Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100
© Maczuga et al.; licensee BioMed Central Ltd. 2012
Received: 29 February 2012
Accepted: 3 July 2012
Published: 24 July 2012
Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.
Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.
Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.
RNA interference (RNAi) is an evolutionary conserved mechanism for regulating gene expression. It plays an important role in defense against viruses but also in development and in normal functioning of the cell [1, 2]. The natural RNAi mechanism functions by endogenous microRNA (miRNA) molecules, which are synthesized in cells as part of longer primary RNA transcripts (pri-miRNAs). Pri-miRNAs are cleaved by the nuclear Drosha-DGCR8 complex to produce miRNA precursors (pre-miRNAs) of 70 nucleotides (nt), which are then transported by Exportin 5 to the cytoplasm and processed by the RNAse III endonuclease family enzyme Dicer to produce a mature miRNA duplex of ~21,22 basepairs (bp). The guide strand of the miRNA enters a multiprotein RNA-induced silencing complex (RISC) where it is used for sequence-specific recognition of target messenger RNA (mRNA). RISC binding to complementary sequences on the target mRNA results in transcript degradation or translational repression .
By introducing molecules that constitute substrates for the endogenous RNAi pathway disease-related mRNA and thus protein translation can be inhibited. RNAi in cells can be induced in different ways. Generally this is achieved by delivery of 20–25 bp-long small interfering RNAs (siRNAs)  which serve as substrates for the RISC complex. Alternatively, siRNAs can be generated by expressing short hairpin RNA (shRNA)  or artificial microRNA (miRNA) structures . Both enter the endogenous RNAi pathway and are processed into mature siRNAs. The crucial difference between shRNAs and artificial miRNAs is in their secondary structure and processing in the RNAi pathway. shRNAs are normally expressed from polymerase III (pol III) promoters and directly generate a mature product which is exported and processed by Dicer, while miRNAs require an additional step of excision from the longer pre-miRNA sequence by the Drosha-DGCR8 complex. Moreover, miRNAs are expressed from polymerase II (pol II) promoters that allow for the use of tissue-specific or regulated expression systems.
To date, shRNA expression from pol III promoters is the most common way to induce RNAi in cells, which provides potent and stable target inhibition. Several pol III promoters are being used for expression of shRNAs, such as H1 or U6, and transcription initiation and termination sites together with the structural requirements for effective expression of the hairpins are well described [7, 8]. However, there are serious disadvantages of pol III expression systems which question their possible application as therapeutic agents. There are cases reporting severe toxicity after administration of high doses of vectors encoding shRNA [9, 10]. Toxicity was shown to be associated with oversaturation of the cellular RNAi machinery and changes in endogenous miRNA expression.
This toxicity problem may be circumvented by using weaker pol II promoter to express shRNAs or by embedding siRNA into artificial miRNA scaffolds. The CMV promoter has been the first pol II promoter shown to express active shRNA and initial requirements for this approach have been established: the shRNA has to be juxtaposed to the transcription start site (TSS) (within 6 nt) and followed by the minimal polyadenylation signal (pA) . When these two conditions were not met, the shRNA was found to be inactive. Additionally, to limit toxicity tissue-specific pol II promoters which restrict expression to the target tissue can be applied. This approach has been used to safely express shRNAs targeting JNK1, JNK2, and PI3 under the control of the prostate-specific antigen pol II promoter in androgen-responsive cancer cells . Furthermore, in vivo toxicity related to high shRNA expression levels from a pol III promoter was abolished when the shRNA was expressed from the liver-specific pol II hAAT promoter and U1 termination signal was used . Incorporation of a siRNA sequence in an artificial miRNA scaffold has been shown to abolish shRNA-induced neuronal cell death and to avoid disruption of the endogenous RNAi pathway [6, 14].
