UCLA

Duchenne muscular dystrophy (DMD) is an x-linked recessive lethal muscle wasting disease with no cure. DMD is often caused by out-of-frame mutations that result in a loss of dystrophin protein. However, there is an allelic and milder form of the disease, Becker muscular dystrophy (BMD), which is typically caused by in-frame mutations that result in somewhat functional dystrophin protein. Therefore, a promising strategy to treat DMD is to utilize gene editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system to convert a DMD mutation into a BMD mutation resulting in a clinically milder phenotype. However, achieving safe and efficient systemic delivery of the CRISPR/Cas9 system remains a significant challenge for DMD as skeletal muscle comprises ~40% of the total body mass. Here, we develop and optimize non-viral nanoparticles and adeno-associated viral (AAV) carriers as approaches to achieve safe and efficient systemic CRISPR/Cas9 delivery. We successfully demonstrate CRISPR/Cas9 delivery in vitro using iteratively optimized polymer-based nanoparticles. While these nanocarriers perform well in vitro, they inefficiently traffic to skeletal muscle after systemic delivery in vivo. In order to improve nanoparticle-mediated delivery to muscle and muscle stem cells, we developed and screened an AAV peptide display library for potential peptide motif ligands that may mediate skeletal muscle and muscle stem cell entry. We identify novel AAV variants highly enriched in skeletal muscle and muscle stem cells. Future work will focus on validating AAV variants and examining whether the muscle-specific peptide motif ligands may improve nanoparticle entry in muscle. While nanoparticles are promising carriers of CRISPR/Cas9 due to their low immunogenicity, AAV carriers are typically administered at high viral doses and often lead to AAV-induced immunotoxicities that pose life threatening risks. Therefore, we characterize AAV-mediated immune responses that arise in response to double dosing AAV in a dystrophic mouse model, in which the first dose effectively immunizes the mice. We reveal the production of AAV-specific antibodies that leads to subsequent activation of the classical complement pathway and induction of pro-inflammatory cytokines only after the second administration of AAV. Single cell RNA-sequencing (scRNA-seq) similarly confirms pronounced activation primarily after the second dose of AAV. Future work will continue to understand and identify targetable genes, pathways, or immune cell subtypes that may facilitate development of mitigation strategies to improve the safety and efficacy of AAV-based therapies.