Fig 2: Representative Western blot analysis of C57BL/6 retinal lysates 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO PROMOTER). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of protein were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted. AK, retinas injected with dual AAV hybrid AK vectors; TS, retinas injected with dual AAV TS vectors; neg, retinas injected with AAV vectors expressing EGFP, as negative controls; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Representative pictures of immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with either dual AAV or with negative control vectors. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.Representative pictures of transmission electron microscopy analysis showing lipofuscin granules content in the RPE of WT and Abca4−/− mice injected with either dual AAV or negative control vectors. The black arrows indicate lipofuscin granules. The scale bar (1.6 μm) is depicted in the figure.Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25 μm2) for each sample. The number ( n) of eyes analyzed is depicted below each bar. The mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05; ** P ANOVA < 0.001. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.Data information: (B-D) WT, BALB/c eyes; Abca4−/− neg, Abca4−/− eyes injected with either AAV vectors expressing EGFP ( n = 2) or the 5′-( n = 3) or 3′-( n = 4) half of the dual AAV hybrid AK vectors, as negative control (neg total n = 9); Abca4−/− AK-ABCA4, mice injected with dual AAV hybrid AK vectors; Abca4−/− TS-ABCA4, mice injected with dual AAV TS vectors.

Fig 3: In vitro knockdown of human RHO-T17M expression.(A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and SaCas9/17-Sg2 were appeared to cut the mutant sequence specifically, the full-length amplicon was 760 bp, the two truncated amplicons were 510 bp and 250 bp, respectively. (C) The cutting efficacy of two sgRNAs with SaCas9 determined by TA and Sanger sequencing in 293T cells. (D) Rhodopsin expression reduction was determined by WB in RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, comparing to the RHOwt cells with 17-Sg1 and -Sg2 plasmid. (E) Densitometric analysis of immunoblots performed on RHOwt and RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, respectively. The experiment was performed in triplicate and presented as mean ± SEM, the significance was calculated using two-tailed paired t-test, ns = not significant, *p<0.05.

Fig 4: Improvement of the retinal function by MC-derived Rho-positive cells in MNU-treated mice.A) Scotopic responses of the ERGs on day 7 after the intravitreal injection of SLCD. ERG waveforms for the 75 mg/kg MNU group are shown in red, and those for the 30 mg/kg MNU group are shown in blue. B) Effect of the compounds on the a-wave in the 30 mg/kg MNU group. All data are presented as the mean ± SEM (DMSO, n = 8; SLCD, n = 12). *p < 0.05, as per Wilcoxon rank sum test.

Fig 5: Differentiation of MCs into Rho-positive cells in MNU-treated mice via intravitreal injection of SLCD.A) Time course of the in vivo experiments using MNU-treated mice. After a single systemic administration of 75 mg/kg MNU, intravitreal injection of SLCD was performed every 3 days. i.p., intraperitoneal injection; TUNEL, TdT-mediated dUTP nick end labeling. B) Immunohistochemistry results of Rho (arrowheads) and DAPI on days 3 and 7 after the intravitreal injection of SLCD into MNU-treated mice. White dotted lines indicate the anterior edge of the retina. Scale bar = 400 μm. C) Magnified images of Rho (red) and PNA (green) expression in (B) (n = 5). Scale bar = 100 μm. D) Quantitative results of the Rho-expressing retinal region in the total retinal length (B) (n = 5). E) Real-time qPCR results of the opsin and Rho expression in the whole retina on day 7 after the intravitreal injection of SLCD (n = 5). F) and G) TUNEL staining (F) and quantification of the TUNEL-positive photoreceptor cells (G) on day 3 after the intravitreal injection of SLCD (n = 5). Scale bar = 100 μm. H) The Rho-positive cells in the ONL (yellow arrowhead) also expressed BrdU on day 7 after the intravitreal injection of SLCD. Scale bar = 50 μm. Magnified images of yellow-framed are shown on the right (n = 5). Scale bar = 20 μm. I) Td-Tomato (red) was co-expressed with Rho (green) in the outer retinal layer on day 7 after intravitreal injection of SLCD into MNU-treated GFAP-td-Tomato mice (n = 5). Scale bar = 50 μm. J) Real-time qPCR results of the expression of Rho and other retinal neuron-specific markers in the td-Tomato-positive cell population on day 7 after the intravitreal injection (n = 5). K) PSD95 (white) was also expressed around Rho-positive cells between INL and ONL. Scale bar = 100 μm. A magnified image of yellow-framed is shown on the right (n = 5). Scale bar = 20 μm. OPL, outer plexiform layer. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, as per Wilcoxon rank sum test.