Bioanalysis of siRNA and Oligonucleotide Therapeutics in Biological Fluids and Tissues

Abstract

This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.

Oligonucleotide-Based Drug Development: Considerations for Clinical Pharmacology and Immunogenicity

“…Several mechanisms may thus affect the immunogenicity of OGN-based therapeutics: (1) the use of modified analogs designed to increase nuclease resistance, (2) linking an OGN to a protein carrier to limit nuclease digestion and enhance cellular uptake, and (3) encapsulation in a pegylated liposome.…”

RNA Interference and Cancer Therapy

“…With more RNAi-based therapies moving from the bench to the clinic, a reliable quantification method is needed to examine the intracellular concentration of siRNA/shRNA in order to evaluate the delivery efficiency and the pharmacokinetics of RNAi drugs. Several bioanalytical approaches are being utilized to determine RNAi effector molecules in preclinical and clinical studies, including quantitative RT-PCR, hybridization assay, HPLC and LC-MS (76). Among them, quantitative PCR has been extensively utilized to investigate pharmacokinetic profiles of RNAi therapeutics in preclinical and clinical development (77)(78)(79)(80).…”

Bioanalytical LC–MS of Therapeutic Oligonucleotides

“…In order to enhance the stability of OGNTs upon administration to the body, the phosphate groups in the backbone are often replaced by phosphorothioates (PS) groups [16]. Other modifications to protect OGNTs from nucleases mostly involve chemical modifications of the sugar moieties, such as, 2´-fluoro-nucleic acids, 2´O-Me-nucleic acids, 2´-O-(2-methoxy) ethyl (MOE)-nucleic acids, locked nucleic acids at the 5´ and 3´ ends and morpholino oligonucleotides (OGNs), where the ribose sugar moiety is replaced with morpholine rings and the anionic phosphodiester linkage is replaced with nonionic phosphorodiamidate groups [17].…”