Bacteria-Mediated Anti-Angiogenesis Therapy

REVIEW Gene therapy for cancer: bacteria-mediated anti-angiogenesis therapy

R Gardlik1,2, M Behuliak2, R Palffy2, P Celec2,3,4 and CJ Li1

Several bacterial species have inherent ability to colonize solid tumors in vivo. However, their natural anti-tumor activity can be enhanced by genetic engineering that enables these bacteria express or transfer therapeutic molecules into target cells. In this review, we summarize latest research on cancer therapy using genetically modified bacteria with particular emphasis on blocking tumor angiogenesis. Despite recent progress, only a few recent studies on bacterial tumor therapy have focused on anti-angiogenesis. Bacteria-mediated anti-angiogenesis therapy for cancer, however, is an attractive approach given that solid tumors are often characterized by increased vascularization. Here, we discuss four different approaches for using modified bacteria as anti-cancer therapeutics—bactofection, DNA vaccination, alternative gene therapy and transkingdom RNA interference—with a specific focus on angiogenesis suppression. Critical areas and future directions for this field are also outlined. Gene Therapy (2011) 18, 425–431; doi:10.1038/gt.2010.176; published online 13 January 2011

Keywords: cancer gene therapy; bacteria; angiogenesis

INTRODUCTION The concept of bacterial oncolytic therapy has been verified in The first modern attempts at using bacteria for therapeutic purposes numerous experimental, as well as clinical studies. Recently, a tumor- were made more than 40 years ago. At this time, it was discovered that targeting auxotrophic strain of Salmonella typhimurium has been bacteria could predominantly replicate in solid tumors.1 However, the shown to completely eradicate primary tumors, as well as cancer first indications of this phenomenon date back to 19th century. These metastases in various mouse models.10,11 However, the most fre- findings remained largely unexplored until the turn of 20th century, quently used anti-cancer approach using bacteria is enzyme-prodrug when oncolytic bacteria capable of lysing host cells were first studied therapy where systemically administered bacteria are used to deliver a by various research groups.2–4 gene/enzyme able to convert systemically administered prodrug into One of the main reasons for the lack of efficacy of radiotherapy and its active compound specifically in tumor tissue.12–14 The utilization of chemotherapy for many solid tumors is presence of hypoxic areas that bacterial systems for therapeutic purposes is further enhanced by are resistant to these interventions. This apparent limitation, however, genetic modifications, which make them a very promising tool for can be exploited for enhancing tumor-targeting, such as the applica- targeted delivery of genes and their products. Specific advantages of tion of obligate or facultative anaerobic bacteria. Several strains of using bacteria for anti-cancer gene therapy include the natural Clostridia, Bifidobacteria and Salmonellae are able to selectively colo- oncolytic potential of some strains/species, direct targeting of tumor nize the hypoxic areas of tumors and destroy the tumor cells, and tissue and the ease of positive regulation/eradication. Moreover, the thereby provide a more selective tumor-targeted therapy.5 Two crucial anti-cancer effect of tumor-targeting bacteria can also be achieved shortcomings of conventional cancer treatments can, therefore, be after oral administration, which may circumvent the use of the potentially overcome by using tumor-targeting bacteria: off-target intravenous route of delivery and its associated adverse events.15 tissue toxicity seen in systemic therapies; and hypoxia-related resis- Nowadays, therapeutics developed on the basis of bacterial carrier tance to conventional therapies. vectors are undergoing a phase where several concepts are being Until recently, nearly all tumor-targeting bacterial strains showed advanced into clinical trials as a sign of maturation of the field. exclusive accumulation in the large central necrotic area of the Several bacterial therapeutic approaches are showing promising results tumor. Necrotic areas are separated from viable tumor cells by a in clinical phase I and II trials. Listeria is being actively developed as barrier of host leukocytes thereby leaving the viable areas of the therapeutic agents using the anticancer-vaccination approach. In a tumor unaffected by the bacteria. Recent studies have shown, Phase I study done in patients with cervical cancer, safety of a live however, that this issue can be overcome by the depletion of attenuated Listeria monocytogenes that secretes the HPV16 E7 anti- neutrophils from the host, which results in bacterial colonization gen in a fusion construct to the Listeria protein Listeriolysin O has of viable and necrotic tumor tissue, and complete eradication of been demonstrated.16 Phase II studies are ongoing. Recently, tumors.6 The historical development and molecular mechanisms Escherichia coli targeting b-catenin has received clearance from US of anaerobic bacteria as tumor targeting vectors are reviewed in detail Food and Drug Administration to initiate clinical trials for treating elsewhere.5,7–9 patients with familial adenomatous polyposis.17 The concept of using

