**5. Strategies to eliminate latent reservoirs**

**4.9. RNA based therapeutics**

to HIV infection, which is discussed later [106].

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

strategies to enhance target cell resilience to HIV infection).

RNA based therapeutic strategies exert their action between the phases of transcription and translation. Numerous RNA based techniques have been developed which are classified by their mechanism of action. They include; inhibitors of messenger RNA (mRNA) translation (antisense oligonucleotides), the agents of RNA interference (RNAi), catalytically active RNA molecules (ribozymes) and RNAs that bind proteins and other molecular ligands (aptamers) [104, 105] (Table-5). These techniques can be utilized for the treatment of any viral disease by engineering specific, complementary inhibitory RNA particles to the viral transcription components. Among these techniques, RNAi and to a lesser extent antisense oligonucleotides, have been tried out to inhibit retroviral replication. As these techniques can also target the host cellular processes, they are being exploited in strategies to increase the resilience of target cells

RNAi is an endogenous mechanism which involves the down regulation of mRNA activity during transcription and post transcription phases using short double stranded RNA (dsRNA) called micro RNA (miRNA), which are about 20-30bp long. The identification of this regulatory process has provoked the interest of controlling unwanted viral replication using exogenously administered specific sequences of short dsRNA. The exogenously administered agents of RNAi therapy include small interfering RNA (siRNA) and short hairpin RNA (shRNA). These agents act on the post transcript mRNA and either cause direct sequence specific cleavage when there is a perfect sequence complementarity match, or lead to translational repression and degradation of mRNA when the interfering RNA sequence is of limited complimentarity to the targeted mRNA. As the siRNA get cleared off after their action, their effects are only transient and need repeated administration similar to agents of chemotherapy. On the other hand the action of shRNA is similar to gene therapy, as they get expressed on promoters and cause long term effects. Various viral and non-viral delivery mechanisms and active targeting strategies have been developed to deliver these active agents into the target cells [107, 108].

Systems employing siRNA and shRNA to target the gene products of tat, rev, nef, env, vif and pol have been designed and evaluated for efficacy [47, 106]. However, the use of this technol‐ ogy against viral replicatory processess is threatened by the genetic variation exhibited by HIV. Very simple mutations allow HIV to escape from the action of both siRNA and shRNA [109, 110]. Four possible solutions are being tried to tackle the problem of these escape mutants. The first attempt is to expand the RNAi technique to simulataneously inhibit multiple HIV mRNA targets similar to the concept of multidrug use. Recent studies have demonstrated that concurrent inhibition of HIV mRNA with three different shRNAs can prevent viral escape *in vitro* [111, 112]. In the second possible solution, inhibitory RNAs with a complete match to the most commonly encountered viral escape sequences are being designed. When used along with the inhibitory RNA of the wild type virus, these could prevent a majority of the mutants from escape [113]. The third solution involves identifying novel, genetically conserved sequences of HIV which do not usually undergo mutation. Targeting these stable sites would favour the success of RNAi [114]. Finally, RNAi techniques are also being designed to restrict the 'genetically more stable' host factors that help in HIV replication (discussed later under

The ability to integrate its nucleic acids with host cell genome and co-exist in a genetically quiescent proviral state is one of the distinct features of HIV, which makes eradication of infection next to impossible. The latent reservoirs of HIV are categorized into two broad groups namely the cellular and the anatomical reservoir. The cellular reservoir comprises the long lived resting CD4 T-cells. With an extended lifespan averaging 400 days, these cells bear the provirus and release the progeny upon activation at a later time [116]. Infection of the cells of the monocyte-macrophage lineage and subsequent compartmentalization of these infected cells into various organs / tissues such as the reticulo-endothelial system, lymph nodes, gastrointestinal tract, brain and lungs, lead to the formation of anatomical reservoirs. Within the macrophages of these anatomical reservoirs, the HIV either remains quiescent inside the chronically infected cells or maintains a continuous low level replication [117]. Thus, the reservoirs act as Trojan horses spilling the progeny virions into circulation at periodic intervals. Although the mechanisms involved in proviral repression are gradually being unraveled, specific factors / agents reactivating the viral replication are yet to be clearly identified. Although early aggressive treatment helps in achieving a functional cure by limiting the reservoir formation, it does not offer a solution to the already established reservoirs. The new strategies and techniques that are being developed for eradicating the established viral reservoirs are subsequently discussed.

