Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution

Abstract

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.

A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability

“…6, 57 AAHI-SC2 appears to elicit strong T cell responses in preclinical studies, possibly due to the combination of the adjuvanting properties of squalene and saRNA, and may provide a foundation for durable and long-term T-cell immunity. Upcoming studies in non-human primates and clinical trials in humans should provide additional data to evaluate the immunogenicity, and particularly the T cell induction, of AAHI-SC2 for comparison to published data on mRNA-1273, BNT162b2, and other SARS-CoV-2 mRNA 6, 7, 58 or saRNA 24, 25, 27, 29, 30 vaccines in development.…”

“…Additional RNA vaccines are currently under development with reported stability data, but these data are sparse and indicate limited, short-term room-temperature stability of days to a few weeks at best. 40, 58, 67 As these reports illustrate, maintenance of RNA integrity during storage and after administration is challenging. RNA molecules are more susceptible to degradation than DNA due to the presence of the 2’ hydroxyl group on the ribose backbone.…”

A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability

“…Additional RNA vaccines are currently under development with reported stability data, but these data are sparse and indicate limited, short-term room-temperature stability of days to a few weeks at best 13,45,65 . As these reports illustrate, maintenance of RNA integrity during storage and after administration is challenging.…”

The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system

“…Indeed, according to recent studies, the larger particles, which are susceptible to contain and deliver a larger amount of mRNA copies per particle and target different cell types, could induce better responses, provided the particle size remains within the limits of lymphatic drainage. 57 , 58 , 59 Furthermore, the better performance of L319 LNPs with UNR seems to be linked to the lack of strong IFN-α induction by this formulation since we observed a negative correlation between the induction of high HI titers and high innate IFN-α production. With L319 LNPs, there was no or little IFN-α detected, even when loaded with UNR, and antibody and T cell responses were high with both UNR and MNR.…”