Interfering ribonucleic acids that suppress expression of multiple unrelated genes
© Passioura et al; licensee BioMed Central Ltd. 2009
Received: 19 November 2008
Accepted: 16 June 2009
Published: 16 June 2009
Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy.
Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated.
Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach.
The different triggers eliciting RNAi all ultimately lead to the formation of short (~21 nucleotide) RNA duplexes termed short interfering RNAs (siRNAs). Complete complementarity between the guide strand and the target mRNA leads to catalytic cleavage of the mRNA and suppresses gene expression . Endogenous microRNAs (miRNAs) are also small duplex RNAs with diverse and critical roles in gene regulation . miRNAs share many of the features of siRNAs including the loading of the guide strand into a RNA induced silencing complex (RISC) . In contrast to siRNAs, mammalian miRNAs do not generally exhibit high complementarity to their cognate target sites. Binding of miRNAs to their target sites may induce target degradation or may prevent translation and reduce gene expression at the protein level . In mammals, miRNAs are thought to bind to partially complementary sites predominantly located in the 3' untranslated regions (UTRs) of target mRNAs [2, 4, 5], thereby enabling the coordinate regulation of genes containing such sites.
Whilst the factors affecting siRNA activity have been extensively studied [3, 6–8], the parameters affecting miRNA-mediated translational suppression have not yet been definitively elucidated. Binding of the 5' end of the guide strand to the target mRNA appears to be critical, with an almost absolute requirement for complementarity at the so-called "seed site" from positions 2–7 (measuring from the 5' end of the guide strand) [9–11]. miRNA target sites appear to be almost exclusively located in the 3'UTRs of target genes [11, 12], and miRNA target sites may be functionally restricted to 3' UTRs, since binding of miRNAs to other sites in the transcript does not induce translational suppression.
The present study demonstrates proof of concept for the design of artificial short RNAs with at least partial complementarity to multiple unrelated transcripts, and which suppress the expression of the corresponding unrelated genes. The interfering RNAs described herein were designed to target expression of VEGF-A and ICAM-1, two genes involved in ocular neovascular disease . We found that for suppression of VEGF-A, the length of complementarity to the seed region, as well as the total complementarity of the guide strand to the target were important determinants of activity. This relationship was not observed for ICAM-1, however this discrepancy appeared to result from specific sequence motifs in those guide strands with high ICAM-1 complementarity. Thus, the length of seed complementarity, and overall complementarity between the guide strand and the target should be considered in the design of multi-target interfering RNAs.
Transcript sequences corresponding to the 3' UTRs of VEGF-A and ICAM-1 (ensembl IDs ENST00000356655 and ENST00000264832, respectively) were used to search for a suitable seed of at least 6 contiguous bases present in both genes. A pool of all possible seeds of 6 bases or greater was generated using the specified length as a window and advancing the window in a stepwise fashion 1 base at a time. Low complexity seeds were eliminated and the pool was further restricted to those for which at least 3 contiguous bases were predicted to bind to an unpaired region in at least 50% of optimal and suboptimal (within -1 kcal/mol of optimal) folded structures (as determined using the Vienna RNA package ).
Two different seeds were selected for experimental testing: one of 12 bases in length and one of 7 bases that was within a genetic context that favoured the design of "consensus target sequences" (comprising the seed sequence and a consensus of the sequence adjacent to the seed sequence from the desired targets, in this instance, VEGF-A and ICAM-1). Multiple consensus target sequences were generated for both seeds by first aligning the target sequences (VEGF-A and ICAM-1) relative to their shared seed sequences. Then, extensions 5' to the seed sequences were proposed to a length of 21 nucleotides. The exact complements of these 21 nucleotide consensus target sequences were assessed in silico for hybridisation to the target sequences, and one sequence for each seed was selected for experimental testing. "Passenger" strands were designed to be complementary to the selected "guide" strands over a duplex length of 19 nt with 3' overhangs of 2 nucleotides (UU added in the case of the passenger strand).
Annealed RNA duplexes or single-stranded RNA oligonucleotides were purchased from Sigma-Proligo. Where required, 100 μM oligonucleotides were annealed in 50 μM Tris, 100 mM NaCl by heating to 90°C and cooling to 4°C over 3–4 hours. Control siRNA sequences are shown in Additional file 1.
The use of RNA duplexes is potentially confounded by off-target inflammatory cellular responses . To confirm the specificity of action of CODEMIR-1 we examined the expression of IFNβ and STAT1. After transfection with CODEMIR-1, no evidence of up-regulation of either of these genes was observed (Additional file 2). In a recent separate study to be reported elsewhere, we studied the activation of TLR7/8 by 207 siRNA sequences when transfected with DOTAP into fresh human PBMCs at concentrations up to 100 nM. CODEMIR-1 belonged to the least active subset of sequences (with IC50 > 100 nM), confirming low propensity of this sequence for activation of RNA-sensing innate receptors.
