Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Directed evolution of liver-detargeted AAV vectors for systemic gene delivery to skeletal muscle and heart

doi: 10.1016/j.omtm.2025.101571

Figure Lengend Snippet: AAV9.AAA.NVG7 has reduced liver biodistribution compared to AAVhu.32 in mice and is comparable in NHP The single vector biodistribution of AAVhu.32 vs. AAV9.AAA.NVG7 with a Spc5.μDys transgene was evaluated by i.v. administration in 10 C57Bl/10ScSn-Dmd mdx /J mice at a dose of 5 × 10 13 vg/kg. Quantification is shown for (A) μDys vg/diploid genome, (B) μDys/TBP mRNA transcripts, and (C) microdystrophin protein expression measured by automated capillary western blot as a percentage of wild-type dystrophin. Gastrocnemius and liver microdystrophin are normalized to wild-type dystrophin levels in gastrocnemius, and heart microdystrophin is normalized to heart wild-type dystrophin levels. Gastrocnemius and heart microdystrophin levels are also normalized to alpha-actinin endogenous control. Two-way ANOVA performed by Šidák’s multiple comparisons test. NHP were dosed with scAAVhu.32 ( n = 3 males) or scAAV9.AAA.NVG7 ( n = 2 males) with a U7.3x-snRNA transgene at a dose of 1 × 10 14 vg/kg. (D) Vector biodistribution and (E) transduction were analyzed at day 92. Skeletal muscle tissues include gastrocnemius, quadriceps, tibialis anterior, biceps, and diaphragm. Heart tissues include atrium, ventricle, and apex. Liver tissues include caudate and left lateral segment. scAAV9 liver vg/diploid genome from Hudry et al. used a dose of 1.1 × 10 14 vg/kg, n = 10 NHP, analyzed at 6 months. Two-way ANOVA performed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. All error bars represent S.D.

Article Snippet: For Jess western blot analysis, the 66–440 kDa fluorescent separation module was used with mouse anti-Dystrophin IgG1 6F11 antibody (Developmental Studies Hybridoma Bank) diluted 1:20 with antibody diluent 2 from the Jess kit (Protein Simple, Bio-Techne).

Techniques: Plasmid Preparation, Expressing, Western Blot, Control, Transduction