# mRNA-based CAR T cell engineering: Unmodified mRNA enables high CAR expression without innate immune activation in T cells

## TL;DR

This study shows that unmodified mRNA can be used to engineer T cells without causing immune activation, making the process more efficient and cost-effective.

## Contribution

The study reveals that T cells do not activate innate immunity when exposed to unmodified mRNA, challenging the need for nucleoside modifications.

## Key findings

- T cells showed no immune activation when exposed to unmodified mRNA, unlike other cell types.
- Unmodified mRNA improved CAR expression and cytotoxic potency in T cells.
- Eliminating nucleoside modifications simplifies and reduces the cost of CAR-T cell manufacturing.

## Abstract

Unmodified, uridine-containing mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To avoid this and enable high expression, modified nucleosides such as N1-methylpseudouridine have become the gold standard for mRNA applications including T cell engineering, albeit at increased cost. Here, immune responses toward mRNA were evaluated across five primary human cell types. Remarkably, T cells, unlike other immune and non-immune cell types tested, exhibited no immune activation by unmodified mRNA. T cell viability and cytokine secretion remained unaffected, regardless of mRNA delivery method via lipid nanoparticles or electroporation. The absence of nucleotide modifications improved expression of chimeric antigen receptor (CAR) in activated T cells and CAR-T cell cytotoxic potency. By eliminating the need for mRNA-nucleoside modification in CAR-T cell engineering, our findings challenge existing paradigms and position mRNA as a non-inflammatory, minimally invasive and highly efficient tool for T cell engineering, while simplifying and reducing manufacturing cost.

Proinflammatory innate immune activation by unmodified mRNA is a limitation, which mandates mRNA-nucleoside modification across applications. However, Drzeniek and colleagues reveal that it is absent in cultured T lymphocytes. This eliminates the risk of inflammatory side effects and functional impairment in mRNA-engineered T cells, such as transient CAR-T cell therapy.