Stimulating Full-Length SMN2 Expression by Delivering Bifunctional RNAs via a Viral Vector

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1. However, a single silent nucleotide difference in SMN2 exon 7 results in the production of a spliced isoform (called SMNDelta7) that encodes a nonfunctional protein. The presence of SMN2 represents a unique therapeutic target since SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bifunctional RNAs that modulate SMN2 splicing. Bifunctional RNAs derive their name from the presence of two domains: an antisense RNA sequence specific to a target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bifunctional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and demonstrate directly a novel therapeutic approach for SMA.

Repair of pre-mRNA splicing

“…It should also be mentioned that some success in cell culture models was reported for a similar bifunctional RNA driven by the U6 promoter, but lacking a snRNA backbone. 149 However, using an almost identical construct, we have not been able to reproduce this result in our hands. 96 The bifunctional RNA was almost undetectable and located in intron 6 of SMN2, whereas the functional moiety was an ESE tail recruiting positive splicing factors.…”

Targeting RNA-Splicing for SMA Treatment

“…This promising perspective seems to be greatly boosted by recent developments in SMN/SMA studies in which technologies such as trans-splicing, bifunctional oligonucleotides, and anti-sense oligonucleotides very specifically increase the inclusion of exon 7 into SMN2 mRNA (Fig. 2) (Baughan et al, 2006;Coady and Lorson, 2010;Coady et al, 2007;Dickson et al, 2008;Hua et al, 2010;Lorson et al, 2010;Osman et al, 2012;Shababi and Lorson, 2011;Shababi et al, 2011;Skordis et al, 2003). In particular, the effectiveness of anti-sense oligos in animals with SMA has excited the scientific community so that SMA may be treatable in the near future.…”

“…Bifunctional oligoribonucleotides effectively increase insertion of exon 7 into SMN2 mRNA. Relative to bifunctional oligoribonucleotides (Baughan et al, 2006;Dickson et al, 2008;Horne and Young 2009;Osman et al, 2012;Skordis et al, 2003;Voigt et al, 2010), the strategy of trans-splicing is new but has offered great promise to correct abnormal splicing of several other genes (Coady et al, 2007;Puttaraju et al, 1999;Rodriguez-Martin et al, 2005). Trans-splicing is based on studies showing that two pre-mRNAs can undergo transsplicing if sufficient homologies between the two pre-mRNAs exist, leading to the first part of the mRNA from exons of premRNA1 and the second part from exons of pre-mRNA2.…”

Progress in therapeutic antisense applications for neuromuscular disorders

“…74 Alternatively, infection with AAV or lentiviral vectors expressing bifunctional RNAs or inducible U7 snRNPs targeting exon 7 and simultaneously recruiting SR proteins enhanced exon 7 inclusion in patient-derived fibroblasts that was accompanied by increased SMN levels. [75][76][77] Further proof of concept for this approach has been presented recently with the generation of transgenic mouse models expressing bifunctional RNAs in an SMN1-negative background, but with a copy of the human SMN2 Figure 2 Exonic and intronic splicing enhancers and silencers. (a) PremRNA splicing is a highly complex process that is orchestrated by the spliceosome, and involves hundreds of different proteins and small nuclear RNAs (snRNAs).…”