Lipid nanoparticle delivery systems for saRNA vaccines

Excitement for RNA-based vaccines is rapidly growing due to their versatility and potential to protect against a range of viral and non-viral pathogens. Self-amplifying RNA (saRNA) vaccines offer the same key advantages as messenger (mRNA) vaccines ― such as rapid development, modular design, and cell-free synthesis ― with an added benefit. The self-replicative properties of these formulations maintain efficacy against disease over time, leading to lower overall dose requirements. Since less RNA is needed for the same level of protection across populations, there’s a reduced manufacturing burden that allows facilities to make 100 times more saRNA versus mRNA, lowering cost and time of production. As a result, distributed/decentralized manufacturing and quick mass immunizations are possible in the event of a pandemic.

Challenges in developing RNA-based drugs or vaccines

There are two main challenges when trying to successfully develop RNA vaccines:

1. RNA is a large, negatively charged molecule and requires a carrier vehicle to get into the cells.
2. RNA is delicate and is likely to break down quickly during process development, manufacturing, and/or formulation development.

Unlike many other small molecule and biological drugs, nucleic acid therapeutics, such as saRNA-based vaccines, require specialized delivery technology. Carefully formulated carriers help avoid degradation and protect the payload cargo to ensure it remains efficacious upon arriving at the target cells. Systematically screening various delivery vehicles and selecting appropriate formulations are therefore critical steps in developing next-generation mRNA vaccines.

Lipid nanoparticle technology helps meet delivery needs for saRNA vaccines

Effective and efficient saRNA delivery systems condense the nucleic acid payload into a cationic carrier that can be taken into a cell without being degraded. So far, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes, and cationic nanoemulsions (Fig 1).

Fig 1. Non-viral saRNA delivery systems

Among all the delivery platforms, LNP has gained the most traction clinically with the recent FDA approval of COVID-19 mRNA vaccines. Now, saRNA vaccines are being studied for use against many infectious diseases, like malaria and COVID-19, with several saRNA LNP vaccine clinical trials currently underway.

The lipid nanoparticle approach to delivering RNA-based vaccines provides:

1. Protection of the RNA cargo during development until it reaches the target cells.
2. Enhanced uptake by antigen-presenting cells.
3. A means to release the RNA cargo from the early endosomes by interacting with the endosomal membrane.

Fig 2. Lipid, polymer, and emulsion-based delivery systems all use cationic groups to mediate condensation of the anionic RNA and delivery across the cell membrane. LNP systems, which have been found to be the most potent vaccine formulation, utilize a pH-sensitive ionizable cationic lipid as the main constituent and are taken up in cells through receptor-mediated endocytosis. In the endosome, the lower pH environment ionizes the cationic lipids, which then interact electrostatically with anionic lipids in the endosomal membrane. These ion pairs cause a phase transition into a porous hexagonal phase (HII) that disrupts the endosome and facilitates the release of the RNA into the cytoplasm.

Understanding the mechanism of LNP formation and delivery

In a study conducted by Imperial College, London, polymeric and lipid nanoparticles were considered in the delivery of self-amplifying RNA vaccines (2). Researchers used saRNA encoding pre-fusion stabilized SARS-CoV-2 spike protein delivered via LNP for a COVID-19 vaccine, and hemagglutinin (HA) glycoprotein for an influenza vaccine.

Dr. Anna Blakney, the study's first author and now an Assistant Professor in the Michael Smith Laboratories and School of Biomedical Engineering at The University of British Columbia (UBC), worked on the development of molecular and biomaterial engineering strategies for the delivery of self-amplifying RNA, under the supervision of Prof. Robin Shattock and Prof. Molly Stevens of Imperial College, London.

During this time, Dr. Blakney and her research team aimed to better understand how the components of nucleic acid delivery formulations interact with the immune system to improve potency and enable clinical translation. They approached Dr. Andy Geall, Chief Development Officer at Replicate Biosciences, and Dr. Anitha Thomas, Director of R&D at Cytiva, seeking guidance on how to experiment with RNA formulation approaches, nanoparticle characterization methods, and a variety of biochemical assays.

Dr. Blakney developed the study using the NxGen™ microfluidic instrument for lipid nanoparticle formation ― the NanoAssemblr Ignite™ system. Dr. Anitha Thomas, who has extensive experience in LNP formulations, gave the research team access to a proprietary LNP portfolio. Their goal was to choose application-specific LNP compositions. Through their study, Dr. Blakney and the team offered an understanding of how LNP can act as an efficient delivery vehicle for saRNA vaccines.

In the study published in the Journal of Controlled Release, researchers extensively investigated the role of lipids and the route of administration (intramuscular versus intranasal) in vaccine delivery (2). Both factors were found to impact the vaccine immunogenicity of influenza hemagglutinin and the SARS-CoV-2 spike protein.

The researchers compared two delivery vehicles for saRNA vaccines ― a lipid nanoparticle and a bio-reducible polymer (pABOL) ― specifically assessing protein expression and immune responses (2). For the LNP delivery system, a proprietary mix of ionizable lipids with two helper lipids was prepared at a total lipid concentration of 25 mM in ethanol. While the polyplex formulation had a higher protein expression, the LNP-encapsulated saRNA exhibited higher humoral and cellular immunity in both vaccine models (influenza hemagglutinin and the SARS-CoV-2 spike glycoprotein).

Although the size of the pABOL polyplexes and LNP were equivalent, the pABOL particles had a net positive surface charge, while the LNP formulations had a neutral surface charge at physiological pH (2). This finding was as expected due to the ionizable cationic lipids comprising the LNPs, which is common for mRNA and saRNA LNP formulations. The researchers concluded that a neutral surface charge could have increased immunogenicity and also found that helper lipids impact immunogenicity. 

While assessing the route of administration, researchers found that both systemic and mucosal antibodies developed in intramuscular (IM) and intranasal (IN) administrations (2); however, the immune responses were 10-fold higher with the IM route of administration.

The above findings suggest that different delivery systems and routes of administration may fill different delivery niches in the field of saRNA or mRNA-based vaccines. Overall, all groups of mice vaccinated with LNP formulations showed superior cellular immunity and reactogenicity. Although more research is needed in the pre-clinical phase, these results indicate that the saRNA formulation with IM and IN route of administration can cause acute, cytokine-driven reactogenicity, allowing for potent humoral and cellular responses.

Fig 3. Development of LNP- mRNA Vaccine

This study emphasizes the importance of LNP delivery vehicles and selecting appropriate formulations to ensure effective therapeutics or vaccines across applications. LNPs, as a clinically validated and scalable technology, have the potential to be the vaccine development technology of the future. Extensive research and investigation can tap the full potential of LNP-delivered saRNA vaccines which are efficient, require a lower dose versus mRNA formulations, and are available for fast vaccine production.