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tRNALys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs
Biochemical and Biophysical Research Communications 398 (2010) 640–646
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
tRNALys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs Victoria Dyer, Abdullah Ely, Kristie Bloom, Marc Weinberg, Patrick Arbuthnot * Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand Medical School, Private Bag 3, Wits 2050, South Africa
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Article history: Received 12 June 2010 Available online 1 July 2010 Keywords: RNAi tRNA Promoter Hepatitis B virus Expression cassette
a b s t r a c t Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNALys3 short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNALys3 transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNALys3 expression cassettes are also effective in vivo. Usefulness of tRNALys3 antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Harnessing the RNA interference (RNAi) pathway to silence pathology-causing genes holds promise for development of new therapies. RNAi is responsible for processing a variety of natural cellular transcripts to effect gene silencing (reviewed in [1]). Maturation and inhibition of target gene expression by micro RNA (miR) is one of the best characterized RNAi functions. Typically Pol II transcribes primary miRs (pri-miRs) which are processed in the nucleus by the microprocessor complex Drosha/DGCR8 to form pre-miR hairpins. These sequences are exported to the cytoplasm where Dicer generates mature miR duplexes. One of the strands, selected by the RNA induced silencing complex (RISC), acts as a guide and directs post transcriptional gene silencing by translational suppression or cleavage of target mRNA. Alternative mechanisms of generating pre-miR, which do not require the microprocessor complex, have been described. These include tRNase Z cleavage of tRNA structure-containing pri-miRs [2], debranching of short introns to form pre-miR-like miRtrons [3,4] and also transcription of natural short hairpin RNA (shRNA) mimics of pre-miRs [5]. Exogenous sequences that have been used to achieve silencing typically include U6 or H1 Pol III promoters to generate mimics of intermediates of the RNAi pathway. Silencing expression cassettes have also been generated * Corresponding author. Fax: +27 (0) 11 717 2395. E-mail address: [email protected] (P. Arbuthnot). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.122
that incorporate Pol II promoters to drive transcription of engineered mono- or polycistronic pri-miR shuttles [6–9]. Although highly effective silencing has been achieved, expansion of the range of control elements with different transcriptional activity and which are suitable for therapeutic application remains important. As a class, tRNA transcriptional regulatory elements provide a wide variety of options from which to select individual Pol III promoters. Importantly some natural tRNA miR genes are bicistronic, and this feature may be adapted to develop combinatorial RNAi for therapeutic application. Availability of a large selection of promoters will also facilitate combinatorial RNAi by enabling multimerization of individual cassettes without risking rearrangement caused by homologous recombination. Other potentially useful properties are that they are smaller and weaker than U6 and H1 promoters. Interestingly tRNA Pol III promoters were used to generate exogenous RNAi activators [10,11] prior to demonstration that tRNA miR genes occur naturally [2]. tRNA miR genes described to date typically contain BoxA and BoxB internal Pol III promoter elements with downstream pre-miR moieties and ‘U’-rich transcription termination sequences[2]. After cleavage of the pri-miR by tRNase Z, the pre-miR is exported from the nucleus and processed by Dicer before effecting target silencing. The arrangement and processing of the natural tRNA pre-miR sequences can be adapted conveniently to engineer expression cassettes that transcribe exogenous RNAi activators. To develop the potential therapeutic utility of exogenous tRNA pre-miR sequences, we have generated
V. Dyer et al. / Biochemical and Biophysical Research Communications 398 (2010) 640–646
tRNALys3 Pol III expression cassettes that target conserved sequences of the X open reading frame (ORF) of the hepatitis B virus (HBV). Assessment shows that the tRNALys3 regulatory sequences that express antiviral shRNAs silence markers of HBV replication efficiently. To our knowledge, these results are the first to demonstrate silencing in vivo by a tRNALys3 transcriptional regulatory element-containing cassette. Efficacy of these exogenous sequences compares favorably with previously described U6, H1 (Pol III) and CMV (Pol II) antiHBV cassettes [7,12,13] and supports the notion that tRNA promoters have potential therapeutic utility.
2. Materials and methods 2.1. Plasmids Modification of the p901-1 plasmid, which contains the tRNALys3 promoter [11], was carried out to generate antiHBV shRNA-encoding sequences. Expression cassettes were produced by inserting annealed forward (tRNALys3 shRNA N F, Supplementary Table 1) and reverse (tRNALys3 shRNA N R, Supplementary Table 1) oligonucleotides at the NruI and PstI sites of p901-1. These oligonucleotides, encoding the numbered (N) antiHBV shRNAs, were complementary to each other and designed to form 50 blunt and 30 sticky ends to be compatible with the NruI and PstI sites of p901-1. The tRNALys3 Mock expression cassette had a Pol III termination signal inserted immediately downstream of the promoter. Propagation of RNA Pol II-driven pri-miRNA-122/5 and -122/10 shuttles has been described [7]. The previously reported two step PCR method of generating the U6 shRNA panel of cassettes [12] was adapted to form H1 shRNA expression cassettes. The H1 promoter was initially amplified from human genomic DNA using PCR with the universal forward (H1 F, 50 -GATCGAATTCACTAGTGA ACGCTGACGTCATCAA-30 ) and reverse (H1 R, 50 -GGATCCGTGGTCTC ATACAGAACTTATAAGATTCCCAAATC-30 ) primers. To incorporate downstream shRNA sequences, the promoter amplicon was amplified with the H1 F and a first reaction reverse primer (H1 shRNA N R1, Supplementary Table 1). These reverse oligonucleotides were complementary to part of the H1 promoter and included sense and loop-encoding sequences of the shRNAs. The amplicon was used as template for a second round of PCR with H1 F and a second reaction reverse primer (H1 shRNA N R2, Supplementary Table 1) that overlapped with the loop sequence, the HBV antisense region and transcription termination signal to constitute the entire shRNA-encoding sequence in the amplified DNA. To form the H1 mock cassettes, a transcription termination signal was inserted immediately downstream of the H1 promoter. The final amplification products were ligated to the PCR cloning vector pTZ57R/T (InsT/Aclone™ Fermentas, MD, USA) and the sequence of the inserts verified using standard automated dideoxy chain termination procedures (Inqaba Biotech., South Africa). The psiCHECK-HBx reporter target vector, which contains an intact HBx target sequence downstream of the Renilla ORF within the psiCHECK (Promega, WI, USA), has been described previously [13]. pCH-9/3091 is an HBV replication competent plasmid and contains a greater than genome length HBV sequence [14]. 2.2. Transfection of cells in culture and analysis of RNAi effecter processing using northern blot hybridization Huh7 cells, maintained as has been described [12] and seeded in CostarÒ 24-well plates (Corning, NY, USA), were at approximately 70% confluence at the time of transfection. Transfections were carried out using Lipofectamine 2000™ (Invitrogen, CA, USA) according to the manufacturer’s instructions and included a total of 1 lg of DNA. This comprised 90 ng of a target vector (psiCHECK-HBx or
