Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which intr
ons 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 i
n which RNA pre-therapeutic molecules (PTMs) are designed to recode a speci
fic pre-mRNA by suppressing cis-splicing while enhancing trans-splicing bet
ween the PTM and its pre-mRNA target. This study examined the feasibility o
f SMaRT as a potential therapy for genetic diseases to correct mutations us
ing cystic fibrosis (CF) as an example. We used several versions of a cysti
c fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing
mutant (Delta F508) pre-mRNA targets and tested this against a number of PT
Ms 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 mRN
A in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT
produced mature CFTR protein, proteins were immunoprecipitated from lysate
s of cotransfected cells and detected by Western blotting and PKA-phosphory
lation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This
proof-of-concept study demonstrates that exon replacement by SMaRT can rep
air an abnormal pre-mRNA associated with a genetic disease.