**9. Other Potential Safety Issues**

When DNA vaccines initially entered into human clinical trials, concern was raised about the theoretical possibility of them causing autoimmunity or that the DNA would integrate into the genome. The rationale for concerns about autoimmunity was that anti-DNA antibodies are a hallmark of various autoimmune diseases. To date, both pre-clinical testing and careful clinical monitoring have shown DNA vaccines to not induce or to worsen auto-immunity, and in fact, human clinical trials employing DNA vaccines for therapy of two autoimmune diseases (diabetes mellitus and multiple sclerosis) gave encouraging results in human clinical trials for a therapeutic benefit of the DNA vaccines [47,62]. For mRNA, a proposed mechanism for possible autoimmune responses is via the induction of type I interferon [63], which may result in both inflammation and possibly autoimmune responses [64]. This includes work showing that the responses seen in mice were similar to those seen in humans for an influenza mRNA vaccine construct via TLR7 and TLR8 in humans and via cytoplasmic RNA sensors in both mice and humans [65].

DNA vaccines did not need to be evaluated by the US National Institutes of Health (NIH) Recombinant Advisory Committee prior to human clinical trials, unlike viral vectors for gene therapy. Nevertheless, significant safety studies were initially required to evaluate the possibility of integration of the plasmid DNA into the host genome. As a result of these studies for both human vaccines [18,66] and for the licensed DNA vaccines for fish [67], as well as the many human studies with DNA vaccines that have demonstrated safety, little concern now exists regarding integration. Comparisons have stated that mRNA offers an advantage because RNA itself cannot integrate into genomic DNA without the presence of the viral elements in a retrovirus that enable such integration (reverse transcriptase and integrase). However, HERVs [68] (human endogenous retroviruses) whose remnants are now permanent parts of human genomes as retrovirus-like sequences comprise up to 8% of the human genome. In addition, some recipients of mRNA drugs or vaccines may be already infected with a retrovirus (e.g., HIV), thus providing a theoretical means for provision of the proteins needed for integration [69,70]. Nevertheless, the risk of integration remains, at this point, extremely unlikely for mRNA, even from a theoretical standpoint, nor is it any longer a significant concern for plasmid DNA. This means that mRNA does not offer any clear advantage compared to plasmid DNA in this regard. From a regulatory perspective, mRNA prophylactic vaccines appear to not be considered gene therapy products [71], similar to DNA vaccines before them.