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pCH-9/3091), 0.9–900 ng of an effecter vector (U6 shRNA, H1 shRNA, tRNALys3 shRNA or pri-miRNA-122 shuttle vectors), 10 ng of the green fluorescent protein (GFP) expression plasmid pCMVGFP [15] and an empty vector (pTZ57R). Forty-eight hours after transfection, cells were viewed using a fluorescence microscope to identify eGFP-positive cells and verify equivalent transfection efficiencies prior to analysis of knockdown efficacy. Luciferase activity in lysates from cells transfected with psiCHECK-HBx was measured using the Dual-LuciferaseÒ Reporter Assay System (Promega, WI, USA). Knockdown of HBV replication was assessed in cells co-transfected with pCH-9/3091 [14] and relevant RNAi effecter plasmids. Culture medium was harvested and HBsAg secretion measured using ELISA with the MONOLISAÒ HBsAg ULTRA kit (Bio-Rad, CA, USA). Assessment of disruptive effects of antiHBV expression cassettes on endogenous miR-16a function was determined as previously described [6]. Northern blot analysis was performed on RNA extracted from cells transfected with the various Pol III shRNA or Pol II miR-122 shuttle cassettes according to previously described methods [7]. Ratios of the guide strand to U6 snRNA signal intensities, determined using scanning densitometry with Image J software (http://rsbweb.nih.gov/ij/), were calculated to compare processing efficiency. 2.3. Assessment of efficacy of silencing of HBV replication by expression cassettes in vivo Mice were injected using the hydrodynamic procedure with a combination of 5 lg pCH-9/3091 [14], and either 5 lg of RNAi expression vector (U6 shRNA 5, H1 shRNA 5, tRNALys3 shRNA 5 or pri-miRNA-122/5 shuttle), or 5 lg of control mock U6, H,1 tRNALys3 or CMV (pCI-neo, Promega, WI, USA) promoter-containing backbone plasmid. Blood was collected 3 and 5 days after injection. Experiments were carried out according to protocols approved by the University of the Witwatersrand Animal Ethics Screening Committee. ELISA for HBsAg levels was performed on serum samples using the MONOLISAÒ HBs Ag ULTRA kit from Bio-Rad (CA, USA). Measurements of circulating viral particle equivalents (VPEs) were determined using real-time quantitative PCR (qPCR), performed on nucleic acids isolated from serum, according to previously described methods [16]. 2.4. Statistical analysis Data are expressed as the mean ± standard error of the mean. Statistical difference was considered significant when the confidence interval was greater than 95% (P < 0.05) and was determined according to the Student0 s two tailed unpaired t-test using the GraphPad Prism software package (GraphPad Software Inc., CA, USA).
3. Results 3.1. Design and processing of antiHBV expression cassettes U6, H1 and tRNALys3 Pol III regulatory elements were employed to drive shRNA expression and the CMV Pol II promoter was used to transcribe pri-miR shuttle sequences. Numbered antiHBV shRNA sequences were selected for this study according to their previously described efficacy against HBV [12]. They included sequences that were highly effective against HBV (numbers 5, 6, 8 and 9) as well as a control (number 10) which did not counter replication of the virus. The structure of the expression cassettes and derived transcripts that incorporate antiHBV guides are illustrated in Fig. 1A and B. The primary transcripts from the tRNALys3 promoter differed from those of U6 and H1 in that the RNAi precursor incor-
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Fig. 1. Expression and processing of antiHBV RNAi activators. (A) Schematic illustration of cassettes with derived transcripts that generate shRNA or pri-miR shuttle sequences. The tRNALys3–shRNA transcript includes tRNA sequences derived from the Pol III promoter element. Numbered different antiviral sequences (N) were incorporated into the expression cassettes. (B) Predicted secondary structures of pri-miR-122, shRNA and tRNALys3 RNAi precursors. The locations of the intended guide strands are indicated in red. (C) Northern blot hybridization analysis to verify processing of the transcripts. Intended antiHBV guide strands of approximately 23 nt in length were detected after analysis of total RNA extracted from cells that had been transfected with the plasmids indicated above each lane. A longer exposure time of the autoradiograph was required to detect the antiHBV guide strand that was generated from the pri-miR-122/5 expression cassette (pri-miR-122/5 LE). To confirm equal loading of RNA onto the gel lanes, blots were stripped and reprobed with a labeled oligonucleotide that was complementary to the U6 snRNA. Ratios of the guide strand to U6 snRNA signal intensities, determined using scanning densitometry, and are indicated.
porated a tRNA sequence that was encoded by the promoter. These tRNALys3 promoter cassettes, with most favorable spacer sequences between tRNA and shRNA elements, were modeled on the arrangement that had previously been optimized for antiHIV sequences [11]. Antiviral sequences expressed from the Pol II CMV promoter cassettes were designed to simulate the structure of naturally occurring liver-specific pri-miR-122. The shuttle sequences included bulges and single stranded regions that were similar to those of the parent template, but the natural guide was substituted with antiHBV sequences [7]. According to the design of the tRNALys3 cassettes, transcripts should initially be cleaved by RNaseZ to remove the tRNA element from the primary transcript [2]. Thereafter, Dicer processing of the hairpin ought to occur in a manner that is similar to that of U6 and H1 shRNA primary transcripts. Northern blot hybridization analysis revealed that guides of appropriate sizes (approximately 23 nt) were detectable, which confirms that processing of the Pol III primary transcripts occurred as anticipated (Fig. 1C). Ratios of the guide strand band intensities relative to those of the U6 snRNA were similar, indicating that processing efficiency was similar for each of the Pol III promoter cassettes. Northern blot analysis of RNA extracted from transfected cells, also demonstrated that the shRNA 6, but not the U6 shRNA 10 transcript, was processed efficiently to produce easily detectable guide (Supplementary Fig. 1). This observation correlates with the poor antiviral efficacy of the shRNA 10 cassettes [7,12]. The concentration of the CMV primiR-122/5 was lower than that of the Pol III guide sequences, and is in accordance with previously reported observations [7].
HBx was inserted downstream of the Renilla luciferase ORF in the psiCHECK plasmid vector that also constitutively expressed Firefly luciferase (Fig. 2A). Varying mass ratios of silencing expression plasmid to reporter vector were transfected into liver-derived Huh7 cells. The normalized ratio of Renilla to Firefly luciferase activity was measured to determine silencing efficacy. Analysis was carried out using the effective antiHBV sequence 5 (Fig. 2B) as well as the control shRNA10 sequence (Fig. 2C), which is not processed efficiently (Supplementary Fig. 1) and does not cause knockdown of HBV targets [7,12]. When compared to mock-treated cells, the Pol III shRNA5 expression cassettes all showed inhibition of reporter gene expression of approximately 80–90%. The CMV pri-miR-122/5 Pol III expression cassette was less effective, and this observation correlates with the lower concentration of mature guide sequence that was detectable following northern blot hybridization analysis (Fig. 1C). Similar efficacy was observed after transfection of expression cassettes encoding shRNA 6, shRNA 8 and shRNA 9 antiviral sequences (Supplementary Fig. 2). Importantly, expression cassettes encoding shRNA 10 and pri-miR-122/ 10 sequences did not achieve silencing of their viral target at all ratios of reporter to RNAi activator-expressing plasmids. These data indicate that silencing is specific and also verify that the tRNALys3 promoter, when used to drive RNAi-activating cassettes, is capable of achieving HBV silencing which was equivalent to that of other promoters.
