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The role of microRNAs in HIV-1 pathogenesis and therapy

Swaminathan, Sanjay; Murray, Daniel D.; Kelleher, Anthony D.

doi: 10.1097/QAD.0b013e328352adca
EDITORIAL REVIEW
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There has been a paradigm shift in our understanding of how protein regulation occurs within mammalian cells in the last 15 years. Our current understanding is that small, noncoding RNA molecules called microRNAs (miRNAs) play a vital role in modulating the translation of mRNAs into protein. Important studies suggest that HIV-1 replication may be restricted by certain host cellular miRNAs, and this in turn may play pivotal roles in host defense and in maintaining latency within resting CD4+ T cells. Conversely, host cellular miRNAs have also been demonstrated to be essential for certain viruses to establish infection and the altered expression of cellular miRNAs in the setting of HIV-1 may also be a factor favoring viral replication. The differential expression of important protective histocompatability locus antigen (HLA) alleles in HIV-1 infection has recently been shown to be regulated by miRNAs. To date, most efforts into finding an effective vaccine to combat HIV-1 have not been successful. Understanding the role that miRNAs may play in HIV-1 pathogenesis may allow a different approach to targeting key small RNAs or the identification of new important protein targets regulated by miRNAs, which may result in a better vaccine construct. The purpose of this review is to look at our current state of understanding of how HIV-1 and the miRNA pathway interact and the possible therapeutic interventions that this knowledge may entail.

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research, Kirby Institute, University of New South Wales, Sydney, Australia.

Correspondence to Sanjay Swaminathan, MBBS, BMedSci, PhD, St Vincent's Centre for Applied Medical Research, 405 Liverpool St, Darlinghurst, New South Wales, Australia, 2010. E-mail: s.swaminathan@amr.org.au

Received 28 November, 2011

Revised 14 February, 2012

Accepted 16 February, 2012

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Introduction

There has been a paradigm shift in our understanding of how protein regulation occurs within mammalian cells in the last 15 years. We know now that small, nonprotein-coding RNA molecules called microRNAs (miRNAs) play a vital role in modulating the translation of mRNAs into protein. Indeed, it is estimated that they target over 50% of mRNA transcripts within the cell at any one point [1]. Most miRNAs are transcribed from noncoding regions and their purpose is to regulate mRNA translation. Currently, more than 1000 human cellular miRNAs are listed in the Sanger miRNA database.

Studies have demonstrated that certain viruses (mostly from the γ-herpes family) also code for miRNAs, which interact with the RNA interference (RNAi) machinery of the cell. The purpose of these viral miRNAs is to enhance viral replication and survival. Host cellular miRNAs have also been demonstrated to be essential for certain viruses to establish infection and may play a role in protecting against infection as well.

In other areas of medicine, especially in oncology, there is much interest in the role that miRNAs play in disease progression and also in their possible utility as prognostic markers. The purpose of this review is to provide a comprehensive overview of our current understanding of how HIV-1 infection and the miRNA pathway interact and the possible therapeutic interventions that this knowledge may engender.

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MicroRNA biogenesis

Most miRNAs are transcribed from noncoding regions of the DNA, but up to 25% of miRNAs are derived from intronic DNA [2–4]. Genes encoding miRNAs are found on all chromosomes, apart from the Y chromosome, and many are in clusters of two to seven genes [5]. Many miRNAs are expressed in concert with protein-coding genes [6], which indicates a common transcriptional control mechanism [7]. However, it has been suggested that more than a quarter of intragenic miRNAs are independently transcribed from their own unique promoter sequences [8]. The biogenesis and processing of miRNAs are outlined in Fig. 1. The RNA polymerase II enzyme produces an initial transcript containing a 5′ cap and 3′ poly (A) tail, called a primary miRNA transcript (pri-miRNA) [9,10]. The pri-miRNA is processed by a microprocessor complex of Drosha and DiGeorge syndrome critical region 8 (DGCR8), which is responsible for cleaving the primary transcript to a stem-loop structure within the nucleus [11,12]. The pri-miRNA transcript is processed into an approximately 70 nucleotide-long precursor miRNA (pre-miRNA), containing a stem-loop structure, a 5′ phosphate and a 3′ two nucleotide overhang. DGCR8 plays a role in accurate Drosha processing by determining the Drosha cleavage site [13]. The pre-miRNA molecule is then transported to the cytoplasm by Exportin V and Ran-GTP [14].

Fig. 1

Fig. 1

In the cytoplasm, Dicer, another RNase III endonuclease, processes pre-miRNAs into approximately 22 nucleotide-long mature miRNAs [15], containing a guide strand (antisense) and a passenger (or sense) strand. Dicer is a highly conserved cytoplasmic enzyme, containing a helicase domain, a Piwi–Argonaute–Zwille (PAZ) domain, a double-stranded RNA-binding domain and two RNase III domains. It associates with transactivation responsive (Tar) RNA-binding protein (TRBP) [16,17] and protein activator of the interferon (IFN)-induced protein kinase (PACT) [18], which aid in the maturation of miRNAs. Dicer cleavage of pre-miRNAs leads to a two nucleotide overhang in the 3′ end of both strands, and following this the guide strand is incorporated into the RNA-induced silencing complex (RISC), whereas the passenger strand is degraded.

