Target Amplification Research Articles

Rapid Multiplex PCR for Species Authentication: Current Prospects and Challenges

Multiplex PCR is a powerful technique that allows simultaneous amplification of multiple DNA targets in a single reaction. It has been widely applied for species authentication in various food matrices, such as meat, fish, dairy, and honey products. Species authentication is important for ensuring food safety, quality, traceability, and compliance with religious, ethical, and environmental standards. However, multiplex PCR also faces some challenges and limitations, such as primer design, optimization, validation, interference, and interpretation of results. This review summarizes the current state of the art of multiplex PCR for species authentication, highlighting its advantages, applications, challenges, and future directions. It also provides some practical guidelines and recommendations for designing and performing multiplex PCR assays for reliable and accurate species identification.

DNA-Binding Magnetic Nanoreactor Beads for Digital PCR Analysis.

Digital PCR (dPCR) is based on the separation of target amplification reactions into many compartments with randomly distributed template molecules. Here, we present a novel digital PCR format based on DNA binding magnetic nanoreactor beads (mNRBs). Our approach relies on the binding of all nucleic acids present in a sample to the mNRBs, which both provide a high-capacity binding matrix for capturing nucleic acids from a sample and define the space available for PCR amplification by the internal volume of their hydrogel core. Unlike conventional dPCR, this approach does not require a precise determination of the volume of the compartments used but only their number to calculate the number of amplified targets. We present a procedure in which genomic DNA is bound, the nanoreactors are loaded with PCR reagents in an aqueous medium, and amplification and detection are performed in the space provided by the nanoreactor suspended in fluorocarbon oil. mNRBs exhibit a high DNA binding capacity of 1.1 ng DNA/mNRB (95% CI 1.0-1.2) and fast binding kinetics with ka = 0.21 s-1 (95% CI 0.20-0.23). The dissociation constant KD was determined to be 0.0011 μg/μL (95% CI 0.0007-0.0015). A simple disposable chamber plate is used to accommodate the nanoreactor beads in a monolayer formation for rapid thermocycling and fluorescence detection. The performance of the new method was compared with conventional digital droplet PCR and found to be equivalent in terms of the precision and linearity of quantification. In addition, we demonstrated that mNRBs provide quantitative capture and loss-free analysis of nucleic acids contained in samples in different volumes.

More than magnetic isolation: Dynabeads as strong Raman reporters toward simultaneous capture and identification of targets

AbstractDynabeads are superparamagnetic particles used for the immunomagnetic purification of cells and biomolecules. Post‐capture, however, target identification relies on tedious culturing, fluorescence staining, and/or target amplification. Raman spectroscopy presents a rapid detection alternative, but current implementations target cells themselves with weak Raman signals. We present antibody‐coated Dynabeads as strong Raman reporter labels whose effect can be considered a Raman parallel of immunofluorescent probes. Recent developments in techniques for separating target‐bound Dynabeads from unbound Dynabeads make such an implementation feasible with high specificity. We deploy Dynabeads anti‐Salmonella to bind and identify Salmonella enterica, a major foodborne pathogen. Dynabeads present major peaks around 1000 and 1600 cm−1 from aliphatic and aromatic C‐C stretching of the polystyrene coating and near 1350 cm−1 from the ɣ‐Fe2O3 and Fe3O4 core, confirmed with electron dispersive X‐ray (EDX) imaging. Minor to no contributions are made from the surface antibodies themselves as confirmed by Raman analysis of surface‐activated, antibody‐free beads. Dynabeads' Raman signature can be measured in dry and liquid samples even at single shot ~30 × 30 μm area imaging using 0.5 s, 7‐mW laser acquisition with single and clustered beads providing a 44‐ and 68‐fold larger Raman intensity compared with the signature from cells. Higher polystyrene and iron oxide content in clusters yields larger signal intensity, and conjugation to bacteria strengthens clustering as a bacterium can bind to more than one bead as observed via transmission electron microscopy (TEM). Our findings shed light on the intrinsic Raman reporter nature of Dynabeads. When combined with emerging techniques for the separation of target‐bound Dynabeads from unbound Dynabeads such as using centrifugation through a density media bilayer, they have the potential to demonstrate their dual function for target isolation and detection without tedious staining steps or unique plasmonic substrate engineering, advancing their applications in heterogeneous samples like food, water, and blood.

