A6 - AAV Vectors - Product Development Manufacturing and Approval Considerations
1067: A Novel, Synthetic DNA Alternative for rAAV Manufacturing
Type: Poster Session
Poster Board Number: 1067
Presentation Details
Session Title: Thursday Posters: AAV Vectors - Product Development Manufacturing and Approval Considerations
The number of FDA-approved gene therapy products using recombinant adeno-associated virus (rAAV) has grown rapidly in the last few years and highlights the key role this vector will play in future gene therapies. However, current manufacturing constraints pose challenges in meeting the increasing demand for rAAV-based therapies. A fundamental challenge is the sourcing of large quantities of plasmid DNA, the essential starting material for conventional rAAV production, which entails long turnaround times and a high probability of alterations in the AAV-specific ITR sequences during the fermentation-based production processes. Furthermore, the bacterial sequences can potentially be packaged, risking the safety profile of the AAV product.
We have developed a novel, optimized synthetic linear double-stranded DNA with customizable hairpin-ended structures for use in rAAV manufacturing. Our production process has several advantages over that of plasmid DNA since it is a one-pot, enzymatic, cell-free, scalable reaction with high yields and rapid production times. The final DNA product is characterized by superior purity and quality, and better product homogeneity.
Interestingly, our customized hairpin-ended structures seamlessly integrate with the function of each synthetic DNA construct. To replicate and package the GOI without the need for any additional unwanted sequence, the hairpin ends of our GOI construct were designed as AAV2 ITR-like hairpins for single strand AAV (ssAAV) or self-complementary AAV (scAAV). To prevent the packaging of AAV structural and replication sequences, the hairpin ends of our RepCap and Helper were designed with synthetic hairpin structures. The absence of bacterial sequences in our DNA material reduces the risk of unwanted DNA sequences in the resulting rAAV product.
Here we show that our synthetic DNA is suitable as input for ssAAV or scAAV manufacturing with a range of serotypes in a conventional triple transfection set. Triple transfection with plasmid constructs was used as reference. We show all rAAV produced from our synthetic DNA to be functionally equivalent to rAAV produced from plasmid. In addition, rAAV production from our novel DNA was comparable or better in yield, percentage of full:empty ratios, particles and infectivity.
Altogether, our results point to our synthetic DNA as a superior alternative for starting material for AAV production compared to conventional plasmid DNA because of the speed and scalability of our process and the reliability and performance of our product.
Plain Language Summary
We have developed a novel synthetic linear double-stranded DNA with customizable hairpin-ended structures and without bacterial sequences for use in rAAV manufacturing. Our production process is a one-pot, enzymatic, cell-free, scalable reaction. Although our DNA is fully synthetic, we can design a synthetic DNA encoding for the gene-of-interest in a way that it can mimic an AAV viral genome and, when transfected into cells, it can be replicated and packaged in full. We have also designed two other synthetic DNA molecules, not compatible with replication and packaging, to encode the RepCap and Helper. In this work, we show that these three synthetic constructs are a superior alternative for rAAV production compared to conventional plasmid DNA
Ileana Guerrini, Roxanne Lourman, Martin Cusack, David Wilson, Luca Distefano, Ivana Pastierikova, Marco Guarrera, Alexander Pekarsky, Jorge Omar Yanez Cuna, Joel de Beer
Anjarium Biosciences AG, Schlieren, Switzerland"
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