DNA Vectors

We have developed a vector system, which is uniquely suited for the genetic modification of cells – it provides persistent expression and episomal maintenance without the use of potentially toxic viral components or the risk of insertional mutagenesis. Additionally, it provides unlimited capacity allowing the unrestricted development of exquisitely designed and endogenously controlled genetic vectors which can comprise entire genomic loci. We have demonstrated the utility of these vectors in vitro, ex vivo and in vivo. The use of eukaryotic chromosomal components allows the design of clinically relevant, episomally sustained replicating DNA vectors that can be used to confer persistent expression of biologically relevant or corrective genes. We were the first to demonstrate the utility of the S/MAR vector system for in vivo application and showed its ability to sustain long-term transgene expression. We also showed by removing the extraneous bacterial sequences from the vector and utilising minicircles we could improve its efficiency and reduce its toxicity. We have recently shown the utility of this vector system to provide persistent genetic modification and phenotypic correction of dividing cells in culture and for ex vivo application. Novel cell-lines can be simply prepared and can be readily utilised for cell marking studies, stem-cell differentiation and in vivo tumour modelling.

We are focused on preparing the next generation of our DNA vectors which have optimised promoters and cloning elements to facilitate the incorporation of new genes and other genetic components. We are currently applying these vectors to a range of established and new projects.

Generation of persistently expressing tumour cell-lines for the development of cancer gene therapy

In this project we are producing DNA vectors using ubiquitous and specific promoters to generate constructs, which are suitable for producing sustained expression of reporter and corrective genes in tumour cell-lines. These genetically modified cells will be used to generate xenograft models and longitudinal studies of gene expression and tumour tracking will be assessed using standard laboratory procedures as well as the utilization of a state of the art bioimaging system. They will then be utilised in the evaluation of anti-tumour genetic therapies.

Development of minimally sized DNA vectors for the genetic modification of stem-cells

We are utilising our DNA technologies to generate an S/MAR minicircle system which addresses the limitations of current nonviral vectors. These vectors will be utilised to safely and persistently genetically modify a range of stem-cells where we will evaluate their genetic integrity through cell division and differentiation. This novel genetic technology will provide improved tools not only for gene therapy but for personalised medicine, stem-cell research and transgenesis.

Utilising DNA Vectors to persistently modify cells without the risk of insertional mutagenesis or transcriptional silencing

Our research is focused on generating novel, next-generation DNA vectors for gene therapy. We have developed a vector system, which is uniquely suited for the genetic modification of cells – it provides persistent expression and episomal maintenance without the use of potentially toxic viral components or the risk of insertional mutagenesis.  Additionally, it provides unlimited capacity allowing the unrestricted development of exquisitely designed and endogenously controlled genetic vectors which can comprise entire genomic loci. We have demonstrated the utility of these vectors in vitro, ex vivo and in vivo. The use of eukaryotic chromosomal components which exploit the interdependence of transcription and replication allows the design of clinically relevant, episomally sustained replicating DNA vectors that can be used to confer persistent expression of biologically relevant or corrective genes.

 

Prototype gene expression plasmid vector

We were the first group to demonstrate the utility of the S/MAR vector system for in vivo application and demonstrated its ability to sustain long-term transgene expression in the livers of mice from a single administration.We also showed by removing the extraneous bacterial sequences from the vector and utilising minicircles we could improve its efficiency and reduce its immunotoxicity. Additionally, we demonstrated for the first time that by generating a version of our vector which contained an in vivo selection marker we could stimulate the episomal replication of S/MAR transgenic DNA in vivo. A great advantage of this vector system is that it can be readily adapted for use in other tissues with the use of specific or ubiquitous promoters.

 

Permanent genetic modification of replicating cells without integration

We have also recently shown the utility of our episomal vectors for the persistent genetic modification and phenotypic correction of dividing cells in culture and for ex vivo application. Stable, genetically modified novel cell-lines can be prepared simply using our vector system and can be readily utilised for cell marking studies, stem-cell differentiation and in vivo tumour modelling.
 

