Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector.

Abstract

Erythropoietin (Epo)-responsive anemia is a common and debilitating complication ofchronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 ,ug of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 ± 0.4% to levels of 64 ± 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 ,ug of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent 10876The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

“…11,[16][17][18][19][20][21] Plasmid DNA delivered directly to muscle generally results in persistent albeit low-level gene expression since skeletal muscle takes up, episomally maintains and expresses plasmid DNA for a time notably longer than other tissues. 12,25,[40][41][42] Furthermore, this method of gene delivery circumvents some of the problems encountered with viral vectors.…”

“…15 Nevertheless, secretion in blood plasma and/or systemic biological activity of the transgene have been confirmed after i.m. delivery of plasmid vectors encoding for erythropoietin, 16 factor VII, 17 rat kallikrein-binding protein (RKBP), 18 vascular endothelial growth factor (VEGF), 19 interleukin 10, 20 and interferon-␥ receptor/IgG1 fusion protein. 21 These studies suggest that i.m.…”

Non-viral gene delivery in skeletal muscle: a protein factory

“…A variety of plasmid backbones containing different promoters have been tested for transgene expression in skeletal muscle with varying success. The most widely used are viral promoters, such as cytomegalovirus (CMV) immediate-early promoter, 28 simian virus, SV40 early promoter 29 and Rous sarcoma virus promoter. 1 These promoters can drive expression in a wide range of cells and tissues eg CMV promoter can be activated in nearly all cell types.…”

A gene therapy for cancer based on the angiogenesis inhibitor, vasostatin

“…In addition, plasmid DNA vectors are easier to construct, can accept large cDNA inserts, and can be prepared as pure chemical solutions without the risk of contamination with wild-type infectious particles. [33][34][35] It has been reported that the delivery of heterologous genes into skeletal muscle can provide the sustained production of proteins in the circulation. 18 Intramuscular injection of plasmid DNA encoding interferon-␣ or endostatin has showed the inhibition of tumor growth.…”