A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Autor:	van Lieshout LP; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Rubin M; Homology Medicines, Inc., 1 Patriots Park, Bedford, MA 01730, USA., Costa-Grant K; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Ota S; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Golebiowski D; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Panico T; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Wiberg E; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Szymczak K; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Gilmore R; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Stanvick M; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Burnham B; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Gagnon J; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Iwuchukwu I; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Yang G; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Ghazi I; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Meola A; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Dickerson R; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Thiers T; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Mustich L; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Hayes A; Homology Medicines, Inc., 1 Patriots Park, Bedford, MA 01730, USA., Rivas I; Homology Medicines, Inc., 1 Patriots Park, Bedford, MA 01730, USA., Lotterhand J; Homology Medicines, Inc., 1 Patriots Park, Bedford, MA 01730, USA., Avila N; Homology Medicines, Inc., 1 Patriots Park, Bedford, MA 01730, USA., McGivney J; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Yin J; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA., Kelly T; Oxford Biomedica Solutions LLC, 1 Patriots Park, Bedford, MA 01730, USA.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Methods & clinical development [Mol Ther Methods Clin Dev] 2023 May 08; Vol. 29, pp. 426-436. Date of Electronic Publication: 2023 May 08 (Print Publication: 2023).
DOI:	10.1016/j.omtm.2023.05.004
Abstrakt:	Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo . The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform. Competing Interests: All authors have received salary with employee stock options and/or restricted stock units during their employment at Oxford Biomedica Solutions LLC or Homology Medicines, Inc. L.P.v.L. and M.S. are inventors of patents associated with the pOXB dual-plasmid system. (© 2023 The Author(s).)
Databáze:	MEDLINE
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