Development of Muscle Optimized Non-viral Gene Editors - Project Summary Duchenne muscular dystrophy is a severe X-linked genetic disorder characterized by progressive muscle degeneration and weakness caused by the loss of dystrophin protein. There are no curative therapies for DMD, and recently approved micro-dystrophin gene therapies have shown modest clinical benefit. Adeno-associated virus (AAV) delivered CRISPR gene editing strategies have achieved high rates of corrective gene editing in mouse and canine models of DMD but rely on dangerously high doses of AAV and result in constitutive expression of immunogenic gene editors. Lipid nanoparticles (LNPs) have emerged as a promising non-viral delivery strategy for gene editing but have lower delivery efficiency in skeletal muscle than AAV. Significant effort has been put towards improving delivery efficiency of LNPs, however, the factors that limit gene editing in skeletal muscle after delivery have not been addressed. Gene editing efficiency is not only determined by the entry of the delivery vehicle into the cell but also by the ability of the gene editor protein to populate all the myonuclei of the myofiber. Myofibers have hundreds or thousands of myonuclei that must be edited to achieve therapeutic benefit, but our preliminary data show that gene editor proteins cannot propagate to myonuclei distant from the site of transfection/transduction. In addition to the issue of low myonuclear propagation, low mRNA stability prevents the accumulation of sufficient levels of gene editor protein to edit most myonuclei. The objective of this project is to develop a muscle-optimized gene editor that can increase gene editing efficiency after non-viral delivery. This objective will be achieved by developing an in-vitro myotube based screening platform to identify modifications to the ABE8e adenine base editor that improve myonuclear propagation. An in-vivo pooled LNP screen will be performed to identify UTRs and codon optimization strategies that improve base editor mRNA stability in skeletal muscle. Modifications identified in these screens will be combined into a muscle-optimized base editor that will be used in LNP-delivered editing of a humanized mouse model of DMD. Completion of these studies will contribute to the development of a non-viral curative therapy for DMD. The insight gained from these studies will provide a basis for developing other muscle-optimized gene therapy cargos. These studies will be carried out in the laboratory of Dr. Eric Olson, PhD at University of Texas Southwestern Medical Center. All studies and career development will be performed under the mentorship of Dr. Olson and Dr. Ning Liu, PhD, pioneers in the field of skeletal muscle gene editing.
