Tandem array

(+)

Non-targeting control

Dual hU6 promoter

Dose:

dSeq212mg1 mg7

hU6hU6

EFS-dSeq212

dSeq212mg1 mg7

hU6

EFS-UBB-dSeq212

Exon 12 excluded

Exon 12 included

EFS-dCasRx

A01271A01270 A01285

Novel RNA-Targeting Gene Therapy Approach for Usher’s Syndrome Type II Retinitis PigmentosaDaniel Gibbs, Rea Lardelli, Greg Nachtrab, Daniela M. Roth, Shawn Lee, Claire Geddes, Alistair Wilson, Nandini Narayan, Dimitrios Zisoulis, Ranjan Batra

Locanabio, Inc. | 3545 John Hopkins Ct Ste 200, San Diego, CA 92121

Ex.13 spliced Usherin

WT. UsherinPDZ1

Transmembranedomain

FN3 repeats

LAM-FGF repeats

LAM-G repeats

LAM-G like domain

LAM-N term domain

FN3 repeats

FN3 repeats

Nuclease inactive dCas13d+single gRNAs show highly efficient Exon 13 skipping of a human USH2A minigene in HEK293T cells

Nuclease inactive dCas13d+dual gRNAs show efficient Exon 12 skipping of a mouse Ush2a minigene in HEK293T cells

AAV8-dCas13d tandem guide vector drives robust dCas13d expression and exon 12 skipping in mouse photoreceptors

Mouse guide 1 (mg1) +mg4 mg5 mg6 mg7 mg8 mg9 NT No guideNT

Exon 13Exon 11 Exon 12

Exon 13Exon 11

Exon 13Exon 11 12

770 bp

*

Partial exon 12 exclusion

Mouse minigene

CONE OPSINRHODOPSIN

CONE OPSINRHODOPSIN

Exon 12 Exon 13 Exon 14U1 U2

Exon 12 Exon 13 Exon 14

Exon 12 Exon 13 Exon 14

USH2A pre-mRNA

USH2A spliced mRNA

Spliceosome intron removal

CD3 CD3

Sub retinal delivery of AAV8-dCAS13d+mg1/mg7 in mouse retina shows preservation of retinal structure and minimal T-cell infiltration

AAV8-dCAS13d+mg1/mg7 Un-injected control retina

dCas13d mRNA RNAScopeDAPI

BaseScopeExon 12 splicedExon 12 intact

Mechanism of action of nuclease inactive CRISPR/ dCas13d mediated skipping of USH2A Exon 13 (Exon 12 in mouse Ush2a)

USHER SYNDROME 2A (USH2A)

§ Syndromic USH2A is characterized by moderate to severe high-frequency hearing impairment and progressive visual loss due to retinitis pigmentosa

§ Caused by mutations in the USH2A gene resulting in a lack of functional Usherin protein

§ Usherin is a very large transmembrane protein important for the structural integrity of the photoreceptor periciliary region and outer segment (Liu et al., 2007, Dona et al., 2018).

§ Mutations in exon 13 account for ~35% of all USH2A cases (~15% of autosomal recessive retinitis pigmentosa)

§ Our nuclease inactive dCas13d mRNA-targeting approach aims to prevent vision loss and restore vision in patients with USH2A exon 13 mutations by mediating in-frame skipping of exon 13

§ 16,000 patients in US, EU, Canada with no approved treatment for vision loss associated with Usher Syndrome

dCas13d mRNARNAScopeDAPI

BaseScopeExon 12 splicedExon 12 intact

AAV8-dCAS13d+mg1/mg7 Uninjected control retina

In-frame stop codon. No protein expressed

Exon 12 Exon 13 Exon 14U1 U2

Exon 12 Exon 13 Exon 14

Exon 12 Exon 14

USH2A pre-mRNA

USH2A exon 13 skippedmRNA

CRISPR/dCas13d inhibition of exon 13 splicing

Normal processing of USH2A Exon 13 mutant mRNA

SR

SR

U1 U2 SR

dCas13d mediated skipping of USH2A Exon 13 mutant mRNA

Ex.13 skipped Usherin

c.2299DG

c.2299DG

c.2299DG

c.2299DG

c.2299DG

dCas13d+gRNA

Exon 12 Exon 14

Exon 12 Exon 14

g8 g1 g2/g3 g6 g7 g4/g5

g7g9

g10

NTg7g6 g8

500 ng dCas13d + 500 ng guide + 50 ng minigene

NTg4g3 g5g2g1Exon 14Exon 13Exon 12

860 bp

208 bp

Exon 14Exon 12

g7g9

dCas13d guide screening identified human g7 as highly potent for Exon 13 skipping

dCas13d g7, g9 and g10 unitary vectors show dose dependent Exon 13 skipping

dCas13d dual guide selection identifies mg1+mg7 as highly potent for mouse Exon 12 skipping

