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

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.

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

a, Using iPSCs derived from a healthy donor (HD^WT), the MYH7 p.R403Q (c.1208 G > A) variant was introduced by CRISPRCas9-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 (HD^403/+) and homozygous genotype (HD^403/403) 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, HD^WT and HD^403/+ 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 HD^WT and HD^403/+ iPSC-CMs as measured by quantitative PCR. Data are mean ± s.d. across four separate differentiations.

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 MYH7^403/+ HCM1 and MYH7^403/+ HCM2 iPSCs.

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 Myh6^h403/h403 mice with ABE-AAV9 or saline. b, Kaplan-Meier curve for Myh6^WT mice (n = 7; 4 male, 3 female), Myh6^h403/+ mice (n = 8; 2 male, 6 female), Myh6^h403/h403 mice (n = 6; 1 male, 5 female), and ABE-treated Myh6^h403/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: Myh6^WT and Myh6^h403/+ mice, >40 days; Myh6^h403/h403 mice, 7 days; AAV LOW Myh6^h403/h403 mice, 9 days (1.3-fold longer, P = 0.0201); AAV HIGH Myh6^h403/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 Myh6^h403/h403 mice and AAV HIGH Myh6^h403/h403 mice, each, compared to Myh6^h403/h403 mice. c, Sanger sequencing chromatograms for a Myh6^h403/h403 mouse and a AAV HIGH Myh6^h403/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 Myh6^h403/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.

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 Myh6^WT mice, Myh6^h403/+ mice, or ABE-treated Myh6^h403/+ 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 Myh6^WT mice compared to Myh6^h403/+ mice (black) and ABE-treated Myh6^h403/+ mice compared to Myh6^h403/+ mice (green). g, Representative M-mode images for Myh6^WT mice, Myh6^h403/+ mice, and ABE-treated Myh6^h403/+ mice at 16 weeks of age.

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 Myh6^h403/+ 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 Myh6^h403/+ 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 Myh6^h403/+ 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 Myh6^WT mice, Myh6^h403/+ mice, and ABE-treated Myh6^h403/+ 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.

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 Myh6^h403/+ mice compared to Myh6^WT mice (top), ABE-treated Myh6^h403/+ mice compared to Myh6^h403/+ mice (middle), and ABE-treated Myh6^h403/+ mice compared to Myh6^WT 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.

Fig. 1 |. In vitro optimization of the ABE system to correct a pathogenic MYH7 variant.

a, A candidate sgRNA, h403_sgRNA, for base editing to correct the MYH7 c.1208G>A (p.R403Q) missense variant. Base editing could convert the neutrally charged glutamine PV back to a positively charged arginine, restoring proper function of the myosin head. The nucleotide sequence highlighted in green corresponds to the adenine base editing window in positions 14–17. b, Eight candidate base editor variants were screened for their efficiencies in correcting the pathogenic adenine to a guanine using the candidate h403_sgRNA within a homozygous MYH7 c.1208G>A iPSC line (HD^403/403). c, DNA editing efficiency of all adenines within the target protospacer in HD^403/403 iPSCs 72 h posttransfection with candidate base editors. Data are mean ± s.d. across three technical replicates. Numbering is with the first base 5′ of the PAM as 1; target pathogenic adenine is position A16.

Fig. 2 |. Analyses of the function of iPSC-CM derived from patients with HCM on base editing correction.

