Extended Data Fig. 8: Generation of the miniXon cassette and assessment of miniXon control of SaCas9 for in vivo gene editing in liver.
From: Regulated control of gene therapies by drug-induced splicing
a, Cartoon depicting the AAV genome size with the SF3B3-Xon and SF3B3-miniXon cassettes. b, Luciferase induction in HEK293 cells transfected with SF3B3-miniXon-luciferase or SF3B3-Xon-luciferase in response to varying doses of LMI070. All samples are normalized to Renilla luciferase activity and are relative to DMSO treated cells. Data are the mean ± s.e.m. of 8 biological replicates (***P < 0.001 versus SF3B3.Xon, two-way ANOVA followed by Bonferroni’s post hoc test). c, Splicing inclusion assays of the LMI070-induced exon at 100 nM LMI070. Pseudo exon inclusion in the Xon cassette was detected using primers flanking the pseudoexon (left) or by priming within the novel exon sequence (right; 4 technical replicates). d, Experimental design. Mice were injected with AAV8-miniXon-SaCas9 plus AAV8-sgAi14-eGFP (1 × 1012 viral genomes, 1:1 ratio) and 2 weeks later dosed with vehicle or LMI070 at 50 mg kg−1 to induce SaCas9 expression and editing of the loxP-STOP cassette (guides: sgA14_1: 5′-CTCTAGAGTCGCAGATCCTC-3′, sgAi14_2: 5′-ACGAAGTTATATTAAGGGTT-3′). One week later, mice were euthanized, and livers processed to assess gene editing by genomic DNA PCR, histology and FACS of isolated hepatocytes. e, Representative FACS analysis of hepatocytes obtained from Ai14 mice after LMI070 or vehicle treatment. The gating/sorting strategy (above), and the percentage of tdTomato expressing cells for each condition (below) is shown (4 biological replicates). f, Representative photomicrographs of liver sections obtained from AAV injected Ai14 mice 1-week after LMI070 treatment. tdTomato expression (red) is evident in LMI070 treated mice (5 mice per group). Scale bars, 100 μm. g, SaCas9 mediated editing of the loxP-STOP cassette in Ai14 mice as detected by PCR assay of liver genomic DNA (3 of 5 mice with guides plus LMI, 2 of 4 mice with guides plus vehicle, 1 of 2 untreated mice are shown). A PCR product of 355 bp size corresponding to the edited Ai14 ROSA Locus was observed in the LMI070-treated mice. h, Sanger sequencing of the 355 bp PCR product confirmed targeted deletion of the loxP-STOP cassette and DNA repair of the Ai14 reporter locus.
