OTS Highlights Advance in Basic and Translational Aspects of Oligonucleotide Research
Authors: John J Rossi
Journal: Molecular Therapy (2011)

It has been more than three decades since it was shown that antisense DNA oligonucleotides could selectively block the expression of targeted genes. Much has happened to the field of oligonucleotide chemistry and applications over this time span. New chemistries accompany an expanding repertoire of oligonucleotide agents. We have also witnessed the discoveries of small interfering RNAs (siRNAs) and microRNAs (miRNAs). The development of a plethora of delivery schemes for these agents has further changed the field. There are now several legitimate oligonucleotide drugs in early- and late-stage clinical trials, and there is heightened interest in the therapeutic application of oligonucleotides due to the addition of RNA interference (RNAi) in our armamentaria. This heightened interest was evidenced at the sixth annual meeting of the Oligonucleotide Therapeutics Society (OTS, http://www.myots.org), which took place 20–23 October at the Laguna Cliffs Resort and Spa in Dana Point, California. The keynote lecture was presented by Andrew Fire, who discussed the roles of RNAi-based mechanisms in controlling pathogen infection and the differences in how RNAi affects this process in lower eukaryotes, plants, and mammals. There were more than 300 attendees, representing academia, industry, and governmental regulatory agencies. As in all scientific fields, new discoveries break old paradigms and create new challenges. The meeting's opening session covered some of the new areas of discovery that impact the application of oligonucleotides as therapeutics. These include new bioinformatics tools and technical approaches that set the stage for consideration of the impact of oligonucleotide therapeutics on global gene expression. Bioinformatic analyses of the transcriptome can aid in predicting the interacting roles of genes and their products. Inhibition of one gene product can have profound implications on the functioning of interacting gene pathways. Studies of gene regulation have progressed remarkably in the past few years with the advent of techniques for characterizing global post-transcriptional regulatory mechanisms. One powerful approach involves a combination of messenger RNA (mRNA) and protein profiling. An interesting new approach to profiling ribosomes along mRNAs presented at the meeting revealed a surprising array of new translational initiation sites in the 5′ UTRs of messages, as well as unexpected codon usage by initiating ribosomes. Long noncoding (lnc) RNAs constitute a large fraction of the transcriptome in higher eukaryotes. These RNAs are expressed from both intergenic and coding regions (antisense to coding transcripts) and have been shown to regulate gene expression via epigenetic chromatin remodeling or direct interactions with protein-encoding mRNAs. It was clear from the meeting's few talks on lncRNAs that this is an exciting, although still emerging, area of gene regulation. miRNAs are endogenously produced RNAs that are 21–23-nucleotides long and target mRNAs for translational inhibition and degradation. The functional importance of these small regulators of gene expression to various diseases was highlighted in several talks at the OTS meeting. New tools for assessing protein mRNA- and miRNA-mediated regulation of gene expression have revealed new global patterns of post-transcriptional gene regulation. We now realize that the 3′ untranslated regions of mRNAs also play important roles in modulating the expression and stability of transcripts via interactions with proteins and miRNAs. Indeed, the loss of miRNA regulation is a key factor in the development and progression of many cancers. This new information should aid in the design of oligonucleotide strategies targeting specific diseases. Emerging therapeutic applications involving miRNAs include supplying synthetic miRNA mimics and using backbone-modified antisense agents to inhibit miRNA function. The founding of the OTS was based on years of research and applications of synthetic DNA antisense oligonucleotides. Until recently, the major formulation for these oligonucleotides was the substitution of non-oxygen-bridging phosphates with sulfur, forming phosphorothioates (PSs). These oligos exhibit stability against nucleases, can be delivered to cells and tissues without carriers, and serve as substrates for the enzyme RNAse H, which cleaves RNA in RNA-DNA duplexes. Despite these attributes, PS oligos have many non-sequence-specific off-target effects. Hence, only one PS oligo has been approved as a drug: Vitrivene, for the treatment of cytomegalovirus retinitis. New backbone modifications that enhance oligo function are necessary to foster their development and application as drugs. At the OTS meeting, several presentations discussed the functional benefits of oligonucleotide backbone modifications that increase their stability and thermodynamic properties. These modifications include 2′-O-methyl, 2′-methoxyethyl, locked nucleic acids, and morpholino and peptide substitutions for the ribose backbones. Each of these has unique properties that increase base-pairing specificity and cellular uptake and hence efficacy. The backbone modifications can be interspersed with blocks of PS-containing bases to trigger RNAse H activity or can be designed to avoid such activity for use as steric block inhibitors. Steric blocking of splice sites or splice-site enhancers can force the splicing apparatus