By Sreenidhi Venkatesh
Since their discovery in the 1980s, the human immunodeficiency viruses (HIV) have taken the medical world by storm due to their ability to induce immune system failure, allowing opportunistic infections and cancers to become even more life-threatening.
Currently, the main form of treatment for HIV is anti-retroviral drugs which target various mechanisms of the virus in an attempt stop it from replicating. While this is an effective way to manage the condition and keep the viral load at low-undetectable levels, it is not a cure. Amongst the various T-cell and gene therapies that are being studied as alternative treatment options for HIV, clustered regularly interspersed short palindromic repeat (CRISPR) is one of the most prominent.
CRISPR/CRISPR associated 9 (CRISPR/Cas9) protein complexes are used to target a section of the genome where it is usually used to induce a double stranded break, which is then repaired by the error-prone non-homologous enjoining leading to silencing/knocking-out that section of the genome. This technique has been studied on various targets to determine its effectiveness in reducing HIV replication.
C-C chemokine receptor type 5 (CCR5) is found on the surface of many lymphocyte cells. They belong to a family of seven transmembrane proteins which are coupled to G proteins, that have many important signalling receptors. These receptors are usually present in naïve T-cells, mature T-cells, some lymphocytes, some neural cells, and epithelial cells. CCR5 was found in ‘membrane raft microdomains’ and this localisation supposedly helped not only chemotaxis of the cell, but also assisted HIV entry into the cell. In a study, it was shown through immunofluorescence that CCR5 molecules in these lipid rafts were closely associated to the CD4+ glycoprotein, which allowed HIV molecules to bind to these co-receptors thus allowing it to enter the cell (Steffens & Hope, 2003). This receptor, along with CD4+, play an essential role in HIV’s mechanism of infection. CCR5 serves as a useful target to ablate in order to eradicate HIV (Lopalco, 2010) (Alkhatib, 2009).
In China, as part of a treatment plan for HIV-infected patients, CRISPR/Cas9 gene edited haematopoietic stem cells were allotransplanted to a patient with HIV-1 and acute lymphoblast leukaemia. Previously it was shown that the only natural resistance to HIV was present in those with the CCR5Δ32 mutation, which caused the CCR5 to develop smaller or be internalised, thereby preventing the virus from binding (Paoli, 2013). For a long time, CCR5 knock-out in human CD4+ T-cells was attempted, however it always had low efficiency and high off-target effects, thus was never viable Xu et al., 2017; Xu et al., 2019).
Xu et al. (2019) developed an optimal CRISPR-Cas9 system to ablate CCR5 by designing and screening single guide RNAs (sgRNAs) from the first exon until the site of the CCR5Δ32 mutation and used multiple screening tools to eliminate sites of non-specific binding. This protocol was used to disrupt CCR5 expression in haematopoietic progenitor stem cells which were used for stem cell transplantation. This resulted in a CCR5 indel mutation with an efficiency of about 18%. As there was uncertainty linked to the long-term persistency of this engraftment, CD34– cells were also transfected. Over the 19 months following the transplantation, it was determined that between 5.2% and 8.28% of the cells in the bone marrow had depleted CCR5. As CD34 is primarily found in early haematopoietic cells, and CCR5 depletion was seen across a range of different mature lymphocytes. This example illustrates that successful engraftment of progenitor haematopoietic stem cells is possible. 7 months after transplantation, anti-retroviral therapy (ART) was stopped to test the persistence of the CRISPR-edited cells. The level of CCR5 disrupted cells rose to its peak level of 4.39% during this period. However, during this period the viral load also began to increase, therefore ART was resumed. While this usage of CRISPR/Cas 9 was not able to eradicate HIV, it is indicative that with the development of a possible system to select for CCR5 knock-out cells, the efficiency of transplants may increase, thereby eliminating the need for ART to control the viral load.
Aside from targeting the cells that HIV infects, the virus itself has been considered a target of interest. HIV has a long terminal repeat (LTR), which encodes its promoter. By targeting this region in the provirus, elimination of the infection has been attempted. CRISPR/Cas9 was used by Ebina et al. (2013) to target the LTR region of the HIV-1 virus in-vitro. This study specifically targeted the NFΚB binding cassettes, leading to inhibition of HIV transcription and replication. The effect of focusing on this target was that the intracellular reservoirs of the virus could be eliminated.