The present study demonstrates proof of concept for the approach of using short interfering RNAs with at least partial complementarity to two target transcripts for suppression of the expression of unrelated genes. Although we have only presented data for two seed sequences in two genes here, we have successfully used the same approach to target unrelated genes implicated in diverse disease states including oncology, virology and inflammation . Thus, this approach can be used as a general technique for the suppression of multiple genes using a single interfering RNA.
Current pharmaceutical research is dominated by a reductionist "one-disease one-target" paradigm. However, the complex nature of many diseases has increasingly led to the realisation that activity against multiple targets may be required for effective treatment . Practically, this can be achieved through the use of multiple agents, and a number of combination drugs have recently become available for the treatment of heart disease and HIV infection, amongst other indications. However, the combination of multiple agents can lead to unforseen interactions . Alternatively, agents with activity against multiple targets can be developed . Indeed, many successful drugs in different areas of medicine (eg clozapine, imatinib) are active precisely because of their promiscuity of action [21, 22]. To date, the RNAi-based drugs that have been investigated have been designed for single specific targets, with efforts taken to reduce non-specific effects [23, 24]. However, in some instances, the processes they target are distinctly polygenic (eg cholesterol metabolism and angiogenesis), and the targeting of multiple genes seems likely to be therapeutically beneficial. Whilst the targeting of multiple genes could be achieved through the use of a mixture of active siRNAs, we believe that using a single active has a number of possible advantages. First, having a single active reduces the complexity of clinical and product development. Secondly, mixtures of siRNA can have disappointing effects because of competition for the RISC machinery . Thirdly, a single active may have reduced off-target effects relative to a pool of actives, since each siRNA has a unique pattern of off-target effects and a mixture may thus increase the magnitude and/or scope of off-target effects.
Our finding that the length of seed complementarity affects CODEMIR activity independent of overall complementarity is surprising given the current understanding of microRNA-target interactions [4, 9, 11]. Mismatches to the target in the central positions of the CODEMIR-1 guide strand decreased VEGF-A suppression at the protein level to 50–60%, and largely abrogated suppression at the RNA level. This suggests that complementarity to the target at the 5'-end and in the centre of the guide strand is sufficient to induce target cleavage, and that CODEMIRs with central mismatches to the targets may act primarily through translational repression. As such, CODEMIRs with longer 5' regions of contiguous complementarity may exhibit higher efficacy. However, suppression of multiple genes related to the same phenotype is likely to have significant advantages over targeting a single gene, and thus CODEMIRs with central mismatches will still be of significant utility.
The present study validates the approach of using a short dsRNA molecule specifically designed with a single guide strand to suppress the expression of multiple unrelated genes implicated in a particular medical condition. We have shown that synthetic duplex RNAs with at least partial complementarity to multiple transcripts are capable of specific suppression of multiple target genes. Given the multi-genic nature of many disease states, such multi-targeting interfering RNAs may offer significant therapeutic benefits relative to single target siRNAs. Our findings also have relevance to the biology of miRNA-target interactions, particularly with respect to the effect of unpaired bases on miRNA-mediated suppression of translation.
Cell culture and transfection
ARPE-19 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal bovine serum and 10 mM glutamine (Gibco). Duplex RNA transfection was performed using Lipofectamine 2000 (Invitrogen), at a ratio of 260 ng siRNA per 1 μL Lipofectamine, according to the manufacturer's instructions. Cells were transfected 24 hours after seeding at a density of 1.25 – 104 cells per cm2 in 96, 48 or 24-well plates. RNA duplex and plasmid co-transfection was performed with 1 μg reporter plasmid (AmCyan based – see below), 1 μg control plasmid (pAsRed-C1 – Clontech) and 40 pmol RNA duplex together complexed with 10 μg Lipofectamine 2000 and applied to cells in a 12 well plate.
ELISA and FACS analysis of gene expression
VEGF-A concentrations in cell supernatants were assayed 48 hours after transfection and 24 hours after stimulation with 130 μM Deferoxamine (Sigma) using a commercially available ELISA kit (R&D Systems) according to the manufacturer's instructions. Cell surface ICAM-1 was assayed by flow cytometry. Cells seeded into 12 well plates were transfected with RNA duplexes 24 hours after seeding. ICAM-1 expression was assayed 48 hours after transfection and 24 hours after stimulation with 1 ng/mL recombinant human Interleukin-1β (R&D systems): cells were trypsinized, stained with 0.5 μg anti-human ICAM-1 mouse IgG1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS, stained with 0.2 μg Phycoerythrin-labelled anti-mouse IgG1 antibody (Becton Dickinson) at 4°C for 20 minutes, washed with PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson). IFNβ production was assayed using a commercially available ELISA (InVitrogen) according to the manufacturer's instructions. For detection of fluorescent reporter expression, mean fluorescence in the reporter channel (FL-1) was normalised to mean fluorescence in the control channel (FL-2). Statistical analysis was performed using Prism software (GraphPad Software Inc.)
