Theoretical basis for stabilizing messenger RNA through secondary structure design

Abstract

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

Stability Modelling of mRNA Vaccine Quality Based on Temperature Monitoring throughout the Distribution Chain

“…Kinetic models describing mRNA degradation have been developed based on firstorder kinetics at physiological pH ranges [21][22][23]. It was also shown that this mRNA degradation reaction follows the Arrhenius behavior [21,24].…”

“…However, the temperature reading from these monitoring devices can feed into computational degradation kinetic models. Therefore, modelling of mRNA degradation kinetics can be feasible [21][22][23][24][25] and with further investigation the impact of temperature exposure profiles on CQAs can be assessed [26][27][28]. Therefore, here solutions are conceptualised to quantify the impact of the distribution conditions, including temperature excursions, on the remaining shelf life and on the stability-related CQAs of these thermolabile mRNA vaccines.…”

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

“…An illustrative example of how deviation to even one factor of the ones mentioned above can compromise mRNA integrity, arises from the rigorous study of SARS-CoV-2 mRNA vaccine stability: under strict “cold-chain” transport conditions a naked RNA molecule encoding the virus’s spike has a calculated half-life of about 900 days, stored in phosphate-buffer saline (pH 7.4) in complete absence of magnesium ions. The reported half-life is predicted to be diminished to just five days, if the product is exposed to temperatures of 37 °C [ 3 ]. Another factor determining stability, is RNA length which is calculated to be inversely correlated with stability against hydrolysis [ 3 ].…”

“…The reported half-life is predicted to be diminished to just five days, if the product is exposed to temperatures of 37 °C [ 3 ]. Another factor determining stability, is RNA length which is calculated to be inversely correlated with stability against hydrolysis [ 3 ]. Attempts at fortification of mRNA stability employing lipid pharmacotechnic formulations are to be considered carefully: utilization of lipids whose cationic head group can lower the pKa of the 2-hydroxyl group residing on the ribose moiety of the mRNA are best avoided since even a 2-unit shift can significantly hasten mRNA hydrolysis [ 185 , 186 ].…”

“…Specifically, the major technological and research innovations over the past decade have enabled mRNA to become a viable therapeutic solution, overcoming some of the associated challenges in its use, such as its short half-life and innate immunogenicity. mRNA’s great potential can manifest clinically through vaccination against infectious diseases and cancers, protein replacement therapies, tissue regeneration and the treatment of genetic diseases [ 1 , 2 , 3 ]. Furthermore, as mRNA technology matures, its convergence with emerging adoptive immunotherapies can materialize into safer and more effective approaches such as the development of state-of-the-art chimeric antigen receptor T-cells ( Figure 1 ).…”