R. Kapsa et al., In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement, HUM GENE TH, 12(6), 2001, pp. 629-642
Targeted genetic correction of mutations in cells is a potential strategy f
or treating human conditions that involve nonsense, missense, and transcrip
tional splice junction mutations. One method of targeted gene repair, singl
e-stranded short-fragment homologous replacement (ssSFHR), has been success
ful in repairing the common Delta F508 3-bp microdeletion at the cystic fib
rosis transmembrane conductance regulator (CFTR) locus in 1% of airway epit
helial cells in culture. This study investigates in vitro and in vivo appli
cation of a double-stranded method variant of SFHR gene repair to the mdx m
ouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR pro
duct was used to repair the exon 23 C-to-T mdx nonsense transition at the X
p21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from ma
le mdx mice. Multiple transfection and variation of lipofection reagent bot
h improved in vitro SFHR efficiency, with successful conversion of mdx to w
ild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci
and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was s
hown to persist for up to 28 days in culture and for at least 3 weeks in TA
. While a high frequency of in vitro gene repair was observed, the lipofect
ion used here appeared to have adverse effects on subsequent cell viability
and corrected cells did not express dystrophin transcript. With further im
provements to in vitro and in vivo gene repair efficiencies, SFHR may find
some application in DMD and other genetic neuromuscular disorders in humans
.