Effectiveness of the Lactococcal Abortive Infection Systems AbiA, AbiE, AbiF and AbiG Against P335 Type Phages

FEMS Microbiology Letters. Apr, 2002  |  Pubmed ID: 12023079

Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined.

AbiA, a Lactococcal Abortive Infection Mechanism Functioning in Streptococcus Thermophilus

Applied and Environmental Microbiology. Dec, 2002  |  Pubmed ID: 12450864

The lactococcal abortive infection mechanisms AbiA and AbiG were introduced into Streptococcus thermophilus 4035, and a range of phages capable of infecting this host were examined for sensitivity to these mechanisms. AbiA proved effective against six phages when examined at a growth temperature of 30 degrees C but had no effect on any of the phages when tested at 37 or 42 degrees C. AbiG failed to affect any of the S. thermophilus phages at 30, 37, or 42 degrees C.

Salmonella Carriage in an Irish Pig Herd: Correlation Between Serological and Bacteriological Detection Methods

Journal of Food Protection. Dec, 2004  |  Pubmed ID: 15633689

Salmonella carriage in pigs represents a serious health problem that undoubtedly contributes to the spread of human disease. Thus, the efficient and reliable testing of farm animals for bacteria such as Salmonella is an important aspect of any efficient control strategy. Serological analysis of 15 meat juice samples detected antibodies against Salmonella in some. but not all, of the animals identified bacteriologically as harboring the pathogen, indicating a lack of correlation between the bacteriological and serological methods used for Salmonella detection. The results suggest that testing by enzyme-linked immunosorbent assay is appropriate at the herd level, with culture methods preferable for individual animal analysis. A novel culture protocol detected Salmonella in the cecal contents of 15 pigs, whereas a method based on the European Standard identified only 9 pigs as being Salmonella-positive. During the study, an unusual finding was the relatively high incidence of Salmonella London carriage in the pigs tested.

Successful Application of Targeted Electrochemotherapy Using Novel Flexible Electrodes and Low Dose Bleomycin to Solid Tumours

Cancer Letters. Feb, 2006  |  Pubmed ID: 15964138

Electroporation is the application of very brief electric pulses to cells or tissues to render the cell membranes transiently and reversibly permeable, facilitating cellular uptake of otherwise impermeant molecules. Flexible electrode arrays were developed which may be used with endoscopic and laparoscopic devices for delivery of therapeutic electroporation. Their efficacy in enhancing the delivery of bleomycin, an impermeant drug, was assessed in vitro and in vivo in both human and murine cancer cell lines, and growing tumours (xenografts). These flexible electrodes consistently and predictably deliver the permeabilising electric pulses requisite for in vivo electroporation, and would be suitable for electrochemotherapy of endoluminal tumours when incorporated into an endoscopic delivery system.

Oral Immune Tolerance Mediated by Suppressor T Cells May Be Responsible for the Poorer Prognosis of Foregut Cancers

Medical Hypotheses. 2006  |  Pubmed ID: 16288967

The poor prognosis of foregut cancers might, in part, be due to the immune tolerising effect of tumour antigens which are shed into the gastrointestinal tract and processed by the gut immune system. This would create a tumour specific tolerance without compromise of global immune functions. Experimental data shows that orally fed cancer tissue induces a non cross reactive attenuation of the cellular anti tumour host responses and confers a growth advantage specific to individual cancers. Although the cellular basis of such pro-tumourogenic responses has yet to be established, it is likely, based on studies of oral tolerance mechanisms, that recruitment of immune suppressive T cells (T(regs)) may be responsible. Abrogation of oral immune tolerance to the tumour by immune based therapy could represent a significant advance in the management of upper gastrointestinal cancers.

