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Genetically Engineered Salmonella typhimuriumfor Targeted Cancer Therapy

Seong Young Kwon and Jung-Joon MinChonnam National University Medical School, Jeonnam, Republic of Korea

KEYWORDS

Salmonella; genetic engineering; gene delivery; genetherapy; attenuation; toxicity; imaging

Abstract

Gene therapy using Salmonella has several possible advantagescompared with other gene therapeutic tools, and Salmonellacan be engineered according to the specific requirements ofeffective cancer therapy. As a delivery vector, Salmonella hasbeen applied to gene therapy via three delivery routes: (1) thedirect transfer of genes, (2) the delivery of proteins expressedby Salmonella, and (3) the transfer of plasmids encoding smallhairpin RNAs. Usually, the therapeutic effect of Salmonellaagainst cancer can be achieved according to several mechan-isms of action, including prodrug-converting enzymes, cyto-toxic agents, and immune response through these deliveryroutes. Salmonella can be engineered for the purpose of precisecontrol of gene expression to maximize the intratumoraleffects while minimizing the systemic toxicity and productionof imaging signal to monitor therapeutic effects. Salmonella isexpected to be a powerful tool that could replace or compen-sate for conventional cancer therapies through genetic engi-neering for “smart” bacteria.

INTRODUCTION

Gene therapy is considered to be one of the mostpromising therapeutic approaches for treating cancer,but the optimized delivery of genes into target cells isa significant issue that hinders successful gene therapy[1,2]. Several different types of delivery vectors (viral,nonviral, and bacterial vectors) have been investigatedin an attempt to resolve this issue [3,4].

Many therapeutic approaches to cancer have usedbacteria because they have distinctive characteristics as

anticancer agents and as delivery vectors during genetherapy [5]. Certain strains of bacteria, such asEscherichia coli [6�10], Salmonella [9,11�13], Clostridium[14,15], Bifidobacterium [16], and Listeria [17], can selec-tively colonize and grow in tumors. A number ofrecent reports have demonstrated that bacteria arecapable of targeting primary tumors and metastases[6�10], and this feature is being exploited by tumor-selective drug delivery [8,17�20].

Salmonella typhimurium has several possible advan-tages compared to the other strains: It can grow in aer-obic or anaerobic conditions that are present in solidtumors; it expresses specialized systems that mediatethe invasion of epithelial cells and macrophages; andthere is a vast body of knowledge and a comprehen-sive understanding of its genetics, which facilitates thegenetic engineering of this bacterium for cancer thera-pies [5,21]. The genetic engineering of Salmonella hasbecome very important for lowering its toxicity andmaximizing its therapeutic efficacy, particularly sincethe 2000s [22].

However, there are still many hurdles to overcomebefore bacterial-mediated cancer therapy is translatedfrom preclinical studies to clinical practice. This chap-ter introduces the genetic engineering of Salmonellaaccording to the specific requirements of effective can-cer therapy, and it summarizes possible directions forfuture clinical applications.

GENETIC ENGINEERING MAP FORCANCER THERAPY

Salmonella species have been investigated ingenetic studies to overcome or compensate for the

443Gene Therapy of Cancer. DOI: http://dx.doi.org/10.1016/B978-0-12-394295-1.00030-5 © 2014 Elsevier Inc. All rights reserved.

problems of conventional therapies, including che-motherapy, radiation therapy, and existing genetherapy methods, particularly in four main areas(Figure 30.1): (1) lowering intrinsic toxicity, (2) pro-moting target cell selectivity, (3) delivering therapeu-tic drugs (cargo drug), and (4) producing imagingsignals [1,5,22�24].

Many different therapeutic approaches based onSalmonella species have been applied in animal models,and most have combined different forms of engineer-ing techniques in these four areas. Thus, Salmonella hasbeen transformed into a combination of geneticallyengineered elements (known as a genetic engineeringmap) for specific purposes.

