Background: Duchenne muscular dystrophy (DMD) results from mutations that prevent the expression of functional dystrophin in muscle fibers. Herpes simplex virus type-1 (HSV-1) represents a potentially useful vector for treatment of DMD because it has the capacity to accommodate the 14-kb full-length dystrophin cDNA and can efficiently transduce muscle cells. We have tested the ability of first- and second-generation replication-defective HSV vectors to deliver full-length dystrophin to dystrophin-deficient mdx muscle cells in vitro and in vivo.
Methods: First-generation replication-defective HSV vectors harboring full-length or truncated (Becker) dystrophin expression cassettes and lacking a single viral immediate-early (IE) gene were constructed and tested by immunofluorescence and immunoblotting for their ability to direct dystrophin expression in infected mdx cells in culture. To reduce vector cytotoxicity and safety concerns, a second-generation dystrophin vector missing additional IE genes was constructed and tested in vitro and in vivo.
Results: Dystrophin expression was observed in infected mdx myotubes in vitro in all cases. Confocal microscopy showed exclusive localization of full-length dystrophin to the cell membrane whereas the Becker variant was also found abundantly throughout the cytoplasm. Dystrophin expression in mdx mice was restored in muscle cells near the site of vector injection.
Conclusion: Highly defective HSV-1 vectors which lack the ability to spread systemically and are greatly reduced in toxicity for infected cells, thus removing an impediment to prolonged transgene expression, can direct the delivery and proper expression of full-length dystrophin whose considerable size is compatible with few other modes of delivery. These vectors may offer a legitimate opportunity toward the development of effective gene therapy treatments for DMD.