The RISC complex is formed by several proteins, including Dicer, Argonaute protein/s (typically Ago 2), TRBP [19], and KH-type splicing regulatory protein (KSRP) [20]. It has been recently been demonstrated that KSRP, a component of both Drosha and Dicer, binds to the terminal loop of miRNA precursors and promotes their maturation. There are four different Ago proteins in mammalian cells and all of these can bind to endogenous miRNAs; however, only Ago2 possesses endonuclease activity [21]. The key region of the guide strand is the seed region, which comprises nucleotides two to eight from the 5′ end of the miRNA. Binding of this seven nucleotide seed to complementary nucleotides found in 3′ untranslated (3′UTR) mRNA transcripts leads to RISC effector function [22]. Matches between the miRNA seed region and complementary mRNA sequences may lead to either mRNA degradation or translational repression. Each miRNA seed sequence may potentially interact with hundreds of mRNA targets. Prior to translation, mRNA exists in a circular form. The 5′ cap that binds eukaryotic initiation factors (eIFs) and the poly (A) tail that binds poly (A)-binding protein (PABP) act in concert to stimulate cap-dependent initiation of translation. Following binding of the seed sequence to matching 3′UTR mRNA sequences, the Ago protein of RISC disrupts the PABP/eIF complex, thus disrupting initiation of translation [23,24]. There is also some evidence to suggest that deadenylation at the 3′ end of the mRNA by RISC also disrupts translation [25].

Cytoplasmic P-bodies (processing bodies) were initially thought to be the location of mRNA degradation [26], which occurs after interaction with RISC. P-bodies contain various enzymes involved in mRNA degradation and RNAi-mediated gene silencing and the silencing complex contains at least an Argonaute protein and GW182 [27]. However, it is not clear whether P-bodies are the site of RNAi silencing or the consequence of RNAi-mediated silencing [28,29]. It may also be possible that mRNA found in P-bodies can leave this compartment and undergo translation through the ribosome.

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Role of microRNAs in immunity

In plants and insects, RNAi has been demonstrated to protect against various viral infections. Viral infections in these lower organisms typically lead to the formation of large transcripts of double-stranded RNA molecules within the cell that are processed down to smaller 21–22 nucleotide RNA molecules by the cell's endogenous RNAi machinery. These smaller RNA species lead to specific degradation of viral RNA.

In mammalian cells, the protective role of miRNAs against pathogens is harder to demonstrate. It is evident that viral infections do alter the miRNA milieu of the cell that is infected. For most infections, it is not clear what the purpose of the altered miRNA milieu is, apart from speculation, that this provides a better environment for the virus to survive and propagate (alternatively it may also reflect a direct or indirect activation of innate immune mechanisms such as the nuclear factor-κB induction of miR-146a in response to Toll-like receptor-4 activation by lipopolysaccharide) [30].

The first reported cellular miRNA to mediate antiviral defense in human cells was miR-32. The retrovirus, primate foamy virus-1 (PFV-1), has many structural features in common with HIV-1, including Gag, Pol and Env proteins and miR-32 appears (by serendipity) to target the open reading frame of a number of key regions of the PFV-1 genome [31]. Whether a cellular miRNA can code for targets within PFV-1, presumably this may occur with other miRNAs and other viruses. There are a number of studies that have used ‘synthetic’ miRNAs or siRNAs targeting key regions of viruses, such as HIV-1, which have demonstrated the utility of this approach [32,33].

The linking of the IFNβ response with the upregulation of certain miRNAs that target the hepatitis C virus (HCV) genome does suggest that there is a link between production of miRNAs and immunity [34]. The type I IFN response is triggered by innate immunity by pathogen pattern receptors located within the cytoplasm of infected cells (i.e. retinoic acid inducible gene -I). Additionally, miR-122 has been shown to be essential for HCV to establish infection and this miRNA is preferentially expressed in hepatic cells [35].

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MicroRNAs and HIV-1 infection

Role in peripheral blood mononuclear cells

The importance of the RNAi machinery in HIV-1 replication was highlighted when Dicer and Drosha were knocked down (using siRNA) in HIV-1-infected peripheral blood mononuclear cells (PBMCs). The result of the decreased expression of these two critical proteins in miRNA processing was a marked increase in HIV-1 replication [36]. This implied that the miRNA pathway played an important role in host control of the virus. More recently, these results have been replicated in 293T cells infected with the HIV-1 molecular clone NL4–3 [37]. In a separate study, the miRNA profile of PBMCs isolated from four different HIV-1-infected patient groups has been performed and compared with uninfected controls. These infected patient groups varied in their levels of CD4+ T-cell counts and HIV-1 RNA viral load. Despite small numbers, each patient group had a set of characteristic miRNAs that were differentially expressed (mostly downregulated compared with healthy controls) between groups and between healthy controls [38]. The study also compared the effect of subjecting PBMCs to an activating stimulus (anti-CD3) and an inactivating stimulus (interleukin-10, IL-10) and profiled these cells with the finding that these stimuli altered the miRNA profile of these cells. Lastly, miR-29a and miR-29b have been shown to be expressed in PBMCs and these miRNAs were able to target sites in the HIV-1 nef region using a luciferase reporter bearing the nef miR-29a site [39]. Although this work did look at the effect of miRNA mimics on HIV replication, the cell line that was used (HEK293 cells) was not derived from T cells. A major drawback of experiments utilizing PBMCs is that it is difficult to pinpoint whether the observed miRNA changes are occurring preferentially in one subset (such as the biologically relevant CD4+ T cell) or reflect changes occurring on a global nature in a number of different cell types.