For semi-quantitative RT-PCR, total cellular RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was performed on 1 μg total RNA using a commercially available kit (First-Strand cDNA Synthesis Kit, Marligen Biosciences). PCR of GAPDH was used to standardise the amounts of starting cDNA. PCR was performed in 1× PCR buffer II (Applied Biosystems) supplemented with 3.75 mM MgCl2, 250 μM each dNTP, 250 nM each primer and 0.125 U/μL AmpliTaq gold (Applied Biosystems). VEGF-A was amplified by 30 cycles of 95°C – 30 seconds, 60°C – 30 seconds and 72°C – 30 seconds, using the primers (5' to 3'): TTC TTG CTG CTA AAT CAC CGA and GAA CAT TCC CCT CCC AAC TCA. GAPDH was amplified by 18 cycles of 95°C – 30 seconds, 65°C – 30 seconds and 72°C – 30 seconds, using the primers (5' to 3'): CTG CTT CAC CAC CTT CTT GAT GTC ATC ATA and GAC CCC TTC ATT GAC CTC AAC TAC ATG GT.
VEGF-A, ICAM-1, IFN β, STAT1 and GAPDH RNA were quantified using a Quantigene® branched DNA assay (Panomics), according to the manufacturer's instructions.
Fluorescent reporter vectors were constructed by cloning target sites into the 3'UTR of the AmCyan1 fluorescent protein gene in the pAmCyan1-C1 vector (Clontech). A stop codon was inserted by cloning the duplex oligodeoxynucleotide pair GAT CTC TCG AGT GAT AGG and AAT TCC TAT CAC TCG AGA into the BglII and EcoRI sites of pAmCyan1-C1. Specific CODEMIR-1 target site reporters were generated by cloning the duplex oligonucleotide pairs AAT TTC CTG TAG ACA CAC CCA CCC ACA TAC and GAT CGT ATG TGG GTG GGT GTG TCT ACA GGA (VEGF-A), and AAT TTG TTA GCC ACC TCC CCA CCC ACA TAC and GAT CGT ATG TGG GTG GGG AGG TGG CTA ACA (ICAM-1) into the EcoRI and BamHI sites of the stop codon-containing pAmCyan1-C1 vector. The full-length VEGF-A 3'UTR (including stop codon) was cloned from ARPE-19 cells by RT-PCR using the primers GGG CTC GAG TGA GCC GGG CAG GAG G (Forward) and GGG GTC GAC TAC GGA ATA TCT CGA AAA ACT (Reverse) and cloned into the XhoI and SalI sites of the pAmCyan1-C1.
Western blot analysis
Total protein lysates were obtained by lysing cells in RIPA buffer (150 mM NaCl, 0.1% sodium dodecyl sulphate, 1% nonidet P-40, 0.5% sodium deoxycholate, 50 mM Tris-Cl, pH 8). Protein concentrations were determined by the Lowry protocol using the Bio-Rad D C Protein Assay. Total proteins (10 μg) were separated by electrophoresis on NuPage 4–12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes. Membranes were blocked with 3% BSA in TBST (10 mM Tris pH 8, 30 mM NaCl, 0.05% Tween) for 20 min at room temperature. After rinsing with TBST twice, anti-STAT1 mouse IgG1 (1:400; Santa Cruz Biotechnology) and anti-β-actin mouse IgG1 (1:2000; Sigma-Aldrich) in TBST-MLK (TBST containing 5% dried skim milk) were added and incubated for 1 hour at room temperature. After 3 – 5 min washes in TBST, membranes were incubated with horseradish peroxidase conjugated anti-mouse Ig (1:2000 in TBS-MLK; Dako Cytomation) for 45 min at room temperature. The membrane was washed in TBST (3 – 5 min) and analysed by chemiluminescence using ECL Western blotting detection (Amersham).
The study was funded and performed with the support of Johnson and Johnson Research Pty Limited.