a, Workflow for reprogramming iPSCs from a healthy donor (HD) and two patients with HCM (HCM1 and HCM2), followed by PV knock-in for the HD line and base editing correction for the HCM1 and HCM2 lines. Isogenic clonal lines were then isolated and differentiated into CMs for downstream analyses of iPSC-CM function. b, HTS to measure editing for all adenines within the on-target protospacer. Target pathogenic adenine is A16. HTS was performed for ABE-treated HCM1^403/+ and HCM2^403/+ iPSCs. c, Quantification of peak systolic force of iPSC-CMs for indicated cell lines. (n = 41 for HD^403/+; 31 for HD^WT; 55 for HCM1^403/+; 57 for HCM1^WT; 41 for HCM2^403/+; and 31 for HCM2^WT). Data are mean ± s.e.m. across three separate differentiations. HD^403/+ iPSC-CMs had a 1.7-fold increase in peak systolic force compared with HD^WT iPSC-CMs (P = 0.0029); HCM1^WT a 2.0-fold decrease compared with HCM1^403/+ (P = 0.000013); and HCM2^WT a 1.6-fold decrease compared with HCM1^403/+ (P = 0.0085). d, Oxygen consumption rate (OCR) as a function of time in indicated cell lines after exposure to the electron transport chain complex inhibitors oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A (AntA) (top), and mean and distribution of values across four timepoints for basal OCR (bottom left) and maximal OCR (bottom right) for indicated cell lines. HD^403/+ iPSC-CMs had a 1.6-fold increase in basal OCR compared with HD^WT iPSC-CMs (P = 0.000018); HCM1^WT a 1.4-fold decrease compared with HCM1^403/+ (P = 0.000019); and HCM2^WT a 1.2-fold decrease compared with HCM1^403/+ (P = 0.0056). For maximum OCR, HD^403/+ iPSC-CMs had a 2.1-fold increase compared with HD^WT iPSC-CMs (P = 0.00020); HCM1^WT a 3.7-fold decrease compared with HCM1^403/+ (P = 0.000016); and HCM2^WT a 2.1-fold decrease compared with HCM1^403/+ (P = 0.00034). Data are mean ± s.d. across three separate differentiations. Each data point is from 14–16 Seahorse assay wells. **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s unpaired two-sided t-test.

Fig. 3 |. Generation of a humanized HCM mouse model.

a, A humanized HCM mouse model was generated by replacing part of the native murine Myh6 genomic sequence with the human MYH7 sequence containing the p.R403Q variant via CRISPR–Cas9 HDR. Sanger sequencing chromatograms show the native Myh6^WT sequence (top), the humanized Myh6^h403/+ mouse model sequence (middle) and the human MYH7 sequence from an HCM patient (bottom). Yellow squares indicate knocked-in single nucleotide polymorphisms (SNPs); Sanger sequencing shows double nucleotide peaks for SNPs. b, Gross histology (top), and Masson’s trichrome staining of coronal (four chamber) (middle) and transverse (bottom) sections of the humanized mouse model for the WT (left), heterozygous (middle) and homozygous (right) genotypes at postnatal day 8. Scale bar, 1 mm. c, Representative Masson’s trichrome, Picrosirius red, and hematoxylin and eosin (H&E) staining of heart sections of the humanized mouse model for the WT (left) and heterozygous (right) genotypes at 9 months of age. Scale bar, 1 mm for ×10 images top, 100 μm for ×10 images middle, 25 μm for ×40 images bottom. Similar results were found in at least three different male mice for each genotype.

Fig. 4 |. Prevention of HCM by dual AAV9 ABE editing of Myh6h403/+ mice.

