Mukherjee ASGCT USH2A Poster Final

In Vivo Proof of Concept for EDIT-102: A CRISPR/Cas9-Based Experimental Medicine for USH2A- Related Inherited Retinal Degeneration Caused by Mutations in Exon 13 P# 719 Swati Mukherjee, Anila Mehta, Dawn Ciulla, James Bochicchio, Georgia Giannoukos, Eugenio Marco, George Bounoutas, Shengfang Jin, Andrea de Erkenez, Cecilia A. Fernandez, Charles F Albright, Kate Zhang, Carrie M. Margulies 11Hurley Street, Cambridge, MA 02141 ׀ .Editas Medicine, Inc

Usher Syndrome Type 2A and EDIT-102 Results

• Mutations in the USH2A gene are a major cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome Type II (USH2A). Patients with USH2A mutation have early hearing loss, progressive peripheral vision loss, and eventual legal blindness. Normalized Productive Editing of USH2A Gene and Cas9 mRNA/gRNA Expression Increased with the Increase of EDIT-102 dose • USH2A gene encodes the transmembrane protein Usherin which is localized in connecting cilia of photoreceptors and believed to play a role in stabilizing the photoreceptor outer segment Dose Response Study Design A. B. C. • c.2299delG mutation in exon 13 is the most common mutation in USH2A gene, causing a frameshift and truncated protein, which leads to ciliary Dosing Dose Number of Groups 10

defect. It is a common cause of inherited retinal degeneration (IRD). Regimen (vg/ml) Retinas 40 10 9 10

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• There are currently no approved disease-modifying therapies for RP caused by USH2A mutations 1 Vehicle 0 10 R

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• We have developed EDIT-102, an experimental medicine for USH2A-related IRD through CRISPR/Cas9-mediated excision of USH2A exon 13. s

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• EDIT-102 is an AAV5 vector expressing S. aureus CRISPR/Cas9 driven by the photoreceptor-specific G protein-coupled receptor kinase 1 (GRK1) g 7 R 20

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Unedited or N M small indels M 5 7 6 EDIT-102 1E+13 31 0 10 10 Vehicle 1e11 3e11 1e12 3e12 1e13 1e11 3e11 1e12 3e12 1e13 1e11 3e11 1e12 3e12 1e13 Dose Concentration (vg/mL) Dose Concentration (vg/mL) Dose Concentration (vg/mL) Figure 4: Dose Response Study For EDIT-102. The figure shows the normalized productive editing rates (Median ± 95% confidence interval) (A), Cas9 mRNA (B) and gRNA (C) expression levels in hUSH2A2299delG KI mice. Each dot represents a single retinal sample and the dotted line represents 10% normalized productive editing of photoreceptors. The level of normalized productive editing in the mouse retina increases with the dose of EDIT-102 from 1e11 to 3e12 vg/mL and decreases at 1e13 vg/ml. We expect a therapeutic benefit when 10% foveal cone photoreceptors are productively edited (Geller 1992, Geller 1993). The dose response of EDIT-102 from 3e11 to 3e12 vg/ml achieve 10% or greater productive editing in the treated mouse EDIT-102 Figure 1: Mechanism of action of EDIT-102. *Ex13 in USH2A retinas. Kozak gene represents any exon 13 mutation that causes IRD, including c.2299delG. EDIT-102 encodes human U6 promoter, guide RNA (gRNA; RSQ9145 and RSQ9265), hGRK1 promoter, SV40 (Simian virus 40) SD/SA (splice 9145 U6 9265 hGRK1 SV40 pA donor/splice acceptor) sequence elements, NLS (nuclear localization U6 SD/SA SaCas9 Total Editing Correlates with gRNA and Cas9 sequence), Sa (Staphylococcus aureus) Cas9 (CRISPR-associated protein 9) Normalized Productive USH2A Gene Editing and Cas9 mRNA/gRNA Increased with Time and pA (polyadenylation signal). EDIT-102 edits on either side of USH2A exon mRNA Expression NLS NLS 13, resulting in removal of exon 13 from the genome and mRNA thereby creating a functional Usherin protein lacking amino acids 723-936. A. 40 Vehicle B. 50 10 3e11 vg/ml 10 3e11 vg/ml Cas9 gRNA expression 1e12 vg/ml Cas9 30 1e12 vg/ml 40 Cas expression 9 3e12 vg/ml Cas9

USH2A Humanized Knock-In (KI) Mouse Models ) 10 Methods 3e12 vg/ml l A e 3e11 vg/ml gRNA v N

