IntroductionThe human immunodeficiency virus

IntroductionThe human immunodeficiency virus (HIV) was first discovered in 1983; since its discovery, the virus has infected more than 70 million people worldwide. According to the World Health Organization, there are currently over 36 million people living with HIV/AIDS worldwide, the highest prevalence being in Sub-Saharan Africa (World Health Organization). Although the virus continues to be an epidemic, the development of HAART has drastically improved the life expectancy of patients with HIV. HAART is a multidrug regimen targeting several different viral components to prevent viral replication and activity. HAART is designed to maintain viral suppression, improve patients’ immune functions, and prevent opportunistic infections that often lead to death (Moore and Chaisson, 1999). The first drug developed to treat HIV was zidovudine, a nucleoside reverse transcriptase inhibitor. However, by itself, this drug was unable to suppress viral activity for extended periods of time. As a result, a combination of drug cocktails was developed. In clinical trials, this combination of drugs showed a 60 % to 80 % reduction in new cases of AIDS, AIDS related deaths, and hospitalizations from opportunistic infections (Bai et al., 2013).
Currently, there are five classes of antiretroviral drugs used to treat HIV. These drugs are classified based on the viral components they inhibit. Nucleoside reverse transcriptase inhibitors are nucleoside analogs that prevent the virus from making DNA from RNA. Non-nucleoside reverse transcriptase inhibitors bind to allosteric sites to prevent reverse transcription. Protease inhibitors impede the production of mature virions by stopping the protease enzyme necessary for cleaving the polyprotein into its functional components. Integrase inhibitors prevent the provirus from integrating into the host cell genome. Fusion inhibitors prevent the HIV virus from binding to and entering the host cell. The recent development of fusion and integrase inhibitors has been proven to be successful in suppressing viral expression; however, there are concerns about their efficacy and adverse effects such as hepatotoxicity (Westby ; van der Ryst, 2005).
Although HAART is very effective in treating patients with HIV, it does not fully restore health and adverse effects associated with lifelong therapy have been noted. HIV is a disease that affects individuals on the genetic level. The biggest drawback to HAART is that it does not treat the underlying cause of this disease – the provirus embedded in the host cell’s genome. Adverse effects have been reported with the use of all antiretroviral drugs and are the most common cause of medication noncompliance (O’Brien et al., 2003). Patients using HAART for long periods of time are at a higher risk for several complications. One complication of HAART is a shorter life span compared to the life span of non-infected populations (Antiretroviral Therapy Cohort Collaboration, 2008). Other complications of long term HAART use include “cardiovascular disease, cancer, osteoporosis, and other end-organ diseases” (Deeks, S.G., 2011). Not only does HAART cause complications in the patients using it, long term therapy in pregnant women can lead to malformations during fetal heart development leading to left heart dysfunction (Lipshultz et al., 2011). Another issue with the continual widespread use of HAART is the development of drug resistant strains of HIV. HIV is a retrovirus with no RNA polymerase proofreading activity, causing mutations to occur very rapidly, and as such, drug resistance will naturally occur. When HIV is not fully suppressed, drug resistance has a higher chance of occurring. This situation frequently occurs when there is a non-compliance with treatment (Colombrini et al., 2008).
Although the current therapies for HIV are effective for extending a patient’s life, they do not address the underlying causes of the disease. Once HAART is halted, individuals will rapidly rebound to their pre-HAART viral burden levels due to activation of proviruses from latent reservoirs (Hatano et al., 2000). Since the cause of the disease is at the genomic level, only genetic therapies can be used to effectively and reliably cure the disease. There are currently four techniques that can be used to cure HIV, one of which is the CRISPR/Cas9 system. These techniques can be used to recognize a specific DNA sequence and perform site specific gene editing.
The CRISPR/Cas9 system is a defense mechanism found in 40% of Bacteria and 90% of Archaea (Grissa et al., 2007). The system serves to protect against foreign genetic elements such as bacteriophages and plasmids that have previously infected the organism. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are regions in the prokaryotic DNA consisting of repeated base pair sequences separated by protospacer sequences (Haft et al., 2005). The very first CRISPR/Cas9 system to be identified was the Type I-E system, which is found in E. Coli. The system most commonly used for genetic editing is a modified version of the Type II CRISPR/Cas system, which is found in Streptotococcus pyogenes (Jinek et al., 2012). CRISPR-associated (Cas) enzyme genes are often found within the CRISPR gene array. The Cas enzymes function as non-specific endonucleases and aid in the synthesis of CRISPR-RNA (Haft et al., 2005).