Improved Luciferase Tagging System for Listeria Monocytogenes Allows Real-time Monitoring in Vivo and in Vitro

Applied and Environmental Microbiology. May, 2007  |  Pubmed ID: 17351089

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

Electrochemotherapy: Aspects of Preclinical Development and Early Clinical Experience

Annals of Surgery. Mar, 2007  |  Pubmed ID: 17435555

To develop an optimized, reproducible system of electrochemotherapy, and to investigate its clinical application in patients with cutaneous or subcutaneous recurrences of inoperable or progressive disease recalcitrant to current anticancer treatments.

Modulation of P21-activated Kinase 1 Alters the Behavior of Renal Cell Carcinoma

International Journal of Cancer. Journal International Du Cancer. Nov, 2007  |  Pubmed ID: 17621631

The p21-activated kinase 1 (Pak1) is a serine/threonine kinase whose activity is regulated by both Rho GTPases and AGC kinase family members. It plays a role in cytoskeletal remodeling and cell motility as well as cell proliferation, angiogenesis, tumorigenesis and metastasis. An involvement of Pak1 in renal cell carcinoma (RCC), which remains highly refractory to chemotherapy and radiotherapy, remains to be investigated. Pak1 expression, phosphorylation and kinase activity were examined in RCC cell lines and human tissue from normal and renal carcinoma. We report increased Pak1 expression and constitutive activity in the membrane and nucleus but not the cytoplasm of resected human RCC. To study a role for Pak1 in RCC, we developed 786-0 clones that expressed either a kinase-active Pak1L83,L86 2 different Pak1 dominant negative mutants, Pak1R299 and Pak1L83,L86,R299 or Pak1 siRNA. The expression of Pak1L83,L86 increased 786-0 proliferation, motility and anchorage independent growth, while the dominant negative mutants and Pak1 siRNA abrogated these effects. In addition, Pak1L83,L86 conferred resistance to 5-fluorouracil with a 40%+/-10% increase in cell viability. Conversely, Pak1L83,L86,R299, Pak1R299 and Pak1 siRNA conferred sensitivity with a 65.2%+/-5.5%, 69.2%+/-3.3% and 73.0%+/-8.4% loss in viability, respectively. Finally, Pak1 plays a role in renal tumor growth in vivo. Only 33% of mice developed tumors in the Pak1L83,L86,R299 group and no tumors developed from Pak1R299 cell challenge. Together these findings point to Pak1 as an exciting target for therapy of renal cancer, which remains highly refractory to existing treatments.

Effective Tumor Treatment Using Optimized Ultrasound-mediated Delivery of Bleomycin

Ultrasound in Medicine & Biology. Mar, 2008  |  Pubmed ID: 17988788

Bleomycin is a nonpermeant, hydrophilic macromolecule with a high intrinsic anticancer cytotoxicity. However, the cytotoxic potential of the drug is restricted by its low membrane permeability. Application of low-intensity ultrasound to growing tumors enhances intracellular delivery of bleomycin after IP or intratumoral administration, thereby potentiating its cytotoxicity. Optimization of ultrasound parameters for in-vivo bleomycin delivery was undertaken, and an effective antitumor effect was demonstrated in solid tumors of both murine and human cell lines. Cell death after treatment was shown to occur by an apoptotic mechanism. The results achieved in these experiments were equivalent to those achieved using electroporation to mediate delivery of bleomycin-electrochemotherapy. We found that, although temperature rises of up to 5 degrees C occur using the optimized ultrasound conditions, this effect is not responsible for the potentiated drug cytotoxicity. This technique could be used with focused ultrasound or with endoscopic ultrasound probes to develop a localized and effective anticancer treatment with little or no systemic toxicity. (E-mail: Geraldc@ccrc.ie).

Viral Vectors in Cancer Immunotherapy: Which Vector for Which Strategy?

Current Gene Therapy. Apr, 2008  |  Pubmed ID: 18393828

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.

