(T1530-06-39) A Two-Part Method for the Separation and Quantification of Free and Encapsulated ASOs by Hybridization ELISA

Purpose: Antisense Oligonucleotides (ASOs) are a promising class of novel therapeutics capable of targeting a wide range of genetic disorders. ASOs are modified, single-stranded oligonucleotides designed to bind specific mRNA sequences and modify expression of the associated protein. By targeting disorders at the mRNA level, ASOs can often produce more effective results than treatments that target downstream protein pathways. As of 2022, the FDA has approved ten ASO treatments, with many more candidates undergoing development and clinical trials. Consequently, methods to detect the presence and quantity of ASOs are becoming increasingly critical as the number of available therapeutics grows. Since ASOs are often administered in lipid nanoparticle (LNP) formulations, there is an additional need to develop strategies for detecting ASOs still contained by LNPs. Here, we describe a two-part quantitative method for detection of an ASO therapeutic in serum via hybridization ELISA. Crucially, this method allows the separation and detection of free ASO in circulation and ASO encapsulated in LNP delivery systems.

Methods: Samples were incubated with proteinase K (Invitrogen) at 0.66mg/mL for 1 hour at 37°C. Exosome precipitation solution (Thermo Fisher) of150 µL was added to 775 µL sample and tubes were inverted 10x prior to incubation at 4°C for 30 minutes. Samples were centrifuged at 10,000 x g for 5 minutes, and supernatant was transferred to a new tube. Supernatant (containing free ASO) was treated with proteinase inhibitor (Thermo Fisher) and incubated for 10 minutes at 37°C, then diluted 1:4 in TE buffer (Thermo Fisher). Pellet (containing encapsulated ASO) was resuspended in 200 µL TE buffer and 200 µL lysis solution (4% Triton X (Thermo Fisher), 2% SDS (Thermo Fisher)) was added. Samples were vortexed and incubated for 15 minutes at 37°C, then diluted 1:10 in TE buffer.

Prior to assay development, capture and detection probes were synthesized with complementarity to the first and second half of the target ASO sequence, respectively. The capture probe included a 5’-conjugated biotin and the detect probe included a 3’-conjugated digoxygenin tag. 200nM capture probe was denatured in hybridization buffer at 95°C for 5 minutes. The reaction was then quenched on ice for 5 minutes to prevent dimerization of probe. Next, capture probe solution 

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