Guiding inactivity of latent HIV reservoirs

From the Jerome lab, Vaccine and Infectious Disease Division

Today, many gene editing techniques use the CRISPR/Cas9 systems. This technique delivers Cas9 nuclease complexed with guide RNA (gRNA) into cells, allowing for cutting of the genome at a specific location that is based on homology with the gRNA. This allows for facile targeted gene editing and has many applications. One such application is HIV, where the goal is to disrupt latent viral genomes already integrated into host DNA in sites that would render them replication incompetent. However, finding universal gRNA to target HIV will be challenging because the virus has such a huge global and intra-host variability. With a latent virus like HIV, complete protection cannot be achieved unless every viral genome is targeted, leaving viral genomes untouched allows for viral rebound. A group led by Dr. Jerome in the Vaccine and Infectious Disease Division at Fred Hutch has been working to target multiple conserved sites that could be potential targets. Their work was recently published in BMC Biology.

The first step in CRISPR/Cas9 success is to identify potential gRNA targets. By using published sequences the group looked for conserved sequences within HIV genes. Each potential hit was ranked by prevalence within an alignment of group M sequences (the major group responsible for global epidemics) and unique gRNA targets were identified for each genomic site. These targets were then tested for predicted activity and ranked against conservation within each group. In order to test predicted activity in vitro, the group used a GFP fusion reporter. This resulted in a weak correlation between predicted and in vitro activity. In addition to activity, the identification of the gRNA targets was determined within HIV sequences from 10 patient samples. This allowed for identification of an intra-host consensus. This data showed that many targets were found within each patient and five target sequences were identified in all 10 patients. One of the five was also highly conserved (87%) within the global population.

Graph shows the predicted effect of therapy on viral reservoir size with varying degrees of efficacy (€). Figure provided by Dr. Roychoudhury

Lastly the group developed a mathematical model to simulate the effect on size and composition of the latent reservoir after weekly dosing of CRISPR/Cas9 therapy. Their model predicted that a single site gRNA was not sufficient at any efficacy/treatment number to achieve a functional cure. Data also suggested that deficient targeting would result in escaped viral genomes that could lead to viral rebound. However, if gRNAs are multiplexed and given together, it is possible (in the model) to achieve 100% coverage after as few as five treatments (see figure). According to first author Dr. Roychoudhury, “By identifying conserved CRISPR target sites at the group and subtype level for HIV, we showed that broadly (globally) conserved sites also tend to be conserved within the host’s reservoir. We used a mathematical model to examine the impact of both target site conservation as well as cutting efficiency on reservoir reduction to ask how many doses of a hypothetical CRISPR-based therapy would be needed to deplete the reservoir to cure thresholds.” In the future, “We hope to apply these methods to identify target sites for other diseases for which gene editing could produce a functional cure, such as HSV and HBV” stated Dr. Roychoudhury.

Roychoudhury P, De Silva Feelixge H, Reeves D, Mayer BT, Stone D, Schiffer JT, Jerome KR. 2018. Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir. BMC Biol, 16(1), 75. PMCID: PMC6040082.

This work was supported by the National Institutes of Health.

Fred Hutch/UW Cancer Consortium faculty members Keith Jerome (Fred Hutch) and Joshua Schiffer (Fred Hutch) contributed to this research.