Dados do Trabalho

Título

A low-cost process of lentiviral vectors production for cell therapy

Introdução

Lentivirus production is a critical stage for the development of several cellular therapies, including chimeric antigen receptor (CAR) T cell generation, since the lentiviral vector is used to introduce the chimeric receptor gene into T cells. The quality of the lentiviral vectors produced can directly affect the effectiveness of the therapy, which may result in low CAR expression or high T cell cytotoxicity, with a consequent reduction in antitumor capacity. Moreover, the production of these vectors usually uses very expensive ultracentrifuges or high cost sedimentation reagents.

Objetivo

In this context, this project aims to optimize a low-cost production of a lentiviral vector (G36/ZsGreen) for CAR T therapy, improving the efficiency of CAR transduction and expression, without compromising T cell viability.

Métodos

Lentiviral vectors will be produced by transient transfection of five plasmids into 293T cells using polyethyleneimine (PEI). Different variables were evaluated for optimizing the concentration of viral particles using a low-cost self-produced reagent that requires lower velocity for sedimentation in the centrifuge. Besides, we tested different values of a multiplicity of infection (MOI); cell densities for virus titration and filtration conditions. The evaluation of CAR expression in transduced cells was done by flow cytometry.

Resultados

Using different MOI values (1-20), we initially found that the T cell viability ranged from 10-40% 48 hours after transduction. Lower MOI values (1-0.0625) were evaluated using a pre-centrifugation condition, verifying that transduction levels varied between 15-25% of positive cells. Under these conditions, the viability levels reached 50-85% after 120 hours.

Conclusões

With this project, we hope to optimize and cheapen the lentiviral vector production for CAR T therapy, improving its effectiveness and contributing to the development of lower-cost and high-efficiency treatments for cancer patients. The project was approved by the institutional review board and CIBio (Technical opinion number 6839/2020).

Palavras-chave

Lentivirus; CAR T; Transduction.