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Introduction
The human immunodeficiency virus (HIV) is estimated to affect 37.9 million people worldwide, of which 1.7 million are children (World Health Organization, 2018). The virus attacks T lymphocytes, cells used by the immune system to protect the body of foreign invaders. HIV uses these cells as a replication machine, leading to a depletion in T cells, therefore weakening the immune system, allowing for opportunistic infections to take over, thus causing autoimmune deficiency syndrome (AIDS) (Bhatti et al., 2016).
Although there are drugs that help with the management of the disease, patients that undergo this treatment develop dependency and several adverse effects. Hence, there is a need to develop new and better therapies for the treatment of HIV (Bhatti et al., 2016).
Gene therapies have been developed and are being used to treat thousands of diseases, however, due to ethical issues and because these therapies are still new, there is not enough evidence of their efficacy in humans. Nevertheless, they show great promise and their efficacy as gene-editing tools is undeniable (Manjunath et al., 2013). Thus, this brings the question, are gene therapies a realistic approach for the treatment of HIV?
Background
HIV is a pathogen that targets the immune systems cells. According to the World Health Organisation (WHO), in 2018, 37.9 million people were estimated to be affected globally, of which 1.7 million are children. Also, 1.7 million new people were affected in the same year (World Health Organization, 2018).
Figure 1. Adapted from World Health Organization, 2018. The number of HIV related deaths.
As shown in figure 1, there were 770.000 HIV related deaths in 2018, among which 100.000 were children (World Health Organization, 2018). Although there has been a significant decline since 2000, the rate of HIV related deaths regression is expected to increase, with the aim of fewer than 400.000 losses by 2030. With the rapid development of new drugs and therapies and a better understanding of the virus, these numbers are realistic.
The Virus
To understand current treatments is important to understand the virus and how is causes pathogenicity. There are two types of HIV viruses, HIV-1 and HIV-2, although both can cause AIDS, HIV-2 is mainly restricted to West Africa and only affects up to 2% of HIV cases, thus, most research and this paper focus on HIV-1, also referred to as HIV (Visseaux et al., 2016).
Figure 2. HIV life cycle: 1. Binding, 2. Fusion, 3. Reverse Transcriptase, 4. Integration, 5. Replication, 6. Assembly, 7. Budding (AIDS info, 2019).
HIV is a lentivirus that binds to T cell receptors. C-C chemokine receptor type 5 (CCR5) is a protein that is present on the surface of white blood cells, acting as a chemokine receptor involved in the immune response. HIV has spike-like proteins on its envelope that mimic these chemokines, therefore aiding attachment of the virus to the immune cells (Sok et al., 2016).
Although CCR5 is the main receptor used by the virus, with the progression of the disease HIV is also able to use C-X-C chemokine receptor type 4 (CXCR4), another chemotactic receptor present on T cells surface (Alkhatib, 2009). After binding (step 1 on figure 2), the HIV fuses with the lymphocyte and releases its genetic material RNA, and proteins into the host cell (step 2 on figure 2). One of these proteins is reverse transcriptase, an enzyme that converts single-stranded RNA into double-stranded DNA, a process known as reverse transcription (step 3 on figure 2). HIVs DNA then translocates to the nucleus where integrase, another viral enzyme, combines the virus genetic material to the cells DNA (step 4 on figure 2). When this occurs, the incorporated HIV DNA is called provirus and may remain inactive for indefinite time manufacturing little or no new copies. This stage is called latency, and the cell is said to be latently infected. When the host cell is stimulated to respond to infection and is activated, HIV takes advantage of its machinery and the provirus is transcribed by RNA polymerase, creating the proteins needed to make new viruses (step 5 on figure 2). Protease, another enzyme inserted into the T cell by HIV, cuts large precursor proteins into smaller proteins that conglomerate with the virus genetic material and form a new virus (step 6 on figure 2). The new virus pushes out of the host cell ( buds ) (step 7 on figure 2) and takes some of the cells envelope to form a protective barrier for the virus. In addition, the virus is also embossed with glycoproteins that will allow it to bind to other cells, starting a new cycle (Barré-Sinoussi et al., 2013). This cycle quickly destroys T lymphocytes, a key participant in the immune system response, thus allowing for opportunistic infections to appear. These infections cause autoimmune deficiency syndrome (AIDS) (Bhatti et al., 2016).
Current Treatment
The first treatment to be developed for HIV was antiretroviral (ART) drugs. According to WHO, 2018, 62% of HIV positive patients take these drugs for the management of the disease. ART drugs are not able to eradicate the infection, however, by acting on different stages of the virus life cycle, they prevent further destruction of the immune system, thus preventing AIDS (Maartens et al., 2014). Nevertheless, ARTs lead to drug resistance, drug abuse and metabolic, central nervous system, gastrointestinal, haematological, psychological, dermatological, musculoskeletal and miscellaneous adverse effects (Bhatti et al., 2016). Therefore, there is a need for new and better treatments.
