Current Gene Therapy - Volume 10, Issue 1, 2010

The concept of exploiting bacterial species as biological gene vectors has existed for some time, and the use of bacteria to deliver therapeutics offers many advantages over other gene delivery approaches. Bacteria fall within the ‘non-viral’ class of delivery systems, investigated primarily for safety reasons, yet the biological nature of bacterial vectors means that many of the inherently beneficial traits of viral vectors are retained. A safety property unique to bacterial vectors is their sensitivity to clinically available antibiotic treatments, presenting control over the vector post-administration, an invaluable property for gene therapy. In terms of GMP-grade vector manufacture, the growth and production of live bacterial cultures has long been a focus of biotech industries both in the production of recombinant proteins and the production of live or killed vaccines. Bacterial vectors for delivery of therapeutic agents would therefore be relatively cheap and straightforward to produce on an industrial scale. In this issue, authors have focused primarily on individual bacterial genera that are particularly suited to specific applications in disease therapy and the reviews highlight the benefits and potential pitfalls of using these agents in human hosts. Because of the widely varying properties of different bacteria, a range of therapeutic strategies exists for bacterial vectors. Invasive pathogens are well suited to intracellular plasmid transfer (‘true’ gene delivery) (see reviews by Moreno et al.; Buttaro and Fruehauf; Tangney and Gahan), but their disease causing potential presents safety fears in terms of usage as systemically administered agents. At the opposite end of the spectrum, apathogenic species lacking the ability to deliver genes internally for mammalian cell expression, may safely be administered (IV, or even orally) to achieve systemic tumour targeting and bacterial expression of agents (cell therapy) (see Morrissey et al. review), or for mucosal therapeutic delivery e.g. to target inflammatory conditions of the gut (Bahey-el-Din et al. review). Furthermore, approaches can overlap, as exemplified by systemic administration of invasive strains of clostridial spores for tumour targeting with cancer cell invasion and bacterial induced oncolysis (see Mengesha et al. review), or oral administration of Salmonella resulting in tumour targeting and gene delivery or for vaccination purposes (see reviews by Moreno et al.; Tangney and Gahan). It is significant that many of the nascent bacterial delivery platforms described in this issue have entered or are currently entering human clinical trials. Use of clostridial species for targeted tumour killing and attenuated S. typhimurium vectors for oral vaccination or tumour gene delivery, represent the most widely applied bacterial vectors at clinical trial level [1-5]. Herein, Buttaro and Fruehauf describe FDA-related aspects of progression of these technologies through the phases of preclinical and clinical testing. They further describe a range of E. coli based vectors that have already been used in human trials. Also Lothar Stiedler and colleagues [6] have used live recombinant L. lactis expressing IL-10 as an active therapeutic in patients with Crohn's disease. It is also encouraging that an attenuated recombinant L. monocytogenes strain has recently been used as an anti-cancer vaccine vector in phase I trials in humans [7] (reviewed in Tangney and Gahan). Certainly, with the development and testing of vectors against cancer and infectious disease [8] we are closer to an era in which recombinant live bacterial vectors will be acceptable for therapeutic or prophylactic use provided they are proved to be safe and efficacious. Despite the massive potential of live bacterial delivery systems, it is clear that in many cases further work is required to limit potential adverse effects and to optimise delivery. Adverse effects may arise through non-specific inflammation triggered by interactions between microbial associated molecular patterns (such as LPS) and cognate toll-like receptors. There may be potential to limit these adverse responses in many cases through mutation of the vector, careful strain selection, coadministration of specific inhibitors or antibiotics and precise calculations of dose. Indeed, bacterial vectors may potentially be administered by oral, intranasal or intravenous routes of inoculation with a diverse range of resultant effects and outcomes (see Bahey-el-din et al., this issue). The concept of an orally administrated delivery vector is particularly attractive. However, for use of recombinant bacteria in humans, particular care must be taken to prevent lateral gene transfer in the gut (Buttaro and Fruehauf) and to limit environmental spread of the vector (Bahey-el-din et al.). With this latter concern in mind, a number of researchers have investigated the concept of ‘biological containment’ whereby the vector is engineered to survive in the host but not in the external environment where specific nutrients are limiting [9]. Overall the development of live bacterial vectors with potential for delivery of therapeutic agents is an exciting area of research that is gaining acceptance by clinicians and regulatory authorities for its potential to deliver positive clinical outcomes. Whilst more needs to be done to improve the safety and efficacy of some systems, this is clearly a technological approach which will yield dividends in the coming years.

Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. The ideal anti-cancer therapy would selectively eradicate tumour cells, whilst minimising side effects to normal tissue. Bacteria have emerged as biological gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. This property enables targeting of both the primary tumour and secondary metastases. In the case of invasive pathogenic species, this targeting strategy can be used to deliver genes intracellularly for tumour cell expression, while non-invasive species transformed with plasmids suitable for bacterial expression of heterologous genes can secrete therapeutic proteins locally within the tumour environment (cell therapy approach). Many bacterial genera have been demonstrated to localise to and replicate to high levels within tumour tissue when intravenously (IV) administered in rodent models and reporter gene tagging of bacteria has permitted real-time visualisation of this phenomenon. Live imaging of tumour colonising bacteria also presents diagnostic potential for this approach. The nature of tumour selective bacterial colonisation appears to be tumour origin- and bacterial species- independent. While originally a correlation was drawn between anaerobic bacterial colonisation and the hypoxic nature of solid tumours, it is recently becoming apparent that other elements of the unique microenvironment within solid tumours, including aberrant neovasculature and local immune suppression, may be responsible. Here, we consider the pre-clinical and clinical data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop a beneficial clinical treatment.

