u003e(d) \u003c/strong\u003eshowing cardiac hypertrophy in 1098hom + Con rats; whereas HDR treatment alleviates cardiac hypertrophy in 1098hom rats.\u003cstrong\u003e (e)\u003c/strong\u003eThe heart weight/body weight ratio (HW/BW) was increased in 1098hom + Con rats. HDR treatment reduced the heart weight-to-body weight (HW/BW) ratio down. \u003cstrong\u003e(f) \u003c/strong\u003eLeft ventricular anterior wall thickness at diastole (LVAW;d) was increased in 1098hom + Con rats, and was alleviated by HDR treatment. WT group: n=8; 1098hom + Con: n=13; 1098hom + HDR: n=6; *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Data were expressed as mean ± standard error of the mean.\u003c/p\u003e","description":"","filename":"figure301.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/c7f7d0b9902e62656dfa9560.jpg"},{"id":29090945,"identity":"12d68bca-3341-4728-8202-1c138f1712ea","added_by":"auto","created_at":"2022-11-15 15:52:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5741775,"visible":true,"origin":"","legend":"\u003cp\u003eHDR treatment improves cardiac function in 1098hom rats.\u003cstrong\u003e \u003c/strong\u003eHDR treatment improved \u003cstrong\u003e(a) \u003c/strong\u003eejection fraction (EF), \u003cstrong\u003e(b) \u003c/strong\u003efractional shortening (FS),\u003cstrong\u003e (c) \u003c/strong\u003emax dp/dt,\u003cstrong\u003e \u003c/strong\u003eand (\u003cstrong\u003ed\u003c/strong\u003e) min dp/dt in 1098hom rats. Biomarkers ANP and BNP were up-regulated in 1098hom + Con rats, and were down-regulated by HDR treatment. Sirius red staining \u003cstrong\u003e(g, h)\u003c/strong\u003erevealed increased fibrosis in 1098hom + Con rats, whereas HDR treatment alleviated cardiac fibrosis. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Data were expressed as mean ± standard error of the mean.\u003c/p\u003e","description":"","filename":"figure401.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/015066c55b035e485deac3bc.jpg"},{"id":29090947,"identity":"cdbd2758-d3df-4162-a9a6-22e55a9e926b","added_by":"auto","created_at":"2022-11-15 15:52:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6959610,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of\u003cstrong\u003e \u003c/strong\u003eHDR editing efficiency. \u003cstrong\u003e(a)\u003c/strong\u003e Sanger sequencing of 1098hom + HDR rat hearts. The upper panel shows the p.W1098x sequence and the donor sequence. The lower panel shows the heteroduplex by HDR editing at the target sites. \u003cstrong\u003e(b) \u003c/strong\u003eDeep sequencing revealed the point substitution frequencies across the target sites. \u003cstrong\u003e(c) \u003c/strong\u003eFrequency of insertion/deletion and precise HDR editing. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Data were expressed as mean ± standard error of the mean.\u003c/p\u003e","description":"","filename":"figure501.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/c4d8e64b956f485aa9c6f978.jpg"},{"id":29092048,"identity":"d36f51a4-f4fa-4ce9-8988-773c5b4f61de","added_by":"auto","created_at":"2022-11-15 16:00:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11399516,"visible":true,"origin":"","legend":"\u003cp\u003eHDR treatment partially restores MYBPC3 protein expression. \u003cstrong\u003e(a) \u003c/strong\u003eImmunofluorescence of heart sections. Green, MYBPC3 protein; blue, cell nucleus. Scale bars, 50µm. \u003cstrong\u003e(b) \u003c/strong\u003eQuantification of percentage of MYBPC3 positive cells by immunofluorescence. Control 1098hom rats did not express detectable levels of MYBPC3, while in rats with HDR treatment, 6.6% of the cardiac myocytes expressed MYBPC3 protein. \u003cstrong\u003e(c, d)\u003c/strong\u003e Immuno blotting and semi quantification of control 1098hom rats and 1098hom rats with HDR treatment. HDR treatment restored MYBPC3 protein expression by 2.12%. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Data were expressed as mean ± standard error of the mean.\u003c/p\u003e","description":"","filename":"figure601.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/de0f16ba9e6cdd1c550eee2c.jpg"},{"id":29092051,"identity":"ccccdb8c-9053-487a-a81a-9cf1e51e3bdf","added_by":"auto","created_at":"2022-11-15 16:00:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1019058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/ccce5549-fd86-40c4-a1f7-edbd41ddde7a.pdf"},{"id":29090944,"identity":"bb120d03-a61a-414c-afc7-803c6ac4237e","added_by":"auto","created_at":"2022-11-15 15:52:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":698042,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2199328/v1/c7487ac755349aba2441efd6.