3.2. AntiHBV shRNA expressed from the tRNALys3 promoter acts efficiently against a reporter target
To determine efficacy of expression cassettes in models of viral replication, the pCH-9/3091 HBV replication competent plasmid [14] was used to transfect liver-derived Huh7 cells in culture or hepatocytes in vivo. pCH-9/3091 has a greater than HBV genome length sequence and the core/pregenome RNA is transcribed from
A dual luciferase reporter gene assay was used to assess the relative silencing efficacy of each of the panel of promoter cassettes.
3.3. Effective inhibition of markers HBV replication by tRNALys3 shRNA 5 in cell culture and in vivo
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Fig. 2. Dual luciferase assay to determine efficacy of active (5) and inactive (10) antiHBV sequences when expressed in each of the panel of four expression cassettes. (A) Schematic illustration of dual luciferase reported plasmid. The HBx target sequence was inserted downstream of the hRLuc ORF. Renilla luciferase activity was used as an indicator of target silencing and efficacy was determined relative to activity of constitutively expressed Firefly luciferase. Varying the mass ratio of the dual luciferase target to RNAi activator plasmids was used to determine relative efficacy of plasmids encoding antiHBV sequence 5 (B) or sequence 10 (C). The target:antiviral plasmid ratio ranged from 1:0.0025 to 1:10. Comparison of normalized Renilla to Firefly luciferase activity was used to determine silencing efficiency.
a CMV promoter. PreS1, PreS2 and N transcripts are produced from downstream viral promoters. Transcription termination of all HBV RNAs occurs at the unique termination signal, and this results in inclusion of the target HBx sequence in all of the viral RNAs (Fig. 3A). Co-transfection of liver-derived Huh7 cells with pCH-9/3091 and expression cassettes encoding shRNA 5 or pri-miR-122/5 sequences revealed a significant and similar decrease in concentration of HBsAg in the tissue culture supernatant. The effect on this marker of HBV replication achieved by tRNALys3 shRNA 5 cassettes was similar to that of the other Pol III sequences which have previously been shown to be highly effective [7,12]. The control vectors that expressed RNAi precursors directed at HBV sequence 10 did not diminish the concentration of HBsAg in the culture supernatants and culture medium HBsAg concentrations were increased in some cases. This observation is in agreement with previous findings, but the reasons for an increase in HBsAg culture supernatant concentration are currently unclear [7,12,13]. Efficient silencing of markers of HBV replication in cell culture prompted investigation of the efficacy against viral replication in vivo. To assess this, mice were subjected to hydrodynamic injection with the pCH-9/3091 replication competent vector together with plasmids containing antiviral expression cassettes. Serum HBsAg and circulating VPEs were measured after blood collection at days 3 and 5 after injection (Fig. 3C). When compared to animals
that were treated with control plasmids that lacked the antiviral RNAi expression cassettes, there were consistent and significant decreases in serum HBsAg concentrations. Similarly, the number of circulating VPEs was also diminished in mice that had received HBV-targeting expression cassettes. The efficacy of the tRNALys3 shRNA 5 vector compared favorably to that achieved by other silencing sequences, and indicates that this transcription control element is effective in vivo. 3.4. Normal function of endogenous miR-16-1 is retained after cotransfection of antiHBV expression cassettes Ensuring that there is minimal if any disruption of the endogenous miR pathway is important for the development of expressed RNAi activators for therapeutic application. To assess possible perturbations of endogenous miR function, we employed a dual luciferase reporter vector in which seven copies of an imperfectly matched endogenous miR-16 target were inserted downstream of the Renilla luciferase ORF (Fig. 4A). The method also utilizes miR sponges as a control to verify de-repressive effects of endogenous miR-16 function [6,17]. A translational suppression by miR16 could be detected sensitively by measuring Renilla/Firefly luciferase activity. miR-16 was selected as it is expressed in a variety of tissues [18] and can be conveniently used to determine disruption of natural miR function. Analysis revealed that co-expression of
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Fig. 3. Inhibition of HBV replication by antiHBV expression cassettes in cultured cells and in vivo. (A) Illustration of the HBV replication competent plasmid, pCH-9/3091, which was used to transfect liver-derived Huh7 cells in culture and hepatocytes in vivo. (B) The concentration of HBsAg was measured in cell culture supernatants following co-transfection of plasmids containing the panel of plasmids encoding effective (5) and control (10) antiviral sequences. Values were normalized relative to the mock transfected groups of cells. (C) Serum HBsAg and circulating viral particle equivalents were measured in mice that were subjected to hydrodynamic injection with each of the panel of expression cassettes. Concentrations of HBsAg and circulating VPEs were determined at days 3 and 5 after hydrodynamic injection. Compared to measurements from mock-treated animals, HBsAg concentrations were significantly decreased (p < 0.05) in all groups at days 3 and 5.
each of tRNALys3 shRNA 5 as well as the other Pol III and Pol II expression constructs within transfected cells did not cause derepression of miR-16 function (Fig. 4B). Control transfection with the miR-16 sponge caused a fivefold decrease in silencing of the reporter target. Collectively these data support the notion that tRNALys3 promoter-controlled expression of HBV silencing shRNA may be used effectively and safely to inhibit viral replication.
