Emerging siRNA Design Principles and Consequences for Biotransformation and Disposition in Drug Development

Abstract

After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking. Advancing ADME knowledge will aid establishment of in vitro–in vivo correlations and pharmacokinetic–pharmacodynamic relationships to optimize candidate selection through discovery and translation. Here, we outline the emerging siRNA design principles and discuss the consequences for siRNA disposition and biotransformation. We propose a conceptual framework for siRNA ADME evaluation, contextualizing the site of biotransformation product formation with PK–PD modulation, and end with a discussion around safety and regulatory considerations and future directions for this modality.

Nonclinical Pharmacokinetics and Absorption, Distribution, Metabolism, and Excretion of Givosiran, the First Approved N-Acetylgalactosamine–Conjugated RNA Interference Therapeutic

“…The GalNAc ligand directs hepatocyte-specific uptake of siRNA via the asialoglycoprotein receptor (ASGPR), which is highly expressed on the surface of hepatocytes (Nair et al, 2014). Givosiran is the first GalNAc-conjugated RNAi therapeutic that has been approved by the U.S. Food and Drug Administration and the European Commission, with the recommended dose of 2.5 mg/kg administered via SC injection once monthly, and currently many more GalNAc-conjugated RNAi therapeutics are in late stage clinical development (Setten et al, 2019;Humphreys et al, 2020).…”

Introducing an In Vitro Liver Stability Assay Capable of Predicting the In Vivo Pharmacodynamic Efficacy of siRNAs for IVIVC

“…Before interacting with a target mRNA molecule and inducing its cleavage, GalNAc-conjguated siRNA must bind ASGPR, be taken up into the cell via endocytosis, escape from endosome, and be loaded into the RISC. 55 , 56 To shed light on our potentially unlikely finding, we looked to the literature and to the specific panel of molecules we used to better understand why liver homogenate stability might be representative of in vivo efficacy.…”

“…Although it is beyond the scope of this work, in the future, it will be important to perform reaction phenotyping to identify the molecular entities responsible for the biotransformation processes leading to the observed metabolites. In a recent review, Humphreys et al 56 compiled a list of known siRNA metabolites and the enzymes and chemical processes that are likely responsible for generating them. Major siRNA-metabolizing enzymes identified in the work include GalNAc cleavage by b-N-acetylglucosaminidase; catabolism by lysosomal hydrolase; internal esterification; cyclization; oxidation by alcohol dehydrogenase and aldehyde dehydrogenase; AGO2-mediated endonuclease hydrolysis; 5 0 /3 0 exonuclease cleavage by XRN1 and XRN2; 3 0 /5 0 exonuclease cleavage by RNR, DEDD, and PDX families and exosomes; and 5 0 phosphorylation and dephosphorylation by kinases, such as CLP1 and phosphatases, respectively.…”

Drug delivery systems for RNA therapeutics

“…Complementing these modifications are phosphate backbone modifications including phosphorothioates, phosphorodiamidate morpholino oligonucleotides (PMO), peptide nucleic acid oligonucleotides (PNA), and nucleobase modifications, such as 5-methylcytosine (m5C). Changing the combination of different chemical modifications of the siRNA has enabled scientists to improve the pharmacokinetic properties, innate immune response and stability 39 . Second, siRNA utilizes RISC, which is endogenous to eukaryotic cells and thus does not require the delivery of large enzymes with nuclease domains.…”