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Homology-Directed Repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertrophic cardiomyopathy (HCM) is a hereditary disease with a global prevalence of approximately 1 in 500 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is characterized by left ventricular hypertrophy, myocardial hypercontractility, reduced compliance, myofibrillar disarray, and fibrosis. Symptoms in patients with HCM range from mild chest pain, syncope, and dyspnea to severe sudden cardiac death (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). HCM is a major cause of sudden cardiac death in young adults, especially young athletes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Current therapy for HCM includes pharmacological intervention represented by \u0026#120573;-blockers or non-dihydropyridine calcium-channel blockers, surgical septal myectomy, alcohol septal ablation, and radiofrequency ablation. These treatments provide some relief, but not a permanent cure.\u003c/p\u003e \u003cp\u003eHCM is predominantly caused by mutations in sarcomere protein encoding genes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Up to 1,500 mutations in 11 genes have been reported as potential causes of HCM. The gene coding cardiac myosin-binding protein C, myosin-binding protein C3 (\u003cem\u003eMYBPC3\u003c/em\u003e), is a major pathogenic gene, accounting for 30\u0026ndash;40% of all HCM mutations (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The \u003cem\u003eMYBPC3\u003c/em\u003e gene encodes a 144-kDa protein located in in the C-zone of the A-band of the sarcomere duplex (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). MYBPC3 protein promotes the structural integrity of the sarcomere by linking myosin, titan and actin. It also regulates cardiac contractility in response to adrenergic stimulation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Variants in \u003cem\u003eMYBPC3\u003c/em\u003e mainly lead to a premature termination codon (PTC), resulting in a defective mRNA unable to express full-length MYBPC3 protein with normal titin and/or the major myosin-binding sites. Patients carrying these variants suffered impaired sarcomere function and developed an HCM phenotype. Studies have shown that correction of \u003cem\u003eMYBPC3\u003c/em\u003e mutations can restore sarcomere function in induced pluripotent stem cell-derived cardiomyocytes. However, \u003cem\u003ein vivo\u003c/em\u003e studies of correcting \u003cem\u003eMYBPC3\u003c/em\u003e mutations to treat HCM have not been reported (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has been proven to be a powerful tool for genetic engineering. To date, the CRISPR/Cas9 system have been implemented as gene therapy for many disease models, such as hearing loss, liver disease and muscle dystrophy(\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, its role in correcting \u003cem\u003eMYBPC3\u003c/em\u003e mutations \u003cem\u003ein vivo\u003c/em\u003e has not been elucidated. In this study, we generated a novel rat model of HCM (referred to as \u0026ldquo;1098hom\u0026rdquo;) that carries the p.W1098x PTC mutation discovered in an HCM pedigree. By employing a CRISPR/Cas9 based homology-directed repair (HDR) strategy in diseased pups, we partially restored MYBPC3 protein expression and alleviated cardiac function. Our work demonstrates that the HDR genome editing strategy is a promising approach for treating HCM associated with \u003cem\u003eMybpc3\u003c/em\u003e mutation, and that this CRISPR-based technique has great potential for treating hereditary heart diseases.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman subjects\u003c/h2\u003e \u003cp\u003e The study was approved by the Ethics Review Board of Tongji Hospital and Tongji Medical College. It complied with the principles of the Declaration of Helsinki. Written informed consent was obtained from individual subjects of the HCM pedigree in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal Experiment\u003c/h3\u003e\n\u003cp\u003e All animal experiments complied with the \u0026ldquo;Guide for the Care and Use of Laboratory Animals\u0026rdquo; published by the United States National Institutes of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). This study was approved by the Institutional Animal Research Committee of Tongji Medical College. The \u003cem\u003eMybpc3\u003c/em\u003e-p.W1098x SD rats were generated by the Beijing Laboratory Animal Research Center of Chinese Academy of Medical Sciences. Experimental rats were housed at the animal care center of Tongji Medical College at 25\u0026deg;C with 12 h/12 h light/dark cycles. Adequate water and food supply were provided throughout the duration of the study. Genotypes of the rats were confirmed with validation primers (\u003cb\u003eSupplementary Information 1.1\u003c/b\u003e). Male homozygous p.W1098x mutant rats and male wild type rats were used in this study.