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Measuring the Action of Oligonucleotide Therapeutics in the Lung at the Cell Type-Specific Level by Tissue Disruption

Helen Graves1, Steven Evans2, Michael Fauler3

  • 1CamPhos Therapeutics, Cambridge, UK.

Methods in Molecular Biology (Clifton, N.J.)
|August 15, 2019
PubMed

Insights

Developing targeted therapies for airways disease requires better preclinical models. Tissue-dissociated cell sorting (TDCS) improves drug evaluation in vivo by analyzing precise cell populations, reducing animal use and enhancing data reliability.

Area of Science:

  • Pharmacology
  • Genomics
  • Respiratory Medicine

Background:

  • Translating DNA and RNA-targeting therapeutics for airways diseases from in vitro to in vivo models remains a significant challenge.
  • Current preclinical models often show reduced drug efficacy (20-60% target knockdown) in lung tissues compared to cell cultures.
  • Improved methods are needed to accurately assess drug pharmacokinetics and pharmacodynamics in specific lung cell subpopulations.

Purpose of the Study:

  • To introduce and validate a Tissue-Dissociated Cell Sorting (TDCS) methodology for analyzing drug effects in precise lung cell subpopulations.
  • To demonstrate the efficiency and reduced animal usage of the TDCS method compared to standard in vivo pharmacology.
  • To provide a detailed protocol for isolating specific lung cell fractions, including epithelia and macrophages.

Main Methods:

  • The TDCS methodology involves mechanoenzymatic disruption of lung tissue followed by cell sorting.
  • A two-stage sorting process is employed: immunomagnetic bead separation followed by flow cytometry.
  • Fluorescent antibodies against surface markers (F4/80, CD11b, CD326) are used for cell identification and isolation.

Main Results:

  • TDCS enables pharmacokinetic and pharmacodynamic analysis in precise lung cell subpopulations.
  • The TDCS method can reduce animal usage by up to 66% due to lower data variance.
  • Specific lung cell fractions (total lung epithelia, lung macrophages, depleted fractions) can be reliably isolated.

Conclusions:

  • TDCS offers a more accurate and efficient preclinical approach for evaluating DNA and RNA-targeting therapeutics in airways disease.
  • This methodology enhances the translation of drug candidates by providing cell-specific drug effect data.
  • The described TDCS protocol facilitates the study of drug responses in defined lung cell populations, potentially accelerating therapeutic development.
Keywords:
AntisenseCell type-specific in vivo pharmacologyFluorescence activated cell sortingLungMagnetic cell sortingMicroRNAOligonucleotidesRNAisiRNA

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