Production of lentiviral vectors under scalable conditions for the generation of CAR-T cells for cell therapy

Author(s)

Mariane Cariati Tirapelle

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-03-15

Examining board members

Kamilla Swiech Antonietto; Fernanda Perpetua Casciatori; Fabiana Testa Moura de Carvalho Vicentini

• Advisor: Kamilla Swiech Antonietto

Abstract

Lentiviral vectors are widely used as vectors for gene modification in cell and gene therapies, such as dose employing CAR-T cells, due to their stability of integration in the cell genome, transduction efficiency and safety. The production of such vectors in monolayer serum-supplemented culture, traditionally used in academia and research centers, has a number of limitations; amongst them incompatibility with a more controlled large-scale production; being the suspension culture of cells in bioreactors desirable. The aim of this project was to produce lentiviral particles in scalable conditions, using cells adapted for serum-free suspension growth. Therefore, HEK293T cells adapted to serum-free suspension culture, were used to produce lentiviral particles that express the synthetic CAR protein anti-CD19 as well as IL-18 and GFP. The first step was to evaluate the production and functionality of lentiviral particles in monolayer serum-supplemented culture as a control condition. In this condition the average production was 8x106 particles/mL. The vector\'s functionality was assessed by modifying T cells to express the CAR molecule. The results indicated that the vector was capable of inducing antitumor response in T cells, which in turn were able to identify and kill CD19 positive cells in vitro. The adapted HEK293T cells had their kinetic and metabolic profile characterized, and satisfactory results were obtained with and without Cellboost supplementation. Nevertheless, the presence of Cellboost inhibited transient transfection and consequently the production of lentiviral particles. Even in the absence of Cellboost, serum-free suspension lentiviral production showed lower results when compared to monolayer serum-supplemented production. To solve that, an optimization of the transfection protocol was performed using Design of Experiments (DoE) - Box-Behnken. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1x106 cells/mL and PEI:DNA ratio of 2.5:1. This protocol was further optimized with the addition of 5mM Sodium Butyrate and resulted in an increase in the production, with an average of 1.5x105 particles/mL. In the present study, it was possible to optimize the protocol for the production of lentiviral particles in serum-free suspension culture, maintaining the functionality and capacity of inducing anti-tumor response in vitro. (AU)

• FAPESP's process: 18/13307-0 - Production of lentiviral vectors in scalable conditions for generation of CAR-T cells for cell therapy
• Grantee: Mariane Cariati Tirapelle
• Support Opportunities: Scholarships in Brazil - Master