In the current study we focused on the structural requirements to determine the optimal configuration for pol II shRNA expression and compared it to the pol II expression of artificial miRNA. To date, no detailed studies on the hairpin positions, transcription termination signal (TTS), and shRNA stem length requirements for efficient pol II shRNA expression have been performed. Varying positions of the hairpin relative to the TSS +5 or +6, stem length of 20, 21, 25, 29 bp and different TTS (pA, U1 and T5) have been tested for a shRNA targeting luciferase (shLuc) and expressed from the pol II CMV promoter. shLuc at +6 location relative to TSS, with stem length of 20 nt with either pA or U1 termination signal was found to yield optimal results. However, the average luciferase inhibition of 40% was still lower than the control pol III H1-shRNA, which inhibited luciferase up to 90%. Additionally, two shRNAs targeting ApoB (shApoB1 and shApoB2) were found inactive when expressed from CMV using the same configuration. In contrast, when the three shRNAs were incorporated in an artificial miRNA scaffold, two were equally active to their respective H1-shRNA controls while expressing much less siRNA molecules. This indicates that using artificial miRNA scaffolds has a greater therapeutic potential than direct expression of shRNAs from pol II promoters.
Design and knockdown efficacy of CMV-shLuc with different transcription start and transcription termination signals
Design and knockdown efficacy of CMV-shLuc with different hairpin stem length
Design and knockdown efficacy of CMV-shRNA and artificial miRNA targeting luciferase or Apolipoprotein B100 (ApoB)
Quantification of siRNA molecules processed from the different shRNA and miRNA scaffolds
Endogenous ApoB mRNA knockdown
There are few examples of successful shRNA expression using pol II promoters. Although several requirements for pol II shRNA expression have been established, optimization and direct assessment of different hairpin stem lengths, TSS and TTS have not been performed. In the current study we evaluated the relevance of these factors by positioning shRNAs targeting luciferase or ApoB at +5 or +6 relative to the CMV TSS and added the T5, pA or U1 TTS at the 3’ end of the hairpins. Additionally, the same siRNA sequences were incorporated in artificial miRNA scaffolds and direct evaluation between the silencing efficacy and processing of pol II-expressed shRNA and miRNA was performed.
The nucleotide distance between the promoter, the shRNA structure and the TTS determines the length and sequence composition of the 5’ and 3’ overhangs of the expressed hairpin. Both ends act as reference points for recognition and proper processing by Dicer . Initial rules published by Xia et al. include shRNA location within 6 nt from the TSS and pA TTS . Later shRNA containing T5 and U1 TTS where also found to be active when substituted for pA [13, 18]. These settings were used in our study to express shRNA from the CMV promoter. The shLuc, shApoB1 and shApoB2 sequences have been previously validated as potent inhibitors of their targets when expressed from the pol III H1 promoter [15, 21]. Surprisingly, all CMV-shLuc constructs induced only a mild target inhibition when the hairpin was placed at +6 TSS and T5 or pA TTS were used. In contrast to previously published data, the use of U1 TTS did not improve silencing efficacy. By shifting the hairpin from +6 to +5 from TSS, thus minimizing the 5’ overhang, no significant improvement was achieved and the effect was not dependent of TTS used. Additionally, the CMV-shApoB1 and CMV-shApoB2 hairpins were ineffective when the initial settings of +6 TSS and pA TTS were used, indicating that those settings cannot be used as a general rule for pol II shRNA expression.
The length of the shRNA hairpin stem has been shown to play an important role in silencing efficacy as it can lead to differential processing into multiple siRNAs [8, 22, 23]. In the current study we optimized the pol II shRNA expression by testing stem lengths of 20, 21, 25, and 29 bp. Slight increase in silencing efficacy was observed only when extending hairpin stem from 20 to 21 bp but not when the stem length was further extended to 25 or 29 bp. Mcintyre et al. performed a similar study by extending the shRNA stem length from 16 to 41 bp and looked at the core placement of the shRNA . While the processing of hairpins was clearly dependent on the stem length, the activity was primarily dependent on the sequence of processed products. Consistent with their data, our results suggest that there is no strict correlation between the increase in stem length and better silencing activity of the shRNAs. Here, we focused only on pol II expression of shRNA but an alternative approach would be to express long hairpin RNA (lhRNA) where the stem length is extended up to 300 bp [24–26]. Using lhRNA allows generating multiple siRNA from a single transcript, which can be used for viral infections or cancer, where multiple sequences have to be targeted. To date, expression of active lhRNA from pol II promoter has not been successful due to inefficient processing of the hairpins by the RNAi machinery, and detailed studies on TSS, TTS and hairpin location, similar to those for shRNA are lacking.
