Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients, which produces low levels of functional protein. Although the SMN2 coding sequence has the potential to produce normal, full-length SMN, approximately 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein lacking the final coding exon (exon 7). SMN2, however, is an excellent therapeutic target. Previously, we developed bifunctional RNAs that bound SMN exon 7 and modulated SMN2 splicing. To optimize the efficiency of the bifunctional RNAs, a different antisense target was required. To this end, we genetically verified the identity of a putative intronic repressor and developed bifunctional RNAs that target this sequence. Consequently, there is a 2-fold mechanism of SMN induction: inhibition of the intronic repressor and recruitment of SR proteins via the SR recruitment sequence of the bifunctional RNA. The bifunctional RNAs effectively increased SMN in human primary SMA fibroblasts. Lead candidates were synthesized as 2'-O-methyl RNAs and were directly injected in the central nervous system of SMA mice. Single-RNA injections were able to illicit a robust induction of SMN protein in the brain and throughout the spinal column of neonatal SMA mice. In a severe model of SMA, mean life span was extended following the delivery of bifunctional RNAs. This technology has direct implications for the development of an SMA therapy, but also lends itself to a multitude of diseases caused by aberrant pre-mRNA splicing.

Repair of pre-mRNA splicing

“…In the second study, SMN levels were also increased in various sections of the spinal cord and a small positive effect on weight development and survival of the mice could be demonstrated. 126 This, so far, has been the only study that has demonstrated a therapeutic effect of an oligonucleotide in a mouse SMA model.…”

“…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. 126 Both of these approaches led to improved exon 7 reinclusion compared to the corresponding simple AONs lacking the functional moiety. They were also tested by intraventricular injections into the brains of SMA mice.…”

A Novel Morpholino Oligomer Targeting ISS-N1 Improves Rescue of Severe Spinal Muscular Atrophy Transgenic Mice

“…However, significant progress in the development of therapeutic strategies has been achieved. These include the following: (1) introducing the exogenous SMN1 gene by viral vectors (Foust et al, 2010;Passini et al, 2010;Valori et al, 2010;Dominguez et al, 2011); (2) increasing the SMN2 transcript and SMN protein by small-molecule drugs, such as histone deacetylase (HDAC) inhibitors (Kernochan et al, 2005;Avila et al, 2007;Garbes et al, 2009;Hauke et al, 2009); (3) transplantation of motor neuron stem cells (Corti et al, 2010); and (4) redirecting SMN2 splicing and increasing the full-length SMN2 transcript by antisense oligonucleotides (AOs) (Skordis et al, 2003;Hua et al, 2007Hua et al, , 2008Hua et al, , 2010Hua et al, , 2011Baughan et al, 2009;Williams et al, 2009;Owen et al, 2011;Passini et al, 2011;Osman et al, 2012;Porensky et al, 2012).…”

Functional correction by antisense therapy of a splicing mutation in the GALT gene

“…8 Effectively, spinal muscular atrophy is a candidate for this therapy, with promising results on patient-derived cell lines that have been already described. [9][10][11] Moreover, many antisense oligonucleotides are already in phase II/III clinical trials, namely AVI4658 (Eteplirsen), a morpholino oligonucleotide for duchenne muscular dystrophy. 12 Classic galactosemia (MIM #230400), which affects about 1/47 000 live-births, results from deficient activity of galactose-1-phosphate uridylyltransferase (GALT, EC 2.7.7.12), an ubiquitous key enzyme in galactose metabolism, essential for nursing infants, as lactose represents their primary carbohydrate source.…”

“…8 Effectively, spinal muscular atrophy is a candidate for this therapy, with promising results on patient-derived cell lines that have been already described. [9][10][11] …”