**6.3. RNA based mechanisms**

stem cells can be effective with a single transplantation as exemplified with replacement

230 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

Both techniques attempt to make the target cells resilient to HIV infection by either modifying the cell surface components required for HIV entry or by altering the intracellular contents which are utilized by the virus during replication, or a combination of both. Strategies involved in limiting viral entry by surface component modification possess certain remarkable advan‐ tages. It has been detected that among all HIV related cell death events, over 95% are caused by apoptosis initiated by the cells immediately after viral entry [149]. Hence, the target cells

With the serendipitous discovery of its curative effects in the Berlin patient, CCR5 which acts as the co-receptor for HIV entry is the most sought-after target for genetic modification. A homozygous deletion of a specific 32 base pair sequence from the CCR5 gene confers complete protection, while a heterozygous deletion of the same nucleic acids confers partial protection from HIV entry, without causing any glaring change in the CD4 T-cell function. All the three systems of gene editing namely the ZFN, the TALEN and the CRISPR/Cas9 system are being evaluated for this purpose and are found to have nearly 50% efficacy in disrupting CCR5 in mature CD4 T-cells and around 25% efficacy in adult haematopoietic stem cells. In this regard, it is intriguing to use induced pluripotent stem (iPS) cells to introduce delta32-like mutation and test their viral resistance. *In vitro* studies of CXCR4 disruption has also shown promising results but this might not serve the purpose *in vivo*, as the deletion is expected to cause functional derangement.[140]. Yet another instance of gene therapy under human trials is the SB-728-T, a zinc finger DNA-binding transcription factor. It binds to the DNA of target cells and disrupts the gene responsible for CCR5 co-receptor production [150]. Apart from the ones mentioned here, there are numerous other techniques of DNA manipulation, RNA based and peptide based techniques being tried to harness the potential of CCR5 alteration in curtailing

The target cells can also be made resistant to HIV infection by increasing the expression of intracellular factors that restrict the viral replication process. TRIM5α, APOBEC3G and tetherin are the well known restriction factors of HIV infection. TRIM5α is a cytoplasmic protein which inhibits HIV by binding with the incoming capsid and preventing further the process of replication. The APOBEC3 family of mRNA editing proteins, especially the APOBEC3G inhibits HIV replication by introducing lethal mutations during reverse tran‐ scription. Tetherin is another host cellular protein involved in HIV restriction by preventing the release of viral progeny from the infected cell.[151] As the vif and vpu proteins of the virus neutralize the effect of APOBEC3G and tetherin respectively, they can be made resistant to their respective viral proteins by introducing point mutations. Single amino acid substitution, D128K in APOBEC3G and T45I in tetherin makes them overcome the viral factors vif and vpu respectively. Gene therapy techniques to increase the expression of TRIM5α and mutated APOBEC3G and tetherin in CD4 T-cells are known to enhance their resilience to HIV infection. Mov 10 and CPSF6 are the newer restriction factors that are being considered for development in this strategy [106, 152]. Recently, the interferon inducible family of proteins called the myxovirus resistance proteins (Mxs) have been identified as potent restriction factors which

can be saved from committing 'suicide' if they are made impervious to viral entry.

therapy [148].

HIV infection (Table 5).

Studies employing RNA based therapeutics such as antisense oligonucleotides, RNAi, ribozymes and aptamers have shown to be effective in down-regulation of CCR5 and subse‐ quent inhibition of HIV replication in humanized mice [106, 157]. Of all the above mentioned technqiues, silencing of CCR5 using RNAi is the most widely studied. RNAi techniques have also been studied for the downregulation of other cellular factors of HIV replication such as CXCR4, CD4, NF-κB, LEDGF/p75 and DDX-3 [158].

RNAi techniques for host cell modulation are devoid of the problem of viral escape mutants that is faced, when the same technique is employed for inhibiting viral targets. However, the adverse effects faced with these techniqiues are due to loss of other essential functions of the target cell as a consequence of cellular factor silencing. Increased susceptibility to West Nile virus infections has been observed with the loss of CCR5 function. CXCR4 downregulation can disrupt the homeostasis of the lymphopoietic pathway as these receptors are essential for the homing of stem cells into bone marrow and their subsequent differentiation into T-cells [159].

Hence, the success of this strategy depends on the identification of suitable cellular co-factors of HIV infection which can be down regulated without altering the normal cellular physiology. In the search for such targets, genome wide profiling studies have revealed numerous previously unknown cellular factors that participate in HIV infection. Subsequent knock down studies have validated the effectiveness of silencing these factors for limiting HIV replication [160-162]. Further studies are needed to reveal the adverse effects following the knock down of these novel cellular factors.

The technique related pitfalls are the same as the ones mentioned earlier, which include the problems faced with delivery of inhibitory RNAs into specific target cells, off-target effects of the inhibitory RNAs and cytotoxicity to the administered cell. As of date, only one RNAi technique has entered clinical trial. The technique was designed to inhibit both viral (tat protein and TAR RNA) and cellular factors (CCR5). However, the trial was terminated in phase-0 and the results were not disclosed [163]. Table-5 broadly summarizes all the nucleic acid based therapeutics that are currently being tried against HIV.


**Table 5.** Nucleic acid based therapeutics for HIV infection
