key: cord-329361-0mpbau1b
authors: Bennasser, Yamina; Yeung, Man Lung; Jeang, Kuan-Teh
title: RNAi Therapy for HIV Infection: Principles and Practicalities
date: 2012-08-16
journal: BioDrugs
DOI: 10.2165/00063030-200721010-00003
sha: 
doc_id: 329361
cord_uid: 0mpbau1b

Inside eukaryotic cells, small RNA duplexes, called small interfering RNAs (siRNAs), activate a conserved RNA interference (RNAi) pathway which leads to specific degradation of complementary target mRNAs through base-pairing recognition. As with other viruses, studies have shown that replication of the HIV-1 in cultured cells can be targeted and inhibited by synthetic siRNAs. The relative ease of siRNA design and the versatility of RNAi to target a broad spectrum of mRNAs have led to the promise that drug discovery in the RNAi pathway could be effective against pathogens. This review discusses the current experimental principles that guide the application of RNAi against HIV and describes challenges and limitations that need to be surmounted in order for siRNAs to become practical antiviral drugs. The practical use of RNAi therapy for HIV infection will depend on overcoming several challenges, including the ability to establish long-term expression of siRNA without off-target effects and the capacity to counteract mutant escape viruses.

RNA interference (RNAi) has emerged as a common in vitro the siRNA duplex is loaded onto RISC to serve as the guide strand while the second strand, the passenger RNA, is degraded. [10] RISC tool for silencing gene expression. [1, 2] The introduction of small uses its guide RNA for base-pairing-mediated recognition of target RNA duplexes of 19 to 21 nucleotides into cells can elicit specific messenger RNA (mRNA). Once this specific hybridization is degradation of complementary gene sequences through base-pairachieved, the PIWI domain of the Ago2 protein within the RISC ing. [3] It is thought that the RNAi pathway serves as part of the complex degrades and silences the targeted substrate mRNA. [11] innate immune defense of eukaryotes against invasion by exoge-Because siRNAs are easy to design and synthesize and because nous nucleic acids. [4] Hence, in plants and Drosophila, when a cell they have sequence specificity for silencing mRNAs, these small is infected by a virus, an RNAi response is triggered by the foreign RNAs potentially represent a future class of antiviral drugs. double-stranded RNA (dsRNA) molecules that originate from the virus. [5] It has been shown recently that retroviruses such as human

Using Small Interfering RNA (siRNA) potentially be engaged into the RNAi pathway of mammalian cells. [6, 7] In a relatively short period of time, RNAi has been shown in An early step in the RNAi response enlists an RNase III enzyme cultured cells to be efficacious against various viruses including called Dicer to bind and cleave long dsRNAs into small duplexes respiratory syncytial virus (RSV), [12] influenza virus, [13] of 19 to 21 nucleotides, termed small interfering RNA (siRNA).

poliovirus, [14] and hepatitis C virus (HCV). [15] For HIV, a variety Dicer-siRNA complex is then recognized by trans activation reof siRNAs have also been reported to be effective in interrupting sponse region (TAR) RNA-binding protein (TRBP) and protein viral infection. Two siRNA strategies have been considered for activator of PKR (PACT), two cellular dsRNA binding proteins.

HIV; the first is to target essential viral genes and the second is to target cellular genes required by HIV for replication (table I) . The multi-protein entity is shuttled through interaction with the argonaute 2 (Ago2) protein into an effector complex, the RNA-To date, HIV sequences that have been targeted include the induced silencing complex (RISC). [8, 9] One of the two strands of structural gag gene, [16] the infectivity factors vif and nef, [3] tat, rev, , a cellular protein involved in multivesicular body formation, [33] and targeting tumor susceptibility gene 101

[TSG101], also called vacuolar protein-sorting protein 23, involved in endocytic trafficking [36] ) 5. other cellular proteins used by HIV-1 for replication such as peptidylprolyl isomerase A (cyclophilin A), [34] the RAS oncogene family member RAB9, [35] and CDK2. [29] In many of the above examples, it should be pointed out that instead of directly introducing siRNA duplexes into cells the effect of which is transient, in many cases, RNAi was expressed using a DNA plasmid expressing a short hairpin RNA (shRNA). It is recognized that the cell's Dicer enzyme can remove the hairpin loop from shRNA molecules, processing them to their siRNA counterparts. Currently, a myriad of systems are available for expressing shRNAs (e.g. under the control of an RNA polymerase III U6 or H1 promoter or an RNA polymerase II CMV promoter) in several vector systems (e.g. adenoviral or lentiviral vector [17, 41] ), allowing for long-term silencing even in nondividing cells.

