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

Journal Article


Abstract


  • Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common ΔF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-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 shown 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 lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

UOW Authors


Publication Date


  • 2001

Citation


  • Kapsa, R., Quigley, A., Lynch, G. S., Steeper, K., Kornberg, A. J., Gregorevic, P., . . . Byrne, E. (2001). In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement. Human Gene Therapy, 12(6), 629-642. doi:10.1089/104303401300057324

Scopus Eid


  • 2-s2.0-0035831649

Start Page


  • 629

End Page


  • 642

Volume


  • 12

Issue


  • 6

Abstract


  • Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common ΔF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-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 shown 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 lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

UOW Authors


Publication Date


  • 2001

Citation


  • Kapsa, R., Quigley, A., Lynch, G. S., Steeper, K., Kornberg, A. J., Gregorevic, P., . . . Byrne, E. (2001). In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement. Human Gene Therapy, 12(6), 629-642. doi:10.1089/104303401300057324

Scopus Eid


  • 2-s2.0-0035831649

Start Page


  • 629

End Page


  • 642

Volume


  • 12

Issue


  • 6