PERSISTENT ERYTHROPOIESIS BY MYOBLAST TRANSFER OF ERYTHROPOIETIN CDNA

A myoblast gene transfer approach was developed to deliver human eryth ropoietin (EPO) systemically. We created stable, high-level EPO-produc ing muscle cell clones by transfecting C2 myoblasts with a plasmid-bea ring human EPO cDNA driven by cytomegalovirus enhancer/promoter and se lection 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 s ecreted EPO. After transplantation of 4 x 10(7) cells from C2-EPO9, th e 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 ce lls, 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 persi stently high (31 +/- 24 mU/ml) at 12 weeks. C2 cells transduced with a retrovirus bearing beta-galactosidase gene were transplanted into nud e mice, which showed X-Gal-positive myofibers in the transplanted area 3 months after the transplantation. These results demonstrate that my oblast gene transfer can successfully deliver functional human EPO cap able of driving sustained erythropoiesis in mice. Thus, long-term EPO delivery for anemic patients may be feasible by myoblast gene transfer .