An enzymatic assay for intracellular lamivudine triphosphate to enable objective antiretroviral adherence monitoring

Abstract

Antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) have overcome significant obstacles in the treatment and prevention of human immunodeficiency virus (HIV). However, poor adherence can lead to adverse patient outcomes, including drug resistance and treatment failure. Adherence measurement is therefore vital for efficacious HIV treatment to ensure dose optimizations and timely interventions. The REverse transcriptase ACTivity with CRISPR-enabled readout (REACTR) assay measures drug levels based on the inhibition of the HIV reverse transcriptase (RT) for deoxyribonucleic acid (DNA) synthesis. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complexes and fluorescence reporters, synthesis can be tracked via enzyme activity in real-time through fluorescence output, with high HIV RT activity producing higher fluorescence. A noticeable gap in the development of REACTR is the lack of testing with other classes of antiretroviral drugs such as lamivudine triphosphate (3TC-TP). To allow for compatibility of 3TC-TP, a new DNA template has been developed to provide an adequate drug inhibition curve, taking length, nucleotide composition, and secondary structures into consideration. Distinguishability of drug concentrations was improved utilizing these new templates, showcasing the tunability of REACTR. For further optimization and exploration of REACTR components, components were tuned to showcase a preference for an imbalance in deoxynucleotide triphosphates (dNTP) at a rate of 1 dCTP per 20 dTAGTP. Early dried blood spot testing has showed adequate results in detecting spiked clinically relevant 3TC-TP concentrations with high specificity and sensitivity. Future developments will move to enable lateral flow readouts and test clinical samples.