Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation

It has been demonstrated that gene transfer by in vivo electroporation of m ouse muscle increases the level of gene expression by more than 100-fold ov er simple plasmid DNA injection. We tested continuous rat erythropoietin (E po) delivery by this method in normal rats, using plasmid DNA expressing ra t Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into th e thigh muscles of rat hind limbs and 100 mu g of pCAGGS-Epo was injected b etween the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 mu g of p CAGGS-Epo, which was delivered to the medial and lateral sides of each thig h. The presence of vector-derived Epo mRNA at the DNA injection site was co nfirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on d ay 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32, The hematocr it levels increased continuously, from the preinjection level of 49.5 +/- 1 .1 to 67.8 +/- 2.2% on day 32 (p < 0.001), In pCAGGS-Epo treated rats, endo genous Epo secretion was downregulated on day 32, In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation di d not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a u seful procedure for the continuous delivery of Epo.