SALMONELLA AS AN EFFECTIVEDELIVERY VECTOR

An ideal vector has to be intrinsically safe in livingsubjects, including humans, and it must accumulateselectively in the target tissue or cell, which facilitatesthe delivery of cargo molecules to specific tissues, cells,or the cellular nucleus. Therefore, the genetic engineer-ing of Salmonella is very important for generating non-virulent strains, enhancing its therapeutic potency, andregulating the host immune response. The geneticmaterial should be transcribed without degradationdue to environmental factors, such as enzymes and theimmune system [3].

Attenuated Salmonella typhimurium

The attenuation of virulence has been achieved byauxotrophic mutations that affect the biosynthesis ofpurines [21,25] or aromatic amino acids [21,25,26], bymutations in the biosynthesis of lipid A [11], or bydeletions of the relA and spoT genes, which generatestrains defective in guanosine 50-diphosphate 30-diphosphate (ppGpp) synthesis [21,24,27].

Salmonella typhimurium VNP20009 was geneticallyengineered by the partial deletion of the msbB and purIgenes [28,29]. The msbB gene is responsible for theaddition of a terminal myristyl group to lipid A. Thelipopolysaccharide (LPS) produced by these lipid Amutants has a much lower capacity for inducingtumor necrosis factor-α (TNF-α) [11,29]. The purI genecan induce a growth requirement for external sourcesof purines [28], and a purI gene mutation results inpurine deficiency and a limited replication rate[22,28].

Salmonella typhimurium A1 is auxotrophic for leucineand arginine, and it was developed to prevent continu-ous infections in normal tissues while maintaining vir-ulence against tumors because it has no otherattenuated mutations compared with S. typhimuriumVNP20009 [30]. To enhance its tumor virulence,S. typhimurium A1 was injected into nude mice thathad been transplanted with an HT-29 human colontumor. The bacteria isolated from the infected tumorwere then cultured, and the re-isolated A1 was desig-nated A1-R. The number of A1-R bacteria attached to

FIGURE 30.1 Genetic engineering processes used for tumor targeting by Salmonella. The application of Salmonella to anticancer therapyhas been studied using genetic engineering processes. Salmonella can be optimized to treat cancer based on a combination of specific engineer-ing requirements, including decreased toxicity, improved selectivity, therapeutic agent loading (cargo drug), and imaging signal generation(known as a genetic engineering map).

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HT-29 human colon cancer cells was approximately sixtimes higher than that of the parent A1 [26].

ppGpp is a stringent signaling molecule that is con-sidered to be a key molecule for the cessation of ribo-some production when a bacterial culture enters thestationary phase [31]. Recently, ppGpp was implicatedin the stationary phase induction of Salmonella pathoge-nicity island type I (SPI-1) genes by the activator hilA,which is a master transcriptional regulator of SPI-1-encoded genes. During the course of infections, it hasbeen suggested that Salmonella encounters variousstressful environments, which are sensed and translatedinto the intracellular signal ppGpp, and this induces theexpression of Salmonella virulence genes, includingSPI-1 genes [32]. A ppGpp-defective Salmonella mutantexhibited significant virulence attenuation, whereas theLD50 of an S. typhimurium mutant that was defective forppGpp synthesis (STΔppGpp) was increased by fiveorders of magnitude, irrespective of the administrationroute [31]. This strain has been investigated in mousemodels [24,33].

Selective Targeting of Tumors

From the standpoint of the interaction between bacte-ria and tumor cells or tissues, tumors have severalunique characteristics compared with normal tissue,including hypoxic or necrotic areas, immune-privilegedmicroenvironments, and abundant nutrients.

As a facultative anaerobe, Salmonella is thought touse several mechanisms to accumulate in tumors, suchas the entrapment of Salmonella in the tumor vascula-ture [34], flooding into tumors after TNF-α-mediatedinflammation [35], chemotaxis toward compounds pro-duced by tumors [36,37], preferential amplification intumor-specific microenvironments [13,37], and protec-tion from immune system clearance [28].

