**7. Increasing the Potency of DNA and mRNA Vaccines**

For DNA vaccines, despite the ease with which preclinical studies demonstrated efficacy for a variety of disease models, the potency in humans proved generally disappointing. This led to a number of approaches to increasing the potency by increasing the amount of protein produced through redesigns of the plasmid. Additionally, adjuvants and other immunostimulants were included (such as cytokines and co-stimulatory molecules) either as recombinant proteins or encoded by plasmid DNA, by various formulations and delivery devices, and by strategies such as prime-boost combinations (generally using plasmid DNA as a prime followed by a heterologous boost with a viral vector or protein). The DNA plasmids themselves were optimized by trying different promoters, adding CpG motifs, (cytosine connected via a phosphodiester bond to guanine-such CpG motifs are pathogen-associated molecular patterns) (see below), codon optimization, etc. As noted above, the initial work by Felgner [6] demonstrated that the expression of protein encoded by plasmid DNA was highest in muscle following intramuscular injection versus expression in other tissues after intravenous or subcutaneous injection. Likewise, immune responses were highest with direct i.m. syringe injection of naked plasmid DNA rather than via intravenous (i.v.), intradermal (i.d.), or subcutaneous (s.c.) injections [7]. Early delivery devices for plasmid DNA included a biolistics gene gun that propelled DNA-coated gold particles into cells [32]. In addition to simple i.m. injection, approaches now include pressurized devices (such as the Biojector® or Stratis®), or electroporation, which supplies an electric current to cause temporary fenestration of membranes to increase the passage of plasmid into the cells and the nuclei.

For mRNA, the areas of continued Research and Development (R&D) efforts for improving the potency of mRNA vaccines are shown in Table 1, which describes efforts similar to those for DNA vaccines. These focus on augmenting delivery of the mRNA and increasing potency via increased stability and greater expression of the protein. Alterations of the mRNA itself include changing the codon usage and the GC (guanine-cytosine) content [5], along with modifications of the other regions, such as the 5 cap, the UTRs, and the poly-A tails. A more detailed description of the efforts to increase the mRNA potency and of delivery formulations that include lipids, nanoparticles, polymers, polycations, and various proprietary entities are presented and reviewed elsewhere with tabular and chemical descriptions [2,11,33,34]. Cells generally take up mRNA by endocytosis, thus efforts are also being made to design delivery systems that increase the endosomal release of the mRNA into the cytoplasm [35]. Certain formulations, such as delivery of a particular encapsulated lipoplex mRNA vaccine, were found to specifically be taken up by dendritic cells via micropinocytosis [36]. As with DNA vaccines, possible immunomodulators added as recombinant proteins or encoded by mRNA are being evaluated [37]. Various routes of injection of mRNA are being explored, including i.m., i.d., s.c., i.v., and intranodal [2], in addition to the ex vivo approach described. Delivery devices such as the gene gun (where mRNA is put onto gold particles) [38] and electroporation are also being explored.

**Table 1.** Continued Research and Development (R&D) Focus for mRNA Vaccines.


Circular RNAs (circRNA) are endogenously expressed and are thought to play roles mainly for gene regulation, with potential activity as tumor antigens [39]. They can be exogenously constructed to produce proteins in cells [40]. These engineered circRNA molecules appear to be more stable and to result in more potent production of protein than linear mRNA. However, the mechanisms for their effects upon gene regulation and other activities are still being explored [41].
