The continual development of resistant viral strains of the Human Immunodeficiency virus (HIV) and the resulting global AIDS pandemic, calls for incessant research directed to develop new strategies to target and suppress this virus. RNA interference (RNAi) is a novel tool being explored for its potential to serve this purpose. Numerous studies demonstrate that RNAi-based strategies can be useful in suppressing HIV infection and replication. This review investigates strategies involving the use of siRNAs and shRNAs to target various viral genes and/or host factor genes that are known to play a role in the virus infection cycle. The review looks into the research done in each of these arenas and attempts to highlight the advantages and flaws of each approach. Further, we attempt to discuss the enigma surrounding viral resistance to RNAi. Despite numerous pre-clinical studies examining the efiiciency of RNAi for viral suppression, RNAi-based therapy has not yet reached the clinic.
Keywords: RNAi, HIV/AIDS, siRNA, shRNA, HHART, gene silencing
Key: AIDS- Acquired Immunodeficiency Syndrome; CDC- Center for Disease Control; HAART- Highly active anti-retroviral therapy; HIV- Human Immunodeficiency Virus; LTRs- Long Terminal Repeats; PTGS- post-transcriptional gene silencing; RNAi- RNA Interference; shRNA- short hairpin RNA; siRNA- small-interfering RNA; RT- Reverse Trancriptase; TGS-transcriptional gene silencing.
As per the CDC HIV Surveillance Report, with more than 35 million people affected by HIV/AIDS worldwide (1), this sexually-transmitted disease has transformed into a global pandemic; thus necessitating research dedicated to explore new treatment options for this debilitating condition. RNA interference technology can serve as a powerful tool for developing a therapy for HIV/AIDS. More than a decade of research in this direction corroborates this new potential approach. This review article focuses on providing a lucid account of the research examining the different RNAi-based strategies that can be used to target HIV.
RNA interference is a post-transcriptional gene silencing mechanism discovered by Andrew Z. Fire and Craig C. Mello in 1998 and was first demonstrated in C. elegans (2). This Nobel Prize-winning discovery was hailed as the ‘breakthrough of the year’ in 2002 by the Science magazine (3). This drew the attention of scientists to the potential applications of RNAi and the first studies on the anti-viral applications of RNAi were carried out for HIV in 2002 (4). The underlying mechanism of RNAi involves the guide RNA strand- dependent silencing of gene expression by either degrading the mRNA or blocking its translation (5).
Current treatment for HIV/AIDS makes use of highly active anti-retroviral therapy (HAART) which involves a combination of three or more anti-viral drugs targeting different viral targets (6). However, although HAART is effective, it has its own set of complications involving drug resistance, toxicities and side-effects and the high cost involved. RNAi is being explored for its potential to be utilized as a therapeutic intervention against HIV viral infection (7). The homology-dependent RNAi technique thus provides the specificity to target various viral mRNA targets i.e. silencing specific viral genes. It may also be directed to target the mRNA transcribed from host genes that are known to be involved in the viral infection cycle. Any of these genes can be targeted by RNAi using either small-interfering RNAs (siRNAs) (8) or gene therapy involving short hairpin RNAs (shRNAs) (9).
Figure 1: Schematic representation of the RNAi mechanism. Reprinted from Figure 3, page 2 (Source: Nobel Media; reference 10)
RNAi is a post-transcriptional gene silencing (PTGS) mechanism which operates endogenously in plants, mammals and some invertebrates. Double-stranded RNA (dsRNA) is known to initiate the process of RNAi by activating the ribonuclease protein Dicer. Dicer is a member of the RNAse III family of dsRNA-specific endonucleases. The dicer cleaves dsRNAs to produce siRNAs which are short double-stranded fragments of 20-25 base pairs with a 2 nucleotide overhang at the 3′ end (11). This structural specificity of the siRNA aids in the formation of the RISC (RNA- induced silencing complex) complex. The siRNA is denatured and the guide strand binds to the RISC complex while the passenger strand is released and subsequently degraded. This guide strand then brings about degradation of complementary mRNA by the Argonaute protein, an endonuclease which is part of the RISC. siRNAs are very specific to their target mRNA. Exogenous introduction of dsRNA or siRNA are effective techniques to knockdown the expression of specific genes (12).
