In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide

Richard J. Bartlett, Sabine Stockinger, Melvin M. Denis, William T. Bartlett, Luca A Inverardi, T. T. Le, Nguyen Thi Man, Glenn E. Morris, Daniel J. Bogan, Janet Metcalf-Bogan, Joe N. Kornegay

Research output: Contribution to journalArticle

137 Citations (Scopus)

Abstract

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)-PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Original languageEnglish
Pages (from-to)615-622
Number of pages8
JournalNature Biotechnology
Volume18
Issue number6
DOIs
StatePublished - Jun 1 2000

Fingerprint

Dystrophin
Oligonucleotides
RNA
Point Mutation
Canidae
Exons
DNA
Repair
Genes
Muscle
Polymerase Chain Reaction
Muscles
Sarcolemma
RNA Splice Sites
Mutation
DNA Mismatch Repair
Duchenne Muscular Dystrophy
Biopsy
Polymerase chain reaction
Transcription

Keywords

  • Canine
  • Chimeric RNA/DNA oligonucleotide
  • Dystrophin
  • In situ RT/PCR
  • Invivo gene repair
  • Quantitative RT/PCR

ASJC Scopus subject areas

  • Microbiology

Cite this

Bartlett, R. J., Stockinger, S., Denis, M. M., Bartlett, W. T., Inverardi, L. A., Le, T. T., ... Kornegay, J. N. (2000). In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nature Biotechnology, 18(6), 615-622. https://doi.org/10.1038/76448

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. / Bartlett, Richard J.; Stockinger, Sabine; Denis, Melvin M.; Bartlett, William T.; Inverardi, Luca A; Le, T. T.; Man, Nguyen Thi; Morris, Glenn E.; Bogan, Daniel J.; Metcalf-Bogan, Janet; Kornegay, Joe N.

In: Nature Biotechnology, Vol. 18, No. 6, 01.06.2000, p. 615-622.

Research output: Contribution to journalArticle

Bartlett, RJ, Stockinger, S, Denis, MM, Bartlett, WT, Inverardi, LA, Le, TT, Man, NT, Morris, GE, Bogan, DJ, Metcalf-Bogan, J & Kornegay, JN 2000, 'In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide', Nature Biotechnology, vol. 18, no. 6, pp. 615-622. https://doi.org/10.1038/76448
Bartlett RJ, Stockinger S, Denis MM, Bartlett WT, Inverardi LA, Le TT et al. In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nature Biotechnology. 2000 Jun 1;18(6):615-622. https://doi.org/10.1038/76448
Bartlett, Richard J. ; Stockinger, Sabine ; Denis, Melvin M. ; Bartlett, William T. ; Inverardi, Luca A ; Le, T. T. ; Man, Nguyen Thi ; Morris, Glenn E. ; Bogan, Daniel J. ; Metcalf-Bogan, Janet ; Kornegay, Joe N. / In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. In: Nature Biotechnology. 2000 ; Vol. 18, No. 6. pp. 615-622.
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