**6.2. DNA manipulation**

CCR5Δ32 deletion and produced T-cells with a truncated chemokine receptor CCR5 conferring resistance to infection with CCR5 utilizing virus. The patient had discontinued HAART after the transplant yet has had no detectable viremia for over five years [141]. The experiences with the Berlin patient, introduced the concept of 'sterilizing cure' which essentially comprises of eradicating all replication competent viruses from the body including the ones inside the latent

Replacement with resilient target cells alone does not suffice to achieve a sterilizing cure. As seen with the Berlin patient, the replacement therapy must be preceded by eradication of latent reservoirs. This has been effectively achieved in the Berlin patient by the myelo-ablative procedures prior to bone marrow transplant and continued by the graft versus host disease

Attempts to replicate the cure of the Berlin patient have not been successful so far. A similar treatment provided for two adults in Boston resulted in a brief period of aviremic state, subsequently followed by rebound viremia in both the patients. A few reasons have been postulated for the failure of sterilizing cure in the Boston patients. The first reason being, the initial myelo-ablative procedure was milder than that which was given to the Berlin patient and hence would have not effectively destroyed the viral reservoirs. Also, the donor cells transplanted to the Boston patients carried a heterozygous CCRΔ32 deletion which might allow HIV infection, when compared to the more resilient homozygous mutant cells trans‐

Although well substantiated, the concept of sterilizing cure still has many lacunae. The exact correlates of protection involved in pre-transplant reservoir ablation, establishment of a graft versus host disease and homozygous versus heterozygous CCR5Δ32 deletion are yet to be identified. It has been observed that CCR5Δ32 deletion is associated with increased suscepti‐ bility to infections with West Nile virus. There may be other serious adverse effects which are to be identified before using defective CCR5 as replacement therapy for HIV infection. Another thought provoking issue is the ability of defective CCR5 in protecting infections caused by

Apart from the above mentioned hurdles, the major setback which could limit the reality of this strategy is the availability of matched donors with the required mutation. Autologous bone marrow transplant has been considered to overcome these stringent requirements of allogenic transplants. Transplants of uninfected haematopoietic stem cells harvested earlier from the same person and made resilient to HIV infection by *in vitro* genetic modification (discussed below) do not confer as much protection as their allogenic counterparts. The poor performance of autologous transplants is attributed to the lack of the 'allo-effect' in clearing the infected reservoirs by establishing a graft versus host disease [145]. Hence it has to be borne in mind that replacement with resilient target cells is not a stand-alone strategy and must be compulsorily preceded by reservoir eradication procedures, in order to achieve an effective

reservoirs.

which occurred following the transplant [142].

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

planted to the Berlin patient [143].

CXCR4 tropic viruses [144].

sterilizing cure.

Techniques of DNA manipulation have immense potential and offer a wide scope in tackling HIV. Apart from their use in peptide delivery and in the shock and kill and the genome editing strategies for reservoir elimination, the different techniques of genetic manipulation can be exploited to confer resilience to the natural target cells against HIV infection. DNA manipu‐ latory techniques can confer CD4 T-cells with resilience to HIV infection by either modification of the natural cellular components which are utilized by HIV or by the administration of engineered genetic material which get expressed to produce HIV inhibitory peptides. The techniques utilized for this purpose include gene therapy and genome editing.

Although the term 'gene therapy' is interchangeably used to denote all the DNA and RNA based techniques, this much earlier developed technique actually involves the delivery of specific genetic elements to the target cells by a suitable vector. This is followed by homologous recombination of the transferred genetic element with the recipient cell genome and its eventual participation in transcription and translation to express the desired phenotype. However attractive it may be, the success of this technique depends on the effective recombi‐ nation of the foreign gene with the target cell genome. Low frequency of recombination is the principal drawback faced with this technology [146].

The more recent gene editing techniques as mentioned earlier, comprises excising specific portions of genetic material from the target cell genome. The principle of this technology makes it a good strategy for reservoir elimination as it can cut off the unwanted proviral genes without any subsequent untoward effect. Although when used for host cell genetic manipulation, the success of these techniques relies on the identification of suitable sub-cellular targets, which on manipulation cause significant impairment of HIV replication without affecting the normal cellular function. These novel techniques are found to have a higher success rate than other dated techniques of gene therapy [140].

Though the functional mechanism differ in the techniques, they still have several processes in common. Firstly, both techniques require an effective carrier-delivery system called vectors which can deposit the genetic elements / editing machinery specifically to the cells that are to be modified. Various viral vectors utilizing adenovirus, baculovirus, canary pox virus or lentivrus have been developed for this purpose. *In vivo* use of viral vectors face the problem of antibody mediated clearance. To overcome this challenge, non viral vectors based on nanoparticles like dendrimers are being developed. Cell electroporation is yet another technique that has been developed for the *in vitro* delivery of nucleic acids into target cells [147]. The second common feature is that, both the techniques can be developed for either *in vitro* or *in vivo* use. The *in vitro* methods involve in harvesting of the cells of interest, genetic modification in laboratory conditions followed by transfusion of the modified cells to the recipient, while the *in vivo* methods rely on vector mediated delivery by active targeting of the cells of interest. Another feature of the genetic manipulation procedures is that they could be performed on either mature cells or stem cells. Modification of mature CD4 T-cells confers protection only during their life span and warrant the need for repetitive transfusions over time. On the other hand, genetically modified stem cells such as the CD34+ haematopoietic stem cells can be effective with a single transplantation as exemplified with replacement therapy [148].

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

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 HIV infection (Table 5).

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 act by inhibiting HIV uncoating [153]. Also, TSG-101 the intracellular protein derivative of tumor susceptibility gene has been identified to inhibit the HIV protein p6 thereby blocking viral budding and release [154]. These proteins could possibly serve as candidates for antiretroviral gene therapy.

Administration and expression of extraneous genetic material can also confer resistance to the target cells against HIV. Much of recent interest is towards the surface modification using the synthetic peptide 'C46'. When expressed on the CD4 T-cell surface, this peptide binds with gp41 of the approaching virions and prevents envelope fusion. Stable expression of C46 can be achieved on CD4 T-cells following delivery of the corresponding gene using retroviral vectors. As the cells made resilient by CCR5 alteration alone remain vulnerable to CXCR4 tropic viruses and the reverse also holds good, C46 can be effectively used to inhibit infection with both of the viral strains [152]. Extraneously administered genetic material coding for dominant negative inhibitory proteins of HIV replication such as the M-10 and those coding for intrabodies and intrakines can also cause favourable intracellular modifications in the Tcells making them resistant to HIV infection [148].

Newer studies advocate the combination of both surface and intracellular modifications of the target cells to obtain improved resilience against HIV [147]. Apart from their role in enhancing resilience to HIV infection, techniques of genetic manipulation are also useful in conferring resistance to viral integration and thereby restricting the reservoir formation [155]. Although the techniques of genetic manipulation has numerous setbacks; poor frequency of recombi‐ nation faced with gene therapy, unwanted off-target effects and double strand break induced apoptosis occurring with gene editing are the principal challenges that have to be overcome. Nevertheless, few of these techniques have entered into clinical trials and give hope for a promising future [152]. Apart from their use in therapeutic strategies, techniques of gene therapy are being evaluated in DNA vaccines for prophylactic use and also in some of the strategies of 'immune therapy' [156].
