Sara A. Collins

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

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.

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

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.

PET imaging for gene & cell therapy.

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 postadministration, 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.

Gene therapy for prostate cancer.

Abstract 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 tumors 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.

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours

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 metasasis 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.

AAV2-mediated in vivo immune gene therapy of solid tumours

BackgroundMany 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.MethodsImmune-competent Balb/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals.ResultsAAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy.ConclusionsOverall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour vasculature and immune cell recruitment.

Control and Augmentation of Long-Term Plasmid Transgene Expression In Vivo in Murine Muscle Tissue and Ex Vivo in Patient MesenchymalTissue

Purpose. 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. Methods. Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. Results. In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. Conclusions. Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

Comparison of DNA Delivery and Expression Using Frequently Used Delivery Methods

Sara A. Collins1,2, David Morrissey1, Simon Rajendran1, Garrett Casey1, Martina F. Scallan2, Patrick T. Harrison3, Gerald C. O’Sullivan1 and Mark Tangney1 1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, Cork, 2Department of Microbiology, University College Cork, Cork, 3Department of Physiology and Biosciences Institute, University College Cork, Cork, Ireland

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

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.

Abstract B03: Development of retroviral replicating vectors expressing codon-optimized nitroreductase for prodrug activator gene therapy in human glioma models

Our studies to date have demonstrated dramatic survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for gene therapy in a variety of preclinical cancer models. RRV-mediated prodrug activator gene therapy using yeast cytosine deaminase (RRV-CD; “Toca 511”) has been evaluated in multi-center Phase I ascending dose trials in patients with recurrent high grade glioma (http://www. clinicaltrials.gov: NCT01156584, NCT01985256, NCT01470794), and based on highly promising evidence of therapeutic benefit, a registrational Phase IIB/III trial has recently been initiated at multiple sites in the United States and Canada (NCT02414165). Translational development of further RRV-based therapeutic agents is also on-going, and we have now developed an RRV encoding E.coli nitroreductase (NTR), a prodrug activator enzyme which converts CB1954 to a potent bifunctional alkylating agent. We constructed RRV encoding wild-type E.coli NTR genes (RRV-NfsA, RRV-NfsB) as well as NTR variants extensively modified to optimize human codon usage and vector stability (RRV-NAO, RRV-NBO). NTR transgene insertion did not affect vector replication, which resulted in increasing NTR expression over time in U87 human glioma cultures for all vectors, but sequence optimization significantly increased genomic stability of the RRV-NAO and RRV-NBO vectors over serial passage. U87 cells fully transduced with the optimized vectors showed higher levels of NTR protein and increased levels of enzymatic activity compared with cells transduced with wild-type vectors. In vitro cytotoxicity was examined by MTS assay after CB1954 treatment of fully transduced U87 cells. Viability was reduced by >80% within 48 hrs in cells transduced with RRV-NAO, which showed the most potent cell killing efficiency and bystander effect among all vectors tested. Significant reduction in luminescence and inhibition of tumor growth was observed in subcutaneous U87-FLuc2 tumors initiated with 2% RRV-NAO transduction followed by intraperitoneal administration of CB1954. In intracerebral U87-FLuc2 orthotopic tumor models, stereotactic intratumoral injection of RRV-NAO and repeated cycles of prodrug treatment also resulted in significant luminescence reduction, and achieved prolonged survival benefit. These results indicate that we have been successful in developing an improved prodrug activator gene with therapeutic efficacy when delivered by RRV in experimental models of human glioma. Further studies are aimed at translational development of improved RRV-NTR vectors for future clinical use both as a stand-alone therapy and in combination with RRV-CD (Toca 511). Citation Format: Sara Collins, Akihito Inagaki, Mark Tangney, Noriyuki Kasahara. Development of retroviral replicating vectors expressing codon-optimized nitroreductase for prodrug activator gene therapy in human glioma models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B03.