**6. Cellular Targets for mRNA and Plasmid DNA Vaccine Delivery**

For many vaccines, antibodies play a key role in protection. The cell that is transfected by the plasmid DNA or the mRNA vaccine does not have to be a professional antigen presenting cell (APC) in order to produce the antigenic protein that stimulates B cells. Cellular immune responses, notably CTLs, are thought to be important for tumor immunotherapy as well as to potentially play a role in protection against certain infectious diseases, e.g., tuberculosis (Tb), HIV, and malaria, or for vaccines effective against multiple strains of a virus, such as influenza, even though CTLs alone would not provide sterilizing immunity. In order for a vaccine to generate MHC Class I-restricted CTLs, the antigen either needs to be produced inside a professional APC or by a cell from which antigen can be cross-presented by an APC to then stimulate CTLs. The most direct way to ensure delivery of the

gene encoding an antigen to a professional APC is to transfect the cells in vitro prior to administering the transfected cells back to the patient. Indeed, the largest number of mRNA clinical trials currently underway, notably for cancer immunotherapy, involve the ex vivo transfection of cells with mRNA encoding tumor antigens followed by re-infusion of the transfected cells into the patient. Because this is a more cumbersome process for making a product than having a non-personalized product in a vial, direct administration of the plasmid DNA or the mRNA to a patient is preferable for convenience, cost, and time.

Plasmid DNA was shown to be effective for stimulating CTLs that were capable of protecting mice against influenza caused by a strain different from the strain from which the encoded antigen was derived [7,27,28]. Because the plasmid DNA, when injected intramuscularly (i.m.), primarily transduced muscle cells rather than professional Antigen Presenting Cells (APCs), the mechanism whereby MHC Class I-restricted CTLs were generated needed explaining. It was found that cross-priming appeared to be a key mechanism for generating CTLs following DNA vaccine immunization, as directly demonstrated by experiments with chimeric mice using bone-marrow derived dendritic cells [27], and because muscle cells were the only cells observed to translate the protein encoded by directly-injected plasmid DNA [6,29]. The efficacy in pre-clinical models raised the hopes that such plasmid DNA-based CTL-inducing vaccines could be developed that would be protective against multiple strains of HIV or influenza [7,30] (so as to produce a "universal" flu vaccine). Currently, existing influenza vaccines depend upon strain-specific antibodies, which result in strain-specific or strain-limited protection. Similarly, mRNA delivered in liposomes was shown early on to be capable of inducing CTLs [9]. The uptake of the mRNA is also mainly by non-immune cells, including muscle cells [31].

Both plasmid DNA and mRNA are also being developed for indications other than vaccines, such as for gene therapy. The delivery of plasmid DNA and mRNA to specific tissues or cells may thus be intentionally directed (in part) by the mode of delivery, the injection route, the formulation, and so on. Both entities are anionic due to the negative charges of the phosphate groups, and various formulations have utilized polycations. Thus, the biodistribution of each molecule depends not simply on the inherent charge and the size of the plasmid or mRNA, but the net charge of all the components of the formulation and the effect of any lipids.