An alternative to optimizing pol II expression of shRNA is to use pre-miRNA scaffolds and replace the mature miRNA sequence with siRNA targeting a gene of interest. Cellular miRNAs are naturally expressed from pol II promoters [1, 27]. Therefore, when shLuc, shApoB1 and shApoB2 were expressed from the pri-mir-155 scaffold the knockdown efficacy was significantly improved. Importantly, artificial miRNA have been found to be less toxic in vitro and in vivo compared to shRNA as they do not disrupt the endogenous RNAi pathway. McBride et al. used this approach to target huntingtin mRNA and to avoid neurotoxicity caused by overexpression of shRNA . Similarly, incorporation of a siRNA against spinocerebellar ataxia 1 protein into an artificial miRNA abolished neuronal cell death observed with a shRNA harboring the same siRNA sequence . In a previous study we have provided evidence for the efficacy of liver-specific expression of artificial miRNA targeting ApoB in vivo and demonstrated its advantages over the pol III-expressed shRNA (Maczuga et al., manuscript submitted). In conclusion, we and others have shown that pol II expression of artificial miRNA scaffolds is a more robust and promising approach than pol II shRNA expression, probably due to the specific structural characteristics of the miRNA.
An additional advantage of the use of artificial miRNAs is that several precursors can be expressed as clusters from longer transcripts, allowing simultaneous targeting of multiple genes . This feature of the miRNA is very important when mutation-prone viruses, such as HIV-1 or HCV, are targeted or when several disease-related genes need to be simultaneously silenced. Moreover, miRNAs can be linked to the 3’ untranslated region of a therapeutic gene, which is an additional advantage for therapeutic applications since it allows co-delivery of a codon-optimized gene together with a miRNA targeting the disease-causing version of the same gene. Such combinational therapy has been shown for alpha1-antitrypsin (AAT) deficiency, in which a hairpin RNA targeting mutated AAT transcript was delivered together with codon-optimized AAT gene .
Quantification of the siRNA molecules expressed from the H1-shRNA, CMV-shRNA or CMV-miRNA constructs was highly specific and revealed differences in the amount of processed siRNA molecules per cell. As expected, highest siRNA amounts were detected when the shRNAs were expressed from the strong H1 promoter. However, when the same shRNAs were expressed from the CMV promoter, 12- to 125-fold less siRNA molecules were detected indicating that either the shRNA was not efficiently transcribed from the CMV promoter or that there was impaired siRNA processing by the RNAi machinery. Surprisingly, the amount of processed siRNA from the CMV-miRNA constructs did not correlate with efficacy. For example, CMV-miLuc yielded 1591 siLuc molecules per cell and was completely ineffective in target knockdown while CMV-miApoB2 yielded only 56 siApoB2 molecules per cell and was highly efficient. A possible explanation for this discrepancy is that the small RNA TaqMan assay detects only one variant of the guide strand and although identical predicted siRNA sequences were incorporated in the CMV-shRNA and CMV-miRNA scaffolds, they can still be processed differently. Indeed, our newest data based on Next Generation Sequencing (NGS) of small RNAs from cells transfected with H1-shApoB2 and CMV-miApoB2 indicates differential processing and different mature sequences from the two scaffolds (Maczuga et al., manuscript submitted). The siRNAs originating from the H1-shRNA were more heterogeneous in cleavage sites and length compared to the products originating from the CMV-miRNA scaffold supporting the notion that Dicer cleavage is less precise than the combination of Drosha and Dicer. Unfortunately, NGS data from pol II-expressed shRNA, which would allow verifying the processed siRNA variants and their abundance, are still not available.