1GI Cancer Laboratory, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia; 3Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia and 4Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Correspondence: Dr R Gardlik, Institute of Pathophysiology, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Slovakia. E-mail: [email protected] Received 18 May 2010; revised 2 August 2010; accepted 4 August 2010; published online 13 January 2011 Bacteria in cancer gene therapy R Gardlik et al 426

Mycobacterium for cancer treatment goes back to the 19th century. models using expression plasmids carrying various cytokine genes, Today, bacillus Calmette-Guerin vaccine is an approved treatment for such as interleukin-12 (IL-12)andGM-CSF,23 IL-4 and IL-18 (see ref. 24) human bladder cancer that is superior to intravesical chemo- or other molecules having role in angiogenesis such as Flt3 ligand.25 therapy and is commonly used as the first-line adjuvant treatment.18 Further, a dual tumoricidal and anti-angiogenic effect of The therapy is known to work through a mechanism of action that S. choleraesuis carrying an expression plasmid containing the includes angiogenesis inhibition, providing a supportive rationale for thrombospondin-1 gene under control of eukaryotic promoter was cancer therapy on the basis of suppression of angiogenesis using observed in a murine model of malignant melanoma.26 However, on bacteria. the basis of the small number of studies using bactofection for cancer Despite recent progress, only a few recent studies on bacterial tumor treatment, it seems that this approach is more suited to targeting other therapy have specifically focused on anti-angiogenic therapy. Although cells and tissues such as the colonic mucosa27 and the lungs.28 effects on the vasculature were observed in most of these studies, these A potential limitation of bactofection for cancer therapy is the fact changes seemed to be a consequence mainly of bacteria-mediated that the effector molecule will likely be expressed exclusively in cells therapy. Bacteria-mediated anti-angiogenesis tumor therapy, however, infected by bacteria, leaving a potentially large population of tumor is a reasonable approach given that solid tumors are often charac- cells untreated. However, if the product of the transgene is secreted terized by increased vascularization. In this review, we summarize outside the target cell, it may still have a therapeutic effect on non- latest research on cancer therapy using genetically modified bacteria infected tumor cells. Bacterial strains, delivery routes, genes, tumors with particular emphasis on the potential of blocking tumor angio- and mouse strains used in studies using bactofection are summarized genesis, and pinpoint critical areas and future directions for this field. in Table 1.

