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. 2023 Jun 22;14:3714. doi: 10.1038/s41467-023-39352-1

Fig. 3. Phenotypic characterization of mice after AAVMYO-ABE treatment.

Fig. 3

a Allele frequency of repaired Rbm20 mRNA in mice treated with AAVMYO-ABE determined by RNA-seq. N = 3 (R636Q), 4 (P635L) and 8 (WT) mice per condition. b, c RBM20 staining in whole heart tissue sections in WT and Rbm20 mutant mice treated with PBS or AAVMYO-ABE. Representative images in (b) and quantification of nuclear and cytoplasmic RBM20 localization in (c). Scale bar: 20 μm. Arrows highlight nuclear restored RBM20 (magenta) and cytoplasmic RBM20 (white) in base-edited mice. Manual quantification of >200 nuclei in 2 mice per condition. d Isoform expression of RBM20 target genes Ttn, Ryr2, Ldb3 and the housekeeping gene Gapdh determined by RT-PCR. N = 2–4 mice per condition. e, f Vertical agarose gel (e) and quantification (f) of titin protein isoforms in WT and Rbm20 mutant mice treated with PBS or AAVMYO-ABE. Based on gel images in Supplementary Fig. 4e. N = 3 mice per condition except for WT and P635L/SpRY where four mice were analyzed. g RNA-seq data showing changes of the ΔPSI values relative to WT in P635L, or R636Q HOM mice injected with saline or base editor. See 'Methods' section (bulk RNA sequencing and analysis) for definition of the three categories. Rescue splice events are labeled in red, all Ttn splice events in blue. N = number of splice events per category. R636Q was sequenced deeper compared to P635L explaining the difference in number of DSGs detected. N = 3 mice per condition except fornWT (4 mice) and P635L SpRY (5 mice). h–j Percentage of ejection fraction (h), LVID (i) and cardiac volume (j) determined by narcosis echocardiography of mutant mice treated with PBS or AAVMYO-ABE. N = 5 mice per condition. Same WT cohort used as in Supplementary Fig. 1m–o (16-week time point). P values obtained from one-way ANOVA with Tukey’s multiple comparison test: ***P < 0.001, **P < 0.01, *P < 0.05, n.s. = not significant. All data were obtained 12 weeks after AAVMYO-ABE injection. Only P635L or R636Q HOM mice were treated. Error bars depict the SEM in all panels.