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Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice

Nature Medicine volume 29, pages 401–411 (2023)Cite this article

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Abstract

The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in β-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease.

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Fig. 1: In vitro optimization of the ABE system to correct a pathogenic MYH7 variant.
Fig. 2: Analyses of the function of iPSC-CM derived from patients with HCM on base editing correction.
Fig. 3: Generation of a humanized HCM mouse model.
Fig. 4: Prevention of HCM by dual AAV9 ABE editing of Myh6h403/+ mice.
Fig. 5: Genomic and transcriptomic changes following dual AAV9 ABE injection in mice.

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper, extended data and supplementary material. Raw and analyzed RNA-seq data generated during this study are available in the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus series accession number GSE201755. DNA sequencing files can be accessed at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) with accession code PRJNA902011. The mm10 reference genome is available at https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/.

Code availability

The MATLAB code used to perform contractile force measurements of iPSC-CMs has been deposited to GitHub: https://github.com/DarisaLLC/Cardio.

References

  1. Maron, B. J. Clinical course and management of hypertrophic cardiomyopathy. N. Engl. J. Med 379, 655–668 (2018).

    Article  PubMed  Google Scholar 

  2. Semsarian, C., Ingles, J., Maron, M. S. & Maron, B. J. New perspectives on the prevalence of hypertrophic cardiomyopathy. J. Am. Coll. Cardiol. 65, 1249–1254 (2015).

    Article  PubMed  Google Scholar 

  3. Trivedi, D. V., Adhikari, A. S., Sarkar, S. S., Ruppel, K. M. & Spudich, J. A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light. Biophys. Rev. 10, 27–48 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Geisterfer-Lowrance, A. A. et al. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell 62, 999–1006 (1990).

    Article  CAS  PubMed  Google Scholar 

  5. Tyska, M. J. et al. Single-molecule mechanics of R403Q cardiac myosin isolated from the mouse model of familial hypertrophic cardiomyopathy. Circ. Res. 86, 737–744 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Sarkar, S. S. et al. The hypertrophic cardiomyopathy mutations R403Q and R663H increase the number of myosin heads available to interact with actin. Sci. Adv. 6, eaax0069 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Koblan, L. W. et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature 589, 608–614 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Suh, S. et al. Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing. Nat. Biomed. Eng. 5, 169–178 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Chemello, F. et al. Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing. Sci. Adv. 7, eabg4910 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Reichart, D. et al. Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice. Nat. Med. (2022).

  13. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Marian, A. J. & Braunwald, E. Hypertrophic cardiomyopathy: genetics, pathogenesis, clinical manifestations, diagnosis, and therapy. Circ. Res 121, 749–770 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic. Acid. Res. 46, W242–W245 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pua, C. J. et al. Genetic studies of hypertrophic cardiomyopathy in Singaporeans identify variants in TNNI3 and TNNT2 that are common in Chinese patients. Circ. Genom. Precis. Med. 13, 424–434 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Toepfer, C. N. et al. Myosin sequestration regulates sarcomere function, cardiomyocyte energetics, and metabolism, informing the pathogenesis of hypertrophic cardiomyopathy. Circulation 141, 828–842 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cohn, R. et al. A contraction stress model of hypertrophic cardiomyopathy due to sarcomere mutations. Stem Cell Rep. 12, 71–83 (2019).

    Article  CAS  Google Scholar 

  23. Vakrou, S. & Abraham, M. R. Hypertrophic cardiomyopathy: a heart in need of an energy bar? Front. Physiol. 5, 309 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P. & Buckingham, M. Developmental regulation of myosin gene expression in mouse cardiac muscle. J. Cell Biol. 111, 2427–2436 (1990).

    Article  CAS  PubMed  Google Scholar 

  25. Geisterfer-Lowrance, A. A. et al. A mouse model of familial hypertrophic cardiomyopathy. Science 272, 731–734 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Ma, S. et al. Efficient correction of a hypertrophic cardiomyopathy mutation by ABEmax-NG. Circ. Res. 129, 895–908 (2021).