Tandem CRISPR nucleases-based lateral flow assay for amplification-free miRNA detection via the designed “locked RNA/DNA” as fuels

Currently, available biosensors based on CRISPR/Cas typically depend on coupling with nucleic acid amplification technologies to enhance their sensitivity. However, this approach often involves intricate amplification processes, which could be time-consuming and susceptible to contamination. In addition, signal readouts often require sophisticated and cumbersome equipment, obstructing the applicability of CRISPR/Cas assays in resource-limited regions. Herein, a tandem CRISPR/Cas13a/Cas12a mechanism (tanCRISPR) has been developed via the designed “Locked RNA/DNA” probe as fuels for the trans-cleavage nucleic acid of Cas13a and activated nucleic acid of Cas12a. Meanwhile, a lateral flow assay (LFA) is designed to combine with this tandem CRISPR/Cas13a/Cas12a mechanism (termed tanCRISPR-LFA), realizing the portable monitoring of miRNA-21. The tanCRISPR could realize the limit of detection at pM levels (266 folds lower than Cas13a-based miRNA testing alone) without the resort to target amplification procedures. Furthermore, the miRNA-21 levels of MDA-MB-231 cell extracts are sensed by tanCRISPR-LFA, which is comparable to qRT-PCR. With the virtues of portability, rapidity, sensitivity, and low cost, tanCRISPR-LFA is amenable for CRISPR/Cas-based biosensing and potential applications in the clinical diagnosis of miRNA-associated diseases.

Aerosol Jet Printing-Enabled Dual-Function Electrochemical and Colorimetric Biosensor for SARS-CoV-2 Detection.

An aerosol jet printing-enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13:guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

Evolution of CRISPR-enabled biosensors for amplification-free nucleic acid detection

CRISPR biosensors enable rapid and accurate detection of nucleic acids without resorting to target amplification. Specifically, these systems facilitate the simultaneous detection of multiple nucleic acid targets with single-base specificity. This is an invaluable asset that can ultimately facilitate accurate diagnoses of biologically complex diseases.

Molecular Recognition-Modulated Hetero-Assembly of Nanostructures for Visualizable and Portable Detection of Circulating miRNAs.

Biomolecular markers, particularly circulating microRNAs (miRNAs) play an important role in diagnosis, monitoring, and therapeutic intervention of cancers. However, existing detection strategies remain intricate, laborious, and far from being developed for point-of-care testing. Here, we report a portable colorimetric sensor that utilizes the hetero-assembly of nanostructures driven by base pairing and recognition for direct detection of miRNAs. Following hybridization, two sizes of nanoparticles modified with single-strand DNA can be robustly assembled into heterostructures with strong optical resonance, exhibiting distinct structure colors. Particularly, the large nanoparticles are first arranged into nanochains to enhance scattering signals of small nanoparticles, which allows for sensitive detection and quantification of miRNAs without the requirement of target extraction, amplification, and fluorescent labels. Furthermore, we demonstrate the high specificity and single-base selectivity of testing different miRNA samples, which shows great potential in the diagnosis, staging, and monitoring of cancers. These heterogeneous assembled nanostructures provide an opportunity to develop simple, fast, and convenient tools for miRNAs detection, which is suitable for many scenarios, especially in low-resource setting.

Droplet digital PCR versus real-time PCR for in-house validation of porcine detection and quantification protocol: An artificial recombinant plasmid approach.

Authenticity and traceability are essential for modern food and medicine inspection, and reliable techniques are important for the trade of halal foods, which reach more than 20 percent of the world market. A sensitive and accurate porcine detection method is required to develop a conformity assessment system that includes laboratory testing for porcine-free certification. This study proposes a procedure that could be incorporated into the development of a standardized control and protocol for real-time PCR (qPCR) methods and their traceability using droplet digital PCR (ddPCR). The design used a recombinant pUC57 plasmid as an amplification target to carry the 97 bp fragment of the porcine ATCB gene. The absolute quantification and linearity assessment showed high precision with R2 values of 0.9971 and 0.9998 for qPCR and ddPCR, respectively. In general, both methods showed comparable results in terms of linearity and detection limit. However, both limit of detection assessments showed high sensitivity, although ddPCR showed a slightly higher sensitivity than that of qPCR, especially at low DNA concentrations. Multiple-sample and inter-participatory testing evaluations revealed a high sensitivity, broad applicability, and robustness of the qPCR method. Therefore, we conclude that based on a recombinant plasmid analysis with a low quantity (less than five copy number), the digital PCR method produced more reliable results. These results could provide scientific information for regulatory authorities, especially those in Indonesia, to consider the development and formulation of a well-established qPCR protocol for porcine detection using expected DNA concentrations.