a, Schematic of dual AAV9 ABE system encoding ABEmax-VRQR base editor halves under the cardiac troponin T promoter (cTnT) and h403_sgRNA to target the human MYH7 p.R403Q variant. b, Experimental outline for intrathoracic injection of Myh6^WT or Myh6^h403/+ mice with saline or dual AAV9 ABE at P0 followed by serial echocardiograms. Chow diet supplemented with 0.1% cyclosporine A (CsA) was given at 5 weeks of age for 11 weeks. c–h, Left ventricular anterior wall thickness at diastole (LVAW;d) (c), left ventricular posterior wall thickness at diastole (LVPW;d) (d), left ventricular internal diameter at diastole (LVID;d) (e) and systole (LVID;s) (f), ejection fraction (EF) (g) and fractional shortening (FS) (h), of Myh6^WT mice, Myh6^h403/+ mice and ABE-treated Myh6^h403/+ mice at 16 weeks of age. n = 5 male mice for each group. Data are mean ± s.e.m. Exact P values can be found in Table 1. i, Representative Masson’s trichrome staining of serial (500-μm interval) transverse sections for Myh6^WT mice, Myh6^h403/+ mice or ABE-treated Myh6^h403/+ mice at 16 weeks of age. Scale bar, 1 mm. j, Ventricular cross-sectional area measurements from n = 3 for Myh6^WT male mice; 5, Myh6^h403/+ male mice; 3, ABE-treated Myh6^h403/+ male mice. Myh6^h403/+ mice had significantly decreased ventricular cross-sectional area (0.679 cm²) compared with Myh6^WT mice (1.32 cm², P = 0.0032) and ABE-treated Myh6^h403/+ mice (1.22 cm², P = 0.0016). Data are mean ± s.d. k, Average wall thickness measurements from n = 3 for Myh6^WT male mice; 5, Myh6^h403/+ male mice; 3, ABE-treated Myh6^h403/+ male mice. Data are mean ± s.d. Myh6^h403/+ mice had significantly increased average wall thickness (1.67 mm) compared with Myh6^WT mice (1.19 mm, P = 0.0094) and ABE-treated Myh6^h403/+ mice (1.31 cm², P = 0.013). l, Heart weight (HW) to tibia length (TL) measurements from n = 5 male mice for each experimental group. Data are mean ± s.d. Myh6^h403/+ mice had significantly increased HW and TL (87.95 mg cm⁻¹) compared with Myh6^WT mice (69.23 mg cm⁻¹, P = 0.022) and ABE-treated Myh6^h403/+ mice (70.48 mg cm⁻¹, P = 0.0082). m, Percentage of collagen area from n = 3 for Myh6^WT male mice; 5, Myh6^h403/+ male mice; 3, ABE-treated Myh6^h403/+ male mice. Myh6^h403/+ mice had significantly increased collagen area (4.33%) compared with Myh6^WT mice (1.45%, P = 0.022) and ABE-treated Myh6^h403/+ mice (1.75%, P = 0.0047). Data are mean ± s.d. *P < 0.05, **P < 0.01 by Student’s unpaired two-sided t-test. LV, left ventricle; RV, right ventricle.

Fig. 5 |. Genomic and transcriptomic changes following dual AAV9 ABE injection in mice.

a, CM nuclei were isolated from the ventricles of 18-week-old Myh6^WT mice, Myh6^h403/+ mice or ABE-treated Myh6^h403/+ mice to assess genomic correction and transcriptomic changes. b, DNA editing efficiency for correcting the pathogenic adenine nucleotide was higher in ABE-treated Myh6^h403/+ mice (32.3%) than Myh6^h403/+ mice (0.15%, P = 0.000359). Data are mean ± s.d. c, Percentage of expressed pathogenic transcripts was lower in ABE-treated Myh6^h403/+ mice (66.9%) compared with Myh6^h403/+ mice (100.0%, P = 0.0216). Data are mean ± s.d. d, Bystander editing in ABE-treated Myh6^h403/+ mice (0.0224%) was not significantly different from saline-treated mice (0.0197%, P = 0.347). Data are mean ± s.d. e, Transcriptome-wide nuclear levels of A-to-I RNA editing was lower in ABE-treated Myh6^h403/+ mice (0.231%) compared with Myh6^WT mice (0.371%, P = 0.0464) and Myh6^h403/+ mice (0.361%, P = 0.0247). Data are mean ± s.d. f, Heat map of 257 differentially expressed genes amongst Myh6^WT or Myh6^h403/+ mice and ABE-treated Myh6^h403/+ mice. Samples and genes are ordered by hierarchical clustering. Data were scaled by the sum of each row and are displayed as row minimum and row maximum. ABE-treated Myh6^h403/+ mice cluster with Myh6^WT mice. Editing efficiency for each ABE-treated Myh6^h403/+ mouse is indicated. g, Top gene ontology (GO) terms associated with the differentially expressed genes in the comparison of h403/+ versus WT (top) and h403/+ ABE-treated versus h403/+ (bottom). P values were obtained using Metascape (Methods). h, Fold-change of Nppa mRNA expression was higher for Myh6^h403/+ mice (2.85) than for ABE-treated Myh6^h403/+ mice (1.31, P = 0.0395) normalized to Myh6^WT mice (1.01, P = 0.0151). Data from RNA-seq and qPCR. Data are mean ± s.d. For all: *P < 0.05 by Student’s unpaired two-sided t-test, n = 3 male mice for each group. NS, not significant.