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 (MDRl) 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 MDRl 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 MDRl mRNA. The ribozyme remained functional despite being tethered to MDRl. Long-term, high-level expression of both the ribozyme and MDRl, 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.