The famous story of the Berlin patient has taken the world in 2008 when it was announced that the first person in the world had been potentially cured of HIV by stem cell therapy. This patient was diagnosed with cancer and HIV, and after undergoing chemotherapy he received stem cell therapy from an HLA-matched donor that carried a homozygous mutation for the CCR5 gene. This meant that the CCR5 protein present on T lymphocytes was not functional and, consequently, HIV was not able to infect the cells, leading to the eradication of the virus (Yukl et al., 2013). Due to the outcome of the Berlin patient, two other people have undergone the same procedure in Boston, however, unlike the Berlin patient, the donors for these two individuals were not homozygous for the CCR5 mutation. Therefore, although there was remission of the virus, the patients had to go back to ART treatment
Finding an HLA-matching donor is challenging, however, to find homozygous CCR5 mutated individual with HLA-matching to every HIV patient is nearly impossible. In addition, this therapy is expensive and difficult to treat in large numbers, and it would be difficult to take to Africa, where most HIV positive patients are (World Health Organization, 2018). Hence, although this therapy has proven to be successful, there is a need for new treatments that are easier and cheaper.
Gene Therapy
In contrast to ARTs and cell therapy, some gene therapies can be replicated, personalised, cheap and stored easily (Manjunath et al., 2013).
Following recent advances in the understanding of genetic mechanisms and development of genetic tools, precise gene engineering becomes a realistic and exciting prospect. Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nuclease (TALENS) and clustered regularly interspaced short palindromic repeats (CRISPR) are novel genetic technologies that enable target specific gene editing.
ZFN
Figure 3. Adapted from BioScope, 2019. Zinc Finger Nuclease structure.
Zinc finger nucleases are artificial restriction enzymes that can cleave DNA. These proteins are composed of a DNA binding domain, and a DNA cleavage domain, as shown in figure 3. The first comprises of zinc finger domains that recognise specific base pairs, and the latter is a non-specific cleavage FokI restriction enzyme. As FokI is not able to target individual regions of the DNA, it needs zinc fingers to target specific sequences (Carroll, 2016).
Most studies focus on disruption of CCR5 and CXCR4 proteins due to their importance in the virus pathogenicity and to the understanding of the pathways involved. According to Manjunath et al., 2013, disruption of CCR5 proteins have been successful in various cell lines, hematopoietic stem cells and primary T cells. Mutated CCR5 proteins by ZFNs are inheritable, therefore, HIV positive patients would only need one set of treatment and potentially be cured (Manjunath et al., 2013).
As previously mentioned, HIV developed strains that can use CXCR4 as a primary target to attach to T cells instead of CCR5. Therefore, although mutated CCR5 proteins have proven to successfully reduce HIV infection, this method is not sufficient and will not work with certain HIV strains that can use CXCR4 as a point of entry in T cells (Yuan et al., 2012). Thus, CXCR4 is also an important target to achieve HIV resistance. Wilen et al., 2011, shows that is possible to use ZFNs to edit both CCR5 and CXCR4 proteins and produce a source of HIV resistant lymphocytes. Furthermore, Yuan et al., 2012, compared the efficacy of ZFN and shRNA for disruption of CXCR4 proteins. It was found that protein mutagenesis was superior with ZFN and that when reintroducing CXCR4 mutated CD4+ T cells into a humanised mouse model, these cells were resistant to HIV. In addition, Sangamo Therapeutics has completed phase II trials that aimed to study the safety of ZFN edited T cells in humans. The trial was a success and ZFN-mediated disruption of the CCR5 gene was safe and well-tolerated (Sangamo Therapeutics, 2015). Thus, ZFN proves to be a viable and safe method for gene editing to prevent and treat HIV infection.
TALENS
Figure 4. Adapted from Yu et al., 2016. TALENS structure.
TALENS are restriction enzymes that can be engineered to cut specific DNA sequences. As shown in figure 4, it composes of transcription activator-like effectors (TALE), the DNA binding domain, and a DNA cleavage domain. TALE are proteins secreted by type III secretion systems in bacteria and are used to infect a host, bind to promoter sequences and activate the respective gene (Boch et al., 2009). The DNA cleavage domain is a non-specific protein that is also used in ZFNs, FokI.
Although TALENS is a popular approach to gene editing, it is challenging to assemble TALE repeats as they present high sequence similarities (Gupta and Musunuru, 2014). Briggs et al., 2012, discusses a technique called a golden-gate assembly, that attempts to overcome this issue, by quickly assembling the repeats in a hierarchical form. This method has also accelerated the cloning process and provided the possibility for large-scale, cost-effective production of TALENS (Briggs et al., 2012).