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Retina (%) Rates Editing Total 0 § Around 17% of USH2A transcripts in 0 10 5 105 106 107 108 109 1010 2299delG SaCas9 & Week 1 Week 3 Week 6 Week 13 1 3 6 13 heterozygous hUSH2A mouse retinal tissue contain human RNA gRNA Time post-dosing (weeks) Expression (Molecules/µg RNA) exon 13, as measured by Nanostring. Extracted RT-qPCR Time post-dosing (weeks) A. Figure 5. Time course study for EDIT-102. The figure indicates the normalized productive editing of USH2A gene (A) and Cas9 mRNA Figure 6: Total USH2A Gene Editing Rates and Cas9 mRNA and Figure 3: Experimental Schematic for EDIT-102 Pharmacology Studies. 2299delG 2299delG and gRNA expression (B) over time. The Vehicle group includes 16 retinas and the dose 3e11, 1e12 and 3e12 vg/ml of EDIT-102 groups gRNA Levels in EDIT-102 Treated hUSH2A KI Mice. Each Mouse USH2A gDNA mRNA Heterozygous hUSH2A KI mice were subretinally injected with EDIT-102 or vehicle. At different time points, retinas were harvested and split equally for DNA & have 47, 48 and 48 retinas. Each point represents an individual mouse eye harvested at various timepoints from 1 to 13 weeks post- data point indicates a single mouse retina processed in EDIT-102 mEx 11 mEx 12 mEx 13 mEx 11 mEx 12 mEx 13 RNA assays. On-target USH2A gene editing was determined using the retinal DNA dosing. dose-response and time-course studies, 1, 3, 6, or 13-week post in a unidirectional targeted sequencing (UDiTaS) method. Expression levels of subretinal injection. Curve fitting between total non-normalized editing Humanized Mouse USH2A gDNA SaCas9 and guide RNA (gRNA) were evaluated using the retinal RNA in a real time and Cas or gRNA expression was fitted using a non-linear regression mEx 11 hEx 13 mEx 13 quantitative polymerase chain reaction (RT-qPCR) assay. The median level of normalized productive editing and expression level of Cas9 mRNA and gRNA, at the doses of 1e12 and 3e12 model using GraphPad Prism. Spearman rank correlation coefficient mEx 11 hEx 13 mEx 13 vg/ml, plateaued at 6 weeks and was maintained at a stable level over 13 weeks post dose. mEx 11 mEx 13 with P-value <0.0001.

Note: mEx 12 = hEx 13 Productive USH2A Gene Editing in EDIT-102 treated Mice § Subretinal injection of 1 μl of AAV5 vector AAV5-GKR1-GFP show 30% transduction of mouse neural retina in previous study (Maeder 2019). B. % Mouse Exon 12 § The total on-target edits is calculated by including large deletions, Conclusions References Acknowledgements 100 % Mouse Exon 11-13 Junction inversions, AAV/plasmid integrations and small indels. % Human Exon 13 § Inversions and deletions were previously defined as productive edits § Pharmacokinetic and pharmacodynamic studies indicated Cas9 and gRNA § Geller AM, Sieving PA. Vision Res. 1993;33(11):1509–24. We would like to thank the following Editas teams for supporting that generate USH2A∆13 mRNA. Normalized productive editing is expression correlated with total editing. Dose response of EDIT-102 from 1e12 to this project: Sequencing, Screening, Sample Management, § Geller AM, Sieving PA, Green DG. J Opt Soc Am A. obtained by multiplying the percentage of productive editing with the 3e12 vg/ml achieved close to 10% or greater normalized productive editing in the Bioinformatics, Computational Biology, Regulators, and Scientific 1992;9(3):472–7. transduction multiplier, as shown below: mouse retinas. Communications. Additionally, we would like to thank Clifford 50 Figure 2: Generation and § Giannoukos G, et al. BMC Genomics. 2018;19(1). Characterization of USH2A humanized Yudoff, Binit Kumar and Mrudula Donepudi for valuable scientific § All animals used in the pharmacological studies demonstrated tolerability as KI mouse model (hUSH2A2299delG). Transduction area of retina: 30% § Liu X,et al. Proc Natl Acad Sci U S A. 2007;104(11):4413–8. insight. Graphic design support was provided by Robert Brown. The control mouse and heterogenous KI Transduction multiplier: 100/30 = 3.3 determined by clinical observations and body weight measurement. § Maeder ML, Stefanidakis M, et al. Nat Med [Internet]. % USH2A mRNA Species USH2A mRNA was isolated and quantified 2019;25(2):229–33. Available from: Disclosures: Normalized Productive Editing = Productive Editing * 3.3 § These data support the continued development of EDIT-102 for the potential Employees and shareholders of Editas Medicine: S.M., A.M., D.C., J.B., 0 by Nanostring. http://dx.doi.org/10.1038/s41591-018-0327-9 Control Het 2299dG treatment of USH2A-related IRD caused by mutations in exon 13. G.G., E.M., S.J., A.D.E., C.F.A., K.Z., and C.M.M.