Abstract

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

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Code availability

Custom analysis software is freely available at the following websites: https://github.com/editasmedicine/uditas https://github.com/editasmedicine/digenomitas

Data availability

Detailed data analysis is available in the Supplementary Tables published with this manuscript. All requests for raw data are promptly reviewed by the CSO or CTO of Editas Medicine, and any data that can be shared will be released tothe requestor. WGS data are available via the NCBI SRA database under accession code PRJNA497602.

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Acknowledgements

We thank B. Tucker and colleagues at the University of Iowa for the LCA10 patient fibroblasts, and the patients for their generous donation of biopsy samples. We thank B. Rabe for advice and technological assistance in establishing retinal explant model. We thank M. McCartney and N. Sprehe from Lions Eye Institute for Transplant and Research for invaluable help and services with human retinal explant experiments, and we thank the donors and their families for their generous donation. We thank our Editas colleagues P. Baciu, D. Balderson and G. Cox for helpful discussions and scientific input, and K. LeClair, W. Jaworowicz and G. Wilmes for helpful discussions and for manufacturing expertise and materials supply. This work was fully funded by Editas Medicine.

Author information

Author notes

  1. These authors contributed equally: Morgan L. Maeder, Michael Stefanidakis.

Affiliations

  1. Editas Medicine, Cambridge, MA, USA

    • Morgan L. Maeder
    • , Michael Stefanidakis
    • , Christopher J. Wilson
    • , Reshica Baral
    • , Luis Alberto Barrera
    • , George S. Bounoutas
    • , David Bumcrot
    • , Hoson Chao
    • , Dawn M. Ciulla
    • , Jennifer A. DaSilva
    • , Abhishek Dass
    • , Vidya Dhanapal
    • , Ari E. Friedland
    • , Georgia Giannoukos
    • , Sebastian W. Gloskowski
    • , Alexandra Glucksmann
    • , Gregory M. Gotta
    • , Hariharan Jayaram
    • , Scott J. Haskett
    • , Bei Hopkins
    • , Joy E. Horng
    • , Shivangi Joshi
    • , Eugenio Marco
    • , Rina Mepani
    • , Deepak Reyon
    • , Terence Ta
    • , Diana G. Tabbaa
    • , Steven J. Samuelsson
    • , Shen Shen
    • , Maxwell N. Skor
    • , Pam Stetkiewicz
    • , Tongyao Wang
    • , Clifford Yudkoff
    • , Vic E. Myer
    • , Charles F. Albright
    •  & Haiyan Jiang
  2. Fulcrum Genomics, Somerville, MA, USA

    • Tim J. Fennell

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Contributions

M.L.M., M.S., C.J.W., D.B., A.G., P.S., V.E.M., C.F.A. and H.J. conceived the strategy and designed experiments. M.L.M., R.B., A.D., A.E.F., S.W.G., J.E.H., S.J., R.M., D.R., S.S. and M.N.S. designed, performed and analyzed in vitro experiments. M.L.M., M.S., G.S.B., H.C., S.S., C.Y. and H.J. designed, performed and analyzed in vivo experiments. S.J.H., C.F.A. and H.J. designed, performed and analyzed immunogenicity experiments. M.L.M., C.J.W., L.A.B., D.M.C., J.A.D., V.D., T.J.F., G.G., G.M.G., H.J., E.M., T.T., D.G.T., T.W. and V.E.M. designed, performed and analyzed specificity studies. B.H. and S.J.S. performed and analyzed histology studies. M.L.M., M.S., C.J.W., C.F.A. and H.J. wrote, and all authors reviewed, the manuscript.

Competing interests

C.J.W., R.B., G.S.B., D.M.C., J.A.D., A.D., V.D., A.E.F., G.G., S.W.G., G.M.G., B.H., S.J., E.M., D.R., T.T., D.G.T., S.J.S., S.S., P.S., T.W., C.Y., V.E.M. and C.F.A. are current employees and shareholders of Editas Medicine. M.L.M., M.S., H.C., L.A.B., D.B., A.G., S.J.H., H.J., J.E.H., R.M., M.N.S. and H.J. are former employees and shareholders of Editas Medicine and were employeed by Editas at the time this work was conducted. T.J.F. is a paid consultant of Editas Medicine. Editas Medicine has filed patents pertaining to the work described in this manuscript.

