GENE EDITING TECHNOLOGIES BASED ON CRISPR-CAS9 SYSTEM FOR THE TREATMENT OF HIV: STUDIES IN VITRO AND IN VIVO

ABSTRACT Retroviruses include two subfamilies, orthoretrovirinae and spumaretrovirinae. The human immunodeficiency virus 1 (HIV-1) belongs to the orthoretrovirinae subfamily and is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1 infects 36.9 million people and 2.6 million children throughout the world. During primary infection HIV converts its RNA genome into DNA, which integrates into the host genome. The cellular environment present at the site of the integration may influence viral transcriptional activity. The sequestration of host transcription factors, the presence of repressor of transcription and nucleosomes and epigenetic modifications on the HIV promoter, or transcriptional modification of Tat are all conditions that influence the formation of long term viral reservoirs. The use of antiretroviral drugs has been proposed as a functional cure to control the viral load but lacks the ability to obtain viral sterilization since antiretroviral drugs can not remove the virus from latently infected cells and anatomical sanctuaries such as brain and the gut associated lymphoid tissue. In recent years gene editing strategies have been largely employed for the treatment of HIV-1. In this present study, we aimed to discover an innovative CRISPR technology specific against the HIV viral genome that can target latently infected cells and be delivered in all tissues. Initially, we performed in vitro analysis, where TZMB-1 cells containing the luciferase gene under the control of LTR were transfected with pCMV-Tat and three plasmids harboring Cas9 under the control of different regions of LTR promoter to evaluate by western blot analysis the minimal LTR promoter region able to activate Cas9 in presence of Tat. TZMB-1 cells were transduced with the lentiviruses, harboring Cas9 or gRNAs specific for the promoter region, and infected with HIV-1 to test, by PCR and luciferase assay, the presence of gene editing. Then PCR and flow cytometric analyses were performed on 2D10 cells, HIV-1 latently infected cells, to test the ability of Tat-induced Cas9 to excise viral DNA. Subsequently, was evaluated the ability of Cas9, in presence of gRNAs, to protect Jurkat cells from viral reinfection by eliminating the virus during the early stages of infection. The second part of our study was performed to test Cas9 and gRNAs specific for HIV-1 LTR and Gag regions in vivo using adeno-associated virus (AAV) as the delivery system. Tissues of HIV-1 transgenic mice and rats and humanized mice were provided by collaborators for evaluation by analyzing DNA and RNA for the presence of viral editing. Results from in vitro experiments showed the ability of Tat to activate the minimal promoter LTR, inducing gene editing in TZMb-I and 2D10 cells. The presence of Cas9 in Jurkat cells induces a reduction of viral RNA of 96% at five days from infection. Studies in vivo showed the presence of viral excision in blood, heart, liver, lung, kidney, spleen and brain in transgenic mice and a reduction of viral RNA in the blood of transgenic rats. Excision of HIV-1 was reported in the spleen, gut associated lymphoid tissue, liver, kidney, lung and brain of humanized mice with complete viral sterilization in 29% of the infected animals that were subjected to antiretroviral treatment. The absence of off-target effects was confirmed by deep sequencing analysis. Together, these data show the ability to create a Cas9-inducible system generating negative feedback against the virus while avoiding persistent Cas9 expression in the cells. The use of AAV vectors in vivo showed high delivery efficiency in the different tissues, obtaining viral sterilization for the first-time. Further experiments on humanized mice and SIV infected monkey models will show this approach combined with ART therapy may have important application for HIV-1 sterilization in clinical trials.