MicroRNA-mediated neuronal detargeting alters astrocyte cell fate conversion trajectories in vivo

Neuronal reprogramming using adeno-associated viruses with a GFAP mini-promoter offers a promising strategy for astrocyte-to-neuron conversion; however, specificity remains a challenge due to off-target transgene expression in endogenous neurons. To address this issue, here we incorporated microRNA-124 target sequences (124T) into a transcriptional cassette containing the GFAP mini-promoter and Ascl1^SA6, a potent reprogramming transcription factor. Lineage tracing via Slc1a3-CreERT and Aldh1l1-CreERT2, used to pre-label astrocytes prior to conversion, confirmed the glial derivation of reprogrammed neuron-like cells, even with 124T. Single-cell transcriptomics identified a transitional cluster emerging from a proliferative astrocyte population with low GSK3 signaling, which branched towards hybrid neuronal and oligodendrocyte fates. Pseudotime trajectory analysis revealed that Ascl1^SA6 drives rapid neuronal transitions, whereas 124T delays conversion and introduces lineage bifurcation. Ascl1^SA6 favors a GABAergic interneuron-like identity, while Ascl1^SA6 -124T biases fate transitions towards an oligodendrocyte-like fate, and to a lesser extent, glutamatergic neuronal-like cells. SeeSawPred linked these distinct trajectories to transcription factor shifts, including Foxo1 in neuron-like cell fates and Stat1 in oligodendrocyte lineages. 124 T thus effectively detargets endogenous neurons, refining target cell specificity, while further guiding reprogramming outcomes. This approach establishes a foundation for precision reprogramming platforms aimed at restoring specific neural cell types.