BACTERIA AFFECTING ANGIOGENESIS DNA VACCINATION Pronounced angiogenesis is one of the hallmarks of solid tumors. It is known that bactofection of plasmids encoding a tumor-expressed Therefore, to search for more efficient anti-cancer drugs, efforts are antigens can lead to induction of humoral and cellular immune being made to block tumor angiogenesis. Despite notable successes response in the host thereby providing protective defense against achieved in studies using oncolytic bacteria for cancer treatment, tumors.29 This approach, termed DNA vaccination, has been success- bacteriolytic therapy in and of itself is often insufficient for complete fully implemented for anti-angiogenic therapy. Oral anti-angiogenic eradication of experimental tumors.19 The idea of using combined bacterial vaccines directed against VEGFR-2 have proved efficicious in therapy with bacteria and angiogenesis inhibition has therefore been animal models of malignant melanoma, colorectal carcinoma and suggested.20 Bacterial infection is often accompanied by increased lung cancer,30 as well as non-cancer diseases like stromal keratitis31 oxygen tension and the generation of immune responses in neighbor- and atherosclerosis.32,33 Furthermore, Salmonella-mediated vaccina- hood of tumor, which is well-perfused and oxygenated. Jia et al.21 tion against murine VEGFR-2 has been successfully combined with successfully addressed this issue by the application of an attenuated, classical gene therapy for the treatment of malignant melanoma.34 In auxotrophic Salmonella strain (unable to induce septic shock) in this study, oral administration of a vaccine strain combined with combination with endostatin (an endogenous angiogenesis inhibitor) direct intratumoral injection of plasmid vector encoding the chemo- in a murine model of malignant melanoma. Separate therapy using kine IP-10 (CXCL10) resulted in a significant synergistic anti-tumor bacteria or endostatin alone had little effect on tumor growth. In effect. Similar suppression of angiogenesis was observed with an oral contrast, combined bacteriolytic and anti-angiogenic therapy proved bacterial vaccine against IL-18 alone and combination with Fos-related very effective at reducing tumor growth, and was associated with antigen 1.35 Bacterial vaccines directed against the apoptosis inhibitor marked tumor necrosis. These data indicate that combined therapy survivin36 and the transforming growth factor-b1 co-receptor endo- using tumor-targeting bacteria that can fight tumors using their glin37 also proved effective for inhibition of tumor angiogenesis. Taken unique metabolic features from poorly perfused tumor areas, (which together, these findings underscore the key role of angiogenesis in are not accessible to systemically administered agents), along with cancer as well as other diseases and, at the same time, highlight the inhibition of angiogenisis to kill residual tumor cells significantly complexity of this essential process. Most DNA vaccination studies for increases the chance of tumor eradication. Also, it is less likely that anti-cancer therapy use attenuated strains of Salmonella whose suit- resistance to such combination therapy will develop. ability has been demonstrated in numerous investigations. Other Below, we discuss four different approaches for using modified bacterial species, however, have also been used such as Pseudomonas bacteria as anti-cancer therapeutics—bactofection, DNA vaccination, aeruginosa.38 For a comprehensive review of the bacterial strains used alternative gene therapy (AGT) and transkingdom RNA interference for DNA vaccination please see the article by Xiang et al.39 (RNAi)—with a specific focus on angiogenesis suppression (Figure 1). Most of the above mentioned vaccines induce a protective CD8+ T-cell-mediated immune response that most likely reflects a break- BACTOFECTION down of peripheral immunological tolerance to self-antigens. The use of bacteria as a vector for the delivery of therapeutic genes to The precise molecular mechanism that regulates this phenomenon, target cells is known as bactofection, and several studies have used this however, remains obscure. The need for further research and deeper approach to deliver genes encoding anti-angiogenic molecules to understanding of these phenomena is highlighted by the fact that tumor cells in vivo. A study was performed using S. choleraesuis strain similar vector strains are currently used for bactofection-based bearing eukaryotic expression plasmid encoding endostatin.22 The approaches and for DNA vaccination. In bactofection and bacterial expression of endostatin was targeted exclusively to tumor tissues gene-therapy approaches, the immune activation may be seen as an colonized by bacteria and significant inhibition of tumor growth undesired side effect, whereas in DNA-vaccination, the immunogenic (40–70%) with decreased intratumoral microvessel density and properties of the carrier-bacteria are a pivotal component of the reduced expression of vascular endothelial growth factor (VEGF) therapeutic effect. was observed. Bactofection using auxotrophic Salmonellae as vectors One of the main advantages of bacterial DNA vaccination as a has also been effectively used for therapy of experimental tumor cancer treatment strategy is the potential for oral application of

