The UK Cystic Fibrosis Gene Therapy Consortium: Nonviral Vector Development

Nonviral Vector Development

E.coli enterobacteria streaked on an agar plate. Plasmid DNA molecules confer antibiotic resistance to the bugs allowing us to grow them in this simple manner.

Plasmid DNA molecules are one of the most widely used tools in molecular biology research. They are derived from naturally occurring molecules in microbes.

These circular double stranded DNA molecules have been manipulated by molecular biologists to allow for the propogation and study of interesting pieces of DNA from a wide variety of sources. Plasmid molecules are available to meet a wide range of needs.

Whether the plasmid is required for a small piece of work, or if it is intended for future clinical use, the process always starts by transforming the molecule into E. coli so it can be replicated by the microbial machinery.

Plasmid molecules purifed from the bacteria is identified by restriction endonuclease analysis and visualised via agarose electrophoresis.

Plasmid molecules purifed from the bacteria is identified by restriction endonuclease analysis and visualised via agarose gel electrophoresis.

Restriction endonuclease enzymes are used cut up DNA and individual fragments can be readily visualised and purified on gels. This has allowed us to create a series of modular plasmid molecules comprised of interchangeable bacterial bacbones, promoter/enhancer regions and transgenes.

This has allowed our group to optimise the elements within our plasmids so they are suitable for clinical use.

A map of our clinical trial plasmid pGM169 (G4 hCEFI soCFTR2).

Over the past few years we have put tremendous effort into improving the design of our plasmid DNA molecules to make them more suitable for clinical studies.

We have developed a range of promoter elements that direct long-term expression in-vivo and have reduced the toxicity of the plasmid by removing elements which cause inflammation in human cells.

This process culminated in the development of our current clinical trial plasmid, pGM169.