PROJECT ABSTRACT Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat and prevent a wide variety of human diseases. The predominate classes of FDA-approved synthetic oligonucleotide therapeutics include antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). siRNAs are duplex RNA containing an antisense (guide) strand and a sense (passenger) strand. The potency of siRNA relies on the ability of the antisense strand to complementarily bind to specific mRNA within the target cell. siRNA drug development requires overcoming the poor pharmacological properties of nucleic acids, including their rapid degradation by ubiquitous nucleases and poor drug delivery to target organs. One chemical modification commonly employed for siRNA involves the conversion of the natural phosphodiester nucleotide linkage into a phosphorothioate (PS) linkage. This modification confers nuclease resistance and enhances protein binding. A by-product of this modification is the PS linkage is chiral creating diastereomers. The number of diastereomers within a PS oligonucleotide scale at a rate of 2n, where n is the number of PS linkages. PS diastereomers are expected to impact the physiochemical and biological properties of oligonucleotide therapeutics. This has important ramifications for drug efficacy and drug dosing. Additional considerations include uncharacterized and/or uncontrolled PS diastereomer compositions adversely impact active ingredient bioequivalence. These are critical for accurate drug labeling for reference listed drug products and development of generic drug options. Thus, characterization of the oligonucleotide PS diastereomer composition is highly consequential. Currently there are five FDA approved siRNAs, four of which have a covalently linked ligand containing three N- acetylgalactosamine (GalNAc) residues to facilitate drug delivery to hepatocytes. These GalNAc-conjugated siRNAs have six terminal PS linkages, and thus the final drug product is a mixture of different diastereomers. Four of the PS linkages are in the antisense strand and two in the sense strand. The number of diastereomers for these GalNAc-conjugated siRNAs is limited (16 for the antisense strand, 4 for the sense strand), which makes it plausible to investigate how each diastereomer behaves and contributes to the overall activity of the drug product. Consequently, the purpose of this proposal is to develop innovative synthetic chemistry, pharmacological assays, and analytical methods to systematically evaluate the diastereomeric composition of LEQVIO (Inclisiran), an FDA-approved, GalNAc-conjugated siRNA drug. This will be achieved by determining the activity of each diastereomer in LEQVIO through stereochemically-controlled synthesis, biological activity assessment using in vitro and animal models, and accurate characterization of the stereochemical structure of each diastereomer. Our developed methods will be directly transfe...