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Role of microRNAs in T cells

Several groups have investigated the role of miRNAs in CD4+ T cells. Triboulet et al.[36] used a time course experiment in HIV-1-infected Jurkat cells to demonstrate that the miR17/92 cluster of miRNAs were downregulated in response to HIV-1 infection. This family of miRNAs targeted the histone acetylase, P300/CBP-associated factor (PCAF), which is an important cofactor for HIV-1 Tat and, therefore, downregulation of this miRNA cluster was suggested to be advantageous to the virus.

Another seminal article in the field was the demonstration that resting CD4+ T cells overexpressed certain miRNAs compared with activated CD4+ T cells. These miRNAs (miR-28, miR-125b, miR-150, miR-223 and miR-382) targeted the 3′ end of HIV-1 mRNA transcripts and were postulated to be one reason why these cells were latently infected [40]. The combination of specific inhibitors of these five miRNAs in resting CD4+ T cells isolated from HIV-1-infected patients undergoing suppressive antiretroviral therapy, resulted in a significant increase in HIV-1 replication. One problem with using multiple miRNA inhibitors to show an effect on viral replication is that it is possible to elicit a number of nonspecific effects as each miRNA has hundreds of targets.

More recently, miR-29a, has been demonstrated to potentially play an important role in regulating viral replication by, first, being highly expressed in CD4+ T cells and, second, being able to target both the 3′ end of HIV-1 transcripts and HIV-1 nef[37,39,41]. Additionally, it seems that P-body depletion enhances HIV-1 replication, providing further evidence that the miRNA system plays a role in modulating HIV-1 infection.

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Role of microRNAs in monocytes

Several recent studies have investigated the role of miRNAs in HIV-1 infection involving monocytes. The first report explored the role of miRNAs that were enriched in resting CD4+ T cells, which were shown to target HIV 3′UTR transcripts. These ‘anti-HIV’ miRNAs (miR-28, miR-150, miR-223 and miR-382) were found to be enriched in monocytes compared with macrophages and said to be a key reason why monocytes were relatively resistant to HIV-1 infection [42]. There were several issues that warranted further exploration with this particular study. Firstly, monocytes have low levels of both CD4 and CCR5 on their cell surface, thus making entry by the virus into the cell problematic [43]. They also express high levels of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G and Tethrin, host factors known to restrict HIV-1 [44,45]. Third, our group has demonstrated high levels of miR-150 and miR-223 (two ‘anti-HIV miRNAs’) in CD4+ T cells isolated from healthy controls compared with HIV-1-infected individuals. The elevated level of these two miRNAs in healthy controls compared with HIV-1-infected individuals is difficult to reconcile with their apparent anti-HIV properties. This finding also draws into doubt whether miR-150 and miR-223 have ‘anti-HIV’ effects in both monocytes and CD4+ T cells or whether these miRNAs are simply downregulated with HIV-1 infection. Lastly, like the study from Huang et al., the use of combined miRNA inhibitors to show an effect raises questions regarding specificity as discussed above [46].

Cyclin T1 is a regulatory subunit of positive transcription elongation factor b (PTEFb), a RNA polymerase II elongation factor and is important in HIV-1 replication, as it is required for Tat transactivation of HIV-1 transcripts. One recent study suggests that monocytes have high levels of miR-198, which targets Cyclin T1, and these levels fall substantially on differentiation to a macrophage phenotype. This interplay between levels of Cyclin T1 and miR-198 levels in monocytes and macrophages has been postulated to be another host restriction factor that prevents infection in monocytes [47].

Lastly, one recent study has suggested that monocytes lack Dicer and have low levels of Drosha, Ago1 and Ago2, but these components are present when monocytes differentiate into macrophages [48]. Low levels of Dicer in monocytes had been described several years earlier as a possible reason for latency in T cells, with the virus exploiting differences in RNAi between T cells and monocytes to its advantage [49]. Dicer expression was not only restricted by miR-106a but also by the HIV-1 viral protein (Vpr). Other groups have shown that Dicer can be targeted by the miRNA let-7b [50], but in this study levels of let-7b were not differentially expressed to explain differences in Dicer expression. The downregulation of Dicer in monocytes may result in a shift toward the processing of piwiRNA (piRNA) rather than miRNA [48]. However, the presence of piRNA in somatic cells is a controversial topic and has yet to be definitively proven. This study raises some interesting issues with regard to the biogenesis of miRNAs in monocytes and whether there are alternate processing pathways for miRNAs. It is also unlikely that the HIV-1-encoded miRNAs, miR-TAR-5p and miR-TAR-3p, are being produced in monocytes, as these miRNA are highly Dicer-dependent [49]. The current understanding of the role of miRNAs in HIV-1 pathogenesis in CD4+ T cells and monocytes is illustrated in Fig. 2.

