Fig. 1: AAV genomic cassette and promoter engineering to drive potent and specific expression.

a In order to validate the assay system, we confirmed transgene expression resulted in proper localization in human MYBPC3^-/- iPSC-CMs transduced with AAV:MYBPC3. Immunofluorescence analysis was performed seven days post-infection (scale bars, 25 µm). b Cassette schematic indicating the genomic size of a standard cassette and the alterations tested. c Human MYBPC3^-/- iPSC-CMs were transduced with AAV6-packaged constructs encoding human MYBPC3 driven by various promoter versions of human cardiac troponin T (TNNT2) (pCard0, pCard1, and pCard2) and harvested 5 days post-infection (n = 1–2/condition). d A head-to-head comparison of the independently packaged versions of the pCard0 and pCard1 constructs. MYBPC3^-/- iPSC-CMs were harvested five days post-infection with CR9-01-packaged constructs (n = 2/condition). e To determine if the optimized promoter also increased expression in vivo and whether selectivity was maintained, adult female mice were retro-orbitally injected with 4E13 vg/kg AAV9 encoding the pCard0 (n = 3) and pCard1 (n = 4) constructs. Heart, skeletal muscle (tibialis anterior), liver, and whole brain samples were harvested two weeks post-injection. P-value per two-sided Student’s t-test with 1.0% False Discovery Rate with Two-stage step-up (Benjamini, Krieger, and Yekutieli). Matched transduction (f), RNA (g), and protein (h) analysis in MYBPC3^-/- iPSC-CMs demonstrated near-WT levels of protein expression at 1 vg/dg compared to parental wild-type control iPSC-CMs one-week post-infection. WT No Inf (n = 3), MYBPC3^-/- No Inf (n = 2), MYBPC3^-/- 1 K (n = 3), MYBPC3^-/- 3 K (n = 3), WT GFP 100 K (n = 3), and WT GFP 100 K (n = 3). 1 K = multiplicity of infection of 1000, or 1000 vector genomes/cell. Data are shown as means ± SEM. Source data are provided as a Source Data file.