title: Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides.
abstract: Antisense oligonucleotide (AON)-induced exon skipping is one of the most promising strategies for treating Duchenne muscular dystrophy (DMD) and other rare monogenic conditions. Phosphorodiamidate morpholino oligonucleotides (PMOs) and 2'-O-methyl phosphorothioate (2'OMe) are two of the most advanced AONs in development. The next generation of peptide-conjugated PMO (P-PMO) is also showing great promise, but to advance these therapies it is essential to determine the pharmacokinetic and biodistribution (PK/BD) profile using a suitable method to detect AON levels in blood and tissue samples. An enzyme-linked immunosorbent assay (ELISA)-based method, which shows greater sensitivity than the liquid chromatography-mass spectrometry method, is the method of choice for 2'OMe detection in preclinical and clinical studies. However, no such assay has been developed for PMO/P-PMO detection, and we have, therefore, developed an ultrasensitive hybridization-based ELISA for this purpose. The assay has a linear detection range of 5-250 pM (R(2)>0.99) in mouse serum and tissue lysates. The sensitivity was sufficient for determining the 24-h PK/BD profile of PMO and P-PMO injected at standard doses (12.5 mg/kg) in mdx mice, the dystrophin-deficient mouse model for DMD. The assay demonstrated an accuracy approaching 100% with precision values under 12%. This provides a powerful cost-effective assay for the purpose of accelerating the development of these emerging therapeutic agents.
authors.affiliation: 1 The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, United Kingdom .
authors.forename: Umar
authors.initials: U
authors.lastname: Burki
authors.affiliation: 1 The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, United Kingdom .
authors.forename: Jonathan
authors.initials: J
authors.lastname: Keane
authors.affiliation: 1 The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, United Kingdom .
authors.forename: Alison
authors.initials: A
authors.lastname: Blain
authors.affiliation: 2 Laboratory of Molecular Biology , Medical Research Council, Cambridge, United Kingdom .
authors.forename: Liz
authors.initials: L
authors.lastname: O'Donovan
authors.affiliation: 2 Laboratory of Molecular Biology , Medical Research Council, Cambridge, United Kingdom .
authors.forename: Michael John
authors.initials: MJ
authors.lastname: Gait
authors.affiliation: 1 The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, United Kingdom .
authors.forename: Steven H
authors.initials: SH
authors.lastname: Laval
authors.affiliation: 1 The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, United Kingdom .
authors.forename: Volker
authors.initials: V
authors.lastname: Straub
mesh_terms: D000818:Animals; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics; D004797:Enzyme-Linked Immunosorbent Assay; D007279:Injections, Subcutaneous; D057230:Limit of Detection; D018101:Mice, Inbred mdx; D060172:Morpholinos / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics
chemical_list: D057846:Cell-Penetrating Peptides; D060172:Morpholinos; D016376:Oligonucleotides, Antisense