Study of transcription factors and application of CRISPR-Cas9 strategy to target intron 1 splice site mutation of a patient with X-linked dilated cardiomyopathy in restoring the dystrophin level in the cardiac muscle cells

Dr Chan, Hoi Shan Sophelia   (Principal Investigator (PI))

Professor Chan Godfrey Chi Fung   (Co-Investigator)

Dr Tan-Un Kian Cheng   (Co-Investigator)

Dr Pijnappel W.W.M. Pim   (Co-Investigator)

24

2017-10-10

2019-10-09

150000

Study of transcription factors and application of CRISPR-Cas9 strategy to target intron 1 splice site mutation of a patient with X-linked dilated cardiomyopathy in restoring the dystrophin level in the cardiac muscle cells

CRISPR/Cas9, Dystrophin gene, Induced Pluripotent Stem Cells, Intron 1 mutation, Transcription Factors, X Linked Dilated Cardiomyopathy

Stem Cell Therapy,Paediatrics

201711159006

Seed Fund for Basic Research for New Staff

2017

Completed

X-linked dilated cardiomyopathy (XLDCM) is a serious condition with high morbidity and mortality. Most of these patients presented with early signs of heart failure and deteriorated quickly despite medical treatment so resulted in early death. Currently there is no curative treatment and heart transplantation is often required. It is therefore an important condition for study researching on treatment and development of therapies. What we know from the literatures are those patients with splice site mutation in intron 1 of the dystrophin gene, developed X-linked dilated cardiomyopathy (XLDCM), a cardiospecific phenotype with progressive dilated cardiomyopathy but minimal skeletal muscles involvement. Cardiac muscle biopsy of this group of affected patients confirmed absence of dystrophin protein expression in the myocardium. The dystrophin protein is a major component of the dystrophin-glycoprotein complex on the muscle cell membrane maintaining the healthy function of the muscle cells. Reduced or absence of dystrophin increases the susceptibility of the muscle cells to the mechanical stress and therefore early muscle cell damage, inflammation, necrosis and eventually fibrosis and fatty replacement in both skeletal and cardiac muscles leading to muscle weakness and dilated cardiomyopathy. Previous studies on the more detail dystrophin isoform expression on this group of patients with XLDCM having splice site mutation in the specific region in intron 1 also confirmed a persistent pattern of the three major dystrophin isoforms expression. Absence of all muscle(M)-, brain(B) and Purkinje(P) dystrophin isoforms expression in the cardiac muscles, but an upregulation of the brain(B) and Purkinji(P) dystrophin isoforms in the skeletal muscles have been consistently found in previous studies. Milasin et al,, isolated the total RNA from frozen endomyocardial and skeletal biopsies of the affected patient, and found the complete absence of all the 3 dystrophin isoforms, namely, muscle, brain and Purkinjee mRNA in the endomyocardial muscles of the affected patient. However, the brain- and Purkinje cell dystrophin mRNA isoforms were detected in the skeletal muscles but not the muscle dystrophin isoform. In another study on two patients with XLDCM, who had mutations again in a similar region with deletion of the 3’-end of exon 1 and part of intron 1, activation of expression for both brain and Purkinje dystrophin isoforms in skeletal muscles was noted again. The findings suggest that the brain and Purkinje dystrophin isoforms have an important ability in maintaining the function of skeletal muscles, and they appear to be crucial in preventing a skeletal myopathy in the two affected males. Bastianutto et al tried to study how these non-muscle dystrophin isoforms (brain and Purkinje dystrophin isoforms) are upregulated in skeletal muscles through the non-muscle promoters. The team studied two patients with XLDCM who had deletions that extended through muscle exon 1 of the dystrophin gene, with preserved first exons of the brain and Purkinje isoforms flanking muscle exon 1. With the deletions removing the M promoter and exon 1, the presence of the dystrophin muscle enhancer 1 (DME1) located in intron 1 was able to lead to an increase in brain and Purkinje promoter activity in the skeletal muscles, but not in the cardiac muscles. Sequences essential for dystrophin gene expression in cardiac muscle is predicted to lie within the region deleted in these 2 patients, while the enhancing elements involved in brain and Purkinje promoter activation in skeletal muscle must be located outside the deletion. What specific regulatory mechanism are involved in this skeletal-muscle-specific activation, and whether other enhancers in addition to DMEI are involved, are important questions that have yet to be answered. Kimura S, et al again identified a mutation in the first exon-intron boundary of the muscle dystrophin isoform gene, c.31+5G>A. The site of mutation is very close to our patient, c31+1G>A. Reverse transcription-PCR analysis again showed that the brain and Purkinje muscle dystrophin isoforms were found in the skeletal muscle of the patient, but not the muscle dystrophin isoform. All muscle, brain and Purkinje isoforms were absent in the cardiac muscle of the patient. Further studies are needed to determine the regulatory mechanism by which the B and P dystrophin isoforms in the heart could be activated. Previous gene editing approach with CRISPR/Cas9 had been applied to patients with different mutations including small to moderate size exons deletion of the dystrophin gene on patients having Duchenne Muscular Dystrophy, a clinical form of dystrophinopathy with predominant progressive skeletal muscle weakness and some also have cardiomyopathy. The studies used CRISPR/Cas9 with different designs on patient-derived induced pluripotent stem cells through both in-vitro studies and in animal studies, and the reported results confirmed encouraging findings These studies have provided evidence that application of CRISPR/Cas9 strategy is a potential treatment approach based on gene editing to dystrophinopathy. On the other hand, there is no previous study using CRISPR/Cas 9 on patients with X -linked dilated cardiomyopathy (XLDCM), a form of dystrophinopathy with predominant cardiac manifestation, or dystrophin gene variants involving splice site mutation. So studies in this area are very much needed. With the above information, this proposed study aims to include two parts: Part 1. To evaluate the underlying mechanism through examination of the expression of different dystrophin isoforms on the disease manifested cardiac and skeletal muscles in a controlled in vitro system. We will focus the study on our patient with c31+1G>A mutation, developed patient-derived induced pluripotent stem cells (iPSCs) and then to differentiate the patient derived iPSCs to the cardiac muscles and skeletal muscles for detail study. We also aim to identify the transcription factors present in skeletal cells that may be responsible in activating the B and P dystrophin isoforms. Subsequently, these transcription factors will be over expressed in cardiomyocytes with the postulation that they may induce expression of the B and P dystrophin isoforms. Part 2. To evaluate the application of gene editing strategy using CRISPR/Cas9 to the patient iPSCs and then study both the patient and the gene-edited iPSC differentiated cardiac muscles and skeletal muscles for dystrophin restoration and dystrophin isoform expression. We would like to prove our hypothesis that gene editing with CRISPR/Cas9 is feasible and can lead to dystrophin restoration in the disease manifested cardiac and skeletal muscles, that is comparable to the cardiac and skeletal muscles from the normal control. We aim to study the feasibility and efficiency of using CRISPR/Cas9 to correct the small 5’ splice site mutation in intron 1 of dystrophin gene, to prevent aberrant splicing and to restore the dystrophin protein expression in the cardiac and skeletal muscle cells.This prove-of-concept study will be the first study to provide evidence of this gene editing approach using CRISPR/Cas9 for XLDCM patients with splice site intron 1 mutation of the dystrophin gene presenting with a cardiospecific phenotype that frequently associated with high mortality unless the patient can undergo heart transplantation. The successful application of this gene editing approach, if proven by this study, will contribute to the knowledge for future development of potential therapeutic investigatory gene-editing treatment for this unique group of patients with high mortality and morbidity due to early progressive cardiomyopathy.