In the quest to developing novel cancer therapies, oncolytic Clostridia are re-emerging as promising candidates due to their ability to specifically target and lyse tumours with great efficacy. Clostridial spores have the innate abilities that exploit the unique tumour microenvironment by directly penetrating, colonising and killing tumour cells. These unique features have prompted many studies to investigate their oncolytic potency. In addition, Clostridia possess a number of characteristics that enable them to be developed as oncolytic gene delivery vectors, such as their unlimited capacity, antibiotic sensitivity, and extracellular existence. Similarly, numerous strategies are being devised and tested to take advantage of these features with modern molecular technologies. In this review, we detail the traits that distinguish Clostridia from other agents, and describe the potential therapeutic effects that Clostridial spores demonstrate, but are not achievable with other treatment modalities. Furthermore, we will also summarize the recent advances in the use of Clostridial spores as viable therapeutic candidates, incorporating the latest progresses in genetic engineering tools, such as ClosTron. Finally, we will highlight some avenues deserving further studies in order to realise the ultimate goal of utilizing oncolytic Clostridia clinically in patients.

Improving the means of drug delivery has become an important field of pharmaceutical research. The development of safe and advanced vectors for gene therapy and other novel therapies will allow for targeted delivery of pharmaceutically active agents and carries promise to improve therapies both through increased efficiency (e.g. improved cellular uptake of the active drug) as well as lower toxicity (e.g. through targeted delivery only to the cells requiring treatment) for a large number of pharmaceutical agents. Here we are reviewing the nascent field using live bacteria as vectors for therapeutic and preventive agents in a wide range of areas, from vaccine purposes to gene therapy and delivery of therapeutic RNA interference. This review focuses particularly on the use of E. coli derived strains for therapeutic delivery.

The food-grade bacterium Lactococcus lactis has been extensively investigated during the last two decades as a delivery vector for therapeutic proteins, DNA and vaccine antigens. The bacterium represents a safe, genetically tractable vector capable of producing heterologous therapeutic proteins at mucosal sites. Here we review recent work in which recombinant L. lactis strains have been exploited as agents to treat inflammatory bowel disease, allergy and cancer. We also describe the ability of L. lactis to deliver proteins with adjuvant potential, vaccines and DNA and discuss the therapeutic possibilities of this approach.

The intracellular pathogen Listeria monocytogenes represents a promising therapeutic vector for the delivery of DNA, RNA or protein to cancer cells or to prime immune responses against tumour-specific antigens. A number of biological properties make L. monocytogenes a promising platform for development as a vector for either gene therapy or as an anti-cancer vaccine vector. L. monocytogenes is particularly efficient in mediating internalization into host cells. Once inside cells, the bacterium produces specific virulence factors which lyse the vaculolar membrane and allow escape into the cytoplasm. Once in the cytosol, L. monocytogenes is capable of actin-based motility and cell-to-cell spread without an extracellular phase. The cytoplasmic location of L. monocytogenes is significant as this potentiates entry of antigens into the MHC Class I antigen processing pathway leading to priming of specific CD8+ T cell responses. The cytoplasmic location is also beneficial for the delivery of DNA (bactofection) by L. monocytogenes whilst cell-to-cell spread may facilitate access of the vector to cells throughout the tumour. Several preclinical studies have demonstrated the ability of L. monocytogenes for intracellular gene or protein delivery in vitro and in vivo, and this vector has also displayed safety and efficacy in clinical trial. Here, we review the features of the L. monocytogenes host-pathogen interaction that make this bacterium such an attractive candidate with which to induce appropriate therapeutic responses. We focus primarily upon work that has led to attenuation of the pathogen, demonstrated DNA, RNA or protein delivery to tumour cells as well as research that shows the efficacy of L. monocytogenes as a vector for tumour-specific vaccine delivery.

The design of efficient vectors for vaccine development and cancer gene therapy is an area of intensive research. Bacteria-based vectors are being investigated as optimal vehicles for antigen and therapeutic gene delivery to immune and tumour cells. Attenuated Salmonella strains have shown great potential as live vectors with broad applications in human and veterinary medicine. An impressively large, and still growing, number of reports published over the last two decades have demonstrated the effectiveness in animal models of Salmonella-based therapies for the prevention and treatment of infectious and non-infectious diseases, as well as cancer. Further, the recent dramatic expansion in knowledge of genetics, biology and pathogenesis of the bacteria allows more rational design of Salmonella constructs tailored for specific applications. However, only few clinical trials have been conducted so far, and although they have conclusively demonstrated the safety of this system, the results on immunogenicity are less than optimal. Thus, more research particularly in target species is required to bring this system closer to human and veterinary use. In this review we first describe some particularities of the bacteria and its relationship with the host that could be on the basis of its success as vector, and then summarize the different strategies used so far to develop Salmonella-based vaccines for infectious diseases as well as for non-traditional indications such as prion and Alzheimer disease vaccination. Finally, we review the many different approaches that employ Salmonella for the design of new therapies for cancer.