Although the history of ONs is intimately connected with that of basic research in molecular biology, we will focus on key areas of direct importance for nucleic acid medicines. Different delivery approaches have recently been reviewed elsewhere.86,148 However, while cellular uptake remains a limitation, it is also clear that several compounds already yield significant treatment effects. This might be overcome by inserting monomers of twisted intercalating nucleic acids, as described for ONs targeting the KRAS promoter.135 So far, TFOs have not been used clinically, but it was recently reported that a TFO targeting the MYC promoter in combination with gemcitabine potentiated the antitumor activity in a mouse model.136, AGOs with capacity to invade into dsDNA and bind to one of the strands via WC base pairing have also been studied. However, there is room for cautious optimism. The early reports on PNA as AGOs have been previously reviewed.134,137 The poor dsDNA invasion (DSI) capacity of PNA-ONs at physiological salt concentrations was somewhat improved by conjugation with the intercalator 9-aminoacridine,138 and using short LNA-containing ONs as openers enhanced invasion into supercoiled DNA.139 For LNA-modified ONs, DSI can occur also at physiological salt concentration, and new LNA-containing AGOs were developed in the form of, for example, the Zorro-LNA. Rocha CS, Wiklander OP, Larsson L, et al.. RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form, Splicing of a critical exon of human survival motor neuron is regulated by a unique silencer element located in the last intron, Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice, Isis Pharmaceuticals reports interim results from ISIS-SMN Rx multiple dose study in children with spinal muscular atrophy, Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts, Correction of disease-associated exon skipping by synthetic exon-specific activators. In this review, we have only briefly addressed the delivery aspect and this remains an important hurdle, since ONs are rather large entities and do not enter into cells by diffusion. The different chemical structures can be found in Supplementary Fig. Sanaker PS, Toompuu M, McClorey G, et al.. Antisense oligonucleotide corrects splice abnormality in hereditary myopathy with lactic acidosis. Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS, Development of therapeutic-grade small interfering RNAs by chemical engineering. History of oligonucleotide therapeutics. 1). Another key discovery in the 1960s was the identification of poly(I:C) as an inducer of interferon in rabbits.9 The biological basis for this observation was not understood at the time, and it took another 34 years before the Toll-like receptor 3 (TLR3) was shown to be the receptor for double-stranded RNA.10 Related to these findings was the notion in 1984 that bacterial DNA has an antitumoral effect.11 Eleven years later it was demonstrated that bacterial CpG triggers the activation of B-lymphocytes,12 and this was followed by the cloning of the corresponding receptor, named TLR9, in the year 2000.13 While the TLR intracellular signaling pathways have been delineated,14 it is interesting to note that short DNA ONs impair TLR3 signaling in both primates and humans, thereby constituting a potential tool for clinically interfering with this receptor-mediated activity.15. Beside this, the most common target organs are the liver and the eye. El-Andaloussi S, Johansson HJ, Lundberg P, et al.. Careers. Zammarchi F, de Stanchina E, Bournazou E, et al.. Antitumorigenic potential of STAT3 alternative splicing modulation. In the first of 13 articles, the strategy is provided for how to synthesize a DNA duplex corresponding in sequence to the major yeast alanine transfer RNA, by the use of overlapping DNA ONs.20 Automated ON synthesis came later, and was developed in the mid-1980s.21,22 Such procedures were developed over a period of several years and many investigators should be credited for these efforts, but owing to space limitations we can only refer to a few major reports. National Library of Medicine recently wrote a very nice overview of different diseases that are targeted by, for example, siRNAs.64. Multistranded synthetic polynucleotide complexes. in 1944,1 followed by the insightful report on the helical structure of DNA,2 paved the way for our current understanding and use of nucleic acids, including the development of oligonucleotide (ON) therapies. The site is secure. Hammond SM, McClorey G, Nordin JZ, et al.. Correlating in vitro splice switching activity with systemic in vivo delivery using novel ZEN-modified oligonucleotides. Martinez et al. However, as compared with antisense approaches for single-stranded RNA, targeting dsDNA is considerably more challenging. The 1970s is also the decade that witnessed what is generally considered as the birth of antisense therapy. This would fulfill the initial conceptual dream that ON medicines, compared to low molecular traditional drugs as well as to biologics, are much more straightforward to both design and develop. Also, the phosphorothioate chemistry, which was developed by Fritz Eckstein,5 remains a highly important modification for most of today's ON drugs, since it both confers protection against degradation and aids in the cellular uptake of ONs.6,7 In the 1960s another 2 modification, 2-O-methyl (2-O-Me), was also synthesized in a laboratory for the first time.8 Although this nucleotide exists naturally in certain endogenous RNA species, it serves as an important ingredient in several synthetic, therapeutic ONs. Goyenvalle A, Griffith G, Babbs A, et al.. Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Aaldering LJ, Tayeb H, Krishnan S, et al.. Smart functional nucleic acid chimeras: enabling tissue specific RNA targeting therapy, Complexity in the therapeutic delivery of RNAi medicines: an analytical challenge, Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics, Fomiversen sodium approved to treat CMV retinitis, Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial, Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes. Just like other ONs, the stability and degradation of the aptamer are major factors influencing their biological effect. Thus, in 1978 Paul Zamecnik and Mary Stephenson reported in two back-to-back articles in the Proceedings of the National Academy of Sciences U.S.A. that the addition of a 13-mer oligodeoxyribonucleotide could inhibit Rous sarcoma virus in infected cell cultures.23,24 The following year, a publication described that RNase H site-specifically cleaves the RNA strand in RNADNA heteroduplexes,25 thereby demonstrating that antisense therapy can work not only by steric blocking, but also through an enzyme-mediated process. The early 1970s mark the beginning of more advanced methods for ON synthesis. Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial. We are grateful to Samir El Andaloussi, Anna Berglf, and Roger Strmberg, Karolinska Institutet; Mark A. Behlke, Integrated DNA Technologies (IDT), Inc.; Peter B. Dervan, California Institute of Technology; Scott Henry, ISIS Pharmaceuticals; Per Trolle Jrgensen, Erik B. Pedersen, and Jesper Wengel, University of Southern Denmark; and Rula Zain, Karolinska University Hospital, for valuable comments. (6) Aptamer, binding alters protein surface. Later, it was discovered that aptamers able to form G-quadruplex structures not only specifically interact with their protein target, but also gain dramatic increases in their serum half-life. This means that substantial further optimization is needed before AGOs have reached the stage where they could enter into clinical trials. The seven biological processes that have been targeted and the corresponding ON therapeutic interventions used are shown in Fig. (3) Micro-RNA (miR) mimic. 1, it is obvious that there are many entirely different approaches being used. In this review we address the development of oligonucleotide (ON) medicines from a historical perspective by listing the landmark discoveries in this field. XVIII. S1 (Supplementary Data are available online at www.liebertpub.com/hum). This phenomenon was first described for the nucleolin-interacting AS1411 aptamer (formerly GRO29A).131. PMC legacy view (7) Splice switching ON (SSO). Received 2015 May 18; Accepted 2015 Jul 4. Cannata F, Brunet E, Perrouault L, et al.. Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification, Gene regulation by antisense transcription, Sequence-selective targeting of duplex DNA by peptide nucleic acids. The landmark discovery of DNA as the hereditary material by Avery et al. Directing HER4 mRNA expression towards the CYT2 isoform by antisense oligonucleotide decreases growth of breast cancer cells. In 1990, duplex ONs were also developed to serve as decoys by sequestering transcription factors.40 In 1992, (L)-ribonucleic acids were synthesized and their properties studied.41 The (L)-type ONs, also known as Spiegelmers, or mirror-image ONs, were recently reviewed.42 The following year ON-mediated splice-switching was developed in the laboratory of Ryszard Kole.43 For this therapeutic approach, RNase H activity is unwanted, since altered splicing, and not degradation, is the aim. Selected through in vitro or cell-based SELEX approaches, a substantial amount of DNA and RNA aptamers have been generated since the early 1990s (Fig. 1). van Deutekom JC, Janson AA, Ginjaar IB, et al.. Local dystrophin restoration with antisense oligonucleotide PRO051, Development of therapeutic splice-switching oligonucleotides. The .gov means its official. The various biological processes that have been targeted and the corresponding ON interventions found in the literature are discussed together with brief updates on some of the more recent developments. Bestas B, McClorey G, Tedebark U, et al.. Design and application of bispecific splice-switching oligonucleotides, Cell-type-specific, aptamer-functionalized agents for targeted disease therapy, Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. In 2001, the first use of CpG ONs in the clinics was reported. This compound was coadministered, in a phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen, as an immunostimulatory phosphorothioate ON adjuvant.46,47 Three years later pegaptanib (brand name: Macugen) became the first FDA-approved aptamer.48 It was used as an antiangiogenic medicine for the treatment of neovascular (wet) age-related macular degeneration and delivered by intravitreous injections. In many cancers the miR pattern is disturbed, and if no additional mutation in known oncogenes exist, a possible treatment can be to block the overexpressed miR with a complementary, synthetic ON, an antagomir. A recent major development in molecular biology is the CRISPR-Cas technology for genome editing, first published in 2012.60 This enzymeRNA complex is dependent on two short RNA sequences, which may also be engineered as a single RNA chimera, dual-tracrRNA:crRNA, or guide RNA, serving as a very efficient tool for editing purposes, and such short RNA species can be synthesized as ONs.