HIV-1 is a notoriously mutable virus. To be effective, antiviral drugs based on sequence hybridization must take into account that HIV-1 mutates its sequence easily. Indeed, HIV-1 can escape env, and TAR RNA. [21, 37, 38] In each instance, sequence-specific siRNAs through nucleotide mutations. For example, Das et al. [42] silencing of viral RNA and transient suppression of HIV replicaobserved that an siRNA targeted to nef rapidly elicited the emertion over a period of 3 to 4 days in single round infection of gence of siRNA-resistant viruses with point mutations in the nef cultured cells have been achieved. However, it is important to note gene. Alternatively, the deletation of an siRNA recognition site that virion-associated HIV-1 genomic RNA that infects cells apcan also allow the virus to evade restriction. [43] Moreover, there is pears to be resistant to RNAi-mediated degradation. [39] This findevidence that HIV-1 can undergo nucleotide substitutions that ing argues that if a goal is to prevent the genesis of integrated induce an alternative RNA folding, thus shielding a previously provirus then, by using siRNA, one should target cellular factors targeted sequence from access by the siRNA. [42] required for early steps of HIV-1 replication rather than viral

To minimize the mutational escape of HIV, one strategy is to sequences.

simultaneously use multiple siRNAs to target discrete sequences. siRNA directed to several HIV-1 relevant cellular factors have

The likelihood that a single viral RNA molecule would mutate all targeted sequences becomes increasingly small as the number of indeed been tested for antiviral efficacy. These include siRNA targets is escalated. [44] This multi-targeting strategy is aided by targeted to:

computational modeling, which can predict how HIV-1 might 1. viral entry (e.g. siRNA against HIV coreceptors chemokine evolve in cells that express multiple antiviral siRNAs. [ [28, 30, 40] ) and CXCR4 have been reported. [46] In addition to evolving siRNA-escape mutations, viruses can target effects can arise from as few as 7 nucleotides of fortuitous also encode suppressor factors that attenuate the cell's RNAi base-pairing between siRNA and mRNA. [62] Considered this way, response. [47, 48] While much remains to be elucidated, HIV-1 ap-a particular siRNA could potentially suppress the expression of its pears to have multifaceted ways to suppress the cell's RNAi intended target and several unintended mRNAs. [63] This off-target machinery. [49] The viral Tat protein appears to work as a protein phenomenon can be a significant drawback should siRNAs be suppressor of RNAi. [50] In addition, the viral RNA, TAR, [51] is also envisioned for in vivo use as human drugs. One anticipates that used by HIV-1 as a suppressor of RNAi. [52, 53] If siRNA-based methods and modifications that minimize off-target hybridization therapy of HIV-1 and other viruses is to be usefully contemplated, would need to be greatly improved to enable further siRNA drug one needs to consider strategies that address virus-encoded RNAi development. [64] suppressors. Despite virally encoded suppressors, there is ample Another unwelcome adverse effect of siRNA use is the potenevidence for RNAi-mediated inhibition of HIV-1 replication. It is tial to inappropriately elicit interferon and inflammatory possible that the suppressors are either synthesized too late or in cytokines. Normally, RNA duplexes shorter than 30 nucleotides in amounts too small to counter the effect of a pre-existing siRNA or length would not be expected to trigger the interferon-linked shRNA.

