Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.

Arnaud F Klein,Genevieve Gourdon,Denis Furling,Guillaume Bassez,Arnaud Ferry,Miguel A Varela,Michael J Gait,Andrey Arzumanov,Dominic Jauvin,Ludovic Arandel,Naira Naouar,Matthew J.A Wood,Lucile Revillod,David Seoane,Ashling Holland,Jack Puymirat

doi:10.1172/jci128205

Abstract

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient–derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Highlights

Myotonic dystrophy (DM1), one of the most common muscular dystrophies in adults [1], is an RNA-dominant disorder caused by the expression of expanded microsatellite repeats located in the 3′ untranslated region (UTR) of the DM1 protein kinase (DMPK) gene [2,3,4]
To test whether advanced Pip6a conjugates enhanced Antisense oligonucleotides (ASOs) delivery and activity in skeletal muscles of DM1 mice following systemic administration, antisense phosphorodiamidate oligomer (PMO) composed of a CAG7 sequence, either naked (PMO) or conjugated to Pip6a peptide (Pip6a-PMO), were injected into the tail vein of human skeletal muscle α actin (HSA)-LR mice
The skeletal muscle–specific expression of CUG expansions (CUGexps) RNAs in HSA-LR mice leads to the formation of ribonuclear foci that sequester MBNL1 proteins, resulting in splicing defects and myotonia, as found in DM1 patients [11, 27]

Summary

CONCISE COMMUNICATION

Systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6aconjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Treated DM1 patient–derived muscle cells showed that Pip6a-PMO-CAG targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1

Introduction

Results and Discussion

Methods

Author contributions