4. Discussion Use of exogenous activators of the RNAi pathway to silence gene expression has become widely applied. Synthetic and expressed activators of RNAi have been used and each category of RNAi effecter has advantages and disadvantages. Compatibility with incorporation into efficient viral vectors and also the sustained production of RNAi-activating transcripts from expression cassettes are particularly useful for the treatment of chronic diseases caused by persistent infection with HBV and HIV. To advance use of tRNA Pol III transcription regulatory elements, we have tested their efficacy against HBV. Persistent infection with this
virus occurs in approximately 6% of the world0 s population and is a significant global public health problem [19]. Complicating cirrhosis and hepatocellular carcinoma are major causes of mortality and morbidity. Currently available treatments rarely achieve a sustained therapeutic effect and demonstration that the virus is susceptible to RNAi-based silencing indicates that this approach has therapeutic potential [12,20,21]. Our data demonstrate that tRNALys3 shRNA cassettes are capable of highly efficient inhibition of HBV replication, which is similar to that of previously characterized antiHBV RNAi-activating expression cassettes. Importantly the effect was observed in vivo in a murine model that simulates HBV infection. Processing of the primary transcripts occurs efficiently and the function of an endogenous miR is not disrupted. Selecting transcription controlling elements to drive production of therapeutic RNAi activators requires careful consideration. Overloading cellular processes that are required for natural miR generation is potentially toxic [22] and utilizing RNAi activators that employ alternative pathways may limit unintended harmful effects. This is particularly important for RNAi-mediated inhibition of viral replication where formation of multiplex cassettes may be required to minimize the risk of target mutation and escape
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Fig. 4. Assessment of silencing function of endogenous miR-16 after co-transfection with antiHBV expression cassettes. (A) Schematic illustration of dual luciferase reporter cassette used to determine miR-16 function. Seven miR-16 targets were inserted in tandem and downstream within the 30 UTR of the Renilla luciferase ORF. (B) Ratio of Renilla to Firefly luciferase activity was measured to assess the effect of antiHBV expression cassettes on endogenous miR-16-1 function. As a control for de-repression of miR-16-1 target silencing, a plasmid encoding a sponge cassette was included.
from silencing. Availability of a broad range of regulatory elements, including the panel of tRNA Pol III promoters, which may be used in antiviral expression cassettes will facilitate advancement of RNAi-based antiviral therapy. Previous reports have shown that tRNA promoters may be incorporated into expression cassettes that inhibit HIV gene expression [11]. Using a similar approach, our study focused on employing a tRNALys3 promoter to express antiHBV sequences, but it is likely that other tRNA promoters with Box A and Box B elements will have similar utility for engineering of gene silencing cassettes. tRNA Pol III promoters have several useful properties that may be applied to therapeutic gene silencing. These include their small size and potential utility in combinatorial RNAi through multimerization and generation of cassettes that simulate natural bicistronic tRNA miR genes [2]. An interesting recent observation has been that the silencing efficacy of tRNA promoters may be enhanced by incorporation of CMV enhancer elements [23]. This suggests that modification of tRNA Pol III promoters with other enhancer elements, so as to confer properties of tissue specificity or inducibility, may be feasible and contribute to the versatility of tRNA Pol III promoters. Acknowledgments The p901 plasmid was generously provided by Lisa Scherer and John Rossi. This work was supported by funding under the Sixth Research Framework Programme of the European Union, Project
RIGHT (LSHB-CT-2004-005276), the South African National Research Foundation (GUNs 68339 and 65495), Medical Research Council, Poliomyelitis Research Foundation and ESASTAP.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.122.
References [1] V.N. Kim, J. Han, M.C. Siomi, Biogenesis of small RNAs in animals, Nat. Rev. Mol. Cell Biol. 10 (2009) 126–139. [2] H.P. Bogerd, H.W. Karnowski, X. Cai, J. Shin, M. Pohlers, B.R. Cullen, A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral microRNAs, Mol. Cell 37 (2010) 135–142. [3] E. Berezikov, W.J. Chung, J. Willis, E. Cuppen, E.C. Lai, Mammalian mirtron genes, Mol. Cell 28 (2007) 328–336. [4] J.G. Ruby, C.H. Jan, D.P. Bartel, Intronic microRNA precursors that bypass Drosha processing, Nature 448 (2007) 83–86. [5] J.E. Babiarz, J.G. Ruby, Y. Wang, D.P. Bartel, R. Blelloch, Mouse ES cells express endogenous shRNAs, siRNAs, and other microprocessor-independent, Dicerdependent small RNAs, Genes Dev. 22 (2008) 2773–2785. [6] A. Ely, T. Naidoo, P. Arbuthnot, Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles, Nucleic Acids Res. 37 (2009) e91. [7] A. Ely, T. Naidoo, S. Mufamadi, C. Crowther, P. Arbuthnot, Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo, Mol. Ther. 16 (2008) 1105–1112.
646
V. Dyer et al. / Biochemical and Biophysical Research Communications 398 (2010) 640–646
[8] Y.P. Liu, J. Haasnoot, O. ter Brake, B. Berkhout, P. Konstantinova, Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron, Nucleic Acids Res. 36 (2008) 2811–2824. [9] L.A. Aagaard, J. Zhang, K.J. von Eije, H. Li, P. Saetrom, M. Amarzguioui, J.J. Rossi, Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs, Gene Ther. 15 (2008) 1536–1549. [10] H. Kawasaki, K. Taira, Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells, Nucleic Acids Res. 31 (2003) 700–707. [11] L.J. Scherer, R. Frank, J.J. Rossi, Optimization and characterization of tRNA– shRNA expression constructs, Nucleic Acids Res. 35 (2007) 2620–2628. [12] S. Carmona, A. Ely, C. Crowther, N. Moolla, F.H. Salazar, P.L. Marion, N. Ferry, M.S. Weinberg, P. Arbuthnot, Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs, Mol. Ther. 13 (2006) 411–421. [13] M.S. Weinberg, A. Ely, S. Barichievy, C. Crowther, S. Mufamadi, S. Carmona, P. Arbuthnot, Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA, Mol. Ther. 15 (2007) 534–541. [14] M. Nassal, The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly, J. Virol. 66 (1992) 4107–4116. [15] M. Passman, M. Weinberg, M. Kew, P. Arbuthnot, In situ demonstration of inhibitory effects of hammerhead ribozymes that are targeted to the hepatitis
[16]
[17] [18] [19] [20]
[21] [22]
[23]
Bx sequence in cultured cells, Biochem. Biophys. Res. Commun. 268 (2000) 728–733. S. Chattopadhyay, A. Ely, K. Bloom, M.S. Weinberg, P. Arbuthnot, Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles, Biochem. Biophys. Res. Commun. 389 (2009) 484–489. M.S. Ebert, J.R. Neilson, P.A. Sharp, MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells, Nat. Methods 4 (2007) 721–726. M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, Identification of novel genes coding for small expressed RNAs, Science 294 (2001) 853–858. P. Arbuthnot, M. Kew, Hepatitis B virus and hepatocellular carcinoma, Int. J. Exp. Pathol. 82 (2001) 77–100. A.P. McCaffrey, H. Nakai, K. Pandey, Z. Huang, F.H. Salazar, H. Xu, S.F. Wieland, P.L. Marion, M.A. Kay, Inhibition of hepatitis B virus in mice by RNA interference, Nat. Biotechnol. 21 (2003) 639–644. A. Shlomai, Y. Shaul, Inhibition of hepatitis B virus expression and replication by RNA interference, Hepatology 37 (2003) 764–770. D. Grimm, K.L. Streetz, C.L. Jopling, T.A. Storm, K. Pandey, C.R. Davis, P. Marion, F. Salazar, M.A. Kay, Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways, Nature 441 (2006) 537–541. M. Weiwei, X. Zhenhua, L. Feng, N. Hang, J. Yuyang, A significant increase of RNAi efficiency in human cells by the CMV enhancer with a tRNAlys promoter, J. Biomed. Biotechnol. (2009) 514287.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