An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

Obtainment and confirmation of intergeneric hybrids between marguerite (Argyranthemum frutescens (L.) Sch.Bip.) and two Rhodanthemum species (R. hosmariense (Ball) B. H. Wilcox, K. Bremer & Humphries and R. catananche (Ball) B. H. Wilcox, K. Bremer & Humphries).

Argyranthemum frutescens (L.) Sch.Bip. and Rhodanthemum gayanum (Coss. & Durieu) B. H. Wilcox, K. Bremer & Humphries are capable of hybridization. To expand flower color variation in this intergeneric hybrid group, we performed crosses using A. frutescens as the seed parent and R. hosmariense (Ball) B. H. Wilcox, K. Bremer & Humphries, R. catananche (Ball) B. H. Wilcox, K. Bremer & Humphries as the pollen parent. One plantlet was obtained from each cross between the white to pale pink-flowered A. frutescens and white-flowered R. hosmariense, and from a cross between the pink-flowered A. frutescens and cream to pale yellow-flowered R. catananche, via ovule culture. The cross with R. hosmariense produced an individual with white to pale pink ray florets, and the cross with R. catananche produced an individual with red ray florets. The flower and leaf shape of the progenies was intermediate between the parents, and other morphological traits were also characterized in the same manner. Morphological observations and a cleaved amplified polymorphic sequence marker-based determination, using the internal transcribed spacer region as a target for amplification and the restriction enzyme Afl II, revealed that both individuals are hybrids between A. frutescens and R. hosmariense, R. catananche. To the best of our knowledge, this is the first study to report that crossbreeding between A. frutescens (seed parent) and R. hosmariense, R. catananche (pollen parent) is possible. Moreover, further development of Argyranthemum breeding, especially that of a series of hybrid cultivars with different flower colors, is expected.

DESAIN PRIMER SECARA IN SILICO UNTUK AMPLIFIKASI GEN tcdA BAKTERI CLOSTRIDIUM DEFFILCILE

Antibiotics are drugs used to fight infections caused by bacteria. C. difficile bacteria is a bacterium that causes Clostridium difficile infection (ICD). Toxin A of C. difficile bacteria is exotoxin A (tcdA) which is a virulence factor that causes disease. Toxin A can damage the intestinal mucosa and cause bleeding. The cost of examining and treating patients with AAD and PMC is very high, so the dosage must be determined correctly. Laboratory tests function to determine the presence or absence of an ICD. The discovery of specific biomarkers is a discovery priority to improve the quality of laboratory tests. Specific primary design is an important step in all types of laboratory examination methods, one of which is molecular examination by PCR. The primary design must have specific properties so as to be efficient at target amplification. This study aims to obtain a primer design that can be used to limit the amplification area of the TcdA gene in silico. This study used a literature study from the NCBI genbank to obtain the tcdA gene sequence of C. defficile bacteria with accession number DQ902560.1. The tcdA gene sequence of C. difficile bacteria was analyzed using Primer3Plus to determine whether there is hairpin and dimer formation. Furthermore, identification was carried out using web-based in silico PCR to determine the number of amplicon and visualization on the electrophoretic gel. The level of tcdA gene homology in the species group in the BLAST test. The results showed that the best primer design for in silico detection of the tcdA gene of C. difficile was the forward primer 5'-GCAGATGCCAAGAGACACAC-3' and the reverse primer 3'-GCAGATGCCAAGACACACAC-5'. The primary design is able to amplify the tcdA gene region with a size of 247 amplicons.

A universal lateral flow assay for microRNA visual detection in urine samples

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription – polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencingand molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with>95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Oxidative stress induces non-apoptotic cell death and release of placental derived mitochondrial DNA