The attenuation of Salmonella by auxotrophy reducesits virulence in mice but also amplifies its targetingcapacity by two or three orders of magnitude intumors compared with the liver [21]. Auxotrophicmutants require exogenous nutrients, so they are gen-erally weakened in mice, which allows them to be con-trolled or eradicated by the host defense mechanisms.Tumors contain actively dividing cells and necroticareas, and they appear to be an environment in whichmetabolites such as purines, pyrimidines, and aminoacids are plentiful, thereby providing a reservoir ofnutrients for bacteria.

Salmonella chemotaxis using surface chemoreceptorsalso supports the targeting mechanism. A study usingknockout bacteria showed that the aspartate receptorinitiates chemotaxis toward viable tumor tissue wherethe serine receptor initiated tissue penetration and the

ribose/galactose receptor directed S. typhimuriumtoward necrotic areas [36].

By contrast, attenuated Salmonella with a partialdeletion of the msbB gene had a lower potential foraccumulating in tumor tissues due to a decline inTNF-α, which could also lead to an influx of bloodinto tumors [5,11,35]. Therefore, an adequate balancebetween attenuation and immunogenicity must beachieved.

Effective Delivery of Genes or Gene-RelatedMaterials

As a delivery vector, Salmonella has been applied togene therapy via three delivery routes [1,2,5,23]: (1) thedirect transfer of genes (or DNA) into the target tissue[38,39], (2) the delivery of proteins that were originallyexpressed by bacteria [18,40�43], and (3) the transferof plasmids encoding small hairpin RNAs (shRNAs)(Figure 30.2) [44,45]. The direct transfer of genes (alsoknown as bactofection) is the bacteria-mediated trans-fer of plasmid DNA into mammalian cells. Bacteriadisrupt and release a plasmid, encoding the therapeu-tic gene after escaping from the blood vessel and enter-ing the target cell. The plasmid is transferred into thecell nucleus and a therapeutic protein, with cytotoxicactivity or immune response, is expressed by the hostcell [23]. Bactofection of mammalian cells can beachieved via the active invasion of nonphagocyticmammalian cells or passive uptake by phagocyticimmune cells [1,39]. Compared with other deliverymechanisms, such as protein delivery, this approachhas benefits and drawbacks. Bactofection may producestronger, more stable expression because it uses themammalian expression system. However, the expres-sion of the transferred genes may be more difficult tocontrol [2]. A poor transfer efficiency could also limitthe therapeutic response because the transferred genesmay be expressed exclusively in infected cells ratherthan being distributed homogeneously in the tissues.Furthermore, plasmids generally cannot replicate inhost mammalian cells [23].

Another approach is the bacterial delivery of thera-peutic agents, in the form of proteins instead of genes.Bacteria can be used as protein production factories, sothey can continue to produce cargo proteins while pro-liferating in the target tissues, either in the extracellularspaces or inside the targeted cells [2,23]. The persistentbacteria can produce the requisite polypeptide in situ,thereby ensuring the localized distribution of a thera-peutic agent without affecting systemic nontargetorgans [23]. This approach may facilitate the specificregulation of protein expression using inducible promo-ters (discussed later). Furthermore, the bacteria do not

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integrate the delivered genetic material into the hostgenome, which greatly decreases the risk of delivery-induced mutagenesis and tumorigenesis [23].

Finally, bacteria can be engineered to deliverplasmid-based shRNAs into tumor cells, where theyproduce small interfering RNA (siRNAs) (also knownas gene silencing) [44,45]. siRNAs induce the degrada-tion of mRNAs transcribed by a specific gene such asBcl2 [44] or the signal transducer and activator of tran-scription 3 (STAT3) gene [45]. The proteins expressedby both genes inhibit apoptosis, and STAT3, in particu-lar, promotes cancer cell growth. The combination ofthese two advanced approaches (i.e., bacterial deliveryand RNA interference) is expected to be a promisingtool for cancer therapy [23].