tRNALys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs Victoria Dyer, Abdullah Ely, Kristie Bloom, Marc Weinberg, Patrick Arbuthnot * Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand Medical School, Private Bag 3, Wits 2050, South Africa
a r t i c l e
i n f o
Article history: Received 12 June 2010 Available online 1 July 2010 Keywords: RNAi tRNA Promoter Hepatitis B virus Expression cassette
a b s t r a c t Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNALys3 short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNALys3 transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNALys3 expression cassettes are also effective in vivo. Usefulness of tRNALys3 antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Harnessing the RNA interference (RNAi) pathway to silence pathology-causing genes holds promise for development of new therapies. RNAi is responsible for processing a variety of natural cellular transcripts to effect gene silencing (reviewed in [1]). Maturation and inhibition of target gene expression by micro RNA (miR) is one of the best characterized RNAi functions. Typically Pol II transcribes primary miRs (pri-miRs) which are processed in the nucleus by the microprocessor complex Drosha/DGCR8 to form pre-miR hairpins. These sequences are exported to the cytoplasm where Dicer generates mature miR duplexes. One of the strands, selected by the RNA induced silencing complex (RISC), acts as a guide and directs post transcriptional gene silencing by translational suppression or cleavage of target mRNA. Alternative mechanisms of generating pre-miR, which do not require the microprocessor complex, have been described. These include tRNase Z cleavage of tRNA structure-containing pri-miRs [2], debranching of short introns to form pre-miR-like miRtrons [3,4] and also transcription of natural short hairpin RNA (shRNA) mimics of pre-miRs [5]. Exogenous sequences that have been used to achieve silencing typically include U6 or H1 Pol III promoters to generate mimics of intermediates of the RNAi pathway. Silencing expression cassettes have also been generated * Corresponding author. Fax: +27 (0) 11 717 2395. E-mail address: [email protected] (P. Arbuthnot). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.122
that incorporate Pol II promoters to drive transcription of engineered mono- or polycistronic pri-miR shuttles [6–9]. Although highly effective silencing has been achieved, expansion of the range of control elements with different transcriptional activity and which are suitable for therapeutic application remains important. As a class, tRNA transcriptional regulatory elements provide a wide variety of options from which to select individual Pol III promoters. Importantly some natural tRNA miR genes are bicistronic, and this feature may be adapted to develop combinatorial RNAi for therapeutic application. Availability of a large selection of promoters will also facilitate combinatorial RNAi by enabling multimerization of individual cassettes without risking rearrangement caused by homologous recombination. Other potentially useful properties are that they are smaller and weaker than U6 and H1 promoters. Interestingly tRNA Pol III promoters were used to generate exogenous RNAi activators [10,11] prior to demonstration that tRNA miR genes occur naturally [2]. tRNA miR genes described to date typically contain BoxA and BoxB internal Pol III promoter elements with downstream pre-miR moieties and ‘U’-rich transcription termination sequences[2]. After cleavage of the pri-miR by tRNase Z, the pre-miR is exported from the nucleus and processed by Dicer before effecting target silencing. The arrangement and processing of the natural tRNA pre-miR sequences can be adapted conveniently to engineer expression cassettes that transcribe exogenous RNAi activators. To develop the potential therapeutic utility of exogenous tRNA pre-miR sequences, we have generated
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tRNALys3 Pol III expression cassettes that target conserved sequences of the X open reading frame (ORF) of the hepatitis B virus (HBV). Assessment shows that the tRNALys3 regulatory sequences that express antiviral shRNAs silence markers of HBV replication efficiently. To our knowledge, these results are the first to demonstrate silencing in vivo by a tRNALys3 transcriptional regulatory element-containing cassette. Efficacy of these exogenous sequences compares favorably with previously described U6, H1 (Pol III) and CMV (Pol II) antiHBV cassettes [7,12,13] and supports the notion that tRNA promoters have potential therapeutic utility.
2. Materials and methods 2.1. Plasmids Modification of the p901-1 plasmid, which contains the tRNALys3 promoter [11], was carried out to generate antiHBV shRNA-encoding sequences. Expression cassettes were produced by inserting annealed forward (tRNALys3 shRNA N F, Supplementary Table 1) and reverse (tRNALys3 shRNA N R, Supplementary Table 1) oligonucleotides at the NruI and PstI sites of p901-1. These oligonucleotides, encoding the numbered (N) antiHBV shRNAs, were complementary to each other and designed to form 50 blunt and 30 sticky ends to be compatible with the NruI and PstI sites of p901-1. The tRNALys3 Mock expression cassette had a Pol III termination signal inserted immediately downstream of the promoter. Propagation of RNA Pol II-driven pri-miRNA-122/5 and -122/10 shuttles has been described [7]. The previously reported two step PCR method of generating the U6 shRNA panel of cassettes [12] was adapted to form H1 shRNA expression cassettes. The H1 promoter was initially amplified from human genomic DNA using PCR with the universal forward (H1 F, 50 -GATCGAATTCACTAGTGA ACGCTGACGTCATCAA-30 ) and reverse (H1 R, 50 -GGATCCGTGGTCTC ATACAGAACTTATAAGATTCCCAAATC-30 ) primers. To incorporate downstream shRNA sequences, the promoter amplicon was amplified with the H1 F and a first reaction reverse primer (H1 shRNA N R1, Supplementary Table 1). These reverse oligonucleotides were complementary to part of the H1 promoter and included sense and loop-encoding sequences of the shRNAs. The amplicon was used as template for a second round of PCR with H1 F and a second reaction reverse primer (H1 shRNA N R2, Supplementary Table 1) that overlapped with the loop sequence, the HBV antisense region and transcription termination signal to constitute the entire shRNA-encoding sequence in the amplified DNA. To form the H1 mock cassettes, a transcription termination signal was inserted immediately downstream of the H1 promoter. The final amplification products were ligated to the PCR cloning vector pTZ57R/T (InsT/Aclone™ Fermentas, MD, USA) and the sequence of the inserts verified using standard automated dideoxy chain termination procedures (Inqaba Biotech., South Africa). The psiCHECK-HBx reporter target vector, which contains an intact HBx target sequence downstream of the Renilla ORF within the psiCHECK (Promega, WI, USA), has been described previously [13]. pCH-9/3091 is an HBV replication competent plasmid and contains a greater than genome length HBV sequence [14]. 2.2. Transfection of cells in culture and analysis of RNAi effecter processing using northern blot hybridization Huh7 cells, maintained as has been described [12] and seeded in CostarÒ 24-well plates (Corning, NY, USA), were at approximately 70% confluence at the time of transfection. Transfections were carried out using Lipofectamine 2000™ (Invitrogen, CA, USA) according to the manufacturer’s instructions and included a total of 1 lg of DNA. This comprised 90 ng of a target vector (psiCHECK-HBx or