    Article  CAS  PubMed  Google Scholar 

  27. Ishikawa, K., Weber, T. & Hajjar, R. J. Human cardiac gene therapy. Circ. Res. 123, 601–613 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zettler, J., Schutz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Prasad, K. M., Xu, Y., Yang, Z., Acton, S. T. & French, B. A. Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Gene Ther. 18, 43–52 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Mendell, J.R. et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs. Mol. Ther. 29, 464–488 (2021).

    Article  CAS  PubMed  Google Scholar 

  31. Teekakirikul, P. et al. Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-beta. J. Clin. Invest. 120, 3520–3529 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pinto, A. R. et al. Revisiting cardiac cellular composition. Circ. Res. 118, 400–409 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Jiang, J., Wakimoto, H., Seidman, J. G. & Seidman, C. E. Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy. Science 342, 111–114 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pare, J. A., Fraser, R. G., Pirozynski, W. J., Shanks, J. A. & Stubington, D. Hereditary cardiovascular dysplasia. A form of familial cardiomyopathy. Am. J. Med. 31, 37–62 (1961).

    Article  CAS  PubMed  Google Scholar 

  35. Fananapazir, L. & Epstein, N. D. Genotype-phenotype correlations in hypertrophic cardiomyopathy. Insights provided by comparisons of kindreds with distinct and identical beta-myosin heavy chain gene mutations. Circulation 89, 22–32 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Ho, C. Y. et al. Genotype and lifetime burden of disease in hypertrophic cardiomyopathy: Insights from the Sarcomeric Human Cardiomyopathy Registry (SHaRe). Circulation 138, 1387–1398 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Carroll, K. J. et al. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9. Proc. Natl Acad. Sci. USA 113, 338–343 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Maron, B. J., Yeates, L. & Semsarian, C. Clinical challenges of genotype positive (+)-phenotype negative (-) family members in hypertrophic cardiomyopathy. Am. J. Cardiol. 107, 604–608 (2011).

    Article  PubMed  Google Scholar 

  39. Green, E. M. et al. A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science 351, 617–621 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Stern, J. A. et al. A small molecule inhibitor of sarcomere contractility acutely relieves left ventricular outflow tract obstruction in feline hypertrophic cardiomyopathy. PLoS ONE 11, e0168407 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ladage, D., Ishikawa, K., Tilemann, L., Muller-Ehmsen, J. & Kawase, Y. Percutaneous methods of vector delivery in preclinical models. Gene Ther. 19, 637–641 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250–265 e216 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tabebordbar, M. et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919–4938 e4922 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Weinmann, J. et al. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat. Commun. 11, 5432 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lompre, A. M. et al. Species- and age-dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals. Dev. Biol. 84, 286–290 (1981).

    Article  CAS  PubMed  Google Scholar 

  47. Desai, M. Y. et al. Study design and rationale of VALOR-HCM: evaluation of mavacamten in adults with symptomatic obstructive hypertrophic cardiomyopathy who are eligible for septal reduction therapy. Am. Heart J. 239, 80–89 (2021).

    Article  CAS  PubMed  Google Scholar 

  48. Saberi, S. et al. Mavacamten favorably impacts cardiac structure in obstructive hypertrophic cardiomyopathy: EXPLORER-HCM Cardiac Magnetic Resonance Substudy Analysis. Circulation 143, 606–608 (2021).

    Article  PubMed  Google Scholar 

  49. Keam, S.J. Mavacamten: First Approval. Drugs 82, 1127–1135 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ho, C. Y. et al. Evaluation of mavacamten in symptomatic patients with nonobstructive hypertrophic cardiomyopathy. J. Am. Coll. Cardiol. 75, 2649–2660 (2020).

    Article  CAS  PubMed  Google Scholar 

  51. Murphy, E. & Steenbergen, C. Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury. Cardiovasc. Res. 75, 478–486 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626–631 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Method. 11, 855–860 (2014).

