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Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides

Lookup NU author(s): Dr Umar Burki, Jonathan Keane, Dr Alison Blain, Dr Steven Laval, Professor Volker StraubORCiD

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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 (2OMe) 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 2OMe 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 (12.5mg/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.

Publication metadata

Author(s): Burki U, Keane J, Blain A, O'Donovan L, Gait MJ, Laval SH, Straub V

Publication type: Article

Publication status: Published

Journal: Nucleic Acid Therapeutics

Year: 2015

Volume: 25

Issue: 5

Pages: 275-284

Print publication date: 17/09/2015

Online publication date: 15/07/2015

ISSN (print): 2159-3337

ISSN (electronic): 2159-3345

Publisher: Mary Ann Liebert, Inc.

URL: http://dx.doi.org/10.1089/nat.2014.0528

DOI: 10.1089/nat.2014.0528

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