Stereochemistry Enhances Potency and Duration of Effect of MALAT1 Antisense Oligonucleotides In Vivo in the Mouse

Abstract

Purpose : Antisense oligonucleotides (ASO) containing phosphorothioate (PS) substitutions, like those currently in clinical development, contain approximately half a million individual stereoisomers at 20-mer length, due to the PS center having two distinct stereochemical configurations. Each ASO mixture therefore contains many non-active isomers that have the potential for unintended biological activities. Using MALAT1 as a surrogate target, we tested the hypothesis that controlling the chirality of PS linkages in the backbones of ASOs to generate stereopure compounds will provide a benefit in potency, distribution and duration of effect in the eye.

Methods : Stereopure compounds targeting the long non-coding RNA MALAT1 were generated using Wave Life Sciences’ proprietary technology that enables precise control of stereochemistry and were evaluated in vitro in iCell neurons under gymnotic conditions. Top compounds were then evaluated in vivo following single intravitreal injection in the mouse eye. Reduction of MALAT1 RNA by qPCR, quantification and distribution of ASO by hybridization ELISA and IHC, and duration of effect are being evaluated over a 6-month period.

Results : Stereopure MALAT1 ASOs were successfully delivered under gymnotic conditions to iCell neurons with top stereopure compounds exhibiting IC₅₀ values approximately 24-fold lower than stereorandom ASOs. Stereorandom ASOs achieved maximal knockdown of 75% in the back and 70% in the front of the eye, which was maintained for 2 weeks and reduced to 50% reduction at one month. However, stereopure ASOs achieved target-specific reduction of 90% in the back and front of the eye as early as 7 days post single intravitreal injection, which was maintained for at least one month thus far. Pharmacokinetic analysis demonstrated stereopure ASOs had greater tissue exposure at 7 days post injection compared with sequence matched stereorandom ASOs.

Conclusions : Stereorandom ASOs contain large numbers of potentially non-active isomers that may have unintended biological activities. Here we demonstrate that controlled chirality to generate a single stereopure compound that can be optimized and provides enhanced potency and duration of effect in vivo for a variety of eye cell types over stereorandom ASOs.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.