Restoration of SMN Function: Delivery of a Trans-splicing RNA Re-directs SMN2 Pre-mRNA Splicing

Abstract

Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients; however SMN2 produces low levels of functional protein due to alternative splicing. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre-messenger RNA (pre-mRNA) splicing occurring between two separate molecules: (i) the endogenous target RNA and (ii) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. SMN trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from a SMN2 mini-gene as well as from endogenous transcripts. Subsequently, recombinant adeno-associated viral vectors were developed that expressed and delivered trans-splicing RNAs to SMA patient fibroblasts. In the severe SMA patient fibroblasts, SMN2 splicing was redirected via trans-splicing to produce increased levels of full-length SMN mRNA and total SMN protein levels. Finally, small nuclear ribonucleoprotein (snRNP) assembly, a critical function of SMN, was restored to SMN-deficient SMA fibroblasts following treatment with the trans-splicing vector. Together these results demonstrate that the alternatively spliced SMN2 exon 7 is a tractable target for replacement by trans-splicing.

Repair of pre-mRNA splicing

“…In particular, Coady and coworkers have developed a therapeutic trans-splicing RNA base-pairing with the intron 6 of SMN2 and containing the SMN1 exon 7 sequence. 151 The final product Figure 4. Scheme depicting splicing correction strategies for SMA based on in vivo expressed rNAs (A) SnrNA-based strategies.…”

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

“…18 SMaRT has been shown to repair defective pre-mRNA molecules in cultured mammalian cells, xenografts and animal models of human disease including cystic fibrosis, 12 hemophilia A (factor VIII deficiency), 13 X-linked CD40 ligand immunodeficiency, 14 human apolipoprotein A1, 15 DNA protein kinase deficiency, 19 epidermolysis bullosa simplex with muscular dystrophy 20 and spinal muscular atrophy. 21 In DM1, a strategy to remove or correct mutant mRNA is appropriate in view of evidence that the accumulation of mutant mRNA in the nucleus is the primary basis of the disease. 3,22 Approaches targeted at the mRNA level to remove mutant mRNA, namely the use of antisense, 23 nuclear-retained hammerhead ribozyme 4 and siRNA, 5 have been shown to be able to remove mutant mRNA in in vitro DM1 cells.…”

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.…”

“…This strategy has been comprehensively explored by Lorson's group during the last few years to correct SMN2 splicing. They optimized trans-splicing strategies and demonstrated that transsplicing increases exon 7 insertion into SMN2 mRNA and extend the life of animals with SMA (Coady and Lorson, 2010;Coady et al, 2007;Shababi and Lorson, 2011;Shababi et al, 2011).…”

“…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.…”