Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells.

Autor:	Patra AT; Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A∗STAR), Singapore 138668, Singapore., Tan E; Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A∗STAR), Singapore 138668, Singapore., Kok YJ; Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A∗STAR), Singapore 138668, Singapore., Ng SK; Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A∗STAR), Singapore 138668, Singapore., Bi X; Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A∗STAR), Singapore 138668, Singapore.; Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore.; Food, Chemical and Biotechnology Cluster, Singapore Institute of Technology, Singapore 138683, Singapore.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Methods & clinical development [Mol Ther Methods Clin Dev] 2024 Jun 08; Vol. 32 (3), pp. 101278. Date of Electronic Publication: 2024 Jun 08 (Print Publication: 2024).
DOI:	10.1016/j.omtm.2024.101278
Abstrakt:	The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production. Competing Interests: The authors declare no competing interests. (© 2024 The Author(s).)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu