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Novel RNA-Targeting Gene Therapy Approach for Usher's

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Novel RNA-Targeting Gene Therapy Approach for Usher's Novel RNA-Targeting Gene Therapy Approach for Usher’s Syndrome Type II Retinitis Pigmentosa Daniel Gibbs, Rea Lardelli, Greg Nachtrab, Daniela M. Roth, Shawn Lee, Claire Geddes, Alistair Wilson, Nandini Narayan, Dimitrios Zisoulis, Ranjan Batra ©2021 Locanabio, Inc Locanabio, Inc. | 3545 John Hopkins Ct Ste 200, San Diego, CA 92121 USHER SYNDROME 2A (USH2A) Nuclease inactive dCas13d+single gRNAs show highly efficient AAV8-dCas13d tandem guide vector drives robust dCas13d expression Exon 13 skipping of a huMan USH2A minigene in HEK293T cells and exon 12 skipping in Mouse photoreceptors dCas13d guide screening identified human g7 as highly potent for Exon 13 skipping Subretinal delivery of AAV serotypes identified AAV8 as the most effective for rod/cone photoreceptor transduction and vector spread § Syndromic USH2A is characterized by moderate to severe high-frequency hearing impairment and progressive AAV5 AAV8 AAV9 AAVrh10 visual loss due to retinitis pigmentosa Exon 12 Exon 13 Exon 14 Native EGFP CAG EGFP WPRE Human minigene g8 g1 g2/g3 g6 g7 g4/g5 DAPI § Caused by mutations in the USH2A gene resulting in a lack of functional Usherin protein 150 • AAV5, 8, 9 or Rh10 vectors were obtAined from SignAgen and delivered by subretinal § Usherin is a very large transmembrane protein important for the structural integrity of the photoreceptor 500 ng dCas13d + 500 ng guide + 50 ng minigene injection to WT BALB/c mice At 1e9 vg/eye • Histology wAs performed 4 weeks post g1 g2 g3 g4 g5 NT g6 g7 g8 NT 860 bp periciliary region and outer segment (Liu et al., 2007, Dona et al., 2018). 100 200µm injection using 12 µm retinal cryosections Exon 12 Exon 13 Exon 14 • Native EGFP expression was compared § Mutations in exon 13 account for ~35% of all USH2A cases (~15% of autosomal recessive retinitis pigmentosa) across serotypes to assess spread and 50 photoreceptor tArgeting AAV8-dCAS13d+mg1/mg7 Uninjected control retina § Our nuclease inactive dCas13d mRNA-targeting approach aims to prevent vision loss and restore vision in 208 bp mg1 mg7 patients with USH2A exon 13 mutations by mediating in-frame skipping of exon 13 % Exon 13 excluded Exon 12 Exon 14 dCas13d mRNA RNAScope dCas13d mRNARNAScope 0 DAPI DAPI g1 g2 g3 g4 g5 g6 g7 g8 nt Non-specific baseline Exon 13 U6 EFS-Ubb dCAs13d WPRE § 16,000 patients in US, EU, Canada with no approved treatment for vision loss associated with Usher exclusion occurs with human guide minigene in HEK293T cells Syndrome • AAV8-U6-mg1/mg7-dCAs13d vector wAs manufActured in-house and delivered by dCas13d g7, g9 and g10 unitary vectors show dose dependent Exon 13 skipping subretinAl injection to WT BALB/c mice At 1e9 vg/eye Exon 12 Exon 13 Exon 14 • Histology wAs performed 4 weeks post LAM-G like LAM-N term LAM-FGF FN3 LAM-G FN3 FN3 Transmembrane Human minigene g7 injection domain domain repeats repeats repeats repeats repeats domain • dCAs13d mRNA expression wAs assessed by g9 200µm 200µm RNAscope probes specific for Ush2a 150 Single vectors expressing PDZ1 g10 • Ush2a splicing wAs Assessed by duplex dCas13d+gRNAs show dose WT. Usherin BaseScope BaseScope BaseScope with probes for spliced dependent Exon 13 sKipping dCas13d+g7g9 dCas13d+g9 dCas13d+g10 Non-targeting Exon 12 spliced Exon 12 spliced (Exon11/13) And intAct (Exon11/12) mRNA 100 g7 ng: 1000 500 250 100 1000 500 250 100 1000 500 250 100 1000 500 250 100 Exon 12 intact Exon 12 intact Exon 12 skipping in treated vs. control photoreceptors 6.0% Ex.13 spliced Usherin 50 5.0% per mm % Exon 13 excluded 4.0% Skipping of USH2A exon 13 results in a minimally truncated but fully functional Usherin 0 3.0% protein missing only Laminin FGF repeats 5-8 100 ng 250 ng 500 ng 1000 ng 2.0% dose of unitary photorecetors 