This application develops a targeted delivery system for the Cas9/guide RNA directed against HIV into both latent and persistently infected cells. Although highly active antiviral therapy (HAART) has greatly increased the life-span of HIV infected individual, it requires life-long treatment due to the presence of integrated copies of the viral genome in long-lived reservoirs, including resting CD4+ memory T cells. Reactivation from latency results in releasing the replication-competent virus, replenishing the viral load within the individual. Excision of the integrated virus is not part of the viral life-cycle. In recent months, the possibility of excisingor inactivating the integrated HIV genome through the use of the CRISPR/Cas9 prokaryotic immune system has become a feasible approach, with the first demonstration of effective excision across LTR sequences with limited off-target effects. The key bottleneck now is the development of means to deliver this system specifically to the HIV infected cells. The approach proposed applies two emerging areas of research of our laboratory. The first develops Murine Leukemia Virus (MLV) virus-like particles (VLP) for the delivery of proteins, rather than genes into cells. We have successfully delivered functional nuclear transcription factors and toxic proteins into cells. This system will be modified to incorporate the Cas9/guide RNAs into MLV VLPs. The second specific aim develops a modified Env system that allows entry through scFv moieties. This approach displays the scFv on the outer viral surface and is stoichiometrically linked with the Env proteins. Selective scFv have been identified which will target both latent as well as persistently infected cells. If successful, the study could lead to the elimination of the viral genome from targeted infected cells. Key to this approach is the inherent versatility of the system, allowing for replacement of both the guide RNA targets as well as the scFv used to direct entry. This system could be generalized and expanded to address a wide range of applications and diseases.
A functional cure for AIDS requires the disabling the proviral copies of the HIV genome within the human DNA. These studies develop a novel mechanism to deliver reagents capable of cleaving and/or excising the integrated viral DNA from latently and persistently infected cells.