Fig. 2

Fig. 2

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Role of microRNAs in the central nervous system

Recently the role of miRNA in HIV-1 infection of neural tissue has begun to be explored more thoroughly. As with all studies involving central nervous system (CNS), there are substantial challenges to conducting these types of studies, including the difficulty of obtaining representative tissue, which in turn results in small sample size and the need to study bulk tissue made up of multiple different cell types, each of which is likely to have its own miRNA profile. With these limitations in mind, profiling of miRNA from the frontal cortices of recently deceased healthy uninfected controls, HIV-1-infected individuals and HIV-1-infected individuals with major depressive disorder (MDD) identified miRNA changes between patient groups. However, there were only three patients in each group and the patient profile was heterogeneous within these groups with one member of the HIV-1-infected group having bacterial leptomeningitis, and in the HIV-1 MDD group, one patient had encephalitis at the time of death. These intercurrent inflammatory processes may have skewed the miRNA profiles in the brains of these individuals [51].

Additionally, there has been a rapid increase in data suggesting that miRNA may play a role in the pathogenesis of HIV-1-induced encephalitis (HIVE). One such study showed that miR-129* and miR-130a, which can target Caspase-6 mRNA, are downregulated in the brains of individuals with HIVE [52]. As a result, the expression of Caspase-6 in these individuals is higher, leading to an increase in apoptosis and causing cellular degeneration and HIVE. A second study proposed that the increased presence of monocyte chemoattractant protein 2 (MCP-2), a ligand of CCR5 and inhibitor of CD4/CCR5 HIV-1 cell entry, in HIV-1 brain cell cultures and in patients with HIVE is due to a decrease in miR-146a [53]. MCP-2 is a target of miR-146a and overexpression of this miRNA prior to HIV-1 infection prevents the release of MCP-2 in infected cells. However, there is no evidence provided that suggests miR-146a influences HIV-1 infection and as this miRNA has also been shown to influence IL-1 and tumor necrosis factorα [30], more work must be done to fully understand its role in the progression of HIVE. Unfortunately, both these studies lack statistical power in determining differences between HIVE brains and controls due to the difficulty in obtaining human brain tissue.

The interplay of Tat, the HIV-1 regulatory protein vital for replication, and miRNA has been explored with relation to progression of HIVE. It is currently accepted that Tat is secreted by HIV-1-infected cells and taken up by uninfected cells, including neurons [54]. This uptake of Tat in neuronal tissue has been postulated as one of the causes of HIV encephalopathy. To investigate this, Tat was overexpressed in rat embryonic cortical neurons, total RNA was extracted and miRNA profiling performed. Tat overexpression led to an increase in miR-128a, which potentially modulated levels of SNAP25, a presynaptic protein important for effective neurotransmission [51]. Clearly, a limitation of this study was the use of rat tissue and its direct relevance to humans with HIVE is questionable. Furthermore, Yelamanchili et al. showed that miR-21 is able to target myocyte enhancer factor 2C (MEF2C) and is upregulated in simian immunodeficiency virus encephalitis (SIVE)/HIVE-infected brains. Further, N-methyl-D-aspartic acid (NMDA) was shown to induce miR-21 expression and when primary human neurons were infected with a lentivirus expressing this miRNA, an increase in outward K+ current was observed in voltage-gated channels. This increase can be interpreted as an early sign of neuronal apoptosis and suggests that the increase in miR-21 is detrimental to neurons. However, there remains a disconnect between miR-21′s targeting of MEF2C and K+ channel disruption as well as how HIV-1 is inducing NMDA in the first place. Therefore, more work must be done before this pathway is properly understood [55]. Another study using a macaque model of SIVE/HIVE showed that miRNAs 26a, 34a, 145 and let-7b can target IFNβ in both human and macaque macrophages, which may lead to an increase in this cytokine in the brains of SIV/HIV-infected individuals contributing to overall CNS inflammation [56].

These studies on neuronal tissue demonstrate miRNAs may play an important role in modulating proteins important in disease progression with the caveat that much more corroborative work in the future is required to confirm these interesting initial findings.

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Effect oF microRNAs on histocompatability locus antigen expression and HIV control

Recently, a polymorphism in a miRNA-binding site with an histocompatability locus antigen (HLA)-C allele has been associated with lower viral loads in infected patients. The insertion/deletion polymorphism in question is located in the 3′UTR of HLA-C mRNA and reduces complementarity to miR-148a, resulting in an increase in HLA-C expression compared with wild-type. It was found that HIV-1-infected individuals with this polymorphism were more likely to have a viral load less than 2000 copies/ml compared with those with wild-type. However, the reason why this increase in HLA-C results in a decrease in viral load is still unknown, but the authors suggest that it may be due to an increased level of antigen presentation to cytotoxic lymphocytes [57].

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Presence of HIV-1 viral microRNAs

The presence of HIV-1 derived viral miRNAs has been a controversial topic and the search for these species has resulted in several disputed findings [58]. The first description of HIV-1-derived miRNAs came in 2004 from a group that found a nef-derived miRNA, named miR-N367, from an in-vitro model of HIV-1 infection [59]. This group claimed that miR-N367 could reduce HIV-1 transcription by targeting the HIV-1 long tandem repeat (LTR) promoter activity through the negative responsive element of the U3 region in the 5′ LTR. However, two other research groups have failed to find this HIV-1 viral miRNA [60,61].

