Engineered CCR2 positive macrophages coordinate immunoregulation with neural regeneration and matrix remodeling after spinal cord injury.

RATIONALE: Spinal cord injury (SCI) triggers a complex secondary injury process characterized by inflammation, neuronal loss, extracellular matrix (ECM) disruption, and limited endogenous repair. Although cell-based therapies hold potential for SCI treatment, their efficacy is often constrained by poor lesion targeting, inadequate persistence after delivery, and limited temporal control over therapeutic factor release.

METHODS

To address these limitations, we developed a macrophage-based mRNA delivery platform by electroporating CCR2 positive (CCR2+) macrophages with ANXA1, GDNF, and CTGF mRNAs. In vitro, we assessed transfection efficiency, cell viability, secretion kinetics of therapeutic proteins, anti-inflammatory activity, and neuroprotective and regenerative effects. In vivo, using a mouse SCI model, we evaluated lesion-site accumulation, inflammatory regulation, tissue repair, electrophysiological recovery, transcriptomic alterations, and behavioral outcomes.

RESULTS

Following electroporation, CCR2⁺ macrophages efficiently expressed ANXA1, GDNF, and CTGF while maintaining high viability, with no marked shift toward CD86- or CD206-associated phenotypes. These engineered macrophages showed enhanced accumulation at the lesion site and sustained therapeutic protein secretion for up to 14 days. In vitro, they protected neuronal cells against oxidative stress-induced injury and promoted neurite outgrowth. In vivo, they attenuated inflammation, improved the local repair microenvironment, and promoted axonal regeneration, remyelination, and ECM remodeling, accompanied by partial recovery of electrophysiological and motor function after SCI. RNA-seq analysis further supported broad changes in pathways related to immune regulation, neural repair, myelination, and matrix remodeling.

CONCLUSIONS

CCR2-enriched macrophages engineered with reparative mRNAs may represent a promising treatment strategy for SCI. By linking CCR2-associated lesion accumulation with multimodal reparative activity, this cell-based platform provides a potential approach for coordinated microenvironmental regulation and tissue repair.