Dynamic expression of ASCL1 drives neurogenesis from infant and adult macaque Müller glia into immature retinal ganglion cells

Strategies to stimulate the regeneration of neurons in the adult central nervous system can offer universal solutions for neurodegenerative diseases. Taking lessons from naturally regenerating species, such as the zebrafish, we have previously shown that vector-mediated expression of proneural transcription factors can stimulate neurogenesis from the resident Müller glia (MG) population in the adult mouse retina, both in vitro and in vivo. To bring this closer to translation, we now show that vector-mediated expression of the proneural transcription factor ASCL1 can reprogram adult macaque MG into functional neurons. To this end, we established purified MG cultures and show they retain a mature transcriptomic profile that correlates with foveal and peripheral MG. Importantly, MG-derived neurons express retinal ganglion cell markers, can fire action potentials and have a transcriptome that overlaps with developing human and adult macaque retinal ganglion cells. To refine this approach for clinical application, we incorporated microRNA-124 target sites in the reprogramming cassette and show that this restricts expression to MG in mixed primary cultures and intact explant cultures of adult macaque retina. Regulating ASCL1 expression with microRNA-124 target sites maintained the reprogramming efficiency from adult MG cultures and improved the yield of RGC-like neurons from infant MG cultures. Most importantly, with this vector cassette we successfully reprogrammed macaque MG from both adult and infant retina into HuC/D+ neurons. Our findings demonstrate that ASCL1 can induce neurogenesis from macaque MG across ages and provide a targeted, effective strategy for potential clinical translation in retinal repair.