In summary, our results demonstrate that it is currently difficult to draw rules for efficient pol II shRNA expression and processing. Optimization of the shRNA stem length, TSS and TTS did not result in better silencing efficacy for any of the CMV-expressed shRNAs. On the other hand, when identical predicted sequences were incorporated in miRNA scaffolds, two out of three sequences resulted in potent inhibition of luciferase reporters and endogenous mRNA. Our data question the application of the previously published rules for pol II shRNA expression as general standards. The rules for design of potent shRNA expressed from pol II promoters are still far from clear and they can be used rather as reference points. Still detailed empirical optimization of hairpin location seems to be required for every target and pol II promoter used. We consider that embedding siRNA in artificial miRNA scaffold and expression from pol II promoter is a better and approach than direct pol II shRNA expression due to the intrinsic characteristics of the miRNA structure rendering it a better substrate for the RNAi machinery.
H1-shRNAs were made by annealing complementary oligonucleotides and ligating them into the BglII and XhoI sites of the pSuper vector (OligoEngine, Seattle, WA, USA). The vector contains a stretch of five thymidines (T5), which acts as the transcription termination signal. Panel of shRNAs were designed to act as positive controls: one shRNAs was designed that target Firefly luciferase (H1-shLuc), two shRNAs were made that target different regions of human ApoB gene (H1-shApoB1 and H1-shApoB2). Additionally two negative controls shRNA were deigned: one that do not target any gene in human and mouse genome (H1-shScr) and one that target the GFP (H1-shGFP). All the sequences of oligonucleotides used in this study are listed in Additional file 1: Table S1.
CMV-shRNA with different termination signals were designed as followed. First different TTS (T5, pA or U1) were annealed from complementary oligonucleotides and ligated into the AccI and SphI sites of pVD23 which includes the CMV promoter. Next, plasmids were digested with SacI and NheI and complementary oligonucleotides containing shRNA targeting GFP (shGFP) or Luciferase (shLuc) were inserted. shLuc oligonucleotides were designed to allow the hairpin transcription to start at +5 or +6 nucleotides after the TSS, which was considered as +1. The final constructs were CMV + 5shLuc-T5, CMV + 6shLuc-T5, CMV + 5shLuc-pA and CMV + 6shLuc-pA. For U1 termination signal only CMV + 6shLuc-U1 was created. As negative controls CMV-shGFP-T5, CMV-shGFP-pA and CMV-shGFP-U1 were made. GFP oligonucleotides were designed that hairpin started at +6 from the TSS.
CMV-shRNAs with different stem length of the hairpin were designed as followed. CMV-shLuc20, CMV-shLuc21, CMV-shLuc25 or CMV-shLuc29 were made by annealing complementary oligonucleotides that create the stem-loop structure of 20, 21, 25 and 29 bp respectively and ligating them into the BamHI and XhoI sites of the pSilencer 4.1 CMV vector (Life Technologies, Grand Island, NY). Constructs contained either the pA or the U1 termination signal. To create the CMV-shApoB1 and CMV-siApoB2, siRNA sequences from shApoB1 and shApoB2 were cloned by annealing complementary oligonucleotides and ligating them into the BamHI and XhoI sites of the pSilencer 4.1 CMV vector with pA termination signal. As the negative control, pSilencer 4.1 CMV control (Life Technologies, Grand Island, NY) plasmid was used, named CMV-shScr.
To create the artificial miRNA expressing vectors, siRNA sequences from shLuc, shApoB1 and shApoB2 were cloned into the pri-mir-155 backbone of pcDNA6.2-GW/EmGFP-miR (Life Technologies, Grand Island, NY) by annealing complementary oligonucleotides and ligation into BamHI- and XhoI-digested pcDNA6.2 plasmid and named respectively CMV-miLuc, CMV-miApoB1and CMV-miApoB2. As the negative control pcDNA6.2-GW/EmGFP-miR-neg control (Life Technologies, Grand Island, NY) was used, named CMV-miScr. The Mfold program was used to determine the secondary structure of all RNA transcripts .