Tripartite Meeting in Gene and Cell Therapy, 2008: Irish Society for Gene and Cell Therapy, British Society for Gene Therapy, and International Society for Cell and Gene Therapy of Cancer

Human Gene Therapy. Oct, 2008  |  Pubmed ID: 18652546

The second annual meeting of the Irish Society for Gene and Cell Therapy was held in Cork, Ireland on May 15 and 16, 2008 (http://crr.ucc.ie/isgct/). The meeting was jointly organized with the British Society for Gene Therapy and the International Society for Cell and Gene Therapy of Cancer. Because of the location of the conference and the co-organization of this meeting with the British and International Gene Therapy societies, the meeting enjoyed a range of talks from some of the major leaders in the field. Particularly notable were the talented molecular and cell biologists from Ireland who have contributed cutting edge science to the field of gene therapy. Topics including cardiovascular disease, repair of single-gene disorders, and cancer gene therapy were discussed with presentations ranging from basic research to translation into the clinic. Here we describe some of the most exciting presentations and their potential impact on imminent clinical gene therapy trials.

Anti-metastatic Effects of Viral and Non-viral Mediated Nk4 Delivery to Tumours

Genetic Vaccines and Therapy. 2009  |  Pubmed ID: 19272140

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods.

Prostate Stem Cell Antigen DNA Vaccination Breaks Tolerance to Self-antigen and Inhibits Prostate Cancer Growth

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2009  |  Pubmed ID: 19337234

Prostate stem cell antigen (PSCA) is a cell surface antigen expressed in normal human prostate and over expressed in prostate cancer. Elevated levels of PSCA protein in prostate cancer correlate with increased tumor stage/grade, with androgen independence and have higher expression in bone metastases. In this study, the PSCA gene was isolated from the transgenic adenocarcinoma mouse prostate cell line (TRAMPC1), and a vaccine plasmid construct was generated. This plasmid PSCA (pmPSCA) was delivered by intramuscular electroporation (EP) and induced effective antitumor immune responses against subcutaneous TRAMPC1 tumors in male C57 BL/6 mice. The pmPSCA vaccination inhibited tumor growth, resulting in cure or prolongation in survival. Similarly, the vaccine inhibited metastases in PSCA expressing B16 F10 tumors. There was activation of Th-1 type immunity against PSCA, indicating the breaking of tolerance to a self-antigen. This immunity was tumor specific and was transferable by adoptive transfer of splenocytes. The mice remained healthy and there was no evidence of collateral autoimmune responses in normal tissues. EP-assisted delivery of the pmPSCA evoked strong specific responses and could, in neoadjuvant or adjuvant settings, provide a safe and effective immune control of prostate cancer, given that there is significant homology between human and mouse PSCA.

International Society for Cell and Gene Therapy of Cancer 2009 Annual Meeting Held in Cork, Ireland

Human Gene Therapy. Jan, 2010  |  Pubmed ID: 20017714

The International Society for Cell and Gene Therapy (ISCGT) of Cancer annual meeting was held from September 2 through September 4, 2009, in Cork, Ireland ( www.iscgt2009.com ). The conference was held in conjunction with the Irish Society for Gene and Cell Therapy third annual meeting, and brought together scientists and clinicians from around the world in a country developing its knowledge economy. Next year's ISCGT meeting will be held in Doha, the capital of Qatar ( www.iscgt.net ), from September 27 through September 29, 2010.

A Novel Listeria Monocytogenes-based DNA Delivery System for Cancer Gene Therapy

Human Gene Therapy. Apr, 2010  |  Pubmed ID: 20105075

Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

Sonoporation Mediated Immunogene Therapy of Solid Tumors

Ultrasound in Medicine & Biology. Mar, 2010  |  Pubmed ID: 20133039

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm(2) at 20% duty cycle for 5 min (60 J/cm(2)). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches.

Tumour Targeting with Systemically Administered Bacteria

Current Gene Therapy. Feb, 2010  |  Pubmed ID: 20156191

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 data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop it into a beneficial clinical tool.

