Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides

May T Aung-Htut,Russell Johnsen,Kerrie Foyle,Steve D Wilton,Sue Fletcher,Iain Comerford

doi:10.1038/s41598-019-49385-6

Abstract

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

Highlights

Since the first report of antisense oligonucleotides (AO) being used to block viral replication, by Zamecnik and Stephenson[1], there was great optimism for their possible application as therapeutics
In vivo validation of splice modulating AO-mediated down-regulation of integrin alpha 4 protein (ITGA4) activity was performed in the experimental autoimmune encephalomyelitis (EAE)[17] mouse model of multiple sclerosis (MS), by injecting peptide conjugated phosphorodiamidate morpholino oligomers (PPMOs) that were designed to induce specific Itga[4] exon skipping, into mice with chronic EAE
The functional full-length protein contains an extracellular domain encoded by exons 1–26, a transmembrane domain encoded by exon and a cytoplasmic domain translated from exon

Summary

Introduction

Since the first report of antisense oligonucleotides (AO) being used to block viral replication, by Zamecnik and Stephenson[1], there was great optimism for their possible application as therapeutics. We describe the development and evaluation of exon skipping AOs designed to induce a non-functional isoform and thereby reduce the activity of integrin alpha 4 protein (ITGA4), known as very late antigen 4 α subunit or CD49d. This work further validated antisense therapy as an alternative strategy for the treatment of MS with ITGA4 as a therapeutic target. In vivo validation of splice modulating AO-mediated down-regulation of ITGA4 activity was performed in the experimental autoimmune encephalomyelitis (EAE)[17] mouse model of MS, by injecting peptide conjugated phosphorodiamidate morpholino oligomers (PPMOs) that were designed to induce specific Itga[4] exon skipping, into mice with chronic EAE. We are optimistic that further refinement of the study, including optimization of the PPMO dosage regimen will confirm the therapeutic potential of the Itga[4] exon 4 skipping strategy

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