Nonviral Vector Development

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

 

Plasmids are circular double stranded DNA molecules that have been manipulated by molecular biologists to study interesting pieces of DNA from a wide variety of sources.  Plasmid molecules are available to meet a wide range of laboratory needs.

Whether the plasmid is required for a simple experiment, or if it is intended for use in a complex clinical trial, the process always starts by introducing the plasmid into laboratory strains of the well-studied bacteria E. coli.  Once inside the bacterium, the plasmid DNA is replicated by the bacterial cellular machinery.

Molecular biologists use specialised enzymes (called restriction endonucleases) that cut plasmid molecules at specific DNA sequences like a pair of scissors to create DNA fragments.  These DNA fragments can be readily visualised and purified on gels.

DNA fragments can be joined using further specialist enzymes (called DNA ligases) which act like molecular glue.  By using these DNA scissors and glue we have created a range of plasmid DNA molecules comprised of interchangeable therapeutic genes, specialist signals to ensure their efficient expression in mammalian cells and alternative antibiotic resistance elements.  These approaches have allowed our group to optimise the necessary elements within our plasmids so they are potent and suitable for clinical use.

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

E.coli streaked on an agar plate. Plasmid DNA molecules confer antibiotic resistance allowing us to select bacteria that contain the plasmids and grow them under simple laboratory conditions

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 (DNA sequences that instruct lung cells to produce therapeutic transgenes such as CFTR) that function for years rather than days;  and have reduced the toxicity of our plasmids by removing elements which cause inflammation in human cells.

Together these approaches led to the development of our current patent protected clinical trial plasmid, pGM169.

Restriction endonuclease analysis of plasmid DNA

Restriction endonuclease analysis of plasmid DNA separated on a DNA gel