HIV viral infection and Current Therapies
The Human Immunodeficiency Virus (HIV) is a known retrovirus that infects helper T-cells, macrophages and dendritic cells, causing Acquired Immunodeficiency Syndrome (AIDS) in humans (13).
The available drugs for HIV/AIDS can be categorized into different categories: Nucleotide Reverse Transcriptase Inhibitors (NRTIs) which are nucleotide analogs, the Non- Nucleotide Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors, Integrase Inhibitors, Fusion Inhibitors and Entry Inhibitors. These classes of anti-viral drugs target different stages of the HIV viral infection cycle (14).
Figure 2: Diagrammatic representation of how the current drugs intervene with HIV infection cycle. Image Source: Reprinted from Figure 2 (Source: Barré-Sinoussi, Ross and Delfraissy, 2013; reference 14)
2. Drug resistance and HAART:
However, the HIV virus is different from other retroviruses since it develops resistance to the anti-viral drugs, when given alone. This occurs since HIV possesses two copies of its single-stranded viral genome and these two strands can undergo recombination during the process of reverse transcription, thus contributing to the evolution of drug resistance and creation of ‘escape mutants’ (15). The mutations conferring the virus with this property of evasion from targeting drugs, arises during the process of reverse transcription. This may occur because the reverse transcriptase possesses the ability to switch template strands during the process, often termed as copy-choice (16). Thus, mutations may occur during strand-switching or may also occur due to mis-incorporated bases by the reverse transcriptase (RT) during the process of reverse transcription of the viral RNA genome (17). The selective pressure exerted by the new environment upon drug exposure, has been shown to select these escape variants (18).
The currently used highly active anti-retroviral therapy (HAART) remains to be the cornerstone for HIV treatment. HAART is a triple-combination therapy involving prescription of three anti-retroviral drugs belonging to two or three of these classes of drugs (14). The underlying rationale for using a combination of drugs is that the environment becomes more challenging for viral propagation, necessitating the need for an evolution of multi-drug resistant virus variants, thus in turn prolonging the emergence of escape mutants.
3. Drawbacks of HAART:
Drug interactions among the HAART anti-retroviral drugs or with other drugs can always lead to complications. These interactions could also lead to reduction in the bio-availability of the drug. One of the major disadvantages of HAART is the necessity to adhere to strict dosage regimens. In case of poor adherence to prescribed drug regimens or because of the above-mentioned drug interactions, the levels of one of the HAART drugs may fall, thus increasing the chances of developing drug resistance. This in turn ends up undermining the major advantage of HAART per se (6).
Besides, HAART-associated drug toxicities can lead to nausea, diarrhea, lipodystrophy and increase risk of cardiovascular disease, atherosclerosis (18). The biggest drawback of HAART is the unaffordability of the treatment (6).
RNAi as a therapy for HIV
1. Genes Targeted:
Synthetically designed siRNAs or shRNA vectors have been used to target various viral genes such as the TAR element, and other protein-coding and regulatory viral genes such as rev, gag, pol, tat, env, vif, nef and reverse transcriptase (19).
Scientists then came up with an alternative approach to target the host genes that are known to be involved in HIV entry and replication processes. The cellular cofactors required for HIV infection cycle are the HIV receptor CD4, the co-receptors CXCR4 and CCR5 and NFκβ which controls many genes involved in inflammation (20). Several studies predict CCR55 to a promising target (4).
Use of small-interfering RNAs (si-RNAs): siRNA molecules are designed and identified, based on computational analysis of the target viral and host genes and/or high-throughput RNAi screening. These siRNA candidate molecules are then validated by mRNA cleavage assays. Novina et al. carried out studies on cell lines using siRNAs directed against the HIV CD4 receptor to prevent entry of the virus and siRNAs to silence the viral gag and nef genes, involved in viral replication. This was one of the preliminary studies which demonstrated the potential of siRNA technology as a plausible treatment option for HIV/AIDS. Naito et al., in 2007, designed several candidate anti-viral siRNA molecules against HIV-1. (21)
Several in vitro and pre-clinical studies have shown that siRNAs can effectively be used to suppress HIV infection. A research study published in the journal Cell, employed a T-cell specific antibody for targeted delivery of siRNA molecules to the T-cells. This pre-clinical study on mice models confirmed the feasibility of siRNA based therapy for HIV suppression. Anti- CCR5 and various anti-viral siRNA oligo-9-arginine peptides conjugated to T-cell specific antibody were demonstrated to control viral replication and T-cell loss (22).
