1. ¹National Institute of Psychiatry and Neurology, Budapest, Hungary
2. ²Biotechnology Research Institute, National Research Council, fa00 Royalmount Avenue, Montréal, QC, Canada H4P 2R2
3. ³Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montréal, QC, Canada H3A 2B4
4. ⁴Department of Neurology, Tzu Chi Medical Center, Hualien, Taiwan
5. ⁵Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA
6. ⁶Department of Neurology, Ludwig-Maximilians University, 81377 Munich, Germany
7. ⁷Respiratory Division, McGill University Health Center and Meakins-Christie Laboratories, McGill University, Montréal, QC, Canada H3A 1A1

Correspondence: George Karpati, Montreal Neurological Institute, 3801 University Street, Montréal, QC, Canada H3A 2B4. Fax: (514) 398 8310. E-mail: george.karpati@mcgill.ca

^*These authors contributed equally to this study.

Received 5 May 2004; Revised 16 June 2004; Accepted 17 June 2004.

Abstract

Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing -galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175–200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.