Fig 2: Visual Acuity Testing Demonstrates Recovery of Optokinetic Response in SOD2-KD Mice Treated with RPE65-Programmed BMDCsC57BL6/J mice received subretinal injection of AAV1-Rz-SOD2 or AAV1-Rz-inactive for 1, 3, or 6 months prior to tail vein injections of naive BMDCs, LV-LacZ, or LV-RPE65. Spatial frequency thresholds were calculated 3 months after systemic delivery of BMDCs using an Optomotor behavioral test of optokinetic response. (A–C) A comparison of average values for photopic activities in response to rotating sinusoidal gratings is shown for each group of mice (n = 5 per group) treated with cells (A) 1 month, (B) 3 months, and (C) 6 months following SOD2 knockdown. Values are expressed as the mean ± SEM.

Fig 3: HDR-mediated genome editing induced by CRISPR-Cas9 in the retina and RPE of rd12 mice.(A) A schematic drawing of the dual AAV strategy for CRISPR-Cas9–mediated therapeutic correction of the nonsense mutation in Rpe65 in rd12 mice. The premature stop codon in Rpe65 (red text) is generated by a C-to-T mutation in exon 3. The U6 and EFS promoters in the AAV vector were used to express the sgRNA together with the donor template and SpCas9, respectively. Green text, synonymous mutations in the donor template. Therapeutic gene correction could be derived from HDR-mediated precise correction (HDR) or in-frame NHEJ (Inf-NHEJ). (B and C) Indel frequencies measured by targeted deep sequencing in the retina (B) and RPE (C) of rd12 mice at 4 weeks after injection of high-dose (a total of 2 × 1011 vg per eye) and low-dose (a total of 2 × 1010 vg per eye) AAV. SpCas9–to–TS4 sgRNA-Rpe65-donor ratios are indicated in parentheses. (D) Correlation between the indel frequency and AAV copy number per diploid cell (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. (E) Frequencies of therapeutic HDR at the site of the C-to-T mutation in the retina and RPE of rd12 mice at 4 weeks after injection. (F) Ratio of HDR to indel frequencies. A high or low dose of SpCas9 and TS4 sgRNA-Rpe65-donor at a 1:1 ratio was injected subretinally (n = 4). (G) Mean values of the number of deep sequencing reads including insertions, deletions, or HDR in AAV-treated RPE (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. Rpe65mut, nonedited; Ins, insertion; Out-f-Del, out-of-frame deletion; Inf-Del, in-frame deletion. (H) Nucleotide sequences showing the Rpe65 donor template and changes induced by the TS4 sgRNA in AAV-treated RPE (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. Violet triangle, position of the DSB induced by the TS4 sgRNA; red text, sequences corresponding to the premature stop codon in rd12 mice; blue text, PAM sequences; green text, synonymous mutations in the donor template; underlined sequences, nucleotides encompassing the premature stop codon and their corresponding amino acid sequences. *P < 0.05; **P < 0.01; ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test (B, C, and E) and Mann-Whitney U test (F).

Fig 4: Genome editing of Rpe65 by CRISPR-Cas9 in vitro.(A) A schematic drawing of Rpe65 in rd12 MEF. Red text, sequence of the premature stop codon; orange arrow, TS4 sgRNA target sequence; blue text, protospacer adjacent motif (PAM). (B) TS4 sgRNA–induced indel frequencies measured by targeted deep sequencing (n = 4). ssODN dose (in microgram scale) are indicated in parenthesis. (C) Correction frequencies in the rd12 mutation measured by targeted deep sequencing (n = 4). (D) The mean values of the number of deep sequencing reads in different categories show the pattern of indels induced by the TS4 sgRNA (n = 4). Of the total reads, about 61% contain deletions. Ins, insertion; Del, deletion; WT, wild type. (E) Mean percent values of different types of in-frame and out-of-frame indels induced by the TS4 sgRNA (n = 4). 3N, one codon; 3N + 1, one codon + 1 nucleotide; 3N + 2, one codon + 2 nucleotides. (F) Nucleotide sequences showing types of editing induced by the TS4 sgRNA and 1.5 µg of ssODN in rd12 MEF (n = 4). Violet triangle, position of the DSB induced by the TS4 sgRNA; red text, sequences of the stop codon in rd12 MEF; blue text, PAM sequences; green text, synonymous mutations in the donor template; underlined sequences, nucleotides encompassing the premature stop codon and their corresponding amino acid sequences. Error bars indicate SEM. **P < 0.01; ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test.

Fig 5: GFP+ BMDCs Expressing RPE65 Colocalize with the Host RPE and Express RPE MarkersOne month after SOD2-KD, animals were treated systemically with naive BMDCs, LacZ-programmed BMDC, or RPE65-programmed BMDCs. (A–C) Representative fluorescent micrographs of flat-mounted posterior cups RPE/choroid (A and B) and paraffin sections (C) were stained for RPE65 (red) (A), CRALBP (B) (red), and GFP (green) to demonstrate colocalization. Note the significantly greater presence of GFP+ cells in animals receiving RPE65-programmed BMDCs. The cross-section of the RPE in (C) shows colocalized green (GFP+) and red (RPE65 or CRALBP+) cells at the RPE layer. Note the percentage of GFP+ cells that are mature and express CRALBP is much higher in the animals receiving RPE65-programmed BMDCs than in untreated control or LacZ-programmed BMDCs.