Reverse Transcriptase Activity with CRISPR (REACTR) Assay for Rapid and User-Friendly Therapeutic Drug Monitoring in...

Area of Science:

• Biotechnology and Biomedical Diagnostics
• Infectious Diseases and Virology
• Pharmacology and Therapeutics

Background:

• Cytomegalovirus (CMV) infection poses severe risks, particularly to infants and immunocompromised individuals.
• Current first-line treatment with ganciclovir faces challenges due to high pharmacokinetic variability, leading to suboptimal exposure or toxicity.
• Monitoring ganciclovir triphosphate (GCV-TP), the active metabolite, is crucial for dose individualization but current measurement methods (LC-MS/MS) are impractical for routine use.

Purpose of the Study:

• To adapt and validate the REverse transcriptase ACTivity with CRISPR (REACTR) assay for measuring GCV-TP.
• To provide a rapid, accessible, and cost-effective alternative to LC-MS/MS for therapeutic drug monitoring of GCV-TP.

Main Methods:

• Adapted the CRISPR-based REACTR assay, leveraging GCV-TP's inhibition of HIV reverse transcriptase.
• Designed custom DNA templates, primers, and CRISPR complexes for GCV-TP detection in buffer and blood.
• Evaluated assay performance using dried blood spots from infants with congenital CMV, comparing results with LC-MS/MS.

Main Results:

• The REACTR assay reproducibly measured clinically relevant GCV-TP concentrations with a simple workflow.
• REACTR measurements showed strong correlation with LC-MS/MS GCV-TP measurements in infant blood samples (Spearman ρ = -0.811; p < 0.0001).
• The assay demonstrated high accuracy in identifying samples above simulated adherence thresholds (AUCs of 0.910 and 0.957).

Conclusions:

• The adapted REACTR assay is a viable and accessible tool for measuring GCV-TP.
• This assay has the potential to significantly improve therapeutic drug monitoring for ganciclovir therapy.
• REACTR offers a promising alternative to LC-MS/MS, especially in resource-limited settings.