Fig. 4. rAAV:HDR vectors rescue liver disease phenotypes in a mouse model of HT-I.
a Schematic of rAAV:HDR:uncleaved and -cleaved vectors (left) and control non-cognate donor or spacer vectors (right) to correct the Fah point mutation in FahPM/PM mice (top). b In vivo experimental regimen to correct the Fah mutation by injection of AAV8 through the tail vein in adult FahPM/PM mice. c Complete rescue of average body weight loss in FahPM/PM mice in treated cohorts injected with rAAV:HDR:uncleaved (red) and -cleaved (green), as compared to control cohorts injected with PBS (black), rAAV:FahDonor:ncSpacer (brown) or rAAV:ncDonor:FahSpacer (blue). Data shown represent the average body weight of each cohort. d Bar graph showing the percentage distribution of NHEJ, HDR, and imprecise NHEJ:HDR mix at Fah in livers of mice 6 weeks after NTBC withdrawal, as measured by NGS sequencing of PCR amplicons from genomic DNA. e Representative images of immunostaining for FAH in liver tissues of negative control and treated cohorts, as indicated below each panel. Scale bar, 100 μm. All mice in each cohort were examined once by this method (see Supplementary Fig. 3). f Levels of Fah mRNA from RNA-seq (transcripts per million) in the livers of FahPM/PM mice. g rAAV copy numbers in HT-I mouse liver tissues. rAAV copies are significantly reduced in rAAV:HDR:cleaved cohorts after NTBC withdrawal using the [U6<>FahDonor] primer pair (left) and [FahDonor<>U1a] primer pair (right). h qRT-PCR analyses with total RNA showing significantly reduced Nme2Cas9 mRNA and sgRNA expression in the rAAV:HDR:cleaved cohort after NTBC withdrawal. Data were presented as mean values ± s.e.m. Sample sizes in panels c, d, f–h: (n = 3 in PBS, rAAV:FahDonor:ncSpacer, rAAV:ncDonor:FahSpacer and pre-NTBC withdrawal rAAV:HDR:cleaved and -uncleaved cohorts; n = 7 in post-NTBC withdrawal rAAV:HDR:cleaved and -uncleaved cohorts). p values are calculated using Student’s t-test (two-sided).