Antisense Oligonucleotide Quantification via Splint-Ligation PCR Assay in Non-Human Primate Central Nervous System Tissues and Biofluids

Abstract Antisense oligonucleotides (ASOs) are chemically modified single-stranded oligonucleotides used to modulate the expression or processing of a target RNA transcript. The development of ASOs to treat human disease requires extensive preclinical studies in animal models. A critical component of these studies is determining the concentration of the ASO in tissues and biofluids, which are used to estimate the distribution, half-life, and dose-response relationship. The methods used to quantify ASOs are often constrained by low sensitivities, poor dynamic ranges, and the use of highly specialized equipment. Here, we describe the development of a Splint-Ligation-based quantitative PCR assay to measure the concentration of ASOs in nonhuman primate tissues and biofluids. Our results show that the Splint-Ligation Assay is highly sensitive and has a broad dynamic range in nonhuman primate central nervous system tissues and biofluids, ranging from picomolar to micromolar concentrations. Overall, our results show that the Splint-Ligation PCR Assay is a reliable, sensitive, and feasible method of ASO quantification. Competing Interest Statement Scott V. Dindot has an equity interest in Ultragenyx Pharmaceutical and is an employee of Ultragenyx Pharmaceutical.