IN-VITRO AND IN-VIVO CHARACTERIZATION OF A 2ND FUNCTIONAL HAIRPIN RIBOZYME AGAINST HIV-1

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-I pol gene. The ribozyme was 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) mu M(-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-I 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. (C) 1995 Academic Press, Inc.