Jacobs Journal of Genetics

A Short Morpholino CAG15 Corrects Aberrant Alternative Splicing of Clcn1 and Serca1 In Vivo

*Shoichi Ishiura
Department Of Life Sciences, The University Of Tokyo, Japan

*Corresponding Author:
Shoichi Ishiura
Department Of Life Sciences, The University Of Tokyo, Japan
Email:cishiura@mail.ecc.u-tokyo.ac.jp

Published on: 2018-08-28

Abstract

Myotonic dystrophy type 1 (DM1) is a hereditary disease associated with multisystemic disorders including myotonia, muscle weakness, and cataracts. DM1 cells express DMPK mRNA harboring expanded CUG repeats, which are responsible for sequestering MBNL1, an RNA-binding protein. MBNL1 regulates alternative splicing of several genes, and sequestration of MBNL1 in the nucleus possibly causes DM-specific symptoms such as myotonia. Therefore, it has been suggested that blocking the interaction between MBNL1 and the RNA CUG repeats would be a promising therapeutic strategy for DM1. A CAG-repeat morpholino oligonucleotide (MO) is expected to bind to expanded CUG repeats in RNA, thus preventing MBNL1 sequestration. In the present report, we optimized the lengths of relevant MOs. We introduced 15-mer, 20-mer, and 25-mer MOs into Neuro 2a cells, which express CUG repeat-containing RNA. We also injected MOs into mice with DM1 (the HSALR line). Such work showed that the 15-mer MO most effectively corrected aberrant splicing of Clcn1 and Serca1. To increase MO permeability, a 15-mer vivo-MO was administered intravenously and resulted in corrected aberrant Clcn1 splicing in the quadriceps of HSALR mice. These results suggest that the short 15-mer MO most efficiently corrects aberrant splicing.

Keywords

Myotonic dystrophy (DM1); Antisense; Clcn1; Serca1; Alternative Splicing

Introduction

Myotonic dystrophy (DM), an autosomal-dominant disorder, is the most common type of adult muscular dystrophy. There are two types of DM: DM1 and DM2. We focused on DM1, which
is caused by expansion of CTG repeats in the 3′ untranslated region (UTR) of the DMPK gene [1]. The symptoms of DM1 patients include myotonia, muscle weakness, cataracts, insulin- resistance, and cognitive impairment [2]. The cells of DM1 patients express DMPK mRNA with expanded CUG repeat sequences; such RNA sequesters MBNL1, an RNA-binding protein [3, 4]. Protein dysfunction causes aberrant splicing of several genes and is thought to trigger certain symptoms such as myotonia. Some abnormal splicings are evident in DM1 patients. Abnormal splicing of CLCN1 is thought to cause electrical discharge in muscles [5]. Abnormal splicing of SERCA1 may affect intracellular Ca2+ homeostasis (Zhao et al., Biochim.Biophys. Acta, in press submitted). MBNL exon 5 is highly conserved between birds and mammals, and its regulation is thought to be important for modulation of MBNL1 function during embryonic development [6].