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Figure 1 Antitumor effect of bacteria colonizing tumor tissue. Auxotrophic bacteria speciﬁcally colonize tumors with necrotic and hypoxic areas (panel on the left). The anticancer effect of bacteria can be exerted in four different ways: (a) Bactofection: after escaping the vessel and entering the target cell, bacteria disrupt and release a plasmid vector encoding the therapeutic gene. The plasmid is transferred into the cell nucleus and the therapeutic protein is expressed by the host cell’s expression system (blue square symbol). (b) DNA vaccination: bacteria deliver therapeutic plasmid into the host cell in a similar way as in bactofection. The host cell may in this case be an antigen-presenting cell. The plasmid encodes a tumor cell-expressed antigen to help prime a T-cell response against the tumor antigen, which is present on the surface of tumor cells leading to induction of humoral and cellular immune response againstthe tumor. (c) Bacterial protein delivery: bacteria, either in extracellular environment or inside tumor cells, express the therapeutic gene directly and serve as protein delivery vehicles (red square symbol). (d) Bacterial delivery of RNA interference: bacteria deliver plasmid-encoding short-hairpin RNAs (shRNAs) or express the shRNAs to induce RNA interference against an oncogene or a tumor-expressed factor.

Table 1 Studies using bacteria-mediated delivery of gene with direct/indirect anti-angiogenic effect: bactofection

Vector Delivery route Gene Tumor Mouse strain Reference

S. choleraesuis i.p. Endostatin B16F10 melanoma C57BL/6 Lee et al.19 i.v. MBT-2 bladder tumor C3H/HeN ML-1 hepatoma Balb/c NOD/SCID S. typhimurium SL3261 oral IL-12 Lewis lung cancer Balb/c Yuhua et al.20 GM-CSF 4T1 breast cancer C57BL/6 S. typhimurium SL3261 oral IL-4 B16F1A melanoma C57BL/6 Agorio et al.21 IL-18 S. typhimurium BRD509 s.c. Flt3L B16F10 melanoma C57BL/6 Yoon et al.22 S. choleraesuis i.p. Thrombospondin-1 B16F10 melanoma C57BL/6 Lee et al.23

Abbreviations: i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous. therapeutic bacterial strains, which was proven to be effective in ALTERNATIVE GENE THERAPY animal studies. Current research is focused on the development of Another means of using bacteria for gene therapy is the so-called AGT clinically relevant oral bacterial vaccines. However, signiﬁcant knowl- approach, which is also known as bacterial protein delivery.40 It is edge gaps still exist and the mechanism of action for oral DNA based on transfer of bacterially expressed therapeutic proteins to the vaccines has not been fully elucidated yet. Recent animal experiments host organism using genetically modiﬁed (transformed) bacteria. investigating angiogenesis-related anti-tumor DNA vaccination studies Unlike bactofection- and DNA vaccination-based methods, the per- are summarized in Table 2. sistence of bacteria in target tissue may be desired in bacterial protein

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Table 2 Studies using bacteria-mediated delivery of gene with direct/indirect anti-angiogenic effect: DNA vaccination

Vector Delivery route Gene Tumor Mouse strain Reference

S. typhimurium SL7207 oral VEGFR-2 (Flk-1) B16G3.26 melanoma C57BL/6 Niethammer et al.27 MC-38 colon carcinoma D121 lung carcinoma CT-26 colon carcinoma S. typhimurium SL7207 oral VEGFR-2 (Flk-1) B16F10 melanoma C57BL/6 Lu et al.31 S. typhimurium RE88 oral IL-18 D2F2 breast carcinoma Balb/c Luo et al.32 IL-18+Fra-1 S. typhimurium RE88 oral Survivin D121 lung carcinoma C57BL/6 Xiang et al.33 Survivin+CCL21 S. typhimurium RE88 oral Endoglin D2F2 breast carcinoma Balb/c Lee et al.34

