Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

Abstract

The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the beginning of a new chapter in modern medicine. However, the rapid rise of mRNA therapeutics has resulted in a regulatory framework that is somewhat lagging. The current guidelines either do not apply, do not mention RNA therapeutics, or do not have widely accepted definitions. This review describes the guidelines for preclinical biodistribution studies of mRNA/siRNA therapeutics and highlights the relevant differences for mRNA vaccines. We also discuss the role of in vivo RNA imaging techniques and other assays to fulfill and/or complement the regulatory requirements. Specifically, quantitative whole-body autoradiography, microautoradiography, mass spectrometry-based assays, hybridization techniques (FISH, bDNA), PCR-based methods, in vivo fluorescence imaging, and in vivo bioluminescence imaging, are discussed. We conclude that this new and rapidly evolving class of medicines demands a multi-layered approach to fully understand its biodistribution and in vivo characteristics.

Keywords: BLI; Biodistribution; Comirnatmy; Distribution; FISH; FLI; Givlaari; Givosiran; Guidelines; Imaging; Inclisiran; LNP; Leqvio; Lipid nanoparticle; Lumasiran; MS; Mass spectrometry; Moderna; Onpattro; Oxlumo; Patisiran; Pfizer/BioNTech; QWBA; RNA therapeutic; RT-qPCR; Regulatory; Spikevax; Vaccine; bDNA; mRNA; mRNA vaccine; siRNA.

Graphical abstract

Fig. 1

Example of QWBA imaging, using ³H-labeling in a rat. Reprinted with permission from Bioanalysis (2015) 7(5), 557–568 as agreed by Newlands Press Ltd.

Fig. 2

Schematic overview of common labeling and detection methods for (m)RNA, proteins and LNPs. QWBA = quantitative whole body autoradiography, IHC Ab = immunohistochemistry antibody, eGFP = enhanced green fluorescent protein, LUC = luciferase, MS = mass spectrometry, RT-qPCR = reverse transcription quantitative polymerase chain reaction, RISC = RNA-induced silencing complex, FISH = fluorescence in situ hybridization. *IHC can also be used to demonstrate target protein knockdown after siRNA-LNP administration.

Fig. 3

(a) Absorption spectra of water, melanin, and oxyhemoglobin. Absorption is minimal in the optical window (white section in both (a) and (b)). (b) Emission spectra of eGFP, miRFP, Cy7, Firefly luciferase (Fluc), Renilla luciferase (hRluc), and NanoLuc (Nluc).