    Article  CAS  Google Scholar 

  56. Correia, C. et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Sci. Rep. 7, 8590 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Kluesner, M. G. et al. EditR: a method to quantify base editing from Sanger sequencing. CRISPR J. 1, 239–250 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Atmanli, A. et al. Cardiac myoediting attenuates cardiac abnormalities in human and mouse models of Duchenne muscular dystrophy. Circ. Res. 129, 602–616 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Kijlstra, J. D. et al. Integrated analysis of contractile kinetics, force generation, and electrical activity in single human stem cell-derived cardiomyocytes. Stem Cell Rep. 5, 1226–1238 (2015).

    Article  Google Scholar 

  60. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Miura, H., Quadros, R. M., Gurumurthy, C. B. & Ohtsuka, M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat. Protoc. 13, 195–215 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Creed, H. A. & Tong, C. W. Preparation and identification of cardiac myofibrils from whole heart samples. Methods Mol. Biol. 2319, 15–24 (2021).

    Article  PubMed  Google Scholar 

  64. Cui, M. & Olson, E. N. Protocol for single-nucleus transcriptomics of diploid and tetraploid cardiomyocytes in murine hearts. STAR Protoc. 1, 100049 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10, 1523 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the members of the E.N.O. laboratory for helpful discussions; J. C. Wu and the Stanford Cardiovascular Institute for providing the HCM patient-derived iPSCs; C. Seidman and H. Wakimoto for suggestions on intrathoracic AAV9 delivery; T. Lanigan, H. Kopera and R. Agate from the University of Michigan Vector Core for rAAV9 production; C. Llamas and P. Mishra from the Children’s Medical Center Research Institute for help with Seahorse assays; J. Cabrera and S. Vargas for graphics; D. Martin from Envigo for custom chow consultation; J. Xu and Y. J. Kim from the Children’s Medical Center Research Institute for performing the Illumina NextSeq sequencing; C. Rodriguez-Caycedo for assistance with iPSCs; the UT Southwestern McDermott Center Sanger Sequencing Core; the UT Southwestern McDermott Center Next-Generation Sequencing Core; the UT Southwestern Flow Cytometry Core; and J. Shelton from the Molecular Histopathology Core for help with histology. This work was supported by grants from the National Institutes of Health (R01HL130253, P50HD087351 and R01HL157281 to E.N.O. and R.B.-D.; F30HL163915 to A.C.C.), the American Heart Association (907611 to A.C.C.), the Foundation Leducq Transatlantic Networks of Excellence in Cardiovascular Research and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). The E.N.O. laboratory is supported by CureHeart, the British Heart Foundation’s Big Beat Challenge Award (BBC/F/21/220106).

Author information

Authors and Affiliations

  1. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Andreas C. Chai, Miao Cui, Francesco Chemello, Hui Li, Wei Tan, Ayhan Atmanli, John R. McAnally, Yu Zhang, Ning Liu, Rhonda Bassel-Duby & Eric N. Olson

  2. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Andreas C. Chai, Miao Cui, Francesco Chemello, Hui Li, Wei Tan, Ayhan Atmanli, John R. McAnally, Yu Zhang, Ning Liu, Rhonda Bassel-Duby & Eric N. Olson

  3. Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Kenian Chen & Lin Xu

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  1. Andreas C. Chai
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Contributions

A.C.C., R.B.-D. and E.N.O. conceptualized the project and designed the experiments. A.C.C., F.C., H.L. and A.A. conducted in vitro iPSC-CM experiments. A.C.C., M.C., F.C., H.L. and Y.Z. conducted in vivo experiments. W.T. performed mouse echocardiography. J.R.M. performed mouse zygote injections. K.C., A.C.C. and L.X. performed bioinformatics analysis. A.C.C., N.L., R.B.-D. and E.N.O. wrote the manuscript.

Corresponding author

Correspondence to Eric N. Olson.