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pCH-9/3091), 0.9–900 ng of an effecter vector (U6 shRNA, H1 shRNA, tRNALys3 shRNA or pri-miRNA-122 shuttle vectors), 10 ng of the green fluorescent protein (GFP) expression plasmid pCMVGFP [15] and an empty vector (pTZ57R). Forty-eight hours after transfection, cells were viewed using a fluorescence microscope to identify eGFP-positive cells and verify equivalent transfection efficiencies prior to analysis of knockdown efficacy. Luciferase activity in lysates from cells transfected with psiCHECK-HBx was measured using the Dual-LuciferaseÒ Reporter Assay System (Promega, WI, USA). Knockdown of HBV replication was assessed in cells co-transfected with pCH-9/3091 [14] and relevant RNAi effecter plasmids. Culture medium was harvested and HBsAg secretion measured using ELISA with the MONOLISAÒ HBsAg ULTRA kit (Bio-Rad, CA, USA). Assessment of disruptive effects of antiHBV expression cassettes on endogenous miR-16a function was determined as previously described [6]. Northern blot analysis was performed on RNA extracted from cells transfected with the various Pol III shRNA or Pol II miR-122 shuttle cassettes according to previously described methods [7]. Ratios of the guide strand to U6 snRNA signal intensities, determined using scanning densitometry with Image J software (http://rsbweb.nih.gov/ij/), were calculated to compare processing efficiency. 2.3. Assessment of efficacy of silencing of HBV replication by expression cassettes in vivo Mice were injected using the hydrodynamic procedure with a combination of 5 lg pCH-9/3091 [14], and either 5 lg of RNAi expression vector (U6 shRNA 5, H1 shRNA 5, tRNALys3 shRNA 5 or pri-miRNA-122/5 shuttle), or 5 lg of control mock U6, H,1 tRNALys3 or CMV (pCI-neo, Promega, WI, USA) promoter-containing backbone plasmid. Blood was collected 3 and 5 days after injection. Experiments were carried out according to protocols approved by the University of the Witwatersrand Animal Ethics Screening Committee. ELISA for HBsAg levels was performed on serum samples using the MONOLISAÒ HBs Ag ULTRA kit from Bio-Rad (CA, USA). Measurements of circulating viral particle equivalents (VPEs) were determined using real-time quantitative PCR (qPCR), performed on nucleic acids isolated from serum, according to previously described methods [16]. 2.4. Statistical analysis Data are expressed as the mean ± standard error of the mean. Statistical difference was considered significant when the confidence interval was greater than 95% (P < 0.05) and was determined according to the Student0 s two tailed unpaired t-test using the GraphPad Prism software package (GraphPad Software Inc., CA, USA).
3. Results 3.1. Design and processing of antiHBV expression cassettes U6, H1 and tRNALys3 Pol III regulatory elements were employed to drive shRNA expression and the CMV Pol II promoter was used to transcribe pri-miR shuttle sequences. Numbered antiHBV shRNA sequences were selected for this study according to their previously described efficacy against HBV [12]. They included sequences that were highly effective against HBV (numbers 5, 6, 8 and 9) as well as a control (number 10) which did not counter replication of the virus. The structure of the expression cassettes and derived transcripts that incorporate antiHBV guides are illustrated in Fig. 1A and B. The primary transcripts from the tRNALys3 promoter differed from those of U6 and H1 in that the RNAi precursor incor-
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Fig. 1. Expression and processing of antiHBV RNAi activators. (A) Schematic illustration of cassettes with derived transcripts that generate shRNA or pri-miR shuttle sequences. The tRNALys3–shRNA transcript includes tRNA sequences derived from the Pol III promoter element. Numbered different antiviral sequences (N) were incorporated into the expression cassettes. (B) Predicted secondary structures of pri-miR-122, shRNA and tRNALys3 RNAi precursors. The locations of the intended guide strands are indicated in red. (C) Northern blot hybridization analysis to verify processing of the transcripts. Intended antiHBV guide strands of approximately 23 nt in length were detected after analysis of total RNA extracted from cells that had been transfected with the plasmids indicated above each lane. A longer exposure time of the autoradiograph was required to detect the antiHBV guide strand that was generated from the pri-miR-122/5 expression cassette (pri-miR-122/5 LE). To confirm equal loading of RNA onto the gel lanes, blots were stripped and reprobed with a labeled oligonucleotide that was complementary to the U6 snRNA. Ratios of the guide strand to U6 snRNA signal intensities, determined using scanning densitometry, and are indicated.
porated a tRNA sequence that was encoded by the promoter. These tRNALys3 promoter cassettes, with most favorable spacer sequences between tRNA and shRNA elements, were modeled on the arrangement that had previously been optimized for antiHIV sequences [11]. Antiviral sequences expressed from the Pol II CMV promoter cassettes were designed to simulate the structure of naturally occurring liver-specific pri-miR-122. The shuttle sequences included bulges and single stranded regions that were similar to those of the parent template, but the natural guide was substituted with antiHBV sequences [7]. According to the design of the tRNALys3 cassettes, transcripts should initially be cleaved by RNaseZ to remove the tRNA element from the primary transcript [2]. Thereafter, Dicer processing of the hairpin ought to occur in a manner that is similar to that of U6 and H1 shRNA primary transcripts. Northern blot hybridization analysis revealed that guides of appropriate sizes (approximately 23 nt) were detectable, which confirms that processing of the Pol III primary transcripts occurred as anticipated (Fig. 1C). Ratios of the guide strand band intensities relative to those of the U6 snRNA were similar, indicating that processing efficiency was similar for each of the Pol III promoter cassettes. Northern blot analysis of RNA extracted from transfected cells, also demonstrated that the shRNA 6, but not the U6 shRNA 10 transcript, was processed efficiently to produce easily detectable guide (Supplementary Fig. 1). This observation correlates with the poor antiviral efficacy of the shRNA 10 cassettes [7,12]. The concentration of the CMV primiR-122/5 was lower than that of the Pol III guide sequences, and is in accordance with previously reported observations [7].
HBx was inserted downstream of the Renilla luciferase ORF in the psiCHECK plasmid vector that also constitutively expressed Firefly luciferase (Fig. 2A). Varying mass ratios of silencing expression plasmid to reporter vector were transfected into liver-derived Huh7 cells. The normalized ratio of Renilla to Firefly luciferase activity was measured to determine silencing efficacy. Analysis was carried out using the effective antiHBV sequence 5 (Fig. 2B) as well as the control shRNA10 sequence (Fig. 2C), which is not processed efficiently (Supplementary Fig. 1) and does not cause knockdown of HBV targets [7,12]. When compared to mock-treated cells, the Pol III shRNA5 expression cassettes all showed inhibition of reporter gene expression of approximately 80–90%. The CMV pri-miR-122/5 Pol III expression cassette was less effective, and this observation correlates with the lower concentration of mature guide sequence that was detectable following northern blot hybridization analysis (Fig. 1C). Similar efficacy was observed after transfection of expression cassettes encoding shRNA 6, shRNA 8 and shRNA 9 antiviral sequences (Supplementary Fig. 2). Importantly, expression cassettes encoding shRNA 10 and pri-miR-122/ 10 sequences did not achieve silencing of their viral target at all ratios of reporter to RNAi activator-expressing plasmids. These data indicate that silencing is specific and also verify that the tRNALys3 promoter, when used to drive RNAi-activating cassettes, is capable of achieving HBV silencing which was equivalent to that of other promoters.
