**2. Bacteria as delivery vectors in gene therapy**

Recombinant bacteria are being considered as an in vivo cell factory that could be used for the delivery of therapeutic genes to target cells. In this process known as "bactofection," a number of bacterial species have been developed as delivery vectors for their application in different therapeutic approaches.

#### **2.1. Attenuated mutant bacteria**

The most known bacteria for such purposes are *Salmonella typhimurium* strains that have proven to be useful in DNA vaccination approach. The strategy is based on the transformation of an attenuated strain with a plasmid DNA bearing the gene of interest. It has been shown that oral administration of such transformants into mice induced a robust immune response against gene-encoding antigen [17]. This study is the first to describe the possible transfer of a plasmid DNA from bacteria to host cells resulting in antigen processing and induction of specific immunity. This DNA vaccination approach has proven to be useful in prophylactic settings against tumor antigens. In the murine melanoma model, it has been shown that oral administration of attenuated *S. typhimurium* harboring gene sequences encoding tumoral peptide epitopes fused to murine ubiquitin gene could confer protection against tumor growth through the induction of a type I protective immunity [18]. This strategy of DNA delivery allowed an optimized antigen processing for vaccine development.

#### **2.2. Naturally occurring nonpathogenic bacteria**

The genus *Clostridium* comprises a group of nonpathogenic species that are strictly anaerobic and largely distributed in the environment. They are able to produce endospores that can selectively germinate under hypoxia. Given these characteristics, wild-type *Clostridium* has been used to target tumors that are known as poorly oxygenated tissues [19,20]. Various experimental studies have reported the usefulness of clostridia in cancer therapy [21–25]. The injection of either whole *Clostridium* or spores into tumor tissues resulted in tumor destruction as a consequence of the multiplication of bacteria within colonized tumors. Subsequently, more elaborate strategies were developed for the potential use of *Clostridium* as a carrier to deliver prodrug converting enzymes into tumor tissues. Following systemic administration of the prodrug, the latter can be locally activated by the enzyme within tumor tissues, hence promoting a targeted effect against cancer cells. Therefore, selective exposure of tumor tissues to the effect of the prodrug is a promising strategy that may have broad applications in clinical studies. Likewise, recombinant spores of *Clostridium* or *Bacillus subtilis* have been used as a model for surface expression of vaccine antigens. This is based on insertion into chromosomal DNA of bacteria of the gene of interest which is fused to a gene encoding a spore surface protein. This stable genetic construction has allowed an efficient assembly and expression of a variety of fused proteins on the surface of the forming spores. The strategy of recombinant spores has been mainly tested for the development of mucosal vaccines [26].

to fill in those blanks [8–14]. As such, microbial vectors are able to not only serve as cell factories for the production of the transgene but also as vehicles that deliver the transgene to specific cells for which they have a naturally high tropism. Gene transduction with recombinant viruses is generally based on the use of an expression cassette encompassing a transgene [8– 11], while in bacteria, the classic approach of gene transfer is based on plasmid-encoded genes [12–14]. The gene of interest must be delivered to the cell's nucleus to allow an efficient manufacturing of the corresponding protein. DNA escape from intracellular bacteria to host cell cytosol may occur following their phagocytosis and lysosomal degradation within the cell. This is, however, not the case for intracellular bacteria that resist or subvert the phagolysosomal processing such as *Salmonella* or *Listeria* [15,16] and for extracellular bacteria that behave as commensals within a specific cellular niche. Commensal bacteria might be, however, of particular interest if the treatment strategy aims at delivering a gene product to targeted cell tissue through a potent delivery machinery. The delivery system used by avirulent vectors is

Recombinant bacteria are being considered as an in vivo cell factory that could be used for the delivery of therapeutic genes to target cells. In this process known as "bactofection," a number of bacterial species have been developed as delivery vectors for their application in different

The most known bacteria for such purposes are *Salmonella typhimurium* strains that have proven to be useful in DNA vaccination approach. The strategy is based on the transformation of an attenuated strain with a plasmid DNA bearing the gene of interest. It has been shown that oral administration of such transformants into mice induced a robust immune response against gene-encoding antigen [17]. This study is the first to describe the possible transfer of a plasmid DNA from bacteria to host cells resulting in antigen processing and induction of specific immunity. This DNA vaccination approach has proven to be useful in prophylactic settings against tumor antigens. In the murine melanoma model, it has been shown that oral administration of attenuated *S. typhimurium* harboring gene sequences encoding tumoral peptide epitopes fused to murine ubiquitin gene could confer protection against tumor growth through the induction of a type I protective immunity [18]. This strategy of DNA delivery

The genus *Clostridium* comprises a group of nonpathogenic species that are strictly anaerobic and largely distributed in the environment. They are able to produce endospores that can selectively germinate under hypoxia. Given these characteristics, wild-type *Clostridium* has been used to target tumors that are known as poorly oxygenated tissues [19,20]. Various

therefore a critical point for optimizing the success of any therapy.

allowed an optimized antigen processing for vaccine development.

**2.2. Naturally occurring nonpathogenic bacteria**

**2. Bacteria as delivery vectors in gene therapy**

therapeutic approaches.

180 Gene Therapy - Principles and Challenges

**2.1. Attenuated mutant bacteria**

Gene therapy in cancer has been also investigated using a food-grade microorganism *Bifidobacterium infantis*, which is a nonpathogenic and anaerobic bacterium that can proliferate in the hypoxic environment of tumor tissues as well. *B. infantis* has been applied as a gene delivery system in various cancer models such as bladder cancer including melanoma [27] thanks to its specific targeting property to the anaerobic environment of tumor cells. This bacterium has been successfully used for antitumor suicide gene therapy in a murine model of renal cell carcinoma [27,28]. This strategy is based on the use of the herpes simplex virus thymidine kinase/ganciclovir system to selectively kill tumor cells. Recombinant bacteria bearing virus thymidine kinase gene can replicate within tumor tissue and locally express the enzyme which, in turn, catalyzes the nontoxic precursor ganciclovir to a toxic form resulting in tumor cell killing through termination of DNA replication.

*Lactococcus lactis* is another food-grade bacterium that has been engineered for gene therapy in inflammatory bowel diseases (IBD). As this bacteria tends to naturally colonize the intestinal epithelium, they were used as vectors for localized delivery of anti-inflammatory mediators. In murine model of induced colitis, oral administration of recombinant *L. lactis* expressing IL-10 [29], IL-27 [30] or anti-TNF nanobody [31] could reduce intestinal inflammation, thereby offering a safe and reliable strategy for the treatment of IBD.
