Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

• Published in: Gene therapy Vol. 7; no. 22; pp. 1885 - 1895
• Main Authors: MANSFIELD, S. G, KOLE, J, PUTTARAJU, M, YANG, C. C, GARCIA-BLANCO, M. A, COHN, J. A, MITCHELL, L. G
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01.11.2000
• Subjects: Biological and medical sciences; Biotechnology; Blotting, Western; Cation Exchange Resins; Cell Line; CFTR protein; Colon - cytology; Cystic fibrosis; Cystic Fibrosis - therapy; Cystic fibrosis transmembrane conductance regulator; Cystic Fibrosis Transmembrane Conductance Regulator - analysis; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Exons; Feasibility Studies; Fundamental and applied biological sciences. Psychology; Gene mapping; Gene therapy; Genetic disorders; Genetic Engineering - methods; Genetic Therapy - methods; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Introns; Kidney - embryology; Lipids; Lysates; mRNA; Mutants; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors - genetics; RNA Splice Sites; Spliceosomes; Spliceosomes - genetics; Splicing; Transfection - methods; transmembrane conductance regulator; Western blotting; Human; Splicing; Respiratory disease; Cystic fibrosis; Genetic disease; Cell line; Messenger RNA; Gene; Deletion; Digestive diseases; Mutation; Cystic fibrosis transmembrane conductance regulator; Repair; Gene therapy; Pancreatic disease
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3301307

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (deltaF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10-24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of cotransfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.