HIV: an endless fight with a virus?

Wim Trypsteen

HIV Translational Research Unit, University of Ghent, Belgium

The human immunodeficiency virus (HIV) has infected over 70 million people worldwide and has been responsible for more than 35 million deaths since the start of the pandemic. In addition, HIV still accounts for approximately two million new infections every year and therefore remains a persistent global health threat [1]. Since its isolation in 1983 and its identification as the causative pathogen for the acquired immunodeficiency syndrome (AIDS), HIV researchers have been challenged with understanding its life cycle, transmission, structure and pathogenic properties in order to overcome this chronic viral disease [2]. Initial research culminated in the production and use of the first antiretroviral drugs (ARVs) in the early 1990s and dramatically improved patient outcome, quality of life and survival [3].

However, despite the success of two decades of newly developed and improved antiretroviral therapy, it still remains impossible to completely eradicate HIV from the human body. As a consequence, patients are condemned to life-long medicinal treatment with its associated adverse events, emerging resistance and high cost. The most important reason for this incomplete clearance and distinctive treatment burden is the existence of a latent reservoir where HIV finds itself in a dormant state integrated in the hosts’ cellular genome, invisible to ARVs and capable of initiating new infection [3].

Therefore, the quest to find an HIV cure is fully ongoing and multiple alternative strategies are being explored. One such promising strategy is targeted gene editing with the clustered regularly interspaced short palindromic repeats with/and CRISPR-associated genes (CRISPR-Cas) system. This editing system is based on a unique antiviral defence system in bacteria and acts as a scissors to introduce DNA mutations/breaks in pathogenic viruses to render them deficient. Researchers have now adapted this system into a modifiable form to target any desired DNA sequence and in this way have opened doors for  HIV cure research to use this natural occurring antiviral defence system against HIV [4,5].

HIV cure research and CRISPR-Cas9 strategies

ARVs have proven their use in blocking different phases of the HIV life cycle and are crucial for controlling HIV infection. None the less, to clear HIV from the body, HIV cure research has to go one step further and aim its arrows at the latent reservoir. In this context, two possible cure scenarios are proposed: a functional cure where HIV is rendered functionally deficient so that patients can control the infection without further therapy (but HIV remains in the human body) and an eradication or sterilising cure where HIV-infected cells are fully cleared from the human body [3].

Currently, two CRISPR-Cas9 strategies are described to aid in the quest for an HIV cure. A first strategy is called viral deactivation where the HIV long terminal repeat (LTR) region is targeted and disrupted to inhibit the transcriptional start of viral replication. The result is that the HIV genome cannot produce its own mRNA/proteins and is not capable of assembling new virions. A second strategy is viral gene disruption where HIV genes are cut out so that no intact or complete viral genome can be formed [5].

CRISPR-Cas9 in action

First proof that CRISPR-Cas9 gene editing could be used to render HIV deficient was provided by researchers at the Laboratory of Viral Pathogenesis, Kyoto University, Japan [6]. They successfully targeted the LTR region of an integrated HIV reporter virus with CRISPR-Cas9 and showed that HIV transcription was deactivated in the main fraction of the cells. Also, in the context of HIV latency, CRISPR-Cas9 showed potential because it could inhibit viral gene expression upon reactivation of the latent virus. Further evidence to confirm these results came from Hu et al. and Zhu et al. These researchers showed that all viral replication and production could be completely blocked by combining multiple LTR-targeting single-guide RNAs (sgRNAs)[7,8]. Interestingly, Hu et al. demonstrated that CRISPR-Cas9 had the same inhibitory effect when it was introduced prior to HIV infection, making cells resistant to HIV infection.

Recently, a further evaluation of CRISPR-Cas9 has been published by Belmonte and co-workers in Nature Communications [9]. The Belmonte group elucidated that CRISPR-Cas9 can also target non-integrated forms of HIV and therefore confirmed the prophylactic antiviral defence system prior to integration. Moreover, experiments were conducted in primary CD4 T cells and haematopoietic stem cells, moving the CRISPR application closer to more physiologically relevant settings.

CRISPR-Cas9 as an HIV drug: the holy HIV grail?

The studies performed so far conclude that CRISPR-Cas9-mediated defence is successful in an HIV cure strategy and that viral deactivation by targeting the LTR proves most efficient. In addition, HIV infection could be blocked when cells are immunised with CRISPR-Cas9, opening doors for a prophylactic application. However, the major risks for implementing CRISPR-Cas9 gene-editing systems as a drug are the adverse consequences of off-target effects caused by introducing involuntary double-strand breaks in the human genome. This has to be closely monitored and it is reassuring to see that these studies show that until now CRISPR-Cas9 is well tolerated by the cells and no off-target cuts have been found.

With the discovery of CRISPR-Cas systems, the field of gene editing is evolving rapidly. In a next phase, researchers are starting to adapt CRISPR-Cas9 molecules to specifically alter the epigenetic state of genes. As a result, the epigenetic environment of the target DNA can be altered, rather than introducing double-stranded DNA breaks. In this way CRISPR-Cas systems could offer a new HIV eradication strategy by reactivating latent virus and  circumventing possible off-target cutting.

All these findings renew hope, and show promise that an HIV cure might one day be possible. However, a long road still lies ahead before CRISPR technology can be used in the clinic (i.e. efficient in vivo delivery systems, clinical trials). Therefore, it will remain to be seen whether this altered form of an ancient bacterial defence system gives us an edge over HIV. It certainly looks promising, as the fight continues to tackle one of our most notorious pathogens.

References

1.                 UNAIDS. Global AIDS epidemic facts and figures. Fact sheet 2014. 2014. Available at: www.unaids.org/en/resources/documents/2014/20140716_FactSheet_en.pdf (accessed May 2015).

2.          Barre-Sinoussi F, Chermann JC, Rey F et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983; 220: 868–871.

3.                 Deeks SG, Lewin SR, Havlir DV. The end of AIDS: HIV infection as a chronic disease. Lancet 2013; 382: 1525–1533.

4.                 Cong L, Ran FA, Cox D et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819–823.

5.                 Saayman S, Ali SA, Morris KV, Weinberg MS. The therapeutic application of CRISPR/Cas9 technologies for HIV. Expert Opin Biol Ther 2015; 15: 819–830.

6.                 Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 2013; 3: 2510.

7.                 Zhu W, Lei R, Le Duff Y et al. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology 2015; 12: 22.

8.                 Hu W, Kaminski R, Yang F et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1infection. Proc Natl Acad Sci U S A 2014; 111: 11461–11466.

9.                 Liao HK, Gu Y, Diaz A et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat Commun 2015; 6: 6413.