a Representative western blottings (W.B.) of homogenates (top panels) with Flag antibody for AAV9-mitoARCUS samples and GFP antibody for AAV9-GFP samples. RFLP “last-cycle hot” PCR analysis (RFLP, bottom panels) of DNA samples from the same injected animals at 6, 12, and 24 weeks PI. Similar analyses were performed for each animal. b Quantification of heteroplasmy shift shown as a percent change in heteroplasmy across all tissues at 6, 12, and 24 weeks PI normalized to brain tissue. Heteroplasmy levels of the heart (H), tibialis anterior (TA), quadriceps (Q), gastrocnemius (G), kidney (K), liver (L), and spleen (Sp) were compared to that of the brain (B) (negative for expression of mitoARCUS). c Quantification by RT-PCR of total mtDNA levels in the skeletal muscle, liver, and brain at 6 and 24 weeks PI using ND1 and ND5 mitochondrial primer/probes normalized to 18S (nuclear DNA). Data are mean ± SEM of n = 4 (with exception of 6 weeks GFP (n = 5) and 24 weeks mitoARCUS (n = 3)). Statistical analysis was performed using two-tailed Student’s t-test.