Lentiviral Vector Bioprocessing

Abstract

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

Adipose-Derived Stem Cells Based on Electrospun Biomimetic Scaffold Mediated Endothelial Differentiation Facilitating Regeneration and Repair of Abdominal Wall Defects via HIF-1α/VEGF Pathway

“…Compared with exogenous addition of cytokines or pretreatment in vitro , target genes transfected into ADSCs using lentiviral overexpression technology can be more precise to regulate the long-term expression of proteins such as VEGF ( Gouvarchin Ghaleh et al, 2020 ). The transfected technology is commonly utilized within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies ( Perry and Rayat, 2021 ). Therefore, genes transfected method was applied for inducing endothelial differentiation of ADSCs in our study.…”

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

“…Continuous-flow centrifugation overcomes this volume limitation and is still being used at a large scale in vaccine manufacturing, especially in the case of influenza vaccine and Japanese encephalitis vaccine. However, it does not translate well for lentiviral vectors, which are subject to a loss of infectivity by ultracentrifugation with or without sucrose gradients [ 41 ]. Continuous-flow centrifugation equipment is also high-maintenance, costly and voluminous.…”

Payload Delivery: Engineering Immune Cells to Disrupt the Tumour Microenvironment

“…Although gammaretroviruses are stable and yield high viral titres, they can only carry relatively small gene constructs. Lentiviruses, in contrast, can accommodate larger gene constructs but at the cost of poor stability and lower viral titres, often requiring a concentration step using ultracentrifugation or a viral capture reagent [131]. The capacity for lentiviral vectors to Different gene delivery tools can be used to engineer immune cells for adoptive cell therapy, including non-viral techniques using either electroporation, CRISPR-Cas9, or transposons (e.g., Sleeping Beauty and piggyBac), as well as viral-based methods utilising adenoviruses, adeno-associated viruses (AAV), or retroviruses (e.g., HIV lentivirus and MLV gammaretrovirus) [129].…”

“…Although gammaretroviruses are stable and yield high viral titres, they can only carry relatively small gene constructs. Lentiviruses, in contrast, can accommodate larger gene constructs but at the cost of poor stability and lower viral titres, often requiring a concentration step using ultracentrifugation or a viral capture reagent [ 131 ]. The capacity for lentiviral vectors to carry larger cargo makes them the ideal candidate for polycistronic gene constructs and thus an important tool in generating the next generation of CAR T cells.…”