A CRISPR Approach for Reactivating Latent HIV-1

Combination antiretroviral therapy (cART) has significantly improved the clinical outcome for HIV-infected patients. Although the treatment is highly effective in driving the viremia to an undetectable level, it does not represent a cure, because a long-lived reservoir of latent virus remains. Persistent proviruses persist in latently infected resting memory CD4⁺ T cells and are found in many tissues, including blood, lymph nodes, central nervous system, and the gut. Eradication of the latent reservoir and the ability to truly cure an infected patient remain a significant challenge.¹ Many options have been explored to reverse latency, and a pharmacological approach is the current favorite. In this issue, Saayman et al.,² Limsirichai et al.³, and Ji et al.⁴ each report their early explorations of a different approach: the use of a modified CRISPR/Cas9 system to reactivate latent HIV-1. These reports provide strong evidence that application of CRISPR/Cas9 can induce specific and effective reactivation of a latent HIV-1 promoter.

The persistence of integrated provirus, despite aggressive therapy with antiretrovirals, represents a major barrier to truly curing HIV infection. The reservoir of latently infected cells is established early in infection within long-lived memory CD4⁺ T cells and persists throughout the life of the patient.^5,6,7 During this latent state, multiple factors associate with the viral long terminal repeat (LTR) and repress transcription.^8,9 Among these factors are the histone deacetylases and other chromatin-modifying proteins that alter the chromatin structure of the integrated viral promoter to prevent access and binding by positive transcription factors.¹⁰ These latently infected cells produce little to no viral proteins and thus escape easy identification as harboring the virus. Latently infected cells do not display any specific target for the immune system or antiviral drugs.

Current attempts to reactivate latent HIV provirus involve the use of protein kinase C activators (i.e., prostratin and bryostatin) and histone deacetylase inhibitors (vorinostat and panobinostat).¹¹ These agents have been shown to efficiently reactivate latent virus and have been proven safe. However, initial attempts to reduce the size of the latent reservoir using such reactivating agents have been unsuccessful.¹² Therefore, a new strategy has been adopted, termed “shock and kill,” in which reactivation will be followed by a different treatment to kill the infected cell. Patients stay on cART to prevent the spread of infection to new cells. It is predicted that activation of virus will coincide with expression of markers that can be used to kill the infected cell either immunologically or through pharmaceutical intervention. Concerns remain and include the inability of clinically achievable levels of drug to reactivate all latent virus, increased susceptibility of T cells to infection following reactivating treatment, and suppression of cytotoxic T lymphocytes following treatment with nonspecific reactivating agents.^13,14,15 Therefore, new approaches that can trigger HIV reactivation with high specificity and low toxicity are needed. The CRISPR/Cas9 system has been shown to be highly specific and may provide an avenue by which greater levels of activation can be achieved without detrimental off-target effects.

Recent approaches to genetic engineering have created systems comprising diverse site-specific DNA-binding domains fused to various nucleases to allow relatively specific gene editing. The modular structure of two popular systems, transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), yields tools in which the nuclease domain can be deactivated and fused to a variety of alternative effector proteins (i.e., activators, repressors, transcription factors) to modify a specific locus, such as the promoter of the HIV provirus. However, the cost and complexity of protein engineering limits the potential of these systems. The recently developed CRISPR/Cas9 system provides a superior alternative to the existing protein-based approaches. The Cas9 protein is targeted to specific loci via co-delivery of an artificially designed single-guide RNA (sgRNA), making this a much more facile system to manipulate and exploit. The CRISPR/Cas9 system has previously been employed to disrupt the HIV-1 genome but not to reactivate latent virus.¹⁶

The three labs employed slightly different approaches in applying CRISPR/Cas9 technology to the reactivation of HIV-1. All of the groups made use of a nuclease-deficient Cas9, referred to as dCas9, which was coupled to different transactivating domains (Table 1 and Figure 1). There are multiple considerations when designing an sgRNA to guide Cas9: inclusion of a terminal protospacer adjacent motif (a short sequence of nucleic acids at the end of the sgRNA that is absolutely required for function) and the avoidance of off-target effects being the most critical. The authors of the studies designed very different sgRNAs that span the U3 (viral promoter) and R (transcription start site) regions of the viral LTR (Table 2). Common themes are found in the articles, such as the strong activation potential of sgRNAs that direct the modified dCas9 to regions in close proximity to nuclear factor-κB (NF-κB) transcription factor binding sites within the LTR. The utility of targeting a transactivating complex to this region (approximately 100 bases upstream of the transcription start site) has been previously demonstrated in other cellular promoters. Transcription of messenger RNA is a highly regulated process involving multiple factors that serve to recruit and activate the RNA Pol II complex. Production of messenger RNA requires that the correct factors are located at the right position at the right time. The observed hotspot at 100 bases upstream of transcription start reveals a general rule about activation of the Pol II complex and may be the reason why the virus has evolved sequence that recruits NF-κB to this specific location.

