Utilizing the CRISPR/Cas9 system to cure HIV-1 infection

Alexander Glen Allen

doi:10.17918/00001028

Human immunodeficiency virus type 1 (HIV-1) remains a large public health problem with over 37 million people currently infected with the virus. In the years before antiretroviral therapy (ART) became the standard of care treatment, HIV-1 was considered a death sentence. ART saved the lives of countless people around the world. While ART has been a transformative therapy, it is unable to cure HIV-1 infection. This means that someone who is infected with HIV-1 has to remain on ART for the rest of their lives. This has caused a number of investigators to evaluate cure strategies for HIV-1. One of the strategies being evaluated has been gene editing using the CRISPR/Cas9 system. The CRISPR/Cas9 system works by a two-component system, there's a guide RNA (gRNA) which binds with complementarity to its target sequence and the Cas9 endonuclease which catalyzes a double-stranded DNA break (DSB). The CRISPR/Cas9-based system has been shown to be effective at binding to and editing and/or excising the integrated HIV-1 provirus in vitro and in vivo. Most of the studies reported haven't been able to track which cells received Cas9 and which cells were still expressing viral proteins to determine if detectable virus is due to lack of delivery or lack of editing. To address this issue, the studies described in this thesis use a two-color system to track which cells received Cas9 therapy and which cells are expressing viral proteins. The data clearly showed that guide RNAs (gRNAs) targeting the transactivation response element (TAR) region of the HIV-1 promoter termed the long terminal repeat (LTR) are effective at stopping viral transcription, with multiple assays demonstrating >95 percent reduction in HIV-1 transcription. Conversely, gRNAs targeting conserved areas of the U3 region were largely ineffective, even when delivered to cells, demonstrating that functional location plays a role in efficacy. In addition, it was observed that other highly conserved regions of the HIV-1 genome including specific locations in Tat were effective in a T-cell model of HIV-1 latency. Given these results, further studies targeted the LTR to identify and test gRNAs that target low diversity regions and have a functional impact on HIV-1 transcription. Although conventionally unfavorable due to potential impact on human promoters, results shown here utilized a computational pipeline and identified gRNA sequences that were predicted and shown experimentally to inactivate HIV-1 transcription by targeting the NF-[kappa]B binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad spectrum (predicted coverage of many known viral sequence quasispecies). Finally, while these studies showed gRNAs were very effective, reduction did not reach 100 percent. It was hypothesized this may be due to the fact that two nucleosomes (nuc-0 and nuc-1) reside within the LTR. The nucleosomes reside where several of the gRNAs target the HIV-1 genome. Studies here showed that chromatin modifying drugs could open the target site for LTR-targeting gRNAs to bind and cleave in the absence of LTR-driven gene activation. Collectively, these thesis studies have provided important results that support the continued utilization of the CRISPR/Cas9-based gene editing system as a potential HIV-1 cure strategy.

Utilizing the CRISPR/Cas9 system to cure HIV-1 infection

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Utilizing the CRISPR/Cas9 system to cure HIV-1 infection

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