- Schwarz DS, Ding H, Kennington L, Moore JT, Schelter J, Burchard J, Linsley PS, Aronin N, Xu Z, Zamore PD: Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide. PLoS Genet. 2006, 2 (9):Google Scholar
- Kloosterman WP, Plasterk RH: The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006, 11 (4): 441-450. 10.1016/j.devcel.2006.09.009.View ArticleGoogle Scholar
- Khvorova A, Reynolds A, Jayasena SD: Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003, 115 (2): 209-216. 10.1016/S0092-8674(03)00801-8.View ArticleGoogle Scholar
- Engels BM, Hutvagner G: Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene. 2006, 25 (46): 6163-6169. 10.1038/sj.onc.1209909.View ArticleGoogle Scholar
- Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS: Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol. 2005, 15 (3): 331-341. 10.1016/j.sbi.2005.05.006.View ArticleGoogle Scholar
- Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD: Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003, 115 (2): 199-208. 10.1016/S0092-8674(03)00759-1.View ArticleGoogle Scholar
- Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T: Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002, 110 (5): 563-574. 10.1016/S0092-8674(02)00908-X.View ArticleGoogle Scholar
- Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A: Rational siRNA design for RNA interference. Nat Biotechnol. 2004, 22 (3): 326-330. 10.1038/nbt936.View ArticleGoogle Scholar
- Brennecke J, Stark A, Russell RB, Cohen SM: Principles of microRNA-target recognition. PLoS Biol. 2005, 3 (3): e85-10.1371/journal.pbio.0030085.View ArticleGoogle Scholar
- Doench JG, Sharp PA: Specificity of microRNA target selection in translational repression. Genes Dev. 2004, 18 (5): 504-511. 10.1101/gad.1184404.View ArticleGoogle Scholar
- Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP: MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007, 27 (1): 91-105. 10.1016/j.molcel.2007.06.017.View ArticleGoogle Scholar
- Lai EC: Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet. 2002, 30 (4): 363-364. 10.1038/ng865.View ArticleGoogle Scholar
- Funatsu H, Yamashita H, Sakata K, Noma H, Mimura T, Suzuki M, Eguchi S, Hori S: Vitreous levels of vascular endothelial growth factor and intercellular adhesion molecule 1 are related to diabetic macular edema. Ophthalmology. 2005, 112 (5): 806-816. 10.1016/j.ophtha.2004.11.045.View ArticleGoogle Scholar
- Hofacker IL: Vienna RNA secondary structure server. Nucleic Acids Res. 2003, 31 (13): 3429-3431. 10.1093/nar/gkg599.View ArticleGoogle Scholar
- Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R: Fast and effective prediction of microRNA/target duplexes. Rna. 2004, 10 (10): 1507-1517. 10.1261/rna.5248604.View ArticleGoogle Scholar
- Marques JT, Devosse T, Wang D, Zamanian-Daryoush M, Serbinowski P, Hartmann R, Fujita T, Behlke MA, Williams BR: A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat Biotechnol. 2006, 24 (5): 559-565. 10.1038/nbt1205.View ArticleGoogle Scholar
- Rivory LRPM, Birkett DJ, Arndt GM, Passioura TJ: Multitargeting Interfering RNAs and Methods of their Use and Design. International Patent Application: WO2007/056826A1Google Scholar
- Imming P, Sinning C, Meyer A: Drugs, their targets and the nature and number of drug targets. Nat Rev Drug Discov. 2006, 5 (10): 821-834. 10.1038/nrd2132.View ArticleGoogle Scholar
- Frantz S: The trouble with making combination drugs. Nat Rev Drug Discov. 2006, 5 (11): 881-882. 10.1038/nrd2189.View ArticleGoogle Scholar
- Morphy R, Rankovic Z: The physicochemical challenges of designing multiple ligands. J Med Chem. 2006, 49 (16): 4961-4970. 10.1021/jm0603015.View ArticleGoogle Scholar
- Meltzer HY: An overview of the mechanism of action of clozapine. J Clin Psychiatry. 1994, 55 (Suppl B): 47-52.Google Scholar
- Pardanani A, Tefferi A: Imatinib targets other than bcr/abl and their clinical relevance in myeloid disorders. Blood. 2004, 104 (7): 1931-1939. 10.1182/blood-2004-01-0246.View ArticleGoogle Scholar
- Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, Lim L, Linsley PS: Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity. Rna. 2006, 12 (7): 1179-1187. 10.1261/rna.25706.View ArticleGoogle Scholar
- Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, et al: RNAi-mediated gene silencing in non-human primates. Nature. 2006, 441 (7089): 111-114. 10.1038/nature04688.View ArticleGoogle Scholar
- Castanotto D, Sakurai K, Lingeman R, Li H, Shively L, Aagaard L, Soifer H, Gatignol A, Riggs A, Rossi JJ: Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res. 2007, 35 (15): 5154-5164. 10.1093/nar/gkm543.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.