Proteomics analysis of cellular components in lentiviral vector production using Gel-LC-MS/MS.

Among the gene therapy vectors developed to date, lentiviral vectors persist in the host and are therefore best suited for long-term gene transfer and gene-replacement therapies. Human immunodeficiency virus (HIV)-1-based lentiviral vector products are currently produced by transient transfection, filtration and ultracentrifugation, and the quality of the resultant vector product is variable. We reason that by identifying host proteins co-produced with viral vectors we may better understand the mechanism of viral vector production, and improve the safety and quality of the resultant products. Our LC-MS/MS studies identified both viral vector proteins and host proteins, including nuclear proteins, elongation factors and chaperone and heat shock proteins (HSP), and confirmed the presence of known HIV-incorporated proteins, e.g. elongation factor-1α, HSP70 and Histone 2A, demonstrating the capability and viability of LC-MS/MS methods for the proteomic analysis of highly complex samples. Evaluation of the functions of the identified components is in progress to understand their implication for product quality and safety. These studies support the development of improved production and characterisation methods and advance the clinical application of lentiviral vector-based products.