OR21

Skeletal muscle organoids for preclinical gene therapy with recombinant AAV vectors

C Lièvre(1,2,3) B Robert(1) E Mozin(1) G Mainieri(1) D L Mack(4,5) B Fraysse(1,2) C Le Guiner(1,2,3) J B Dupont(1,2)

1:Inserm UMR 1089; 2:Université de Nantes; 3:CHU de Nantes; 4:University of Washington; 5:Institute for Stem Cells and Regenerative Medicine (ISCRM)

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease caused by mutations in the DMD gene, coding for dystrophin. Its deficiency causes muscle degeneration in young boys and premature death. Among possible therapeutic approaches, gene therapy using vectors derived from the adeno-associated virus (AAV) is promising. Efficiency and safety of this approach has been shown in animal models. However, during clinical translation, the efficiency was lower and several patients have developed serious adverse events that were not anticipated in preclinical studies. Therefore, new experimental models are needed to better predict the efficacy and toxicity of AAV vectors.

In this context, we developed a preclinical testing platform for DMD gene therapy using skeletal muscle organoids derived from human induced pluripotent stem cells (hiPSCs). We used a reporter AAV vector to determine the most efficient vector delivery strategy and the minimal dose for successful organoid transduction. We observed that transgene expression was maintained over 4 weeks, demonstrating that our engineered muscle model allows for medium-term monitoring of AAV transduction. We also compared the efficiency of 13 AAV serotypes in organoids at different stages of maturation. Interestingly, our results indicate that organoids subjected to mechanical tension to improve their maturation, are better suited to predict the efficiency of AAV variants recently engineered to target muscle cells. Altogether, our study demonstrates the relevance of a preclinical AAV vector screening platform based on human muscle organoids derived from hiPSCs.