The unique sequences found in the protospacer region between CRISPRs are foreign genetic material from phages and plasmids that have previously infected the organism. It seems that different CRISPR/Cas systems use different methods to acquire the spacer region (Horvath et al., 2012). Type I and Type II CRISPR-Cas systems rely on Cas1 and Cas2 enzymes to capture foreign genetic material while Type III does not (Yosef et al., 2012). Bioinformatics analysis of the spacer regions revealed that they were not randomly selected from the phage genome but were always adjacent to a short segment of DNA which became known as PAMs. PAMs, or Protospacer Adjacent Motifs, are 3 to 4 bp in length and play a role in the acquisition of foreign DNA and the maintenance of correct spacer length. They are also required for the specific targeting of Cas enzymes to foreign DNA. Cas enzymes will not bind to the target DNA sequence if that sequence is not adjacent to a PAM (Shah et al., 2013). The bacteria use the PAMs as a means to distinguish self genome from non-self genome.
The CRISPR immune response involves two steps, synthesis of the CRISPR-RNA or crRNA and crRNA guided interference. The CRISPR gene array along with all the unique spacer sequences is transcribed as a single long mRNA. In Type II CRISPR/Cas systems, this mRNA then base pairs with a trans-activation crRNA or TacrRNA to form a pre-crRNA/TacrRNA hybrid. This hybrid is then cleaved by RNaseIII and processed to form mature crRNA/TacrRNA or guide-RNA (Cong et al., 2013). This guide-RNA or gRNA contains one CRISPR and a unique spacer sequence complementary to a fragment of foreign genetic material. When a Cas enzyme associates with the gRNA hybrid, it is able to target the foreign genetic material that is complementary to the unique spacer sequence (Gasiunas et al., 2012).
Type II CRISPR-Cas system is the simplest CRISPR/Cas system, requiring only three components to function. The CRISPR/Cas9 system that is widely used in gene therapy is a modified version of the Type II CRISPR/Cas system. The TacrRNA and crRNA are ligated together to create a single piece of chimeric RNA, reducing the complexity to a two component system (Mali et al., 2013). This simplicity has made the type II CRISPR/Cas system more attractive for use in human gene therapy.

Results
The CRISPR/Cas9 system has been studied in many different cell types, including T cells and pluripotent stem cells infected with the HIV-1 virus. Based on the experiments that were analyzed for this systemic review, the data showed that by using CRISPR/Cas9 on HIV infected cells, it is possible to mutate the virus, leading to inhibition of viral replication and infectiveness. The cells with the mutant HIV virus can then produce daughter cells that are resistant to HIV. Thus, CRISPR/Cas9 can be used to treat and potentially eliminate the HIV virus from infected cells.
Zhu and colleagues (2015) demonstrated this effect by creating latent HIV cells that contained the full length viral DNA. 10 target sites for the Cas9 endonuclease were chosen, including 5 in the pol gene and 2 in the exon of the tat/rev gene. To demonstrate the effectiveness of the Cas9 endonuclease to cleave and mutate the HIV1 DNA at the target sites, the cells were treated with TNF-alpha to allow for viral expression, which was measured by the levels of GFP expression. Cells that were targeted by the Cas9 endonuclease showed a 5-fold decrease in GFP expression when compared to wild type cells that did not contain Cas9 (Figure 1A, 1B). When the levels of HIV-1 p24 antigen were measured using ELISA, viral production was decreased in DNA regions targeted by Cas9 compared to wild type cells (Figure 1C,1D). HIV-1 suppression was achieved in multiple HIV-1 DNA regions that were targeted by Cas9. Three different guide sequences (gRNAs) were cotransfected with Cas9 to recognize specific DNA sites within the genome of the HIV-1 virus. All 3 gRNA sequences included the target site for the tat/rev genes. When gene expression and viral production levels were analyzed in the gRNA sequences, there was a decrease in the number of GFP positive cells and a 24-fold decrease in viral production (Figure 1G, 1H). This finding demonstrates that the tat and rev genes of the HIV-1 virus can be specifically targeted by CRISPR/Cas9 to produce mutant HIV-1 viruses that have no viral activity.
Figure 2 shows that by using the CRISPR/Cas9 system to target the HIV1 LTR regions, it is possible to inhibit expression of the HIV1 provirus not only in the transcriptionally active provirus but also in the latent integrated provirus. Mutant HIV1 viruses were created, with 81.8% of the mutants containing a deletion in the LTR sequence while 9% of the mutants had insertions in the LTR sequence (Ebina et al., 2013).

