Cell Membrane Vesicles Encapsulating <i>Il24</i> mRNA With Enriched CD6 Display Exhibit Enhanced Targeted Antitumor Efficacy

ABSTRACT mRNA‐based therapeutics offer significant potential for cancer treatment owing to their ability to induce transient, non‐integrating expression of therapeutic proteins. However, effective and tumour‐specific delivery remains a major barrier. In this study, we report a cell membrane vesicle (CMV)‐based delivery platform for targeted mRNA therapy, employing engineered CMVs enriched with the surface molecule CD6 for the selective delivery of Il24 mRNA to CD166‐overexpressing tumour cells. CMVs were derived from NIH‐3T3 cells via cytochalasin B induction and genetically modified to express CD6, enabling biomimetic targeting through the CD6–CD166 axis. Il24 mRNA was loaded into CD6‐CMVs through a digitonin‐assisted permeabilisation strategy, achieving high encapsulation efficiency and sustained intracellular release. In vitro , CD6‐CMVs facilitated enhanced uptake in CT26 cells, leading to elevated IL‐24 expression, activation of apoptotic and autophagic pathways, and suppression of migration and invasion. In mouse colorectal cancer and squamous carcinoma models, CD6‐CMVs/ Il24 mRNA demonstrated effective tumour targeting, increased intratumoral IL‐24 translation, and potent antitumor efficacy, with minimal off‐target accumulation or systemic toxicity. Notably, this treatment promoted immune cell infiltration and reshaped the tumour microenvironment towards a pro‐inflammatory state. This study presents a scalable, low‐immunogenicity CMV platform capable of targeted mRNA delivery and cytokine expression, offering a promising strategy for precision immunotherapy for solid tumours.