A second HIV-1 viral siRNA was described in 2005 derived from the Rev response element (RRE). The RRE is a highly structured RNA element that aids in the nuclear export of HIV-1 mRNA. This finding was disputed by the laboratory of Cullen [58], which argued that the proposed siRNA structure does not correlate with the known structure of the RRE from both in-vitro and in-vivo experiments. Their argument was that the sense and antisense strands of the proposed siRNA were shown to be separated by 197 base pairs and, thus, unlikely to form a functional siRNA.

The TAR element is a structured RNA that is located at the 5′ end of all HIV-1 transcripts. TAR is critical for Tat-mediated transactivation of viral gene expression, and is needed for efficient viral gene expression. Functional viral miRNAs have been described, which are derived from asymmetrical processing of the HIV-1 TAR element by two separate groups [49,62]. More recently, the HIV-1 TAR miRNA has been shown to downregulate host genes ERCC1 and IER3, which are important for apoptosis and cell survival, thus giving HIV-1-infected cells a survival advantage by preventing cell death [63]. Interestingly, HIV-1 TAR RNA appears to modulate host/viral miRNA production by sequestering TRBP, an essential component of the RISC complex. The inhibition of the RISC complex presumably also gives HIV-1 a survival advantage and again points to an important role of the RNAi machinery in control of the virus [64]. More recently, deep sequencing of HIV-1-infected SupT1 T cells has also demonstrated the presence of HIV-1-derived viral miRNAs and a novel HIV-1 TAR miRNA. The authors suggest that new technologies, such as deep sequencing, now allow for the identification of viral small RNAs, whereas in the past, due to their low abundance, this had been difficult to detect [65].

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Manipulation of microRNAs for the treatment of HIV-1 infection

Current HIV treatment is able to control viral replication and disease progression; however, there is still no cure. Additionally, antiretroviral drugs are associated with a number of cumulative side-effects (such as affecting lipid profiles), which makes alternate ways of controlling HIV-1 infection worth pursuing. Several studies have shown that overexpression of certain endogenous miRNAs in an in-vitro setting can significantly reduce HIV-1 replication. These miRNAs include miR-29a and miR-29b [37] as well as miRNAs from the miR-17/92 cluster, particularly miR-17–5p and miR-20a [36]. Upregulating the expression of miR-28, miR-125b, miR-150, miR-223 and miR-382 [40] (miRNAs that have been shown to be increased in resting CD4+ T cells and that can target HIV-1 mRNA transcripts) in CD4+ T cells from patients with HIV-1 infection may also be a possible way of using endogenous miRNAs to target HIV-1 infection. The drawback with overexpressing particular miRNAs is that they usually have multiple targets and little is known of their normal function in T cells and, hence, overexpression of these miRNAs may lead to unintended, off-target and unanticipated side-effects. Table 1[36,37,39–42,46,47,52,53,55] summarizes the key miRNAs that, if manipulated, may play an important role in modulating disease response and outcome.

Table 1

Table 1

The presence of unique HIV-1-specific viral miRNAs presents an opportunity to specifically target these molecules to inhibit viral replication with the prospect of minimal off-target effects of therapy (off-target effects are discussed below). The difficulty with this approach is that little is known of the function of these small RNAs and, hence, the effect of blocking these miRNAs may not impact significantly with viral replication.

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Delivery of small RNAs

Many different approaches have been used to deliver RNAi to relevant tissues [66]. There remain a number of key issues that require attention when applying RNAi to human disease treatment. The main issue is targeting the tissue of interest without affecting nonrelevant tissues or organs. Another main concern with RNAi therapeutics is to limit off-target effects, which have the potential to derail the benefits of target gene regulation. Most of the approaches described have been with siRNA (double-stranded RNA with perfect homology to the mRNA that they are targeting). However, miRNA delivery potentially will have similar issues and is likely to be more prone to off-target or nonspecific effects because of the broad range of proteins potentially influenced by a single miRNA.

Delivery of small RNAs can be divided into nonselective and specific delivery methods. Some of these delivery methods can overlap depending on modifications, such as coating nanoparticles (nonspecific delivery) with monoclonal antibodies that can target specific tissues. This approach has been recently used to deliver specific miRNAs to neural tissue [67]. One way to deliver these small RNAs is to inject these molecules directly into the blood or into target organs. Other nonselective delivery methods include conjugation of small RNAs to a cholesterol group [68] or packaging small RNAs into a liposomal particle to improve cellular uptake [69]. miRNA mimics have been delivered systemically by complexing them with a neutral lipid emulsion that appears to deliver these miRNA mimics to lung tumors in mice [70]. Polymers have been used successfully to deliver small RNAs to target tissues as well [71]. Recently, stable nucleic acid lipid particles have been used to deliver small RNAs that are coated with polyethylene glycol polymers. One novel delivery vector that has been developed modifies single-stranded circular oligodeoxynucleotides in order to encode primary miRNA mimics [72]. These nonselective methods are often suitable in cases where direct injection into a target tissue is appropriate. The half-life of miRNAs is short, so these delivery systems have to be incorporated into a repeated dosing regimen or dosed as a depot reagent.