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Competing interests

E.N.O. is a consultant for Vertex Pharmaceuticals and Tenaya Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Generation of isogenic HD403/+ and HD403/403 iPSCs by homology-directed repair.

a, Using iPSCs derived from a healthy donor (HDWT), the MYH7 p.R403Q (c.1208 G > A) variant was introduced by CRISPR-Cas9-based homology-directed repair (HDR) using SpCas9, a sgRNA (spacer sequence colored in green, PAM sequence colored in gold), and a single-stranded oligodeoxynucleotide (ssODN) donor template containing the PV. A heterozygous genotype (HD403/+) and homozygous genotype (HD403/403) were isolated. Chromatograms highlighting insertion of the PV and corresponding amino acid changes are shown for indicated genotypes. Red arrows indicate coding nucleotide 1208 and amino acid 403. b, Sanger sequencing chromatogram showing no insertion of the PV on the highly homologous MYH6 gene. Red arrow indicates coding nucleotide 1211 and amino acid 404. c, HDWT and HD403/+ iPSCs readily differentiate into CMs. Cardiac troponin I (cTnI, green) highlights CMs; nuclei (blue) are marked by DAPI (4′,6-diamidino-2-phenylindole). Scale bar, 25 μm. Similar ability for iPSCs to differentiate into CMs was found in at least three separate differentiations for each genotype. d, Ratio of MYH7 to MYH6 gene expression in HDWT and HD403/+ iPSC-CMs as measured by quantitative PCR. Data are mean ± s.d. across four separate differentiations.

Source data

Extended Data Fig. 2 Computationally determined off-target sites for h403_sgRNA with ABEmax-VRQR.

a, Genomic loci of eight candidate off-target (OT) sites (left) and alignments of eight candidate off-target sequences to the on-target protospacer (right). Nucleotides that match the protospacer are indicated with a vertical dash. Nucleotides that differ are shown for each site. Numbering of nucleotides in protospacer starts with the nucleotide immediately 5′ of the PAM as nucleotide 1. b, HTS to measure editing for all 58 adenines within the protospacers of the top 8 CRISPOR-identified candidate off-target loci. HTS was performed for ABE-treated MYH7403/+ HCM1 and MYH7403/+ HCM2 iPSCs.

Source data

Extended Data Fig. 3 Comparison of predominantly expressed mouse and human myosin heavy chain sequences.

Homology comparison for mouse α-myosin heavy chain (Myh6) and human β-myosin heavy chain (MYH7) at the amino acid level (top) and DNA sequence level (bottom) around glutamine 403. The h403_sgRNA is illustrated in green and the PAM sequence is illustrated in yellow. The pathogenic c.1208 G > A variant is located at position 16 within the canonical base editing window of positions 14–17, counting the adenine nucleotide immediately 5′ of the PAM as position 1.

Extended Data Fig. 4 Validation of a dual AAV9 ABE system in mice.

a, Injection details for treating Myh6h403/h403 mice with ABE-AAV9 or saline. b, Kaplan-Meier curve for Myh6WT mice (n = 7; 4 male, 3 female), Myh6h403/+ mice (n = 8; 2 male, 6 female), Myh6h403/h403 mice (n = 6; 1 male, 5 female), and ABE-treated Myh6h403/h403 mice at a low (AAV LOW, n = 3; 1 male, 2 female) or high dose (AAV HIGH, n = 5; 4 male, 1 female). Median lifespans: Myh6WT and Myh6h403/+ mice, >40 days; Myh6h403/h403 mice, 7 days; AAV LOW Myh6h403/h403 mice, 9 days (1.3-fold longer, P = 0.0201); AAV HIGH Myh6h403/h403 mice, 15 days (2.1-fold longer, P = 0.0014). *P < 0.05, **P < 0.01 by log-rank (Mantel–Cox) test for AAV LOW Myh6h403/h403 mice and AAV HIGH Myh6h403/h403 mice, each, compared to Myh6h403/h403 mice. c, Sanger sequencing chromatograms for a Myh6h403/h403 mouse and a AAV HIGH Myh6h403/h403 mouse showing 35% on-target editing of the target pathogenic adenine at the cDNA level. d, Four-chamber sectioning and Masson’s trichrome staining of a AAV HIGH Myh6h403/h403 male mouse at 15 days of age. No other replications were possible as all other pups (5 total) were cannibalized before hearts could be collected.

