Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease.

New targets for the immunotherapy of colon cancer-does reactive disease hold the answer?

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women, posing a serious demographic and economic burden worldwide. In the United Kingdom, CRC affects 1 in every 20 people and it is often detected once well established and after it has spread beyond the bowel (Stage IIA-C and Stage IIIA-C). A diagnosis at such advanced stages is associated with poor treatment response and survival. However, studies have identified two sub-groups of post-treatment CRC patients--those with good outcome (reactive disease) and those with poor outcome (non-reactive disease). We aim to review the state-of-the-art for CRC with respect to the expression of cancer-testis antigens (CTAs) and their identification, evaluation and correlation with disease progression, treatment response and survival. We will also discuss the relationship between CTA expression and regulatory T-cell (Treg) activity to tumorigenesis and tumor immune evasion in CRC and how this could account for the clinical presentation of CRC. Understanding the molecular basis of reactive CRC may help us identify more potent novel immunotherapeutic targets to aid the effective treatment of this disease. In this review, based on our presentation at the 2012 International Society for the Cell and Gene Therapy of Cancer annual meeting, we will summarize some of the most current advances in CTA and CRC research and their influence on the development of novel immunotherapeutic approaches for this common and at times difficult to treat disease.

Bacterial vectors for imaging and cancer gene therapy: a review.

The significant burden of resistance to conventional anticancer treatments in patients with advanced disease has prompted the need to explore alternative therapeutic strategies. The challenge for oncology researchers is to identify a therapy which is selective for tumors with limited toxicity to normal tissue. Engineered bacteria have the unique potential to overcome traditional therapies' limitations by specifically targeting tumors. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally, either external to (non-invasive species) or within tumor cells (pathogens). Pre-clinical and clinical investigations involving bacterial vectors require relevant means of monitoring vector trafficking and levels over time, and development of bacterial-specific real-time imaging modalities are key for successful development of clinical bacterial gene delivery. This review discusses the currently available imaging technologies and the progress to date exploiting these for monitoring of bacterial gene delivery in vivo.

The emerging role of viruses in the treatment of solid tumours.

There is increasing optimism for the use of non-pathogenic viruses in the treatment of many cancers. Initial interest in oncolytic virotherapy was based on the observation of an occasional clinical resolution of a lymphoma after a systemic viral infection. In many cancers, by comparison with normal tissues, the competency of the cellular anti-viral mechanism is impaired, thus creating an exploitable difference between the tumour and normal cells, as an unimpeded viral proliferation in cancer cells is eventually cytocidal. In addition to their oncolytic capability, these particular viruses may be engineered to facilitate gene delivery to tumour cells to produce therapeutic effects such as cytokine secretion and anti -tumour immune responses prior to the eventual cytolysis. There is now promising clinical experience with these viral strategies, particularly as part of multimodal studies, and already several clinical trials are in progress. The limitations of standard cancer chemotherapies, including their lack of specificity with consequent collateral toxicity and the development of cross-resistance, do not appear to apply to viral-based therapies. Furthermore, virotherapy frequently restores chemoradiosensitivity to resistant tumours and has also demonstrated efficacy against cancers that historically have a dismal prognosis. While there is cause for optimism, through continued improvements in the efficiency and safety of systemic delivery, through the emergence of alternative viral agents and through favourable clinical experiences, clinical trials as part of multimodal protocols will be necessary to define clinical utility. Significant progress has been made and this is now a major research area with an increasing annual bibliography.

Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation.

Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm(3) tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.

We investigated if the range of efficacy of a gene-based immunotherapy of solid tumours against systemic disease could be extended when used as a neoadjuvant to surgery. Hundred percent mice whose subcutaneous tumours were surgically removed 4 days post-electroporation with GM-CSF and B7-1 plasmid were systemically resistance to tumour rechallenge. In mice bearing both subcutaneous and hepatic tumours, neoadjuvant treatment of the primary tumour resulted in significant reduction in, or elimination of, hepatic tumour growth, with a significant increase in survival, indicating that control of the primary tumour by immunotherapy was not a prerequisite for containment of systemic disease.

Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

Local gene therapy of solid tumors with GM-CSF and B7-1 eradicates both treated and distal tumors.

Gene therapy-induced expression of immunostimulatory molecules at tumor cell level may evoke antitumor immune mechanisms by recruiting and enhancing viability of antigen-processing cells and specific tumoricidal lymphocytes. The antitumor efficacy of a plasmid, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and the B7-1 costimulatory immune molecule, delivered into growing solid tumors by electroporation was investigated. Murine fibrosarcomas (JBS) growing in Balb/C mice (

Non-viral in vivo immune gene therapy of cancer: combined strategies for treatment of systemic disease.

Many patients with various types of cancers have already by the time of presentation, micrometastases in their tissues and are left after treatment in a minimal residual disease state [Am J Gastroenterol 95(12), 2000]. To prevent tumour recurrence these patients require a systemic based therapy, but current modalities are limited by toxicity or lack of efficacy. We have previously reported that immune reactivity to the primary tumour is an important regulator of micrometastases and determinant of prognosis. This suggests that recruitment of specific anti-tumour mechanisms within the primary tumour could be used advantageously for tumour control as either primary or neo-adjuvant treatments. Recently, we have focused on methods of stimulating immune eradication of solid tumours and minimal residual disease using gene therapy approaches. Gene therapy is now a realistic prospect and a number of delivery approaches have been explored, including the use of viral and non-viral vectors. Non-viral vectors have received significant attention since, in spite of their relative delivery inefficiency, they may be safer and have greater potential for delivery of larger genetic units. By in vivo electroporation of the primary tumour with plasmid expressing GM-CSF and B7-1, we aim to stimulate immune eradication of the treated tumour and associated metastases. In this symposium report, we describe an effective gene based approach for cancer immunotherapy by inducing cytokine and immune co-stimulatory molecule expression by the growing cells of the primary tumour using a plasmid electroporation gene delivery strategy. We discuss the potential for enhancement of this therapy by its application as a neoadjuvant to surgical excision and by its use in combination with suppressor T cell depletion.

Combined electric field and ultrasound therapy as a novel anti-tumour treatment.

The permeabilising effects of electric pulses on cell membranes and the use of ultrasound energy of various intensities, for both thermal effects and enhancement of drug and gene delivery, have led to extensive research into the potential applications of these systems in the development of novel anti-cancer treatments. In the present study we have demonstrated for the first time that the application of brief electric pulses 'sensitises' tumour cells to the effects of low intensity ultrasound. The studies were conducted in human tumours established in athymic nude mice and in many instances resulted in the reduction of tumour mass. The combined electric field and ultrasound approach (CEFUS) was applied in vivo to a murine colon adenocarcinoma (C26) and a human oesophageal adenocarcinoma (OE19). The experiments performed demonstrated the anti-tumour effects of the combined therapy. Varying the electrosensitisation parameters used (voltage, waveform, electrode type) contributed to optimise the procedure. Exponential electric pulses with a peak of 1000 V/cm were initially used, but square wave pulses (1000 V/cm, 1 ms, x2, 1 Hz) were found to be just as effective. All ultrasound application parameters were kept constant during the study. The growth rate of C26 tumours treated with CEFUS was significantly reduced with respect to untreated controls at day 7 (96% of average initial tumour volume in CEFUS group versus 615% for controls, P < 0.05). Similar reduction was observed in OE19 tumours treated with CEFUS by day 4 (82% versus 232%, P < 0.032). Our preliminary data suggest that this novel technology could potentially be of wide application in clinical practice for the treatment of solid tumours and is worth further investigation.

Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract.

