Direct Gene Transfer into Skeletal Muscle In Vivo : Factors Affecting Efficiency of Transfer and Stability of Expression

Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or β-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA. The technique of direct gene transfer into muscle by intramuscular injection of plasmid DNA has been improved by introducing a preinjection procedure. Although the levels of expression are still insufficient to consider human trials of gene therapy for treatment of inherited primary myopathies, this method offers an excellent animal model to evaluate physiological regulation of gene expression in muscle. In addition, direct gene transfer may provide a simple and safe method to deliver recombinant proteins to the circulation in order to treat other (i.e., nonmuscle) inherited disorders.