ART is the utilization of the antiretroviral drugs referred to as antiretrovirals (ARVs). ARVs do not treat the virus, it just slows it down. If the combination of the different ARVs is used, it is known as Highly Active Antiretroviral Therapy (HAART). Undoubtedly, Art has decreased the number of deaths due to HIV. But the major limitation of this therapy is that it gradually creates resistance in HIV against ARVs and do not eliminate the latent provirus in the infected cells (Martin Vogel et al., 2010). Other limitations include high cost during the lifelong antiretroviral therapy, monitoring issues while using ARVs, taking a lot of tablets on daily basis and toxicity associated with high usage of antiretroviral drugs. Although, these days only one tablet is available that contains combinations of the drugs that target the HIV on different points during its replication cycle. ARV are usually grouped as fusion inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs) and protease inhibitors (PI) (Wang et al., 2018).
Tre-recombinases are the mutated enzymes derived from Cre-recombinases which was obtained from P1 bacteriophage. Tre-recombinases identify special LTR present in HIV genome and subsequently eliminate the integrated HIV provirus in infected HIV individuals. PreS2 is a surface antigen of hepatitis B virus. A translocation motif derived from this surface antigen was fused to Tre-recombinase enzyme for the delivery into the host cell. Using lentiviral vectors, Tre-recombinases was also delivered to CD4+ T cells ex vivo. These tailored enzymes were under the control of a TAR sequence hence limiting the expression to Tat-expressing cells. Although these results are very promising, yet there is no certainty whether there will be enough Tat to stimulate Tre-recombinase functionality (Saayman et al., 2015).
ZFNs are the nucleases that are made by fusing two domains i.e. a zinc-finger DNA binding domain and a DNA-cleavage domain. ZFNs are genetically engineered to manipulate the DNA repair machinery and to alter the specific DNA in the whole genome. ZFNs have also been utilized to alter the HIV LTR sequences leading to alteration of the provirus and ultimately its deactivation. T cells, that contains HIV1 integrated into their genome, were treated with ZFN that targeted the TAR sequence present in HIV LTR region. The result was that the provirus has been eradicated completely from the genome of T cells (Qu et al., 2013). But the major clinical disadvantage of Tre-recombinases and ZFNs is that the enzymes themselves select the specificity of the site which needs to be cleaved by them. To change the cleavage site, either further genetic engineering techniques are required for protein engineering or in vitro directed evolution will be an essential part of the trial. This additional step makes ZFNs and Tre Recombinases not the best option for clinical utilization.
TALENs consist of two components. One is TAL effectors (DNA binding protein) and the other is nuclease (DNA cleavage domain). TALENs recognize the specific DNA sequence by DNA/Protein recognition method and introduces a double stranded break in the DNA. TALENs are engineered to identify LTR located on both ends of HIV genome and produce a double stranded break in the provirus. Because non-homologous end joining repair method of the infected cell is error-prone thus it produces the mutation in the provirus leading to the deactivation of the latent HIV incorporated in the CD4+ T cells of the infected individuals (Wang et al., 2018). Researchers have also fused the catalytic site of an endonuclease enzyme called FokI to a binding domain of zinc finger making a combination of TALE/FokI this was known as TALEN. The mechanism of these TALENS were same. They bind to a specific target on the DNA and cleaves the DNA. The natural repairing system of the cell undergoes non-homologous end joining (NHEJ) repair or homology directed repair (HDR). NEHJ mechanism was used to disrupt the genes in HIV leading to the permanent deactivation of the latent HIV provirus and HDR was used to edit the gene desirably i.e. correction or insertion of exogenous DNA into the targeted site (T. Li et al., 2011).
Clustered Regularly Inter-spaced Short palindromic repeats (CRISPR) of small DNA sequences present in bacteria and archaea bacteria provide adaptive immunity to them. These are small repeats of the DNA fragments that are interspaced with other short DNA fragments of viral origin called spacer DNA (viral DNA fragments implanted into a CRISPR locus). This spacer DNA are actually the sequences of the viruses called proto-spacers that have previously infected the bacteria, since the bacteria have CRISPR adaptive system, so they made the protospacers a part of their own DNA in the form of Spacers. Spacers plus short palindromic repeats of DNA makes CRISPR. Along with the CRISPR locus are the genes of CRISPR-associated proteins known as Cas proteins (Jiang & Doudna, 2017). The most famous is Cas-9 that belongs to class 2 family of Cas proteins and only require single-subunit effector. (Table 02). When a bacteriophage attacks the bacteria, the bacteria encodes cas genes making Cas proteins along with crRNA and trans-acting CRISPR RNA (tracrRNA). These RNAs guides the Cas protein to foreign DNA, recognizes it and cleaves the viral DNA saving the bacteria (Kim et al., 2017). This CRISPR/Cas9 system can be engineered to edit and knockout or knockdown the genes from the genome of any organism. CRISPR/Cas9 can be used to target the provirus present in the HIV infected individuals and the provirus can be deactivated permanently (Wang et al., 2018).
Table 2 Comparison between three gene editing tools i.e. ZFN, TALEN and CRISPR/Cas9 (Kim et al., 2017).
|Target site size, bp||18–36||30–40||22|
|Cytotoxicity||Variable to high||Low||Low|
|Design availability||More complex||Complex||Simple|
|Ease of multiplexing||Low||Low||High|