\u003c/p\u003e\n\u003ch3\u003eT7e1 Assay\u003c/h3\u003e\n\u003cp\u003eThe T7E1 detection kit was from New England Biolabs (#M0302). Guide RNA (gRNA) sequences were cloned into the px458 vector (a plasmid expressing spCas9 and gRNA backbone, bought from Addgene, RRID: Addgene_48138). Rat fibroblast cells 208F were transfected with the gRNA-px458 vector for genome editing. Genome DNA was extracted and amplified by PCR. Heteroduplex DNA was obtained by denaturation/renaturation of the following 25 \u0026micro;l mixture: 20 \u0026micro;l purified genomic PCR sample, 2.5 \u0026micro;l of 10X NEB buffer 2 and 2.5 \u0026micro;l ddH2O using the following conditions: 95\u0026ordm;C for 10 min, 95\u0026ordm;C to 85\u0026ordm;C (-1.0\u0026ordm;C/s), 85\u0026ordm;C for 1 min, 85\u0026ordm;C to 75\u0026ordm;C (-0.2\u0026ordm;C/s), 75\u0026ordm;C for 1 min, 75\u0026ordm;C to 65\u0026ordm;C (-0.2\u0026ordm;C/s), 65\u0026ordm;C for 1 min, 65\u0026ordm;C to 55\u0026ordm;C (-0.2\u0026ordm;C/s), 55\u0026ordm;C for 1 min, 55\u0026ordm;C to 45\u0026ordm;C (-0.2\u0026ordm;C/s), 45\u0026ordm;C for 1 min, 45 \u0026ordm;C to 35 \u0026ordm;C (-0.2\u0026ordm;C/s), 35\u0026ordm;C for 1 min, 35\u0026ordm;C to 25\u0026ordm;C (-0.2\u0026ordm;C/s), 25\u0026ordm;C for 5 min, hold at 4\u0026ordm;C. After denaturation/renaturation, 0.5 \u0026micro;l T7E1 enzyme was added to cut the heteroduplex and the mixture was incubated for 1 hour at 37\u0026ordm;C. 1.5% agarose gel electrophoresis was performed for undigested and T7E1 digested PCR products to analyze the mismatch frequency.\u003c/p\u003e\n\u003ch3\u003eAav9 Delivery\u003c/h3\u003e\n\u003cp\u003eThe spCas9-AAV vector was generated by cloning the spCas9 transgene from the px458 vector into the AAV2/9 backbone. The gRNA-donor-AAV vector was generated by cloning the U6-gRNA-donor sequence into the AAV2/9 backbone. Cloning details and AAV9 production were previously described (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Rat pups were injected with AAV9 at postnatal day 3 using a 30-gauge syringe retro-orbitally (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The wild type rats were allocated to the WT group; the 1098hom littermates were randomly allocated to receive different AAV9 treatments. Each rat received a total of 5 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e vg SpCas9-AAV9 plus 5 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e vg gRNA-donor-AAV9 (or BbsI-donor-AAV9).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eRat myocardium was fixed with 4% paraformaldehyde, embedded with paraffin, and sliced into heart sections. After dewaxing and tissue autofluorescence quenching, the sections were blocked with 5% donkey serum (Biossci Biotechnology, Wuhan, China)/TBST, followed by incubation with MYBPC3 antibody, ɑ-actinin antibody and GFP antibody in 5% donkey serum/TBST at 4\u0026deg;C overnight. The MYBPC3 antibodies are sc-137237 from Santa Cruz Biotechnology (Dallas, Texas, USA) or bs-9868R-Cy3 from Bioss Antibodies (Beijing, China). ɑ-actinin (11313-2-AP) and GFP (66002-1-Ig) antibodies are from Proteintech (Wuhan, China). The sections were then incubated with secondary antibodies (1:200, biossci biotechnology, Wuhan, China) and for 1 hour. Nuclei were stained with DAPI (AR1176, BOSTER Biological Technology, Wuhan, China).\u003c/p\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eVevo 1100 Imaging System (Visual Sonics Inc., Toronto, Canada) was used for echocardiography examination. Rats were anesthetized with isoflurane (21% in oxygen). The rats were maintained under a controlled temperature of 22\u0026deg;C and a 12-hours-light- 12-hours-dark photoperiod. Echocardiographic parameters were obtained with a 30 MHz transducer.\u003c/p\u003e\n\u003ch3\u003eHemodynamics\u003c/h3\u003e\n\u003cp\u003eAfter anesthetization, a pressure\u0026ndash;volume catheter (Millar 1.4Fr, SPR 835, Millar Instruments, USA) was inserted into the left ventricle through the right carotid artery. After stabilization, the readout signals were analyzed using the PVAN software (Millar Instruments, Houston, Texas, USA).