Introduction: The placenta, a metabolically active tissue, forms a critical interface between the developing fetus and mother. Stressors such as oxidative stress contribute to placental inflammation, cell death, and release of proinflammatory factors into the maternal circulation. Extracellular mitochondrial DNA (mtDNA) is a proinflammatory factor that can be found in blood circulation in response to cellular stress. Circulating cell-free mtDNA increases during healthy pregnancy and is dysregulated in pregnancies with preeclampsia. Mechanisms underlying the release of mtDNA into circulation from the placenta remain unclear. The objective of this study is to elucidate the relationship between oxidative stress, cell death, and the release of mtDNA from the placenta. We hypothesized that human placental cells exposed to oxidative stressors release mtDNA into the extracellular space via cell death-dependent mechanisms. Methods: BeWo cells (ATCC ® CCL-98), a model of placental trophoblast, were treated with the oxidative stressors, rotenone (0.8, 5, 10, 25, 50 μM) or antimycin A (10, 50, 100, 320 μM) and their respective vehicles for 4 hours. Supernatants were collected and the following experiments were performed: absolute real-time qPCR quantification with TaqMan™ probes and chemistry to quantify intracellular and extracellular mtDNA (amplification target: MT-ND5gene) and nuclear DNA (nDNA), assays to measure reactive oxygen species, flow cytometry to assess cell death mechanisms. Results: Exposure of placental cells to antimycin A induced the release of mtDNA (p&lt;0.0001) and nDNA (p=0.024), whilst intracellular mtDNA copy number was unaffected (p=0.62). Antimycin A (320 μM) reduced cell viability (vehicle: 64.44 ± 5.46% vs antimycin A: 18.14 ± 5.78%, p=0.0005), increased non-apoptotic cell death (vehicle: 10.39± 3.11% vs antimycin A (100, 320 μM): 30.51 ± 4.43%, 40.16± 5.08%, p=0.0001), and did not affect apoptosis (p=0.11). In addition, there was no change in the release of mtDNA (p=0.43) or nDNA (p=0.82), cell viability (p=0.94), non-apoptotic cell death (p=0.52), or apoptosis (p=0.85) in cells treated with rotenone. Conclusions: Placental cells exposed to oxidative stressors undergo non-apoptotic cell death and release mtDNA. These responses are only observed at high concentrations of antimycin A, a pharmacological inhibitor of complex III of the electron transport chain (ETC), but not rotenone, an inhibitor of complex I of the ETC, suggesting the presence of cytoprotective or rescue mechanisms against oxidative stressors in the placenta. NIH R01 HL146562, AHA 22PRE-900431, AHA 22POST-903250, NIH T32 AG 020494 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Highly Selective, Single-Tube Colorimetric Assay for Detection of Multiple Mutations in the Epidermal Growth Factor Receptor Gene

Many closed-tube methods are designed to detect DNA biomarkers. However, the utility of biomarkers such as a DNA mutation related to personalized medicine is limited as the operation of expensive detection instruments requires well-trained technicians. Therefore, we developed a simple and cheap colorimetric assay based on aggregation of silica-gold nanoparticle-modified probes, with linking probes, to detect mutations. This method consists of target amplification, sequence identification, and aggregation of the silica-gold nanoparticle-modified probes. All reactions are controlled by one individual and proceed sequentially, in a single tube, with no manual intervention. Approximately 10 copies of target DNA were detected with this assay, using 12 hot-spot mutations in exon 19 of EGFR gene as the example. In artificial samples, 0.1% mutant DNA can be distinguished from wild-type genomic DNA. The technology was tested on 104 clinical samples, which included 29 samples that were positive for an exon 19 deletion. The data were consistent with amplification refractory mutation system PCR, with the exception of one weakly positive sample, which was confirmed to be positive by digital PCR. The limit of detection of this colorimetric assay was verified to be better than that of amplification refractory mutation system PCR, and it provides a tool to discriminate multiple mutations in EGFR gene in clinical samples.

Development of a high-resolution mass-spectrometry-based method and software for human leukocyte antigen typing.

The human leukocyte antigen (HLA) system plays a critical role in the human immune system and is strongly associated with immune recognition and rejection in organ transplantation. HLA typing method has been extensively studied to increase the success rates of clinical organ transplantation. However, while polymerase chain reaction sequence-based typing (PCR-SBT) remains the gold standard, cis/trans ambiguity and nucleotide sequencing signal overlay during heterozygous typing present a problem. The high cost and low processing speed of Next Generation Sequencing (NGS) also render this approach inadequate for HLA typing. To address these limitations of the current HLA typing methods, we developed a novel typing technology based on nucleic acid mass spectrometry (MS) of HLA. Our method takes advantage of the high-resolution mass analysis function of MS and HLAMSTTs (HLA MS Typing Tags, some short fragment PCR amplification target products) with precise primer combinations. We correctly typed HLA by measuring the molecular weights of HLAMSTTs with single nucleotide polymorphisms (SNPs). In addition, we developed a supporting HLA MS typing software to design PCR primers, construct the MS database, and select the best-matching HLA typing results. With this new method, we typed 16 HLA-DQA1 samples, including 6 homozygotes and 10 heterozygotes. The MS typing results were validated by PCR-SBT. The MS HLA typing method is rapid, efficient, accurate, and readily applicable to typing of homozygous and heterozygous samples.

Hierarchical Porous Metal-Organic Framework as Biocatalytic Microreactor for Enzymatic Biofuel Cell-Based Self-Powered Biosensing of MicroRNA Integrated with Cascade Signal Amplification.