delivery. Persisting bacteria produce the therapeutic polypeptide challenging to genetically engineer this bacterial strain mainly because in situ, thus ideally providing a local distribution of drug without of the presence of several inherent plasmids. In addition, the over- increasing its systemic levels. As with bactofection, AGT is mostly used whelming majority of literature about E. coli Nissle 1917 studies for treatment of tumors and uses primarily oncolytic and discusses its beneficial use for gastrointestinal indications. Thus, tumor-colonizing bacterial strains of Clostridia, Bifidobacteria or more studies are needed to uncover its potential for anti-tumor Salmonellae.2,4 Both Clostridia and Bifidobacteria have been success- therapy. However, it seems that other live bacterial vectors might be fully used for ‘enzyme-prodrug’ therapy.41 Owing to their obligate more suitable for systemic intravenous therapy of cancer. anaerobic nature, ability to form spores and ease of transformation, Another approach being investigated to improve regulated gene Clostridia are especially suited to AGT for tumors.42,43 Further, their expression as well as the targeting of bacteria to the hypoxic (that is, ability to colonize tumors in vivo can be enhanced by deletion of genes poorly vascularized) regions of tumors is the use of hypoxia responsive associated with basal oxygen tolerance, such as that encoding super- promoters in tumor-colonizing Salmonellae.51,52 Asubsetofpromo- oxide dismutase.44 The main advantage of Bifidobacteria, on the other ters preferentially activated inside tumors has been identified recently, hand, is that they are non-pathogenic. Li et al.45 successfully used suggesting that there are additional regulatory mechanisms beside Bifidobacterium adolescentis as a vector for the expression of endostatin those hypoxia-related.53 This is particularly interesting in light of within tumors. These authors showed that a strong inhibition of recent findings regarding the ability of tumor-targeting bacteria to angiogenesis was able to significantly inhibit local tumor growth. colonize both the necrotic and well-perfused areas of tumors.6,54 These In another study, Bifidobacterium longum was shown to efficiently results further enhance the potential of using bacteria for anti-tumor deliver the anti-angiogenic protein endostatin to murine liver tumors gene therapy, by providing a wider variety of promoters capable of and induce anti-tumor activity.46,47 Furthermore, the anti-tumor tumor targeting and regulated gene expression. Also, bacteria have an effect was enhanced by co-administration of the same bacterial strain advantage over viruses when used for cancer gene therapy as the expressing tumor necrosis factor-related apoptosis inducing ligand.48 therapy can be readily terminated using antibiotics. Moreover, bacteria These findings emphasize the need for combination therapy, in which do not integrate genetic material into host genome, significantly multiple anti-tumor pathways are inhibited. reducing the risk of treatment-induced mutagenesis and tumori- A key aspect of the studies investigating in vivo AGT is the ability to genesis. In situ production of cytokines by bacteria represents a cost- specifically regulate bacterial expression of the therapeutic protein. effective and safe alternative mainly to systemic administration, which Precise temporal and spatial control is necessary to maximize the can be associated with unwanted side effects. However, in recent study therapeutic effect and minimize the any potential adverse effects. One using Listeria monocytogenes as a vehicle for tumor specific gene and of the key tools in this area is the use of inducible promoters that are protein delivery, bactofection proved to be more efficient than AGT.55 activated only in the presence of a specific exogenously administered An important study was performed recently by Loeffler et al.56 that compound (for example, isopropyl thiogalactoside and arabinose). engineered attenuated S. typhimurium to improve their oncolytic Exogenous regulation of therapeutic gene expression can also be activity by stably incorporating a gene encoding LIGHT, a tumor further improved by the use of bacteria carrying inducible suicide necrosis factor-family cytokine known to promote tumor rejection. genes. An important study by Loessner et al. has demonstrated the LIGHT has various roles in immune response mechanisms including ability to regulate the expression of therapeutic genes from plasmids stimulation of B and T cell as well as natural killer cell migration. in vivo.49 In this study, intravenously applied S. typhimurium carrying More importantly, LIGHT may have direct anti-angiogenic effects a plasmid containing green fluorescent protein (GFP)geneunder through activation of chemokine CXCL9 and CXCL10 receptors control of arabinose (pBAD) promoter were able to selectively known for triggering the processes leading to angiogenesis inhibition. colonize tumors, but only expressed green fluorescent protein after In a follow-up study, the same authors demonstrated an anti-tumor 57 administration of L-arabinose. Bacteria-mediated transfer of therapeu- effect of IL-18 producing Salmonellae. Besides its well-known effect tic proteins to tumors using an L-arabinose expression induction on the immune system, IL-18 also acts as a suppressor of angiogenesis system has also been reported for E. coli Nissle 1917, a probiotic by direct inhibition of fibroblast growth factor 2-induced endothelial bacterial strain that can colonize humans.50 The results of this study cell proliferation and, thus, inhibits various pathways involved in significantly advance the exploitation of this gut-trophic strain and tumor pathogenesis. Taken together, these studies provide evidence provide a good example of the therapeutic potential of AGT. However, that intravenously administered, non-virulent genetically modified despite the excellent safety profile of E. coli Nissle 1917, it is quite bacteria, can be efficiently exploited as vehicles for local generation