Listeria Monocytogenes As a Vector for Anti-cancer Therapies

Current Gene Therapy. Feb, 2010  |  Pubmed ID: 20158470

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.

Optimised Electroporation Mediated DNA Vaccination for Treatment of Prostate Cancer

Genetic Vaccines and Therapy. 2010  |  Pubmed ID: 20181099

Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer.

Editorial [Hot Topic: Bacterial Vectors for Gene & Cell Therapy]

Current Gene Therapy. Feb, 2010  |  Pubmed ID: 20230364

Orally Administered Bifidobacteria As Vehicles for Delivery of Agents to Systemic Tumors

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jul, 2010  |  Pubmed ID: 20389288

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.

Gene Therapy for Prostate Cancer

Postgraduate Medicine. May, 2010  |  Pubmed ID: 20463426

Cancer remains a leading cause of morbidity and mortality. Despite advances in understanding, detection, and treatment, it accounts for almost one-fourth of all deaths per year in Western countries. Prostate cancer is currently the most commonly diagnosed noncutaneous cancer in men in Europe and the United States, accounting for 15% of all cancers in men. As life expectancy of individuals increases, it is expected that there will also be an increase in the incidence and mortality of prostate cancer. Prostate cancer may be inoperable at initial presentation, unresponsive to chemotherapy and radiotherapy, or recur following appropriate treatment. At the time of presentation, patients may already have metastases in their tissues. Preventing tumor recurrence requires systemic therapy; however, current modalities are limited by toxicity or lack of efficacy. For patients with such metastatic cancers, the development of alternative therapies is essential. Gene therapy is a realistic prospect for the treatment of prostate and other cancers, and involves the delivery of genetic information to the patient to facilitate the production of therapeutic proteins. Therapeutics can act directly (eg, by inducing tumor cells to produce cytotoxic agents) or indirectly by upregulating the immune system to efficiently target tumor cells or by destroying the tumor's vasculature. However, technological difficulties must be addressed before an efficient and safe gene medicine is achieved (primarily by developing a means of delivering genes to the target cells or tissue safely and efficiently). A wealth of research has been carried out over the past 20 years, involving various strategies for the treatment of prostate cancer at preclinical and clinical trial levels. The therapeutic efficacy observed with many of these approaches in patients indicates that these treatment modalities will serve as an important component of urological malignancy treatment in the clinic, either in isolation or in combination with current approaches.

Gene Therapy for Cancer: Dairy Bacteria As Delivery Vectors

Discovery Medicine. Sep, 2010  |  Pubmed ID: 20875340

The prime obstacle to achieving an effective treatment for cancer is that of eradicating tumors without harming healthy organs and cells of the patient. The concept of utilizing biological agents for delivery of therapeutic genes to patients to kill cancer cells has been under investigation for two decades, which exploits the natural ability of disease causing microbes to invade human cells. Safety-modified versions of pathogenic viruses or bacteria can deposit genes and induce production of anti-cancer agents upon administration to tumors and promising clinical trial successes have been achieved with various types of gene delivery vehicles. Bacteria present an attractive class of gene vectors, possessing a natural ability to grow specifically within tumors following intravenous (IV) injection. Several species such as Clostridium and Salmonella have been examined in clinical trials. However, as foreign, disease-causing bugs, their inherent toxicity has outweighed therapeutic responses in patients, despite efforts to reduce toxicity through genetic modification. A promising alternative exploits non-pathogenic bacterial species that have an existing natural relationship with humans. Our recent study (Cronin et al., 2010) has demonstrated that IV injection or ingestion of a species of probiotic bacterium, Bifidobacterium breve, in high numbers, results in trafficking of the bacteria throughout the body and accumulation specifically within cancerous tissue.

AAV2-mediated in Vivo Immune Gene Therapy of Solid Tumours

Genetic Vaccines and Therapy. 2010  |  Pubmed ID: 21172020

Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy.

