Zhou, Yu, Sach, Tina, Ong, Joseph, Lim, Ting-an, Berecz, Zoltan, Deniston, Colin, Milicic, Goran, Tsai, Connie, Kandepalli, Taryn Jessica, Langeslay, Derek and Qin, Qiang (2024) Adeno-associated virus serotype 2 with proteolytic cut by trypsin remains intact and potent. Gene therapy. ISSN 1476-5462; 0969-7128

Abstract

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity, immunogenicity, and play important roles in receptor binding, viral escape from the endosome, and transportation of viral DNA to the nucleus. In the journey of AAV vector, AAV vectors can be exposed to different proteolytic environments inside the production cells, during the cell lysis step prior to downstream purification, within endosome, and finally inside the cell nucleus. Therefore, evaluation of AAV stability using a proteolytic approach can provide valuable information for engineering AAV vectors for AAV-based gene therapy development. The stability of a modified AAV serotype 2 (AAV2) capsid proteins was evaluated via a proteolytic approach using trypsin and other proteases. Proteolytic digestion of the AAV2 capsid with trypsin results in clips of the capsid proteins at C-terminus as confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and reversed-phase LC-MS. It was found that the AAV2 capsid with clips not only remains structurally intact, as confirmed by native (non-denaturing) methods including SEC, thermos stability testing, and Cryogenic electron microscopy (cryo-EM), and also remains potent, as confirmed in a potency assay. This finding reveals that the icosahedral three-dimensional structural arrangement of AAV capsid proteins can protect the clipped fragment being released from the capsid, such that the AAV capsid remains intact allowing for the functionality to be maintained to deliver the DNA in the host cell. The finding is valuable for engineering AAV capsids to develop AAV-based gene therapy.

Item Type:	Article
Keywords:	AAV, capsid proteins, CE-SDS, cryo-EM, LC-MS, potency, proteolytic digestion, SDS-PAGE, SEC, thermo stability
Date Deposited:	31 Dec 2024 00:45
Last Modified:	31 Dec 2024 00:45
URI:	https://oak.novartis.com/id/eprint/53001