Therapeutic base editing alleviates restrictive cardiomyopathy

Figures

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  Figure 1. Development of ABE for correcting the Tnni3R193H/R193H mutation (A) A schematic diagram illustrating the screening process for identifying the optimal candidate to correct the Tnni3 c.578G>A mutation. Different ABE and sgRNA plasmid pairs were co-transfected into the 293T cells containing the mutation. Three days post-transfection, genomic DNA was extracted and subjected to PCR amplification followed by sequencing analysis to determine the most effective ABE-sgRNA combination. (B) A heatmap showing the editing efficiency for different sgRNAs targeting the A>G repair at the Tnni3 c.578G>A site. The red arrow highlights the most efficient combination of ABE and sgRNA, as determined by the highest repair efficiency. (C) The highest editing efficiency was achieved with sgRNA8, as demonstrated by the detailed editing within the defined editing window. These results were derived from three independent replicate experiments, ensuring the reproducibility and reliability of the findings. Data are presented as mean ± SEM. (D) A schematic depiction of ABE8e in combination with sgRNA8, designed using SpRY-HF1, for the correction of pathogenic mutations associated with RCM. This design…

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  Figure 2. Tnni3R193H/R193H mice recapitulate restrictive cardiomyopathy (A) Sanger sequencing analysis of the Tnni3 transgene in Tnni3R193H/R193H transgenic homozygous mice, where the native arginine (R) at position 193 has been substituted with a histidine (H) in the pathogenic mutant allele. (B) Representative heart images from wild-type and Tnni3R193H/R193H mice. In contrast to wild-type hearts, the Tnni3R193H/R193H mice display significant morphological changes, including pronounced dilation of both the left and right atria, indicative of cardiac remodeling associated with the mutation. These alterations are consistent with the pathological features observed in restrictive cardiomyopathy. Scale bars: 1 mm. (C) Representative echocardiographic images of WT and Tnni3R193H/R193H mice showing atrial dilatation without significant ventricular remodeling. LA, left atrium; LV, left ventricle; RV, right ventricle. (D) Representative cardiac Doppler echocardiography and tissue Doppler imaging tracings from wild-type (WT; left) and Tnni3R193H/R193H mice (right). Distinct early (E) and late diastolic (A) waveforms, with altered E/A ratio indicating impaired ventricular filling; ECG: green traces…

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  Figure 3. Intrathoracic ABE treatment corrects Tnni3R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual AAV9 vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following the AAV9-ABE treatment. As in (C), gene editing efficiency in the livers of Tnni3R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

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  Figure 4. Intrathoracic ABE treatment alleviates RCM in adult Tnni3R193H/R193H mice (A) Representative photographs of hearts from wild-type (WT), mock-treated (Mock), and ABE-treated Tnni3R193H/R193H mice (Treated) at 12 weeks post-treatment. Scale bars: 1 mm. (B) Representative echocardiographic images of WT, Mock, and ABE-treated Tnni3R193H/R193H mice. Note, Mock mice exhibit marked dilation of both left and right atria, which is significantly reduced in the ABE-treated mice. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. (C–F) Quantification of echocardiographic parameters: (C) left atrial end-diastolic volume (LA volume; d), (D) E/A ratio, (E) E/e' ratio, (F) isovolumic relaxation time (IVRT). Note, all parameters in treated mice are restored to near-WT levels, indicating improved diastolic function. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA). ns, not statistically significant. Data are presented as mean ± SEM. (G) Serum CK-MB levels in WT (n = 6), Mock (n = 5), and Treated (n = 5) mice were determined via an enzymatic assay. ∗p < 0.05, ∗∗p < 0.01 (one-way ANOVA). ns, not statistically significant. Data are presented as mean ± SEM. (H) Hematoxylin and…