Persistent erythropoiesis by myoblast transfer of erythropoietin cDNA.

A myoblast gene transfer approach was developed to deliver human erythropoietin (EPO) systemically. We created stable, high-level EPO-producing muscle cell clones by transfecting C2 myoblasts with a plasmid-bearing human EPO cDNA driven by cytomegalovirus enhancer/promoter and selection by G418. Eleven clones secreted EPO into the media as detected by radioimmunoassay. In vitro bioassay using the EPO-dependent human leukemic cell line UT-7/Epo confirmed the functional activity of the secreted EPO. After transplantation of 4 x 10(7) cells from C2-EPO9, the highest producing clone, the hematocrit increased from 43.4 +/- 2.8 to 56.1 +/- 2.7 (%) in 2 weeks in C3H mice that are syngeneic to C2 cells, and from 44.6 +/- 3.0 to 71.2 +/- 7.9 in nude mice. The increased hematocrit gradually returned to the basal level in 4-5 weeks in C3H mice, while it was sustained for at least 12 weeks in nude mice. Human EPO concentrations in the sera from transplanted nude mice were persistently high (31 +/- 24 mU/ml) at 12 weeks. C2 cells transduced with a retrovirus bearing beta-galactosidase gene were transplanted into nude mice, which showed X-Gal-positive myofibers in the transplanted area 3 months after the transplantation. These results demonstrate that myoblast gene transfer can successfully deliver functional human EPO capable of driving sustained erythropoiesis in mice. Thus, long-term EPO delivery for anemic patients may be feasible by myoblast gene transfer.