Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

• Published in: International journal of molecular sciences 2021-10, Vol.22 (21), p.11499
• Main Authors: Strasser, Lisa, Boi, Stefano, Guapo, Felipe, Donohue, Nicholas, Barron, Niall, Rainbow-Fletcher, Alana, Bones, Jonathan
• Format: Article
• Language: English
• Subjects: adeno-associated virus (AAV); Automation; Cell culture; Cell cycle; Cell death; Cell Line; Chromatography; Chromatography, Reverse-Phase; Dependovirus - genetics; Endocytosis; Endocytosis - genetics; Endocytosis - physiology; Expression vectors; Gene Expression - genetics; Gene Expression Regulation - genetics; Gene Ontology; Gene therapy; Genetic Therapy - methods; Genetic Vectors - genetics; Genomes; HEK293 Cells; Humans; label-free quantitation; LC-MS/MS; Liquid chromatography; Lysosomes; Lysosomes - metabolism; Manufacturing; Mass spectrometry; Mass spectroscopy; Metabolism; Plasmids; Productivity; Protein expression; Proteins; Proteolysis; Proteome - genetics; Proteomes; Proteomics; Quantitation; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Scientific imaging; Signal Transduction - genetics; SP3; Tandem Mass Spectrometry; Transfection - methods
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222111499

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.