The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-γ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

This review will explore the four major pillars required for design and development of an saRNA vaccine: Antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). We report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to messenger RNA (mRNA), thus minimizing the required dose. However, previous delivery strategies were optimized for siRNA or mRNA and do not necessarily deliver saRNA efficiently due to structural differences of these RNAs, thus motivating the development of saRNA delivery platforms. Here, we engineer a bioreducible, linear, cationic polymer called ‘pABOL’ for saRNA delivery and show that increasing its molecular weight enhances delivery both in vitro and in vivo. We demonstrate that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo, and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics.

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two-dose heterologous vaccination regimens than single-dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1⁺ CD4 T cells, which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.