CFTR mRNA Based Gene Therapy for cystic fibrosis (CF)

Abstract

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease which affects over 70, 000 people in the world. The responsible gene for CF was identified in 1989, so called CFTR (cystic fibrosis transmembrane conductance -regulator), and CF is caused by over 1300 mutations in CFTR. Among these mutations, deletion phenylalanine 508 (dF508) causes more than 90% cases of CF. Main pathological features are thick and dehydrate airway mucus, high sweat Cl- etc, and major site of CF pathology is lung, accompanying infection by S. aureus and P.aureginosa. Life expectancy is around 30 years. CFTR is localized in chromosome 7q31.2, comprises 27 exons, and the tanscripts is approximately 6.5 kb. CFTR protein is polytopic integral membrane glycoprotein composed of 1480 amino acids, and functions as channel proteins to conduct both Cl- and HCO3- in the apical membrane of epithelial cells. Thus far, many strategies of gene therapy for CF have been investigated. They can be mainly grouped into viral and non-viral strategy. We have worked on mRNA non-viral strategy for CF gene therapy since this year, 2007. There are several advantages to use this strategy: 1. the nuclear membrane, which is major obstacle for DNA non-viral vector can be circumvented. 2. the risk of insertion of mutagenesis can be avoided. 3. the repeated application is possible. 4. vector-induced immunogenicity is avoidable. 5. it is also effective on non-dividing cells. In order to reach a high level of protein, we are currently establishing effective vectors to produce mRNA in vitro. It has been already reported that modified cap structure, ARCA (Anti-reverse cap analogs) and long poly A lead to strong and long lasting expression. We are going to examine if mRNA which is satisfying these conditions is very sufficient for the cure by biochemical analysis, first of all using CHO cell line and CFBE41o- harbouring homozygously dF508.