PBAE nanoparticle-mediated delivery of ASCL1 and NGN2 genes for astroglia-to-neuron reprogramming to remodel glial scar for spinal cord injury repair.

Abstract

Background: Irreversible loss of neuronal cells elicited by neurotraumatic injuries or neurodegenerative disorders is particularly devastating due to the limited regenerative capacity of the central nervous system (CNS). Cell reprogramming-based therapies have emerged as promising therapeutic avenues for neuronal replenishment. However, their therapeutic potential in neural regeneration still faces formidable challenges, including risks of viral vector gene delivery, potential damage from cell transplantation, and significant glial scar (GS) formation following CNS injury. Therefore, developing an optimal approach that simultaneously replaces lost neurons and overcomes these persistent obstacles is crucial for neural regeneration and functional recovery.

Methods: We engineered a non-viral gene delivery platform using biodegradable poly(β-amino ester) (PBAE) nanoparticles (NPs) to effectively co-deliver plasmids encoding proneural transcription factors ASCL1 and NGN2 directly to astroglia (ATG) within GS region, in combination with neural induction. The biochemical and physiological properties of reprogrammed ATGs were characterized both in vivo and in vitro. The therapeutic potential of PBAE-A/N delivery was assessed in spinal cord injury (SCI) animal models through behavioral evaluations. Finally, the molecular mechanisms underlying ASCL1/NGN2-mediated ATG-to-neuron reprogramming were investigated.

Results: PBAE-mediated delivery of ASCL1/NGN2 plasmids effectively reprogrammed resident ATGs within GSs into functional neurons, as evidenced by the acquisition of neuronal morphology and biochemical phenotype (neuronal marker expression), loss of ATG characteristics, scar remodeling, and functionality indistinguishable from those of genuine neurons, including specialized calcium signaling, synaptic activity, and action potential firing. Critically, local administration of PBAE-ASCL1/NGN2 NPs into the GS region of the injured spinal cord significantly ameliorated neurological deficits. Mechanistically, this reprogramming event likely involved the modulation of downstream targeting signaling mediated by Cend1, RanBPM, and Dyrk1, along with crosstalk with the Notch1/Cyclin D1 axis.

Conclusions: This study demonstrates that PBAE-mediated ASCL1/NGN2 delivery enables in situ reprogramming of ATG into functional neurons while actively dissolving GSs, thereby addressing both neuronal loss and GS barriers in CNS repair. The identified Cend1/RanBPM/Dyrk1 signaling and its crosstalk with Notch1/Cyclin D1 axis provide mechanistic insights into the events. Collectively, this work presents a novel therapeutic alternative for CNS repair and neurodegeneration by simultaneously replacing lost neurons and eliminating endogenous GSs through in situ cell reprogramming.