Gene Electrotransfer Results in a High-Level Transduction of Rat Skeletal Muscle and Corrects Anemia of Renal Failure

Abstract

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: Inhibition of primary tumor growth and metastatic spread

“…19 The efficiency of gene transfer by electroporation is greater by several orders of magnitude than that of passive DNA transfer (see Fig 1). Electrotransfer of the endostatin gene into normal tissues ( muscle ) could also be achieved (Fig 4) and several previous studies have indicated that electrotransfer into muscle of other therapeutic genes such as those encoding interleukin -12 (e.g., Hanna et al 34 ) and erythropoietin ( e.g., Rizzuto et al 35 ) or FGF 1 ( e.g., Mir et al 36 ) is very effective. Transfer of the endostatin gene into muscles resulted in augmented levels of circulating endostatin ( Fig 4) and diminished the number of metastatic foci in mouse lungs ( Fig 5) ( see also Blezinger et al 17 ).…”

Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase – increased expression with reduced muscle damage

“…This concern has recently been addressed by the development of in vivo electrotransfer techniques that have routinely achieved a 10-to 100-fold increase in expression. [8][9][10]15 The LacZ reporter gene was used in present studies, as its expression is easily quantifiable by histochemistry and ELISA thus allowing an assessment of both the number of positive fibres and the overall levels of expression. Samples were taken at 5 days after injection, as this represents the peak of ␤-galactosidase expression in these animals following direct injection alone.…”

“…Previous reports of high efficiency electrotransfer have also noted the association of muscle damage with efficient gene transfer. 10 Other studies, however, have failed to comment on the muscle histology 9,15 although in some cases the figures showed evidence of muscle damage. 8 Hyaluronidase has previously been used to improve the efficiency of virus-based in vivo gene transfer methods.…”

“…Hence, where plasmid gene transfer into skeletal muscle is carried out for induction of an immune response (genetic vaccination), or for systemic delivery (eg apoE, factor IX, erythropoietin), the damage caused when using current electrotransfer protocols may not be significantly detrimental. 11,[13][14][15] However, for treatment of muscle disease by gene replacement, for example, in the case of muscular dystrophies, it is essential to ensure the survival of muscle fibres while maximising the number of transfected fibres.…”

In vivo NMR imaging evaluation of efficiency and toxicity of gene electrotransfer in rat muscle

“…The signal intensity is determined experimentally in water from a phantom with calibrated concentrations of Gd-DOTA, in acquisition conditions identical to those used in vivo. From the measurements obtained in the water phantom and from muscle relaxation times, the equation linking the signal increase R ¼ (SiÀSm)/Sm to the concentration of Gd-DOTA C is C ¼ 0:099R þ 0:154R 2 We here assume that So is not modified, and that the influence of T2 variation can be neglected since TE value is short. This is an acceptable approximation at value of electric field under 200 V/cm where T2 and T1 variations induced by oedema are weak.…”