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bioRxiv · 10.64898/2025.12.08.693028

REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring

Chernoske, W. A.·Singh, M. A.·Lin, C. H.·Chang, M. M.·Craig, C. A.·Park, A. H.·Rower, J. E.·Olanrewaju, A. O.
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Abstract

BackgroundCytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. Although effective at treating CMV, the first-line drug - ganciclovir (GCV) - has high rates of pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular concentrations of ganciclovir triphosphate (GCV-TP) - GCVs active anabolite - are associated with dose-dependent neutropenia and could enable dose individualization to improve treatment efficacy and reduce adverse effects. However, GCV-TP is currently measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) which is impractical for routine use, especially in resource-limited settings, because of its high cost, labor-intensiveness, and need for specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. MethodsWe leveraged our earlier work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously also inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. We evaluated the assays analytical performance with 40 dried blood spots from infants with congenital CMV. ResultsREACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (r = -0.7891; p<0.0001). ConclusionsThis study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV.

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Chernoske, W. A., Singh, M. A., Lin, C. H., Chang, M. M., Craig, C. A., Park, A. H., Rower, J. E., Olanrewaju, A. O.. 2025-12-08. REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring. https://doi.org/10.64898/2025.12.08.693028

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