An antisense oligonucleotide efficiently suppresses splicing of an alternative exon in vascular smooth muscle in vivo

Targeting alternative exons for therapeutic gain has been achieved in a few instances and potentially could be applied more broadly. The myosin phosphatase (MP) enzyme is a critical hub upon which signals converge to regulate vessel tone. Alternative exon 24 of myosin phosphatase regulatory subunit (Mypt1 E24) is an ideal target as toggling between the two isoforms sets smooth muscle sensitivity to vasodilators such as nitric oxide (NO). This study aimed to develop a gene-based therapy to suppress splicing of Mypt1 E24 thereby switching MP enzyme to the NO-responsive isoform. CRISPR/Cas9 constructs were effective at editing of Mypt1 E24 in vitro; however, targeting of vascular smooth muscle in vivo with AAV9 was inefficient. In contrast, an octo-guanidine conjugated antisense oligonucleotide targeting the 5' splice site of Mypt1 E24 was highly efficient in vivo. It reduced the percent splicing inclusion of Mypt1 E24 from 80% to 10% in mesenteric arteries. The maximal and half-maximal effects occurred at 12.5 and 6.25 mg/kg, respectively. The effect persisted for at least 1 mo without toxicity. This highly effective splice-blocking antisense oligonucleotide could be developed as a novel therapy to reverse vascular dysfunction common to diseases such as hypertension and heart failure.NEW & NOTEWORTHY Alternative exon usage is a major driver of phenotypic diversity in all cell types including smooth muscle. However, the functional significance of most of the hundreds of thousands of alternative exons has not been defined, nor in most cases even tested. If their importance to vascular function were known these alternative exons could represent novel therapeutic targets. Here, we present injection of Vivo-morpholino splice-blocking antisense oligonucleotides as a simple, efficient, and cost-effective method for suppression of alternative exon usage in vascular smooth muscle in vivo.