Properties of Stereodefined Variants of Nusinersen

In the pharmaceutical industry, stereopure drugs are generally preferred over isomeric mixtures to reduce the risks of side effects and to simplify pharmacodynamics (PD) and pharmacokinetics (PK) properties. This reasoning applies equally to antisense oligonucleotides (ASOs). All current ASO drugs are modified to provide appropriate PK/PD properties. Among the modifications, phosphorothioate (PS) groups provide essential nuclease resistance and advantageous biodistribution properties. However, PS groups are chiral, with R p- and S p-centers that are challenging to incorporate stereoselectively during solid-phase synthesis, and thus current ASO drugs are diastereoisomeric mixtures. Here, we describe the synthesis of stereodefined variants of the approved drug nusinersen (Spinraza), a 2′- O -methoxyethyl-modified oligonucleotide that redirects splicing of SMN2 during treatment for spinal muscular atrophy (SMA). We found that a single 3′- S p linkage stabilizes nusinersen from degradation in mouse tissue homogenates. Although R p-nusinersen has higher target-binding affinity than S p-nusinersen, it is not more active than parent nusinersen in patient-derived cells. Finally, we show mixed results from comparing stereodefined nusinersen with parent nusinersen in a murine model of SMA. While our data do not provide definitive evidence for the superior performance of stereodefined nusinersen, we conclude that it is likely equally effective as its parent ASO, while bearing the pharmacological advantages of a single stereoisomer.