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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.

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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.

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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).

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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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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.

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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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