A Novel Approach To Block HIV-1Coreceptor CXCR4in Non-toxic Manner

The epidemic of human immunodeficiency virus type1(HIV-1) continues to cause millions of new infections and deaths annually with no effective vaccine available. The current therapy against HIV-1infection, highly active antiretroviral therapy (HAART) utilizing a combination of drugs, has proven markedly successful in controlling AIDS progression and in reducing the risk of opportunistic disease and mortality. However, with prolonged use, severe side effects and the emergence of drug-resistant HIV-1strains remain major issues to be addressed. Gene therapy based on RNA interference (RNAi) offers a promising alternative or adjuvant to current chemotherapy due to advantages that include the possibility of a one-time treatment and long-term viral inhibition particularly if hematopoietic stem cells are targeted.For HIV gene therapy, both viral genes and cellular factors have been considered as potential therapeutic targets. Considering that inhibition of HIV-1using RNAi targeting viral genes can be abolished by viral escape, targeting cellular factors essential for HIV-1replication offers an alternative strategy as emergence of viral escape mutants is less likely. A major concern in using this strategy is that cellular factor suppression may lead to adverse effects on cell metabolism. The only reported and confirmed safe cellular factor for therapeutic intervention is CCR5, which is one of the coreceptors for HIV entry. However, suppression of CCR5can only block R5-tropic HIV (CCR5coreceptor usage) entry but not X4-tropic HIV (CXCR4coreceptor usage), while suppression of CXCR4is considered to induce undesired side effects due to the loss of its natural function. To overcome this dilemma, in this study, we developed a novel approach using a single lentiviral vector to express simultaneously dual shRNA to silence endogenous CXCR4expression and a shRNA-resistant CXCR4mutant possessing the most possible natural functions of CXCR4and reduced HIV-1coreceptor activity. Via this approach we achieved the replacement of endogenous CXCR4by CXCR4mutant P191A that could compensate the functional loss of endogenous CXCR4and significant reduction of HIV-1replication by60%. Moreover, we demonstrated the construction and evaluation of the lentiviral vectors using two construction strategies:additional promoter strategy and picomaviral2A peptide-based strategy, finding the latter with significant advantages. In conclusion, this novel approach to block HIV-1coreceptor CXCR4without impairing its normal function provides a new strategy for CXCR4-targeted HIV/AIDS gene therapy. Recently, a flourishing number of host cellular factors have been described that might represent potential therapeutic intervention targets to combat HIV-1infection. These strategies, however, are fraught with the problem that the vast majority of these potential targets are essential for cell normal functions, and that suppression of these host factors may impair cell viability. In light of these considerations, the approach developed in this study provides potential universal applications to down-modulate a cellular factor in nontoxic manner for HIV/AIDS gene therapy.