Gene-based vaccination was born in 1990 after Wolff et al.
demonstrated the local uptake and expression of exogenously injected plasmid DNA and in vitro
transcribed messenger RNA (mRNA),1
followed shortly thereafter by the demonstration that injected nucleic acids could promote immune responses to encoded antigens.2,3
Both DNA and mRNA-based vaccines share many advantages over alternative protein, peptide, or live vector-based vaccine strategies in terms of safety, manufacturability and suitability for long-term storage, and the ability to promote broad cytotoxic T-cell responses.4,5
Since the first demonstrations, DNA-based vaccines have undergone extensive preclinical and clinical testing, while mRNA-based therapies remained less thoroughly investigated. However, DNA vaccines have failed to show potency in large-animal models and humans, in discord from results in small-animal studies,6
which may reflect in part the difficulty of overcoming not only the barrier posed by the plasma membrane of cells but also the need to transport DNA through the nuclear membrane of non-dividing cells. Vaccines based on mRNA, by contrast, require nucleic acid delivery only to the cytosol, thereby allowing the transfection of quiescent and post-mitotic cells that comprise the majority of target cells in vivo
To deliver macromolecules intracellularly, synthetic vectors are preferred over approaches based on viral vectors for their low cost, ease of large-scale production and potential for improved safety.9–12
To date, strategies reported for non-viral delivery of mRNA for vaccines and gene therapy applications include injection of naked mRNA,1,4
lipoplexes or liposome-entrapped mRNA,15,16
bolistic delivery via gene gun,17,18
, particulate carrier-mediated delivery19
Recently, clinical trials in which human patients were vaccinated with naked or protamine-complexed mRNA against tumor antigens via intradermal injections were completed, demonstrating feasibility, lack of toxicity and promising responses based on clinical and immunological read-outs.23,24
However, more efficient transfection in vivo
, and the ability to deliver these nucleic acids non-invasively and/or to mucosal sites, would be expected to enhance the prospects of this strategy for vaccination.
A variety of polymer and/or lipid-based drug delivery systems with the capability to disrupt endosomes have been developed that might be applicable for mRNA vaccine delivery25–29
, but systems that achieve efficient cytosolic delivery or transfection in vivo
with minimal cytoxicity are still sought. For vaccine applications, cytosolic delivery of mRNA into dendritic cells (DCs), immune cells that play a key role in the initiation of adaptive immune responses30
, is desired but the transfection of these cells poses a significant challenge as synthetic agents generally achieve only 10–35% transfection of these cells in vitro
We previously reported a pH-responsive core-shell nanoparticle system prepared by sequential emulsion polymerization of a secondary-amine-containing monomer (diethlyaminoethyl methylacrylate) forming a pH-responsive core, followed by a second monomer (aminoethyl methacrylate) forming a hydrophilic corona.34,35
The core-shell particle structure enabled the physical and compositional segregation of the functions for the particle into an endosome-disrupting pH-responsive core and a shell whose composition could be separately tuned to facilitate particle targeting, cell binding, and/or drug binding. These particles efficiently delivered associated protein or oligonucleotide cargos to the cytosol of dendritic cells through endosomal disruption mediated by the core polymer via the proton-sponge effect, while maintaining low cytotoxicity by sequestering the cationic charge and hydrophobicity of the polymer core within a more hydrophilic polymeric shell. However, a limitation of this proof-of-concept system is its lack of biodegradability, which hinders clinical translation.
Here we adapted our core-shell design approach to a fully degradable system comprised of a pH-responsive poly(β-amino-ester) (PBAE) core and a phospholipid shell. As a first step toward enhanced mRNA vaccines, we show that negatively-charged mRNA can be adsorbed via electrostatic interactions onto the surface of these cationic nanoparticles, protecting the nucleic acids from degradation in serum. Lipid-enveloped PBAE particles disrupted endosomes and delivered mRNA into the cytosol of DCs with minimal cytotoxicity, leading to in vitro transfection of a DC clone at levels comparable to the best reports for DC transfection from the literature. Importantly, mRNA-loaded lipid-enveloped particles also promoted in vivo transfection following non-invasive intranasal delivery, suggesting their potential utility for mRNA vaccine formulations.