(M1430-06-32) Development of Sensitive Method for Antisense Oligonucleotide Therapeutics Using SIMOA Technology

Purpose: Antisense oligonucleotides (ASOs) are short, synthetic, oligonucleotides that have the potential to target specific gene products, offering a potent means of gene expression regulation. They represent a promising therapeutic avenue for a range of diseases, including neurodegenerative conditions, inflammatory disorders, and cancer. As of late 2021, around 15 oligonucleotides have gained FDA approval and approximately 60 entered clinical trials. Current methodologies for ASO analysis, such as liquid chromatography-mass spectrometry (LC-MS)³, ligand binding assays (LBA), and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)², often involve a balance between sensitivity and specificity inherent to each method. The sensitivity of ASO analysis becomes crucial when detecting low doses or in complex matrices like tissue or cerebrospinal fluid (CSF). Advancements in ligand binding assays offer opportunities to enhance sensitivity for detecting trace amounts of ASOs. In this study, we developed a sensitive LBA on the single molecule arrays (SIMOA) technology⁴ targeting MALAT1 (Metastasis-associated lung adenocarcinoma transcript)¹ due to its widespread expression and relevance across various CNS cell types and peripheral tissues. MALAT1 serves as a benchmark target for ASO studies due to its ubiquitous tissue expression and limited cellular function. This robust bioanalytical method aims to enhance our understanding of the pharmacokinetic-pharmacodynamic (PKPD) relationship of ASOs and can be adapted for similar oligonucleotide projects with comparable requirements.

Methods: In our previous work we have already established the hybridization ELISA on the Electrochemiluminescence (ECL) platform. To further improve the analytical sensitivity, we used the digital detection platform based on SIMOA. The target analyte is immobilized on paramagnetic beads functionalized with the capture probe. The biotinylated probe binds to the extended complementary extension on the capture probe and forms complex with streptavidin-β-galactosidase (SβG). The enzyme labelled beads generate florescence when suspended in the resorufin-β-D-galactopyranoside (RGP) solution that is captured by the CCD camera. The data was analyzed using 5PL regression and weighting 1/Y2 using SoftMax Pro software from Molecular Devices.

Results: After overcoming various challenges and optimizing several assay parameters like hybridization temperature, probe concentration, nuclease selection activity, and incubation time, we successfully developed hybrid ELISA to selectively measure MALAT1 ASO. The assay has an increased sensitivity with a lower limit of quantitation of 3.4 pM in rat plasma which is 10-fold more sensitive compared to ECL. The quantitation and robustness of the assay was confirmed by running inter and intra-day assays for accuracy and precision that was within 20% (Table1). The specificity of the assay was evaluated by assessing a negative control and synthetic metabolites of ASO. The assay is selective towards N-1 and 2 at 5’ with decreased reactivity towards N-1 and 2 at 3’ (Figure 1).

Conclusion: This study highlights the successful use of the Single Molecule Array (SIMOA) technology in the analysis of Antisense Oligonucleotides against MALAT1. The hybridization assay performed on the SIMOA platform exhibits noteworthy enhancement in the sensitivity, enabling the detection of the ASO at the picomole level in both rat plasma and cerebrospinal fluid. By employing optimized probe design and refinement techniques, this approach holds promise as a widely applicable method for the oligonucleotide drug analysis.

References: 1. Thayer, M. B. et al. Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics. Sci. Rep. 9, 3566–3566 (2019).
2. Cheng, A. et al. Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo. Oligonucleotides 19, 203–208 (2009).
3. Basiri, B. & Bartlett, M. G. LC-MS of oligonucleotides: Applications in biomedical research. Bioanalysis 6, 1525–1542 (2014).
4. https://www.quanterix.com/simoa-technology/

Precision and Accuracy of MALAT1 ASO in Rat Plasma and Cerebrospinal fluid

Specificity of synthetic metabolites with the probes.