But, siRNAs do not persist in the cellular milieu for extended periods of time, thus necessitating repetitive delivery of the siRNA molecules. The dosage needed in order to ensure sufficient quantities of the siRNAs also depends on the ability of the siRNA to suppress its target genes (4).
Use of short-hairpin RNAs (shRNAs): Site-specific delivery of siRNAs and maintaining an effective concentration of the siRNA molecules are seemingly big challenges when it comes to developing a commercial therapy for humans. Here, short-hairpin RNAs (shRNAs) serve as rescue guards. shRNAs are structurally similar to siRNAs except that they have a loop at one end of the molecule. They major advantage over siRNAs is that, shRNAs can be used for a stable expression of silencing molecules, rather than the transient siRNA silencing.
The procedure, as illustrated in the following figure, involves incorporating the DNA coding for the shRNA into the host cell’s nucleus. This is brought about by transduction of cells with lentiviral vectors containing plasmids having genes coding for one or more shRNAs. The stably transfected cells are then selected for and introduced into the host system. These cells tend to persist in the host organisms, as they express shRNAs which confer them protection from viral infection. The shRNAs produced are cleaved by the dicer, like dsRNA, to produce molecules similar to the siRNAs. These then bring about silencing of corresponding genes by the same mechanism as siRNAs.
Figure 3: Diagrammatic representation of the shRNA-mediated gene therapy (HSCs: Hematopoetic stem cells)
Several studies have evaluated the efficacy of multiple shRNA coding plasmids. These could include either shRNAs that target the viral genes or the host co-factor genes (7). Studies expressing two (23) and four (9) different shRNAs have successfully shown inhibition of viral infectivity and prevention of viral escape. These shRNA coding constructs can also be made with different promoters based on the requirement of a spatial or temporal control of expression (9).
shRNA- mediated gene therapy involving hematopoetic stem cells could face ethical and social challenges at the clinical phases. Besides, another drawback is overexpression, which can saturate the endogenous RNAi machinery and thus hinder the normal RNAi regulatory function in the cells (24). It is worth mentioning a recent study carried out by Herrera-Carrillo et al., which solved the quandary surrounding the impact of unprotected cells in such shRNA-mediated gene therapy. The general scientific community was of the opinion that addition of unprotected cells (i.e. the cells that have not received the plasmids with the genes encoding the shRNAs) in a gene therapy, enhances the emergence of viral resistance to the shRNAs. However, the said study demonstrated that even in the presence of unprotected cells, expressing multiple shRNAs could successfully prevent viral escape and thus provides strong evidence that unprotected cells accelerated the evolution of the virus (25).
2. Benefits and Limitations:
The first reported study of an emergence of RNAi-resistant HIV escape variants was published in 2005. siRNAs targeting the nef viral gene were unable to inhibit the virus thus exhibiting emergence of RNAi-resistant viral escape variants. Further, sequence examination of these escape variants revealed that these variants emerged as a result of substitution or deletion mutations within the nef sequence or because of mutations in other regions which cause a change in the secondary structure of the RNA, in turn making the nef sequence unavailable for binding to the siRNA/shRNA (26).
The strategy that can be employed to overcome this hurdle is the use of multiple different siRNA/ shRNA molecules targeting different genes. Therefore, as discussed in detail in the previous sections, selecting an optimal combination of several distinct RNA inhibitors can potentially serve as an effective therapeutic technique in overcoming this hurdle (7).
Another recent study by scientists at the University of California, Berkeley, uncovered a new mechanism by which escape mutants might cause an indirect “cross-resistance” to shRNAs targeting two distinct regions of the viral genome. They found four HIV escape mutants, with mutations in the promoter regions in the Long Terminal Repeats (LTRs) of the viral genome that led to increased expression of viral genes, thus escaping the effect of gene silencing by the shRNAs. This is an indirect resistance to shRNAs which target distinct sequences i.e. not the LTRs. However, their study evaluated shRNA monotherapies and combinatorial therapies wherein two different shRNA molecules were introduced at a given time (27). Therefore, a multiple shRNA expression approach could still be able to overcome this problem of cross-resistance. This same study also provided another solution to this problem wherein they demonstrated that RNAi enhancing small molecules when combined with the anti-viral RNA, were able to supress the RNAi-resistant mutants (27). The RNAi enhancing small molecule used in the study by Shah et. al facilitated the interaction between the RNAs (siRNA/shRNA) and the trans-activation-responsive region RNA-bindingprotein (TRBP). TRBP along with the Dicer protein is responsible for the processing and loading of the shRNAs and siRNAs onto the RISC (27).
