Antisense Oligonucleotides Containing Conformationally Constrained 2′,4′-(N-Methoxy)aminomethylene and 2′,4′-Aminooxymethylene and 2′-O,4′-C-Aminomethylene Bridged Nucleoside Analogues Show Improved Potency in Animal Models

Abstract

To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.

An Overview of Sugar‐Modified Oligonucleotides for Antisense Therapeutics

“…16) was reported in which the O-atom in the LNA was replaced with a methoxyamino group [82]. ASOs containing N-MeO-amino BNA and LNA residues showed similar T m values [82]. In vitro and in vivo activity, and the toxicity profile of PTEN gapmer ASO containing NMeO-amino BNA was also reported.…”

“…Interestingly, N-MeO-amino BNA and LNA ASOs showed similar in vitro potency. Even though the toxicity profile of N-MeOamino BNA ASO was superior to the corresponding LNA ASO, its in vivo potency was 2.6-fold less [82].…”

“…Recently N-(methylamino)oxy BNA (Fig. 18) was also reported [82] [NMe], and N-Me-aminooxy BNA were also reported [82]. The BNA NC [NMe] ASO was twofold less active, and 2',4'-BNA NC [NH] was significantly less active than the corresponding LNA ASO [82].…”

“…The N-Me-amino BNA ASO was also less active (ca. fourfold) [82]. A PTEN ASO containing BNA NC [NMe] (ED 50 6 mg/kg) was twofold more potent than N-Meaminooxy BNA ASO (ED 50 11 mg/kg) [81].…”

In vitro selection of BNA (LNA) aptamers

“…Among those, 2',4'-BNA COC , 2',4'-BNA NC , and 2',4'-BNA NC(Me) were found to exhibit further improved nuclease resistance while still retaining binding affinities to single-stranded RNA and/or double-stranded DNA as a target. [28][29][30][31] The first model of BNAs, i.e., 2',4'-BNA/LNA has already reached clinical trials. Santaris Pharma A/S in Demark has lowers blood LDL-C levels because of the inhibition of ApoB synthesis in liver.…”