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Table 3 Studies using bacteria-mediated delivery of protein with direct/indirect anti-angiogenic effect: alternative gene therapy

Vector Delivery route Gene Tumor Mouse strain Reference

B. adolescentis i.v. Endostatin Heps liver cancer Balb/c Li et al.42 B. longum i.v. Endostatin H22 hepatoma Kunming Xu et al.43 B. longum oral Endostatin Heps liver cancer Balb/c Fu, Li et al.44 HepG2 Nude S. typhimurium VNP20009 i.v. LIGHT D2F2 breast carcinoma Balb/c Loeffler et al.53 IL-18 CT-26 colon carcinoma C57BL/6 Loeffler et al.54 CCL21 Lewis lung carcinoma Loeffler et al.55 S. typhimurium BRD509 i.p. IL-2 B16F1 melanoma C57BL/6 Al-Ramadi et al.57

Abbreviations: i.v., intravenous; i.p., intraperitoneal.

Table 4 Studies using bacteria-delivered RNA interference inhibiting angiogenesis-related genes: transkingdom RNAi

Vector Delivery route Gene Tumor Mouse strain Reference

E. coli BL21DE3 i.v. b-catenin SW480 colon carcinoma Balb/c Xiang et al.62 S. typhimurium LH430 i.v. STAT3 RM-1 prostate cancer C57BL/6 Zhang et al.64 S. typhimurium SL3261 oral Bcl-2 B16F10 melanoma C57BL/6 Yang et al.65

Abbreviation: i.v., intravenous.

