A critical step in gene electrotransfer: The injection of the DNA

Abstract

In gene electrotransfer, DNA must be injected before the electric pulses delivery. This is a critical step in the success of the gene transfer. Because plasmid molecule has a very high molecular weight, even large amounts of plamid (in mg) correspond to a few number of molecules that are highly diluted if injection is made intravenously. Therefore, injections are often made in the target tissue. We show the way in which that this intramuscular, intratumoral or intrahepatic injection is performed can largely impact on the result of gene transfer, even (and mainly) in the absence of electric pulses (1). Indeed, the simple injection of DNA into muscles is known to result in the expression of the injected genes, even though at low and variable levels. We report that this variability in DNA expression is partly dependent on the injection speed. The acceleration of the injection speed from values around 2 μl/s up to ones around 25 μl/s (depending on the tissue) results in a significant increase in gene expression in skeletal muscle (280 times on an average) and in liver (50 times) and a non-significant sevenfold increase in tumors. Heparin, which inhibits the spontaneous uptake of the injected DNA, also inhibits the increases related to the injection speed. However, at the highest injection speed, this inhibition is not total because very fast injections provoke a direct permeabilization of the cells. This “hydroporation” could be similar to the permeabilization found in the hydrodynamics method based on the fast intravascular injection of a huge volume of DNA. Neither the “hydroporation” nor the heparin-inhibitable uptake mechanism induce histologically detectable lesions. There is a limited muscle cell stress independent of the injection speed. Heterogeneity in the injection speed might thus be an explanation for the variability in DNA expression after simple injection. Our data stress the importance of the DNA injection step in the electrotranfer procedures. This conclusion is important because gene electrotransfer is gaining momentum both in the preclinical and clinical stages (2) as a safe and efficient non-viral method for gene therapy.