Fig. 6: In vivo physiology and chemistry of Rpe65 correction.
a, ABE RNP LNP degradation kinetics after subretinal injection into WT mice; anti-Cas9 western blot analysis of RPE/choroid/sclera and neural retina lysates collected at the indicated hours post injection. b,c, Next-generation sequencing of genomic DNA (b) and of transcripts (c) to document Rpe65-editing outcomes after treatment with ABE RNP LNP or PE RNP LNP. d, Anti-RPE65 western blot analysis of RPE/choroid/sclera lysates from WT, untreated rd12 and ABE-RNP-LNP-treated rd12. e, High-performance liquid chromatography (HPLC) quantification of 11-cis-retinal in whole eyes from dark-adapted untreated rd12 mice, ABE-RNP-LNP- and PE-RNP-LNP-treated rd12 mice and WT mice. f, RPE flatmounts of rd12 untreated, ABE-RNP-LNP-treated rd12 and WT mice stained for RPE65 (green) and counterstained with ZO-1 (magenta) and DAPI (blue). Scale bar, 50 µm. g, Scotopic flash ERG a-wave (left) and b-wave (right) amplitudes for rd12 mice treated with ABE RNP LNP or PE RNP LNP, compared to untreated rd12 and WT mice. h, PLR after a 101.2 W m−2 stimulus for ABE-RNP-LNP- and PE-RNP-LNP-treated rd12 mice, compared to untreated rd12 and WT mice. Data quantified as pupil diameter constriction post stimulus compared to pupil diameter pre-stimulus in dark-adapted animals. Representative frames (left) and summarized data (right). Scale bar, 1 mm. i, Representative SC (left) and V1 (right) responses from WT mice (black), rd12 mice treated with free ABE RNP (green), ABE RNP LNP (purple), or PE RNP LNP (red) and untreated rd12 mice (orange). The mice received 1 µl of 2.5 µM ABE RNP (560 ng) or 2.2 µM PE RNP (655 ng) per eye. All data plotted as mean ± s.d. Data in e and g were analysed using one-way analysis of variance (ANOVA) with Kruskal–Wallis test; in h, using one-way ANOVA with Dunnet’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NSP ≥ 0.05. Uncropped blots are available as Source data.