Corresponding author

Correspondence to Morgan L. Maeder.

Extended data

  1. Extended Data Fig. 1 Splicing reporter assay demonstrates functionality of deletions and inversions.

    a, Schematic depicting design of GFP reporter constructs to determine functionality of edits and ability to correct splicing. Cytomegalovirus promoter drives split GFP interrupted by a portion of CEP290 intron 26. Correct splicing is required to reconstitute GFP. b, Quantification of GFP expression (mean fluorescence intensity), normalized to PGK-mCherry also present on reporter plasmid, by flow cytometry following transfection into human U2OS cells. n = 3 independent transfections performed on separate days. Bars represent mean and standard deviation.

  2. Extended Data Fig. 2 In vitro validation of editing strategy in primary patient fibroblasts.

    a, Targeted deletion at the CEP290 locus in primary patient fibroblasts transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs, as quantified by Droplet Digital PCRR. Left, cell line IVS26#36, n = 4 independent transfections performed on different days, error bars represent standard deviation. Right, cell line IVS26#35, n = 1. b, Quantification of WT 26-27 (blue) and mutant 26-X-27 (orange) CEP290 mRNA transcripts in IVS26#35 primary patient fibroblasts by qRT–PCR. CEP290 expression is normalized to beta-actin. n = 2 independent transfections performed on different days, qRT–PCR run in triplicate. Line represents mean. c, representative western blot from IVS26 primary patient fibroblasts transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs. Experiment was performed twice in each cell line with similar results. d, Quantification of CEP290 full-length protein expression in IVS26#36 and -#35 cell lines transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs. Expression level fold change over control calculated by densitometry of western blot bands and normalized to control. n = 4 biological replicates (two different transfections on different days, each in the two different cell lines), error bars represent mean and standard deviation. Source data Source data

  3. Extended Data Fig. 3 Specificity profiling of CEP290 gRNAs 323 and 64.

    a, Digenome-Seq was used to identify cuts across the genome. The positive control, SpCas9-EMX1, showed dose responsiveness with an increased number of cut sites as RNP concentration increased. No cut sites, other than the on-target site, were identified for guide 64 and only one cut site was detected at the highest concentration (1,000 nM) for guide 323. That candidate off-target site was located in intron 2 of gene CTD-2058B24.2, an 'anti-sense RNA' with no ascribed function; the cut site is close to, but distinct from, an annotated DNase-hypersensitive region. The raw data associated with this figure are available in Supplementary Tables 6 and 7. b, Targeted amplicon sequencing of candidate off-target sites in cell lines nucleofected with plasmids and human retinal explants transduced with EDIT-101. Off-target sites are plotted on the x axis and grouped into categories (on-target sites, detection below the lowest level of detection, and detection above the lowest level of detection with no change relative to control), then sorted by mean indel percentage. The y axis is a log scale plot of indels detected at the predicted cut site ±2 bases. The raw data associated with this figure are available in Supplementary Table 8. c, Summary table of candidate off-target site-targeted sequencing. See detailed list in Supplementary Table 8.

  4. Extended Data Fig. 4 Transduction and editing efficiency of mouse neural retina by subretinal injection of 1 μl of AAV5 vectors.

    a, A representative image of a flat-mounted retina from an HuCEP290 IVS26 KI mouse administered AAV5-GKR1-GFP. The red line outlines the retina, and the GFP-positive area is colored white. b, Total editing rates, as quantified by UDiTaS, in genomic DNA isolated from either total retinal cells or fluorescent-activated cell sorter-isolated GFP-positive retinal cells following subretinal injection of EDIT-101 and AAV5-GRK1-GFP in mice. Each bar represents one mouse eye.

