Ying Poi Liu

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 (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.

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.

Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs

Small virus-derived interfering RNAs (viRNAs) play an important role in antiviral defence in plants, insects and nematodes by triggering the RNA interference (RNAi) pathway. The role of RNAi as an antiviral defence mechanism in mammalian cells has been obscure due to the lack of viRNA detection. Although viRNAs from different mammalian viruses have recently been identified, their functions and possible impact on viral replication remain unknown. To identify viRNAs derived from HIV-1, we used the extremely sensitive SOLiDTM 3 Plus System to analyse viRNA accumulation in HIV-1-infected T lymphocytes. We detected numerous small RNAs that correspond to the HIV-1 RNA genome. The majority of these sequences have a positive polarity (98.1%) and could be derived from miRNAs encoded by structured segments of the HIV-1 RNA genome (vmiRNAs). A small portion of the viRNAs is of negative polarity and most of them are encoded within the 3′-UTR, which may represent viral siRNAs (vsiRNAs). The identified vsiRNAs can potently repress HIV-1 production, whereas suppression of the vsiRNAs by antagomirs stimulate virus production. These results suggest that HIV-1 triggers the production of vsiRNAs and vmiRNAs to modulate cellular and/or viral gene expression.

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.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.

Design of extended short hairpin RNAs for HIV-1 inhibition.

RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs.

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.

Dicer-independent processing of short hairpin RNAs

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

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.

Preclinical In Vivo Evaluation of the Safety of a Multi- shRNA-based Gene Therapy Against HIV-1

Highly active antiretroviral therapy (HAART) has significantly improved the quality of life and the life expectancy of HIV-infected individuals. Still, drug-induced side effects and emergence of drug-resistant viral variants remain important issues that justify the exploration of alternative therapeutic options. One strategy consists of a gene therapy based on RNA interference to induce the sequence-specific degradation of the HIV-1 RNA genome. We have selected four potent short hairpin RNA (shRNA) candidates targeting the viral capside, integrase, protease and tat/rev open-reading frames and screened the safety of them during human hematopoietic cell development, both in vitro and in vivo. Although the four shRNA candidates appeared to be safe in vitro, one shRNA candidate impaired the in vivo development of the human immune system in Balb/c Rag2-/-IL-2Rγc-/- (BRG) mice. The three remaining shRNA candidates were combined into one single lentiviral vector (LV), and safety of the shRNA combination during human hematopoietic cell development was confirmed. Overall, we demonstrate here the preclinical in vivo safety of a LV expressing three shRNAs against HIV-1, which is proposed for a future Phase I clinical trial.Highly active antiretroviral therapy (HAART) has significantly improved the quality of life and the life expectancy of HIV-infected individuals. Still, drug-induced side effects and emergence of drug-resistant viral variants remain important issues that justify the exploration of alternative therapeutic options. One strategy consists of a gene therapy based on RNA interference to induce the sequence-specific degradation of the HIV-1 RNA genome. We have selected four potent short hairpin RNA (shRNA) candidates targeting the viral capside, integrase, protease and tat/rev open-reading frames and screened the safety of them during human hematopoietic cell development, both in vitro and in vivo. Although the four shRNA candidates appeared to be safe in vitro, one shRNA candidate impaired the in vivo development of the human immune system in Balb/c Rag2−/−IL-2Rγc−/− (BRG) mice. The three remaining shRNA candidates were combined into one single lentiviral vector (LV), and safety of the shRNA combination during human hematopoietic cell development was confirmed. Overall, we demonstrate here the preclinical in vivo safety of a LV expressing three shRNAs against HIV-1, which is proposed for a future Phase I clinical trial.

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.