Viruses, particularly lentiviral vectors, can also be used to incorporate shRNA (small hairpin RNA) transgenes into cells of interest. The shRNA can encode miRNA genes and, once incorporated into the genome, will be transcribed and processed in similar ways to endogenous miRNAs. There appears to be several advantages of using shRNA to knockdown gene expression. One major advantage of using shRNA is that only a small number of integrated shRNA are required for continuous and prolonged gene knockdown without repeated dosing. Also, shRNA appears to be more efficiently loaded onto RISC compared with siRNA. Strong immune responses to the type of viral vector or prior immunity to pathogens such as adenovirus may limit the use of certain viral vectors. An example of the issues linked to the adenoviral delivery platform is the failed STEP trial, which aimed to deliver HIV-1 antigens to HIV-1-negative hosts in the hope of developing an effective immune response against HIV-1. Those patients administered the vaccine construct with high preexisting adenoviral antibody titres had a higher rate of HIV-1 acquisition compared with patients with low titres – a finding that is currently being heavily investigated [73–75].

Examples of selective delivery systems already developed include aptamers, antibody fragments and nanoparticles. Aptamers are structured RNA ligands that bind to specific cell surface receptors and can be conveniently covalently linked to small RNA molecules [76]. The binding of aptamers to cell surface receptors leads to release of the small RNA once the aptamer is internalized into the cell. Antibody fragments have also been used as a specific delivery method for small RNAs. The fragment antigen-binding (Fab) portion of an antibody contains the unique antigen binding site and this Fab fragment has been conjugated to positively charged protamines that, in turn, link to the negatively charged small RNA molecules [77]. Nanotechnology has allowed the development of nanoparticles that have also been used with success to deliver small RNAs in vivo[78]. These nanoparticles can be coupled with cell type-specific ligands, such as antibodies, and carry small RNAs to the tissue of interest [79]. Pseudotyping of lentiviruses to contain proteins that target molecules also has the potential to target specific tissues with a small RNA of interest [80].

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Off-target effects of microRNAs used as therapeutics

Each miRNA may have hundreds of mRNA targets with the possibility that manipulating miRNA levels in-vivo will cause unintended adverse events. These ‘off-target’ effects remain a major impediment to future clinical applications. The seed region of a candidate miRNA may not only just bind to complementary mRNAs of the intended target mRNA leading to translational repression but may also possibly bind to other mRNA targets leading to unintended gene silencing effects. The introduction of exogenous single-stranded or double-stranded RNA can lead to the upregulation of type I IFNs, which in general terms lead to an antiviral phenotype that may not lead to the desired outcome.

Adverse events using RNAi therapeutics have already been noted. In one study in mice which utilized 49 adeno-associated virus shRNAs (directed to six targets within the liver), 36 of these 49 shRNAs were associated with an adverse event due to the shRNA itself, including 23 shRNAs that eventually led to death mostly due to acute liver failure a [81]. The authors speculated that there may have been oversaturation of endogenous small RNA pathways and that this can be reduced by keeping the shRNA dose to a minimum.

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Conclusion

In the last decade, it has become clear that miRNAs play a vital role in regulating gene expression. Compared with other areas of medicine, such as cancer research in which there have been major insights into understanding the role between miRNAs and disease, important interactions between HIV-1 infection and miRNAs have only started to emerge. The major benefits regarding the interplay between HIV-1 and miRNAs with regard to diagnostics, prognostics and therapeutics are still to be realized. miRNAs play an important regulatory role in normal homeostasis with regard to maintaining protein expression within cells. They are also clearly altered in cells infected with HIV-1, contributing to the pathological decline in function. However, it is impossible to characterize all HIV-1-infected cells in the same way, as it appears that each individual cell type has a unique miRNA profile. In some situations and within certain cell types, they have a potential beneficial role in combating HIV-1. The presence and function of HIV-1-derived viral miRNAs remain to be fully elucidated, but there is much enthusiasm that newer, more sensitive detection techniques (such as deep sequencing) will give more insight into their potential role in pathogenesis. Several lines of evidence suggest that manipulating miRNAs may provide a novel approach to treating HIV-1 infection. Potential therapeutic uses of miRNAs may be hampered by off-target effects from seed region matches to mRNAs unintentionally targeted. Although the future is promising, the use of small RNA therapeutics still has a number of limitations that must be overcome.

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Acknowledgements

S.S., D.D.M and A.D.K. reviewed the literature. D.D.M. prepared the diagrams with assistance from S.S. and A.D.K. The manuscript was written by S.S. with input from D.D.M and A.D.K.

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Conflicts of interest

There are no conflicts of interest.

