SuperCult® Skeletal Muscle Cell Growth Medium Kit

Background

This study describes tandem promoter monocistronic expression cassettes packaged in a single AAV vector that combine hepatic and extra-hepatic tissue-specific transgene expression to prevent anti-transgene immunity. Validated in infantile Pompe disease, the tandem liver-muscle promoter (LiMP) and liver-neuron promoter (LiNeuP) drove specific transgene expression in target tissues of immunocompetent Gaa−/− mice without eliciting anti-GAA antibodies. Neonatal AAV8 or AAV9 gene therapy with LiMP provided persistent therapeutic GAA expression and corrected glycogen accumulation, cardiomegaly and muscle strength at low vector doses.

Key Findings

Tandem promoters combining the liver ApoE-hAAT enhancer-promoter with the muscle spC5.12 promoter (LiMP) or the neuronal hSYN promoter (LiNeuP) were engineered within the <5 kb AAV packaging limit and verified in hepatocyte, myoblast and neuronal cell lines. Systemically delivered AAV vectors using LiMP or LiNeuP drove specific hGAA mRNA expression in liver plus muscle or CNS, respectively, and, unlike the ubiquitous CAG or muscle-only C5.12 promoters, prevented anti-hGAA IgG formation in adult immunocompetent Gaa−/− mice; LiMP also reduced pre-existing anti-GAA antibodies. In neonate Gaa−/− mice, AAV8- or AAV9-LiMP-sec-hGAA at doses 10–50-fold lower than previously reported produced persistent hepatic and muscle enzyme expression, supra-physiological muscle GAA activity, glycogen clearance in heart, diaphragm, triceps, quadriceps, spinal cord and brain, corrected cardiomegaly and rescued muscle strength without anti-GAA immunity, whereas single tissue-specific promoters or LiNeuP failed.

Contribution

Creative Bioarray provided the human skeletal muscle myoblasts (CSC-C3196) and the SuperCult Skeletal Muscle Cell Growth Medium Kit used to confirm in vitro that the tandem LiMP promoter retains transcriptional activity in human muscle cells, supporting translation of the tandem promoter design to human muscle.