Abstract:The potency of second generation antisense oligonucleotides (ASOs) in animals was increased 3-to 5 -fold (ED 50 ≈ 2-5 mg/kg) without producing hepatotoxicity, by reducing ASO length (20-mer to 14-mer) and by employing novel nucleoside modifications that combine structural elements of 2′-O-methoxyethyl residues and locked nucleic acid. The ability to achieve this level of potency without any formulation agents is remarkable and likely to have a significant impact on the future design of ASOs as therapeutic agents.Antisense technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.1 The most advanced second generation antisense oligonucleotides (ASOs) are chimeric phosphorothioate 2,3 (PS) modified oligonucleotides, which have a central DNA region of 8-16 nucleotides, flanked on the 5′ and 3′ ends with five to two 2′-O-methoxyethyl (MOE) residues.4 This "gapmer" design supports RNase H mediated degradation of target mRNA due to the central DNA region. The flanking MOE residues increase hybridization to complementary mRNA and further stabilize the oligonucleotide toward enzymatic degradation. The PS backbone not only provides stabilization to nucleases but also confers a substantial pharmacokinetic benefit by increasing the binding to plasma proteins. This prevents rapid renal excretion of the ASO and facilitates binding to other acceptor sites which promote uptake to tissues. 5,6 There are currently multiple second generation ASOs in development for a variety of disease indications including hypercholesteremia, diabetes, and cancer, among others. One particular compound, mipomersen (ISIS 301012), targeting apolipoprotein B (ApoB), 7 reduced LDL cholesterol by 6-41% in normal volunteers at doses ranging from 50 to 400 (mg/kg)/ week in a phase I clinical trial. 8 Ongoing phase II clinical trials with mipomersen have further substantiated clinical efficacy and demonstrated an attractive safety profile for this drug. The improved performance of second generation ASOs in animals can be attributed in part to the higher affinity of MOE (B, Figure 1) residues (∆T m ≈ 1.5°C/incorporation), 10 which typically translates to increased binding affinity for the biological receptor (complementary mRNA). To probe if further increases in affinity could enhance the potency of gapmer ASOs, we replaced MOE residues in the wings with bicyclic nucleic acids such as 2′,4′-methylene bridged nucleic acid (BNA) 11 commonly called LNA (D, locked nucleic acids, Figure 1). 12 While this substitution resulted in improved potency of some ASOs in animals, it was accompanied by a significant increase in the risk for hepatotoxicity. 13 In contrast, the MOE modification employed in second generation ASOs is well tolerated in a variety of animal models and has also demonstrated an excellent safety profile in human clinical trials.14 Our previous study with LNA gapmers had indicated that the motifs that provided the greatest increase in potency of LNA containing ASOs were gapmer desi...

We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5′-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.