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