Latently infected cell models stably transduced with an inducible HIV-1 targeting gene editing system: characterisation and effect on viral replication

HIV-1 infection is an ongoing pandemic that currently counts almost 38 million living patients, being thus classified by the World Health Organization as one of the ten greater global health threats. Antiretroviral therapies (ARTs) are very effective in keeping the viremia levels under a detectable threshold, significantly improving patients’ survival and life-quality; however, with the classical pharmacological approach, it is impossible to reach and eradicate the proviral DNA that remains transcriptionally inactive at the level of the so-called HIV-1 latent reservoir. Thus, in case of treatment interruption, a viral rebound can be observed within few weeks, making it necessary for the patient to proceed with a life-long therapy. In recent years, gene editing tools have opened up new possibilities to promote the removal of the virus from the latent reservoir and, specifically, multiple studies have shown, both in vivo and in vitro, promising results using the CRISPR-Cas9 systems. The current study is part of a wider proof-of-principle project that aims to develop a cell-activation-inducible CRISPR/Cas9 HIV-1 targeting vector, for the inactivation of the cellular CCR5 gene (coding for the host cell co-receptor) and of two viral functional genes (tat/rev and gag) selectively in reactivated latently infected cells. The peculiarity of the system’s design is thus the presence of an inducible promoter that regulates the expression of the nuclease to limit its editing effects to re-activated latent reservoir, with a consequent increase in selectivity and decrease of potential off-target effects. Following the system’s validation in a non-infected cell model (Human CD4+ T-lymphoblastoid Jurkat cells), in this project we tested the system’s functionality in latently infected cell models, specifically in U1 and J-Lat 10.6 cells, mirroring respectively the promonocytic and lymphoid reservoir found in patients on long-term HAART. We generated U1 and J-Lat clones stably transduced with the test vector (showing the Cas9 under the control of a previously characterized synthetic inducible promoter, NFAT-regulated), and we assessed the Vector Copy Number associate to each of them. Moreover, in U1 stably transduced clones, upon induced cellular reactivation with Bryostatin-1, we carried out molecular investigations of Cas9 expression (in terms of both mRNA and protein), then we confirmed the biological effect of the system, highlighting a reduction in HIV-1 replication upon cellular stimulation and, finally, we investigated at the genomic level the U1 gene edited cells to assess on-target effects.