Figure 4.

Five MiniPromoters delivered by subretinal and intravitreal injection in adult mice showed co-labeling with a ganglion cell marker. MiniPromoters, each driving EmGFP-WPRE in rAAV9, were injected directly into adult mouse eyes, and harvested 4 weeks later. (A) Subretinal injection led to expression in the ganglion cell layer (GCL) and the inner nuclear layer (INL). (B) Intravitreal injection of MiniPromoters Ple320 (POU4F1), Ple321 (TUBB3), Ple344 (TUBB3), and Ple346 (NEFM) led to robust expression in retinal ganglion cells (RGCs), as indicated by co-staining with the ganglion cell marker RBPMS (RNA-binding protein with multiple splicing), and a few cells in the INL. MiniPromoter Ple345 (NEFL) led to robust expression only in RGCs. (C) Quantification of endogenous EmGFP fluorescence intensity in RBPMS-positive cells showed that Ple321 (TUBB3) and Ple344 (TUBB3) were not statistically different from the small chicken beta-actin promoter/CMV enhancer (smCBA) ubiquitous control (for histology see Supplementary Fig. S3). However, Ple345 (NEFL) drove significantly stronger endogenous EmGFP expression in RGCs compared to the smCBA ubiquitous control. Dose for all injected eyes was 2.00 × 10¹⁰ genome copies (GC)/eye. **p > 0.01; IPL, inner plexiform layer; n, number of cells counted; n.s., not significant; ONL, outer nuclear layer; OPL, outer plexiform layer. Green, anti-GFP; red, anti-RBPMS; yellow, merge. Scale bar, 20 μm.