\u003c/p\u003e\n\u003ch3\u003eSgrna Design And Plasmid Cloning\u003c/h3\u003e\n\u003cp\u003eSgRNAs are designed using the CCTop tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://crispr.cos.uni-heidelberg.de/)(13, 14)\u003c/span\u003e\u003cspan address=\"https://crispr.cos.uni-heidelberg.de/)(13, 14)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. For \u003cem\u003ein vitro\u003c/em\u003e studies, an sgRNA expressing backbone plasmid, BK23264, was constructed by cloning the sgRNA expressing scaffold into the backbone of pEGFP-C1 plasmid. For in vivo study, sgRNA backbone and Cas9 were cloned into two separate AAV plasmids and processed to AAV9 virus packaging.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eRat myocardium were homogenized using Protein or IP lysate buffer (Beyotime Technology, Shanghai, China) containing 1:100 protease inhibitor and 1:100 phosphatase inhibitor. Protein concentrations were determined using the Bicinchoninic Acid Assay Kit (Boster, Wuhan, China). After denaturation, tissue lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and blocked with 5% BSA in TBST. After incubation with primary antibody for 12-16h at 4℃ and incubation with secondary antibody for 2h at room temperature, the membrane was developed with ECL system (Advansta, California, USA). The antibodies are MYBPC3 (sc-137237, Santa Cruz Biotechnology, Dallas, Texas, USA) and GAPDH (AC002, ABclonal Technology, Wuhan, China).\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003e208F and H9c2 cells were cultured in MEM supplemented with 10% FBS. Transfection was performed with 3\u0026micro;l Lipofectamine 2000 (Life Technologies, Carlsbad, CA) and 2\u0026micro;g of total plasmid each well of a 12-well culture plate.\u003c/p\u003e\n\u003ch3\u003eRna Extraction And Qrt-pcr\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Reverse-transcription was performed using MultiScribe system (ABI, Waltham, MA) for complementary DNA (cDNA). 7900HT Fast Real‐Time PCR System was used for real‐time PCR. The primer sequences are listed in Supplementary XX.\u003c/p\u003e\n\u003ch3\u003eIsolation Of Genomic Dna\u003c/h3\u003e\n\u003cp\u003eGenome DNA were isolated from the cultured cell or myocardium using TIANamp Genomic DNA Kit (DP304, TIANGEN BIOTECH, Beijing, China) according to the manufacturer\u0026rsquo;s protocols. Briefly, the myocardium tissue was cut into pieces and digested with Proteinase K in buffer GA overnight at 56℃. Genomic DNA was sedimented and retained in a spin column. Elute the column with buffer TE and centrifuge. Genomic DNA was thus obtained.\u003c/p\u003e\n\u003ch3\u003eDeep Sequencing\u003c/h3\u003e\n\u003cp\u003eThe primer details for deep sequencing were listed in \u003cb\u003eSupplementary Information 4\u003c/b\u003e. Genomic DNA was extracted and was PCR amplified by locus-specific primers targeting on- or off-target sites using AlleleID version 6 (PREMIER Biosoft). A pair of 8 base unique barcodes were added to 5\u0026rsquo; end of the forward and reverse primers of each sample. The PCR products were purified using Universal DNA Purification Kit (DP214, TIANGEN BIOTECH, Beijing, China). All barcoded amplicons in an equal molar ratio were pooled together and processed on the Illumina MiSeq platform by 2\u0026times;250 paired-end sequencing.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Comparisons among groups were performed by unpaired Student\u0026rsquo;s t-test or one-way analysis of variance using SPSS version 20 (IBM) or GraphPad Prism version 5. Biostatistics significance was accepted at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of HCM rat model and designation of gene editing strategy\u003c/h2\u003e \u003cp\u003eThe p.W1098x variant in \u003cem\u003eMYBPC3\u003c/em\u003e was discovered in a Chinese Han HCM pedigree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This three-generation pedigree consists of two family members who presented with HCM. The proband (II:2) is a 33-year-old man diagnosed with HCM eighteen years ago. He suffered from chest pain and syncope. Echocardiography revealed a ventricular septum thickness of 16 mm. High-throughput sequencing identified a c.3293G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation causing the p.W1098x variant in the proband. Subsequent investigation revealed that the proband\u0026rsquo;s mother (I:2) is also an HCM patient carrying the same variant. The p.W1098x variant is located within the 30th exon and converts the tryptophan 1098 (Trp, W) codon to a premature termination codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). While there have been a few sporadic reports of HCM cases with this variant (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), there is no functional evidence for this variation in ClinVar database.