SALMONELLA AS ADVANCEDTHERAPEUTIC STRATEGIES

As mentioned previously, Salmonella can deliver vari-ous types of therapeutic agents into target tissues viadifferent delivery mechanisms. In addition, clinical andpreclinical studies have been conducted to validate theeffectiveness of bacteria-mediated drug delivery accord-ing to their mechanisms of action in cancer. These stud-ies have included prodrug-converting enzymes,

cytotoxic agents, cytokines, and tumor-specific antigensor antibodies (Table 30.1). These therapeutic strategieshave been investigated using the appropriate deliveryroutes—that is, gene transfer or protein delivery.

Prodrug Conversion Strategies

Prodrug-converting enzymes (or suicide enzymes)aim to improve the therapeutic efficiency (benefit vs.toxic side effects) of cancer chemotherapy [46]. A gene,encoding an enzyme that is not naturally expressed inthe host, is introduced into the target cells by Salmonellaand the transformed cells express the enzyme [46].

Salmonella typhimurium has been engineered toexpress suicide enzymes, such as cytosine deaminase(CD) [47] and herpes simplex virus thymidine kinase(HSV-TK) [21], and administration of the appropriateprodrugs (5-fluorocytosine or ganciclovir, respectively)leads to selective localization of the active forms, lead-ing to tumor growth suppression or regression [47].

Cytotoxic Approaches to Controlling CancerCells

This strategy involves the introduction of anti-angiogenic or cytotoxic genes and native bacterial toxicproteins, which kill cancer cells [1,5].

FIGURE 30.2 Delivery mechanisms for genetically engineered Salmonella. Salmonella can be used as a delivery vector to introduce genesor gene products into target cells or tissues via three mechanisms. (A) Salmonella can transfer genes into the target cell in the form of a plasmidthat encodes the therapeutic gene. The plasmid is released from disrupted Salmonella after infecting the target cell, and it is transferred intothe cellular nucleus. The therapeutic protein is expressed by the target cell’s expression system. (B) Salmonella can deliver therapeutic proteinsinstead of genes, where the therapeutic protein is expressed by the Salmonella expression system inside or outside the target cell.(C) Salmonella can also transfer plasmid-based small hairpin RNAs (shRNAs) into the target cell nucleus or express the shRNAs directly. TheshRNAs are processed into small interfering RNAs (siRNAs), which induce the degradation of mRNAs transcribed from specific genes.

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The direct transfer of anti-angiogenic genes such asendostatin [48] and thrombospondin-1 [49] can killcancer cells by preventing new blood vessel formationand blocking the nutrient supply. Salmonella has beenused as the carrier to deliver cytotoxic genes thatencode the second mitochondria-derived activator ofcaspases (SMAC) and tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), which can induceapoptosis in many types of cancer cells [50].

Furthermore, bacterial toxic proteins, such as cytoly-sin A (also known as HlyE) [24,40], and other cytotoxicproteins, such as FAS ligand [43], TNF-α, or TRAIL [51],can be delivered to cancer cells using engineeredSalmonella, where they induce apoptosis. In particu-lar, the extracellular domain of TRAIL is a potentapoptotic agent in tumor cells, whereas it hasminimal toxicity against normal cells. AttenuatedSalmonella has been used to express TRAIL under thecontrol of a prokaryotic promoter, resulting in tumorgrowth reduction [51].

Activation of the Immune System UsingCytokines

Unlike normal cells, cancer cells are generally toler-ant of their host’s immune system, and they preventimmune cells from recognizing them as cancer cells.Conventional therapies such as chemotherapy or radia-tion therapy are limited when increasing the dose toeliminate all tumor cells because they can cause severeside effects in normal tissues. Immune-based therapy(immunotherapy) using cytokines is considered a pow-erful therapeutic tool, but it has several problems,including a short in vivo duration and severe sideeffects after the systemic administration of cytokines[52,53]. Therefore, immunotherapy using genetically

engineered bacteria may be a promising tool for over-coming the limitations of existing therapeutic methods.

Immunotherapy using Salmonella includes the trans-fer of specific genes encoding cytokines and the deliv-ery of cytokines expressed by bacteria, which canenhance the anticancer effects of various lymphocytes,such as B cells, T cells, and natural killer cells [1].

