Preclinical Characterization of an Anti-tat Ribozyme for Therapeutic Application

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Abstract
A hammerhead ribozyme retrovial construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme “escape mutants” occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tesed in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual. This study attempts to address the issues including ribozyme efficacy in inhibition of a variety of human immunodeficiency virus type 1 (HIV-1) strains, impact of ribozyme expression on HIV-1-infected peripheral blood lymphocytes (PBLs), potential generation of ribozyme escape mutants, and impact of ribozyme on quasi-species of HIV-1. Retroviral vector carrying an anti-HIV-1 ribozyme was constructed and its efficacy against HIV-1 was validated in several in vitro systems. In a multiple passage assay, no escape mutants were detected and a HIV-1 quasi-species was abolished in ribozyme-expressing T cells.