Self-replicating (self-amplifying) RNA replicons are genetically engineered viral RNA molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions (infectious virus particles). Self-replicating subgenomic RNA replicons or DNA-launched RNA replicons (i.e., DNA-encoded replicons) are routinely delivered into host cells using either electroporation (in vitro) or its combination with injection (e.g., subcutaneous, intramuscular) in vivo. The application of electroporation for in vitro conditions has virtually no disadvantages, whereas in vivo electroporation has two major problems associated with it. First, in vivo electroporation lacks reproducibility. Second, in vivo electroporation is quite painful, meaning that in addition to the pain associated with injection there will be the electroporation-associated pain.
Chapter 2: Identifying the most promising RNA vaccine formulation
The lipid-based delivery approach – the domination and the drawback
Twelve major delivery methods for mRNA vaccines are listed in the review article1. There are ten delivery methods, which contain a component other than RNA. For at least six methods, this component is lipid. Thus, the majority, i.e., at least 60%, of current RNA vaccine formulation modalities contain lipid molecules.
Invitrogen™ Lipofectamine™ (Thermo Fisher Scientific) cationic lipid-based transfection reagents are expensive, efficient, and widely used for DNA (2000, LTX/PLUS) and siRNA (RNAiMAX) delivery into eukaryotic cells typically growing on plastic in cell culture incubators. Lipofectamine type of compounds are referred to as non-viral, or lipoplex-type, delivery vectors.
There are limited amount of works that compare the transfection efficiency of viral and non-viral vectors in a direct and quantitative manner. Two studies, however, shed light on striking differences in transfection efficiency associated with viral and non-viral delivery methods. In particular, it was demonstrated that to achieve a comparable to adenovirus-mediated transgene expression, the Lipofectamine reagent had to deliver 3 orders of magnitude (~7,000-fold) more DNA copies into the cell2,3. It was further found that endosomal escape of Lipofectamine-formulated DNA was nearly as efficient as that of adenovirus2. Subsequent analysis showed that transfection efficiency difference seen between Lipofectamine- and adenovirus-based DNA delivery was due to decreased transcription (16-fold) and decreased translation (460-fold) efficiencies associated with the lipid-based delivery method3. Furthermore, it was demonstrated that Lipofectamine inhibited the test tube mRNA translation approximately 20-fold3.
Given the data presented above, it becomes clear that lipid stays associated with DNA after its delivery both into cytosol and into nucleus of the cell. This conclusion can be equally well applied to mRNA or replicon RNA delivery. As a result, the lipid-DNA, lipid-mRNA, and lipid-(replicon RNA) complexes cannot be efficiently utilized by cellular transcription and translation machineries, which leads to severely impaired efficiency of the above-mentioned machineries.
Cationic polymers/nanoemulsions and peptides – difficulties with entry
Another 30% of current mRNA vaccine formulation modalities which contain at least a single component that is not RNA can be roughly grouped as purely cationic delivery vehicles. This “cationic” group includes cationic polymer, cationic nanoemulsion, and polycationic peptide called protamine1. The major drawback of purely cationic or polycationic formulation is their stickiness to the cell membrane as demonstrated for nona-arginine peptides4, which can serve as a model for the protamine-based mRNA delivery.
It has been demonstrated that protamine-formulated non-replicating mRNA vaccines stored at temperatures in the -80°C to +70°C range for several months were stable and preserved their protective capacity in mice after intra-dermal injection5. The subsequent follow-up first-in-human phase 1 clinical trial demonstrated clearly that the variation of the same anti-rabies mRNA vaccine fails to induce functional antibodies when injected by a needle-syringe6. In the same study, however, it was demonstrated that physical needle-free gas- or liquid jet-driven methods that disrupt the skin barrier and damage dermis do lead to the appearance of boostable functional antibodies against the rabies antigen. Consequently, when applied via an injection the non-replicating protamine-formulated mRNA vaccines does not work in humans and physical methods akin to electroporation have to be used.
The situation described above for non-replicating polycationic protamine-based mRNA vaccine implies that there is a bottleneck that does not allow the mRNA to be translated. In other words, it follows that in humans, protamine-mRNA vaccine does not enter the cells after conventional needle-syringe injection. Similar consequences are also observed after subcutaneous injection of nona-arginine(R9)-type cell-penetrating peptide(CPP)-formulated DNA vaccines in swine – DNA is not internalized by the cells of dermis, whereas injection coupled with electroporation leads to powerful and boostable immune responses. The most advanced CPPs are typically covalently linked to fatty acids like stearyl moiety, however, it does not boost their activity in vivo.
In this Solution, NextGenRnD proposes novel RNA vaccine formulation that: (i) is based on FDA compliant materials; (ii) should be compatible with encapsulation of RNA molecules ranging in size from 700 nucleotides (nts) to approximately 24,000 nts; (iii) can be lyophilized and resuspended when necessary; (iv) can be used for subcutaneous or intramuscular delivery; (v) is low cost, i.e., the cost of materials for 0.5 milligram (mg) RNA encapsulation is approximately $1; (vi) is neither lipid-based nor depends on cationic polymers/nanoemulsions or peptides; (vii) does not require electroporation.