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 preclinical 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.
Highlights
Pharmacokinetic and pharmacodynamic assessment of oligonucleotide and other DNA or RNA-targeting therapeutics in vivo typically involves bioanalytical procedures carried out at tissue level
The problem occurs because the levels of an RNA target measured in an extract from a whole lung lobe reflect the average level of that RNA molecular species across the pool of different cell types that constitute the lung tissue
The extent to which target RNA levels may change on account of oligonucleotide treatment in a given cell is, as a minimum, a function of oligonucleotide transfection efficiency: this efficiency varies between cell types
Summary
Pharmacokinetic and pharmacodynamic assessment of oligonucleotide and other DNA or RNA-targeting therapeutics in vivo typically involves bioanalytical procedures carried out at tissue level. Research on oligonucleotide drugs for respiratory diseases involves agents that work as toll like receptor agonists, splice modulating antisense, in vitro transcribed RNA therapeutics including long noncoding RNAs, mediators of DNA and RNA editing, and RNA interference modulators (microRNA mimics, siRNA, and microRNA inhibitors) This is on account of the recent regulatory approvals of the antisense drugs mipomersen (familial hypercholesterolemia), inotersen (hereditary transthyretinmediated amyloidosis), nusinersen (spinal muscular atrophy; drugs developed by Ionis Pharmaceuticals), and eteplirsen (Duchenne’s muscular dystrophy; developed by Sarepta Therapeutics), the siRNA drug patisiran (hereditary transthyretin-mediated amyloidosis; developed by Alnylam Pharmaceuticals), as well as unprecedented advances involving the development and use of an antisense drug for a single patient harboring a unique mutation, known as “n of 1 medicine,” as exemplified by milasen [10]. With the emergence of single cell ‘omics, the anticipated parallelization, and cost reduction of these newer bioanalytical approaches, we look forward to TDCS-based in vivo pharmacology delivering highly potent, safe, and targeted airway therapeutics
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