Abstract
The past decade has seen the development of new technologies capable of editing the genome that have naturally led to exploring their therapeutic application for the treatment of many disorders. Among those, Duchenne muscular dystrophy (DMD) represents an ideal candidate for gene editing primarily due to the large size of dystrophin, the gene responsible for the disease, which limits the use of gene replacement approaches. Critical in the evaluation of the efficacy of the treatment is the development of a method that can accurately quantitate the frequencies of gene repair obtained in the dystrophin gene at both the genomic level as well as the mRNA level. The mdx 5cv mouse model of DMD offers an ideal system to precisely determine the frequencies of gene repair. Here we describe the methods used for determining those frequencies and the limitations associated with the use of gene correction for the treatment of DMD. Clinical approaches to muscle disorders using ssODNs will heavily rely on the optimization of the technology and will have to take into consideration the safety, efficacy and cost of the procedure in vision of systemic delivery of the therapeutic treatment.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Rando TA, Disatnik MH, Zhou LZ (2000) Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc Natl Acad Sci U S A 97:5363–5368
Bartlett RJ, Stockinger S, Denis MM, Bartlett WT, Inverardi L, Le TT, thi Man N, 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. Nat Biotechnol 18:615–622
Bertoni C, Rando TA (2002) Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides. Hum Gene Ther 13:707–718
Bertoni C, Morris GE, Rando TA (2005) Strand bias in oligonucleotide-mediated dystrophin gene editing. Hum Mol Genet 14:221–233
Bertoni C (2008) Clinical approaches in the treatment of Duchenne muscular dystrophy (DMD) using oligonucleotides. Front Biosci 13:517–527
Kayali R, Bury F, Ballard M, Bertoni C (2010) Site directed gene repair of the dystrophin gene mediated by PNA-ssODNs. Hum Mol Genet 19:3266–3281
Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500
SantaLucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–1465
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Rando TA, Blau HM (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125:1275–1287
Chomczynski P, Mackey K (1995) Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19:942–945
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Schmittgen TD, Livak KJ (2008) Analyzing qPCR data by the comparative C(T) method. Nat Protoc 3:1101–1108
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Mygind T, Birkelund S, Birkebaek NH, Ostergaard L, Jensen JS, Christiansen G (2002) Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae. BMC Microbiol 2:17
Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112–115
Acknowledgments
The author would like to thank Farnoosh Nik-Ahd for technical assistance. This work was supported by funds from the Muscular Dystrophy Association (USA).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bertoni, C. (2014). Methods for the Assessment of ssODN-Mediated Gene Correction Frequencies in Muscle Cells. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-761-7_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-760-0
Online ISBN: 978-1-62703-761-7
eBook Packages: Springer Protocols