**11. Summary and Conclusions**

In summary, despite all the excitement over pre-clinical efficacy of mRNA, it should be remembered that in many ways, the mRNA field is recapitulating what occurred with plasmid DNA 20+ years ago, when seemingly almost any disease could be prevented or treated in pre-clinical animal disease models with the administration of an unformulated plasmid encoding a key antigen [1]. Therefore, one must keep in mind that pre-clinical immunogenicity or even protection/therapy, and human immunogenicity are low hurdles and are not predictive of human efficacy. One reason this is so challenging is that, for many of the diseases under evaluation, scientists do not know which immune response or combination of immune responses and which antigen targets are the crucial elements for efficacy; the vaccine technology alone is not the only piece of the puzzle. Table 5 summarizes the main advantages and disadvantages of mRNA vaccines with a comparison to DNA vaccines.

**Table 5.** Advantages and Disadvantages of mRNA Vaccines (and comparison to DNA vaccines).

#### Advantages:


Disadvantages:

	- a. Decreased stability of mRNA
	- b. Decreased translation into protein
	- c. Effects upon desired type of immunity
	- a. Finding the optimal delivery formulation/device for humans may be challenging given the unknown predictability of animal models (as with DNA vaccines, although DNA vaccines are much further advanced in clinical trials with different formulations and delivery devices for a number of different diseases)

Reported Phase I clinical trial results for mRNA vaccines are encouraging, although only the results of the first rabies mRNA vaccine have been published in the peer reviewed literature. The results for the human Metapneumovirus + Parainfluenza virus 3, and the Respiratory syncytial virus (RSV) phase I studies were announced via press release, thus details are not available. The target for the RSV vaccine mRNA was not publicly disclosed at the time the Phase I study was initiated, and to this date, the study appears to not be listed on clinicaltrials.gov. Whether the immune responses are at sufficient levels or have the types of needed immune responses and the necessary duration to result in protective efficacy is unknown and is not necessarily predicted by the Phase I studies.

One should also not ignore the reported toxicities seen with the rabies mRNA vaccine [55] that included limited systemic AEs for the majority of patients (78%) and even grade three AEs in ~10% of patients following doses of 80–400 μg mRNA via different routes, although the conclusions were that the vaccine was generally safe. It is not known whether the pre-clinical hepatic toxicity that proved to be a "no go" result for a particular Crigler-Najjar mRNA candidate is relevant to the mRNA vaccine studies from the same company, because, despite the low doses used, the doses and mRNA formulation for vaccine studies may be different. This is in comparison to DNA vaccine clinical trials

where 4 mg doses of DNA i.m. with boosts have been used in a variety of clinical trials with limited systemic symptoms [83–85] while generating good immune responses.

Just as DNA vaccines, after more than 25 years since the first publication of preclinical protective efficacy, are still a work in progress in improving potency and finding the right antigens and targets, there remain challenges for mRNA to become clinical products. For both DNA and mRNA vaccines (and monoclonal antibodies and bi-specific antibodies before them), a simple concept may have a challenging path to reality, and the technology may not be totally generic. mRNA may be even more complex than plasmid DNA because of the modifications (modified nucleosides) plus the formulations needed for stability, delivery, and the need to control the innate immunostimulatory activity of the mRNA. However, it also offers advantages in terms of manufacture that avoids the need for any animal or cellular products. The hope is that once the fundamental key challenges are solved for both plasmid DNA and mRNA, the clinical successes will come rapidly, although that has not occurred for moving from the veterinary licensed products for DNA vaccines into humans, demonstrating how much still needs to be understood, not just about the technologies but about the diseases that are being treated or prevented.