Several different types of cytokines and growth factorshave been investigated via the direct transfer of genes.These molecules, including FLT3L [54], interleukin-12(IL-12) [53,55,56], and granulocyte�macrophage colony-stimulating factor (GM-CSF) [53], can activate severaltypes of lymphocytes.

Salmonella has also been studied with respect to thedelivery of cytokines produced intrinsically by the bac-teria. These include IL-2 [57�59], IL-18 [42], CCL21[41], and LIGHT [18]. These cytokines induce T cells,natural killer cell proliferation (IL-2 or IL-18), dendriticcell growth (LIGHT), and leukocyte and neutrophilinfiltration (LIGHT or CCL21), and they control themigration of immune cells (CCL21) [18,41,42,57�59].

Tumor-Specific Antigens

The engineering of Salmonellae to produce a cancervaccine is another type of immunotherapy used tobreak the preexisting tolerance of the immune systemwith regard to tumor-specific antigens. Several mem-bers of the Salmonella genus have been used in vaccina-tion strategies in which they serve as DNA or antigencarriers [1].

DNA vaccine delivery by genetically engineeredSalmonella can inhibit the growth of several types oftumors that express specific antigens, such as α-feto-protein [60] and vascular endothelial growth factor(VEGF) receptor 2 [61]. Antibodies against these

TABLE 30.1 Applications of Genetically Engineered Salmonella in Advanced Therapeutic Strategies

Therapeutic Strategy Engineering Approach Related Genes or Molecules References

Prodrug activation Introduction of a gene encoding aprodrug-converting enzyme into targetcells

CD, HSV-TK [21,46,47]

Cytotoxic effect Introduction of cytotoxic genes orproteins to kill cancer cells

Endostatin, thrombospondin-1, SMAC,TRAIL, cytolysin A, FAS ligand, TNF-α

[24,40,43,48�51]

Immune Activation

Cytokines Delivery of genes encoding cytokine(s)expressed by Salmonella to enhance theanticancer effects of immune cells

FLT3L, IL-12, GM-CSF, IL-2, IL-18,CCL21, LIGHT

[18,41,42,52�59]

Tumor-specific antigens(Ags)

Delivery of genes encoding tumor-specific Ags, or the Ags themselves, tosensitize immune cells via antibodyformation

α-Fetoprotein, VEGFR, PSA, NY-ESO-1,CPV, RAF-1

[60�65]

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antigens can prevent tumor formation, tumor growth,and angiogenesis [60,61].

In addition to DNA delivery, vaccine therapy basedon antigen delivery has also been investigated usingSalmonella. Immune cells may be sensitized and theformation of tumors that express specific antigens canbe prevented by the delivery of these antigens, such asprostate-specific antigen (PSA) [62], NY-ESO-1 [63],canine parvovirus (CPV) [64], and RAF-1 [65].

PRECISE CONTROL OF SALMONELLA

A major hurdle that must be overcome to realize thefull potential of cytotoxic drug delivery, using engi-neered bacteria such as Salmonella, is their toxicity tonontumor tissues. Following intravenous administration,these bacteria tend to localize to the liver and spleen ini-tially [7,9]. Thus, the constitutive expression of a cyto-toxic drug would result in unavoidable severe hepatic orsplenic injuries. The most desirable system would be onein which gene expression is controlled in a manner thatmaximizes the intratumoral effects while minimizing thesystemic toxicity [34]. Indeed, inducible systems that uti-lize three different external gene “triggers” have beendeveloped for this purpose, as follows [5]: The pBADsystem, which is an L-arabinose-responsive system inwhich expression is tightly regulated via the regulatoryprotein AraC [24,33,66�68]; the salicylate system alsoachieves tight regulation via a signaling cascade thatamplifies gene expression [69]; and the γ-irradiationsystem, in which γ-irradiation-induced DNA damageactivates RecA to promote autoproteolysis of therepressor LexA and the subsequent induction of geneexpression [70,71].

