Technology

The novel "RBPS-IVR Technology" is based on the coordinated consecutive steps of in vivo recombination, in vivo restriction and the subsequent chromatographic purification to remove residual impurities. The inducible sequence specific recombination process results in mc-DNA containing the gene of interest for the desired applications and miniplasmids carrying the unwanted bacterial backbone sequences. Triggered by this recombination based minicircle-DNA production step the expression of an endonuclease is initiated. The restriction sites of this endonuclease are located on the parental plasmid and - after recombination/minicircle production - on the miniplasmid. Upon expression of the endonuclease, plasmids carrying the corresponding sites are linearised and subsequently further degraded by host exonucleases. Therefore, after recombination and in vivo restriction/degradation the host cell harbours almost exclusively minicircle-DNA. Remaining trace amounts of residual parental plasmids can subsequently be removed by routine DNA chromatography techniques optimised for minicircle production.


Developmental status

The RBPS-Technology has been successfully tested and scaled up using GMP compliant high cell density fermentation and chromatographic purification processes established in a common project with
BIASeparations, Eurogentec and the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology.


 
  Minicircle-DNA carrying a GFP expression cassette:
Standard miniprep of GFP mc-DNA produced at lab scale
lane 1: Parental Plasmid digested with XhoI
lane 2: GFP mc-DNA digested with XhoI
lane 3: GFP mc-DNA
 
Transfection of C2C12 cells with GFP mc-DNA shows prolonged fluorescence:
(Courtesy of Ludwig Boltzmann Institute for Experimental and Clinical Traumatology)
The mouse myoblast cell line C2C12 was transfected with either mc-DNA or a conventional plasmid both carrying the same GFP expression cassette under the control of the CMV promoter.
A: C2C12 cells transfected with GFP mc-DNA 21 days after transfection
B: C2C12 cells transfected with a conventional GFP plasmid 21 days after transfection
 
 
  Transfection of C2C12 cells with luciferase mc-DNA shows in-creased enzyme activity:
(Courtesy of Ludwig Boltzmann Institute for Experimental and Clinical Traumatology)
The mouse myoblast cell line C2C12 was transfected with either 1µg mc-DNA or a conventional plasmid both carrying the same luciferase expression cassette under the control of the CMV promoter.