  5. Extended Data Fig. 5 Comparison of human CEP290 gRNAs and NHP surrogate guides.

    HuCEP290 IVS26 KI mice were subretinally injected with 1 μl of either EDIT-101 (blue) or NHP surrogate vector (VIR067, orange) at varying doses. Total editing was quantified by UDiTaS. Each point represents a single mouse eye and error bars represent mean and standard deviation. There was no significant difference found between EDIT-101 and VIR067 at any dose.

  6. Extended Data Fig. 6 Localization of AAV genomes and SaCas9 protein in NHPs subretinally injected with VIR026.

    a,b, Detection of AAV5 vector genome in photoreceptor cells of NHP retina from animals treated with either 1 × 1011 vg ml–1 (a, animal no. I16464) or 1 × 1012 vg ml–1 (b, animal no. I16467) and assayed at 13 weeks post-dosing. In situ hybridization with a probe specific for the vector genome shows positive staining enriched in the outer nuclear layer (red, arrow). The area of positive staining was quantified on ×20 stitched tiles. Scale bar, 100 μm. The experiment was performed on six retinas from six different animals treated with VIR026. c,d, Anti-Cas9 immunohistochemistry in monkey no. I6467 showing positive staining in the photoreceptor nuclear layer (arrows) within the bleb region (c) but not outside of the bleb on the opposite side of the optic nerve (d). ×20 stitched tiles. Scale bar, 50 μm. The experiment was performed on six retinas from six different animals treated with VIR026. e, Detection of AAV5 vector genome in photoreceptor cells of NHP 1012 OD showing positive staining encompassing the foveal area (red). ×20 stitched tiles. OD: right eye.

  7. Extended Data Fig. 7 Immunogenicity assessment of AAV5-CRISPR/Cas9-based in vivo genome editing in NHPs.

    a,b, Antibodies against AAV5 capsid protein (a) and Cas9 protein (b) were measured in sera from the study animals using a Luminex bead-based assay. Results are presented as concentration of IgG against AAV5 and Cas9 in each individual animal’s serum at each time point. All samples were run in triplicate. c,d, ELISpots were performed to measure Cas9-specific CD8 T-cell responses (c) and Cas9-specific CD4 T-cell responses (d). Peripheral blood mononuclear cells from individual animals were stimulated with Cas9 peptide pools and assayed for IFN-γ production. Data are presented as the number of IFN-γ spot-forming cells per million cells.

  8. Extended Data Fig. 8 Inhibition of anti-Cas9 and anti-AAV5 antibody binding with excess antigen.

    a,b, To confirm antibody specificity, animal serum was pre-incubated with excess AAV5 capsid protein, 1 × 1013 viral particles ml–1 (a) or excess Cas9 protein, 160 µg ml–1 (b). Reduction in antibody binding, denoted by a decrease in median fluorescence intensity, was measured using the Luminex bead platform.

  9. Extended Data Fig. 9 Ocular tolerability.

    Scoring of anterior and posterior changes based on modifications of the Standardization of Uveitis Nomenclature, Hackett–McDonald and Semi-quantitative Preclinical Ocular Toxicology Scoring systems.

  10. Extended Data Fig. 10 Representative fundus image.

    Representative fundus images from animal no. 1005 (treated OD with EDIT-101 at 1 × 1012 vg ml–1 and OS with vehicle) at pre-test, post-dosing and days 8 and 59, showing submacular bleb formation. Fundus imaging was performed with similar results for all 16 NHPs reported in this study, and is detailed in Supplementary Table 9. OD: right eye; OS: left eye.

Supplementary Information

  1. Supplementary Information

    Supplementary Results

  2. Reporting Summary

  3. Supplementary Tables

    Supplementary Tables 1–12

Source data

  1. Extended Data Fig. 2

    The unprocessed gel image for the CEP290 portion of Extended Data Figure 2c

  2. Extended Data Fig. 2

    The unprocessed gel image for the GAPDH portion of Extended Data Figure 2c

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https://doi.org/10.1038/s41591-018-0327-9