**4. Issues to overcome for better translating the generated proofs-of-concept to effective treatments in human**

Bodies of evidence clearly indicate that bacterial vectors are a promising strategy for gene delivery. Many experimental investigations have shown proof-of-concept examples of the feasibility of such an approach, yet steps forward are still needed not only to translate these concepts into effective treatments for humans but also to find the perfect delivery system for each disease situation.

For safety reasons, nonpathogenic food-grade bacteria remain more attractive as live vectors for vaccine and therapeutic strategies. Some concerns exist, however, about targeting issues which is crucial for optimal efficiency. The best example is the potential use of *Lactobacillus lactis* for the delivery of IL-10 in the treatment of IBD. As this anti-inflammatory cytokine has a pleiotropic immunosuppressive effect, it is particularly crucial to target the inflammatory site while preserving healthy tissues. On the other hand, studies related to the potential application of some microbial vectors in gene therapy are on hold for safety issues. Although research on attenuated bacteria has led to significant progresses in gene therapy, there remain some limitations that preclude their use in immunocompromised populations as well as in infants. The challenge is how to emphasize the benefits while controlling the disadvantages of these microbial vectors. With this regard, recent studies highlighted new lines of develop‐ ment of TTSS-based delivery in avirulent vectors. Interestingly, the gene locus coding for TTSS of *Vibrio parahaemolyticus* has been cloned into a nonpathogenic *E. coli* K-12 strain and shown to be efficient in the delivery of heterologous peptides. The generation of a nonpathogenic *E. coli* displaying an active TTSS is an important step that opens the way for applicability of TTSSdependent delivery of foreign molecules [58]. In the same way, it has been shown that bacterial minicells derived from aberrant cell division of a mutant strain of *S. typhimurium* may assemble functional TTSS. These nonreplicating nanoparticles were shown to deliver antigen by the TTSS and to promote Th1 immune response, thereby offering an alternative strategy of antigen delivery platform for vaccine and immunotherapeutic developments [59].
