SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.

The RNA interference (RNAi) pathway can be exploited using short hairpin RNAs (shRNAs) to durably inactivate pathogenic genes. Prediction of optimal target sites is notoriously inaccurate and current approaches applied to HIV-1 show weak correlations with virus inhibition. In contrast, using a high-content model for disrupting pre-existing intramolecular structure in the HIV-1 RNA, as achievable using high-resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing information, we discovered strong correlations between inhibition of HIV-1 production in a quantitative cell-based assay and very simple thermodynamic features in the target RNA. Strongest inhibition occurs at RNA target sites that both have an accessible "seed region" and, unexpectedly, are structurally accessible in a newly identified downstream flanking sequence. We then used these simple rules to create a new set of shRNAs and achieved inhibition of HIV-1 production of 90% or greater for up to 82% of designed shRNAs. These shRNAs inhibit HIV-1 replication in therapy-relevant T cells and show no or low cytotoxicity. The remarkable success of this straightforward SHAPE-based approach emphasizes that RNAi is governed, in significant part, by very simple, predictable rules reflecting the underlying RNA structure and illustrates principles likely to prove broadly useful in understanding transcriptome-scale biological recognition and therapeutics involving RNA.

RNAi-inducing lentiviral vectors for anti-HIV-1 gene therapy.

RNA interference (RNAi)-based gene therapy for the treatment of HIV-1 infection provides a novel antiviral approach. For delivery of RNAi inducers to CD4+ T cells or CD34+ blood stem cells, lentiviral vectors are attractive because of their ability to transduce nondividing cells. In addition, lentiviral vectors allow stable transgene expression by inserting their cargo into the host cell genome. However, use of the HIV-1-based lentiviral vector also creates specific problems. The RNAi inducers can target HIV-1 sequences in the genomic RNA of the lentiviral vector. As the RNAi-inducing cassette contains palindromic sequences, the lentiviral vector RNA genome will have a perfect target sequence for the expressed RNAi inducer. Vectors encoding microRNAs face the putative problem that the vector RNA genome can be inactivated by Drosha processing. Here, we describe the design of lentiviral vectors with single or multiple RNAi-inducing antiviral cassettes. The possibility of titer reduction and some effective countermeasures are also presented.

Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs.

BACKGROUND: RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence. For durable inhibition of HIV-1 replication the emergence of such escape viruses must be controlled. Here we present a strategy that anticipates HIV-1 escape by designing 2nd generation short hairpin RNAs (shRNAs) that form a complete match with the viral escape sequences. RESULTS: To block the two favorite viral escape routes observed when the HIV-1 integrase gene sequence is targeted, the original shRNA inhibitor was combined with two 2nd generation shRNAs in a single lentiviral expression vector. We demonstrate in long-term viral challenge experiments that the two dominant viral escape routes were effectively blocked. Eventually, virus breakthrough did however occur, but HIV-1 evolution was skewed and forced to use new escape routes. CONCLUSION: These results demonstrate the power of the 2nd generation RNAi concept. Popular viral escape routes are blocked by the 2nd generation RNAi strategy. As a consequence viral evolution was skewed leading to new escape routes. These results are of importance for a deeper understanding of HIV-1 evolution under RNAi pressure.

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies.

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

Optimization of shRNA inhibitors by variation of the terminal loop sequence.

Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.

Lentiviral vector engineering for anti-HIV RNAi gene therapy.

RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy.

Combinatorial RNAi against HIV-1 using extended short hairpin RNAs.

RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses.

Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAs.

BACKGROUND: RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. METHODS: In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. RESULTS: Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. CONCLUSIONS: These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.

Towards a durable RNAi gene therapy for HIV-AIDS.

BACKGROUND: RNA interference (RNAi) can be employed as a potent antiviral mechanism. OBJECTIVE: To discuss RNAi approaches to target pathogenic human viruses causing acute or chronic infections, in particular RNAi gene therapy against HIV-1. METHODS: A review of relevant literature. RESULTS/CONCLUSIONS: The future of antiviral RNAi therapeutics is very promising. RNAi was discovered only a decade ago, and although we are still in the early days, the first clinical trials are already ongoing.

Probing the sequence space available for HIV-1 evolution.

We designed a novel experimental approach to probe the sequence space available for HIV-1 evolution. Selective pressure was put on conserved HIV-1 genomic sequences by means of RNA interference (RNAi). Virus escape was monitored in many parallel cultures, and we scored the mutations selected in the RNAi target sequences. The experimentally induced sequence variation closely resembles the sequence variation of natural HIV-1 strains. This indicates that we actually mapped a restricted area of sequence space compatible with virus replication.

Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron.

