Page 72 - Ebook-Book JCMS 2025

Enhancing Lentiviral Based Transduction Efficiency of CD19
CAR-T Cells Using mRNA Technology
Proudphat Jumnongjit 1,2,3*, Supannikar Tawinwung1,3,4, Koramit Suppipat1,3,5, Thanyavi Chinsuwan1,3
1 Cellular Immunotherapy Research Unit, Faculty of Medicine, Chulalongkorn University
2 Master of Science Program in Research for Enterprise Faculty of Pharmacy, Chulalongkorn University
3 Thailand Hub of Talents in Cancer Immunotherapy
4 Department of Pharmacology and Physiology, Faculty of Pharmaceutical Science, Chulalongkorn
University
5 Department of Research Affairs, Faculty of Medicine, Chulalongkorn University
Background: Methods: Results: Conclusions: *Corresponding Author E-mail: proudphat4114@gmail.com
Abstract
The Chimeric Antigen Receptor (CAR) T cell is a novel therapy for cancer treatment.
Lentiviral transduction is the most common strategy for CAR T cell manufacturing.
However, lentiviral transduction still has some limitations in achieving high efficiency in
CAR T cells because the low-density lipoprotein (LDL) receptor, which serves as the cellular
receptor for vesicular stomatitis virus G protein (VSV-G) envelop of lentivirus vector, is
expressed at low levels on T cells. Upregulation of the LDL receptor on T cells may improve
the efficiency of VSV-G lentiviral transduction. This holds significant promise for increasing
yield in CAR T cell manufacturing.
In this study, we generated mRNA LDLR using in vitro transcription (IVT) then transfected
into activated T cells. The LDLR expression was measured, then T cells were transduced
with the CD19-CAR lentiviral vector. The efficiency and expression of CAR were determined
by flow cytometry.
Flow cytometric analysis showed mRNA LDLR expression levels, with transfection
resulting in 80.2 ± 9.3 % compared to 47.9 ± 15.5 % in non-transfected T cells. Next, LDLR
mRNA-transfected T cells exhibited significantly higher CD19 CAR surface expression of 6.3
± 0.09 %, 31.4 ± 15.6 %, and 47.4 ±15.9 %, compared to 13.6 ± 1.2 %, 48.3 ± 19.8 % and
62.5 ± 9.0 % in non-transfected T cells at MOI 0.3, MOI 1, and MOI 3 respectively. Moreover,
there is no significant difference in cytotoxicity to Nalm-6 cell line between LDLR mRNA
transfected and non-transfected CD19 CAR T cells.
In this study, we successfully enhanced lentivirus-based CD19 CAR T cell transduction
efficiency by upregulating LDLR using mRNA. LDLR mRNA transfected CD-19 CAR T cells
demonstrated higher efficeincy and potent cytotoxic activity, providing a promising outlook
for increasing the efficiency of CAR T cells in manufacturing.
70 Joint Conference in Medical Sciences 2025