Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides
| Autor: | M J Gait, Steven H. Laval, Liz O'Donovan, Umar Burki, Straub, Jonathan Keane, A. Blain |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Biodistribution
Duchenne muscular dystrophy Injections Subcutaneous Peptide Enzyme-Linked Immunosorbent Assay Cell-Penetrating Peptides Conjugated system Biochemistry Morpholinos Limit of Detection Drug Discovery Genetics medicine Methods Animals Elisa method Molecular Biology chemistry.chemical_classification Chemistry Oligonucleotide Oligonucleotides Antisense medicine.disease Molecular biology Exon skipping Morpholino Oligonucleotides Mice Inbred mdx Molecular Medicine |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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