Development and optimization of skeletal muscle delivery vehicles

Project Summary/Abstract Correction of genetic muscle diseases, such as the muscular dystrophies, is a unique problem due to the distribution of muscle throughout the body in inaccessible locations. Current clinical trials for Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, involve gene therapy strategies that utilize AAV

serotypes to deliver micro-Dystrophin (µDys). While AAV gene therapies are promising, use of lentiviruses that have some desirable characteristics are not considered for skeletal muscle gene therapy. AAVs contain protein capsids that mediate entry into cells, in contrast to lentiviruses that have a membrane envelope derived from

budding off from host cells. Enveloped viruses utilize membrane fusion to enter cells, which is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated

myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We tested if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We used a pseudotyping platform, a

general process where envelope proteins are altered to change the tropism of the virus, to engineer Myomaker and Myomerger on the membrane of lentiviruses. We found that these muscle fusogenic lentiviruses leads to specific transduction of skeletal muscle and that locally and systemically injected virions can deliver micro-

Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. In addition to lentiviruses, extracellular vesicles are another type of membrane vehicle being considered for gene therapies. Extracellular vesicles are a heterogenous group of membrane particles released

from most cell types and can contain factors important for homing and entry. We have also uncovered a system where Myomaker and Myomerger are present on extracellular vesicles and these vehicles exhibit delivery of material to muscle cells. We will explore these novel membrane vehicles that harness the intrinsic properties of

myogenic membranes. Specifically, we propose to: 1) identify an optimal dosing strategy for lentiviruses pseudotyped with Myomaker and Myomerger 2) characterize these novel lentiviruses and molecularly dissect their fusion mechanism with muscle cells 3) develop extracellular vesicles engineered with Myomaker and

Myomerger that target skeletal muscle. Successful completion of these studies will provide unique insight into these novel vectors specific for skeletal muscle, which have the potential to complement limitations of current viral-based gene therapies.