title: Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene.
abstract: 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.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20895, USA.
authors.forename: C G
authors.initials: CG
authors.lastname: Lee
authors.forename: K T
authors.initials: KT
authors.lastname: Jeang
authors.forename: M A
authors.initials: MA
authors.lastname: Martin
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: M M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: 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
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016337:RNA, Catalytic