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References

1. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009; 19:92–105.
2. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:281–297.
3. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res 2004; 14:1902–1910.
4. Kim YK, Kim VN. Processing of intronic microRNAs. EMBO J 2007; 26:775–783.
5. Tanzer A, Stadler PF. Evolution of microRNAs. Methods Mol Biol 2006; 342:335–350.
6. Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nat Struct Mol Biol 2011; 18:302–308.
7. Morlando M, Ballarino M, Gromak N, Pagano F, Bozzoni I, Proudfoot NJ. Primary microRNA transcripts are processed co-transcriptionally. Nat Struct Mol Biol 2008; 15:902–909.
8. Corcoran DL, Pandit KV, Gordon B, Bhattacharjee A, Kaminski N, Benos PV. Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data. PLoS One 2009; 4:e5279.
9. Borchert GM, Lanier W, Davidson BL. RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 2006; 13:1097–1101.
10. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J 2004; 23:4051–4060.
11. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 2004; 18:3016–3027.
12. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 2003; 425:415–419.
13. Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, et al. The microprocessor complex mediates the genesis of microRNAs. Nature 2004; 432:235–240.
14. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science 2004; 303:95–98.
15. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409:363–366.
16. Daniels SM, Melendez-Pena CE, Scarborough RJ, Daher A, Christensen HS, El Far M, et al. Characterization of the TRBP domain required for dicer interaction and function in RNA interference. BMC Mol Biol 2009; 10:38.
17. Haase AD, Jaskiewicz L, Zhang H, Laine S, Sack R, Gatignol A, et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 2005; 6:961–967.
18. Lee Y, Hur I, Park SY, Kim YK, Suh MR, Kim VN. The role of PACT in the RNA silencing pathway. EMBO J 2006; 25:522–532.
19. Wang HW, Noland C, Siridechadilok B, Taylor DW, Ma E, Felderer K, et al. Structural insights into RNA processing by the human RISC-loading complex. Nat Struct Mol Biol 2009; 16:1148–1153.
20. Trabucchi M, Briata P, Garcia-Mayoral M, Haase AD, Filipowicz W, Ramos A, et al. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 2009; 459:1010–1014.
21. Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 2004; 15:185–197.
22. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136:215–233.
23. Walters RW, Bradrick SS, Gromeier M. Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression. RNA 2010; 16:239–250.
24. Fabian MR, Mathonnet G, Sundermeier T, Mathys H, Zipprich JT, Svitkin YV, et al. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol Cell 2009; 35:868–880.
25. Beilharz TH, Humphreys DT, Clancy JL, Thermann R, Martin DI, Hentze MW, et al. MicroRNA-mediated messenger RNA deadenylation contributes to translational repression in mammalian cells. PLoS One 2009; 4:e6783.
26. Sheth U, Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 2003; 300:805–808.
27. Liu J, Rivas FV, Wohlschlegel J, Yates JR, 3rd, Parker R, Hannon GJ. A role for the P-body component GW182 in microRNA function. Nat Cell Biol 2005; 7:1261–1266.
28. Eulalio A, Behm-Ansmant I, Schweizer D, Izaurralde E. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol 2007; 27:3970–3981.
29. Omer AD, Janas MM, Novina CD. The chicken or the egg: microRNA-mediated regulation of mRNA translation or mRNA stability. Mol Cell 2009; 35:739–740.
30. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A 2006; 103:12481–12486.
31. Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, et al. A cellular microRNA mediates antiviral defense in human cells. Science 2005; 308:557–560.
32. Liu YP, Berkhout B. Lentiviral delivery of RNAi effectors against HIV-1. Curr Top Med Chem 2009; 9:1130–1143.
33. Subramanya S, Kim SS, Manjunath N, Shankar P. RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA? Expert Opin Biol Ther 2010; 10:201–213.
34. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 2007; 449:919–922.
35. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 2005; 309:1577–1581.
36. Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, et al. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science 2007; 315:1579–1582.
37. Nathans R, Chu CY, Serquina AK, Lu CC, Cao H, Rana TM. Cellular microRNA and P bodies modulate host-HIV-1 interactions. Mol Cell 2009; 34:696–709.
38. Houzet L, Yeung ML, de Lame V, Desai D, Smith SM, Jeang KT. MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1) seropositive individuals. Retrovirology 2008; 5:118.
39. Ahluwalia JK, Khan SZ, Soni K, Rawat P, Gupta A, Hariharan M, et al. Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication. Retrovirology 2008; 5:117.
40. Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, et al. Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes. Nat Med 2007; 13:1241–1247.
41. Hariharan M, Scaria V, Pillai B, Brahmachari SK. Targets for human encoded microRNAs in HIV genes. Biochem Biophys Res Commun 2005; 337:1214–1218.
42. Wang X, Ye L, Hou W, Zhou Y, Wang YJ, Metzger DS, et al. Cellular microRNA expression correlates with susceptibility of monocytes/macrophages to HIV-1 infection. Blood 2009; 113:671–674.
43. Naif HM, Li S, Alali M, Sloane A, Wu L, Kelly M, et al. CCR5 expression correlates with susceptibility of maturing monocytes to human immunodeficiency virus type 1 infection. J Virol 1998; 72:830–836.
44. Bishop KN, Holmes RK, Sheehy AM, Malim MH. APOBEC-mediated editing of viral RNA. Science 2004; 305:645.
45. Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 2008; 451:425–430.
