Scarless circular mRNA-based CAR-T cell therapy elicits superior antitumor efficacy

Area of Science:

• Biotechnology
• Immunotherapy
• Molecular Biology

Background:

• Messenger RNA (mRNA)-based transient expression of chimeric antigen receptors (CARs) offers safety advantages over viral vectors for CAR-T-cell therapies.
• Conventional linear mRNAs exhibit instability, limiting protein expression duration and therapeutic efficacy.
• Challenges remain in optimizing mRNA stability and protein expression for advanced cellular immunotherapies.

Purpose of the Study:

• To engineer a novel platform for synthesizing stable, scarless circular mRNAs (cmRNAs) for enhanced CAR expression.
• To compare the expression magnitude and durability of cmRNAs versus linear mRNAs in CAR-T cells.
• To evaluate the functional antitumor efficacy of cmRNA-based CAR-T cells in vitro and in vivo.

Main Methods:

• Development of a high-efficiency permuted intron exon (PIE) platform for scarless cmRNA synthesis.
• Transfection of primary human T cells with engineered cmRNAs encoding anti-CD19 and anti-GPRC5D CARs.
• Assessment of CAR expression levels, duration, T cell activation, and exhaustion markers.
• Evaluation of in vitro cytotoxicity, cytokine release, and in vivo tumor models.

Main Results:

• cmRNAs demonstrated significantly increased magnitude and duration of CAR expression compared to linear mRNAs.
• cmRNA-based CAR-T cells exhibited superior in vitro antigen-specific cytotoxicity and cytokine release.
• Transcriptomic analysis revealed sustained T cell activation and absence of exhaustion signatures in cmRNA-treated cells.
• In vivo studies showed enhanced tumor elimination and prolonged survival benefits with cmRNA-based CAR-T cells.

Conclusions:

• Scarless circular mRNA (cmRNA) represents a next-generation modality for potent and durable CAR expression.
• The cmRNA platform streamlines manufacturability and potentially reduces innate immune sensing.
• cmRNA technology holds significant promise for advancing cellular immunotherapy and precision medicine.