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the efficacy of genetic engineering for the treatment of a \u003cem\u003eMYBPC3-\u003c/em\u003eassociated HCM missense variant, we generated the p.W1098x rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Different from human, heterozygous rats with the p.W1098x allele and the wild-type allele do not exhibit an HCM phenotype (\u003cb\u003eTable S1\u003c/b\u003e). However, homozygous rats carrying both mutant p.W1098x alleles developed cardiac hypertrophy and reduced cardiac function, reproducing the phenotype of the HCM patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e,f,g). Therefore, homozygous p.W1098x mutant rats (1098hom) were used as the HCM model in this study.\u003c/p\u003e \u003cp\u003eAn HDR genome editing strategy was designed to correct the stop codon back to the original TGG (Trp, W) codon. To achieve efficient genome editing, we designed 3 guide RNAs (gRNA) to target exon 30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). These gRNAs were cloned into plasmid with a SpCas9 backbone and transfected into 208F rat fibroblast. Genomic DNA was extracted from 208F cells, and target PCR products were generated for the T7E1 assay and Sanger sequencing. T7E1 endonuclease detected mismatched double-stranded DNA and cut it into fragments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u003cb\u003eand Figure S1\u003c/b\u003e, gRNA1 showed the highest editing activity and was selected for \u003cem\u003ein vivo\u003c/em\u003e gene editing studies. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, a dual AAV9 strategy were employed, with one virus carrying the gRNA1 plus donor sequence, and the other virus carrying the SpCas9 sequence. The unedited BbsI clone site was used as a negative control sequence. Five synonymous mutations were introduced into the donor sequence at the gRNA target site to avoid repeated editing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Detailed information of gRNA targeting and donor sequence were provided in \u003cb\u003eSupplemental Methods\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlleviation Of Cardiac Hypertrophy And Cardiac Function By Hdr Treatment\u003c/h3\u003e\n\u003cp\u003eRat pups were divided into 3 groups: wild type (WT), 1098hom rats receiving control treatment (1098hom\u0026thinsp;+\u0026thinsp;Con), and 1098hom receiving HDR treatment (1098hom\u0026thinsp;+\u0026thinsp;HDR). AAV9 viruses were injected retro-orbitally at postnatal day 3. The rats were equally fed and raised for 6 months without any other interventions. Six months later, echocardiographic, hemodynamic, biochemical and heart histochemical analysis were performed for all the rats. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, control 1098hom rats had enlarged hearts and thickened ventricular wall, as assessed by heart/body weight ratio and left anterior ventricular wall thickness. HDR treatment alleviated the cardiac hypertrophy of 1098hom rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next assessed cardiac function by echocardiography and \u003cem\u003ein vivo\u003c/em\u003e hemodynamics. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, cardiac function was impaired in control 1098hom rats. Ejection fraction (EF) was 61% in control 1098hom rats compared with 78% in the WT group. Fractional shortening (FS), max dP/dt and min dP/dt were also decreased in control 1098hom rats. HDR treatment partially restored the impaired cardiac function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b,c,d \u003cb\u003eand Table S2\u003c/b\u003e). In addition, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), biomarkers of heart failure that were elevated in control 1098hom rat hearts, were normalized by HDR editing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f). Sirius red staining of heart section revealed increased cardiac fibrosis in control 1098hom rat, which was attenuated by HDR treatment. There was no significant difference in cardiac hypertrophy and cardiac function between 1098hom rats with and without control AAV treatment (\u003cb\u003eTable S3\u003c/b\u003e). Together, these observations revealed that HDR treatment partially improved the HCM phenotype in 1098hom rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHdr Treatment Partially