RNA interference (RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication. However, HIV-1 can escape from RNAi-mediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, multiple small interfering RNAs (siRNAs) against conserved viral sequences should be combined. Ideally, these RNA inhibitors should be expressed simultaneously from a single transgene transcript. In this study, we tested a multiplex microRNA (miRNA) expression strategy by inserting multiple effective anti-HIV siRNA sequences in the miRNA polycistron mir-17-92. Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and HIV-1. We show that an antiviral miRNA construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shRNA) construct. When combined in a polycistron setting, the silencing activity of an individual miRNA is strongly boosted. We demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral siRNAs from the polycistronic miRNA transcript. These combined results indicate that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens.

Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition.

Human immunodeficiency virus type 1 (HIV-1) replication in T cells can be inhibited by RNA interference (RNAi) through short hairpin RNA (shRNA) expression from a lentiviral vector. However, for the development of a durable RNAi-based gene therapy against HIV-1, multiple shRNAs need to be expressed simultaneously in order to avoid viral escape. In this study, we tested a multiple shRNA expression strategy for different shRNAs using repeated promoters in a lentiviral vector. Although highly effective in co-transfection experiments, a markedly reduced activity of each expressed shRNA was observed in transduced cells. We found that this reduced activity was due to recombination of the expression cassette repeat sequences during the transduction of the lentiviral vector, which resulted in deletions of one or multiple cassettes. To avoid recombination, we tested different promoters for multiple shRNA expression. We compared the activity of the human polymerase III promoters U6, H1, and 7SK and the polymerase II U1 promoter. Activities of these promoters were similar, irrespective of which shRNA was expressed. We showed that these four expression cassettes can be combined in a single lentiviral vector without causing recombination. Moreover, whereas HIV-1 could escape from a single shRNA, we now show that HIV-1 escape can be prevented when four shRNAs are simultaneously expressed in a cell.

Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference.

RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but-in contrast to what is observed with nef-no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.

Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions.

BACKGROUND: HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. METHODS: Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RESULTS: RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. CONCLUSIONS: Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.

Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). The use of siRNA and shRNA as antiviral therapeutic is limited because of the emergence of viral escape mutants. This problem is theoretically prevented by intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, thus reducing the chance of viral escape. However, gene constructs encoding lhRNA molecules face problems with delivery to the right cells in an infected individual. In order to solve this problem, we constructed an HIV-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the Nef gene (HIV-lhNef). RESULTS: Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells. CONCLUSION: Expression of the lhNef hairpin within the HIV-1 genome results in potent trans-inhibition of wild-type HIV-1. Although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. This may have implications for the development of virus strains to be used as live-attenuated virus vaccines.

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference.

BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.

Silencing of HIV-1 with RNA interference: a multiple shRNA approach.

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against conserved HIV-1 regions. Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication. We show, for the first time, that expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA compared to single shRNA vectors. Thus, their combined expression results in a much stronger inhibition of virus production. Moreover, when we infected cells transduced with a double shRNA viral vector, virus escape was delayed. These results confirm that RNAi has great potential as an antiviral gene therapy approach and support our efforts to develop this strategy for treatment of HIV-1-infected individuals.

Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry.

Antimicrobial peptides are found in a number of body compartments and are secreted at mucosal surfaces, where they form part of the innate immune system. Many of these small peptides have a broad spectrum of inhibitory activity against bacteria, fungi, parasites, and viruses. Generally, the peptide's mode of action is binding and disruption of membranes due to its amphipathic properties. Histatin 5 is a salivary peptide that inhibits Candida albicans, an opportunistic fungus that causes oropharyngeal candidiasis in a majority of human immunodeficiency virus type 1 (HIV-1)-infected patients progressing towards AIDS. Previously, we increased the fungicidal properties of histatin 5 by replacing amino acids in the active domain of histatin 5 (Dh-5) (A. L. Ruissen, J. Groenink, E. J. Helmerhorst, E. Walgreen-Weterings, W. van't Hof, E. C. Veerman, and A. V. Nieuw Amerongen, Biochem. J. 356:361-368, 2001). In the current study, we tested the anti-HIV-1 activity of Dh-5 and its derivatives. Although Dh-5 inhibited HIV-1 replication, none of the peptide variants were more effective in this respect. In contrast, one of the derivatives, Dhvar2, significantly increased HIV-1 replication by promoting the envelope-mediated cell entry process. Most likely, Dhvar2 affects membranes, thereby facilitating fusion of viral and cellular membranes. This study shows that modification of antimicrobial peptides in order to improve their activity against a pathogen may have unpredictable and unwanted side effects on other pathogens.

A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs.

RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this technology as a therapeutic tool against pathogenic viruses. Indeed, it has been shown that siRNAs can be used effectively to inhibit virus replication. The focus of this review is on RNA interference strategies against HIV-1 and how this new technology may be developed into a new successful therapy. One of the hallmarks of RNAi, its sequence specificity, also presents a way out for the virus, as single nucleotide substitutions in the target region can abolish the suppression. Strategies to prevent the emergence of resistant viruses have been suggested and involve the targeting of conserved sequences and the simultaneous use of multiple siRNAs, similar to current highly active antiretroviral therapy. We present an additional strategy aimed at preventing viral escape by using a second generation of siRNAs that recognize the mutated target sites.