Gene transfer to muscle using herpes simplex virus-based vectors

The main goal of gene therapy for Duchenne muscular dystrophy (DMD) is to restore dystrophin into as many muscle cells as necessary to be therapeutic. Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. The viral genome is large and can accommodate multiple or large non-viral genes including the full length dystrophin. Here we report on the use of a replication defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. Our initial experiments showed that HSV-1 can efficiently infect and express a foreign reporter gene in myoblasts and myotubes in vitro. Furthermore, the intramuscular inoculation of HSV-1 resulted in transduction of a significant number of muscle fibers in newborn mice and some muscle fibers in adult animals. We have attempted to exploit these features to develop new HSV mutant vectors for dystrophin gene delivery to DMD muscle, however two impediments to using this virus for muscle gene delivery have to be overcome: namely viral cytotoxicity and the differential transducibility with HSV-1 throughout the development of muscle fibers. To solve the first problem, virus mutants deleted for the immediate early (IE) genes (ICP4, ICP22, ICP27 and UL41) were constructed and the multiple deleted virus was greatly reduced in cytotoxicity relative to our first generation HSV vector strains. Current work is aimed at incorporating full length dystrophin under muscle specific promoter (muscle creatine kinase MCK) into these new viral vectors. To address the second problem we have analysed by immunohistochemistry the spreading of the HSV-1 in newborn versus adult muscles to determine whether mature basal lamina which surrounds the adult muscle fibers blocks the HSV-1 entry into the mature muscle fibers.