Functional in vivo gene transfer into the myofibers of adult skeletal muscle

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Abstract

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for β-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.

Section snippets

Methods

Plasmids and DNA preparation. The plasmids VR1012 (pCMV-Snap25-GFP, a gift of Prof. T. Pozzan), pCMV-Luc (kindly provided by Dr. M. Cantini), and pCMV-β-gal (pCMV-β-gal, Clontech), containing the cytomegalovirus (CMV) promoter, were grown in DH5α cells and purified using the Plasmid Maxi Kit (Qiagen, Crawley, UK) following the supplier protocol. Identity was confirmed by agarose gel electrophoresis of both uncut and restriction digested plasmids. Contamination with RNA was absent and all the

Influence of electrode shape and electric field on in vivo transfection of mouse muscle fibers

Placed at each side of the tibialis anterior muscle, the spatula-like electrodes shown in Fig. 1 induced much less damage and higher transfection than standard external plates or needle electrodes. These new electrodes delivered low voltage stimulation pulses that induced muscle twitching without lesion of muscle tissue (myofibers and vessels). Three to five days after electroporation, high levels of expression were present (Figs. 2A and B). The entire section of TA showed transfected

Discussion

Viral vectors are highly efficient in gene transfer; on the other hand, serious concerns regarding the possibility of insertional mutagenesis and induction of the host immune response limit their clinical desirability. Several genetically modified viruses, such as retrovirus, herpes simplex virus, Epstein–Barr virus, adenovirus (AdV), and adeno-associated virus (AAV), have been tested for gene delivery into muscle. Of these, AdV and AAV have been found to be most efficient so far for

Acknowledgements

This work was supported in part by institutional funds of the Italian C.N.R. Institute of Neuroscience, Unit for Neuromuscular Biology and Physiopathology, and of the Italian M.U.I.R. (ex 60% to U.C.), Laboratory of Applied Myology, Department of Biomedical Science, University of Padua. We are grateful to Dr. Karen Gustafson for a revision of the English manuscript.

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