Comprehensive assessment of the diastereomer composition of LEQVIO (Inclisiran) to determine how chemical synthesis impacts biological activity

PROJECT ABSTRACTNucleic acids are an increasingly popular platform for the development of biotherapeutics to treat and prevent awide variety of human diseases. The predominate classes of FDA-approved synthetic oligonucleotidetherapeutics include antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). siRNAs are duplexRNA containing an antisense (guide) strand and a sense (passenger) strand. The potency of siRNA relies onthe ability of the antisense strand to complementarily bind to specific mRNA within the target cell. siRNA drugdevelopment requires overcoming the poor pharmacological properties of nucleic acids including their rapiddegradation by ubiquitous nucleases and poor drug delivery to target organs. One chemical modificationcommonly employed for siRNA involves the conversion of the natural phosphodiester nucleotide linkage into aphosphorothioate (PS) linkage. This modification confers nuclease resistance and enhances protein binding. Aby-product of this modification is the PS linkage is chiral creating diastereomers. The number of diastereomerswithin a PS oligonucleotide scale at a rate of 2n where n is the number of PS linkages. PS diastereomers areexpected to impact the physiochemical and biological properties of oligonucleotide therapeutics. This hasimportant ramifications for drug efficacy and drug dosing. Additional considerations include uncharacterizedand/or uncontrolled PS diastereomer compositions adversely impact active ingredient bioequivalence. Theseare 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. Currentlythere 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-conjugatedsiRNAs 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 diastereomersfor these GalNAc-conjugated siRNAs is limited (16 for the antisense strand 4 for the sense strand) which makesit plausible to investigate how each diastereomer behaves and contributes to the overall activity of the drugproduct. Consequently the purpose of this proposal is to develop innovative synthetic chemistrypharmacological assays and analytical methods to systematically evaluate the diastereomeric composition ofLEQVIO (Inclisiran) an FDA-approved GalNAc-conjugated siRNA drug. This will be achieved by determiningthe activity of each diastereomer in LEQVIO through stereochemically-controlled synthesis biological activityassessment using in vitro and animal models and accurate characterization of the stereochemical structure ofeach diastereomer. Our developed methods will be directly transferable to other synthetic oligonucleotides andwill greatly facilitate the development and assessment of generic oligonucleotide drug products.