46. Swaminathan S, Zaunders J, Wilkinson J, Suzuki K, Kelleher AD. Does the presence of anti-HIV miRNAs in monocytes explain their resistance to HIV-1 infection?. Blood 2009; 113:5029–5030.author reply 5030–5021.
47. Sung TL, Rice AP. miR-198 inhibits HIV-1 gene expression and replication in monocytes and its mechanism of action appears to involve repression of cyclin T1. PLoS Pathog 2009; 5:e1000263.
48. Coley W, Van Duyne R, Carpio L, Guendel I, Kehn-Hall K, Chevalier S, et al.Absence of Dicer in monocytes and its regulation by HIV-1. J Biol Chem 2010; 285:31930–31943.
49. Klase Z, Kale P, Winograd R, Gupta MV, Heydarian M, Berro R, et al. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 2007; 8:63.
50. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature 2008; 455:58–63.
51. Eletto D, Russo G, Passiatore G, Del Valle L, Giordano A, Khalili K, et al. Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a. J Cell Physiol 2008; 216:764–770.
52. Noorbakhsh F, Ramachandran R, Barsby N, Ellestad KK, LeBlanc A, Dickie P, et al. MicroRNA profiling reveals new aspects of HIV neurodegeneration: caspase-6 regulates astrocyte survival. FASEB J 2010; 24:1799–1812.
53. Rom S, Rom I, Passiatore G, Pacifici M, Radhakrishnan S, Del Valle L, et al. CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells. FASEB J 2010; 24:2292–2300.
54. Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, et al. Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med 2000; 6:1380–1387.
55. Yelamanchili SV, Chaudhuri AD, Chen LN, Xiong H, Fox HS. MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological disease. Cell Death Dis 2010; 1:e77.
56. Witwer KW, Sisk JM, Gama L, Clements JE. MicroRNA regulation of IFN-beta protein expression: rapid and sensitive modulation of the innate immune response. J Immunol 2010; 184:2369–2376.
57. Kulkarni S, Savan R, Qi Y, Gao X, Yuki Y, Bass SE, et al. Differential microRNA regulation of HLA-C expression and its association with HIV control. Nature 2011; 472:495–498.
58. Cullen BR. Is RNA interference involved in intrinsic antiviral immunity in mammals?. Nat Immunol 2006; 7:563–567.
59. Omoto S, Ito M, Tsutsumi Y, Ichikawa Y, Okuyama H, Brisibe EA, et al. HIV-1 nef suppression by virally encoded microRNA. Retrovirology 2004; 1:44.
60. Lin J, Cullen BR. Analysis of the interaction of primate retroviruses with the human RNA interference machinery. J Virol 2007; 81:12218–12226.
61. Pfeffer S, Sewer A, Lagos-Quintana M, Sheridan R, Sander C, Grasser FA, et al. Identification of microRNAs of the herpesvirus family. Nat Methods 2005; 2:269–276.
62. Ouellet DL, Plante I, Landry P, Barat C, Janelle ME, Flamand L, et al. Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 2008; 36:2353–2365.
63. Klase Z, Winograd R, Davis J, Carpio L, Hildreth R, Heydarian M, et al. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression. Retrovirology 2009; 6:18.
64. Bennasser Y, Yeung ML, Jeang KT. HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP. J Biol Chem 2006; 281:27674–27678.
65. Schopman NC, Willemsen M, Liu YP, Bradley T, van Kampen A, Baas F, et al. Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs. Nucleic Acids Res 2012; 40:414–427.
66. Castanotto D, Rossi JJ. The promises and pitfalls of RNA-interference-based therapeutics. Nature 2009; 457:426–433.
67. Hwang DW, Son S, Jang J, Youn H, Lee S, Lee D, et al.A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA. Biomaterials 2011; 32:4968–4975.
68. Wolfrum C, Shi S, Jayaprakash KN, Jayaraman M, Wang G, Pandey RK, et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 2007; 25:1149–1157.
69. Hughes J, Yadava P, Mesaros R. Liposomal siRNA delivery. Methods Mol Biol 2010; 605:445–459.
70. Trang P, Wiggins JF, Daige CL, Cho C, Omotola M, Brown D, et al.Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther 2011; 19:1116:1122.
71. Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, et al. RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. Mol Ther 2006; 14:476–484.
72. Seidl CI, Ryan K. Circular single-stranded synthetic DNA delivery vectors for microRNA. PLoS One 2011; 6:e16925.
73. Benlahrech A, Harris J, Meiser A, Papagatsias T, Hornig J, Hayes P, et al. Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1. Proc Natl Acad Sci U S A 2009; 106:19940–19945.
74. Hutnick NA, Carnathan DG, Dubey SA, Makedonas G, Cox KS, Kierstead L, et al. Baseline Ad5 serostatus does not predict Ad5 HIV vaccine-induced expansion of adenovirus-specific CD4+ T cells. Nat Med 2009; 15:876–878.
75. O’Brien KL, Liu J, King SL, Sun YH, Schmitz JE, Lifton MA, et al. Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans. Nat Med 2009; 15:873–875.
76. McNamara JO, 2nd, Andrechek ER, Wang Y, Viles KD, Rempel RE, Gilboa E, et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 2006; 24:1005–1015.
77. Song E, Zhu P, Lee SK, Chowdhury D, Kussman S, Dykxhoorn DM, et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 2005; 23:709–717.
78. Zhang W, Yang H, Kong X, Mohapatra S, San Juan-Vergara H, Hellermann G, et al. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med 2005; 11:56–62.
79. Wang Y, Li Z, Han Y, Liang LH, Ji A. Nanoparticle-based delivery system for application of siRNA in vivo. Curr Drug Metab 2010; 11:182–196.
80. Maurice M, Verhoeyen E, Salmon P, Trono D, Russell SJ, Cosset FL. Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide. Blood 2002; 99:2342–2350.
81. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441:537–541.
Keywords:

CD4+ T cell; HIV-1; microRNA; monocytes; neurons; peripheral blood mononuclear cells

© 2012 Lippincott Williams & Wilkins, Inc.