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A novel platform for the direct profiling of circulating cell-free ribonucleic acids in biofluids

Periodic Reporting for period 1 - diaRNAgnosis (A novel platform for the direct profiling of circulating cell-free ribonucleic acids in biofluids)

Reporting period: 2021-01-01 to 2023-05-31

Circulating cell-free ribonucleic acids (ccfRNAs) have shown significant potential as innovative cancer biomarkers over the past decade, with applications for screening, prognosis, and treatment monitoring. However, despite successful identification and validation through research and clinical trials, their use in clinical diagnostics is limited due to challenges such as high costs, complex sample preparation, and intricate methodologies. The diaRNAgnosis project aims to overcome these challenges by identifying and validating ccfRNA signatures, developing the ODG Platform for direct ccfRNA profiling, and validating this platform with actual clinical samples. Initially focusing on testicular germ cell tumour (TGCT) and prostate cancer (PCa), the project aspires to eventually extend its approach to other cancers, thereby revolutionizing cancer diagnostics.
The diaRNAgnosis project made significant progress during this period:
Discovery of ccfRNA Biomarkers: The team validated miRNA-371 as a biomarker for testicular germ cell tumours (TGCTs) and identified potential infertility-related biomarkers. Nine differentially expressed microRNAs were discovered, three of which were confirmed via Real-Time PCR. The team found potential RNA biomarkers for prostate cancer (PCa), including long non-coding RNAs and miRNAs, overexpressed in urine samples from PCa patients.
ODG Platform prototypes: Two prototypes were developed using fluorescence and chemiluminescence protocols. Prototype 1 uses a time-gated detection approach for miRNA detection with photoluminescent-tagged SMART bases, whereas Prototype 2 enhances the chemiluminescent signal with silver and gold nanoparticles. Both performed satisfactorily, but Prototype 2 was selected for future development due to its superior Limit of Detection.
Reagent Development: Reagents were designed and synthesized to enhance the assays on the prototypes and target the newly identified or confirmed ccfRNA biomarkers. A particular success was the synthesis of a SMART base labelled with an Eu(III) luminophore and the successful functionalization of Dynabeads with DGL-Probes. These will be tested with the ODG Platform to investigate further other discovered or validated biomarkers, including long non-coding RNAs.
DiaRNAgnosis aims to develop a biomedical diagnostic tool to identify new circulating cell-free RNA (ccfRNA) biomarkers, particularly for testicular germ cell tumours (TGCT) and prostate cancer (PCa) which are major causes of male cancer-related morbidity. Conventional serum tumour markers for these cancers, such as AFP, hCG, LDH for TGCT and PSA for PCa, often exhibit low accuracy and sensitivity. ccfRNAs have shown greater sensitivity in some contexts but their use in clinical diagnostics has been limited due to challenges in absolute quantification of RNA. DiaRNAgnosis intends to overcome these limitations by further developing established ccfRNA-based cancer biomarkers and discovering new ones, using a new technological platform to transform ccfRNA research into innovative clinical diagnostic products, thereby enhancing patient healthcare and cancer diagnostics.
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