3.2. AntiHBV shRNA expressed from the tRNALys3 promoter acts efficiently against a reporter target
To determine efficacy of expression cassettes in models of viral replication, the pCH-9/3091 HBV replication competent plasmid [14] was used to transfect liver-derived Huh7 cells in culture or hepatocytes in vivo. pCH-9/3091 has a greater than HBV genome length sequence and the core/pregenome RNA is transcribed from
A dual luciferase reporter gene assay was used to assess the relative silencing efficacy of each of the panel of promoter cassettes.
3.3. Effective inhibition of markers HBV replication by tRNALys3 shRNA 5 in cell culture and in vivo
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Fig. 2. Dual luciferase assay to determine efficacy of active (5) and inactive (10) antiHBV sequences when expressed in each of the panel of four expression cassettes. (A) Schematic illustration of dual luciferase reported plasmid. The HBx target sequence was inserted downstream of the hRLuc ORF. Renilla luciferase activity was used as an indicator of target silencing and efficacy was determined relative to activity of constitutively expressed Firefly luciferase. Varying the mass ratio of the dual luciferase target to RNAi activator plasmids was used to determine relative efficacy of plasmids encoding antiHBV sequence 5 (B) or sequence 10 (C). The target:antiviral plasmid ratio ranged from 1:0.0025 to 1:10. Comparison of normalized Renilla to Firefly luciferase activity was used to determine silencing efficiency.
a CMV promoter. PreS1, PreS2 and N transcripts are produced from downstream viral promoters. Transcription termination of all HBV RNAs occurs at the unique termination signal, and this results in inclusion of the target HBx sequence in all of the viral RNAs (Fig. 3A). Co-transfection of liver-derived Huh7 cells with pCH-9/3091 and expression cassettes encoding shRNA 5 or pri-miR-122/5 sequences revealed a significant and similar decrease in concentration of HBsAg in the tissue culture supernatant. The effect on this marker of HBV replication achieved by tRNALys3 shRNA 5 cassettes was similar to that of the other Pol III sequences which have previously been shown to be highly effective [7,12]. The control vectors that expressed RNAi precursors directed at HBV sequence 10 did not diminish the concentration of HBsAg in the culture supernatants and culture medium HBsAg concentrations were increased in some cases. This observation is in agreement with previous findings, but the reasons for an increase in HBsAg culture supernatant concentration are currently unclear [7,12,13]. Efficient silencing of markers of HBV replication in cell culture prompted investigation of the efficacy against viral replication in vivo. To assess this, mice were subjected to hydrodynamic injection with the pCH-9/3091 replication competent vector together with plasmids containing antiviral expression cassettes. Serum HBsAg and circulating VPEs were measured after blood collection at days 3 and 5 after injection (Fig. 3C). When compared to animals
that were treated with control plasmids that lacked the antiviral RNAi expression cassettes, there were consistent and significant decreases in serum HBsAg concentrations. Similarly, the number of circulating VPEs was also diminished in mice that had received HBV-targeting expression cassettes. The efficacy of the tRNALys3 shRNA 5 vector compared favorably to that achieved by other silencing sequences, and indicates that this transcription control element is effective in vivo. 3.4. Normal function of endogenous miR-16-1 is retained after cotransfection of antiHBV expression cassettes Ensuring that there is minimal if any disruption of the endogenous miR pathway is important for the development of expressed RNAi activators for therapeutic application. To assess possible perturbations of endogenous miR function, we employed a dual luciferase reporter vector in which seven copies of an imperfectly matched endogenous miR-16 target were inserted downstream of the Renilla luciferase ORF (Fig. 4A). The method also utilizes miR sponges as a control to verify de-repressive effects of endogenous miR-16 function [6,17]. A translational suppression by miR16 could be detected sensitively by measuring Renilla/Firefly luciferase activity. miR-16 was selected as it is expressed in a variety of tissues [18] and can be conveniently used to determine disruption of natural miR function. Analysis revealed that co-expression of
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Fig. 3. Inhibition of HBV replication by antiHBV expression cassettes in cultured cells and in vivo. (A) Illustration of the HBV replication competent plasmid, pCH-9/3091, which was used to transfect liver-derived Huh7 cells in culture and hepatocytes in vivo. (B) The concentration of HBsAg was measured in cell culture supernatants following co-transfection of plasmids containing the panel of plasmids encoding effective (5) and control (10) antiviral sequences. Values were normalized relative to the mock transfected groups of cells. (C) Serum HBsAg and circulating viral particle equivalents were measured in mice that were subjected to hydrodynamic injection with each of the panel of expression cassettes. Concentrations of HBsAg and circulating VPEs were determined at days 3 and 5 after hydrodynamic injection. Compared to measurements from mock-treated animals, HBsAg concentrations were significantly decreased (p < 0.05) in all groups at days 3 and 5.
each of tRNALys3 shRNA 5 as well as the other Pol III and Pol II expression constructs within transfected cells did not cause derepression of miR-16 function (Fig. 4B). Control transfection with the miR-16 sponge caused a fivefold decrease in silencing of the reporter target. Collectively these data support the notion that tRNALys3 promoter-controlled expression of HBV silencing shRNA may be used effectively and safely to inhibit viral replication.
4. Discussion Use of exogenous activators of the RNAi pathway to silence gene expression has become widely applied. Synthetic and expressed activators of RNAi have been used and each category of RNAi effecter has advantages and disadvantages. Compatibility with incorporation into efficient viral vectors and also the sustained production of RNAi-activating transcripts from expression cassettes are particularly useful for the treatment of chronic diseases caused by persistent infection with HBV and HIV. To advance use of tRNA Pol III transcription regulatory elements, we have tested their efficacy against HBV. Persistent infection with this
virus occurs in approximately 6% of the world0 s population and is a significant global public health problem [19]. Complicating cirrhosis and hepatocellular carcinoma are major causes of mortality and morbidity. Currently available treatments rarely achieve a sustained therapeutic effect and demonstration that the virus is susceptible to RNAi-based silencing indicates that this approach has therapeutic potential [12,20,21]. Our data demonstrate that tRNALys3 shRNA cassettes are capable of highly efficient inhibition of HBV replication, which is similar to that of previously characterized antiHBV RNAi-activating expression cassettes. Importantly the effect was observed in vivo in a murine model that simulates HBV infection. Processing of the primary transcripts occurs efficiently and the function of an endogenous miR is not disrupted. Selecting transcription controlling elements to drive production of therapeutic RNAi activators requires careful consideration. Overloading cellular processes that are required for natural miR generation is potentially toxic [22] and utilizing RNAi activators that employ alternative pathways may limit unintended harmful effects. This is particularly important for RNAi-mediated inhibition of viral replication where formation of multiplex cassettes may be required to minimize the risk of target mutation and escape
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Fig. 4. Assessment of silencing function of endogenous miR-16 after co-transfection with antiHBV expression cassettes. (A) Schematic illustration of dual luciferase reporter cassette used to determine miR-16 function. Seven miR-16 targets were inserted in tandem and downstream within the 30 UTR of the Renilla luciferase ORF. (B) Ratio of Renilla to Firefly luciferase activity was measured to assess the effect of antiHBV expression cassettes on endogenous miR-16-1 function. As a control for de-repression of miR-16-1 target silencing, a plasmid encoding a sponge cassette was included.
