Nonviral Vector Development

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Overview of plasmid cloning

Introduction

Due to vast array of tools and protocols that have been developed over the past 40+ years DNA is surprisingly easy to manipulate in the lab.

Our basic goal in vector development is to take functional pieces of DNA and combine them together to produce a DNA molecule that can express a gene independently of the host cell genome.

We do this by combining elements which drive the expression of genes (promoters and enhancer) with gene sequences (CFTR or reporter genes). Within this simple challenge there is almost unlimited cope for optimisation and development.

However on its own these expression cassettes are useless unless they are combined with elements that allow there production in E. coli so all plasmids have a backbone sequence comprised of an antiobiotic resistance marker and an origin of replication which allows for replication in E. coli. The finished circular piece of DNA is called a plasmid.

E. coli is used because its cheap to grow in culture, divides rapidly and purifying DNA from the bugs is relatively easy.

 

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.
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.

Manipulating plasmid DNA

Plasmid DNA molecules are one of the most widely used tools in molecular biology research.

Derived from naturally occurring molecules in microbes, these circular double stranded DNA molecules have been manipulated by molecular biologist to allow for the propogation and study of interesting pieces of DNA from a wide variety of sources and 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.

 

Purification of plasmid DNA


Lysis of an E.coli culture (visualised by blue-white colour change) to allow purification of plasmid DNA.

E.coli colonies like those pictured above are selected and inoculated into growth media which allows them to rapidly divide and form a dense culture or 'broth'. At the lab scale this allows us to make several miligrams of plasmid DNA from 400 ml cultures at a time using commercially available kits. The purified plasmid DNA can then undergoes some QA/QC checks (below) and can be tested in a wide range of models. For clinical DNA production we have worked with VGXi in Houston to massively scale up the quality and scale of production so we can manufacture >50g of plasmid ready for use in human studies.

 

 

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 electrophoresis.

Restriction endonuclease enzymes are used cut up DNA and individual fragments can be readily visualised and purified on agarose gels. This has allowed us to create a modular series of plasmid molecules comprised of interchangeable bacterial backbones, 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).
A map of our clinical trial plasmid pGM169 (G4 hCEFI soCFTR2).

Optimisation of plasmid DNA

Over the past few years we have put tremendous effort into improving the design of our plasmid DNA molecules to make the 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 (Hyde et al 2008).

 

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

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