to skip exons that contain frame-shift- or stop-codon-encoding mutations. Bypassing the inclusion of these exons results in truncated, but functional, proteins. This application has been particularly effective in Duchenne's muscular dystrophy. siRNAs are short, double-stranded duplexes 21–23 bp long that trigger sequence-specific degradation of targeted mRNAs via perfect Watson–Crick base pairing to target mRNAs and activation of the RNAi pathway. Small inhibitory RNAs are being developed as therapeutic agents for a variety of diseases. A major challenge for therapeutic applications of siRNAs is efficient delivery to diseased cells and tissues. Promising new delivery options include complexation with cell-penetrating peptides, cellular receptor targeting using chimeric siRNA aptamers, development of combinatorial library–derived polymers that bind siRNAs, and even the use of selective, enzymatically reversible backbone modifications that abrogate the need for delivery vehicles. The breadth of new options, particularly for the targeted approaches, was highlighted at the meeting, given that delivery is deemed the missing link for therapeutic application of siRNA. SiRNAs targeting promoter regions of tumor suppressor genes have also been shown to activate transcription via induced epigenetic changes in the local chromatin state of the promoter. A novel application for this type of RNA (termed small activating RNA) for induction of pluripotent stem cells via activation of reprogramming genes was reported at the meeting. Previous studies have shown that siRNAs with certain sequence motifs can activate Toll-like receptors, which in turn stimulate type I interferon gene expression. Alternatively, certain structural motifs in siRNAs can trigger other intracellular receptors for RNA, such as RIG I, and elicit interferon-regulated gene induction. At one time this effect was considered a liability for siRNA-based applications, but, as described in two key presentations, the synergy between siRNA-directed inhibition of gene expression and activation of the interferon pathways may prove advantageous for the treatment of viral infections and cancers. The more traditional application of antisense oligonucleotides is the triggering of RNAse H–mediated destruction of the mRNA to which the antisense oligo is paired. RNAse H cleaves the mRNA hybridized to the antisense oligo without destroying the DNA component, allowing the DNA to recycle and target multiple mRNAs. The DNA component must have a negatively charged backbone for this cleavage to take place. “Gapmers” are oligos with backbone modifications on the 5′ and 3′ flanks of a negatively charged core containing either diester or PS linkages. Several presenters at the meeting focused on the mechanism of this effect and the development of strategies to use noncharged backbones in the flanking region to enhance base pairing with the target while maintaining the negatively charged PS core. These gapmers are stable in serum, are internalized into cells in the absence of carriers, and provide more efficient base pairing to targeted transcripts, resulting in greater potency of target inhibition. Several presentations were devoted to the development of aptamers, which are in vitro evolved RNA or DNA oligomers that bind to target ligands with a high degree of specificity. One speaker described tumor cell killing triggered by a bivalent aptamer that targets a prostate tumor cell–specific receptor as well as a costimulatory ligand receptor on tumor-infiltrating T cells. Two separate applications of aptamers for treatment of HIV-1 infection were described. In one, an aptamer targeting the HIV envelope neutralizes the virus but also serves as a vehicle for delivery of therapeutic siRNAs to infected cells. In another approach, an aptamer targeting HIV-1 Pol (reverse transcriptase) and delivered via an integrated viral vector was effective in blocking virus entry and infection. Selectively inactivating aptamers is important to prevent unwanted side effects and toxicity. One presentation demonstrated that RNA aptamers being developed as anti–blood clotting agents can be quickly and irreversibly inactivated in vivo through intravenous injection of cationic polymers such as dendrimers and cyclodextrins. The meeting closed after a session focused on the development and testing of oligonucleotide agents and strategies for diseases ranging from cancer to viral infection. Because antisense base pairing is a two-dimensional design strategy, many proteins that are not druggable by small molecules are now potential drug targets via oligonucleotides that function by sequence-specific pairing and inhibition of the cognate target mRNAs. As noted above, this inhibition takes place via various mechanisms, including RNAi activation, RNAse H–mediated cleavage of mRNAs, and steric blockage of protein–RNA interactions. miRNAs are also becoming attractive targets for oligonucleotide therapies given the potent inhibition of miRNA function with backbone-modified oligonucleotides with greater stability and efficacy. Both early- and late-phase clinical trials are under way for each type of oligonucleotide agent discussed at this conference. Although no blockbuster results were presented, the advances in both the basic and translational aspects of the field left the participants with anticipation for exciting results in the coming months and years ahead and the sense that the field is rapidly progressing toward broader clinical testing and applications of the oligonucleotide-based drugs.