Also, Suzuki et al. provide a new interesting aspect to the whole scenario. Their study provides clue to transcriptional gene silencing (TGS) by siRNAs. Their results indicate that siRNAs targeted to the NF-κβ-binding regions of 5’- LTR promoter region of HIV induces chromatin modification and heterochromatinization which then leads to transcriptional repression (28). This conclusion followed their findings wherein siRNA-directed TGS correlated with the recruitment of chromatin-modifying repressor complexes (28). The exact mechanism of how the induction and recruitment of the chromatin modifying machinery takes place is not clear. siRNA induced TGS resembles HIV-1 latency observed in vitro (28). Plausibly, this mechanism could also be exploited for overcoming viral resistance to RNAi.
RNA Interference is an endogenous gene expression regulatory mechanism documented in animals, plants and certain invertebrates. Introduction of exogenous dsRNA can mimic the effect of endogenously produced dsRNA, thus utilizing the cellular machinery to bring about silencing of the desired genes. This constitutes the mechanism of action of the exogenously introduced siRNA. shRNAs are however different, in that they are produced by cells stably transduced with plasmid expression vectors capable of producing the shRNA molecules. This in turn constitutes the RNAi-based gene therapy. Either of these techniques can be employed to target specific genes. This property of RNA interference can be exploited for various applications. The HIV/AIDS pandemic demands research directed to find innovative ways to suppress the HIV infection. This review highlights how siRNA and shRNA based approaches can be used to target different viral genes directly. The host genes which play a role in viral infection such as the genes coding for receptors that the viral particles bind to, may also be targeted. Many studies in the last decade have served to support the potential of RNAi as a therapeutic intervention for HIV infection. The emergence of RNAi resistant viral mutants, questions the feasibility of this new approach over the existing treatment options. More research is required to clear the existing perplexity over RNAi escape variants. Such studies will help to develop strategies for new combinatorial therapies involving RNAi for HIV treatment.
Several anti- sense RNA based studies are in different phases of clinical trials, however, studies involving RNAi-base treatments involving either shRNA and siRNA are yet in the pre-clinical phase (29). Additionally, the future studies directed to improving our understanding of delivery vehicles for a targeted delivery of siRNAs, can further strengthen the scope of siRNAs being used for treatment against HIV/AIDS and several other diseases.
- CDC (2013).HIV Surveillance Report: Diagnoses of HIV Infection and AIDS in the United States and Dependent Areas, 25.
- Fire A., Xu S., Montgomery M. K., Kostas S.A., Driver S.E. & Mello C.C., (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, pp.806-811.
- Jennifer Couzin (2002). “BREAKTHROUGH OF THE YEAR. Small RNAs Make Big Splash”.Science 298 (5602): 2296–2297.
- Rossi, J. (2006). RNAi as a treatment for HIV1 infection. BioTechniques, 40(S4), pp.S25S29.
- Meister, G. and Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature, 431(7006), pp.343-349.
- Peters, B. and Conway, K. (2011). Therapy for HIV: Past, Present, and Future. Advances in Dental Research, 23(1), pp.2327.
- Jasmijn von Eije, K. and Berkhout, B. (2009). RNA-interference-based gene therapy approaches to HIV type-1 treatment: tackling the hurdles from bench to bedside. Antivir Chem Chemother., 19(6):221-33.
- Novina, C., Murray, M., Dykxhoorn, D., Beresford, P., Riess, J., Lee, S., Collman, R., Lieberman, J., Shankar, P. and Sharp, P. (2002). siRNAdirected inhibition of HIV1 infection. Nature Medicine, 8(7), pp.681-686.
- Brake, O., Hooft, K., Liu, Y., Centlivre, M., Jasmijn von Eije, K. and Berkhout, B. (2008). Lentiviral Vector Design for Multiple shRNA Expression and Durable HIV1 Inhibition. Mol Ther, 16(3), pp.557564.
