Continuous Erythropoietin Delivery by Muscle-Targeted Gene Transfer Usingin VivoElectroporation

Abstract

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit 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, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar–Kyoto rats

“…The peak serum Epo levels following the tail vein injection of 800 mg of pCAGGS-Epo were 100-fold greater than the levels reached following the muscle-targeted gene transfer of 400 mg of pCAGGS-Epo by in vivo electroporation. 28,29 In the present study, the peak serum vIL-10 levels following the tail vein injection of 800 mg of pCAGGS-vIL-10 were at least three-fold greater than the levels reached following intravenous gene transfer with an adenovirus vector, peaking at 165.47101 ng/ml 1 day after the gene transfer. 30 Thus, our previous and present studies demonstrate that gene transfer into the liver by the hydrodynamics-based transfection method is easily applicable to the rat, which is more than 10 times larger than the mouse.…”

“…45 Plasmids were grown in Escherichia coli DH5a cells, and prepared using a Qiagen EndoFree plasmid Giga kit (Qiagen GmbH, Hilden, Germany), as described previously. 28 The empty pCAGGS plasmid was used as a control.…”

Inhibition of mast cells by interleukin‐10 gene transfer contributes to protection against acute myocarditis in rats

“…In earlier studies, it has been shown that gene transfer into muscles by electroporation can be used to deliver cytokines systemically [28,29]. In the present study, we investigated whether IL-10 gene transfer by electroporation protected rats from acute myocarditis and whether the effect of IL-10 involved the modulation of mast cells.…”

“…Mouse IL-10 cDNA cloned after amplification by polymerase chain reaction was inserted into the unique Xho I site between the cytomegalovirus immediate early enhancerchicken b-actin hybrid promoter and rabbit b-globin poly A site of the pCAGGS expression plasmid [28,29]. The resulting plasmid, pCAGGS-IL-10, was grown in Escherichia coli DH5a and prepared using plasmid purification columns (Qiagen, Tokyo, Japan).…”

Viral and non-viral gene therapy partially prevents experimental cisplatin-induced neuropathy

“…Our findings are consistent with previous reports, showing a decrease in the systemic production of various proteins with time following muscle-targeted gene therapy using adenoviral vectors 10,21 or naked DNA injection and muscle electroporation. 8,22,23 This suggests that a single injection is not sufficient for human therapeutic applications as cisplatin therapy generally involves repeated treatment over several months. In the case of adenoviral vectors, the immune reaction against viral proteins constitutes the main limitation preventing long-term expression, 21 and reducing the efficacy of subsequent repeat injections.…”