**3. Type III delivery system: A promising strategy for targeting intended cell tissues**

A broad spectrum of pathogenic bacteria (*Salmonella*, *Shigella*, *Yersinia*, *Pseudomonas*,...) use the type III secretion system (TTSS) to deliver their effector molecules to the membrane or into the host cell's cytosol to subvert the signaling pathways [32,33]. Most of the effector proteins are produced and stored inside bacteria before their secretion by the TTSS upon contact with host cells [34]. This elaborate process allows the bacteria to optimize the function of delivered molecules and, therefore, to resist the host defense mechanisms and proliferate within their niche. The potential of TTSS in gene therapy has been investigated in various experimental models for localized delivery of vaccine antigens or therapeutic molecules.

#### **3.1. Application in immunoprophylaxy**

The first attempt in using the TTSS for the delivery of heterologous antigens for vaccine purposes was performed with attenuated *Salmonella*. It has been shown that recombinant *Salmonella* harboring a heterologous gene from pathogenic microorganisms fused to a *Salmonella* effector protein-encoding gene or to a small DNA sequence coding for bacterial signal peptide was able to deliver the hybrid protein into the host cytosol [35]. When injected into mice, these recombinant bacteria induced a protective cytotoxic T lymphocyte (CTLs) response against infection. Thus, the engagement of the hybrid protein by the TTSS allows their subsequent engagement by the major histocompatibility class-I pathway and generation of CTLs that are required for effective immunity against intracellular pathogens [36–38]. The use of the TTSS vaccination approach has been proven to work in different infectious models. In parasitic models of *Plasmodium berghei* infection, TTSS-dependent delivery of a dominant CD8 epitope by *Salmonella* conferred protection from infection in mice [39]. In a similar way, *Yersinia* has been used in vaccination studies in murine models to deliver antigens from a pathogenic protozoan parasite *Entamoeba histolytica* [40]. In this model, it has been shown that TTSS can mediate the delivery of high-molecular-weight antigen that induced significant protection against infection through promoting specific type 1 immune response.

The experimental approach of the bacterial TTSS in vaccination studies has been investigated in cancer models as well. Studies in mice indicated that oral administration of recombinant *Salmonella* expressing tumor antigens induced CD8+ T cell-mediated control of tumor progres‐ sion [41,42]. *Pseudomonas aeruginosa* was also evaluated as a live attenuated vector for TTSS delivery of antigen in antitumor vaccine experiments. Inoculation of recombinant *Pseudomo‐ nas* delivering ovalbumin to mice was shown to induce specific CD8+ T cell response that was associated with a significant resistance against ovalbumin-expressing tumor [43]. These experimental investigations underline the efficacy of this delivery system in antitumor immunoprophylaxy.

Besides their role in the delivery of heterologous antigens, bacterial vectors present major advantages over nonmicrobial adjuvant vaccines in that they are endowed with the ability to induce innate immunity through pathogen-associated molecular patterns (PAMPs). These specific microbial motifs include lipoproteins, lipopolysaccharides, single-strand RNA, and nonmethylated DNA sequences that can trigger the maturation process of antigen-presenting cells through binding to their specific Toll-like receptors and consequently induce the pro‐ duction of inflammatory cytokines [44]. This is particularly interesting for vaccination strategies aiming to optimize the protection efficacy [45].

