Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy.
AAV; liver; shRNA; miRNA; ApoB; familial hypercholesterolemia
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.
Schistosome; Luciferase; RNA interference; U6 gene; Vector-based RNAi; Promoter
RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.
RNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.
Our modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.
Aim: To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE.
Methods: First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models.
Results: Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-β1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated.
Conclusion: These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications.
RNA interference; microRNA(miRNA); heparanase; metastasis; safety
RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.
Reverse genetics studies gene functions by altering a gene and observing the consequences. A powerful method of reverse genetics in mammals is gene knockout by homologous recombination, which mutates a gene to prevent its functional expression. Using this method, investigators have revealed the functions of many genes. However, this method is relatively complex, time-consuming, and costly. In addition, this method is limited to studies in mice because it is not well established in other mammalian species. The authors of this study tested an alternative method using RNA interference (RNAi), which is a widely conserved mechanism in eukaryotes and can mediate gene-specific silencing. These investigators used RNA polymerase II (Pol II) to express a short hairpin RNA (shRNA) that triggers destruction of the mRNA-encoding Mn superoxide dismutase (SOD2) in transgenic mice. These mice exhibit phenotypes that were typical in Sod2 knockout mice, including elevated levels of oxidative stress in various tissues, fat deposition in liver and muscles, dilated cardiomyopathy, and premature death. These results open the door of RNAi to a wide array of well-established Pol II transgenic strategies and offer a technically simpler, cheaper, and quicker alternative to gene knockout for reverse genetics in mice and other mammalian species.
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.
The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45–82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.
RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.
We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.
In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.
The degradation of a selected mRNA species by RNA interference requires a high degree of homology between the short interfering or short hairpin RNA (si or shRNA) and its target. Recent reports have demonstrated that the number and location of nucleotide mismatches affect the activity of si/shRNA. Here, we systematically examined the effect of single nucleotide mutations in all 21 positions of an effective shRNA that targets the gag gene of HIV-1. We found that all mutant shRNAs exerted RNAi activity but were less effective in gene silencing compared to the wild-type gag shRNA. The most pronounced reduction in function was observed with mutations in the central and 5′ regions of the shRNA. Our results demonstrate that optimal gene silencing requires perfect homology between shRNA and the chosen target, but that a variable degree of silencing occurs, depending upon the precise location of nucleotide mismatches.
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.
RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.
Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNALys3-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3′ tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.
Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 106 PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 × 109 PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.
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.
RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.
To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.
The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
Intradermal administration of DNA vaccines encoding luciferase represents a convenient method to assess gene expression in vivo. Gene silencing by intradermal gene gun administration of DNA encoding short hairpin RNA (shRNA) may represent an effective technique for the specific knockdown of gene expression in vivo. In the current study, we characterized luciferase gene expression over time in vivo by noninvasive bioluminescence imaging. Furthermore, we characterized in vivo luciferase gene silencing with DNA encoding shRNA targeting luciferase. We also characterized human papillomavirus type 16 (HPV-16) E7-specific CD8+ T cell immune responses in mice immunized with E7 DNA and DNA encoding shRNA targeting Fas ligand (FasL), a key proapoptotic signaling protein. Our results indicated that coadministration of DNA encoding shRNA targeting luciferase significantly reduced luciferase expression in mice intradermally administered luciferase DNA. Furthermore, we observed that mice vaccinated with E7-expressing DNA coadministered with DNA encoding shRNA targeting FasL generated significantly enhanced E7-specific CD8+ cytotoxic T cell responses as well as potent therapeutic antitumor effects against E7-expressing tumors. Thus, intradermal administration of DNA encoding shRNA represents a plausible approach to silence genes in vivo and a potentially useful tool to enhance DNA vaccine potency.
Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.
Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.
By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.
The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.
Progress in constructing biological networks will rely on the development of more advanced components that can be predictably modified to yield optimal system performance. We have engineered an RNA-based platform, which we call an shRNA switch, that provides for integrated ligand control of RNA interference (RNAi) by modular coupling of an aptamer, competing strand, and small hairpin (sh)RNA stem into a single component that links ligand concentration and target gene expression levels. A combined experimental and mathematical modelling approach identified multiple tuning strategies and moves towards a predictable framework for the forward design of shRNA switches. The utility of our platform is highlighted by the demonstration of fine-tuning, multi-input control, and model-guided design of shRNA switches with an optimized dynamic range. Thus, shRNA switches can serve as an advanced component for the construction of complex biological systems and offer a controlled means of activating RNAi in disease therapeutics.
aptamer; mathematical modelling; RNA; shRNA; synthetic biology
We have developed a stable RNA interference (RNAi) delivery system that is based on the Frog Prince transposable element. This plasmid-based vector system combines the gene silencing capabilities of H1 polymerase III promoter-driven short hairpin RNAs (shRNA) with the advantages of stable and efficient genomic integration of the shRNA cassette mediated by transposition. We show that the Frog Prince-based shRNA expressing system can efficiently knock down the expression of both exogenous as well as endogenous genes in human cells. Furthermore, we use the Frog Prince-based system to study the effect of knockdown of the DNA repair factor Ku70 on transposition of the Sleeping Beauty transposon. Transposon-mediated genomic integration ensures that the shRNA expression cassette and a selectable marker gene within the transposon remain intact and physically linked. We demonstrate that a major advantage of our vector system over plasmid-based shRNA delivery is both its enhanced frequency of intact genomic integration as well as higher target suppression in transgenic human cells. Due to its simplicity and effectiveness, transposon-based RNAi is an emerging tool to facilitate analysis of gene function through the establishment of stable loss-of-function cell lines.
RNA interference; short hairpin RNA; Frog Prince; Sleeping Beauty; nonviral gene transfer; stable gene knockdown; transposon-based gene delivery
In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose. However, induction of long-term shRNA expression can also cause toxicities by inducing off target effects and interference with the endogenous micro RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities. In this review we describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections.
RNAi; shRNA; miRNA; viral infection; lentivirus
The RNA polymerase III (pol III) type III promoters U6 and 7SK are routinely used to express short hairpin RNA (shRNA) molecules from a DNA construct. In this study, we identified, characterised and compared the porcine 7SK promoter in porcine (homologous) and non-porcine (heterologous) derived cell lines. The porcine 7SK small nuclear RNA (snRNA) was identified by alignment with known sequences and further characterisation of the upstream regions determined the presence of typical RNA pol III sequence motifs. The porcine 7SK (po7SK) promoter was cloned and a one-step PCR strategy used to construct shRNA expression cassettes. The 7SK promoter activity was quantified by knockdown of the exogenous reporter gene encoding the enhanced green fluorescent protein (EGFP). Results indicated the po7SK promoter was functional in both homologous and heterologous cells lines. The identification and characterisation of the porcine RNA pol III promoter will contribute to the area of RNAi delivery and further develop our understanding of RNA promoter structure and function.
RNAi; 7SK; U6; pol III; shRNA
RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox.