Slide 1: Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Genome Editing
Therapeutic Correction of an LCA- Causing Splice Defect in the CEP290 Gene by CRISPR/Cas- Mediated Genome Editing Maxwell N. Skor editasmedicine.com © 2017 Editas Medicine 1
Slide 2: CEP290 in Ciliary Trafficking and Phototransduction
CEP290 in Ciliary Trafficking and Phototransduction WT Photoreceptor LCA10 Photoreceptor Degenerated discs Discs Protein trafficking Outer segment Inner segment Outer segment CEP290 No protein Connecting cilium trafficking Inner segment It is estimated that visual acuity can be achieved with ~10% of
Slide 3: Gene Editing to Repair CEP290 Splicing Defect
Gene Editing to Repair CEP290 Splicing Defect Exon 26 128bp Exon 27 Cys998X mRNA Exon 26 Exon 27 Correct CEP290 protein Transcription and splicing CEP290 (IVS26 c.2991 + 1655 A>G) * TAGT AATTGTGAGT DNA Exon 26 Exon 27 Gene Editing Edited DNA Exon 26 Exon 27 Transcription and splicing mRNA Exon 26 Ex
Slide 4: Targeted Deletion in Patient Fibroblasts
Targeted Deletion in Patient Fibroblasts gRNA1 gRNA2 Exon 26 * Exon 27 Targeted deletion of SaCas9 IVS26 mutation-containing region of intron 26 gRNA1 gRNA2 gRNA1 gRNA2 Unedited Exon 26 Exon 27 allele * gRNA1 gRNA2 Exon 26 Exon 27 * IVS26 mutation Targeted deletion Exon 26 Exon 27 Nucleofect IVS26 f
Slide 5: Editing Restores CEP290 Expression in Patient Fibroblasts
Editing Restores CEP290 Expression in Patient Fibroblasts CEP290 mRNA CEP290 CEP290 mRNAmRNA WT 5 5 WT WT Mut Mut Mut **** **** **** 4 4 **** **** **** **** **** **** **** **** **** **** **** Relative CEP290 mRNA 3 3 Expression * 2 **** 2 **** **** **** **** **** **** **** **** **** **** **** **** *
Slide 6: Total Editing Events Include Inversions
Total Editing Events Include Inversions Inversion gRNA1 gRNA2 Exon 26 * Exon 27 Indels Indels Deletion 80 Large Inversions % Editing of Total Alleles 70 18 Large Deletions 60 Small Indels UDiTaS sequencing method was 50 used to accurately quantify all 40 gene editing events in U2OS cells 40 (Please
Slide 7: Construction of GFP Reporter Construct: Is the Inversion Event Functional?
Construction of GFP Reporter Construct Is the Inversion Event Functional? gRNA1 gRNA2 WT CEP290 G GFP CMV FP intron 26 CEP290 CEP290 ex26 SD ex27 SA gRNA1 gRNA2 IVS26 CEP290 * CMV G FP G 128bp FP intron 26 CEP290 CEP290 ex26 SD ex27 SA Correct Splicing as Determined by GFP Expression 20 18 16 14 Rel
Slide 8: Targeted Deletions and Inversions Correct Splicing
Targeted Deletions and Inversions Correct Splicing Deletion CMV G FP GFP CEP290 CEP290 ex26 SD ex27 SA Inversion CMV G gRNA2 * gRNA1 FP ? CEP290 CEP290 ex26 SD ex27 SA Correct Splicing as Determined by GFP Expression 20 18 16 14 Proof of concept that we are able to Inversions reach >15% are producti
Slide 9: In vivo CEP290 Editing in Non-Human Primates
In vivo CEP290 Editing in Non-Human Primates Cyno macaque injected sub-retinally with 4E11 vg of AAV5-NHPCEP290gRNAs-GRK1-SaCas9 Vehicle 40 GCL Inversions 40 IPL Deletions 7.01 in Retina AAV Insertions INL 15% 15.47 30 Indels 30 8.46 OPL % Editing % Editing ONL 3.03 20 20 6.46 5.89 % Editing 40 3.43
Slide 10: Developing a Human Retinal Explant System
Developing a Human Retinal Explant System 3 mm Remove Neural punches Retina Obtain Human Eyes 3-5 hrs Post-Mortem Histology Retina were collected 28 days post-transduction UDiTaS In a 24-well format Transduced retina © 2017 Editas Medicine 10
Slide 11: Preliminary Data Demonstrating Editing of CEP290 in Human Retinal Explants
Preliminary Data Demonstrating Editing of CEP290 in Human Retinal Explants Human retina transduced with AAV5-GRK1-GFP High ONL Low % Editing INL High dose 28 day Ctrl P1 P2 P3 P4 P5 Low dose = 1e11 vg Targeted CEP290 gene editing in High dose = 5e11 vg mature human photoreceptors © 2017 Editas Medic
Slide 12: Towards a Therapy for LCA10
Towards a Therapy for LCA10 ▪ LCA10 patient fibroblast experiments demonstrate proof of concept for a CRISPR- based gene editing approach to treat LCA10 caused by the IVS26 splice mutation. ▪ In vivo experiments support pre-clinical development of a LCA10 therapeutic. ▪ Development of a human retina
Slide 13: Acknowledgements
Acknowledgements © 2017 Editas Medicine 13