A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Summary

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Abstract

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-ϒ. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

Introduction

Cancer is an increasingly important threat to human health, with an annually estimated 23.6 million newly diagnosed cases and 10 million deaths globally¹. Surgical treatment combined with radiation and chemotherapy remains the gold standard of care for various types of localized noninvasive and invasive cancers^2,3,4. Although the traditional systematic treatment has achieved tremendous success in managing early-stage cancers, it is very toxic and has limited effect on metastatic and hematological cancers⁵. Patients with these ca....

Protocol

The PBMCs used here were previously isolated from whole blood from the Stanford Hospital Blood Center according to the Institutional Review Board (IRB)-approved protocol (13942) using the Ficoll-Paque density gradient described before⁴². The participants' informed consent was not applicable as the blood we used to collect the PBMCs was obtained commercially from the Stanford Hospital Blood Center.

1. Preparation of CAR mRNA

1. Template preparation
   NOTE: Either a linearized plasmid or a PCR fragment can serve as a template to synthesize CAR mRNA in vitro, provided they con....

Discussion

In this study, we describe a detailed nonviral, non-integrating approach for generating transient CAR-T cells and provide technical procedures for assessing CAR-T cell function. This approach avoids the use of conventional retroviral and lentiviral vectors for permanent and random CAR transgene delivery, instead leveraging electroporation to efficiently introduce in vitro modified CAR mRNA into T cells. This method enables high-level, transient CAR expression on the T cell surface, significantly reducing the ris.......

Materials

Name	Company	Catalog Number	Comments
7-AAD viability dye	Biolegend	420404
ACEA Novocyte flow cytometer	Agilent	NovoCyte 3000
AIM-V medium	Gibco	12055083
APC goat anti-human IgG F(ab')2 antibody	Jackson ImmunoResearch Laboratories	109-136-097
BglII Restriction Enzyme	New England BioLabs (NEB)	R0144L
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog	NEB	S1411S
DMEM, high glucose	Gibco	11965092
Dynabeads Human T-Activator CD3/CD28 beads	Gibco	11131D
E-Plate 96	Agilent	5232376001
Ethanol	Sigma	459844-4L
FBS	Lonza.com	14-503F
HiScribe SP6 RNA Synthesis Kit	New England BioLabs (NEB)	E2070S
Human IL-2 Recombinant Protein	Gibco	15140122
Millennium RNA Markers	Invitrogen	AM7150
Monarch RNA Cleanup Kit (500 μg)	NEB	T2050L
N1-Methylpseudo-UTP	Trilink	N-1081-10
Neon Transfection Instrument	Invitrogen	MPK5000
Neon Transfection System 100-μL Kit	Invitrogen	MPK10096
Penicillin-Streptomycin (10000 U/mL)	Gibco	14-503F
RPMI 1640 Medium	Gibco	11875135
Trypsin-EDTA (0.05%), phenol red	Gibco	25300120
UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)	Invitrogen	15593049
xCELLigence Real-Time Cell Analysis (RTCA) instrument	Agilent	RTCA MP
ZymoPURE II Plasmid Midiprep Kit	Zymo Research	D4201