Dados do Resumo

Título

Optimization of a low-cost process of lentiviral vectors production to obtain cell therapy products

Introdução

Lentiviral vectors (LVs) are important tools for biomedical research and gene therapy, allowing the integration of genetic material into the host genome, with stable expression of the insert by target cells. Cell therapy using T lymphocytes (TLs) expressing a chimeric antigen-targeting receptor (CAR) has recently emerged as a promising therapeutic strategy against cancer. The quality of the LVs 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, producing these vectors usually uses expensive ultracentrifuges or high-cost sedimentation reagents.

Objetivo

In this context, this project aims to optimize an efficient low-cost method to produce lentiviral vectors for CAR T cell 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 to optimize the concentration of viral particles using a low-cost, self-produced reagent that requires lower velocity for sedimentation in the centrifuge. We also tested different reagents and protocols for titration and filtration conditions. The evaluation of CAR expression in transduced cells was performed by flow cytometry.

Resultados

Initially, we compared the application or not of filtration during the production process, finding that the lentiviral yield ranged from 1.3 – 6.6 x10^7 TU/mL, with better results when unfiltered. The conditions of activation or non-activation of T lymphocytes with anti-CD3/CD28 beads resulted, respectively, in 1.2x10^7 TU/mL and 3.4x10^7 TU/mL. Different CAR-codifing lentiviral constructs (A716/ZsGreen and G36/ZsGreen) were used to evaluate the use of the dNTP reagent and the Fetal Bovine Serum depletion during titration, verifying that the yield ranged from 2.0 – 4.0 x10^7 TU/mL.

Conclusões

With this project, we hope to optimize and cheapen the production of lentiviral vectors 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).

Financiador do resumo

FAPESP 2023/03631-3 e 2018/17656-0

Palavras Chave

CAR T; low-cost lentiviral production; cell therapy vector