CRISPR/Cas9 can target the latent HIV provirus in multiple types of human cells. Myeloid lineage cells are the primary cell types that harbor HIV-1 in the brain. Hu and colleagues (2014) showed that the HIV provirus can be targeted and disrupted in these cells. They designed a gRNA that targets the U3 region in the LTR. CHME5 microglial cells were transfected with a plasmid containing the gRNA and Cas9. Analysis of the cells showed high disruption efficiency of the genomically integrated HIV-1 provirus but there were some off target site cleavages. In Figure 3A, (right) it can be seen that the HIV-1 genome was successfully excised from chromosome Xp11.4. (Left) Using PCR, it is further seen that CRISPR/Cas9 was able to successfully remove the HIV-1 genome that was once integrated into the chromosomal DNA. They also demonstrated that Cas9/gRNA genome editing can be used to immunize cells against HIV-1 infection. In Figure 3B, it can be seen through PCR that cell clones containing Cas9 showed reduced expression of LTR regions where Cas9 excised the LTR sequence (Hu et al, 2014).
The HIV1 proviral DNA contains duplicate LTR regions on both ends of the integrated viral genome, allowing for CRISPR/Cas9 to cleave both LTR regions and remove the integrated viral genome from the host cell. In Figure 4, using quantitative PCR, CRISPR/Cas9 was again shown to reduce the LTR region GFP expression. The amount of DNA in the LTR region was decreased compared to the wild type cells not treated with CRISPR/Cas9. About 32% of the provirus was excised from the host cell genome after the CRISPR/Cas9 endonuclease was introduced into the mutant cells (Ebina et al, 2013).

HIV resistant cells can be created by using lentiviral vectors expressing CRISPR/Cas9, allowing for the modification of HIV1 susceptible human CD4 T cells by disrupting the CCR5 gene making the T cells resistant to HIV 1 infection (Wang et al, 2014). Ye and colleagues (2014) also investigated whether a CCR5 mutant stem cell could be induced to produce monocytes and macrophages that were resistant to HIV-1 infection. After 24 days of culture, monocytes and macrophages (CD45+ CD14+ and CD14+ Cd11b+) were obtained from both parental wild type stem cells and the mutated cells infected with HIV-1. Phytohemagluttinin was used to stimulate the primary CD 4+ T cells and by day 16, HIV-1 viral replication could be detected in the monocytes and macrophages induced from the wild-type parental stem cells but no viral activity was detected in the cell lines derived from the mutated CCR5 stem cells (Figure 5). These results demonstrate that by using CRISPR/Cas9, pluripotent stem cells with a homozygous mutation for CCR5 can be created allowing for differentiation of monocytes and macrophages that are resistant to HIV-1.
Similarly, Liao and colleagues (2015) were able to engineer human-induced pluripotent stem cells that expressed HIV-targeted gRNA/?Cas9. The modified stem cells differentiated into HIV reservoir cell types and showed resistance to HIV infection. This opens the possibility that a gene editing based vaccine strategy may be effective in eradicating integrated HIV-1 genome and newly packaged proviruses in cells.
Discussion
CRISPR/Cas9 is an endonuclease that recognizes a specific DNA site. When bound to the target site, cleavage of the DNA by the endonuclease creates double stranded DNA breaks, which can be repaired using nonhomologous end joining to produce insertions and deletions at the target DNA site, producing mutations within the target sequence (Zhu et al., 2015). By using CRISPR/Cas9, it is possible to create site specific mutations within the HIV-1 viral genome that results in reduced viral expression and viral production (Liao et al., 2015). This decrease in viral activity can be achieved in both transcriptionally active viral cells and latent viral cells integrated within the infected cell (Ebina et al., 2013). Mutations within the latent HIV cells prevents reactivation and infection of the virus (Hu et al., 2014). An important use of this ability of CRISPR/Cas9 to create mutations within the HIV-1 viral genome was demonstrated in multiple studies where CRISPR/Cas9 was used to create mutations in the CCR5 gene of the HIV-1 virus (Wang et al., 2014 & Li et al., 2015). Resultant clonal cells containing these mutations were found to be resistant to HIV-1.

