Injectable CRISPRa-microspheres for targeted A20 activation rescue age-related osteogenic impairment via senescence mitigation.

Abstract

Age-related osteogenic failure in bone defect repair remains a significant clinical challenge, primarily due to persistent chronic inflammation-induced stem cell senescence. To address this, we engineered injectable CRISPRa-based gene-editing microspheres (GEMs), utilizing microfluidic-synthesized lipid nanoparticles (cLNPs) to co-deliver dCas9-VP64/sgRNA. This platform allows for precise spatiotemporal activation of tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) within bone marrow stromal cells (BMSCs), effectively reprogramming the senescence-osteogenesis axis. Our study identifies A20 as a key regulator of the senescence-associated secretory phenotype (SASP) and osteogenic impairment in aged BMSCs. In vitro, GEMs reduced senescence markers (p16 and p21) by over 30 %, while increasing osteogenic gene expression (RUNX2 and ALP) by 4 ∼ 5-fold, and suppressed inflammatory cytokines IL-6 and TNF-α by more than 30 %. In vivo, in aged mice with critical-sized bone defects, GEMs achieved a significant bone regeneration and promoted vascularization 3.1 times faster (CD31⁺ staining) compared to controls. This GEM system offers a promising, clinically viable strategy for recalibrating age-related skeletal disorders, demonstrated by the precise targeting of host stem cells in situ and achieving approximately 80 % defect healing in aged bone defect models.