#### **5.1. Targeted drug delivery to the reservoirs**

Apart from attempts to limit viral replication in actively infected CD4 T-cells, nanotechnology methods are widely studied for the elimination of the latent viral reservoirs. Strategies include passive and active targeting and the use of surface moieties which enhance the penetration of biological barriers by the nanoparticles.

Upon systemic administration, most of the nanoparticles are instantaneously opsonized with plasma proteins and rapidly cleared off from circulation by the phagocytosis. The macro‐ phages of the reticulo-endothelial system are the principal cells which are involved in the degradation and clearance of the nanoparticles [118]. This forms the basis of passive targeting which aims at achieving high concentrations of anti-retroviral agents in the reticulo-endothe‐ lial reservoir as a consequence of phagocytosis of the drug loaded nanoparticles. Studies have demonstrated the achievement of higher drug concentrations in the reticulo-endothelial cells following the administration of liposomes, nanocapsules or polymeric nanoparticles loaded with anti-retroviral drugs [119]. Likewise, passive targeting of the lymphatic reservoir can be achieved by incorporation of the nanoparticles with lipids such as phosphatidylcholine and cholesterol or by surface coating of the nanocarriers with polyethylene glycol [120, 121].

Apart from passive targeting, specific drug delivery to the anatomical reservoirs can be accomplished by active targeting strategies. Nanocarriers tagged on their surface with galactose or mannose residues or anti-HLA-DR monoclonal antibodies, effectively localize in the reticulo-endothelial system which have abundant receptors for these ligands such as the galactose and lectin receptors and the HLA-DR determinant of MHC-II respectively. Active targeting of the cellular reservoirs is based on the aforementioned principle of employing nanocarriers with homing ligands to viral components on infected cell surface. The infected resting CD4 T-cells containing HIV envelope glycoproteins on their surface attract the drug loaded nanoparticles coated with recombinant CD4 molecules or Fab fragments of monoclonal anti-gp120 antibodies [13].

Nanotechnology also offers a solution for the problem of poor drug penetration in certain anatomical sites. One such anatomical reservoir is the brain, where the infected microglial cells rest safely with the protection conferred by the blood brain barrier against the system‐ ically administered anti-retroviral agents. The blood brain barrier functions not only by preventing the permeation of the circulating anti-retroviral compounds into the brain tissue, but also by efflux of a considerable portion of the compounds that have managed to cross through. With the ability to increase the crossing and reduce the efflux of drugs, nanotechnol‐ ogy methods help in reservoir elimination by overcoming the privilege offered by the blood brain barrier [122].

Nanotechnology methods that increase the drug transport across the blood brain barrier function by mimicry of natural substrates, utilization of cell penetrating peptides or by active targeting of molecules of abundance in neuronal vasculature with suitable surface ligands. The non-ionic surfactant 'polysorbate-80' has found to be an effective enhancer of drug delivery to the brain using the principle of substrate mimicry. Upon systemic administration, the drug loaded nanoparticles coated with polysorbate-80 adsorb various apolipoproteins in circulation to form a complex that mimics lipoproteins. Presuming them as natural substrates, the blood brain barrier permits the entry of these complexes by receptor mediated transcytosis resulting in delivery of the drug to the brain tissue [123]. Nanoparticles coated with cell penetrating peptides such as the HIV-1 tat peptide has shown enhanced efficacy in crossing the blood brain barrier. As the microvasculature of the brain is rich in receptors such as transferring receptor, low-density lipoprotein receptor and β2 receptors, their respective ligands such as transferrin, apolipoprotein-E and β2 agonists are considered possible candidates for active targeting.

The use of nanotechnology for reservoir elimination poses some peculiar problems in addition to other drawbacks mentioned earlier. The effectiveness of nanotechniques that rely on antiretroviral drugs to eliminate the reservoir is questionable as these agents are lethal to the virus only during active replication and do not affect the inactive provirus. The final clearance of the nanoparticles from the anatomical sites, especially from the brain is not clearly known and accumulation overtime with repeated doses can possibly lead to neurotoxicity. Acquisition of more knowledge through carefully designed clinical trials is needed so that the potential of this technology could be put into adequate use [122].