Corrected P.w1098x Mutation And Restored Mybpc3 Protein Expression\u003c/h3\u003e\n\u003cp\u003eThe functional and histological studies described above revealed therapeutic effects of the HDR treatment. To verify the efficacy of genome editing by HDR strategy, we performed Sanger sequencing on rat heart tissue samples, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. A considerable number of alleles was edited according to the donor template, as indicated by the changes in peak pattern. Of interest to us, the p.W1098x mutation was also partially corrected by A\u0026thinsp;\u0026gt;\u0026thinsp;G conversion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the precise editing efficiency, we performed deep sequencing, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb shows the proportion of base substitution of 5 synonymous mutations, and the p.W1098x position were about 25%. However, the precise genome editing efficiency was much lower. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, though insertions/deletions were found in 20.6% of all reads, the proportion of precise HDR editing was 3.56%. Our data was consistent with previous studies, indicating the high efficiency of NHEJ and low efficiency of precise editing by HDR strategy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, we assessed the frequency of off-target edits. We predicted the potential off-target sites of gRNA1 using the CCTop tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://crispr.cos.uni-heidelberg.de/\u003c/span\u003e\u003cspan address=\"https://crispr.cos.uni-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and amplified the sequence of top 10 potential off-target sites. Deep sequence revealed that no obvious off-target sites were detected (\u003cb\u003eTable S4, S5, and Figure S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWe also performed immunostaining of the myocardium to detect MYBPC3 protein expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, control 1098hom rats did not express detectable levels of MYBPC3, while in rats with HDR treatment, 6.6% of the cardiac myocytes expressed MYBPC3 protein. Immunoblotting revealed MYBPC3 protein in the myocardium of HDR treated 1098hom rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Together, these data indicate that the HDR strategy partially corrected the p.W1098x mutation and restored MYBPC3 protein expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we first demonstrated that the p.W1098x variant in \u003cem\u003eMYBPC3\u003c/em\u003e causes HCM by establishing a 1098hom rat model. Next, we used a CRSIPR/Cas9 strategy to correct the p.W1098x variant using HDR, which partially restored MYBPC3 protein expression levels. Furthermore, HDR treatment also attenuated cardiac hypertrophy and cardiac function in 1098hom rats. Our study suggests that CRISPR/Cas9 may be a potential treatment for HCM. This is the first study \u003cem\u003ein vivo\u003c/em\u003e to reveal the therapeutic potential of HDR in treating inherited heart disease.\u003c/p\u003e \u003cp\u003eThe HDR pathway is an ideal way of precise gene engineering. With efficient HDR, it is theoretically possible to program the genome with any desired change. Prior studies have shown that HDR has great potential for the treatment of genetic diseases such as hereditary tyrosinemia (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), OTC deficiency(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), and inherited blindness(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). For myopathy treatment, nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA template corrected 5.4% of the dystrophin gene and improved animal strength in the \u003cem\u003emdx\u003c/em\u003e mouse model of Duchenne\u0026rsquo;s muscular dystrophy(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Collectively, these studies demonstrated the potential of an HDR strategy for disease treatment.\u003c/p\u003e \u003cp\u003eThe biggest obstacle to the application of HDR is its low efficiency in postmitotic cell types. Since most somatic cells are nondividing, the efficiency of HDR repair is naturally low. Adult mammalian heart, which is thought to be postmitotic, has only 1% of its cardiomyocyte renewed at the age of 20 and 0.3% at the age of 70, as measured by the integration tests of \u003csup\u003e14\u003c/sup\u003eC (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The mammalian heart, on the other hand, retains its ability to regenerate for some time after birth. Mouse cardiomyocytes exit the cell cycle after about a week of birth, while human heart cardiomyocyte regeneration continues for years(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Retention of cardiomyocyte regeneration provides a time window for therapeutic gene editing of postnatal hearts.