Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts

Tristan H Coady,Travis D Baughan,Christian L Lorson,Marco A Passini,Monir Shababi,Juan Valcarcel

doi:10.1371/journal.pone.0003468

Tristan H Coady, Travis D Baughan + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0003468

Copy DOI

Journal: PloS one	Publication Date: Oct 22, 2008
Citations: 108	License type: CC BY 4.0

Affiliation: University of Missouri

Abstract

RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.

Highlights

Trans-splicing has recently been envisioned as a potential therapeutic intervention for a variety of genetic diseases
Since pre-mRNA splicing is a highly dynamic process, we hypothesized that competition exists between the trans-splicing event and the naturally strong SMN2 exon 8 splice site
SMN levels in the co-transfected cells were comparable to SMN levels detected in the unaffected control fibroblasts, 3814 cells. These results demonstrate that endogenous SMN transcripts can be re-directed by trans-splicing and that SMN levels can be significantly increased by the antisense oligonucleotides (ASO)/trans-splicing strategy in a relevant disease context

Summary

Introduction

Trans-splicing has recently been envisioned as a potential therapeutic intervention for a variety of genetic diseases. Trans-splicing is a natural, albeit infrequently utilized process in mammals, maximizing efficiency is central to developing trans-splicing therapeutics [7]. This strategy relies upon nuclear pre-mRNA splicing occurring between two different molecules: 1) the mutant endogenous RNA and 2) the exogenous therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. The critical distinction between the two genes occurs at the RNA processing level: SMN1 produces full-length transcripts, while SMN2 primarily produces an alternatively spliced transcript lacking the final coding exon [10]. A single silent C to T nonpolymorphic nucleotide difference is responsible for disrupting a critical splice enhancer element in SMN2 exon 7 [11,12]

Methods

Results

Conclusion