protein kinase R pathway. However, recently it was found that some GU-rich sequences, such as 5′-UGUGU-3′and 5′-GUC-CUUCAA-3′, which are toll-like receptor (TLR) immunostimu-

latory motifs, can trigger an interferon response [65] even when presented in duplexes shorter than 30 nucleotides. To minimize The siRNA machinery used in eukaryotic cells is the same as nonspecific interferon responses, judicious care must be exercised that used to process a class of endogenous small RNAs called in the sequence design of siRNA drugs. microRNAs (miRNAs). miRNAs are important regulators of at least 30% of cellular genes, including those involved in development, signal transduction, apoptosis, cell proliferation, and 4. Development of Antiviral siRNA Drugs: Strategies tumorigenesis. [54] [55] [56] [57] To date, 474 human miRNAs have been de-and Limitations scribed, with more likely to be discovered (see miRNA database [58] ). [59] Recent optimism regarding the possibility of siRNA drugs The mechanism of miRNA action and the relevance of arises from studies that indicate that intravenous injections of an miRNAs to HIV-1 infection have been reviewed elsewhere [47, 60] siRNA directed against Fas can protect mice from liver injury and and will not be elaborated further here. What needs to be empha-fibrosis in two models of autoimmune hepatitis [66] and from findsized is the fact that the shared factors for shRNA, siRNA, and ings that intranasal administrations of separate siRNAs protected miRNA processing are saturable entities. Because miRNAs are mice from RSV infection [67] and Rhesus macaques from SARS essential to cellular metabolism and viability, if the machinery for coronavirus infection. [68] siRNA application to the mouse genital miRNA genesis is diverted to handle exogenous siRNA or tract has also been found to be protective against lethal herpes shRNA, then a dearth of miRNAs can potentially manifest as simplex virus type 2 infection. [69] These preliminary positive findcellular toxicity. This hitherto unexpected finding was indeed ings have prompted phase I studies to be initiated in humans. To demonstrated in a recent study of in vivo overexpression of shRNA date, three clinical trials have been completed in humans and show in mice. [61] In that study, severe liver toxicity and the death of 150 no significant siRNA toxicity. [70] siRNAs are currently in clinical mice occurred within 2 weeks of shRNA treatment. The explana-investigation for two diseases: age-related macular degeneration tion for the fatalities was the saturation by shRNA of the pathway (AMD) caused by an overexpression of the vascular endothelial normally used in the liver to process miRNAs. Hence, the high growth factor (VEGF) and respiratory infection caused by the shRNA expression in the mouse liver overwhelmed the export RSV. pathway (i.e. exportin-5) needed to transport precursor miRNAs At this time, there is no siRNA drug for HIV-1. As outlined from the nucleus into the cytoplasm. The interruption of proper above, there are several issues unique to the targeting of viral transport of miRNA precursors led to reduced levels of mature genes which do not apply to the targeting of cellular genes. miRNAs with resulting cellular toxicity.

However, a question that is relevant to all siRNA applications is Apart from the problem of pathway saturation, off-target ef-how best to deliver siRNA into virus-infected cells. In this regard, fects of siRNAs raise additional concerns. On-target effects ema-several methods are being considered. For example, in HCV nate from perfect complementarity between guide-RNA and target infections, a procedure that couples siRNA to cholesterol [71] al-mRNA (i.e. 19 of 19 matched nucleotide base-pairings), while off-lows for efficient targeting of hepatocytes, the cells preferentially infected by virus. Elsewhere, a novel strategy using antibody-sette, which was shown to be effective in inhibiting HIV replicamediated siRNA delivery has been proposed for HIV-1-infected tion [22] and a doxycycline-dependent lentivector. [73] Going forcells. [18] Whether these biochemical delivery techniques can dis-ward, a major challenge is to perfect vectors that are wholly silent tribute siRNAs effectively to in vivo compartments awaits further for shRNA expression unless specifically triggered by HIV-1 verification.

infection. There are also virus-based approaches to deliver shRNA; how-Despite the optimized delivery of siRNA and shRNA into cells, ever, this type of gene therapy approach does raise issues related to the ability of viruses to mutate and escape from RNAi is a biosafety. [72] A major focus for HIV-1-infected cells has been the significantly daunting problem. A multi-modal approach may use of lentiviral vectors, which have the ability to infect nondivid-have to be adopted in order to minimize viral escape. [44] For ing cells, such as resting T-cells, macrophages, and progenitor example, shRNA targeted to Rev can be used at the same time as a hematopoietic cells (CD34+ cells ogies has been proposed for HIV-1 therapy. [75] Here, the strategy is 

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