Retrovirally expressed anti-HIV ribozymes confer a selective survival advantage on CD4(+) T cells in vitro

To date, a selective advantage of cells expressing anti-HIV ribozymes has n ot been shown. This study was undertaken to determine whether such a select ive advantage can be demonstrated in vitro. A retroviral vector coding for a hairpin ribozyme targeting the HIV 5' LTR and for the low affinity nerve growth factor receptor (LNGF-R Delta) was designed. Since we demonstrated b y RT-PCR that the amount of ribozyme transcripts was highly correlated with the level of surface LNGF-R Delta expression, the vector was utilized to a ssess ribozyme expression by flow cytometry. Transduced Hut78 and primary C D4(+) T cells were purified and subsequently mixed with unmodified cells. A fter HIV challenge the percentage of ribozyme expressing cells in the cell mixture was monitored by flow cytometry. Twenty-one days after HIV infectio n the proportion of ribozyme expressing CD4(+) T cells was 2.6 times higher in comparison to cells with the control vector. CD4(+) T cells with a stro ng ribozyme expression conferred a 7.4-fold selective advantage at day 21 a nd a 11.7-fold at day 28. For Hut78 cells a selective advantage was detecte d exclusively for strongly ribozyme expressing cells. As a mechanism underl ying the selective advantage an inhibition of HIV induced apoptosis was sho wn. These results demonstrate that anti-HIV ribozymes are able to confer a selective survival advantage and indicate that the protective effect is dep endent on the amount of ribozyme expression.