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 × 107 cells from C2-EP09, 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 β-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. This paper describes the feasibility of a myoblast gene transfer approach to deliver human erythropoietin (EPO) systemically as a possible alternative therapy for various types of anemia currently treated with the frequent administration of recombinant human EPO. A stable high-level EPO-producing C2 muscle cell clone was created that met three essential requirements: (i) sustained and (ii) sufficient delivery of (iii) functionally active EPO transgene product for a therapeutically significant systemic response. The transplantation of this clone resulted in persistent, marked erythropoiesis in nude mice with sustained high serum EPO concentrations for 3 months. This technology could be extended to the use of primary myoblasts or to heterologous muscle cell transplantation with an implantable immunoisolation device. Thus, long-term EPO delivery for anemic patients may be feasible by myoblast gene transfer.