of anti-angiogenic agents in tumors. The same authors recently into nude mice. This approach is termed transkingdom RNAi, as published two additional experimental studies using immunocompe- this study provided the first evidence of transfer of RNAi effector tent mice models of cancer, in which they demonstrated in vivo the molecules between bacteria and mammals in vivo. In another study, anti-tumor effect of attenuated S. typhimurium producing the chemo- attenuated S. typhimurium were also successfully tested as a vector for kine CCL21, and proapoptotic cytokine Fas ligand suggesting the in vivo transfer of MDR1 siRNA to tumors.66 Combined oncolytic and molecules also possess angiostatic properties.58,59 Interestingly, a siRNA therapy using systematically administered attenuated S. typhi- pronounced anti-tumor effect of IL-2-producing S. typhimurium murium expressing siRNA against tumor transcription factor STAT3 was also recently shown in a mouse model of melanoma.60 Moreover, has also been evaluated.67 Both of these studies demonstrated higher this effect was clearly correlated with decreased tumor angiogenesis. In efficiency for combined therapy compared with treatment with non- fact, previously unknown anti-angiogenic effects have recently been RNAi-inducing bacteria. In addition to being used to produce and discovered for a variety of molecules involved in the immune response deliver silencing short-hairpin RNA, bacteria can also been exploited and cellular apoptosis mainly due to extensive ongoing research in the to deliver eukaryotic short-hairpin RNA-expression plasmids to field of cancer therapy. Bacteria in gene therapy, differences between mammalian tumor cells.17 Oral administration of S. typhimurium bactofection and AGT, advantages and disadvantages, as well as bearing an short-hairpin RNA-expression plasmid against the anti- specific application of both approaches are discussed in detail in our apoptotic protein bcl-2 led to a marked delay in tumor growth, and review article.40 A detailed summary of experimental studies using prolonged survival in a murine melanoma model.68 To date, however, AGT is provided in Table 3. transkingdom RNAi has not been used to directly target angiogenesis, In spite of great success in a preclinical setting, the application of although some of the targets mentioned above (b-catenin, STAT3 and bacteria for human tumor therapy has not been particularly effica- bcl-2) are known to augment angiogenesis through their effects on the cious, although this approach was well tolerated in most studies.61–63 expression of various proangiogenic factors.69–71 Anti-tumor effects of In light of above-mentioned findings, however, we would suggest that knocking down these targets may, in part, be related to suppression angiogenesis would be a meaningful target for further experimental of blood vessel formation. However, a number of studies using and clinical studies of bacteria-mediated anti-cancer therapy, particu- ‘non-bacterial’ application of siRNA against VEGF,72,73 HIF-1a74 larly if used in conjunction with oncolytic strains of bacteria. and other angiogenesis-related molecules75 indicates that inhibition of endothelial cell proliferation and new blood vessel formation are TRANSKINGDOM RNAi a viable target for RNAi-based treatment of cancer, as well as other A promising new approach for bacteria-mediated anti-cancer therapy diseases.76 Although transkingdom RNAi clearly provides a new is the combination of two distinct methodologies: bacteriotherapy and avenue to explore, further studies will be required to uncover all its RNAi. Bacteria that were engineered to produce and deliver short potential applications. Table 4 provides a brief summary of studies interfering RNA (siRNA) represent a novel tool for the efficient using transkingdom RNAi in tumor therapy. induction of RNAi in host cells.64 This concept was pursued inde- pendently by Zhao et al.65 and Xiang et al.17 Zhao et al. showed that CONCLUSION siRNAs produced by invasive E. coli strain could induce RNAi in Inhibition of angiogenesis is of great importance from experimental, mammalian cell cultures. The first in vivo demonstration of transking- pathophysiological and especially clinical point of view. However, dom RNAi was provided by Xiang et al. who showed that invasive further research is needed to fully delineate the most effective way E. coli capable of producing b-catenin short-hairpin RNA could to target angiogenesis for cancer treatment. The application of new induce RNAi in colonic epithelial cells and colonic tumors xenografted approaches using bacteria for the transfer of therapeutic genes or the

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Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium. Int J Cancer Drug Administration to initiate clinical trials for treating patients 2001; 94: 438–443. with familial adenomatous polyposis,17 suggesting the feasibility and 24 Agorio C, Schreiber F, Sheppard M, Mastroeni P, Fernandez M, Martinez MA et al. Live attenuated Salmonella as a vector for oral cytokine gene therapy in melanoma. JGene promise of bacteria-based therapy for cancer. Med 2007; 9:416–423. 25 Yoon WS, Choi WC, Sin JI, Park YK. Antitumor therapeutic effects of Salmonella typhimurium containing Flt3 Ligand expression plasmids in melanoma-bearing mouse. CONFLICT OF INTEREST Biotechnol Lett 2007; 29:511–516. The authors declare no conflict of interest. 26 Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther 2005; 12: 175–184. ACKNOWLEDGEMENTS 27 Castagliuolo I, Beggiao E, Brun P, Barzon L, Goussard S, Manganelli R et al. Engineered E. The authors are grateful to Andrew Keates, PhD for kindly revising the coli delivers therapeutic genes to the colonic mucosa. Gene Therapy 2005; 12: manuscript. Roman Gardlik is supported by grant of National Scholarship 1070–1078. Programme of the Slovak Republic. 28 Larsen MD, Griesenbach U, Goussard S, Gruenert DC, Geddes DM, Scheule RK et al. Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli. Gene Therapy 2008; 15: 434–442. 29 Xiang R, Lode HN, Chao TH, Ruehlmann JM, Dolman CS, Rodriguez F et al. An autologous oral DNA vaccine protects against murine melanoma. 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