The emerging field of CRISPR/cas9-based genetic engineering is an exciting frontier. With uses in almost all aspects of gene engineering and modification, some researchers are investigating the technology and its abilities to combat the human immunodeficiency virus (HIV). Since approximately 2013, research in the field has been mostly in vitro in nature, and is operating under the assumption that the disruption of HIV reservoirs can result in complete clearance of infection. This review outlines recent important milestones in the field, with three specific methods for reservoir elimination: HIV receptor disruption, HIV proviral disruption, and shock and kill-based methods. Research in the field is ongoing, with only a few studies currently in pre-clinical testing. From this timeline, it is likely that methods for the elimination of HIV in vitro will begin clinical trials in approximately 10-15 years, and that a true cure to HIV will be seen as a combination of various types of gene therapies, and antiretroviral pharmaceuticals.
The Human Immunodeficiency Virus (HIV) is a scourge that affects millions of people worldwide and causes widespread dysfunction of the immune system. Without treatment, the virus will cause clinical progression to a state known as acquired immunodeficiency syndrome (AIDS).9 A patient with AIDS, defined as having a CD4+ T-lymphocyte count of less than 200 cells / mL of blood, becomes susceptible to opportunistic infections and neoplasms that are regularly cleared by the immune system during regular surveillance.9 These opportunistic infections can easily maim or kill patients with the syndrome, making the contraction of HIV a potentially fatal condition. With treatment however, patients with HIV can lead full and relatively normal lives.7 The gold standard treatment is highly active antiretroviral therapy (HAART), consisting of a cocktail of drugs targeted to a patient’s specific viral strain. Most commonly nucleoside / nucleotide reverse transcriptase inhibitors, this regimen can be as simple as one pill a day, allowing patients who can afford the treatment to live normal lives.7 With success in treatment, a patient’s immune system will clear most HIV-infected cells in the body, bringing viral load (the amount of HIV viral RNA in the blood) to undetectable levels with current PCR-based diagnostic tests.7 While undetectable in this state, a patient is not cured of HIV infection, as proviral HIV DNA remains embedded in the nuclei of so called “reservoir” cells: areas of persistent HIV infection despite treatment.8
These reservoir cells are most notably found in the brain, gastrointestinal tract, lymph, and blood tissues, with a handful of other cell types found across the body.6 Reasons for persistent infection are unknown at best, with hypotheses focusing on drug penetration and proviral integration sites in specific cell types. What is theorized however, is that the elimination of these reservoirs can lead to an increased resistance of novel HIV infection, and in some cases the complete elimination of HIV from a system in vitro.1 The goal of using CRIPSR/cas9 technology in this field is therefore ultimately the elimination or modification of these reservoir cells, to remove, disable, or prevent novel infection of the HIV virus. The entire field operates on the assumption that the combination of current gold-standard treatment (HAART) and the elimination/disabling of reservoirs could be enough to effectively cure a patient of their HIV infection.
Research in this field has been highly varied, with most trials operating at the bench top stage of research. The modalities of reservoir elimination / disruption can be broadly classified into three areas:
This review focuses on primary research representing both the most recent innovations in the field, and those with the strongest scientific data to support HIV eradication in their respective models.
Xu et al is a promising trial in the pre-clinical stages, that has demonstrated efficacy in preventing novel HIV infection and the decrease of viral load.1 The Xu et al study successfully used a CRISPR/spCas9 (a modified cas9) system to target and alter the D32 mutation site in the CCR5 gene. The mutation was performed in a human hematopoietic stem cell (HSC) model, which when mutated and compared to controls showed full self-renewal and differentiation capabilities. This was of notable importance as it demonstrated that editing this specific mutation had no effect on regular stem cell homeostasis, making it a prime candidate for transplantation. Then engrafted into immunodeficient mice, the mutant HSC cells and their T-lymphocyte derivatives were able to confer resistance to HIV infection when challenged with a CCR5-tropic strain of HIV-1. Overall levels of HIV RNA were observed to be reduced following the viral challenge as compared to control (wild-type) CCR5 engrafted animals.
