A spotlight on plasmid and minicircle DNA starting materials for AAV vectors
Join our lunch symposium on Wednesday, Oct 23rd, 1:30 p.m., Meeting Room 1 about challenges and
opportunities in AAV-based Gene Therapy and get insights from our experienced speakers.
Organized by PlasmidFactory GmbH
For AAV vector production, a broad range of developments is reported in vector design, cell culture conditions and chromatographic purifications. Many of these important breakthroughs have successfully pushed the volumetric yield as well vector product quality in the last few years. However, due attention is often not paid to the starting materials involved in any AAV vector production – the plasmid DNA carrying ITR-flanked cargo, the packaging genes and the adenoviral helper genes. The specific design of these constructs can have direct consequences to the AAV vector product. Based on the philosophy that good products can only come from good materials, a high titer and well-defined quality of recombinant AAV vectors would require correspondingly well-defined high quality starting material in the form of plasmid or minicircle DNA. In this symposium, we would like to draw attention to certain important features that one would want to look at when working with those humble and often overlooked molecules where it all starts from – the plasmid DNA.
Firstly, the conventional triple transfection protocol for AAV production has been a hallmark scientific development that formed the basis of realizing the goal of engineering the wild type virus into a recombinant vector particle. Reducing the number of plasmid constructs required for an industrial AAV vector production offers advantages in process optimization, development as well as reducing manufacturing costs involved and thus offers to considerably bring down the price of the final gene therapy product. The recent trend has been to combine features in any of the different combinations possible. One of the earliest developments in this direction came from German Cancer Research Centre through the creation of the pDG series of plasmids. This led to the development of a 2-plasmid system for AAV production which has been successfully used by researchers around the world for several years. Scientists at Reithera in Italy have used this system and followed a rational optimization plan for their conditions and will be reporting their findings during this lunch symposium.
Top quality dishes require top quality ingredients: Plasmid & minicircle DNA for your AAV needs!
The 2-plasmid system consists of a transfer vector carrying the ITR sequences which flank the gene cargo. The cargo could either be a reporter gene or a therapeutic gene for the intended application. The second plasmid is a Helper and Packaging plasmid containing the rep and cap genes as well as the adenoviral helper genes. Further important aspects to discuss include the possibility to deliver large quantities in High Quality Grade and as in-stock (off the shelf) products which would greatly ease manufacturing challenges during vector production. The use of HQ plasmid DNA allows for the GMP manufacturing of AAV vector products up to the early-stage clinical trial phases.
One major drawback in using plasmid DNA as a transfer vector is the issue of retropackaging of plasmid backbone sequences into a fraction of the recombinant AAV vectors. Significant results were first reported from workers at University of Cologne, wherein the value of using minicircle DNA devoid of prokaryotic backbone sequences became evident in that the resulting AAV vector preparations were essentially free of any prokaryotic DNA contamination. This represented a tremendous leap in AAV vector quality and sets the stage for future vector preparations suitable for clinical grade materials.
Finally, we examine the Inverted Terminal Repeats of AAV in greater detail. This crucial genetic structure needs to be faithfully supplied in the plasmid template to be reliably built into the rAAV genome. Failure to do so results in sub-optimal AAV yields and packaging efficiency, show researchers at the Bielefeld University. These elements are challenging to propagate in prokaryotic hosts by their inherent complex repeat structure, often leading to truncated versions co-existing with a dwindling population of original length molecules. Recent advances have made it possible to dependably maintain the ITR length in the plasmid transfer vector and thus give rise to better performing AAV vectors containing full, wild-type length ITRs.
Visitors to this symposium are expected to benefit from a confluence of ideas and people surrounding the AAV vector manufacturing space, including both academia and industry. With all eyes on vector manufacturers to match up and deliver the promise of affordable gene therapy, this event placed rightfully during the ESGCT Annual Congress is expected to provoke new ideas and inspire critical thinking on these lines that will shape the next round of AAV vector manufacturing advances.
Chairs:
Dr. Eduard Ayuso, DINAMIQS, Schlieren, Switzerland
Dr. Marco Schmeer, PlasmidFactory GmbH, Bielefeld, Germany
Programme:
Dr. Marco Schmeer, Head of Project Management, PlasmidFactory GmbH, Germany
Welcome address
Prof. Dr. Hildegard Büning, MHH, Hannover, Germany
Improving the quality of AAV vector preparations by exchanging plasmids by minicircles
Dr. Michele Gentile, ReiThera srl, Rome, Italy
Optimization of the Two-Plasmid System for AAV Production
Prof. Dr. Kristian Müller, Bielefeld University, Germany
A Hitchhiker‘s Guide to ITR Stability
Dr. Ram Shankar, Manager R&D, PlasmidFactory GmbH, Germany
ITR maintenance and in-depth analysis for AAV transfer plasmids
Questions and discussion
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