Intranasal CRISPR- lipid nanoparticles targeting MAPK9 reduce neuroinflammation after traumatic brain injury

Abstract Traumatic brain injury (TBI) triggers a sustained neuroinflammatory response driven by activated microglia, which contributes to secondary injury and long-term neurological dysfunction. Therapeutic reprogramming of microglial activation from a pro-inflammatory (M1-like) to a reparative (M2-like) phenotype represents a promising strategy; however, the lack of cell-specific targeting within an injured brain has limited clinical translation. Here, we developed a targeted gene-editing nanotherapy to modulate post-traumatic innate immune responses. Lipid nanoparticles (LNPs) encapsulating CRISPR-Cas12a components were engineered to target mitogen-activated protein kinase-9 (MAPK9), a key regulator of pro-inflammatory signaling, and were conjugated with an Iba-1 antibody (Iba-1-CRISPR-LNPs) to enable selective targeting of microglia. In vitro , MAPK9 editing in primary macrophages inhibited M1 polarization and promoted an M2-like phenotype, leading to reduced production of pro-inflammatory cytokines. In a TBI mouse model, intranasal administration of Iba-1-CRISPR-LNPs achieved efficient delivery to the injured brain, with selective localization in Iba-1+ microglia. MAPK9 CRISPR targeting significantly attenuated microglial activation, reduced central and peripheral inflammatory responses, and decreased pro-inflammatory cytokine levels. Importantly, this approach demonstrated a favorable safety profile, with no detectable toxicity across major organs. Collectively, these findings establish a non-viral, intranasal CRISPR-based strategy for cell-specific modulation of neuroinflammation following TBI. Targeted genome editing of MAPK9 effectively reprograms microglial activation and attenuates acute inflammatory responses, highlighting its potential as a promising and translationally relevant therapeutic platform for TBI and related neuroinflammatory disorders.