In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozymewas modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes ( k cat/ K m = 75 min −1 μM −1. Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.