Implantation of recombinant rat myocytes into adult skeletal muscle: a potential gene therapy.
The ability of skeletal muscle to regenerate provides an excellent therapeutic entry, via genetic engineering, for correcting diseases of skeletal muscle and other tissues. We have used a retrovirus to transfer the cDNA for the human multidrug transporter, encoded by the MDR1 gene, into the genomes of the rat muscle cell line L6 and into primary rat myocytes. The MDR1 gene confers drug resistance to cells, and thus serves as a selectable marker in vitro. In cultured cells, the retroviral promoter-driven human MDR1 cDNA was shown to be stable in the presence or absence of drug selection or muscle cell fusion. MDR1 mRNA was synthesized, as shown by RNA blot analysis and in situ hybridization. The protein product was localized to the plasma membrane of transduced myocytes and myotubes by immunofluorescence. As a model for skeletal muscle gene therapy, transduced L6 myocytes were implanted into the tibialis anterior muscle of Wistar rats. The retroviral sequences of the human MDR1 gene and its mRNA were present in the muscles of Wistar rats 5 days, but not 12 days, after implantation, possibly because of immunorejection. On the other hand, the human MDR1 cDNA was stable in the tibialis anterior muscle of nude mice, which are incapable of immunorejection, at least 4 weeks after implantation of myocytes. Immunosuppression of Wistar rats with cyclosporine A delayed immunorejection of recombinant myocytes, and MDR1 cDNA and mRNA was detected 3-4 weeks after implantation. In situ hybridization revealed that injected recombinant myocytes remain in discrete foci in adult rodent skeletal muscle and express MDR1 mRNA for at least 30 days in nude mice and cyclosporine-treated rats.
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
A
A
Salminen
H F
HF
Elson
L A
LA
Mickley
A T
AT
Fojo
M M
MM
Gottesman
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002459:Cell Fusion; D002478:Cells, Cultured / Q000637:transplantation; D003524:Cyclosporins / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004351:Drug Resistance; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006085:Graft Survival / Q000187:drug effects; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D009132:Muscles / Q000637:transplantation*; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D003524:Cyclosporins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D004247:DNA