New Publication - Not All Transgene Sequences Are Equal

Friday, April 8th 2016

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Biomaterials.jpgTransgene sequences free of CG dinucleotides lead to high level, long-term expression in the lung independent of plasmid backbone design. Biomaterials (2016), In Press.

Blog Post:- Minicircles V Plasmids. Time to think again.

This new publication by Bazzani, Pringle et al looks at different plasmid vector designs to try an understand what factors are important and what are not. It follows on from a series of other papers that we have produced demonstrating that plasmids completely free of  CpG dinucleotides result in long term expression in our model systems.

Such a vector, pGM169 became the basis for our MD clinical trial that was published last year. Making plasmids completely free of a very common DNA sequence is a challenge. So in this study we broke down the plasmid into different parts to try and see if the entire sequence had to be 'CpG-free' or not. 

We found, that in the context of vectors based on our clinical trial plasmid, the most important element was the actual transgene sequence itself. It does not appear to mater what design or sequence composition is used for the plasmid backbone sequence, long term expression of the transgene is dependent on this sequence being CpG-free. Whenever we put conventional CpG containing transgenes into our plasmids, the duration of expression was very poor.

pGM169_pGM292_Small.jpg 

We looked at the duration of expression from plasmids with CpG-free transgenes V plasmids with normal CpG rich transgenes (View in new tab ).

Similarly, other researchers have found that minicircle plasmids with the entire backbone sequence removed, perform better to conventional plasmids. Here we found this not to be the case and minicircles (which are much more expensive to produce) do not appear to have any advantage over our conventional plasmid designs.

This work is interesting because people would not assume that short transgene sequences could have such an impact on the control of gene expression which is generally controlled by the promoter sequences in the plasmid. However, we believe the mechanism is due to methlyation of CpG sequences in the transgene that lead to its suppression. We don't have any direct evidence of this yet as its incredibly difficult to detect, and infact we have looked for it in the past and found no evidence of this following gene transfer (Pringle et al 2005).

pGM169_pGM292_Graph_Small.jpg

The duration of expression from plasmids with a CpG-Free transgene was always superior to that of a normal transgene, regardless of backbone design (View in new tab ).

 

At next month's ASGCT Conference Prof Deborah Gill will present a talk on this work:- Minicircles Are Similar To Plasmids In Providing High Level, Long-Term Expression In The Lung

Further Reading.


 

Human airway liquid interface cultures transduced with a lentivirus expressing Luciferase.

 

E.coli from a large scale industrial production of our clinical trial plasmid pGM169.

 

A cake that only some of us got to enjoy!

 

Light microscope image of a human airway liquid interface cultures. Dark patches are mucous.

 

DNA fragments being cut from an agarose gel exposed to UV.

 

Pellets of DNA following precipitation.

 

Schematic diagram of the large human airways.