LacZGene Transfer to Skeletal Muscle Using a Replication-Defective Herpes Simplex Virus Type 1 Mutant Vector

Abstract

Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. 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. The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter. The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells. UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly β-galactosidase production. Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo. Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks. This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression. Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals. To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice. These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer. These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications. Herpes simplex virus type 1 (HSV-1) represents an important gene delivery system that can transduce post-mitotic cells such as muscle fibers. Our initial experiment showed that HSV-1 can efficiently infect and express a reporter gene in myoblast and differentiated myotubes in cell culture as well as newborn myofibers in vivo. Currently, we are attempting to exploit these features to develop HSV mutant vectors for dystrophin gene delivery to Duchenne muscular dystrophy (DMD) muscles. However, as observed with viral systems, the use of HSV-vector mediated gene expression has been limited by transient transgene expression and the low transduction efficiency of mature myofibers. The differential transduction with HSV-1 has been attributed to the gradual maturation of the basal lamina, which acts as a barrier in mature myofibers. The transient transgene expression is attributed to cytotoxic as well as immunological barriers related to the HSV-1 infection. Because the development of strategies to improve the efficiency of HSV-1 gene transfer to muscle has to overcome these impediments, several drugs or agents that can permit an adequate permeabilization of the basal lamina are being tested. In addition, virus mutants expressing genes to elude the immune response and deleted for multiple immediately early genes that contribute to viral toxicity are under investigation.