Progress and Perspectives in the Development of Lentiviral Vector Producer Cells

Abstract

After two decades of clinical trials, gene therapy demonstrated effectiveness in the treatment of a series of diseases. Currently, several gene therapy products are approved and used in the clinic. Lentiviral vectors (LVs) are one of the most used transfer vehicles to deliver genetic material and the vector of choice to modify hematopoietic cells to correct primary immunodeficiencies, hemoglobinopathies, and leukodystrophies. LVs are also widely used to modify T cells to treat cancers in immunotherapies (e.g., chimeric antigen receptors T cell therapies, CAR-T). In genome editing, LVs are used to deliver sequence-specific designer nucleases and DNA templates. The approval LV gene therapy products (e.g., Kymriah, for B-cell Acute lymphoblastic leukemia treatment; LentiGlobin, for 𝜷-thalassemia treatment) reinforced the need to improve their bioprocess manufacturing. The production has been mostly dependent on transient transfection. Production from stable cell lines facilitate GMP compliant processes, providing an easier scale-up, reproducibility and cost-effectiveness. The establishment of stable LV producer cell lines presents, however, several difficulties, with the cytotoxicity of some of the vector proteins being a major challenge. Genome editing technologies pose additional challenges to LV producer cells. Herein the major bottlenecks, recent achievements, and perspectives in the development of LV stable cell lines are revised.

Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models

“…This section describes the production of lentiviral particles using pCMV-VSV-G envelope and psPAX2 packaging plasmid, which can be used with 2nd and 3rd generation lentiviral vectors. Transfected HEK293T cells will produce lentiviral particles which accumulate in the culture medium supernatant ( Ferreira et al., 2020 ) and can be used directly for transduction of N/TERT-1 keratinocytes CRITICAL: Cells should be actively growing and not cultured for many weeks before use when seeded. HEK293T cells should be ∼80–90% confluent on the day of transfection.…”

Chimeric Antigen Receptor-T Cells: A Pharmaceutical Scope

“…The approval of LV gene therapy products ( e.g ., Kymriah/Novartis) increased their demand and has created the need to improve their large-scale manufacture to clinical-grade LV ( Lester et al, 2018 ). Nevertheless, the LVs production has challenges, such as their inherent cytotoxicity, low stability, and the dependency on transient transfection impact, both upstream and downstream processes that are reflected in low yielding and cost-ineffective compared to another viral vector ( Ferreira et al, 2021 ). The level of GMP compliance required for the manufacture of LV is a combination of diverse factors including regulatory expertise, compliant facilities, validated and calibrated equipment, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and quality by design approach ( Levine et al, 2017 ; Dasgupta, et al, 2020 ).…”

“…The level of GMP compliance required for the manufacture of LV is a combination of diverse factors including regulatory expertise, compliant facilities, validated and calibrated equipment, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and quality by design approach ( Levine et al, 2017 ; Dasgupta, et al, 2020 ). In addition, part of the commercialization success of this gene therapy product is the establishment, standardization, and implementation of stable cell lines to produce LVs facilitating GMP-compliant processes, providing an easier scale-up, reproducibility, biosafety, and cost-effectiveness ( Ferreira et al, 2021 ).…”

CAR-T in Cancer Treatment: Develop in Self-Optimization, Win-Win in Cooperation

“…Currently, viral vector-mediated semi-random DNA integration is the main approach for expressing CAR structures on T cells [ 107 ]. However, the complex and expensive manufacturing processes of viral vectors, cumbersome production, quality control steps of viral vector-transfected CAR-T, and inevitable long-term monitoring of unanticipated side effects also limit its wide clinical application.…”