LONG-TERM EXPRESSION OF ERYTHROPOIETIN IN THE SYSTEMIC CIRCULATION OFMICE AFTER INTRAMUSCULAR INJECTION OF A PLASMID DNA VECTOR

Erythropoietin (Epo)-responsive anemia is a common and debilitating co mplication of chronic renal failure and human immunodeficiency virus i nfection. Current therapy for this condition involves repeated intrave nous or subcutaneous injections of recombinant Epo, In this report, we describe the development of a novel muscle-based gene transfer approa ch that produces long-term expression of physiologically significant l evels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDN A under the transcriptional control of the cytomegalovirus immediate e arly (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 mu g of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits f rom preinjection levels of 48 +/- 0.4% to levels of 64 +/- 3.3% 45 day s after injection, Hematocrits in these animals remained elevated at g reater than 60% for at least 90 days after a single i.m. injection of 10 mu g of pVRmEpo, We observed a dose-response relationship between t he amount of plasmid DNA injected and subsequent elevations in hematoc rits, Mice injected once with 300 mu g of pVRmEpo displayed 5-fold inc reased serum Epo levels and elevated hematocrits of 79 +/- 3.3% at 45 days after injection. The i.m. injected plasmid DNA remained localized to the site of injection as assayed by the PCR, We conclude that i.m. injection of plasmid DNA represents a viable nonviral gene transfer m ethod for the treatment of acquired and inherited serum protein defici encies.