#### **3.2. Application in therapeutic development**

Optimal efficiency of any microbial vector in gene therapy relies particularly on its ability to deliver a sufficient amount of the drug to targeted cell tissues while preserving healthy tissue. The fact that *Shigella* specifically colonizes the colon and activates the TTSS upon contact with epithelial cells prompted their use as a candidate for localized delivery of anti-inflammatory mediators in inflammatory bowel diseases. Ulcerative colitis and Crohn's disease are charac‐ terized by the massive production of inflammatory cytokines such as TNF-α and IL-1β that mediate colon tissue destruction. Anti-inflammatory recombinant IL-10 was used successfully for treatment of IBD, although high doses and repeated administrations were necessary for minimal therapeutic efficacy [46–50]. *Bacteria* TTSS-mediated delivery of IL-10 may offer a good alternative of treatment targeting the colon. The proof-of-principle of this strategy was shown in inflammatory models of *Shigella* infection. When IL-10 was fused to a bacterial signal peptide, the hybrid protein was shown not only to be delivered by the TTSS of *Shigella* but also to be biologically active. Injection of IL-10 recombinant *Shigella* to mice induced a marked reduction of inflammatory symptoms as compared to wild-type *Shigella* and this was associ‐ ated with a significant local reduction of TNF-α, a major inflammatory cytokine [51]. IL-1 receptor antagonist is a natural inhibitor that antagonizes the inflammatory potential of IL-1β. Imbalance between IL-1β and IL-1 receptor antagonist is associated with acute intestinal inflammation [52,53]. In keeping with this, it has been shown that delivery of recombinant IL-1 receptor antagonist in the intestine blocks IL-1β-mediated colitis in rabbits [54]. Localized delivery of IL-1 receptor antagonist by the TTSS of *Shigella* was shown to be as efficient as IL-10 in reducing the inflammatory symptoms within invaded tissues [51]. As outlined elsewhere, the treatment of experimental colitis could be partially achieved using IL-10 recombinant *Lactobacillus* that colonizes all the intestine. Nevertheless, *Shigella* may provide a useful alternative as a live vector thanks to its specific targeting to the site of IBD, the colon. Yet, due to safety concerns, this is possible only with the use of highly attenuated *Shigella* that can be biologically contained [55]. Furthermore, the efficiency of such an approach awaits additional insight into experimental intestinal models of *Shigella* [56,57]. Taken together, the use of bacterial TTSS for localized delivery of immunogenic antigens or therapeutic molecules may offer alternative options in improving the effectiveness of gene therapy.

niche. The potential of TTSS in gene therapy has been investigated in various experimental

The first attempt in using the TTSS for the delivery of heterologous antigens for vaccine purposes was performed with attenuated *Salmonella*. It has been shown that recombinant *Salmonella* harboring a heterologous gene from pathogenic microorganisms fused to a *Salmonella* effector protein-encoding gene or to a small DNA sequence coding for bacterial signal peptide was able to deliver the hybrid protein into the host cytosol [35]. When injected into mice, these recombinant bacteria induced a protective cytotoxic T lymphocyte (CTLs) response against infection. Thus, the engagement of the hybrid protein by the TTSS allows their subsequent engagement by the major histocompatibility class-I pathway and generation of CTLs that are required for effective immunity against intracellular pathogens [36–38]. The use of the TTSS vaccination approach has been proven to work in different infectious models. In parasitic models of *Plasmodium berghei* infection, TTSS-dependent delivery of a dominant CD8 epitope by *Salmonella* conferred protection from infection in mice [39]. In a similar way, *Yersinia* has been used in vaccination studies in murine models to deliver antigens from a pathogenic protozoan parasite *Entamoeba histolytica* [40]. In this model, it has been shown that TTSS can mediate the delivery of high-molecular-weight antigen that induced significant

models for localized delivery of vaccine antigens or therapeutic molecules.

protection against infection through promoting specific type 1 immune response.

*Salmonella* expressing tumor antigens induced CD8+

strategies aiming to optimize the protection efficacy [45].

**3.2. Application in therapeutic development**

immunoprophylaxy.

The experimental approach of the bacterial TTSS in vaccination studies has been investigated in cancer models as well. Studies in mice indicated that oral administration of recombinant

sion [41,42]. *Pseudomonas aeruginosa* was also evaluated as a live attenuated vector for TTSS delivery of antigen in antitumor vaccine experiments. Inoculation of recombinant *Pseudomo‐ nas* delivering ovalbumin to mice was shown to induce specific CD8+ T cell response that was associated with a significant resistance against ovalbumin-expressing tumor [43]. These experimental investigations underline the efficacy of this delivery system in antitumor

Besides their role in the delivery of heterologous antigens, bacterial vectors present major advantages over nonmicrobial adjuvant vaccines in that they are endowed with the ability to induce innate immunity through pathogen-associated molecular patterns (PAMPs). These specific microbial motifs include lipoproteins, lipopolysaccharides, single-strand RNA, and nonmethylated DNA sequences that can trigger the maturation process of antigen-presenting cells through binding to their specific Toll-like receptors and consequently induce the pro‐ duction of inflammatory cytokines [44]. This is particularly interesting for vaccination

Optimal efficiency of any microbial vector in gene therapy relies particularly on its ability to deliver a sufficient amount of the drug to targeted cell tissues while preserving healthy tissue.

T cell-mediated control of tumor progres‐

**3.1. Application in immunoprophylaxy**

182 Gene Therapy - Principles and Challenges