\u003c/p\u003e \u003cp\u003eIn this study, the CRISPR/Cas9 HDR strategy was used to restore the expression of MYBPC3 protein by correcting 3.6% of the p.W1098x variant as evaluated by deep sequence. It is interesting that such a small portion of corrected cardiomyocytes would improve cardiac function, which is consistent with previous studies. For instance, expression of 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1% and 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% of dystrophin by cardiomyocytes improved cardiac symptoms in a mouse model of muscular dystrophy(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Low levels of dystrophin (3%-15%) are sufficient to delay the onset of cardiomyopathy (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Disruption of about 2\u0026ndash;6% of mutant alleles restores heart morphology and function in PRKAG2 transgenic mice with glycogen-storage cardiomyopathy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). These findings suggest that relatively low editing efficiencies are sufficient to produce therapeutic effects.\u003c/p\u003e \u003cp\u003eThe major cause of HCM are variants in genes encoding sarcomere components, such as the \u003cem\u003eMYBPC3\u003c/em\u003e and \u003cem\u003eMYH7\u003c/em\u003e genes (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Individuals carrying homozygous, double or triple mutations have more severe disease progression (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). At present, there is no effective treatment for HCM caused by truncating variants in \u003cem\u003eMYBPC3\u003c/em\u003e. Our study has established an HCM rat model of the homozygous p.W1098x variants in \u003cem\u003eMybpc3\u003c/em\u003e. The heart function of these 1098hom rats was restored by CRISPR/Cas9 genome editing, indicating that CRISPR/Cas9 is a good tool for the treatment of HCM. A limitation of this study is that the inability of gRNA to distinguish between the mutant and wild-type alleles. In patients who carry a pathogenic sequence that is heterozygotes, it is important to target the mutant allele while maintaining the integrity of the wild-type allele. This might be more like the real world, where disease-causing allele could not be specifically targeted by current Cas9 variants. For homozygous mutations, HDR strategy would be a better choice.\u003c/p\u003e \u003cp\u003eIn conclusion, we applied the CRSIPR/Cas9 HDR strategy to correct a truncating \u003cem\u003eMybpc3\u003c/em\u003e variant, restored MYBPC3 protein expression, and improve cardiac function of HCM rats. Our work demonstrates that HDR strategy is a promising therapy for the treatment of HCM associated with \u003cem\u003eMYBPC3\u003c/em\u003e mutations, and that CRISPR technology has great potential for treating hereditary heart diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eD. W. Wang, L. Ni and J. Nie designed the study. J. Nie and Y. Han conducted the experiments, analyzed data and completed the\u0026nbsp;manuscript. Z. Jin, W. Hang and H. Shu conducted the animal experiments. Z. Wen performed the echocardiography examination of the animals.\u003c/p\u003e\n\u003ch2\u003eSources of Funding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China [No. 82100401, 82070354, 81470519, 81630010] and Huazhong University of Science and Technology Academic Frontier Youth Team (No. 2019QYTD08).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the Ethics Review Board of Tongji Hospital and Tongji Medical College. It complied with the principles of the Declaration of Helsinki. Written informed consent was obtained from individual subjects of the HCM pedigree in this study. All animal experiments complied with the \u0026ldquo;Guide for the Care and Use of Laboratory Animals\u0026rdquo; published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996). This study was approved by the Institutional Animal Research Committee of Tongji Medical College.\u003c/p\u003e\n\u003ch2\u003eDisclosures\u003c/h2\u003e\n\u003cp\u003eThe authors claimed no conflict of interest.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I, Maron MS. Hypertrophic cardiomyopathy: present and future, with translation into contemporary cardiovascular medicine. Journal of the American College of Cardiology. 2014;64(1):83-99.