Barcoded dsDNA Detection of AR-V7 Splicing Variants
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Solution Overview
Problem
Current methods for predicting resistance to androgen receptor-targeted therapies in prostate cancer patients are limited by the scarcity of detectable circulating tumor cells, making it difficult to effectively use RNA splicing variants like AR-V7 for treatment selection and prognosis.
Innovation Solution
A method involving the extraction of nucleic acids from biofluids, where single-strand RNA is barcoded and converted to double-strand DNA, allowing for the detection of genetic alterations such as AR-Vs, mutations, and copy number variations using Next Generation Sequencing and other technologies, independent of CTC presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If circulating tumor cells (CTCs) are used for detecting RNA splicing variants such as AR-V7, then predictive information for treatment selection can be obtained, but the detection is limited by the scarcity of detectable CTCs in most patients
Solution Approach 1:
The patent uses cell-free RNA (cfRNA) as an intermediary substance to detect AR-V7 splicing variants indirectly. Instead of requiring direct detection of rare CTCs, the method extracts and analyzes cfRNA from plasma, which contains RNA transcripts including AR-V7 variants. This intermediary approach allows detection of the biomarker of interest without needing to isolate actual tumor cells, thereby overcoming the limitation of low CTC abundance while maintaining detection sensitivity.
2Ease of operation
If non-invasive biofluid sampling is used for genetic alteration detection, then patient comfort and serial assessment capability are improved, but the concentration of target nucleic acids in biofluids is extremely low
Solution Approach 1:
The patent employs preliminary enrichment and concentration steps before analysis. The method includes extracting nucleic acids from biofluids, converting ssRNA to dsDNA with barcodes, and performing pre-amplification or enrichment of target sequences before final detection. These preliminary actions concentrate the extremely low abundance of target nucleic acids from biofluids to levels suitable for accurate genetic alteration detection, while preserving the non-invasive sampling advantage.
3Measurement precision
If RNA-based detection methods are used to detect splicing variants like AR-V7, then treatment resistance prediction accuracy is improved, but RNA is more labile and difficult to isolate and stabilize compared to DNA
Solution Approach 1:
The patent changes the physical and chemical parameters of RNA to improve stability and ease of handling. Specifically, the method converts labile single-stranded RNA (ssRNA) into more stable double-stranded DNA (dsDNA) through reverse transcription, while incorporating barcodes during this conversion process. This parameter change from RNA to DNA format maintains the ability to detect splicing variants (since the barcode encodes the original RNA sequence information) while dramatically improving nucleic acid stability and reducing isolation complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the non-invasive detection of genetic alterations in prostate cancer patients, predicting resistance to AR-targeted therapies with increased sensitivity and specificity, thereby informing treatment decisions.
Implementation Method 1
barcoding ss RNA and converting ssRNA to dsDNA wherein the dsDNA is a barcoded dsDNA
Data Source
AI summary
Disclosed are systems and methods for detecting genetic alterations comprising androgen receptor gene splice variants (AR-Vs), mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient. The systems and methods are similarly applicable to the detection of gene alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof of other genes of interest. The streamlined methods improve the consistency and simplicity of non-invasive detections of biomarkers.