ABSTRACT: Objectives: HIV-1 multi-drug resistance has necessitated a continued search for therapeutics which prevent or delay the emergence of resistance to co-administered agents and/or which retain activity in antiretroviral experienced patients. We have examined, in an in vitro cell culture model, the potential role for combined gene-pharmacological therapy as a strategy for the treatment of drug-resistant HIV-1 and as a means for maintaining drug susceptibility in wild-type virus.
Methods: Recombinant pClneo expressions vectors (Promega) were constructed that contain either (I) a Rev responsive element (RRE) decoy or (ii) an HIV-1 RT antisense gene amplified onto the 3' end of the green fluorescent protein gene (GFP). Antisense RNA produced by the latter gene targets the mRNA of the lamivudine-resistant HIV-1 RT at codons 182-185. Gene expression was driven by the human cytomegalovirus immediate early promoter. Plasmids were lipofected into MT-2 T cells. Stably transfected MT-2 cells were G418-selected. The HIV-1 strains/clones, including a wild-type clinical isolate (HIV-1906) and a lamuvidine-resistant clone containing the M184V RT coding region mutation (HIV-1 strain xxLA1M84V), were used to challenge the MT-2 cell lines maintained in culture with or without lamivudine at the IC50 (0.1 µg/ml) at varying m.o.i. (0.1-0.001). Supernatants were harvested for p24 antigen assay and stored for future genotypic characterization.
Results: HIV-1 inhibition, as reflected by p24 antigen level reduction and syncytium formation, was demonstrated in MT-2 cells expressing the RRE decoy or RT antisense. HIV-1906 (wild-type) infection (m.o.i. 0.001) of control MT-2 cells without lamivudine, for example, resulted in peak p24 antigen levels of 265,000 pg/ml versus 5 pg/ml in RRE decoy-expressing cells and undetectable levels with decoy plus lamuvidine (2 weeks post-infection). To study the effect on drug-resistant virus, MT-2 cells expressing the lamuvidine resistance genotype antisense yielded a p24 antigen level of 440 pg/ml versus 77360 pg/ml (6 days post-infection) in control cells when challenged with the lamuvidine-resistant HIV-1 xxLA1M184V.
Conclusions: Combination gene-drug therapy may represent a novel approach toward the treatment of HIV-1 infection as well as a method for studying molecular pathogenesis of antiretroviral drug resistance.