Strong et al., 2015, discuss the use of TALENS as a potential treatment for HIV. In this study, it was shown that this technique could be used to mutate both CCR5 and CXCR4 genes (Strong et al., 2015). However, in contrast to ZNF, TALENS are large proteins, this causes challenges regarding delivery. Bergmann, 2014, discusses TALENs delivery methods and shows that most of the commonly used vector systems, for example, lentiviral and adenoviral vectors, fail in providing an appropriate capacity of two TALEN cassettes in one vector (Bergmann, 2014). TALENS are also easier to design and construct, nonetheless, unlike ZFN that each finger recognises 3 amino acids, each TALE repeat recognises only one amino acid. TALE repeats also present high sequence similarity, this causes a challenge in assembling them in the same compound (Gupta and Musunuru, 2014). Thus, it is possible to conclude that although TALENS are effective at mutating vital genes for HIV, further preventing disease progression and prevention, there is still a need for further research.
CRISPR
Clustered regularly interspaced palindromic repeat, also known as CRISPR, is a genetic engineering tool that was firstly used by bacteria (Ran et al., 2013). Bacteria can keep small pieces of invading viruses DNA and use it as an identification tool in case there is an invasion by the same or a similar virus. If this situation occurs, bacteria can recognise the virus and create a guide RNA from the virus genetic material, to guide CRISPR associated protein 9 (Cas9) to the sequence of interest and cleave the virus DNA. Thus, creating a break in the double-stranded molecule. Scientists have taken advantage of this mechanism and created a genetic engineering tool (Ran et al., 2013).
Figure 5. Adapted from Vox, 2018. CRISPR
Scientists can create a guide RNA (gRNA) in the laboratory. As shown in figure 5, gRNA consists of a CRISPR RNA (crRNA), that recognises the sequence of interest, and a trans-activating crRNA (tracrRNA). This sequence is used by endonuclease Cas9, which cleaves DNA at the recognised site. This technique can be used to disrupt a gene and therefore create a knockout, or to insert a new gene of interest (Ran et al., 2013).
Wang et al., 2018 explain in great detail how CRISPR has been used to study and treat HIV. The meta-analysis discusses how, much like for ZFN and TALENS, most research focuses on targeting CCR5 and CXCR4 receptors, however, Wang and colleagues have focused in CRISPR application strategies that target HIVs viral genome instead of cellular sequences (Wang et al., 2018). Their research studies two approaches. In the first method a gRNA and Cas9 are inserted into infected cells to attack the viral DNA, and in the second CRISPR is introduced into uninfected cells that should act as a defence in case of an HIV invasion. Both methods reduced viral presence in immune cells, proving CRISPR efficacy in fighting HIV (Wang et al., 2018). However, further studies need to be completed as different gRNAs showed dissimilar suppression levels, allowing the virus to escape from inhibition from most cultures. Guide RNAs targeting strongly conserved sequences prevented HIV replication for longer periods, thus, future studies should focus on these sequences (Wang et al., 2018).
CRISPR is a gene-editing tool that is easier and faster to make than ZFN and TALENS, however, there are serious ethical concerns regarding editing the human genome. In 2018, a Chinese scientist used CRISPR to mutate the CCR5 protein on unborn twin girls (Normile, 2019). These girls are the first gene-edited babies. This project was not published until the editing was complete and concerns regarding the twins cognitive function have arisen as the CCR5 gene is also involved in memory and the brains ability to form new connections (Normile, 2019) (Lee et al., 2009).
Conclusion
To conclude, HIV is a mortal disease that can be managed by ARTs and has been potentially cured in the Berlin patient by cell therapy (Maartens et al., 2014)(Yukl et al., 2013). However, gene therapies are a more realistic approach for the treatment of the infection as they can be replicated, personalised, cheap and stored easily (Manjunath et al., 2013).
ZFN, TALENS and CRISPR are tools with high genome editing efficiency. They work by causing double-strand breaks in the genome and present various degrees of specificity and efficiency. ZFNs are the most studied. They have been reviewed in clinical trials and humanised models, having proved efficacy and safety (Yuan et al., 2012). In contract, TALENS are larger proteins that have delivery issues, however, they present minimal toxicity and off-target editing. Further studies to assess safety and efficacy in HIV-positive animal models is needed (Strong et al., 2015). CRISPR, unlike TALENS and ZFN, is controlled by RNA-DNA interactions. This offers an easy design, easy prediction regarding off-target effects, high specificity and is a method well suited for high-throughput. In addition, CRISPR is also able to target multiple genes simultaneously. Nevertheless, as this is a newer technique, further studies need to assess the safety and off-target predictability (Wang et al., 2018).
Thus, although HIV is manageable nowadays, there is still a need for a cure, and, with further research, gene editing tools present a realistic approach to the eradication of the disease.
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