Similar results were seen directed towards CXCR4, another receptor protein HIV uses to enter a target cell.4 Targeting the conserved sites of the CXCR4 gene with a lentiviral vector, the study by Hou et al showed exceptional promise in not only the knocking down of the CXCR4 receptor, but in lowering rates of HIV-1 infection and proviral activity.4 Using a GHOST X4 cell line, ten sgRNA targets were chosen and modified with CRISPR/cas9 technology. Insertion/Deletions (INDELs) were confirmed with a T7 endonuclease assay, which showed a reduction in CXCR4 expression of 70-80% from controls. Hou et al then applied the same procedure to a GHOST X4 reporter line, a specific line with a green fluorescent protein (GFP) surface marker tied to HIV-1 Long Terminal Repeat (LTR) gene activity. Using flow cytometry, 4 days post-HIV infection a complete loss of GFP production was seen in CRISPR-modified cells vs. non-modified controls. Indicating no HIV-1 activity, this result was confirmed with standard real time polymerase chain reaction (rtPCR/qPCR) and enzyme linked immunosorbent assay (ELISA) methods. CRISPR-modified cells showed a significant decline in viral RNA 4-5 days post infection, confirming the ability of CRISPR-mediated CXCR4 ablation to reduce incidence of HIV infection in an in vitro based line.
The concept of reactivating latent reservoirs so they can be killed with gold standard HAART is not new. Researchers have been exploring methods for the reactivation of reservoirs for many years with pharmaceutical approaches.5 These approaches however have not been met with much success, as many have broad off target effects, are not specific, or are poorly regulated in their action.5 CRISPR/cas9, due to its highly specific nature, gives rise to an approach that eliminates all these previous challenges.
Using a newer iteration of CRISPR/cas9 technology known as a nuclease-deficient mutant (dCas9) based Synergistic Activation Mediator (SAM) system, research by Zhang et al was able to successfully reactivate latent reservoirs in a highly specific method.5 By engineering and subsequently pairing a single guide RNA (sgRNA) with a series of transcription activation proteins known as a MS2-p65-HSF1 complex, researchers targeted the HIV-1 LTR region of proviral DNA in latently infected cells. Using these transcriptional activation proteins and the sgRNA molecules of perceived importance researchers transfected cells of various origins, most notably the TZM-bI line (a cell line providing qualitative viral information via a luciferase reporter assay). Once transfected, luciferase activity was measured with a standard luciferin-luciferase assay (with amount of bioluminescence corresponding to the amount of HIV activity). This found that the system with sgRNA targeting was able to reactivate latent viral reservoirs in a specific and effective manner. More notably, the reactivation of these reservoirs induced apoptosis from the over accumulation of viral proteins (the kill method in shock & kill).
A second study by Saayman et al also took advantage of the nuclease-deficient cas9 (dCas9) technology, pairing it once again with a transactivation domain (VP64), a component of the MS20-p65-HSF1 complex seen in the previous trial.6 Targeting 23 specific sites of the LTR region of the HIV-1 genome, the sgRNA dCas9-VP64 complex was delivered by means of nucleofection (an electroporation-based technique) to an immortalized CD4+ t-lymphocyte line. It was determined that there was an increase of 3-40 fold in latent viral transcription in vitro with low off-target effects as measured by a fluorescent reporter (mCherry). It was predicted by these authors however that the degree of reactivation is not comparable in vitro to in vivo, and furthermore highly variant upon the type of cell receiving the treatment. A similar transfection procedure was therefore performed on many cell types to establish average models of transcriptional activation with the technology. Focusing on seven in vitro associated cell lines, rates of HIV proviral reactivation ranged once again from a few to 48 fold, with specific cell lines responding better to treatment. It is likely however that shock and kill will be highly variable in an in vivo environment, depending on intrinsic genetic makeup of the patient, integration site of HIV, type of cell being targeted, and other environmental factors.
Methods that result in the death of the cell along with the virus are acceptable in certain populations, but cell populations that perform essential roles or have limited renewal capabilities require a more finessed approach.
