Gp130/Stat3 agonism increases regeneration and improves AAV-CRISPR delivery to dystrophic skeletal muscle in vivo


Translational Research

Poster Number: 176


Michael Hicks, PhD, University of California, Irvine, Lily Gane, BS, University of California, Los Angeles, Mandee Yang, University of California, Los Angeles, Matt Romero, PhD, University of California, Los Angeles, Diana Bieccera, University of California, Los Angeles, Ekaterina Mokhonova, University of California, Los Angeles, Melissa Spencer, PhD, University of California, Los Angeles, Denis Evseenko, PhD, University of Southern California, Ben Van Handel, PhD, University of Southern California, April Pyle, PhD, University of California, Los Angeles

CRISPR/Cas9 gene editing holds promise for a broad range of genetic diseases. For Duchenne Muscular Dystrophy (DMD), delivery of gene editing machinery to both the skeletal myofibers and satellite cells (SCs) could improve long-term efficiency, as myofibers regenerated by the gene edited SC pool could restore dystrophin after multiple rounds of injury. However, gene editing of SCs is inefficient, and thus we set out to regulate SCs numbers to improve in vivo gene editing. We identified a novel gp130/Stat3 agonist (423F) that induced in vitro proliferation of freshly isolated mdx SCs by 50-fold within 3 days. To test 423F efficiency in vivo, we crossed mdx or mdx/Dba2 mice to Cre-inducible Pax7-ZsGreen lineage tracing mice and demonstrated 423F treatment increased ZsGreen+ myofibers within 2 weeks. 423F also increased Pax7+ cell numbers at sites of regeneration, and newly formed myofibers in vivo as measured by embryonic myosin. Single cell RNA seq in mdx mice treated with 423F identified shifts in SC and inflammatory cell populations indicative of a regeneration phenotype. We next sought to optimize 423F for use in combination with adeno-associated virus (AAV) gene delivery. Using Ai9 tdTomato reporter mice and AAV to deliver Cre recombinase, we determined that SC targeting could be increased by 4-fold in young mice and by 20-fold in activated Itga7+Cd34+Cd104+ SCs. Encouraged by these data we next treated pups in humanized DMD mdx mice with 423F and injected AAVs containing Cas9 and gRNAs targeting human DMD exons 45-55 to restore the reading frame of mutated human dystrophin in vivo. After 5 weeks, we demonstrated significantly increased dystrophin expression in multiple skeletal muscles and improvements to tetanic muscle force using Aurora force transducers. Collectively, these data highlight transient activation of SCs leading to increased regeneration in combination with CRISPR/Cas9 gene editing leads to therapeutic increases of dystrophin production in vivo in mdx mouse models.