- “The 2006 Nobel Prize in Physiology or Medicine – Popular Information”.org.Nobel Media AB 2014. Web. 28 Apr 2015. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2006/popular.html
- Guo, P., Coban, O., Snead, N., Trebley, J., Hoeprich, S., Guo, S., & Shu, Y. (2010). Engineering RNA for Targeted siRNA Delivery and Medical Application.Advanced Drug Delivery Reviews, 62(6), 650-666. doi:10.1016/j.addr.2010.03.008
- Aagaard, L., & Rossi, J. (2007). RNAi therapeutics: principles, prospects and challenges.Advanced Drug Delivery Reviews, 59(2), 75–86.
- Abbas, W. and Herbein, G. (2012). Molecular Understanding of HIV-1 Latency. Advances in Virology, 2012, pp.1-14.
- Barré-Sinoussi, F., Ross, A. and Delfraissy, J. (2013). Past, present and future: 30 years of HIV research. Nat Rev Micro, 11(12), pp.877-883.
- Nora, T., Charpentier, C., Tenaillon, O., Hoede, C., Clavel, F. and Hance, A. (2007). Contribution of Recombination to the Evolution of Human Immunodeficiency Viruses Expressing Resistance to Antiretroviral Treatment. Journal of Virology, 81(14), pp.7620-7628.
- Hwang, C., Svarovskaia, E. and Pathak, V. (2001). Dynamic copy choice: Steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching. Proceedings of the National Academy of Sciences, 98(21), pp.12209-12214.
- Chen, J., Rhodes, T. and Hu, W. (2005). Comparison of the Genetic Recombination Rates of Human Immunodeficiency Virus Type 1 in Macrophages and T Cells. Journal of Virology, 79(14), pp.9337-9340.
- ter Hofstede, H.J.M., Burger, D.M., Koopmans, P.P. (2003). Antiretroviral therapy in HIV patients: aspects of metabolic complications and mitochondrial toxicity. The Netherlands Journal of Medicine, 61 (12), pp. 393-403.
- Jacque, J.M., Triques, K., Stevenson, M., (2002). Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438.
- Ambesajir, A., Kaushik, A., Kaushik, J. and Petros, S. (2012). RNA interference: A futuristic tool and its therapeutic applications. Saudi Journal of Biological Sciences, 19(4), pp.395-403.
- Naito, Y., Nohtomi, K., Onogi, T., Uenishi, R., Ui-Tei, K., Saigo, K. and Takebe, Y. (2007). Optimal design and validation of antiviral siRNA for targeting HIV-1. Retrovirology, 4(1), p.80.
- Kumar, P., Ban, H., Kim, S., Wu, H., Pearson, T., Greiner, D., Laouar, A., Yao, J., Haridas, V., Habiro, K., Yang, Y., Jeong, J., Lee, K., Kim, Y., Kim, S., Peipp, M., Fey, G., Manjunath, N., Shultz, L., Lee, S. and Shankar, P. (2008). T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice. Cell, 134(4), pp.577-586.
- ANDERSON, J. and AKKINA, R. (2005). 439. CXCR4 and CCR5 shRNA Transgenic Macrophages Are Functionally Normal and Resist HIV-1 Infection. Molecular Therapy, 11, pp.170-170.
- Wheeler, L. (2014). Silencing Sexually Transmitted Infections: Topical siRNA-Based Interventions for the Prevention of HIV and HSV. Infectious Diseases in Obstetrics and Gynecology, 2014, pp.1-11.
- Herrera-Carrillo, E., Liu, Y. and Berkhout, B. (2013). The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS. Mol Ther, 22(3), pp.596-606.
- Westerhout, E. (2005). HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Research, 33(2), pp.796-804.
- Shah, P., Pham, N. and Schaffer, D. (2012). HIV Develops Indirect Cross-resistance to Combinatorial RNAi Targeting Two Distinct and Spatially Distant Sites. Mol Ther, 20(4), pp.840-848.
- Suzuki, K., Juelich, T., Lim, H., Ishida, T., Watanebe, T., Cooper, D., Rao, S. and Kelleher, A. (2008). Closed Chromatin Architecture Is Induced by an RNA Duplex Targeting the HIV-1 Promoter Region. Journal of Biological Chemistry, 283(34), pp.23353-23363.
- Zeller, S. J., and Kumar, P. (2011). RNA-based Gene Therapy for the Treatment and Prevention of HIV: From Bench to Bedside. Yale Journal of Biology and Medicine, 84 (3), pp. 301-309.