Antisense oligonucleotide induction of progerin in human myogenic cells.

Yue-Bei Luo,Abbie M Adams,Frank L Mastaglia,Sue Fletcher,Steve D Wilton,Chalermchai Mitrpant,Russell D Johnsen

doi:10.1371/journal.pone.0098306

Abstract

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the ‘natural’ ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2′-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.

Highlights

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disease caused by mutations in lamin A gene (LMNA) that activate a cryptic splice site in exon 11 [1]
Antisense oligonucleotides (AOs) targeting the pre-mRNA from 30 bases downstream of the cryptic splice site to the donor site, were able to induce some LMNA D150 transcript production, as assessed by RT-PCR (Figure 2)
Transfection of AOs that anneal to the acceptor site or the first 120 bases upstream of the cryptic splice site did not have any obvious effect on the splicing of LMNA exon 11 (Figure 2B)

Summary

Introduction

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disease caused by mutations in LMNA that activate a cryptic splice site in exon 11 [1]. Induction of this inappropriate alternative splicing leads to the loss of 150 bases from the end of exon 11, and results in the translation of a truncated protein isoform, progerin. We report the use of two different types of splice-switching AOs to redirect processing of exon 11 of LMNA, so as to enhance expression of the progerin isoform in human myogenic cells and generate an in vitro model of premature muscle ageing

Experimental Procedures

Results

Discussion