Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Abstract

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered “impurities” in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

On the Relationship of Viral Particles and Extracellular Vesicles: Implications for Viral Vector Technology

“…Measures for a more regulated handling of EVs have been discussed [ 68 , 69 ]. Thus, addressing the balance of EV and VI in VV-based therapies may be a way to optimize the efficacy and safety of VV-mediated biomedical strategies [ 70 ]. Due to persisting issues regarding, e.g., immunogenicity of viral vectors, exosomal vectors may prove to be a more secure alternative to classic VVs, depending on a better understanding of EV biogenesis, sub-classes and functionalities.…”

Critical Assessment of Purification and Analytical Technologies for Enveloped Viral Vector and Vaccine Processing and Their Current Limitations in Resolving Co-Expressed Extracellular Vesicles

“…UF/DF is also a method of choice in the purification of retroviral and lentiviral vectors using 100 to 300 kDa membranes [ 43 , 44 ]. UF/DF has also been employed in the field of EVs, especially for its scalability advantage [ 22 , 45 ].…”

“…However, specificity has not been demonstrated for efficient separation of EVs from viruses. Indeed, tetraspanins were also associated with viruses, such as CD63 with retroviral vectors and CD81 with lentiviral vectors [ 22 , 23 ]. Distinctive markers have yet to be accurately identified, and they may differ depending on the expression system and the produced enveloped virus.…”

“…However, PCR techniques are based on specific primers, which have to be designed in such a way that they are specific to the measured particles. In Do Minh et al [ 22 ], primers used in ddPCR targeted the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which is commonly used in viral vector design to enhance transgene expression, the presence of WPRE thus indicating the presence of the viral genome. The cell line used in the study expressed the green fluorescence protein (GFP) transgene constitutively.…”

“…Few studies have been designed to compare viruses to coproduced EVs in cell culture produced systems using omics approaches [ 21 , 22 ]. Sviben et al compare mumps and measles produced in Vero cells to the coproduced EVs, while Do Minh et al compare lentiviral vectors to coproduced EVs in a HEK293-derived cell line.…”