Directed HIV-1 Evolution of Protease Inhibitor Resistance by Second-Generation Short Hairpin RNAs

Abstract

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PIresistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches. Worldwide, more than 35 million individuals are infected with human immunodeficiency virus type 1 (HIV-1), and each year approximately 3 million persons are newly infected. Despite the major success of antiretroviral therapy (ART) that delays or prevents the onset of disease, HIV-1 infections remain incurable, and attempts to make a vaccine have so far proved unsuccessful (14). The emergence of drug-resistant viruses remains a major problem for some patients, especially those who exhibit suboptimal therapy adherence.The RNA interference (RNAi) mechanism is a posttranscriptional gene silencing process that holds promise as a new antiviral strategy. RNAi is induced by double-stranded RNA (dsRNA) that is processed by the RNAi machinery into small interfering RNAs (siRNAs). The siRNAs are designed with perfect base pairing complementarity to the target RNA sequence and trigger cleavage of the targeted mRNA (5, 8). HIV-1 can be inhibited effectively and specifically by RNAi in vitro. Many HIV-1 genes have been targeted by transfected siRNAs or intracellular expressed short hairpin RNAs (shRNAs), and combinatorial RNAi strategies can durably inhibit HIV-1 replication (2,11,31). Antiviral drugs and siRNAs can also be combined (9). One successful RNAi-based approach concerns the use of second-generation shRNAs designed to target the favorite escape variants that are selected under pressure of first-generation shRNAs, thus skewing virus evolution (23). In this study, we designed second-generation shRNAs to counter the evolution of clinically relevant drug-resistant HIV-1 variants.We investigated the potential of combining two anti-HIV strategies. Protease inhibitors (PIs) that successfully suppress HIV-1 replication were combined with second-generation shRNAs to block the favorite viral escape routes. To do so, we first designed second-generation shRNAs and tested them in repo...

Potential mechanisms for cell-based gene therapy to treat HIV/AIDS

“…Figure 5 lists the major advantages and disadvantages of the different combinatorial RNAi strategies [138]. One could also combine RNAi gene therapy with other RNA-based inhibitors [106] or conventional antiretroviral drugs [139,140]. For instance, we demonstrated that 'secondgeneration' shRNAs can be combined with Protease inhibitors to avoid the evolution of clinically relevant drug-resistance mutations in the Protease encoding gene [140].…”

Gene Therapy Strategies to Block HIV-1 Replication by RNA Interference

“…Leonard et al reported that a combination of RNAi attack with antiretroviral drug did enhance the antiviral activity [ 61 ]. We recently demonstrated that second-generation shRNAs can be combined with Protease inhibitors to skew virus evolution, imposing an evolution block or triggering the selection of less fi t virus variants [ 62 ]. The combination of two siRNAs against the viral Gag mRNA and the cellular CCR5 mRNA provided additive inhibition [ 63 ].…”

Combinatorial CRISPR-Cas9 and RNA Interference Attack on HIV-1 DNA and RNA Can Lead to Cross-Resistance

“…One can even set up nucleic acid selection scenarios that influence the evolution of certain protein mutants. For instance, we used RNAi strategies to skew the evolution toward certain drug-resistant HIV-1 variants that are less fit (36). Other strategies forced the selection of intersubtype recombinants under imposed RNAi pressure (37).…”