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

Abstract

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-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.

Intracellular application of hairpin ribozyme genes against hepatitis B virus

“…Previous work with hairpin ribozymes against HIV have revealed that adding a tetraloop to the loop 3 region can enhance the catalytic efficiency (k cat /K m ) of a ribozyme. 13 It is noteworthy that the addition of a tetraloop to either BR1 or BR3 increases the catalytic efficiency by increasing k cat , without significantly altering K m . Based on our studies with HIV, the in vitro catalytic efficiency of each ribozyme usually serves as a good indicator of its catalytic efficiency in cells (unpublished data).…”

“…Some regions of a hairpin ribozyme can be modified for its optimized cleavage activity for each individual ribozyme. [9][10][11][12][13] those regions (Figure 1b). 12,13 Modification in the loop 3 ribozyme target site was chosen due to high sequence region of BR1 and BR3 resulted in improved RNA cleavconservation between different HBV strains.…”

“…stabilizing 'tetraloop' motif. 13 For this reason, tetraloops were used to replace loop 3 of BR1 and BR3 and the resulting in vitro activities were compared with the protoEffects of anti-HBV ribozymes in liver cell culture The effectiveness of anti-HBV ribozymes as therapeutic type BR1 and BR3 (Figure 3). Cleavage time-course experiments (see Materials and methods) with each agents in vivo will depend on their ability to function in a complex cellular environment.…”

“…This retroviral vector was chosen due to its successful application in delivering ribozymes against HIV. 13 As the first step of testing the principle of ribozyme gene therapy for the treatment of HBV infection, we employed transient DNA co-transfection to facilitate the rapid test of intracellular ribozyme efficacy. Following individual transfections into the human hepatocellular carcinoma cell line Huh7, each ribozyme was expressed at similar levels as determined by RNase protection (Figure 4), considering that the different sizes of each detected ribozyme RNA were due to the specific design of the probe (Materials and methods and Figure 4b).…”

“…Some regions of a hairpin ribozyme can be modified for its optimized cleavage activity for each individual ribozyme. [9][10][11][12][13] Correspondence: J Barber Received 13 September 1996; accepted 10 March 1997…”