title: Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.
abstract: 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.
authors: Intronn, LLC, Durham, NC 27701, USA.
authors: S G
authors: SG
authors: Mansfield
authors: J
authors: J
authors: Kole
authors: M
authors: M
authors: Puttaraju
authors: C C
authors: CC
authors: Yang
authors: M A
authors: MA
authors: Garcia-Blanco
authors: J A
authors: JA
authors: Cohn
authors: L G
authors: LG
authors: Mitchell
mesh_terms: D015153:Blotting, Western; D002411:Cation Exchange Resins; D002460:Cell Line; D003106:Colon / Q000166:cytology; D003550:Cystic Fibrosis / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000235:genetics*; D005091:Exons; D005240:Feasibility Studies; D005818:Genetic Engineering / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000196:embryology; D008055:Lipids; D012322:RNA Precursors / Q000235:genetics*; D022821:RNA Splice Sites; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017381:Spliceosomes / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: C505032:CFTR protein, human; D002411:Cation Exchange Resins; D008055:Lipids; C086724:Lipofectamine; D012322:RNA Precursors; D022821:RNA Splice Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator