Antisense Oligonucleotide Quantification via Splint-Ligation PCR Assay in Nonhuman 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 (NHP) tissues and biofluids. Our results show that the Splint Ligation Assay was highly sensitive across central nervous system (CNS) tissues and biofluids (as low as 100 pM in NHP CNS tissue and 1 pM in NHP plasma), with broad linear dynamic ranges. Overall, our results show that the Splint-Ligation PCR Assay is a reliable, sensitive, and feasible method of ASO quantification.