The Use of Listeria Monocytogenes As a DNA Delivery Vector for Cancer Gene Therapy

Bioengineered Bugs. Jul-Aug, 2010  |  Pubmed ID: 21327062

Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

Bacteria As Vectors for Gene Therapy of Cancer

Bioengineered Bugs. Nov-Dec, 2010  |  Pubmed ID: 21468205

Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.

Targeting of Breast Metastases Using a Viral Gene Vector with Tumour-selective Transcription

Anticancer Research. May, 2011  |  Pubmed ID: 21617219

Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression.

Induction of Effective Antitumor Response After Mucosal Bacterial Vector Mediated DNA Vaccination with Endogenous Prostate Cancer Specific Antigen

The Journal of Urology. Aug, 2011  |  Pubmed ID: 21683415

The induction of systemic immune responses against antigenic targets that are over expressed by cancer cells represents a powerful therapeutic strategy to target metastatic cancer. We generated specific antitumor immune responses in a murine model of prostate cancer by oral administration of an attenuated strain of Salmonella typhimurium containing a plasmid coding for murine prostate stem cell antigen.

Preclinical Evaluation of Gene Delivery Methods for the Treatment of Loco-regional Disease in Breast Cancer

Experimental Biology and Medicine (Maywood, N.J.). Apr, 2011  |  Pubmed ID: 21444371

Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.

In Vivo Optical Imaging in Gene & Cell Therapy

Current Gene Therapy. Jan, 2012  |  Pubmed ID: 22263919

Integral to the development of all gene therapy technologies is the ability to monitor gene delivery, in terms of distribution, levels and kinetics of vector transgene expression. This can be achieved to some extent at the preclinical level through use of traditional ex vivo analytical methods, but these hold several drawbacks, not least the requirement for death of experimental subjects for such end-point assays. Real-time In vivo analysis of reporter gene expression empowers the investigator with the ability to non-invasively assess gene delivery over time, as well as host responses to vector administration and therapeutic interventions. While there exist several technologies for such small animal monitoring, imaging of light emission from luminescent or fluorescent reporters has become the mainstay of preclinical imaging for gene therapy research. Optical imaging strategies represent powerful yet cost-efficient and convenient systems compared with alternative methods. Through tagging of vector and/or cells or interest with suitable reporter genes, both vector and host responses can be assessed in rapid, high-throughput analyses, providing spatial, temporal and quantitative read-out, without the need for radioactivity. In this review, we discuss the current state-of-the-art for optical technologies, describe related approaches employed in gene therapy research for a wide range of diseases, and outline the potential for this imaging modality in the progression of gene therapy as a medicine.

PET Imaging For Gene & Cell Therapy

Current Gene Therapy. Jan, 2012  |  Pubmed ID: 22263921

As the interest in gene therapy increases, the development of an efficient and reliable means to monitor gene delivery and expression in patients is becoming more important. An ideal imaging modality would be non-invasive, allowing for repeated imaging, thus validating stages subsequent to vector administration and allowing for the improvement of clinical protocols. Positron Emission Tomography (PET) has been employed for some time in clinical imaging and has in more recent years been adapted to enable imaging in small animal models, including gene therapy models for a range of diseases. PET imaging is based on the detection of trace quantities of positron-emitting molecular probe within cells post-administration, permitting imaging of target molecules in vivo, and numerous tracers have been developed for a wide range of applications, including imaging of reporter gene activity. Use of radiolabelled substrates that interact with specific transgene proteins, has identified a number of reporter genes that are suitable for imaging vector mediated gene delivery and expression in both pre-clinical and clinical situations. These reporter genes enable non-invasive analysis of the location, level and kinetics of transgene activity. Among the various imaging modalities in existence, the PET approach displays arguably the optimum characteristics in terms of sensitivity and quantitation for in vivo gene expression measurements. Given the existing availability of PET scanning equipment and expertise in hospitals, this imaging modality represents the most clinically applicable means of analysing gene therapy in patients. This review outlines the principles of PET imaging in the context of gene and cell therapy at both pre-clinical and clinical levels, comparing PET with other relevant modalities, and describes the progress to date in this field.

