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Technologies for Gene Therapy

Our vision is to build upon and develop strengths primarily in the pre-clinical and underpinning resources areas with a view to develop gene therapy research outputs for translation within 5-10 years. In addition, we have existing strengths particularly in the areas of gene delivery using the most recent viral and non-viral vectors that have been demonstrated to be effective in achieving targeted and permanent or transient gene transfer. Importantly, to support our expertise in vector development and production, we have developed specific skills in vector safety and have exploited this by developing a robust genotoxicity platform with a solid tumour phenotype, which is an area of active research at Brunel. This work has benefitted enormously from our collaboration with Dr Manfred Schmidt at the Division of Oncology DKFZ, Heidelburg, who support our safe vector insertion site (IS) research and recent MRC funding.

The area that we are now developing is the expansion of our viral vector-based repertoire and expertise to include gene therapy studies that utilise adeno-associated virus (AAV) serotypes, herpes simplex virus type 1 (HSV-1), adenovirus (Ad) along with our current lentiviral systems (LV).

Ad and LVs.

These vectors are currently in use in our laboratories. Ad systems have been developed with the Brunel Institute of Bioengineering in order to produce quality vector preparations with aqueous two phase separation (ATPS) technology. We have identified several ATPS (PEG/Potassium phosphate combinations) that already generate high titre Ad preps avoiding expensive CsCl gradient ultracentrifugation. This technology is being scaled up using suspension culture cell production systems for the generation of oncolytic vectors. This will be in collaboration with the Institute of Cancer at Barts and the London, Queen Mary’s School of Medicine and Dentistry.

We have screened LV systems for efficient delivery of therapeutic genes to specific target tissues in vivo. We also aim to use the recently developed avian leucosis sarcoma virus (ALSV) because of the good safety integration profile of this LV.

To validate our vector preparations and validate our LV backbones for purity and to continue our research into the potential side effects of LV components such as VSV-G glycoproteins and the LV integrase on host cells, we have initiated a collaboration with Yuan Zhao at the National Institute for Biological Standards and Control (NIBSC).

AAV

We recognize that AAV vectors are becoming highly important to achieve widespread gene transfer. Serotypes of AAV are highly effective at targeting neuronal tissues and we plan to use these to correct diseases involving triplet repeat expansions, such as fragile X syndrome with Gus Alusi at the Institute of Cancer at Barts and the London, Queen Mary’s School of Medicine and Dentistry. We also plan to develop new state-of-the-art vectors with novel promoters (such as synapsin-1 for highly neuronal specific expression), novel production methods, and innovative genotoxicity testing that will enhance the safety and efficacy of future gene therapies.

HSV-1

We have recently initiated collaboration with Dr Fillip Lim at the Department of Molecular Biology, University of Madrid, Spain. Together we plan to generate neuron targeting HSV-1 carrying reporter and frataxin genes for the correction of Friedreich ataxia. This vector will also be validated for the correction of fragile X syndrome.

Non-virus vectors

Our non-virus vector research has entered a new phase where we are producing virus components from yeast. This work is enabling the study of these components to understand the mechanisms of virus infection at Brunel.

Vector Genotoxicity Studies (Themis)

We have developed a highly sensitive model of LV mediated reverse genetics that also reproducibly develops hepatocellular carcinoma (HCC) in mice at high frequency with virtually zero HCC background. Using this model, EIAV and FIV LVs induce HCC at frequencies of 80%-40%, respectively whereas HIV-1 LV does not. Our model capitalises on using the property of LV preference for actively transcribed genes. In this model LV delivery is to fetal or neonatal mice where genes that are transcribed are involved in proliferation, differentiation and growth and considered cancer genes if active after birth. Thus far, we have identified several LV IS RefSeq genes in clonal tumours that we believe are involved or associated with liver cancer. We have also found that all the genes we have identified are controlled by known microRNAs involved in HCC. This powerful reverse genetic approach will identify additional genes involved in HCC and test LV safety.

Most recently we have develop a novel humanized model system to develop vectors that show vector safety with the absence of genotoxic side effects. This work is supported by Innovate UK MRC NC3rs funding along with Brunel pilot project support funds.

Gene Therapy of Friedreich Ataxia Mouse models (Themis and Pook)

Friedreich ataxia (FRDA) is an inherited progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia (DRG) and the cerebellum. The disease is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron-sulphur cluster (ISC) biogenesis and low frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species (ROS) and sensitivity to oxidative stress.

We have a good expertise in the use of cell and mouse models for the study of FRDA and we have a keen interest in targeting peripheral sensory neurons. With recent funding from Ataxia UK, FARA US and FARA Australasia, we have shown that LV-meditated frataxin gene delivery reverses genome instability in FRDA patient and mouse model fibroblasts. Ataxia UK has recently provided support to show gene therapy in the FRDA mouse model.