Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides

May Aung-Htut,Kane Greer,Kristin Ham,Loren Flynn,Sue Fletcher,Ianthe Pitout,Steve Wilton,Craig McIntosh

doi:10.3390/ijms20205030

Abstract

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone.

Highlights

Splice switching antisense oligonucleotides (AOs) are gaining interest as therapeutics for a wide variety of inherited and acquired diseases, with the approvals of Eteplirsen and Nusinersen to treat Duchenne muscular dystrophy and spinal muscular atrophy, respectively [1,2,3]
We provide reference AOs optimised to modulate ubiquitously expressed human gene transcripts that may be employed as controls to monitor the efficiency of transfection, RNA extraction, and RT-PCR amplification
We have frequently found that selective AO cocktails, which include two or more AOs used in conjunction for a given exon target, mediate exon skipping in a synergistic manner, while each AO transfected alone is ineffective [18,29]

Summary

Introduction

Splice switching antisense oligonucleotides (AOs) are gaining interest as therapeutics for a wide variety of inherited and acquired diseases, with the approvals of Eteplirsen and Nusinersen to treat Duchenne muscular dystrophy and spinal muscular atrophy, respectively [1,2,3]. The original AO that induced specific dystrophin exon 23 skipping in mdx mouse muscle was directed to the donor splice site [17], AOs targeting the same coordinates of the human dystrophin transcript were completely ineffective [18]. Targeting the human dystrophin exon 51 donor splice site with AOs of different lengths and chemistries did not induce any exon skipping. One such control is an AO designed to induce skipping of exon 3 from the ITGA4 transcript, a widely expressed gene in many different cell types. The cells were transfected with 100 nM ITGA4 H3A (+ 30 + 49), a 2-OMe PS AO that induces skipping of exons 3 and 4 from the ITGA4 gene transcript, using three different lipid-based transfection reagents (Figure 3A). When both AOs were transfected at nM, the lower efficiency of ITGA4 H3A (+ + 75) compared to ITGA4 H3A (+ 41 + 65) was evident

Discussion

Cell Culture

Findings

RT-PCR