High Resolution in Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting

PloS One. 2012  |  Pubmed ID: 22295120

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E.coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (IV) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post IV-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

Editorial: in Vivo Imaging & Gene Therapy

Current Gene Therapy. Feb, 2012  |  Pubmed ID: 22348524

Polymer Coatings for Delivery of Nucleic Acid Therapeutics

Journal of Controlled Release : Official Journal of the Controlled Release Society. Jul, 2012  |  Pubmed ID: 22366547

Gene delivery remains the greatest challenge in applying nucleic acid therapeutic for a broad range of diseases. Combining stability during the delivery phase with activation and transgene expression following arrival at the target site requires sophisticated vectors that can discriminate between cell types and respond to target-associated conditions to trigger expression. Efficient intravenous delivery is the greatest single hurdle, with synthetic vectors frequently found to be unstable in the harsh conditions of the bloodstream, and viral vectors often recognized avidly by both the innate and the adaptive immune system. Both types of vectors benefit from coating with hydrophilic polymers. Self-assembling polyelectrolyte non-viral vectors can achieve both steric and lateral stabilization following surface coating, endowing them with much improved systemic circulation properties and better access to disseminated targets; similarly viral vectors can be 'stealthed' and their physical properties modulated by surface coating. Both types of vectors may also have their tropism changed following chemical linkage of novel ligands to the polymer coating. These families of vectors go some way towards realizing the goal of efficient systemic delivery of genes and should find a range of important uses in bringing this still-emerging field to fruition.

Control and Augmentation of Long-term Plasmid Transgene Expression in Vivo in Murine Muscle Tissue and Ex Vivo in Patient Mesenchymal Tissue

Journal of Biomedicine & Biotechnology. 2012  |  Pubmed ID: 22811595

In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues.

Pre-treatment with Bifidobacterium Breve UCC2003 Modulates Citrobacter Rodentium-induced Colonic Inflammation and Organ Specificity of Infection

Microbiology (Reading, England). Aug, 2012  |  Pubmed ID: 22902730

Citrobacter rodentium, which colonizes the gut mucosa via formation of attaching and effacing (A/E) lesions, causes transmissible colonic hyperplasia. The aim of this study was to evaluate if prophylactic treatment with Bifidobacterium breve UCC2003 can improve the outcome of C. rodentium infection. Six week old Albino C57BL/6 mice were pre-treated for 3 days with B. breve, challenged with bioluminescent C. rodentium and administered B. breve or PBS-C for 8 days post infection; control mice were either administered B. breve and mock infected with PBS, or were mock treated with PBS-C and mock infected with PBS. C. rodentium colonisation was monitored by bacterial enumeration from faeces and by a combination of both 2D bioluminescence imaging (BLI) and composite 3D diffuse light imaging tomography with microCT imaging (DLIT-microCT). At day 8 post infection, colons were removed and assessed for crypt hyperplasia, histology by light microscopy, bacterial colonisation by immunofluorescence, and A/E lesion formation by electron microscopy. Prophylactic administration of B. breve did not prevent C. rodentium colonisation or A/E lesion formation. However, this treatment did alter C. rodentium distribution within the large intestine and significantly reduced colonic crypt hyperplasia at the peak of bacterial infection. These results show that B. breve could not competitively exclude C. rodentium, but reduced pathogen-induced colonic inflammation.

DNA Vaccination for Prostate Cancer, from Preclinical to Clinical Trials Where-we Stand?

Genetic Vaccines and Therapy. Oct, 2012  |  Pubmed ID: 23046944

ABSTRACT: Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.