Durable Control of HIV-1 Using a Staphylococcus aureus Cas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

Abstract

CRISPR/Cas9 gene editing has the potential to revolutionize the clinical management of HIV-1 infection, and may eliminate the need for antiretroviral therapy (ART). Current gene therapies attempt to either excise HIV-1 provirus or target HIV-1 entry receptors to prevent infection of new cells. Using a viral dynamic model, we determined that combining these two interventions, in the presence or absence of ART, significantly lowers the gene editing efficacy thresholds required to achieve an HIV-1 cure. To implement this dual-targeting approach, we engineered a single lentiviral vector that simultaneously targets multiple highly-conserved regions of the provirus and the host CXCR4 coreceptor, and developed a novel coculture system enabling real-time monitoring of latent infection, viral reactivation, and infection of new target cells. Simultaneous dual-targeting depleted HIV-1-infected cells with significantly greater potency than vectors targeting either virus or host independently, highlighting its potential as an HIV-1 cure strategy.

High-resolution Inference of Multiplexed Anti-HIV Gene Editing using Single-Cell Targeted DNA Sequencing

“…According to mathematical models, more than 99% of total provirus in people living with HIV needs to be inactivated to achieve a functional cure. 16 Provirus inactivation requires editing on several sites due to viral genetic diversity and plasticity, which may result in the generation of escape mutants considering the exceptional adaptability of HIV. 37 Moreover, it is unlikely that 99% of infected cells will be targeted by the CRISPR-Cas9 system: some macrophages for example can infiltrate deep into tissues, where delivery of the CRISPR payload can be very challenging.…”

“…In this case, mathematical models predict that inactivating provirus in 60% of the infected cells and reducing the pool of susceptible cells by 68% is enough to achieve a functional cure. 16 So far, no method has been proposed to rigorously assess the frequencies of such events in vitro or in vivo. Here, we demonstrate that single-cell encapsulation and targeted next generation sequencing, in combination with a specific bioinformatic pipeline, is a reliable and powerful method to characterize antiviral gene editing in both cell lines and primary cells.…”

“…Thirty-six primer pairs cover the HIV genome from position 30 to position 9516 based on the HIV HXB2 reference genome (Figure 1). This includes the 5' long terminal repeat (LTR) region, which has been targeted specifically in several HIV gene therapy strategies, 16,[26][27][28][29][30]44 allowing detection of edits in highly conserved regions such as the Primer Binding Site (PBS) and packaging signals (PSI sequences).…”

“…Drawing from lessons learned during the development of combination ART, a minimum of three distinct genome sites may need to be targeted to prevent evolution of resistance. 16 Similarly, the neutralization of a single HDF supporting HIV replication is unlikely to completely protect cells from infection, suggesting that multiple HDFs will likely need to be targeted simultaneously to achieve a functional or sterilizing cure for HIV infection. 16 Even when considering a single HDF locus, genes exhibiting codominance between alleles will likely require biallelic knockout to fully eliminate generation of the encoded protein and, in this manner, block viral infection.…”

“…[12][13][14] The Cas9 enzyme interacts with the scaffold sequence of a guide RNA (gRNA) complementary to a target sequence, enabling knockout of a targeted gene in a highly specific manner. 15 HIV gene therapy-based cure strategies in development typically aim to excise the HIV provirus directly, [16][17][18][19][20][21][22] or target host dependency factors (HDFs) including viral entry receptors that support viral persistence. [23][24][25] In recent years, a number of preclinical HIV cure studies have showed promising results using this technology.…”