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.
Hamamori
Y
Y
Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles 90033.
Samal
B
B
Tian
J
J
Kedes
L
L
D000818:Animals; D018076:DNA, Complementary / Q000008:administration & dosage / Q000235:genetics*; D004920:Erythropoiesis; D004921:Erythropoietin / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006085:Graft Survival; D006400:Hematocrit; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008819:Mice, Nude; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000097:blood; D012190:Retroviridae / Q000235:genetics; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D001616:beta-Galactosidase