Several studies, including Wang et al. (2014), demonstrated that while single gRNAs had the ability to inactivate the provirus, they were often able to escape from the single gRNA cleavage and thereby allowing for the virus to spread. A solution to combat this was combinatorial CRISPR/Cas9 gene-editing. This involved using an all-in-one adeno-associated virus (AAV) combined with multiplex sgRNAs. When sgRNA/SaCas9 AAV-DJ8 was injected into Tg26 mice, a significant reduction in viral replication was observed (Xiao, Guo & Chen, 2019). Similarly, when the same complex was injected into HIV-1 positive mice with humanised bone marrow, liver, and thymus, cleavage of the provirus was observed. The reason that this combinatorial method worked was due to a single vector with duplex sgRNA having greater excision efficiency as compared to two vectors with a sgRNA on each (Lebbink et al., 2017). Moreover, AAVs are small viruses which are very infectious to mammalian cells, and these allowed for compact packing and delivery of this genetic material into cells (Xiao, Guo & Chen, 2019). They went on to show that a quadruplex sgRNAs/Cas9 would maximise the indel mutations caused and thereby reduce the possibility of the virus escaping, and increase the efficiency of excision (Yin et al., 2017). Although the usage of multiplex complexes has shown to have efficacy in a murine model, the implications of it need to be explored in a human model as the off-target effects of the sgRNAs chosen are yet to be determined.
As previously mentioned, the only use of CRISPR/Cas9 in treating HIV-positive humans has been in editing stem cells prior to transplantation, which does not influence HIV reservoirs in other cell lineages. Possible therapeutic methods that do reduce the viral load in cells have not yet been tested on humans due to possible off-target effects. An area that is challenging to target with drugs but is susceptible to the virus is the brain. The brain’s immune system is guided by microglia, and these cells have CCR5 receptors and CD4 receptors, which is indicative of them being targets for HIV-1. In order to overcome this problem, a novel idea of using magneto-electric nanoparticles (MENPs) has been researched which targeted the LTRs of the HIV (Kaushik et al., 2019).
Kaushik et al. (2019) made MENPs from of BaTiO3 (BTO) and CoFe2O4 (CFO). These complexes form a piezoelectric shell with a magnetic core, which are characteristics that makes it ferromagnetic which is essential for on-demand drug release. By inducing a magnetic field, these particles can be moved around the body to targeted areas. A special characteristic of the MENPs is that they have a ‘quantum mechanically induced non-zero magnetoelectric effect (ME)’, which means that the dipole moments of these particles can be controlled by a local magnetic field, and their magnetic moments can be controlled by a local electric field. This can be manipulated in order to control the release of a drug (Stewart et al., 2018). In the case of the gRNA/Cas9-MENP complex, the release was controlled by an alternating current (AC) magnetic field system. When an AC magnetic field is applied, the core (made of CFO) undergoes a change due to strain, which is absorbed by the piezoelectric shell made of BTO. These cause pressure waves. When the MENP is a certain distance away from the phospholipid layer, these pressure waves can lead to poration of the cell membrane, causing an increased uptake of the nanoparticles across the blood brain barrier (BBB) (Kaushik et al., 2019).
The efficacy of this system was evaluated by using a BBB model which was placed in an electromagnetic coil. After alternating magnetic stimulation, the LTR levels were found to be significantly reduced. Overall, this study showed that it might be possible to use electromagnetism to non-invasively deliver CRISPR/Cas9 across the brain to target HIV-1 reservoirs. While a previous study by Kaushik et al. (2019) illustrated that MENPs can safely be delivered to the mouse brain without affecting its function, this combination along with CRISPR/Cas9 has not been investigated before. Therefore, further exploration of this method must be conducted in animal models to test its efficacy and viability before determining whether it can be used as treatment for HIV in humans.
Overall, the investigation of CRISPR to treat HIV has proven to be fruitful and has shown that there is great potential for targeting not only the sites that it affects, but to targeting the virus itself as well. It is essential that techniques such as using MENPs are considered with great detail as they will not only revolutionise the targeted delivery and eradication of HIV in various reservoirs but will also serve to deliver other drugs to various parts of the body in a non-invasive manner. In conclusion, the unfortunate rapid spread of HIV along with the development of CRISPR has given the biomedical research community an opportunity to explore personalised gene-editing in therapeutics further which will revolutionise the mechanism of drug delivery and its efficacy.
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