Table 1. Comparison of activation domains used.

Figure 1.

Different strategies used to reactivate via CRISPR/dCas9. To function as an activator the nuclease deficient Cas9 must be fused to an activating domain. In these studies multiple activating domains are used. (a) dCas9-VP64 is the dCas9 protein fused to the VP64 (four tandem copies of the Herpes Simplex Virus VP16 activator) to initiate transcription through recruitment of TATA Binding protein, TFIIB and the CBP/P300 chromatin remodeling complex. (b) dCas9 can also be engineered to include histone remodeling proteins such as P300. The configuration induces modification of the chromatinized DNA to a state more inductive of transcription. (c) dCas9-VPR is comprised of VP64 and two new domains, P65 that activates NFkB and the Epstein-Barr Virus transactivator (Rta) that recruits Sp1 and Oct1. (d) Modification of the guide RNA, termed sgSAM, and co-delivery of the MS2-P65-HSF1 allows the modified guide RNA to recruit the dCas9 protein as well as MS2-P65-HSF1 protein. This additional fusion protein activates NFkB (thru P65) and adds the ability of the heat shock factor 1 (HSF1) to recruit Sp1. (e) the SunTag system contains a dCas9 fused to a repeated epitope tag. This repeated tag allows the dCas9 protein to recruit twenty-four copies of an activating protein made up of green-fluorescent protein (for visualization) and the VP64 activator.

Table 2. Summary of effectiveness in targeting various regions of HIV-1 for activation.

The commonly used JLat model, which simulates HIV-1 latency by using Jurkat T cells containing integrated but transcriptionally inactive proviruses, is employed by each laboratory group, allowing some direct comparison between the findings. Examination of the reactivation of transcription in JLat clones 9.2 and 10.6 provides convincing evidence that activation by a modified CRISPR/dCas9 system can be at least as effective as vorinostat. Limsirichai et al.³ additionally show evidence that this approach synergizes with vorinostat and prostratin treatment for increased activation. Saayman et al.² and Ji et al.⁴ both provide analysis of off-target effects and conclude that their approaches do not cause significant changes in gene expression or toxicity in the infected T cells.

Examination of the three bodies of work reveals interesting differences as well. Each group chose a slightly different system for initial screening, often relying on alteration of a transiently transfected LTR reporter system. Saayman et al. describe the use of a LTR-mCherry-IRES-Tat (LChIT) latency reporter system.² This construct expresses the mCherry reporter under control of the viral promoter and includes a Tat positive-feedback loop that mimics the reactivation seen with intact provirus. The authors use LChIT to construct a population of latency reporter cells, a technique that is of potential use in myriad latency studies. Ji et al. employ a SunTag system (Figure 1) that allows a single dCas9 protein to recruit over 20 copies of the VP64 activation domain for increased activity.⁴ Limsirichai et al. use the added approach of constructing a dCas9 fused to the histone acetyltransferase protein P300.³ This approach is designed to acetylate the histones and open the chromatin to allow transcription to occur. Using dCas9-P300 they showed that targeting the nucleosome known as Nuc0 in the LTR can drive activation, supporting the intriguing conclusion that remodeling of this nucleosome may be an alternative approach to reversing latency. This is an interesting finding, as most work to date has focused on the nucleosome that is associated with the transcription start site (termed Nuc1) and not Nuc0, which is found upstream.

These innovative studies offer good insight into the prospects of employing a CRISPR-mediated latency-reversing approach. The key benefits are the specificity, potency, and flexibility of these systems. However, further challenges remain. As a first step, lentiviral delivery systems for dCas9, such as those constructed by both Ji et al. and Limsirichai et al., need to be tested in a primary cell latency model or by a quantitative viral outgrowth assay on patient samples. Indeed, as with all gene therapy approaches, delivery is a consideration that must be addressed for any therapeutic application of the CRISPR/Cas9 system. The big question is whether activation induced by this system can induce useful cell killing. The specificity of CRISPR/Cas9 probably means that these approaches will not inhibit cytotoxic T-lymphocyte responses, as was observed with vorinostat, thus rendering these systems ideal candidates to be employed alongside immune-mediated methods of clearance.