Gene Transfer into Skeletal Muscles by Isogenic Myoblasts

Abstract

The best way to overcome immunorejection in heterologous myoblast transfer (HMT) is by the use of immunodeficient and/or highly immunosuppressed mice as hosts. The same may be attained by autologous myoblast transfer (AMT). In this paper, we describe myoblast transfer in mdx and normal mice where the donor myogenic cells originated from highly inbred litter mates that are considered to be isogenic and thus the procedure is analogous to AMT. The myoblasts were marked in vitro with Rous Sarcoma Virus (RSV)–luciferase (Lux) or RSV–β-galactosidase (LacZ) reporter genes through transduction mediated by an autonomously replication-defective recombinant human adenovirus. This permitted us to follow their fate after transplantation. mdx and normal mice were irradiated with 20 Gray gamma rays; necrosis and regeneration were induced by intramuscular notexin prior to myoblast injection. In both mdx and normal mice, the expression of luciferase rapidly declined after the injection implying that a large portion of the injected myoblasts were lost by 48 hr, due to undetermined cause(s). The surviving, injected myoblasts well-mosaicized large groups of host fibers but only in the immediate vicinity of the injection. Substantial expression of the reporter gene continued up to 1 month post-transplantation in normal mice, but there was a gradual decline and eventual disappearance of the reporter gene expression in mdx mice. This latter phenomenon was due to the ongoing intense necrosis of muscle fibers in mdx. There was no evidence of immunorejection. These experiments indicate that even in the absence of immunorejection, myoblast transfer suffers from important negative features: major loss of myoblasts within 48 hr after the injection and lack of significant spread of the injected cells from the injection site in the host muscle. These factors, plus the limited proliferative and fusion capacity of Duchenne muscular dystrophy (DMD) myoblasts, make them less than an ideal vector for the dystrophin cDNA for dystrophin gene replacement therapy in DMD. Transplantation of isogenic myoblasts into skeletal muscle is a convenient way of determining the efficiency of myoblast transfer free of the risk of immunnrejection. The donor cells were highly enriched primary myoblasts derived from satellite cells of litter mates of mdx and a highly inbred normal mouse strain. The myoblasts were efficiently transduced during culturing by replication-defective recombinant human adenovirus containing either RSV-LacZ or RSV-Lux reporter genes. Lux gene expression has rapidly declined by 80% within 48 hr after injection in normal muscle, presumably due to death of the injected myoblasts. The surviving myoblasts well mosaicized the host muscle fibers but only in the vicinity of the injection. In mdx muscle, the reporter gene expression declined and disappeared by 28 days due to ongoing necrosis of muscle fibers, but it remained stable over 1 month in normal mice. These experiments indicate that, even without immunorejection, the efficiency of AMT is still limited for replacing missing structural proteins in skeletal muscle fibers.