Source data

Extended Data Fig. 5 Serial echocardiograms following dual AAV9 ABE editing of Myh6h403/+ mice.

a–f, Left ventricular anterior wall thickness at diastole (a) left ventricular posterior wall thickness at diastole (b), left ventricular internal diameter at diastole (c) and systole (d), ejection fraction (e), and fractional shortening (f), of Myh6WT mice, Myh6h403/+ mice, or ABE-treated Myh6h403/+ mice from 8–16 weeks of age. n = 5 male mice for each group. Exact P values can be found in Table 1. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 by Student’s unpaired two-sided t-test for Myh6WT mice compared to Myh6h403/+ mice (black) and ABE-treated Myh6h403/+ mice compared to Myh6h403/+ mice (green). g, Representative M-mode images for Myh6WT mice, Myh6h403/+ mice, and ABE-treated Myh6h403/+ mice at 16 weeks of age.

Source data

Extended Data Fig. 6 Genomic and proteomic analysis of select tissues following dual AAV9 ABE editing.

a, Viral copy numbers for the N terminal AAV and C terminal AAV were quantified from the right atrium (RA), right ventricle (RV), left atrium (LA), left ventricle (LV), lung, liver, spleen, and quadriceps muscle (Quad) from ABE-treated Myh6h403/+ mice at 16 weeks of age. b, The percentage of A to G editing was determined by HTS of genomic DNA in the RA, RV, LA, LV, lung, liver, spleen and Quad from ABE-treated and saline-injected Myh6h403/+ mice. c, The percentage decrease in mutant transcripts in the RA, RV, LA, and LV was determined by HTS of cDNA from ABE-treated and saline-injected Myh6h403/+ mice. The percentage decrease was greater in the RV (22.7%, P = 0.0202) and the LV (26.7%, P = 0.00157) compared to the LA (12.9%). d, Cardiac myofibrils were isolated from Myh6WT mice, Myh6h403/+ mice, and ABE-treated Myh6h403/+ mice, run on a 4–20% polyacrylamide gel, and stained with Coomassie G-250. Key sarcomeric proteins are marked, including titin, myosin heavy chain (MHC), myosin binding protein C (MyBP-C), actin, cardiac troponin T (cTnT), cardiac tropomyosin (cTm), and cardiac troponin I (cTnI). Sizes for ladder markings are in kDa. Relative protein amounts for each key sarcomeric protein are normalized to WT. Data are mean ± s.d. *P < 0.05 by Student’s unpaired two-sided t-test, n = 3 male mice for each group.

Source data

Extended Data Fig. 7 RNA-sequencing analysis of dual AAV9 ABE editing of Myh6h403/+ mice.

Volcano plot showing fold-change and p-value of genes up-regulated (red) and down-regulated (blue) in Myh6h403/+ mice compared to Myh6WT mice (top), ABE-treated Myh6h403/+ mice compared to Myh6h403/+ mice (middle), and ABE-treated Myh6h403/+ mice compared to Myh6WT mice (bottom). P-adjust < 0.01 and fold-change > 2.0 were used for cutoffs; p-adjust values are calculated by two-tailed Wilcoxon rank-sum test. n = 3 male mice for each group.

Source data

Supplementary information

Supplementary Information

Supplementary Note 1, Figs. 1 and 2 and Table 1.

Reporting Summary

Source data

Source Data Fig. 1

Numerical source data.

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Chai, A.C., Cui, M., Chemello, F. et al. Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice. Nat Med 29, 401–411 (2023). https://doi.org/10.1038/s41591-022-02176-5

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