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Elimination of HIV-1 Genomes from Human T-lymphoid
Cells by CRISPR/Cas9 Gene Editing
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http://www.nature.com/articles/srep22555
March 4 2016
Our findings address key barriers to this goal, as we developed CRISPR/Cas9 techniques that eradicated integrated copies of HIV-1 from human CD4+ T-cells, inhibited HIV-1 infection in primary cultured human CD4+ T-cells, and suppressed viral replication ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells of HIV-1+ patients. They also address a further key issue, providing evidence that such gene editing effectively impedes viral replication without causing genotoxicity to host DNA or eliciting destructive effects via host cell pathways. Prior studies using gene editing based on zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems prompted much interest in their potential abilities to suppress viral infection, either by altering virus receptors or introducing mutations in the viral genome (for review see26,30). All these studies suggest that gene editing strategies can be engineered for targeting specific regions of the viral genome and once efficiently delivered to infected cells, their robust antiviral activity effectively suppresses viral replication. However, there are several important issues that require close attention including the careful design of the targeting strategy that achieves the highest levels of specificity and safety with optimum efficiency of editing.
When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical to understand that not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection. Thus, there is a high likelihood that rebounding viral infections will be contained by the resistant cells. Still, some formidable challenges remain before this type of strategy can be implemented. First, it will be important to maximize elimination of viral sequences from patients. This will require analysis of the HIV-1 quasi-species harbored by patients’ CD4+ T-cells and design of suitable, i.e. personalized CRISPRs. Second, improved delivery of CRISPR/Cas9 will be required to target the majority of circulating T-cells. In summary, our novel ex vivo findings that our lentiviral delivery-based approach reduced HIV-1 DNA copy numbers and protein levels in PBMCs of HIV-1 infected patients provides strong proof-of-concept evidence that CRISPR/Cas9 can be effectively utilized as part of HIV Cure strategies.
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Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing
Abstract
We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5' and 3' LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.
Introduction
AIDS remains a major public health problem, as over 35 million people worldwide are HIV-1-infected and new infections continue at steady rate of greater than two million per year. Antiretroviral therapy (ART) effectively controls viremia in virtually all HIV-1 patients and partially restores the primary host cell (CD4+ T-cells), but fails to eliminate HIV-1 from latently-infected T-cells1,2. In latently-infected CD4+ T cells, integrated proviral DNA copies persist in a dormant state, but can be reactivated to produce replication-competent virus when T-cells are activated, resulting in rapid viral rebound upon interruption of antiretroviral treatment3,4,5,6,7,8. Therefore, most HIV-1-infected individuals, even those who respond very well to ART, must maintain life-long ART due to persistence of HIV-1-infected reservoir cells. During latency HIV infected cells produce little or no viral protein, thereby avoiding viral cytopathic effects and evading clearance by the host immune system. Because the resting CD4+ memory T-cell compartment9 is thought to be the most prominent latently-infected cell pool, it is a key focus of research aimed at eradicating latent HIV-1 infection.
Recent efforts to eradicate HIV-1 from this cell population have used primarily a “shock and kill” approach, with the rationale that inducing HIV reactivation in CD4+ memory T-cells may trigger elimination of virus-producing cells by cytolysis or host immune responses. For example, epigenetic modification of chromatin structure is critical for establishing viral reactivation. Consequently, inhibition of histone deacetylase (HDAC) by Trichostatin A (TSA) and vorinostat (SAHA) led to reactivation of latent virus in cell lines10,11,12. Accordingly, other HDACi, including vorinostat, valproic acid, panobinostat and rombidepsin have been tested ex vivo and have led, in the best cases, to transient increases in viremia13,14. Similarly, protein kinase C agonists, can potently reactivate HIV either singly or in combination with HDACi15,16. However, there are multiple limitations of this approach: (i) since a large fraction of HIV genomes in this reservoir are non-functional, not all integrated provirus can produce replication-competent virus17; (ii) total numbers of CD4+ T cells reactivated from resting CD4+ T cell HIV-1 reservoirs, has been found by viral outgrowth assays to be much smaller than the numbers of cells infected, as detected by PCR-based assays, suggesting that not all cells within this reservoir are reactivated18; (iii) the cytotoxic T lymphocyte (CTL) immune response is not sufficiently robust to eliminate the reactivated infected cells19 and (iv) uninfected T-cells are not protected from HIV infection and can therefore sustain viral rebound.
These observations suggest that a cure strategy for HIV-1 infection should include methods that directly eliminate the proviral genome from the majority of HIV-1-positive cells, including CD4+ T-cells, and protect cells from future infection, with little or no harm to the host. The clustered, regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease has wide utility for genome editing in a broad range of organisms including yeast, Drosophila, zebrafish, C. elegans, and mice, and has been applied in a broad range of in vivo and in vitro studies toward human diseases20,21,22,23,24. Recently we modified the CRISPR/Cas9 system to enable recognition of specific DNA sequences positioned within the HIV-1 promoter spanning the 5' long terminal sequence (LTR)25,26. Using this modified system, we now demonstrate excision of integrated copies of the proviral DNA fragment from a latently HIV-1-infected human T-lymphoid cell line, completely eliminating HDAC inhibition-elicited viral production. Results of whole-genome sequencing and comprehensive bioinformatic analysis ruled out any genotoxicity to host cell DNA. Further, we found that lentivirally-delivered CRISPR/Cas9 reduces viral replication upon HIV-1 infection of primary cultured CD4+ T-cells. The results point toward this approach as a promising potential therapeutic avenue to eradicating HIV-1 from T reservoir cells of host patients, to prevent AIDS re-emergence.
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