Additive and antagonist effects of therapeutic gene combinations for suppression of HIV-1 infection

Abstract

A previously described Moloney-based vector expressing a double copy anti- tat antisense tRNA (DC-tRNA-AT) (Biasolo et al., 1996. J. Virol. 70, 2154–2161) was modified to increase the copy number of the antisense molecule and to target the intra-cytoplasmic localization of the HIV genome. To this end, an anti-U5 hammerhead ribozyme, engineered as a hybrid small adenoviral VAI RNA (VAIα), was inserted into the vector as a single molecule or in combination with the double copy anti- tat sequence. The retroviral vector expressing only VAIα (DC-VAIα) inhibited HIV-1 replication to an extent comparable to that of DC-tRNA-AT. A more effective inhibition was produced by the vector expressing multiple copies of the anti- tat antisense (DC-6tRNA-AT). This higher effectiveness correlated with anti- tat stochiometry, i.e. with the absolute number of therapeutic molecules being produced on a per cell basis at the steady state. Surprisingly, when the tRNA-AT and VAIα genes were combined in the same vector (DC-AT-VAIα), an enhancement of viral replication was noticed. This study indicates that it is possible to potentiate the antiviral activity of a retroviral vector by increasing the steady-state level of the therapeutic molecule. Results also show that the combined expression of two singularly active therapeutic RNAs can have antagonistic rather than synergistic effects.