Reporter constructs that were used for luciferase knockdown are Firefly luciferase pGL4 (Promega, Madison, WI) and pRL Renilla (Promega, Madison, WI) under control of the CMV promoter. Luc-ApoB1 reporter, containing in its 3’ UTR a fragment of 239 nucleotides of human ApoB (NM_000384, 9362–9600) sequence was made, by cloning of the ApoB sequence, that was PCR amplified with primers pr565f and pr566r in the XhoI and NheI sites of pGL4 plasmid. For The Luc-ApoB2 containing in the 3’UTR of siCheck2 vector (Promega, Madison, WI) 1851 nucleotides of the last exon from the human ApoB has been described previously.
Cell culture and transfections
The human embryonic kidney (HEK)293 T and mouse hepatoma Hepa1-6 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY) containing 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin, at 37°C and 5% CO2. For luciferase assays, endogenous ApoB knockdown assays, and siRNA expression analysis, cells were seeded in 96- or 24-well plates at a density of 3*104 or 1.2*105 cells per well, respectively, in DMEM one day prior transfection. Transfections were performed with Lipofectamine 2000 reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions.
Cells were co-transfected with 100 ng shRNA or miRNA expressing plasmid and respectively 2,5 ng Firefly luciferase or Luc-ApoB1 and 0,5 ng Renilla or 50 ng Luc-ApoB2 reporter. Transfected cells were assayed at 48 hr post-transfection in 20 μl 1x passive lysis buffer (Promega, Madison, WI) Firefly and Renilla were measured luciferase activities with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). For luciferase knockdown calculations, H1-shLuc was used as a positive control. CMV-shScr, CMV-shGFP or CMV-miScr served as negative controls and were set at 100%. For luciferase- and ApoB1- and ApoB1 targeting constructs Firefly luciferase expression was normalized to Renilla luciferase expression. For ApoB2-targeting constructs Renilla luciferase expression was normalized to Firefly luciferase expression since the siCheck2 plasmid was used.
RNA isolation and quantitative real-time RT-PCR
To determine endogenous ApoB mRNA knockdown by shApoB and miApoB constructs in vitro, Hepa1-6 cells were transfected with 1 μg shApoB1, shApoB2, miApoB1 or miApoB2 and total RNA was isolated from cells 48 hr post-transfection using the Nucleospin kit (Clontech, Mountain View, CA). Genomic DNA (gDNA) was removed by DNase treatment using TURBO DNase (Ambion, Austin, TX). First strand cDNA was reverse transcribed using random hexamer primers with the Dynamo kit (Finnzymes, Espoo, Finland). Real time PCR amplification was performed with ApoB- and beta actin-specific primers (Additional file 1: Table S1). PCR reaction conditions were: 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The assays were performed on ABI 7000 or ABI 7500 (Applied Biosystems, Foster City, CA). ApoB expression levels were normalized to beta actin as an internal control, and the relative gene expression 2-ΔΔCt method of Livak and Schmittgen was used for analysis of PCR data .
siRNA detection by small RNA TaqMan assay
RT reactions for siLuc, siApoB1 and siApoB2 expression were performed with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) using 10 ng RNA isolated from cells transfected with shRNA or miRNA expression plasmids and 3 μl custom-made specific RT-stem-loop primers (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The TaqMan assay was done in 20 μl using 1,33 μl cDNA, 1-μl custom-made siRNA-specific primer with FAM-labeled fluorogenic probe (Applied Biosystems) and 10 μl TaqMan 2× Universal PCR Master Mix (Applied Biosystems). For determining the assay specificity, 10 ng of the indicated oligo or expression plasmid was added to the TaqMan reaction. Amplification of the beta actin gene was used as a RNA quality and loading control. siRNA copy number per cell was calculated based on the amplification plot of a dilution series of synthetic siRNA standards (IDT, Coralville, IA). Tenfold dilutions (100 pg – 1 fg) of the guide strand of the synthetic siRNA oligo were used to create a standard line. The copy number of each dilution was calculated according to the formula 1 mol = 6.02 × 1023 molecules. The trend line value of the standard line was used to calculate the siRNA copy number per cell, assuming 15 pg RNA per cell .
This work was carried out and funded by uniQure biopharma b.v.
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