Antiretroviral Gene Therapy

The discovery of the CRISPR/Cas9 system has undeniably revolutionized the world of gene therapy. We are developing a CRISPR platform to excise the proviral HIV genome as an HIV eradication approach. However, several challenges need to be addressed before this strategy can be tested in the clinic: First, guide RNAs (gRNAs), the RNA molecules that direct Cas9 to its editing target, must account for circulating strain diversity within and across HIV-infected individuals; second, multiple sites must be targeted simultaneously in order to guard against Cas9-induced viral escape mutants; and third, off-target editing of the host genome must be eliminated. Working with Dr. Bette Korber at Los Alamos National Lab, we adapted her HIV vaccine immunogen design algorithm to identify highly-conserved gRNA targets across the HIV genome, and are now targeting these sites using a lentiviral vector engineered in our lab that encodes an “enhanced” Cas9 nuclease with increased specificity. To simultaneously target several genome sites, we are utilizing polycistronic tRNA-gRNA (PTG) multiplexing and self-cleaving ribozymes.

As of now, we have identified and tested 29 Cas9 gRNAs that were highly conserved across the entire HIV-1 M-group. Five of the 29 gRNAs (highlighted in red with reference to the HIV genome) allowed for highly efficient editing of the HIV provirus, targeting >50% of integrated genomes in CD4+ T cells:

HIV genome editing across sites greatly reduced the production and infectivity of progeny virus upon latency reversal, in some cases completely eliminating detectable virus production. Importantly, no off-target editing has been observed. As avoiding off-target effects is critical to the clinical potential of any CRISPR approach, we have just established a collaboration with the Bay Area startup Mission Bio to apply their Tapestri single-cell DNA analysis platform to detect Cas9-induced mutations with unprecedented sensitivity.