Astrocytes, the major glial cell of the brain, are a key example for the proviral-DNA disruption method of HIV eradication.2 These cells are the primary target of HIV-1 in the central nervous system (CNS) and due to their number and location behind the blood-brain-barrier, act as a strong reservoir and formidable challenge to eradication. A recent study by Kunze et al however has shown significant effects in proviral disruption of astrocytes without significant cell death.2 Eradication of HIV-1 proviral DNA in this population is of importance not only for reservoir elimination, but the elimination of HIV-associated neurological disorders (HANDs) which can affect persons with the virus. Targeting once again the LTR region of the HIV genome, five sgRNA molecules were designed and inserted into AAV vectors. Simultaneously, a HEK293T cell line was transfected with a plasmid containing HIV-1 genetic data and a luciferase reporter, and the cells transfected with the sgRNA AAV vectors. From the results gained by this initial assay, two sgRNA targets were chosen and packaged with CRISPR/cas9 machinery and the respective sgRNA molecules into AAV vectors. Using a modified model of human neural stem cells containing a transcriptionally silent (latent) HIV-1 provirus, cells were transfected with the CRISPR/cas9-sgRNA AAV vectors. Next, transcription was artificially activated with TNF-alpha, and levels of HIV RNA measured in controls and transfected cells. Cells that had undergone the CRISPR/cas9-sgRNA transfection showed a remarkable reduction in viral RNA production as opposed to cells that had no transfection when challenged with TNF-alpha. What was ultimately found was the ability of the AAV vector to not only effectively deliver the editing system to astrocytes, but to down regulate transcription. These results effectively demonstrated the ability of CRISPR/cas9 technology to specifically and permanently knock down genes such as the LTR region of HIV – necessary for viral production. Such a feat would allow the finessed treatment of HIV-1 infected cell populations that cannot be killed without widespread systemic effects.
A possible cure involving target-cell receptor disruption could be the replacement of a patient’s hematopoietic stem cell population with a mutant version. With good results in the studies by Xu et al and Hou et al, showing HIV-1 viral resistance, low off-target effects, and the maintenance of self-renewal and differentiation of progenitor cells, this solution poses ideal for the elimination of blood-based and lymphatic based reservoirs (or any derivative of the hematopoietic stem cell). While this solution doesn’t directly address reservoirs, the eventual repopulation with a mutant version resistant to novel infection could prevent a patient from developing HIV-associated conditions like AIDS without the use of medication. There exists however the possibility for a patient’s HIV strain to mutate and develop an infection route around the CCR5 or CXCR4 mutations, so this technique would be unwise to be used without tandem therapies like HAART, or other forms of gene therapy. With knowledge of astrocytes as a potent HIV reservoir, and the potential of HIV-1 infection to cause neurological conditions, while autologous transplantation might remove the main lethal threat of HIV, the potential for neurodegeneration could still exist for patients.2 As it stands however, the excision, modification, and transplantation of a patient’s own cells is a commonly explored theme in the field of tissue engineering, making it a treatment more likely to enter clinical stages sooner.
In a study by Lebbink et al, it is hypothesized that a combinatory approach is not only beneficial, but necessary in full eradication of infection from a patient.3 It is hypothesized that in order to prevent escape variants and viral mutation, multiple loci on a gene must be targeted simultaneously by CRISPR-cas9 systems.
It is entirely likely that with this upcoming technology and current drug protocols, a clinical cure for HIV infection will be seen within 30 years (enough time to allow further experimental elaboration, preclinical, and clinical testing). From the state of research into eradication and viral mutation, it is likely the cure will be a combinatory approach of not only pharmaceuticals and genetic editing, but combinations of types of genetic editing themselves, ranging from autologous transplantation to multi-loci proviral targeting.3 As previously mentioned, treatment of one line of cells while allowing another to persist untreated could have unintentional consequences. There will likely be no one specific cure for HIV infection, but a patient-specific, strain-specific approach highly dependent on intrinsic genetic factors. As knowledge in the field of CRISPR/cas9 targeting of proviral DNA progresses, it is possible this technology will not only be used to clear all strains and clades of HIV infection, but all possibilities of viral infections regardless of type or origin. Quite simply, CRISPR/cas9 shows tremendous promise.