An episomally maintained MDR1 gene for gene therapy.
Potential applications of the MDR1 multidrug transporter in gene therapy include protecting sensitive bone marrow cells against cytotoxic drugs during cancer chemotherapy and serving as a dominant selectable marker when coexpressed with a corrective passenger gene. To address safety concerns associated with integrating viral systems, such as retroviruses, we tested the feasibility of maintaining a nonvirally delivered MDR1 gene (pEpiHaMA) episomally. An MDR1 vector containing the Epstein-Barr virus (EBV) origin of replication (OriP) and its nuclear retention protein (EBNA-1) was transfected into human (KB-3-1) cells. MDR1 was expressed at a higher level in cells carrying the episomal vector, pEpiHaMA, compared with the vector lacking sequences needed for episomal maintenance (pHaMA). Furthermore, more drug-resistant KB-3-1 colonies were obtained on selection after transfection with pEpiHaMA. These observations correlated with longer maintenance of episomes in cells transfected with pEpiHaMA. In addition, episomes could still be recovered for more than 1 month from tumor explants in nude mice that were injected with pEpiHaMA-liposome complexes after drug selection, suggesting that these constructs can be maintained extrachromosomally in vivo.
Lee
C G
CG
Department of Biochemistry, National University of Singapore, Singapore.
Vieira
W D
WD
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D019450:Genes, MDR / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018952:Antigens, CD34; D019309:Epstein-Barr Virus Nuclear Antigens; D008081:Liposomes; C417029:EBV-encoded nuclear antigen 1