Rescuing CFTR expression of mutation specific polymorphisms in Cystic Fibrosis patients — ASN Events
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  2. Poster Session 2
  3. Rescuing CFTR expression of mutation specific polymorphisms in Cystic Fibrosis patients

Rescuing CFTR expression of mutation specific polymorphisms in Cystic Fibrosis patients (#242)

Kelly M Martinovich 1 2 3 , Anthony Kicic 1 2 4 5 , Sue Fletcher 3 6 , Stephen D Wilton 3 6 , Stephen M Stick 1 2 4 5 , on behalf of AREST-CF 1 5 7 8
  1. Telethon Kids Institute, Subiaco, WA, Australia
  2. School of Paediatrics and Child Health, The University of Western Australia, Nedlands, WA, Australia
  3. Centre for Comparative Genomics, Murdoch University, Perth, WA, Australia
  4. Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Nedlands, WA, Australia
  5. Department of Respiratory Medicine, Princess Margaret Hospital for Children, Subiaco, WA, Australia
  6. Western Australian Neuroscience Research Institute and Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Perth, Western Australia, Australia
  7. Department of Respiratory Medicine, Royal Children’s Hospital, Melbourne, Victoria, Australia
  8. Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

Background

 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations result in cystic fibrosis (CF) disease and a broad spectrum of clinical phenotypes are observed in CF patients. Some patients show a severe disease phenotype with pulmonary dysfunction and pancreatic insufficiency, while others present with a mild ‘CF-like’ disease or congenital bilateral absence of the vas deferens (CBAVD). Currently, over CFTR 2000 mutations have been identified and one mutation, Arg117His affects the conductivity of the CFTR channel and can result in a mild or severe phenotype, influenced by co-location of the mutation and an intron 9 polymorphism, a poly T tract varying from 5 to 9 nucleotides [1]. The shorter ‘5T’ allele weakens the intron 9 acceptor site and promotes exclusion of exon 10 from the mature CFTR transcript, resulting in a non-functional CFTR channel, leading to a more severe disease [2-4]. Manipulation of CFTR pre-RNA splicing using antisense oligonucleotides (AOs) is a potential therapy for those CF patients with this particular mutation [5].  This study explores antisense therapy to correct abnormal splicing of CFTR RNA and improve CFTR function.

Aim

To develop splice modulating antisense oligonucleotides to rescue CFTR function in CF patients that carry Arg117His and a 5T polymorphism in intron 9.

Methods

AOs targeting CFTR intron 9, around the specific 5T polymorphism, were designed and transfected into monolayer primary airway epithelial cells [6] from a CF patient [7] harbouring this disease-associated polymorphism. Altered splicing was assessed by RNA extraction and amplification of the CFTR transcript by RT-PCR. The resultant amplicons were fractionated on agarose gels to visualise the transcripts, before and after treatment with splice-switching AOs. The ratios of full-length product to the products missing exon 10 were determined using densitometry, and AOs that enhanced exon 10 inclusion in the mRNA identified. AOs that increase the levels of full length CFTR transcript will then be assessed for CFTR function using an Ussing Chamber and CF patient primary epithelial cells grown at the Air-liquid interface.

 

We propose that corrected splicing of the CFTR 5T allele will improve function in CF patients carrying selected mutations, either alone or in combination with current therapeutics.

  1. Chu, C.S., et al., Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium. EMBO J, 1991. 10(6): p. 1355-63.
  2. Kiesewetter, S., et al., A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet, 1993. 5(3): p. 274-8.
  3. Chu, C.S., et al., Extensive posttranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts of the cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis. J Clin Invest, 1992. 90(3): p. 785-90.
  4. Chu, C.S., et al., Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet, 1993. 3(2): p. 151-6.
  5. Fletcher, S., et al., Antisense suppression of donor splice site mutations in the dystrophin gene transcript. Mol Genet Genomic Med, 2013. 1(3): p. 162-73.
  6. Kicic, A., et al., Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma. Am J Respir Crit Care Med, 2006. 174(10): p. 1110-8.
  7. Garratt, L.W., et al., Determinants of culture success in an airway epithelium sampling program of young children with cystic fibrosis. Exp Lung Res, 2014. 40(9): p. 447-59.
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