1.0% 200µm 200µm % g7 g9 g10 non-targeting 12 exclusion Exon with retina 0.0% AAV8-dCas13d-mg1/mg7 uninjected MechanisM of action of nuclease inactive CRISPR/ dCas13d Mediated Nuclease inactive dCas13d+dual gRNAs show efficient Exon 12 Sub retinal delivery of AAV8-dCAS13d+mg1/mg7 in mouse retina shows skipping of USH2A Exon 13 (Exon 12 in Mouse Ush2a) skipping of a Mouse Ush2a minigene in HEK293T cells preservation of retinal structure and minimal T-cell infiltration Normal processing of USH2A Exon 13 mutant mRNA dCas13d dual guide selection identifies mg1+mg7 as highly potent for mouse Exon 12 skipping AAV8-dCAS13d+mg1/mg7 Un-injected control retina CONE OPSIN CONE OPSIN USH2A pre-mRNA Exon 12 Exon 13 Exon 14 mg6 mg8 mg4 mg9 mg1 RHODOPSIN RHODOPSIN U6 EFS-Ubb dCAs13d WPRE c.2299DG Exon 11 Exon 12 Exon 13 Mouse minigene mg5 mg7 • AAV8-U6-mg1/mg7-dCAs13d vector wAs manufActured in-house and delivered by subretinal 150 Mouse guide 1 (mg1) + injection to WT BALB/c mice At 1e9 vg/eye U1 U2 SR U1 U2 SR Spliceosome intron removal Exon 12 Exon 13 Exon 14 NT mg4 mg5 mg6 mg7 mg8 mg9 NT No guide • Immunohistology wAs performed 4-weeks post injection on 12 µm retinal cryosections D c.2299 G • 100 Exon 11 Exon 12 Exon 13 Cone photoreceptors were immunolAbeled with the 770 bp anti-cone opsin pAb (AB5405. 1:500) • Rod photoreceptors were immunolAbeled with the USH2A spliced mRNA Exon 12 Exon 13 Exon 14 In-frame stop codon. No protein expressed 50 Partial exon 12 exclusion anti-rhodopsin mAb (MAB5216. 1:500 ) Exon 11 12 Exon 13 200µm 200µm • T Cells were immunolAbeled using the Anti-CD3 mAb D c.2299 G % Exon 12 excluded (E4T1B. 1:250) 0 * exon 12 exclusion CD3 CD3 nt Exon 11 Exon 13 References dCas13d mediated skipping of USH2A Exon 13 mutant mRNA 1. Dona M, Slijkerman R, Lerner K, Broekman S, mg1 + nt no guide mg1 + mg1mg4 + mg1mg5 + mg1mg6 + mg1mg7 + mg1mg8 + mg9 Wegner J, Howat T et al. Usherin defects lead USH2A pre-mRNA Exon 12 Exon 13 Exon 14 150 guide combinations to early-onset retinal dysfunction in UnitarY 1: UnitarY 2: UnitarY 3: UnitarY control: c.2299DG dCas13d dCas13d dCas13d dCas13d zebrafish. Experimental eye research. 2018; Dual U6 promoter Dual U6 promoter Tandem arraY U6 non-targeting 173:148-159. 100 EFS-UBB-dSeq212 dCas13d+gRNA mg1+EFS-dSeq212 mg7 mg1+ mg7 mg1+ mg7 EFScontrol-dCasRx guide 2. Liu X, BulgakoV OV, Darrow KN, Pawlyk B, unitary 1 Adamian M, Liberman MC, et al. Usherin is dose: (ng):Dose: 1000A01270 500 250 100 1000A01271 500 250 100 1000A01285 500 250 100 (+) 1000 500 250 100 CRISPR/dCas13d inhibition U2 SR required for maintenance of retinal Exon 12 U1 Exon 13 Exon 14 unitary 2 Exon 12 of exon 13 splicing 50 included photoreceptors and normal deVelopment of c.2299DG unitary 3 cochlear hair cells. Proc Natl Acad Sci USA % Exon 12 excluded 200µm 200µm non-targeting 2007; 104(11):4413 8. USH2A exon 13 sKipped 0 Exon 12 Exon 14 Ex.13 sKipped Usherin Exon 12 mRNA 100 ng 250 ng 500 ng 1000 ng excluded dose of unitary Non-targeting control hU6 hU6 hU6 mg1 mg7 dSeq212 mg1 mg7 dSeq212 Summary and DualConclusions hU6 promoter Tandem array • In vitro expression of nuclease inactive dCas13d+single gRNAs result in dose dependent skipping of USH2A exon13 with >98% efficacy in HEK293T cells expressing a human USH2A minigene • Dual gRNAs are required for mouse exon 12 skipping and result in dose dependent skipping of Ush2a exon12 with >98% efficacy in HEK293T cells expressing a mouse Ush2a minigene • Subretinal delivery of single AAV8 vectors packaging dCas13d with a tandem gRNA array targeting mouse Ush2a mRNA resulted in a >10-fold increase in photoreceptors with Exon 12 skipping compared to controls and was well tolerated 4wks post injection in mouse retina • These findings support the development of RNA-targeting gene therapies using highly efficacious CRISPR/dCas13d exon skipping in USH2A for patients with exon 13 mutations..
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