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Doggybone™ DNA: an advanced platform for AAV production

Scale-Out and -Up Strategies

Kinga Karbowniczek, Paul Rothwell, Jon Extance, Sarah Milsom, Vera Lukashchuk, Kevin Bowes, Daniel Smith & Lisa Caproni

Recombinant adeno-associated virus (AAV) represents one of the most promising delivery vehicles for genetic medicines. However, the manufacture of plasmid DNA for the production of AAV presents a number of significant challenges, including scalability, fidelity, mis-incorporation of plasmid-derived DNA sequences, high costs and long lead times for GMP production. Touchlight has developed a novel, rapid, in vitro, enzymatic technology for multi-gram scale GMP manufacture of DNA that addresses all of the issues of DNA manufacture for AAV production. The process combines the use of two enzymes; Phi29 DNA polymerase and a protelomerase to generate covalently closed, linear DNA constructs known as doggybone™ DNA or dbDNA™. The process is rapid, cost effective, of high fidelity and eliminates antibiotic resistance genes. Here we present a case study, with data generated by Cobra Biologics as part of an on-going Innovate UK-funded collaboration, demonstrating that AAV particles can be produced using dbDNA™ with total and genomic titres equivalent to AAV particles made with plasmid DNA. In parallel experiments, with an academic collaborator, we have seen in vivo expression of reporter genes after administration of AAV vectors manufactured using dbDNA™ (unpublished data). As such, we believe that dbDNA™ has the potential to resolve a number of significant challenges in the production of AAV vectors at both clinical and commercial scale.

DOI: 10.18609/cgti.2017.074
Submitted for review: August 29, 2017
Citation: Cell Gene Therapy Insights 2017; 3(9), 731-738.
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