Research Group DNA Vectors

Dr. Richard Harbottle

Schematic depiction of the episomal replication and maintenance of a scaffold matrix attachment region (S/MAR) DNA vector associated with Chromatin Looped Domains (CLD). SAF-A: Scaffold attachment factor protein A; ?: Auxiliary transcription/ replication proteins.
© dkfz.de

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 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.

Contact

Dr. Richard Harbottle
DNA Vectors (F160)
Deutsches Krebsforschungszentrum
Im Neuenheimer Feld 280
69120 Heidelberg
Tel: +49 6221 424978

Selected Publications

  • Wong S.P. and Harbottle R.P. (2013) Genetic modification of dividing cells using episomally maintained S/MAR DNA vectors Molecular Therapy Nucleic Acids 2, e115; doi:10.1038/mtna.2013.40
  • Argyros O., Wong S.P., Gowers K. and Harbottle R.P. (2012) Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modeling. PLoS ONE 7(10): e47920. doi:10.1371/journal.pone.0047920
  • Wong S.P., Argyros O., Howe S.J. and Harbottle R.P. (2011) Systemic gene transfer of non-viral plasmids to neonatal mice Journal of Controlled Release Mar 30;150(3):298-306
  • Argyros O., Wong S.P., Constantinos F., Tolmachov O., Waddington S.N., Howe S.J., Niceta M., Coutelle C. and Harbottle R.P. (2011) Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver Journal of Molecular Medicine Journal of molecular medicine (Berlin, Germany)
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