We are currently focused on preparing the next generation of our DNA vectors which have optimised promoters and cloning elements to facilitate the incorporation of new genes and other genetic components. We are also applying these vectors to a range of established and new projects.

 

Generation of persistently expressing tumour cell-lines for the development of cancer gene therapy

In this project we will produce DNA vectors using ubiquitous and specific promoters to generate constructs, which are suitable for producing sustained expression of reporter and corrective genes in tumour cell-lines.  After establishment of the vector tumours will be induced in mice using these cells and longitudinal studies of gene expression and tumour tracking will be assessed using standard laboratory procedures as well as the utilization of a state of the art bioimaging system. 
The advantages of developing a genetic marker within the tumour are that their size and development might more easily be tracked and the efficacy of therapeutic interventions such as gene or cell therapy protocols might be more easily monitored. We will utilise these modified cells as a tool to evaluate a range of anti-tumour genetic therapies developed by collaborators in the UK. These novel gene-silencing molecules will be formulated into nanoparticles and delivered into xenograft tumour models and their therapeutic efficacy evaluated. 
Additionally we will also generate bi-cistronic DNA vectors which permanently correct the genetic of tumour cells. These novel cell lines will provide us with a unique tool to investigate the mechanisms of tumorigenesis and a greater insight into the phenotypic correction of cancer cells.

 

Development of minimally sized DNA vectors for the genetic modification of stem-cells

Despite considerable progress over the last 10 years, the development of safer, more efficient and persistently-expressing genetic vectors remains one of the main strategic tasks of gene therapy research and is the crucial prerequisite for its successful clinical application. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production and great versatility. They do have limitations namely: the transient nature of gene expression and the consequences of the immunotoxicity of bacterial DNA which is mainly due to the presence of extraneous motifs in the plasmid vector. 
We have developed a novel non-viral episomally replicating DNA vector, which provides persistent transgene expression without the limitation of insertional mutagenesis. 
We have also developed a novel minicircle-vector system in which the bacterial moiety of a production-plasmid is eliminated in the minicircle-producing bacterial strain. The reduction of the vector to the core sequences required for mammalian expression has several advantages; the amount of potentially immunotoxic sequences are depleted also the number of therapeutically functional sequences per milligram of DNA is increased, thus enhancing transgene expression after transfer into a target cell. 
The aim of this project is to combine these two technologies and build a minicircle system to generate a novel vector, which addresses the limitations of current non-viral vectors. These next-generation vectors will be utilised to safely and persistently genetically modify a range of stem cells where we will evaluate their genetic integrity through cell division and differentiation. This novel genetic technology will provide improved tools not only for gene therapy but for personalised medicine, stem-cell research and transgenesis.

 

Utilising DNA Vectors to Develop a Prophylactic Gene Therapy for the Kidney Cancer Caused by Birt-Hogg-Dubé Syndrome

The aim of this project is to apply these vectors for the preventative treatment of BHD. 
BHD syndrome predisposes patients to second-hit mutations in the BHD gene - folliculin and they develop hair follicle hamartomas, lung cysts, and an increased risk for renal neoplasia which leads to bilateral, multifocal renal tumors. 

Much is yet to be learnt about the molecular mechanisms of this disease and although great breakthroughs have recently been made in the identification of its genetic cause the only current treatment for the renal tumours is repetitive surgical intervention. Unfortunately, even with a complete resection of the tumour the risk of spontaneous oncogenesis in the remaining renal tissue is unremitting. 
We propose to utilize the current knowledge of the genetic cause of the disease and our expertise in renal pathology and gene delivery vector design to develop a prophylactic genetic therapy for BHD. 
We have recently demonstrated that the cancer phenotype can be permanently corrected in cells isolated from a patient's tumour. Our hypothesis is that if a protective, mutation-proof copy of folliculin could be introduced into the renal cells of BHD patients these cells would become resistant to BHD-associated renal neoplasia. 
 

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