Enzymatic biofuel cells have become powerful tools in biosensing, which however generally suffer from the limited loading efficiency as well as low catalytic activity and poor stability of bioenzymes. Herein, the hierarchical porous metal-organic frameworks (MOFs) are synthesized using tannic acid (TA) for structural etching, which realizes co-encapsulation of glucose dehydrogenase (GDH) and nicotinamide adenine dinucleotide (NAD+ ) cofactor in zeolitic imidazolate framework (ZIF-L) and are further used as the biocatalytic microreactors to modify bioanode. In this work, the TA-controlled etching can not only expand the pore size of microreactors, but also achieve the reorientation of enzymes in their lower surface energy form, therefore enhancing the biocatalysis of cofactor-dependent enzyme. Meanwhile, the topological DNA tetrahedron is assembled on the microreactors, which acts as the microRNA-responsive "lock" to perform the cascade signal amplification of exonuclease III-assisted target recycling on bioanode and hybridization chain reaction (HCR) on biocathode. The proposed self-powered biosensor has achieved a detection limit as low as 2 aM (6 copies miRNA-21 in a 5µL of sample), which is further successfully applied to identify cancer cells and clinical serums of breast cancer patients based on the different levels of miRNA-21, holding great potential in accurate disease identification and clinical diagnosis.

Local governments' economic growth target pressure and bank loan loss provision: Evidence from China

This paper aims to investigate whether and how the top-down amplification of economic growth targets affects banks' loan loss provision (LLP). We find that banks in prefecture-level cities with higher economic growth target pressure (EGTP) will withdraw more LLPs, indicating that they will accrue reserves prospectively to deal with potential credit losses. We further find that EGTP reduces credit allocation efficiency and increases future loan losses, and banks' risk prevention motivation is the mechanism for accruing LLPs. It's also shown that fiscal pressure and promotion pressure will amplify EGTP's effect, while reduced government intervention will weaken banks' risk prevention motivation. Overall, our paper provides systematic evidence on how banks respond to the negative impact of EGTP through loan loss provisioning.

Abstract LB255: Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products

Abstract The FDA Guidance to Industry on Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products states the importance of longitudinal testing of gene products introduced into human subjects. Depending on the delivery mechanism, the therapeutic gene product may or may not integrate into the genome. Of particular interest are gene-product integrations near proto-oncogenes which might lead to malignancies. The FDA LTFU guidance states that recipients of an integrating gene therapy modality should be tracked for 15 years, while those receiving a non-integrating therapy modality should be tracked for 5 years. Therefore, advanced analytical methods are needed to identify, quantify, and track integration events across the genome. Here, we provide a comprehensive evaluation of methods leveraging next-generation sequencing approaches for genome-wide analysis of lentiviral integration events. Our analysis employed well-characterized standards consisting of varying copy number and known integration sites. ​ The approaches we characterized can be bucketed into two major groups: PCR amplification approaches and target capture-based approaches. All methods detected true positives with strong correlation to theoretical integration site dosage levels down to 1% allele frequency. Comparatively, PCR amplification-based approaches have lower data requirement per sample suggesting higher sensitivity, greater molecular capture, and lower limit of detection compared with target enrichment-based approaches. Target enrichment-based approaches can afford the flexibility to capture the integrated vector, which is of interest for characterizing partial integration events. While all methodologies performed well in our study, the choice of assay (or assays) for testing will depend on numerous factors including but not limited to the viral vector system and construct and starting material availability. Citation Format: Jeffrey M. Otto, Yongjun Fan, Guanhui Bao, David Corney, Ben Niu, Qiu Yu, Baursha Khan, Laure Turner, Chris Mozdzierz, Haythem Latif, Ginger Zhou. Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB255.

Mechanism of Resistance to the WDR5 Inhibitor in MLL-Rearranged Leukemia

Drug resistance is a major problem often limiting the long-term effectiveness of targeted cancer therapeutics. Resistance can be acquired through mutations or amplification of the primary drug targets or activation of bypass signaling pathways. Considering the multifaceted function of WDR5 in human malignancies, WDR5 has emerged as an attractive drug target for the discovery of small-molecule inhibitors. In this study, we investigated if cancer cells might develop resistance to a highly potent WDR5 inhibitor. We established a drug-adapted cancer cell line and discovered that WDR5P173L mutation occurs in the resistant cells, which confers resistance by preventing target engagement of the inhibitor. This work elucidated the WDR5 inhibitor's potential resistance mechanism in a preclinical study as a reference for future study in the clinical stage.