Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene.
Ribozymes as anti-HIV-1 agents hold promise for the treatment of AIDS. They can be delivered into cells either exogenously or through an expression system. For effective protection against HIV-1, sufficient and sustained amounts of the antiviral ribozymes must be delivered into target cells. The coexpression of a dominant selectable marker with ribozymes would serve to enrich for cells containing the molecular antiviral and facilitate prolonged expression of these ribozymes. The multidrug resistance gene (MDR1) is a potential clinically relevant selectable marker and offers many advantages over other known dominant selectable markers, including the use of diverse pharmacologically characterized drug or drug combinations for selection. Harvey sarcoma-based retroviral vectors encoding the MDR1 multidrug transporter with a hammerhead ribozyme targeted to highly conserved sequences within the HIV-1 U5 LTR segment have been constructed in a bicistronic format. The internal ribosome entry site (IRES) from encephalomyocarditis virus was used to initiate translation of the MDR1 mRNA. The ribozyme remained functional despite being tethered to MDR1. Long-term, high-level expression of both the ribozyme and MDR1, as evident by RT-PCR and FACS analysis, was observed in a human T cell line containing the construct selected with vincristine, a cytotoxic substrate for the multidrug transporter.
Lee
C G
CG
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20895, USA.
Jeang
K T
KT
Martin
M A
MA
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D002460:Cell Line; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005822:Genetic Vectors; D016325:HIV Long Terminal Repeat; D006251:Harvey murine sarcoma virus / Q000235:genetics; D006801:Humans; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016337:RNA, Catalytic