<scp>REverSe TRanscrIptase</scp> chain termination (<scp>RESTRICT</scp>) for selective measurement of nucleotide analogs used in <scp>HIV</scp> care and prevention

Olanrewaju, Ayokunle; Sullivan, Benjamin P.; Gim, Alicia; Craig, Cosette A.; Sevenler, Derin; Bender, Andrew T.; Drain, Paul K.; Posner, Jonathan D.

doi:10.1002/btm2.10369

Cited by 3 publications

(2 citation statements)

References 74 publications

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“…We first tested using CasPART for therapeutic drug monitoring. Measuring antiretroviral drug concentrations in patient samples can enable identification of interindividual differences in drug levels based on missed doses, drug-drug interactions, or differences in drug pharmacokinetics due to age, pregnancy, or body size [55,56]. As a proof-of-concept, we tested measuring rilpivirine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in ART regimens.…”

Section: Resultsmentioning

confidence: 99%

“…However, prior HIV RT activity assays with PCR-based readout showed that as low as 10% mutant mixed with wild type HIV RT can be detected [62]. The substrate sequence in CasPART could also be systematically designed to match the targeted NRTI drug, to maximize enzyme inhibition events and potentially further favor the detection of MUT RT [56]. Future work is needed to investigate the sensitivity of CasPART in detecting the MUT proportion and optimize assay conditions such as drug concentrations accordingly.…”

Section: Caspart Assay For Phenotypic Characterization Of Hiv Rt Enzymementioning

confidence: 99%

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Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas

Wang,

Kline,

Gilligan-Steinberg

et al. 2024

Preprint

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Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25μL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.

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Section: Resultsmentioning

confidence: 99%

Section: Caspart Assay For Phenotypic Characterization Of Hiv Rt Enzymementioning

confidence: 99%

Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas

Wang,

Kline,

Gilligan-Steinberg

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention

Brainerd,

Singh,

Tatka

et al. 2024

Anal Bioanal Chem

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Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout

Singh,

Chang,

Wang

et al. 2024

Preprint

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Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR’s sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

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REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention

Cited by 3 publications

References 74 publications

Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas

Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas

REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention

Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout

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