Directed transcriptional gene silencing to control HIV-1 replication and reactivation from latency

Eradicating HIV-1 from an infected individual remains one of the main quests in HIV-1 research. However, the latent reservoir has presented an unexpected barrier, with its many different faces and hiding places. Current reactivation strategies, while limited in success, have enlightened researchers toward the complexity of the latent HIV-1 provirus, highlighting the need for alternate approaches. This study explores an alternative strategy able to specifically target actively replicating and latent HIV-1 provirus. The strategy is based on the manipulation of a natural cellular mechanism that suppresses gene expression in eukaryotes, known as Transcriptional Gene Silencing (TGS), through the use of effector molecules called short interfering RNAs (siRNAs). Firstly, through an extensive screening approach in search of effective siRNAs targeting the HIV-1 promoter, the novel siRNA-143 was discovered, and observed to be able to potently supress HIV-1 for a prolonged time. A comprehensive evaluation of its mode of action, in comparison to the previously characterised siRNA-PromA, further confirmed siRNA-143 indeed induced the characteristic epigenetic marks of TGS, H3K27me3 and H3K9me3, at the HIV-1 promoter, with predominantly undetectable off-target effects. Secondly, by using a model of HIV-1 latency that strongly responded to a variety of stimuli, it was demonstrated that stable expression of siRNA-143 or siRNA-PromA robustly prevented HIV-1 from reactivation despite supra-physiological or -pharmacological doses of stimuli. Finally, disruption of induced TGS in siRNA-143 or siRNA-PromA stable-expressing Jurkat E6 cells through infection with excessive amounts of HIV-1, verified the siRNAs execute a strong and specific pressure on viral replication and that escape mutants can develop under this extreme circumstance of virus challenge.This thesis identified the novel siRNA-143 target, which has the potential to be used in a single or combinatorial gene therapy approach with siRNA-PromA, for the treatment and prevention of HIV-1 infection and more importantly to prevent reactivation from latent reservoirs