Opportunity
The current landscape of RNA expression analysis faces significant limitations, particularly in clinical diagnostics. Traditional next-generation sequencing (NGS) methods for RNA quantification suffer from low efficiency in targeting clinically relevant genes, poor accuracy for low-expression but critical genes (e.g., ALK fusion that is important in precision medicine), high costs due to reliance on whole-transcriptome analysis, and incompatibility with common clinical samples like formalin-fixed paraffin-embedded (FFPE) tissues. Existing techniques, such as RT-qPCR or digital PCR, lack the multiplexing capability needed for comprehensive profiling. Additionally, immune checkpoint inhibitor (ICI) therapies, while revolutionary in cancer treatment, benefit only 20–30% of patients who rely on reliable diagnostic biomarkers like PD-L1 protein expression. Varoius PD-L1 immunohistochemistry (IHC) assays are semi-quantitative, use inconsistent cutoff values, and detect different epitopes, leading to variable results. This highlights the urgent need for a precise, high-throughput RNA quantification method to improve ICI response prediction and personalized medicine.
Technology
The patent introduces a novel method called Anchored Ligation Priming for Highly Accurate RNA Expression (Alpha-RNA). This NGS-based approach simultaneously enriches target DNA and RNA sequences, calculating the RNA-to-DNA ratio for each target to quantify expression levels. Key steps include:
- Reverse Transcription: Converts RNA to cDNA, preserving the original genomic DNA (gDNA) mixture.
- Adapter Ligation: Attaches universal sequencing adapters to both cDNA and gDNA ends.
- PCR Amplification: Uses gene-specific primers near exon-intron boundaries to amplify target sequences. A unique innovation is the use of a single primer to enrich both genomic DNA (containing introns) and cDNA (spliced exons), enabling accurate RNA/DNA ratio calculations by canceling out primer efficiency biases.
- NGS and Ratio Calculation: Quantifies cDNA reads relative to gDNA reads, providing normalized expression levels without relying on housekeeping genes.
This method overcomes limitations of traditional RNA-seq by offering high accuracy for low-abundance transcripts, compatibility with degraded FFPE samples, and cost-effective multiplexing (100+ targets). It also integrates seamlessly with standard NGS workflows, eliminating the need for additional sample processing.
Advantages
- High Efficiency: Targets clinically relevant genes with precision.
- Accuracy: Measures low-expression genes (e.g., ALK) reliably.
- Cost-Effective: Focuses on actionable genes, avoiding whole-genome sequencing.
- Broad Applicability: Works with FFPE tissues, liquid biopsies (blood, urine), and other challenging samples.
- Multiplexing: Supports >100-plex assays for comprehensive profiling.
- Standardization: Eliminates variability from housekeeping gene normalization.
Applications
- Cancer Diagnostics: Quantifies biomarkers like PD-L1, GZMA, and tumor microenvironment genes to predict ICI response.
- Therapeutic Monitoring: Guides immunotherapy decisions by identifying patients with high CD274 (PD-L1) and low GZMA expression.
- Research Tools: Enables study of alternative splicing (e.g., CD274 exon variants linked to poor prognosis).
- Liquid Biopsies: Analyzes RNA/DNA ratios in blood or urine for non-invasive cancer detection.
- Companion Diagnostics: Validated for NSCLC subtypes (adenocarcinoma, squamous cell carcinoma).
