Measuring the action of oligonucleotide therapeutics in the lung at the cell type specific level by Tissue Disruption and Cell Sorting (TDCS): In vivo cell type-specific oligonucleotide PK/PD

Helen Graves, Steve Evans, Michael Fauler, Manfred Frick, Sterghios Moschos

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

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Abstract

The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the clinic. For example, classical pre-clinical approaches routinely report 20-60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro. Preparation of monocellular suspensions of Tissues by mechanoenzymatic Disruption followed by Cell Sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.
Original languageEnglish
Title of host publicationOligonucleotide-based Therapies
EditorsOlof Gissberg, Rula Zain, Karin Lundin
Place of PublicationNew York, NY
PublisherSpringer
Pages187-203
ISBN (Electronic)9781493996704
ISBN (Print)9781493996698
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Number2036
ISSN (Print)1064-3745

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