Abstract
The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8–15 nucleotides and by the incorporation of high affinity RNA binders such as 2′, 4′-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2′,4′-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4–5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.
Highlights
Antisense oligonucleotide (ASO) therapeutics are emerging as the third platform for drug development after small molecules and biologics, with more than 100 compounds in clinical development
Because short phosphorothioate backbone ASO have lower plasma protein binding than longer ASO [9,11] we hypothesized that they would undergo greater renal clearance in vivo, with a corresponding reduction in uptake into liver and other tissues, and a loss of therapeutic efficacy
In order to test the properties of the multi-targeting oligonucleotides (MTOs) design, we performed a series of studies with various ASO, starting with a previously reported 8mer shortmer blocking miR122 [8,14]
Summary
Antisense oligonucleotide (ASO) therapeutics are emerging as the third platform for drug development after small molecules and biologics, with more than 100 compounds in clinical development. There are two standard singlestranded ASO designs that act by different mechanisms of action [1,2,3]. ‘gapmer’ ASOs contain a DNA center that activates RNase H to cleave the target RNA: this core generally is flanked by 5 and 3 wings of nucleotides with ribose modifications such as 2 - methoxyethyl (MOE) or LNA which do not support RNase H cleavage, but which confer an increased affinity for hybridization. ‘shortmer’ and ‘mixmer’ ASOs act by steric blocking, and contain ribose modifications distributed in such a manner that the ASO does not activate RNase H. Several groups have reported that the incorporation of LNA or cET makes it possible to use shorter gapmer ASOs of less than 15 nucleotides in length [4,5,6,7]: steric blocking shortmer ASOs can be as short as 8 nucleotides long [8]
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