SUMMARY In spinal cord injury (SCI), the scar-forming reactive astrocytes with upregulated GFAP proliferate aberrantly near the injury site, allowing themselves as a prime target 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 report that the scar-forming reactive astrocytes are selectively transdifferentiated into neurons with 87% efficiency and 96% specificity via TRANsCre-DIONE, a combination of the split-Cre system under two different promoters of GFAP and Lcn2 and a Cre-loxP-dependent inversion and expression of Neurog2 under the strong EF1α promoter. After SCI, TRANsCre-DIONE caused transdifferentiation into Isl1-positive motor neurons, reduced astrogliosis, enhanced regeneration in surrounding cells, and a significant motor recovery. Our study proposes TRANsCre-DIONE as the next-generation therapeutic approach for patients suffering from SCI. Highlights TRANsCre-DIONE converts reactive astrocyte into neuron by over-expression of Neurog2 Reactive astrocytes are targeted using split-Cre under two promoters, GFAP and Lcn2 TRANsCre-DIONE reduces reactivity, replaces dead neurons and alleviates symptom of SCI Transdifferentiated-neurons are GABA+ in the striatum and Isl1+ in the spinal cord

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