Quantification of exon skipping in Duchenne muscular dystrophy by qRT-PCR

Karen Anthony, Jennifer Morgan, Francesco Muntoni

Research output: Contribution to journalAbstractResearchpeer-review

Abstract

Antisense oligonucleotide (AON)-mediated exon skipping of
the dystrophin gene has emerged as a promising therapeutic
intervention for Duchenne muscular dystrophy (DMD) which
is currently undergoing phase II clinical trials. AON efficacy
is traditionally first assessed by nested RT-PCR due to the
low abundance of dystrophin. However, semi-quantitative end
point detection is extremely variable, time consuming and relies
on imprecise size discrimination. A quantitative assessment of
dystrophin exon skipping has not been performed in any of the
published studies. Here we describe the development of a Taqman
qRT-PCR assay for skipped and unskipped dystrophin targets and
highlight its use to relatively determine the percentage of exon
skipping in DMD patients treated intra-muscularly with AON
AVI-4658. This valuable assay could be used for the systematic
selection of lead AON sequences, and allows for the accurate
assessment of AON efficiency and the stratification of the rate
of response in different patients recruited into clinical trials.
Furthermore, applications for qRT-PCR across the wider field of
the dystrophinopathies include the determination of dystrophin
transcript levels between Becker muscular dystrophy patients with
the same deletion but different levels of protein, adding an exciting
new contribution to existing efforts to quantify protein levels in
these patients.
Original languageEnglish
JournalNeuromuscular Disorders
Publication statusPublished - 2011

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Duchenne Muscular Dystrophy
Antisense Oligonucleotides
Dystrophin
Exons
Polymerase Chain Reaction
Phase II Clinical Trials
Proteins
Clinical Trials
Genes

Cite this

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title = "Quantification of exon skipping in Duchenne muscular dystrophy by qRT-PCR",
abstract = "Antisense oligonucleotide (AON)-mediated exon skipping ofthe dystrophin gene has emerged as a promising therapeuticintervention for Duchenne muscular dystrophy (DMD) whichis currently undergoing phase II clinical trials. AON efficacyis traditionally first assessed by nested RT-PCR due to thelow abundance of dystrophin. However, semi-quantitative endpoint detection is extremely variable, time consuming and relieson imprecise size discrimination. A quantitative assessment ofdystrophin exon skipping has not been performed in any of thepublished studies. Here we describe the development of a TaqmanqRT-PCR assay for skipped and unskipped dystrophin targets andhighlight its use to relatively determine the percentage of exonskipping in DMD patients treated intra-muscularly with AONAVI-4658. This valuable assay could be used for the systematicselection of lead AON sequences, and allows for the accurateassessment of AON efficiency and the stratification of the rateof response in different patients recruited into clinical trials.Furthermore, applications for qRT-PCR across the wider field ofthe dystrophinopathies include the determination of dystrophintranscript levels between Becker muscular dystrophy patients withthe same deletion but different levels of protein, adding an excitingnew contribution to existing efforts to quantify protein levels inthese patients.",
author = "Karen Anthony and Jennifer Morgan and Francesco Muntoni",
year = "2011",
language = "English",
journal = "Neuromuscular Disorders",
issn = "0960-8966",
publisher = "Elsevier",

}

Quantification of exon skipping in Duchenne muscular dystrophy by qRT-PCR. / Anthony, Karen; Morgan, Jennifer; Muntoni, Francesco.

In: Neuromuscular Disorders, 2011.

Research output: Contribution to journalAbstractResearchpeer-review

TY - JOUR

T1 - Quantification of exon skipping in Duchenne muscular dystrophy by qRT-PCR

AU - Anthony, Karen

AU - Morgan, Jennifer

AU - Muntoni, Francesco

PY - 2011

Y1 - 2011

N2 - Antisense oligonucleotide (AON)-mediated exon skipping ofthe dystrophin gene has emerged as a promising therapeuticintervention for Duchenne muscular dystrophy (DMD) whichis currently undergoing phase II clinical trials. AON efficacyis traditionally first assessed by nested RT-PCR due to thelow abundance of dystrophin. However, semi-quantitative endpoint detection is extremely variable, time consuming and relieson imprecise size discrimination. A quantitative assessment ofdystrophin exon skipping has not been performed in any of thepublished studies. Here we describe the development of a TaqmanqRT-PCR assay for skipped and unskipped dystrophin targets andhighlight its use to relatively determine the percentage of exonskipping in DMD patients treated intra-muscularly with AONAVI-4658. This valuable assay could be used for the systematicselection of lead AON sequences, and allows for the accurateassessment of AON efficiency and the stratification of the rateof response in different patients recruited into clinical trials.Furthermore, applications for qRT-PCR across the wider field ofthe dystrophinopathies include the determination of dystrophintranscript levels between Becker muscular dystrophy patients withthe same deletion but different levels of protein, adding an excitingnew contribution to existing efforts to quantify protein levels inthese patients.

AB - Antisense oligonucleotide (AON)-mediated exon skipping ofthe dystrophin gene has emerged as a promising therapeuticintervention for Duchenne muscular dystrophy (DMD) whichis currently undergoing phase II clinical trials. AON efficacyis traditionally first assessed by nested RT-PCR due to thelow abundance of dystrophin. However, semi-quantitative endpoint detection is extremely variable, time consuming and relieson imprecise size discrimination. A quantitative assessment ofdystrophin exon skipping has not been performed in any of thepublished studies. Here we describe the development of a TaqmanqRT-PCR assay for skipped and unskipped dystrophin targets andhighlight its use to relatively determine the percentage of exonskipping in DMD patients treated intra-muscularly with AONAVI-4658. This valuable assay could be used for the systematicselection of lead AON sequences, and allows for the accurateassessment of AON efficiency and the stratification of the rateof response in different patients recruited into clinical trials.Furthermore, applications for qRT-PCR across the wider field ofthe dystrophinopathies include the determination of dystrophintranscript levels between Becker muscular dystrophy patients withthe same deletion but different levels of protein, adding an excitingnew contribution to existing efforts to quantify protein levels inthese patients.

M3 - Abstract

JO - Neuromuscular Disorders

JF - Neuromuscular Disorders

SN - 0960-8966

ER -