from silencing. Availability of a broad range of regulatory elements, including the panel of tRNA Pol III promoters, which may be used in antiviral expression cassettes will facilitate advancement of RNAi-based antiviral therapy. Previous reports have shown that tRNA promoters may be incorporated into expression cassettes that inhibit HIV gene expression [11]. Using a similar approach, our study focused on employing a tRNALys3 promoter to express antiHBV sequences, but it is likely that other tRNA promoters with Box A and Box B elements will have similar utility for engineering of gene silencing cassettes. tRNA Pol III promoters have several useful properties that may be applied to therapeutic gene silencing. These include their small size and potential utility in combinatorial RNAi through multimerization and generation of cassettes that simulate natural bicistronic tRNA miR genes [2]. An interesting recent observation has been that the silencing efficacy of tRNA promoters may be enhanced by incorporation of CMV enhancer elements [23]. This suggests that modification of tRNA Pol III promoters with other enhancer elements, so as to confer properties of tissue specificity or inducibility, may be feasible and contribute to the versatility of tRNA Pol III promoters. Acknowledgments The p901 plasmid was generously provided by Lisa Scherer and John Rossi. This work was supported by funding under the Sixth Research Framework Programme of the European Union, Project
RIGHT (LSHB-CT-2004-005276), the South African National Research Foundation (GUNs 68339 and 65495), Medical Research Council, Poliomyelitis Research Foundation and ESASTAP.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.122.
References [1] V.N. Kim, J. Han, M.C. Siomi, Biogenesis of small RNAs in animals, Nat. Rev. Mol. Cell Biol. 10 (2009) 126–139. [2] H.P. Bogerd, H.W. Karnowski, X. Cai, J. Shin, M. Pohlers, B.R. Cullen, A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral microRNAs, Mol. Cell 37 (2010) 135–142. [3] E. Berezikov, W.J. Chung, J. Willis, E. Cuppen, E.C. Lai, Mammalian mirtron genes, Mol. Cell 28 (2007) 328–336. [4] J.G. Ruby, C.H. Jan, D.P. Bartel, Intronic microRNA precursors that bypass Drosha processing, Nature 448 (2007) 83–86. [5] J.E. Babiarz, J.G. Ruby, Y. Wang, D.P. Bartel, R. Blelloch, Mouse ES cells express endogenous shRNAs, siRNAs, and other microprocessor-independent, Dicerdependent small RNAs, Genes Dev. 22 (2008) 2773–2785. [6] A. Ely, T. Naidoo, P. Arbuthnot, Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles, Nucleic Acids Res. 37 (2009) e91. [7] A. Ely, T. Naidoo, S. Mufamadi, C. Crowther, P. Arbuthnot, Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo, Mol. Ther. 16 (2008) 1105–1112.
646
V. Dyer et al. / Biochemical and Biophysical Research Communications 398 (2010) 640–646
[8] Y.P. Liu, J. Haasnoot, O. ter Brake, B. Berkhout, P. Konstantinova, Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron, Nucleic Acids Res. 36 (2008) 2811–2824. [9] L.A. Aagaard, J. Zhang, K.J. von Eije, H. Li, P. Saetrom, M. Amarzguioui, J.J. Rossi, Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs, Gene Ther. 15 (2008) 1536–1549. [10] H. Kawasaki, K. Taira, Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells, Nucleic Acids Res. 31 (2003) 700–707. [11] L.J. Scherer, R. Frank, J.J. Rossi, Optimization and characterization of tRNA– shRNA expression constructs, Nucleic Acids Res. 35 (2007) 2620–2628. [12] S. Carmona, A. Ely, C. Crowther, N. Moolla, F.H. Salazar, P.L. Marion, N. Ferry, M.S. Weinberg, P. Arbuthnot, Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs, Mol. Ther. 13 (2006) 411–421. [13] M.S. Weinberg, A. Ely, S. Barichievy, C. Crowther, S. Mufamadi, S. Carmona, P. Arbuthnot, Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA, Mol. Ther. 15 (2007) 534–541. [14] M. Nassal, The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly, J. Virol. 66 (1992) 4107–4116. [15] M. Passman, M. Weinberg, M. Kew, P. Arbuthnot, In situ demonstration of inhibitory effects of hammerhead ribozymes that are targeted to the hepatitis
[16]
[17] [18] [19] [20]
[21] [22]
[23]
Bx sequence in cultured cells, Biochem. Biophys. Res. Commun. 268 (2000) 728–733. S. Chattopadhyay, A. Ely, K. Bloom, M.S. Weinberg, P. Arbuthnot, Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles, Biochem. Biophys. Res. Commun. 389 (2009) 484–489. M.S. Ebert, J.R. Neilson, P.A. Sharp, MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells, Nat. Methods 4 (2007) 721–726. M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, Identification of novel genes coding for small expressed RNAs, Science 294 (2001) 853–858. P. Arbuthnot, M. Kew, Hepatitis B virus and hepatocellular carcinoma, Int. J. Exp. Pathol. 82 (2001) 77–100. A.P. McCaffrey, H. Nakai, K. Pandey, Z. Huang, F.H. Salazar, H. Xu, S.F. Wieland, P.L. Marion, M.A. Kay, Inhibition of hepatitis B virus in mice by RNA interference, Nat. Biotechnol. 21 (2003) 639–644. A. Shlomai, Y. Shaul, Inhibition of hepatitis B virus expression and replication by RNA interference, Hepatology 37 (2003) 764–770. D. Grimm, K.L. Streetz, C.L. Jopling, T.A. Storm, K. Pandey, C.R. Davis, P. Marion, F. Salazar, M.A. Kay, Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways, Nature 441 (2006) 537–541. M. Weiwei, X. Zhenhua, L. Feng, N. Hang, J. Yuyang, A significant increase of RNAi efficiency in human cells by the CMV enhancer with a tRNAlys promoter, J. Biomed. Biotechnol. (2009) 514287.