Summary

Messenger RNA (mRNA) vaccines provide several potential advantages over conventional vaccines including faster and more economical development and manufacture of new vaccines, as well as the ability to easily engineer the antigen. Additionally, mRNA vaccines are potentially safer than live or attenuated vaccines because they are completely synthetic. Moderna Therapeutics (Cambridge, USA) have been led the field in developing mRNA vaccines, using lipid nanoparticles (LNPs) to encapsulate and deliver mRNA for Zika and Influenza vaccines, some of which are in clinical trials. In a paper published in Molecular Therapy Nucleic Acids (Available online February 7, 2019, and in print April 2019), they endeavor to optimize LNP formulations for intramuscular (IM) delivery of vaccines. A large body of work exists for optimizing LNP formulations for intravenous (IV) injection, particularly for delivery of small interfering RNA. Intramuscular injection is the preferred route of administration for vaccines. Many factors affect the potency of the nucleic acid being delivered including the length of the nucleic acid, the formulation of lipids in the LNP, the route of administration, and the cells in which the nuclieic acid needs to be active. Hence the challenges of delivering an mRNA vaccine are unique and a thorough study of possible optimizations are required. Furthermore, immune response is crucial to the effectiveness of mRNA vaccines but activation of inflammatory responses can lead to unwanted side effects. To that end, a panel of biodegradable ionizable lipids designed to be cleared rapidly following mRNA delivery were developed by Moderna.

The authors set about optimizing formulations by screening 30 different LNPs featuring different biodegradable ionizable cationic lipids and compared them to the canonical ionizable cationic lipid MC3 in terms of immunogenicity and expression of a reporter gene (Luciferase) following IM administration. Expression levels were also compared with IV administered formulations. 5 formulations were selected for futher study on the basis of improved egen expression over MC3. Tolerability was tested in a rat model and expression and immunogenicity were tested in a non-human primate (NHP) model. These studies required a large number of small formulations for in vivo screening as well as a larger volume of fewer formulations for testing in larger animal models. Moderna formulated these using the NanoAssemblr platform which is ideal for rapid, consistent, and inherently scalable LNP formulation.

Several surprising findings were reported on the basis of these results: (1) Good expression after IV administration does not predict good expression by IM administration. (2) The highest protein expression from IM injection was achieved with ionizable lipids with higher pKas compared to IV administration. (3) An optimal pKa of the ionizable lipid for Immunogenicity after IM administration is between 6.6-6.8. (4) Improved antigen expression did not necessarily lead to better immune response.  (5) mRNA vaccines do not necessarily require strong adjuvant response to induce potent immune response

With these results, the authors identified one ionizable cationic lipid in particular, which they call Lipid H, that led to improved tolerability, reduced innate immune stimulation, improved endosomal escape of the mRNA payload while maintaining immune titers comparable to MC3. This systematic approach to optimizing mRNA-LNPs has shed considerable light on improving the tolerability and potential effectiveness of mRNA vaccines for IM administration. There is more to be optimized and understood about the differences between IM and IV administrations and the role of the innate immune response in the effectiveness of a vaccine. Importantly, this study points to ways in which formulation can further be optimized. For instance, a better understanding of how features of the cationic lipids correlate with differences in uptake and payload escape in different cells and can further improve IM delivery. The NanoAssemblr platform is an efficient and powerful tool for making a large number of LNP formulations for in vivo screening and to scale lead candidates up for late-stage testing and optimization. 

The paper is open access and available online through the publisher’s site.

Abstract 

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency.