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Gene therapy: an integrated approach for neoplastic treatment

Final Report Summary - GIANT (Gene therapy: an integrated approach for neoplastic treatment)

The ultimate aim of the GIANT project was to provide an international resource to apply innovative technologies for modification of existing gene therapy vectors, focusing on increased targeting and decreased vector immunogenicity by 'stealthing'. The project would address the current shortcomings in gene therapy for human prostate cancer by developing and testing vectors both in a pre-clinical and clinical setting. This translational action would serve as a template for the development of innovative treatments based on gene therapy, which could be applied in generic terms to other tumour types, using appropriate, tumour-specific targeting. The overall objective of GIANT was the production of an optimally re-targeted gene transfer vector with minimal immunogenicity that would be both clinically tested and ethically approved for prostate cancer therapy.

The technology for genetic targeting of adenovirus vectors to prostate cancer cells was used to generate several vectors for pre-clinical evaluation. New tumour-specific vectors with enhanced potency or specificity were generated and evaluated. In parallel, approaches were implemented for developing new test systems for functional characterisation of these viruses. This yielded the development and implementation of new technologies that may reduce and replace animal experimentation, e.g. a system for growth of human tumours in chicken eggs, and the extraction of antigen-specific immunoglobulins from commercially available human immunoglobulin preparations. The development of chemically detargeted and re-targeted adenoviral vectors focused on overcoming the barriers for vector delivery with the ultimate goal to achieve efficient in vivo vector delivery. Strategies for polymer coating of adenovirus in combination with ligand-mediated targeting were further developed and optimised. An example was the Bungarotoxin system that proved to be a flexible way of targeting and re-targeting of vectors. For the development of binders against novel prostate cancer-specific antigens, several strategies were developed for the incorporation of single-chain variable fragment (scFv) and ingle-chain T-cell receptor (scTCR), including camelised antibodies.

Vectors were made bio-responsive via coating with endosomal pH responsive or thermoresponsive polymers. Specificity was added to these vectors by introducing prostate cancer specific binders to the coating. GIANT project placed a heavier workload on the partners whose main task was to test vectors generated in the project in clinically relevant models. Comparisons of the ability of GIANT vectors to transduce two-dimensional (2D) and three-dimensional (3D) cellular models of prostate cancer revealed a number of significant differences between different cell types, but most significantly, confirmed the problems encountered in the mice models with PC346C of the ability of various vector types to diffuse attach and penetrate complex structures. Such difficulties were not experienced with 2D monolayer cultures and this phenomenon was not related directly to receptor expression levels on complex tissues. Rather, the bioavailability of receptors was critical, in relation to injection points and adhesion contacts between cells. The second main testing task was to produce critical pre-clinical data for the clinical trial safety dossier. The consortium decided to carry out pre-clinical testing in a range of relevant primary human cell cultures, grown from tissues, which were likely to be infected at detectable levels in a clinical setting. The results of this dossier were collated for presentation and reveal the exquisite specificity of the GIANT virus Ad(I/PPT-E1A). Even in human hepatocytes, which are extremely sensitive to infection by AdWT, there was no evidence of viral replication and toxicity at viral multiplicities of infection less than 500 viral particles / cell, a multiplicity which would only rarely be achieved in patients' livers.