Patients that carry a homozygous deletion in the CCR5 gene have been reported to be resistant to HIV infection. CRISPR/Cas9 can be used to create a vaccine for HIV-1 that targets the CCR5 gene within T cells and macrophages. By using CRISPR/Cas9 to create site specific breaks in the CCR5 gene of T cells and macrophages, it is possible to create deletions within the gene, producing cells that are homozygous for the deletion in the CCR5 gene. Subsequent daughter cells will also contain the deletion in the CCR5 gene, resulting in daughter cells are effectively resistant to the HIV virus.
CRISPR/Cas9 can also be used to effectively excise the latent HIV-1 virus from the genome of the infected cells (Khalili et al., 2015). In a previous experiment, Mandal and colleagues (2014) were able to excise the CCR5 gene from CD 34+ hematopoietic stem cells. Clonal stem cell colonies all showed a deleted CCR5 gene making these cells immune to HIV-1. This finding suggests that it is possible to remove the HIV genome from infected cells, essentially curing the cells of the virus.
Transient suppression of genes can be used in combination with other gene therapies in the treatment of HIV. Qi and colleagues (2013) created a Cas9 mutant that lacks endonuclease activity. The mutant Cas9 can still bind to guide RNA to generate a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system is called CRISPR interference (CRISPRi); it can be used to repress multiple target genes simultaneously, and its effects are reversible. It has been demonstrated that CXCR4 targeting CRISPRi causes significant suppression of CXCR4 in HeLa cells rendering them temporarily immune to CXCR4 tropic-HIV (Gilbert et al., 2013). By using CRISPR/Cas9, it may be possible to eliminate the HIV provirus from infected cells, while also producing uninfected stem cells that are immune to the virus, leading to a possible cure for HIV in the near future.
Limitations
The CRISPR/Cas9 system is a relatively new technology and there are some road bumps that need to be overcome. The major safety concerns that arise in gene therapy are off-target effects. What factors affect gRNA/Cas9 specificity is not fully understood. Some studies have shown that CRISPR/Cas9 can have substantial off-target effects. In vitro studies show that the Cas9 enzyme is lax in binding specifically at positions distal from the protospacer-adjacent motif region (Cong et al., 2013). The gRNA also only targets about 20 nucleotides; in large genomes, the lower specificity of the gRNA has the potential to create more off-target sites. Furthermore, the CRISPR/Cas system has been known to bind to DNA by non-Watson–Crick base pairing which results in unwanted mutations and cytotoxicity (Jiang et al., 2013). When Cradick and his team (2013) attempted to disrupt CCR5 in HEK-293 T cells using CRISPR/Cas9, they found that their CCR5-targeting CRISPR/Cas9 system induced off-target cleavage in CCR2, with mutation rates ranging from 5% to 20%.
Future Directions
The experiments included in this review studied the effects of CRISPR/Cas9 in vitro. The results obtained from these experiments have not been tested on human subjects. While the CRISPR/Cas9 system works in stem cells and T cells created specifically for the experiments conducted, the results may not be valid when these experiments are replicated in patients. Future studies need to demonstrate the effects of CRISP/Cas9 in clinical trials using human patients.

The major hurdle in gene therapy is target specificity and the potential for off target effects. Most gene editing approaches have some degree of off-target cleavage. However, gene therapy is still relatively new and it is constantly being improved upon and optimized for safe use in humans. The use of gene therapy has the potential to complement conventional antiretroviral therapies and enhance their effects. No clinical trials test the possibility of using a combination of gene therapy approaches in the treatment of HIV. Given the success achieved with combined drugs in HAART, it makes sense to devise treatment plans that use a combination of different gene therapy techniques along with HAART.
Conclusion
Given the current health and financial cost of the HIV epidemic, it is imperative to continue to pursue a variety of treatment strategies. Many protein and RNA based gene therapy options are available for use in the treatment of HIV infections. Although there are some short comings for the CRISPR/Cas9 system, it is a powerful technology for manipulating human genes. gRNA can easily and quickly be created at a very cheap price. Escape mutations by HIV won’t hamper this system as much as it would a system that uses a protein based domain for DNA recognition. CRISPR/Cas9 can also be used to target multiple strains of HIV. In addition, gRNA isn’t fused to Cas9 and Cas9 can function with all gRNAs, giving the system a great deal of flexibility. Given that it has only been a few years since CRISPR/Cas9 first started to be used for gene editing, it is very likely to be improved for both efficiency and to reduce off-target effects. From the data collected in this review, CRISPR/Cas9 looks promising for the treatment of HIV.