Figure 1.

Design of the AAV Vectors, Assessment of Processing, and Effect on Retinal Function of miR-204 Subretinal Injection in Wild-Type Mice

(A) Schematic representation of expression cassettes used for AAV vector production. (B) miRNA expression profile analysis on the LCM-collected photoreceptor layers of three animals injected subretinally with AAV.CMV.miR204 (miR204) and AAV.CMV.EGFP (Control) at PN30 and sacrificed 3 weeks later. Expression levels were determined by TaqMan Real-Time PCR on total RNA and normalized against small nucleolar RNA (snoRNA)234. Levels of miR-124, a retinal-expressed miRNA, were not affected, suggesting that miR-204 administration does not interfere with the global miRNA processing machinery in the retina. Error bars are SEM. (C) ERG responses at PN30 of C57BL/6 mice injected at PN14 with AAV.CMV.miR204 (n = 8) or miR204MUT or AAV.CMV.EGFP (n = 8). No differences were observed. Statistical significance was assesed with an unpaired Student’s t test against control vector. (D) Immunolabeling for photoreceptor markers (M Opsin, S Opsin, Cone Arrestin; in red) on retinal sections from wild-type eyes injected with AAV.CMV.miR204. Nuclei were counterstained with DAPI (blue). Control virus distribution (coinjected at a 1:10 ratio with AAV.CMV.miR204) is shown by the EGFP fluorescence (green). Evident differences in retinal structure and expression of PR markers were not observed. ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium. Scale bar: 50 μm.