Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2

Abstract

Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

“…In general, silent mutations should mimic the codon usage of the original codon as closely as possible. It should be noted that there is evidence in the literature that silent mutations may have unintended consequences on transcription, splicing, and/or translation (e.g., Biro, 2008;Doktor et al, 2014;Nielsen et al, 2007). This must be balanced against the possibility of Cas9 re-cutting and introducing cis-located indels.…”

Developing therapies for spinal muscular atrophy

“…Pigs lack the intron splicing silencer in exon 7 and have modified splicing of SMN1 , so they are not a good model for splice modulation therapies. 48 However, Burghes and colleagues have developed a porcine model using intrathecal delivery of scAAV9-mediated SMN mRNA knockdown that recapitulates the SMA phenotype of motor neuron loss and axon degeneration, as determined by assessment of motor unit function with electromyography (EMG), compound motor action potential (CMAP), and motor unit number estimation (MUNE). 49 Further, the phenotype is rescued by administration of human SMN1 via scAAV9-SMN, providing evidence that this is a very useful pre-clinical model for gene therapy approaches.…”