therapeutic correction of an lca- causing splice defect in the … · 2020-05-14 · © 2017 editas...

1© 2017 Editas Medicine© 2017 Editas Medicineeditasmedicine.com

Therapeutic Correction of an LCA-Causing Splice Defect in the

CEP290 Gene by CRISPR/Cas-Mediated Genome Editing

Maxwell N. Skor

2© 2017 Editas Medicine

CEP290 in Ciliary Trafficking and PhototransductionO

uter

seg

men

tIn

ner s

egm

ent

WT Photoreceptor LCA10 Photoreceptor

Connecting cilium

Proteintrafficking

CEP290

Discs

1.Geller, Sieving and Green, J. Opt. Soc. Am., 19922.Geller and Sieving, Vision Res., 1993

It is estimated that visual acuity can be achieved with ~10% of functioning photoreceptors1,2

No protein trafficking

Inne

r seg

men

tO

uter

seg

men

t

Degenerateddiscs


Gene Editing to Repair CEP290 Splicing Defect

Exon 26 Exon 27

CEP290 (IVS26 c.2991 + 1655 A>G)

DNA AATTGTGAGTTAGT *

Transcription and splicing

mRNACorrect CEP290 protein

Exon 26 Exon 27 Cys998X128bp

Exon 26 Exon 27

mRNA

Transcription and splicing

Correct CEP290 proteinExon 26 Exon 27

Exon 26 Exon 27Edited DNA

Gene Editing


Targeted Deletion in Patient Fibroblasts

**** P < 0.0001*** P < 0.001

Guide RNA Pairs

% D

elet

ion

Rat

e

Ctrl A B C D E F G

SaCas9

gRNA1 gRNA2

Exon 26 Exon 27gRNA1 gRNA2

*IVS26 mutation

Nucleofect IVS26 fibroblasts with SaCas9 and gRNAs

Exon 26 Exon 27gRNA1 gRNA2

*

Exon 26 Exon 27

Targeted deletion of IVS26 mutation-containing

region of intron 26

Exon 26 Exon 27

gRNA1 gRNA2

*

Quantify deletion rate by ddPCR

Uneditedallele

Targeted deletion


Editing Restores CEP290 Expression in Patient Fibroblasts

Relative CEP290 Protein

Expression

Relative CEP290 mRNA

Expression

A B C D E F G GFP 0

1

2

3

4

5 WTMut****

**** ********

****

**** ****

****

****

****

********

****

CEP290 mRNA

Guide RNA Pairs **** P < 0.0001

A B C D E F G GFP 0

1

2

3

4

5 WTMut****

**** ********

****

**** ****

****

****

****

********

****

CEP290 mRNA

Guide RNA Pairs **** P < 0.0001

A B C D E F G GFP 0

1

2

3

4

5 WTMut****

**** ********

****

**** ****

****

****

****

********

****

CEP290 mRNA

Guide RNA Pairs **** P < 0.0001 ****P < 0.0001

Ctrl A B C D E F G

CEP290 mRNA

A B C D E F G GFP0

1

2

3

4

Guide RNA Pairs

CEP290 Protein

A B C D E F G GFP0

1

2

3

4

Guide RNA Pairs

CEP290 Protein

A B C D E F G GFP0

1

2

3

4

Guide RNA Pairs

CEP290 Protein

Ctrl A B C D E F G

Guide RNA Pairs

Guide RNA Pairs

CEP290 Protein


Total Editing Events Include Inversions

Exon 26 Exon 27

gRNA1 gRNA2

Deletion

Inversion

IndelsIndels

17

40

18

0

10

20

30

40

50

60

70

80

1

Large InversionsLarge DeletionsSmall Indels

% E

ditin

g of

Tot

al A

llele

s

UDiTaS sequencing method was used to accurately quantify all

gene editing events in U2OS cells(Please see Eugenio Marco’s Poster

for more details on UDiTaS)

*


Construction of GFP Reporter ConstructIs the Inversion Event Functional?

G

CEP290 ex26 SD

FPCMV

CEP290 ex27 SA

WT CEP290 intron 26

gRNA1 gRNA2GFP

G

CEP290 ex26 SD

FPCMV

CEP290 ex27 SA

IVS26 CEP290 intron 26

gRNA1 gRNA2G FP* 128bp

Correct Splicing as Determined by GFP Expression

RelativeGFP Expression

0

2

4

6

8

10

12

14

16

18

20

WT IVS26 Deletion Inversion

IVS26 mutation leads to aberrant splicing and a

non-functional GFP-signal


Targeted Deletions and Inversions Correct Splicing

Correct Splicing as Determined by GFP Expression

RelativeGFP Expression

G

CEP290 ex26 SD

FPCMV

CEP290 ex27 SA

Inversion

gRNA1gRNA2 *

G

CEP290 ex26 SD

FPCMV

CEP290 ex27 SA

Deletion GFP

Proof of concept that we are able to reach >15% functional editing in NHPInversions are productive

editing events

?

0

2

4

6

8

10

12

14

16

18

20

WT IVS26 Deletion Inversion


In vivo CEP290 Editing in Non-Human Primates

Proof of concept that we are able to reach >15% productive editing in NHP bulk retina and potentially as

high as 50% productive editing in photoreceptors

4.62 5.7710.48

16.671.25

1.80

5.21

5.89

1.301.61

3.43

8.46

1.741.92

3.03

7.01

0

10

20

30

40

6wk 6wk 13wk 6wk 6wk 13wk

GRK low GRK high

InversionsDeletionsAAV InsertionsIndels

% E

ditin

g

15.47

6.46

3.533.04

*

4.62 5.7710.48

16.671.25

1.80

5.21

5.89

1.301.61

3.43

8.46

1.741.92

3.03

7.01

0

10

20

30

40


GRK low GRK high


% E

ditin

g

15.47

6.46

3.533.04

*

Cyno macaque injected sub-retinally with 4E11 vg of AAV5-NHPCEP290gRNAs-GRK1-SaCas9Vehicle

AAV5-NHPCEP290gRNAs-GRK1-Cas9

RPEPOS

ONL

OPL

INL

IPL

GCL

RPE

POS

ONL

OPL

INL

IPL

GCL

Cas9

40X

40X

4.62 5.7710.48

16.671.25

1.80

5.21

5.89

1.301.61

3.43

8.46

1.741.92

3.03

7.01

0

10

20

30

40


GRK low GRK high


% E

ditin

g

15.47

6.46

3.533.04

*

15%

% E

ditin

g in

Ret

ina


Developing a Human Retinal Explant System

Transduced retinaIn a 24-well format

Remove NeuralRetina

3 mm punches

Obtain Human Eyes 3-5 hrsPost-Mortem

Histology

UDiTaS

Retina were collected 28 dayspost-transduction


Preliminary Data Demonstrating Editing of CEP290 in Human Retinal Explants

% E

ditin

g

Low dose = 1e11 vgHigh dose = 5e11 vg

Ctrl P1 P2 P3 P4 P5

Low

High

Targeted CEP290 gene editing in mature human photoreceptors

High dose28 day

ONL

INL

Human retina transduced with AAV5-GRK1-GFP


▪ 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 retinal explant assay demonstrates efficient editing in

mature human photoreceptors, which enables ongoing specificity studies in the therapeutically relevant cell type

Towards a Therapy for LCA10

CEP290 gene editing therapeutic has the potential to have a major impact on vision in LCA10 patients


Acknowledgements

therapeutic correction of an lca- causing splice defect in the … · 2020-05-14 · © 2017 editas...

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