AIMS: To identify lactic acid bacteria (LAB) of porcine intestinal origin with anti-Salmonella activity. METHODS AND RESULTS: Samples were obtained from pig faeces and caeca and screened for the presence of anti-Salmonella LAB. The 11 most promising isolates were identified as belonging to the genera Lactobacillus and Pediococcus. The LAB exhibited large variation in their ability to survive in simulated gastric juice at pH 1.85. While Lactobacillus johnsonii species survived at levels of 80% for up to 30 min, Lactobacillus pentosus species declined to <0.001% in that time. All isolates tolerated porcine bile at a concentration of 0.3% (w/v), with some isolates capable of growth in the presence of up to 5% (w/v) bile. The ability of the LAB isolates to prevent Salmonella invasion of intestinal epithelial HT-29 cells varied, with reductions of between 30% (Lact. pentosus) and 80% (Lactobacillus murinus spp.) observed. CONCLUSIONS: LAB of porcine origin were observed to survive simulated passage through the GIT and inhibit growth of Salmonella and its invasion of the intestinal epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that some porcine intestinal LAB isolates may offer potential as probiotics for the reduction of Salmonella carriage in pigs.

Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis.

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular gamma-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.

Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824.

The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated. Glucose was used preferentially to maltose, when both substrates were present in the medium. Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme II(Mal) and a maltose 6-phosphate hydrolase.

Analysis of a catabolic operon for sucrose transport and metabolism in Clostridium acetobutylicum ATCC 824.

The utilization of sucrose by Clostridium acetobutylicum ATCC 824 was investigated. Sucrose was found to be transported via a phosphoenol-pyruvate (PEP)-dependent phosphotransferase system (PTS) and a metabolic pathway identical to that previously identified in C. beijerinckii, was established. The genes encoding the proteins of this pathway were identified from the C. acetobutylicum genome sequence, in the order scrAKB encoding Enzyme II of the sucrose PTS, fructokinase and sucrose 6-phosphate hydrolase respectively. While the pathway for sucrose metabolism is conserved between C. acetobutylicum and C. beijerinckii, the operons show considerable differences in organization and regulatory elements. The C. acetobutylicum scr operon contains the elements of an antiterminator-mediated regulation mechanism, typical of the BgIG family of regulators. The scrT gene, located upstream of scrA encodes an antiterminator that is preceded by a transcription terminator, which is overlapped by a classical ribonucleic antiterminator (RAT) sequence. We also propose the existence of a new variant RAT-like sequence which overlaps a terminator between scrT and the downstream structural genes.

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Identification of a DNA region from lactococcal phage sk1 protecting phage 712 from the abortive infection mechanism AbiF.

Bacteriophage 712 is a small isometric-headed phage which is sensitive to the lactococcal abortive infection mechanism AbiF. Its 29.6-kb DNA genome was characterized by restriction mapping and transcriptional analysis. Construction of a gene bank of lactococcal phage sk1, which is insensitive to the action of AbiF, in Lactococcus lactis containing AbiF resulted in the identification of a 324-bp DNA fragment which reduced the effect of the abortive infection mechanism on phage 712. Analysis of this region provided evidence that the action of AbiF is related to the cos ends of small isometric-headed phages. Sequence analysis of a 3.2-kb segment containing the middle operon and the cos ends of phage 712 genome allowed comparison of this part of the phage 712 genome with the equivalent sequences of four other small isometric-headed phages.

Expression, regulation, and mode of action of the AbiG abortive infection system of lactococcus lactis subsp. cremoris UC653

The abortive infection system AbiG is encoded by the lactococcal plasmid pCI750. The abiG locus (consisting of two genes, abiGi and abiGii) was examined by Northern blot analysis, revealing two transcripts of approximately 2.8 and 1.5 kb which were homologous to the two gene-specific probes. A transcriptional start site was mapped upstream of abiGi, and it appeared that the two genes were cotranscribed, resulting in the 2.8-kb transcript. The smaller transcript may be the result of independent transcription of abiGii within abiGi or of the presence of a weak terminator within abiGii. The locus was shown to be constitutively expressed. Evidence is presented for the possible existence of a second Abi mechanism on pCI750. Examination of phage sk1 RNA synthesis demonstrated that both the subcloned AbiG and, to a greater extent, pCI750 inhibited this process. pCI750 also severely inhibited synthesis of both early and late phage c2 transcripts, while the presence of the subclone resulted in a reduction in late transcript synthesis only.

Note: sucrose transport and metabolism in Clostridium beijerinckii NCIMB 8052.

Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetonebutanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed.

A gene system for glucitol transport and metabolism in Clostridium beijerinckii NCIMB 8052.

The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted.

Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis.

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.