\u003c/li\u003e\n\u003cli\u003eMarian AJ, Braunwald E. Hypertrophic Cardiomyopathy: Genetics, Pathogenesis, Clinical Manifestations, Diagnosis, and Therapy. Circulation research. 2017;121(7):749-70.\u003c/li\u003e\n\u003cli\u003eLopes LR, Zekavati A, Syrris P, Hubank M, Giambartolomei C, Dalageorgou C, et al. Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing. 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Biochimica et biophysica acta Molecular cell research. 2019.\u003c/li\u003e\n\u003cli\u003eLong C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351(6271):400-3.\u003c/li\u003e\n\u003cli\u003evan Putten M, van der Pijl EM, Hulsker M, Verhaart IE, Nadarajah VD, van der Weerd L, et al. Low dystrophin levels in heart can delay heart failure in mdx mice. Journal of molecular and cellular cardiology. 2014;69:17-23.\u003c/li\u003e\n\u003cli\u003eXie C, Zhang YP, Song L, Luo J, Qi W, Hu J, et al. Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome. Cell research. 2016;26(10):1099-111.\u003c/li\u003e\n\u003cli\u003eCarrier L, Mearini G, Stathopoulou K, Cuello F. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology. Gene. 2015;573(2):188-97.\u003c/li\u003e\n\u003cli\u003eProndzynski M, Mearini G, Carrier L. Gene therapy strategies in the treatment of hypertrophic cardiomyopathy. Pflugers Archiv : European journal of physiology. 2019;471(5):807-15.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2199328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2199328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVariants in myosin-binding protein C3 (\u003cem\u003eMYBPC3\u003c/em\u003e) gene are a main cause of hypertrophic cardiomyopathy (HCM), accounting for 30\u0026ndash;40% of the total number of HCM cases. Gene editing represents a potential permanent cure for HCM. The aim of this study was to investigate whether genome editing of \u003cem\u003eMYBPC3\u003c/em\u003e using the CRISPR/Cas9 system \u003cem\u003ein vivo\u003c/em\u003e could rescue the phenotype of rats with HCM. We generated a rat model of HCM (\u0026ldquo;1098hom\u0026rdquo;) that carried an \u003cem\u003eMybpc3\u003c/em\u003e premature termination codon mutation (p.W1098x) discovered in a human HCM pedigree. On postnatal day 3, the CRISPR/Cas9 system was introduced into rat pups by a single dose of AAV9 particles to correct the variant using homology-directed repair (HDR). Analysis was performed 6 months after AAV9 injection. The 1098hom rats didn\u0026rsquo;t express MYBPC3 protein and developed an HCM phenotype with increased ventricular wall thickness and diminished cardiac function. Importantly, CRISPR HDR genome editing corrected 3.56% of total mutations, restored MYBPC3 protein expression by 2.12%, and normalized the HCM phenotype of 1098hom rats. Our work demonstrates that the HDR strategy is a promising approach for treating HCM associated with \u003cem\u003eMYBPC3\u003c/em\u003e mutation, and that CRISPR technology has great potential for treating hereditary heart disease.\u003c/p\u003e","manuscriptTitle":"Homology-Directed Repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-15 15:52:15","doi":"10.21203/rs.3.rs-2199328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-12-06T10:03:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-12-05T17:20:54+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-11-27T01:15:56+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-11-24T16:31:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-22T19:50:30+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-15T17:17:16+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-13T01:49:20+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-11-13T01:47:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-11T13:26:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-11T13:25:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Gene Therapy","date":"2022-10-24T15:36:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0651098a-bf7d-4b81-8695-bf8e8e55fc2f","owner":[],"postedDate":"November 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":16923905,"name":"Health sciences/Diseases/Cardiovascular diseases"},{"id":16923906,"name":"Biological sciences/Biological techniques/Genetic engineering"}],"tags":[],"updatedAt":"2023-01-23T17:32:06+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-15 15:52:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2199328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2199328","identity":"rs-2199328","version":["v1"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}