Extended Data Figure 4. Optimizing sup-tRNA expression cassette for in vivo rAAV delivery.

a, Schematic showing AAV9 vector constructs expressing 1x, 2x, or 4x sup-tRNA^Tyr. Nucleotide sequences are shown in Supplementary Table 4. b, Workflow to study in vivo rAAV9 delivery of various sup-tRNA^Tyr expression cassettes in the Idua^W401X/W401X knock-in mice (KI/KI). c, IDUA enzymatic activity in liver (n=5, 4, 6, 4 as indicated by individual circles) and heart (n=5, 5, 5, 4 as indicated by individual circles) tissue lysates derived from mice treated by indicated AAV9 vectors. Data are mean ± s.d. Statistical analysis was performed by one-way ANOVA followed by two-sided Dunnett’s multiple comparisons test against the untreated group. d, Representative denaturing gel electrophoresis of extracted rAAV vector genomes of the 1x, 2x, 4x constructs of three technical replicates. Predicated vector genome sizes are labeled at the top. Black arrowheads: intact vector genomes; red arrows: truncated vector genomes. e, Characterization of serum IDUA activity (n=4 per group), urine GAGs (n=7 or 4 per group), liver IDUA activity (n=3 or 5 per group), and liver GAGs (n=3 or 5 per group) in KI/+ and wildtype (+/+) mice. Statistical analysis was performed by two-sided Student t-test. *p < 0.05, **p < 0.01, ns: not significant.