1000 500 250 100 1000 500 250 100 1000 500 250 100 1000 500 250 100

Exon 11 Exon 12 Exon 13

AAV5 AAV8 AAV9 AAVrh10

CAG EGFP WPRE

EFS-Ubb dCas13d WPREU6

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

6.0%

AAV8-dCas13d-mg1/mg7 uninjected

% p

hoto

rece

tors

per m

m

retin

a w

ith E

xon

12 e

xclu

sion

Exon 12 skipping in treated vs. control photoreceptors

EFS-Ubb dCas13d WPREU6

• AAV5, 8, 9 or Rh10 vectors were obtained from Signagen and delivered by subretinal injection to WT BALB/c mice at 1e9 vg/eye

• Histology was performed 4 weeks post injection using 12 µm retinal cryosections

• Native EGFP expression was compared across serotypes to assess spread and photoreceptor targeting

• AAV8-U6-mg1/mg7-dCAs13d vector was manufactured in-house and delivered by subretinal injection to WT BALB/c mice at 1e9 vg/eye

• Histology was performed 4 weeks post injection

• dCas13d mRNA expression was assessed by RNAscope probes specific for Ush2a

• Ush2a splicing was assessed by duplex BaseScope with probes for spliced (Exon11/13) and intact (Exon11/12) mRNA

mg1 mg7

dCas13d+g7 Non-targeting

mg1mg9mg4mg8mg6

mg7mg5

©2021 Locanabio, Inc

Skipping of USH2A exon 13 results in a minimally truncated but fully functional Usherinprotein missing only Laminin FGF repeats 5-8

dCas13d+g9 dCas13d+g10

Human minigene

Human minigene

exon 12 exclusion

Subretinal delivery of AAV serotypes identified AAV8 as the most effective for rod/cone photoreceptor transduction and vector spread

1000 500 250 100 1000 500 250 100 1000 500 250 100 1000 500 250 100

Summary and Conclusions• 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.

• AAV8-U6-mg1/mg7-dCAs13d vector was manufactured in-house and delivered by subretinal injection to WT BALB/c mice at 1e9 vg/eye

• Immunohistology was performed 4-weeks post injection on 12 µm retinal cryosections

• Cone photoreceptors were immunolabeled with the anti-cone opsin pAb (AB5405. 1:500)

• Rod photoreceptors were immunolabeled with the anti-rhodopsin mAb (MAB5216. 1:500 )

• T Cells were immunolabeled using the anti-CD3 mAb(E4T1B. 1:250)

Native EGFPDAPI

References1. Dona M, Slijkerman R, Lerner K, Broekman S,

Wegner J, Howat T et al. Usherin defects lead to early-onset retinal dysfunction in zebrafish. Experimental eye research. 2018; 173:148-159.

2. Liu X, Bulgakov OV, Darrow KN, Pawlyk B, Adamian M, Liberman MC, et al. Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells. Proc Natl Acad Sci USA 2007; 104(11):4413 8.

Unitary control: dCas13d

U6 non-targeting control guide

ng:

dose: (ng):

Unitary 2:dCas13d

Dual U6 promoter mg1+ mg7

Unitary 3: dCas13d

Tandem arraymg1+ mg7

Unitary 1:dCas13d

Dual U6 promoter mg1+ mg7

Exon 13

Exon 13

200µm 200µm

200µm 200µm

200µm 200µm

g1 g2 g3 g4 g5 g6 g7 g8 nt0

50

100

150

guide

% E

xon

13 e

xclu

ded

mg1 +

nt

mg1 +

mg4

mg1 +

mg5

mg1 +

mg6

mg1 +

mg7

mg1 +

mg8

mg1 +

mg9 nt

no guid

e0

50

100

150

guide combinations

% E

xon

12 e

xclu

ded

100 ng 250 ng 500 ng 1000 ng0

50

100

150

dose of unitary

% E

xon

12 e

xclu

ded

unitary 1

unitary 2

unitary 3

non-targeting

100 ng 250 ng 500 ng 1000 ng0

50

100

150

dose of unitary

% E

xon

13 e

xclu

ded

g7 g9 g10 non-targeting

200µm 200µm

200µm

Non-specific baseline Exon 13 exclusion occurs with human minigene in HEK293T cells

Single vectors expressing dCas13d+gRNAs show dose dependent Exon 13 skipping