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Rz1-7 is a multimeric hammerhead ribozyme targeting seven unique sites within the human CCR5 mRNA that is active in vitro. Mouse stem cell virus-based MGIN and human immunodeficiency virus (HIV)-1-based HEG1 vectors were used to express Rz1-7 in a human CD4+ T lymphoid cell line. Stable transductants expressed Rz1-7, which was further shown to be active, since CCR5 mRNA and surface CCR5 protein expression levels decreased. High levels of progeny virus were produced when the transduced cells were challenged with an X4-tropic HIV-1 (NL4-3) strain, suggesting that Rz1-7 expression does not affect X4-tropic virus replication. When the transduced cells expressing Rz1-7 were challenged with the R5-tropic HIV-1 (BaL) strain, 99-100% inhibition of progeny virus production was observed for the duration of the experiment (approximately 2 months). When the cells were precultured for 2-3 months prior to HIV-1 infection, inhibition was more prominent in cells transduced with MGIN-Rz1-7 than with HEG1-Rz1-7. Inhibition occurred at the level of viral entry, as no HIV-1 DNA could be detected. These results demonstrate that Rz1-7 confers excellent inhibition of R5-tropic HIV-1 replication at the level of entry. Therefore, we anticipate that this multimeric ribozyme will be beneficial for HIV-1 gene therapy.

Inhibition of human immunodeficiency virus-1 entry using vectors expressing a multimeric hammerhead ribozyme targeting the CCR5 mRNA