TRANsCre-DIONE transdifferentiates scar-forming reactive astrocytes into functional motor neurons

In spinal cord injury (SCI), the scar-forming reactive astrocytes with upregulated glial fibrillary acidic protein (GFAP) proliferate aberrantly near the injury site, representing a potential cellular source for transdifferentiation into neurons to replenish dead neurons. However, the conventional use of GFAP promoter to target reactive astrocytes has two inherent problems: inadvertent conversion of normal astrocytes and low efficiency due to progressive weakening of promoter activity during transdifferentiation. Here, we present TRANsCre-DIONE, a dual-promoter split-Cre system combining GFAP and Lcn2 regulatory elements with Cre-dependent Neurog2 expression under the EF1α promoter, enabling selective targeting of scar-forming reactive astrocytes. This approach achieved 87% conversion efficiency and 96% specificity in vivo. After SCI, TRANsCre-DIONE caused transdifferentiation into Isl1-positive and ChAT-positive motor neurons, reduced astrogliosis, enhanced regeneration in surrounding cells, and a significant motor recovery. These findings suggest that TRANsCre-DIONE enables efficient and selective astrocyte-to-neuron conversion and represents a promising strategy for SCI repair.