Fig. 5. Assessment of genome editing and plasma lipids when targeting PCSK9, Pcsk9 and Angptl3 with single-AAV ABEs in vivo.

a, Strategy for assessing base editing and plasma analytes in AAV-treated mice. b, Bulk liver editing efficiencies at human PCSK9, and mouse Pcsk9 and Angptl3 (n = 3–5 mice; each dot represents one mouse, error bars represent s.e.m.). Human PCSK9 editing was performed using humanized PCSK9 mice, whereas mouse Pcsk9 and Angptl3 editing was performed at the endogenous mouse loci of wild-type C57BL/6J mice. AAV was administered by retro-orbital (RO) injection at 6–8 weeks of age at a dose of 1 × 10¹¹ vg per mouse. c, Dose-dependent base editing for dual SpABE8e and single SaKKH-ABE8e at mouse Pcsk9 exon 1 splice donor. The total AAV dose administered is indicated below each set of bars in vg per mouse. The dual SpABE8e editing data are reported in another publication from our group². Each dot represents a different mouse (n = 5). d, Direct comparison of editing efficiencies of dual-AAV8 intein-split SaKKH-ABE8e and single-AAV8 SaKKH-ABE8e targeting the Pcsk9 exon 1 donor site in bulk liver at two doses. The total AAV dose administered is indicated below each set of bars in vg per mouse, ***P = 0.0004. Each dot represents a different mouse (n = 4). For b-d, liver tissue was harvested at four weeks post injection and analyzed by HTS. e, Plasma PCSK9 protein in humanized mice treated with 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e. f, Plasma Pcsk9 protein in C57BL/6J mice treated with either 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e, dual-AAV8 SpABE8e or non-targeting control, ***P = 0.0001. g, Plasma Angptl3 protein in C57BL/6J mice treated with 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e or non-targeting control, **P = 0.0027. h, Plasma total cholesterol in humanized mice treated with 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e. i, Plasma total cholesterol in C57BL/6J mice treated with either 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e, dual-AAV8 SpABE8e or non-targeting control, ***P = 0.0007. j,k, Plasma total cholesterol (j, ***P = 0.0007) and plasma triglycerides (k, *P = 0.0118) in C57BL/6J mice treated with 1 × 10¹¹ vg single-AAV8 SaKKH-ABE8e or non-targeting control. For e–k, dots represent individual mice and error bars represent s.e.m. of n = 5 different mice. Significance was calculated for c and d using two-way unpaired t-test. Significance for f, g and i–k was calculated using two-way repeated measures ANOVA with Tukey’s or Sidak multiple comparisons, as applicable, and is shown for the week 4 timepoint for all graphs except for f, in which week 3 significance is shown as week 4 protein levels did not reach statistical significance. In all instances, non-targeting control is dual-AAV8 SpABE7.10 with sgRNA targeting mouse Dnmt1, an unrelated site in the mouse genome, administered at the same timepoint, route and dose.