Potent and nontoxic antisense oligonucleotides containing locked nucleic acids

Abstract

Insufficient efficacy and͞or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a highaffinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA͞DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA͞DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA͞DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.A ntisense oligonucleotides designed according to straightforward base-pairing rules have been useful in functional genomics efforts, and there also has been recent clinical progress in developing antisense drugs (1-5). The key objective in the field, however, remains the identification of oligonucleotide analogs that provide the possibility to achieve high in vivo efficacy in the absence of significant toxicity (1-3).To date, all human antisense studies, as well as the vast majority of studies on other species, have relied on the use of phosphorothioate DNA analogs (where one nonbridging phosphate oxygen has been replaced). Although phosphorothioates are markedly more resistant to degrading enzymes than DNA, their DNA-binding capacity (relating to potency when used as antisense agents) is low, and they are well known to cause nonspecific protein binding, largely because of their polyanionic nature. The latter phenomenon contributes to a toxicity profile that limits many applications (6, 7). For example, when injected into the brain, phosphorothioates can cause severe tissue damage, especially with repeated or prolonged administration schedules (7,8). Such phosphorothioate-induced toxic reactions are thought to be reduced but not absent in second-generation antisense agents, like mixed backbone oligonucleotides (containing phosphorothioates in combination with oligodeoxyribonucleotides or oligoribonucleotides) (9).Interestingly, conformational restriction has been successfully applied in recent years to the design of high-affinity oligonucleotides. Several analogs containing bi-and tricyclic carbohydrate moieties have displayed enhanced duplex stability (10-20) and most notably so locked nucleic acids (LNA) (Fig. 1). LNA induces unprecedented increases in the thermal stability (melting temperature, T m ) of duplexes toward complementary DNA and RNA (⌬T m ͞LNA monomer ϭ ϩ 3 to ϩ 11°C compared with the corresponding DNA reference). By virtue of their bicyclic structure, the furanose ring of the LNA monomers is locked in a 3Ј-endo conformation, thus structurally mimicking the standard RNA monomers. Moreover, LNA͞LNA duplex formation has been shown to constitute the most stable...

Specific RNP capture with antisense LNA/DNA mixmers

“…LNA nucleosides carry a ribose ring that is "locked" by a methylene bridge connecting the 2 ′ -O atom and the 4 ′ -C atom. By "locking" the molecule, the LNA is constrained into a conformation that facilitates Watson-Crick base-pairing, increasing the duplex melting temperature by 2°C-10°C per LNA nucleotide (Wahlestedt et al 2000). Our hybrid LNA/DNA probes were designed to permit selective, highly stringent hybridization conditions in spite of their limited (∼20 nucleotide [nt]) lengths.…”

Synthesis and Properties of Isobicyclo‐DNA

“…2'-OMe [27] ) as well as the sterically constrained (e.g. LNA [28] ; tcDNA [9a] ) do not activate RNase H. [26] From this point of view it is not surprising that iso-bc-DNA is unable to do so.…”

Nano-sized carriers in gene therapy for peritoneal fibrosisin vivo

“…Antisense oligonucleotides are short, artificial synthetic 15–25 nt oligonucleotides [55], which include phosphorothioate linkages that confer nuclease resistance to enhance intracellular stability [55]. BNAs are modified RNA nucleotides including a molecule with a five- or six-membered bridged structure with a fixed C3'-endo sugar puckering [56]. BNAs increase the binding affinities to target oligonucleotides and transgene stability [56].…”

“…BNAs are modified RNA nucleotides including a molecule with a five- or six-membered bridged structure with a fixed C3'-endo sugar puckering [56]. BNAs increase the binding affinities to target oligonucleotides and transgene stability [56]. Intraperitoneal injection of naked HSP47 antisense oligonucleotides inhibited peritoneal fibrosis in a rat model, associated with reduced expression levels of HSP47, types I and III collagen, and α-SMA, as well as reducing the number of infiltrating macrophages in a rat peritoneal fibrosis model [41].…”