(T1030-03-17) Optimized and Scalable LNP Composition for RNA Vaccine Delivery

Purpose: Lipid nanoparticle encapsulated RNA (RNA-LNP) vaccines have played a pivotal role in the global response to the COVID-19 pandemic, with over two billion people vaccinated with RNA-LNPs worldwide (1). With proven safety and efficacy, in addition to other benefits including scalability and cost-effectiveness, RNA-LNPs are now at various stages of development against a variety of other infectious diseases including respiratory syncytial virus (RSV), influenza and HIV (2). LNPs typically consist of four main components including a phospholipid, cholesterol, PEGylated lipid and, most critically, an ionizable lipid. However, access to potent ionizable lipids is limited and this, in addition to the significant know-how required to formulate and scale LNPs, present real barriers to entry in this field. To overcome these challenges, we developed and optimized a novel ionizable lipid and a corresponding LNP composition for the delivery of RNA in vaccine applications. Using a scalable mixing platform, we produce RNA-LNP vaccine candidates using mRNA and self-amplifying RNA (saRNA) encoding Influenza A virus (A/California/07/2009 (H1N1)) haemagglutinin, and SARS-CoV-2 spike proteins, respectively, and investigate their physicochemical attributes, in vitro potency, and in vivo immunogenicity.

Methods: RNA-LNPs were prepared on the NanoAssemblr® Ignite™ system (Precision NanoSystems ULC) using a custom lipid mix including an ionizable lipid from the Precision NanoSystems Lipid Portfolio, encapsulating different RNA including 1) Cleancap® eGFP mRNA (TriLink), 2) H1N1 HA PNI mRNA, 3) eGFP PNI saRNA and 4) nCoV PNI saRNA (Precision NanoSystems ULC). Unless otherwise stated, RNA-LNPs were prepared at a total flow rate of 20 mL/min, a flow rate ratio of 3:1, and a N/P ratio of 8. Organic solvent was removed by dilution with a physiological buffer (pH 7.4) and ultrafiltration using 30 kDa Amicon® filters (Millipore Sigma). LNPs were stored at -80°C in a cryopreservation buffer for up to 6 weeks. RNA-LNP size and polydispersity were determined by dynamic light scattering using a Zetasizer™ (Malvern Panalytical), and encapsulation efficiency by Ribogreen™ (Thermo Fisher Scientific). In vitro protein expression was determined on HEK-293 cells by flow cytometry following treatment with eGFP mRNA and saRNA-LNPs. In vivo immunogenicity was investigated by intramuscular injection of BALB/c mice with SARS-CoV-2 saRNA and haemagglutinin mRNA LNPs, with a booster dose on day 28. Tail bleeds were performed on day 21 and mice were culled by cardiac puncture on day 42 followed by spleen removal. IgG titers and HA inhibition were assessed using sera, and cytokine response using splenocytes and a 12-plex LEGENDplex™ MU Th cytokine panel (Biolegend). To demonstrate the scalable nature of both the novel LNP composition and the mixing process, we took our lead SARS-CoV-2 saRNA-LNP vaccine candidate and prepared batch sizes of 100 mL at 48 L/h using the Nanoassemblr® commercial formulation system (Precision NanoSystems ULC).

Results: The novel LNP composition, combined with a scalable mixing process, resulted in high quality RNA-LNPs using different RNA with varying chain length (up to 12k nucleotides) and structures, with reproducible particle sizes of 60-90 nm, polydispersity indices < 0.2, RNA encapsulation >90% and GFP transfection of 70-80%. In addition to the robustness of the LNPs with different RNA payloads, we observed stability of the LNPs when stored at -80°C in cryopreservation buffer, with no detrimental effects on physicochemical attributes and in vitro potency observed over 6 weeks. In vivo immunogenicity studies were completed using SARS-CoV-2 saRNA and HA mRNA-LNPs. A strong humoral and cellular immune response was mounted against both antigens, with antigen specific IgG titers of 106 ng/mL observed, and IFNγ responses >100-fold higher than naïve mice. Finally, RNA-LNPs prepared on a commercially relevant scale were found to be comparable to those prepared on the benchtop scale with respect to physicochemical attributes and immunogenicity.

Conclusion: In this study we developed and optimized a potent and scalable LNP composition for vaccine applications, demonstrated using different RNA coding for clinically relevant antigens. All the RNA-LNPs had high-quality physicochemical attributes, and strong biological activity in vitro and in vivo. Consequently, we envisage this new LNP composition will have utility for a wide-range of preclinical drug development activities including antigen screening, and in vivo proof-of-concept studies. In addition, we have de-risked the scale-up of this composition and the mixing process, showcasing how these technologies can be used to accelerate drug development timelines.

References: 1. Verma M, Ozer I, Xie W, et al. The landscape for lipid-nanoparticle-based genomic medicines. Nat Rev Drug Discov. 2023 Jan 10.
2. Gote V, Bolla PK, Kommineni N, et al. A comprehensive Review of mRNA vaccines. International journal of molecular sciences. 2023 Jan 31;24(3):2700.