Another strategy is to design bacteria that sensehypoxia using the fumarate and nitrate reduction(FNR) regulator [40]. FNR is an oxygen-responsivetranscription factor that is naturally present inSalmonella [72]. In the absence of oxygen, iron sulfideclusters induce the formation of FNR homodimers thatbind to specific DNA sequences and promote tran-scription. In the presence of oxygen, the clusters andFNR homodimers disassemble, thereby reducing tran-scription. Arrach et al. developed a reporter systemthat was tested in tumor-bearing mice [73]. The twomost active promoters, pflE and ansB, both containedFNR binding sites, and they are known to be oxygendependent. Leschner et al. also screened an S. typhi-murium promoter-trap library to identify promotersthat drive gene expression exclusively in cancerous tis-sue [74]. Twelve genomic fragments were identifiedthat appeared to be necessary for tumor specificity.

In the near future, a study will be performed inwhich a specific promoter induced by external signals

will activate the expression of an anticancer agentwhile being monitored using molecular imaging tech-niques in a theranostic strategy (image-guided molecu-lar therapy).

ENGINEERED BACTERIA FOR IMAGINGSTUDIES

The visualization of bacteria has several roles in pre-clinical and clinical applications of bacteria-mediatedcancer therapies. Bacterial imaging allows the monitor-ing of the location, magnitude, and timing of geneexpression by the bacterial gene expression or by thebacterial delivery system, all of which are importantparameters for predicting and measuring the effects oftreatments.

Bacterial imaging techniques have been developedusing three major imaging modalities: optical fluores-cence or bioluminescence imaging, positron emissiontomography (PET), and magnetic resonance imaging(MRI). For optical imaging applications, bacteria areengineered by transforming them with plasmids con-taining genes for bioluminescence (luxCDABE operonfrom Photobacterium leiognathi) [9,24] (Figure 30.3) orfluorescent proteins [13]. These optical methods arevery efficient for identifying the tumoral localization inmice, although they have limited utility in clinicalapplications because of the poor penetration of lightsignals through tissues.

FIGURE 30.3 Multimodal imaging of bacterial-mediated cancer

therapy. Engineered S. typhimurium expressing bacterial luciferaseand HSV1-TK was injected intravenously into a CT26 murine coloncancer-bearing BALB/c mouse. Three days after bacterial injection,optical bioluminescence imaging was performed. Following the opti-cal imaging study, microPET images were acquired after intravenousinjection of [18F]FHBG (200 μCi). The luminescence activity andradioactivity were observed in the bacterial-injected mouse (bottom).A negative control mouse injected with PBS is also shown (top).

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Salmonella typhimurium VNP20009 has been engi-neered to express the PET reporter gene herpessimplex thymidine kinase (HSV1-tk), and it phosphor-ylates and sequesters a radiolabeled nucleoside(purine) analog, 20-fluoro-1-β-D-arabino-furanosyl-5-iodouracil (FIAU), which can be visualized by PET[75]. HSV1-TK-expressing bacteria can also be visual-ized using the pyrimidine analog, 9-[4-[18F]fluoro-3-(hydroxymethyl) butyl] guanine [18F]FHBG)(Figure 30.3). Instead of using an exogenous reportergene such as HSV1-tk, an endogenous thymidinekinase from the probiotic E. coli Nissle 1917 can beused for PET imaging to phosphorylate and sequestera radiolabeled nucleoside analog; however, the imag-ing signal has been reported to be very weak [76].

Magnetotactic bacteria, such as Magnetospirillummagneticum AMB-1, can be used to enhance the utilityof MRI during tumor localization [77]. They producemagnetite particles [78] and accumulate in tumors bysearching for the low-oxygen conditions that promotetheir growth [79].

Bacterial imaging is expected to have significantvalue, particularly in tumor-specific gene triggering,because it can provide real-time information about thelocation and abundance of bacteria before switchingon target gene expression.

CLINICAL APPLICATIONS ANDCHALLENGES

Despite the many positive results reported in pre-clinical studies, no significant progress has beenreported in clinical trials since the first trial in 2002[80�82]. Numerous challenges remain before bacteriacan be used in the clinic, including limited drug pro-duction, intrinsic bacterial toxicity, targeting efficiency,genetic instability, and their combination with othertherapies [5].

Safety of Bacterial Therapy

Low toxicity is essential for clinical applications,and several studies of attenuated Salmonella haveshown that its safety is acceptable in multiple animalspecies [25] and in clinical trials [80�82]. Moreover,the reduction of toxicity is also important for the thera-peutic efficacy because low toxicity can increase themaximum tolerable dose.

Toxicity is usually affected by the immune responseafter systemic administration, as well as the innatebacterial virulence. Salmonella typhimurium VNP20009was attenuated by chromosomal deletion of the purIand msbB genes, and several clinical studies were

conducted using this strain with advanced cancerpatients. These studies showed that intravenousadministration of a large amount of S. typhimuriumVNP20009 was well tolerated, and this strain wasfound to be a safe delivery vector in humans [80�82].However, further investigations are necessary to deter-mine a guideline maximum tolerable dose because thisdose will differ according to the patient’s characteris-tics and cancer type (e.g., even a low degree of patho-genicity may be serious for immunocompromisedcancer patients).

Genetic Instability

Genetic instability is another factor that may need tobe overcome when addressing safety problems becausemutations can create unexpected phenotypes that areharmful to humans. Genetic stability can also affecttherapeutic effectiveness because the level of mutationis a limiting factor that determines how long Salmonellapersists in tumor cells.

Genetic stability might be improved by incorporat-ing targeted genes in the Salmonella chromosome or bylimiting homologous recombination and horizontalgene transfer [5], but very few studies support thesehypotheses. Therefore, further investigations into thegenetic instability of genetically engineered Salmonellain large mammals or nonhuman primates are neces-sary before proceeding to clinical trials.

Efficacy of Bacterial Therapy

Identifying the optimal concentration of therapeuticagents, both bacterial and cargo drugs, can contributesubstantially to tumor regression and prolonged sur-vival [5], and it is affected by multiple extrinsic orintrinsic factors related to bacteria.

The extrinsic factors that affect the concentration ofa drug in the target tissue (cancer) depend on the tar-geting efficiency. Salmonella can colonize distal tumorregions that are inaccessible to conventional chemo-therapy, but many studies have indicated that its tar-geting efficiency is too variable to expect a therapeuticeffect in humans. Several clinical trials were not satis-factory in terms of the preferential localization ofSalmonella within tumor lesions [22,82].

The targeting efficiency should be improved by fur-ther exploration of new techniques, such as syntheticbiology to engineer bacteria with enhanced endogenouschemoreceptors [36] but with a reduced immune reac-tion and lower innate virulence [5]. For example, theessential Salmonella virulence factors required to evadethe host’s immune system have been determined byscreening knockout models of virulence genes that may

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induce uptake into cells, promote intracellular replica-tion, and stimulate cargo production [83].

Intrinsic factors are also associated with the processof gene expression, including the gene copy number,promoter strength, optimized codons, bacterial metab-olism, mRNA secondary structure [84], and syntheticribosome binding sites [85].

CONCLUSIONS

Many investigations show that genetically engi-neered Salmonella has great potential as a tool for anti-cancer therapy, but many hurdles still need to beovercome. Previous clinical trials involved advanced-stage cancer patients, and they were inadequate forverifying safety and efficacy. However, a number ofclinical and preclinical trials (http://clinicaltrials.gov)have been performed using genetic engineering techni-ques to elucidate the mechanisms whereby bacteria killcancer cells. Novel biological techniques, such as syn-thetic biology and systems biology, are beingemployed to accelerate the development of “smart”bacteria with enhanced targeting efficiency, sophisti-cated control of drug production, efficient delivery ofdrugs, and minimal toxicity. Salmonella therapy isexpected to be a powerful tool that could replace orcompensate for conventional cancer therapies, if allthese problems and hurdles can be overcome.

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