In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement

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 .