Abstract: A potency release assay for measuring the potency of drug product composition comprising self-amplifying mRNA (SAM) that encodes at least one immunogenic polypeptide or at least one therapeutic peptide and a non-viral delivery system is described. In one embodiment the drug product is a vaccine comprising SAM and a non-viral delivery system such as SAM/lipid nanoparticle (LNP) delivery system, a Cationic Nanoemulsion (CNE) delivery system, or another SAM delivery system. It is demonstrated that the potency of a SAM drug product can be assessed in an in vitro system, at the RNA amplification stage (agnostic assay), by measuring the amount of double-stranded RNA (dsRNA) in cells which have been transfected with the SAM in the drug product. Thus, dsRNA can be used as a surrogate endpoint for potency. It is demonstrated that there is a very high correlation between total dsRNA in a cell culture transfected with the SAM and the potency of the SAM based drug product.

Abstract: Provided herein are RNA that: are collectively self-amplifying in an intracellular environment, comprise N1-methyl-pseudouridines and uridines, and have a mole percentage or mole proportion of the N1-methylpseudouridines to the total of the uridines and the Ni-methylpseudouridines or a mole ration of the N1-methylpseudouridines to the uridines.