Future Network Talks #21

Tools for gene therapy and genetic vaccines made in Germany: Plasmid and Minicircle DNA

Minicircles are plasmid derivatives engineered to be devoid of bacterial sequence elements such antibiotic resistance genes or replication origin. In this way, they represent a meaningful adaptation of the idea of a gene delivery vehicle – without the problematic and unnecessary sequence elements. Apart from the reduction in size compared to the original plasmid, the trimming away of excess sequence enables the molecule to better evade host immune responses ultimately being better poised to circumvent gene silencing scenarios post-transfection. Moreover, with the removal of the resistance genes, these constructs are a safe bet from a regulatory standpoint going forward to the next generation of gene therapies.

Thanks to the reduction in size, for example, the amount of DNA required for nucleofecting T-cells can be reduced. Our collaborators used a non-viral sleeping beauty-based transposition to generate CAR-T cells whereby a higher transposition rate was noticed while using minicircles as compared to plasmids. The reduced DNA toxicity experienced by the cells contributed ultimately to a higher yield of CAR-modified T cells. These minicircles were produced in High Quality grade allowing for the GMP production of such CAR T cells which have actually entered the clinic.

When used for AAV production, minicircles offer certain unique advantages. The absence of undesired backbone sequences removes the possibility of these sequences being falsely packaged into the transfer vectors. In one of the studies by our collaborators, a head-to-head comparison with the use of plasmids revealed up to a four-fold increase in vector genome yield.

Minicircles are versatile in their range of applications. Recently, our collaborators working on Parkinson disease models reported that minicircle constructs coding for short hairpin RNA and packaged into extracellular vesicles could effectively downregulate alpha synuclein expression in the brain, intestine and spinal cord thus paving the way for future strategies to halt disease progression at early stages.