Jan Weiler

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

MicroRNAs (miRNAs) are endogenous 19–25 nucleotide RNAs that have recently emerged as a novel class of important gene-regulatory molecules involved in many critical developmental and cellular functions. miRNAs have been implicated in the pathogenesis of several human diseases, such as neurodegenerative disorders, cancer, and more recently in viral and metabolic diseases. Unraveling the roles of miRNAs in cellular processes linked to human diseases will lead to novel opportunities for the regulation of protein function and will help to evaluate their potential for therapeutic intervention. Approaches to interfere with miRNA function in vitro and in vivo based on synthetic anti-miRNA oligonucleotides (AMOs) are discussed in this review.

Design of a genome-wide siRNA library using an artificial neural network

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.

Combination of siRNA-directed Gene Silencing With Cisplatin Reverses Drug Resistance in Human Non-small Cell Lung Cancer

One of the most challenging aspects of lung cancer therapy is the rapid acquisition of multidrug-resistant (MDR) phenotype. One effective approach would be to identify and downregulate resistance-causing genes in tumors using small interfering RNAs (siRNAs) to increase the sensitivity of tumor cells to chemotherapeutic challenge. After identifying the overexpressed resistance-related antiapoptotic genes (survivin and bcl-2) in cisplatin-resistant cells, the siRNA sequences were designed and screened to select the most efficacious candidates. Modifications were introduced in them to minimize off-target effects. Subsequently, the combination of siRNA and cisplatin that gave the maximum synergy was identified in resistant cells. We then demonstrated that the combination treatment of the selected siRNAs and cisplatin encapsulated in CD44-targeting hyaluronic acid (HA)-based self-assembling nanosystems reversed the resistance to cisplatin and delayed the tumor growth significantly (growth inhibition increased from 30 to 60%) in cisplatin-resistant tumors. In addition, no abnormalities in body weights, liver enzyme levels or histopathology of liver/spleen tissues were observed in any of the treatment groups during the study period. Overall, we demonstrate that the combination of siRNA-mediated gene-silencing strategy with chemotherapeutic agents constitutes a valuable and safe approach for the treatment of MDR tumors.

Regulating the regulators: mechanisms controlling the maturation of microRNAs

MicroRNAs (miRNAs) are small noncoding RNAs that control diverse cellular and developmental events through repression of large sets of target mRNAs. Regulated transcription of the genes encoding miRNAs by RNA polymerase II promotes specific expression patterns of individual miRNAs. However, recent studies have established that substantial regulation of mature miRNA accumulation also occurs after transcription. Here, we review the mechanisms of such post-transcriptional regulation, with a particular focus on examples where molecular mechanisms or physiological principles are beginning to emerge. Elucidating these mechanisms will increase our understanding of gene regulation and provide new insights into causes of miRNA misexpression in diseases such as cancer.

Specific inhibition of the rat ligand-gated ion channel P2X3 function via methoxyethoxy-modified phosphorothioated antisense oligonucleotides.

P2X3 is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dose-dependent, sequence-specific downregulation of the rat P2X3 receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P2X3 inhibition. The functional response of the P2X3 receptor was examined using whole-cell voltage clamping. Upon application of 10 microM of a nonspecific agonist, alpha,beta-methylene-ATP (alphabeta meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P2X3 receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides

Abstract Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.

Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin

In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA.

Effective Exon Skipping and Dystrophin Restoration by 2′-O-Methoxyethyl Antisense Oligonucleotide in Dystrophin-Deficient Mice

Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

Improving gene silencing of siRNAs via tricyclo-DNA modification

Small interfering RNAs (siRNAs) can be exploited for the selective silencing of disease-related genes via the RNA interference (RNAi) machinery and therefore raise hope for future therapeutic applications. Especially chemically modifed siRNAs are of interest as they are expected to convert lead siRNA sequences into effective drugs. To study the potential of tricyclo-DNA (tc-DNA) in this context we systematically incorporated tc-DNA units at various positions in a siRNA duplex targeted to the EGFP gene that was expressed in HeLa cells. Silencing activity was measured by FACS, mRNA levels were determined by RT-PCR and the biostability of the modifed siRNAs was determined in human serum. We found that modifications in the 3-overhangs in both the sense and antisense strands were compatible with the RNAi machinery leading to similar activities compared to wild type (wt) siRNA. Additional modifications at the 3-end, the 5-end and in the center of the sense (passenger) strand were also well tolerated and did not compromise activity. Extensive modifications of the 3- and the 5-end in the antisense (guide) strand, however, abolished RNAi activity. Interestingly, modifications in the center of the duplex on both strands, corresponding to the position of the cleavage site by AGO2, increased efficacy relative to wt by a factor of 4 at the lowest concentrations (2nM) investigated. In all cases, reduction of EGFP fluorescence was accompanied with a reduction of the EGFP mRNA level. Serum stability analysis further showed that 3-overhang modifications only moderately increased stability while more extensive substitution by tc-DNA residues significantly enhanced biostability.