Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Nermin Ibreljic; Benjamin E Draper; Carl W Lawton

doi:10.1016/j.omtm.2024.101188

Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Mol Ther Methods Clin Dev. 2024 Jan 17;32(1):101188. doi: 10.1016/j.omtm.2024.101188. eCollection 2024 Mar 14.

Authors

Nermin Ibreljic^{1

2}, Benjamin E Draper³, Carl W Lawton¹

Affiliations

¹ Department of Chemical Engineering, University of Massachusetts Lowell, One University Avenue, Lowell, MA 01854, USA.
² Sarepta Therapeutics, 55 Blue Sky Drive, Burlington, MA 01803, USA.
³ Megadalton Solutions, 3750 E. Bluebird Lane, Bloomington, IN 47401, USA.

Abstract

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Keywords: adeno-associated virus; anion exchange chromatography; capsid melting temperature; capsid titer; charge detection-mass spectroscopy; differential scanning fluorimetry; gene therapy; genome ejection melting temperature; triple transfection; viral genome titer.