Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients’ cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.
Triazole-linked morpholino (TLMO) oligonucleic acids were synthesised using the CuI catalysed (3 + 2) azide–alkyne cycloaddition (CuAAC) reaction.
Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.
Background RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. Results Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. Conclusion The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.
Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.
Introduction: While the FDA has approved over 20 targeted therapies for non-small cell lung cancer (NSCLC), less than half of patients in the US undergo comprehensive biomarker testing to determine if they would benefit from these agents. This gap in testing is compounded in ex-US and minority populations. There are multiple barriers to access for biomarker testing, many of which stem from the complexity and high costs of current solutions, which limit testing to large, centralized laboratories. Alongside this, tissue requirements are high, turnaround times (TAT) for results typically take weeks, and reimbursement is uncertain. We demonstrate how ASPYRE can address these limitations by enabling ultra-sensitive biomarker testing using existing staff and widely available instrumentation. The technology is based on four enzyme steps (Silva et al 2020), the last of which is monitored on a real-time PCR instrument. ASPYRE-Lung targets 111 markers in 11 genes, including 77 DNA variants (substitutions and indels), and 34 RNA variants (fusions and exon skipping) across 24 wells, with a TAT of less than <4 hours from extracted nucleic acids to result. Experimental procedures: Four test sites were provided with ASPYRE reagents and contrived samples including Seracare ctDNA Mutation Mix v2 and a panel consisting of synthetic RNA oligonucleotides diluted in a background of normal lung RNA. The Seracare sample included variants in BRAF, EGFR, ERBB2, and KRAS at 0.25% variant allele fraction. The RNA sample included fusions in RET, ROS1 and NTRK1, with each fusion present at a mean of 6 copies in a background of 1 ng lung RNA. Operators from a range of educational backgrounds were provided a set of written Instructions for Use, with no additional training. Summary of data: The ASPYRE-Lung test was set up and performed at each test laboratory in as little as one day, using their existing real-time PCR instrument. The results demonstrated detection of the expected variants with consistent performance characteristics across the test sites and between users. Importantly, the sensitivity of detection was in line with, or superior to, the current gold standard. Conclusions: Biofidelity’s breakthrough ASPYRE technology enables a dramatic simplification of workflows, and the ultra-sensitive detection of actionable biomarkers using existing real-time PCR instruments. This work has demonstrated the ease with which independent laboratories can implement comprehensive multi-gene assays based on ASPYRE technology, utilizing their existing staff and infrastructure. This stands in stark contrast to alternative solutions, which typically require substantial investments in new instrumentation, skilled staff, IT infrastructure and bioinformatics. Taken together, ASPYRE promises to remove key barriers in cancer biomarker testing, enabling all patients to access the right treatment at the right time. Citation Format: Paulina Powalowska, Nicola Potts, Brandon A. Smith, Kala F. Schilter, Honey V. Reddi, Lan Beppu, Jerald Radich, Charles Massie, Quentin Vicentini, Tom Brown, Robert Osborne, Barnaby Balmforth. External user testing of Biofidelity’s ASPYRE-Lung assay demonstrates breakthrough capabilities and ease of use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4105.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.