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Development of a Novel Vector for Cancer Gene Therapy for Clinical Application

Final Report Summary - ORAL-GT-VECTOR (Development of a Novel Vector for Cancer Gene Therapy for Clinical Application)

Background: Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. Gene or cell therapies are realistic prospects for the treatment of cancer and other diseases, and involve the delivery of genetic information to a tumour to facilitate the production of therapeutic proteins. A novel strategy for targeted cancer treatment employs bacterial gene therapy.
Our recent research has shown that oral administration of Bifidobacteria to mice results in translocation from the Gastro-Intestinal Tract (GIT) with subsequent homing to and replication specifically in tumours, providing high-level gene expression confined to tumours. These findings indicate potential for treatment and/or detection of cancer via ingestion of non-pathogenic bacteria.
Project Aims: i) Scientific- This project set out to further improve this technology and validate its potential for clinical use as a safe, non-invasive, tumour-targeting vector for cancer therapeutics or diagnostic agents.
ii) Training- Further skill sets are required to progress this research. These involve availing of state-of-the art expertise in cancer gene therapy and diagnostic techniques, as well as experience in advancing preclinical research to clinical trial. Lack of activity in Ireland constrains the logical progression of the research of the applicant and his institution.
This fellowship facilitated the progression of both these aims, though enabling the candidate to spend 18 months at the University of California Los Angeles (UCLA), prior to returning to the host institution in Ireland. Prof. Noriyuki Kasahara’s laboratory (outgoing host) at UCLA is internationally renowned for cancer gene therapy research and is part of the Digestive Diseases Research Center, which specialises in GIT biology. UCLA also hosts the Crump Institute for Molecular Imaging, making this host uniquely suited to the proposed research in developing a gastrointestinal bacterial vector for cancer gene therapy and/or diagnosis. These centres are within a clinical facility with active clinical trials, in a biotechnology industry rich region.
Objectives: The scientific workpackages for the first 18 months (at UCLA) involved a) investigation of the Mechanism of Gastrointestinal Egress, b) therapeutic application of the technology in clinically relevant tumour models, and c) development of imaging technology for the bacteria. d) A further complementary training aim of the fellowship involved training of the candidate in biotech commercialisation.
The workpackages for the return 12 months (at UCC in Ireland) involved e) further mechanistic studies on the translocation phenomenon and studies on immune responses to the bacteria; f) investigating regulatory requirements for administrating the bacteria to cancer patients, and g) investigating bacterial GMP.

Results:
a) Investigation of the Mechanism of Gastrointestinal Egress:
B.breve translocation in mice has now been validated in 3 different animal vivaria (UCLA, UCC, Caliper).
Multiple cytokine analyses were performed on GIT samples from UCC2003-fed and PBS-fed athymic mice. Only IFNγ levels differed between the groups, suggesting a possible IFNγ induced increase in epithelial permeability.
Optical imaging and bacterial recovery from different regions of the murine intestine demonstrated where probiotic bacteria (B.breve or E. coli nissle) were concentrated (see figure).
Ex vivo electron microscopy of murine GIT tissue from mice +/- oral feeding of B.breve indicated no observable differences in physiology.
Overall, these assays did not elucidate the underlying mechanism of bacterial translocation, but did serve to rule out several possibilities.

b) Therapeutic application of the technology in clinically relevant tumour models: All tumour types examined were colonised by our probiotic strains examined (B16 melanoma, FaDu pharyngeal, U87 glioma, LLC Lewis lung carcinoma).
In addition to the proposed MDA-7/IL24 therapeutic delivery by B.breve (which proved only partially effective), we also investigated other therapeutics, which induced better tumour reduction, namely, the immune upregulating cytokine IL21, and the prodrug activating enzyme Nitroreductase, as delivered by either probiotic E. coli nissle or B.breve.

c) Development of imaging technology for bacteria: We originally proposed to examine PET imaging of our bacteria. Results with PET were poor, and we instead opted to develop an optical imaging strategy, which proved significantly better, for both intratumoural and GIT imaging of bacteria (see figures). Optical Imaging has recently been validated in a clinical cancer setting, and location in California permitted a new partnership with Caliper-Perkin Elmer, the world leaders in optical imaging. Several publications have followed with these industrial collaborators.

d) Training in biotech commercialisation: While in Los Angeles, facilitated by Dr. Kevin Scanlon, I engaged with the LA Tech Coast Angels, the largest angel investment organization in the U.S. attending their monthly screening sessions, where tech companies pitched for investment.

e) Further mechanistic studies on the translocation phenomenon and studies on immune responses to the bacteria:
Translocation studies - The potential effect of B. breve on gut permeability was examined using Ussing chamber studies. Sections of GIT from treated and control mice were isolated and physiological alterations in gut permeability examined in the Ussing chamber. No significant difference between mice gavaged with B. breve or with control PBS were detected, indicating that B. breve does not cause a permanent or substantial increase in GIT permeability
Immune studies - We have determined the immune responses to vector administration in mice, through measurement of multiple cytokines in peripheral blood. No dramatic increase/toxic levels was observed with any bacteria examined.
It has also been validated, by our collaborator, that the EPS encapsulation layer of B. breve UCC2003 is necessary for protection against host immune responses, validating our initial hypothesis.

f) Investigating regulatory requirements for administrating the bacteria to cancer patients: We have initiated developing a clinical study involving ingestion of probiotic bacterial suspensions, by cancer patients with renal metastases. Ethical approval applications were prepared and approval has been awarded from the Clinical Research Committee of the Cork Teaching Hospitals (CREC).

g) Investigating bacterial GMP: Rather than attempt to perform cGMP work ourselves, we explored an alternative strategy, involving direct outsourcing. We have explored partnerships with suitable bodies in Ireland and the UK.

Overall, this fellowship strengthened Europe’s collaborative links with world-leading groups, and equipped the researcher with skills for his future research.
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