Discovery of EDIT-101 for the Treatment of Leber’s ...€¦ · presentation represent the...

1© 2018 Editas Medicine© 2018 Editas Medicineeditasmedicine.com

Discovery of EDIT-101 for the

Treatment of Leber’s Congenital

Amaurosis Type 10

Precision Genome Editing with Programmable Nucleases

January 2018

Charlie Albright

Chief Scientific Officer

2© 2018 Editas Medicine

Forward Looking Statements

This presentation contains forward-looking statements within the

meaning of the “safe harbor” provisions of The Private Securities

Litigation Reform Act of 1995. All statements, other than statements

of historical facts, contained in this presentation, including statements

regarding the Company’s strategy, future operations, future financial

position, future revenue, projected costs, prospects, plans, and

objectives of management, are forward-looking statements. The

words ‘‘anticipate,’’ ‘‘believe,’’ ‘‘continue,’’ ‘‘could,’’ ‘‘estimate,’’

‘‘expect,’’ ‘‘intend,’’ ‘‘may,’’ ‘‘plan,’’ ‘‘potential,’’ ‘‘predict,’’ ‘‘project,’’

‘‘target,’’ ‘‘should,’’ ‘‘would,’’ and similar expressions are intended to

identify forward-looking statements, although not all forward-looking

statements contain these identifying words. Forward-looking

statements in this presentation include statements regarding the

Company’s goals of submitting of an IND for the LCA10 program by

mid-2018, initiating an LCA10 natural history study in mid-2017,

achieving preclinical proof-of-concept for additional programs and

establishing alliances. The Company may not actually achieve the

plans, intentions, or expectations disclosed in these forward-looking

statements, and you should not place undue reliance on these

forward-looking statements. Actual results or events could differ

materially from the plans, intentions and expectations disclosed in

these forward-looking statements as a result of various factors,

including: uncertainties inherent in the initiation and completion of

preclinical studies and clinical trials and clinical development of the

Company’s product candidates; whether interim results from a clinical

trial will be predictive of the final results of the trial or the results of

future trials; expectations for regulatory approvals to conduct trials or

to market products; availability of funding sufficient for the Company’s

foreseeable and unforeseeable operating expenses and capital

expenditure requirements; and other factors discussed in the “Risk

Factors” section of the Company’s most recent Quarterly Report on

Form 10-Q, which is on file with the Securities and Exchange

Commission, and in other filings that the Company may make with the

Securities and Exchange Commission in the future.

In addition, the forward-looking statements included in this

presentation represent the Company’s views as of the date of this

presentation. The Company anticipates that subsequent events and

developments will cause its views to change. However, while the

Company may elect to update these forward-looking statements at

some point in the future, it specifically disclaims any obligation to do

so. These forward-looking statements should not be relied upon as

representing the Company’s views as of any date subsequent to the

date of this presentation.


Acknowledgements

Shannon Boye

Sanford Boye

Tyler McCullough

Paul Gamlin

C. Douglas Witherspoon Zuben Sauna


▪ Editing corrects splicing defect in cells from

LCA10-IVS26 patients and restores full-length

mRNA and protein

▪ EDIT-101 gRNAs are highly specific

▪ EDIT-101 and NHP-specific construct

efficiently edit photoreceptors in transgenic

mice and NHPs, respectively

▪ Immunogenicity studies suggest low levels of

anti-Cas9 antibodies in humans

Towards a CRISPR Medicine for LCA10

CEP290 gene editing medicine has the potential to have a

major impact on vision in LCA10 patients


CEP290 Essential for Ciliary Trafficking and

Phototransduction

Ou

ter

se

gm

en

tIn

ne

r se

gm

en

t

WT Photoreceptor LCA10 Photoreceptor

Connecting cilium

Protein

trafficking

CEP290

Discs

No protein

trafficking

Ou

ter

se

gm

en

t

Degenerateddiscs

Inn

er

se

gm

en

t


Photoreceptors Survive in LCA10 Patients

▪ It is estimated that visual acuity can be achieved with ~10%

of functioning photoreceptors1,2

Boye et al., PLOS ONE 2014

1.Geller, Sieving and Green, J. Opt. Soc. Am., 1992

2.Geller and Sieving, Vision Res., 1993

Outer nuclear layer

= photoreceptors


Gene Editing to Repair CEP290 Splicing Defect

DNA

mRNA

Protein

Exon 26 Exon 27*IVS26

Exon 26 Exon 27

323 64

Deletion

Inversion

*IVS26

CEP290 with IVS26 mutation CEP290 corrected with EDIT-101

Exon 26 Exon 27Exon 26 Exon 27X

Transcription

Translation

p.Cys998X

Prematurely truncated

and non-functional

Full length,

functional

CEP290


Identification of gRNAs that Remove IVS26 Mutation

128bp

cryptic

exon

*

IVS26

mutation

Alu Repeat

1000 bp upstream of Alu

41 NNGRRT PAMs

31 gRNAs (various lengths) tested

1000 bp downstream of mutation

29 NNGRRT PAMs

20 gRNAs (various lengths) tested

37 gRNA pairs tested

7 gRNA pairs selected

Exon 27Exon 26

0

5

10

15

20

25

%D

ele

tio

n

Guide RNA Pairs

A B C D E F G Ctrl


Comprehensive Specificity Assessment for Genome

Editing Medicines: METHODS

DETECTION

Discovery Verification

SC

OP

E

Small DNA

changes(within a single

chromosome, up to

~100 base pairs)

In silico modelingMeasure effect of

enzyme activity on

‘discovered’ sites

(Targeted PCR and

UDiTaS)

Biochemical approaches

(Digenome)

Cellular approaches

(GUIDE-Seq)

Large DNA

changes(within or between

chromosomes)

In silico modelingMeasure effect of

enzyme activity on

‘discovered’ large

changes

(ddPCR and UDiTaS)

Cellular approaches

(UDiTaS)


Robust Specificity Analysis in Multiple Model Systems

Identifies Molecules with No Detectable Off Targets

gRNA

Genome-Wide Cell BAsed Discovery Assay

(GUIDE-seq)

Targeted Measurement

Assays

Cell Type

1

Cell Type

2

Cell Type

3

Cell Type

4

# Off-

target

sites

Cell 1

# of sites

assayed

Cell 4

# of sites

assayed

# Off-

target

sites

11 ✓ ✓ ✓ ✓ 4 3 6 1

64 ✓ ✓ ✓ ✓ 0 63 83 0

323 ✓ ✓ ✓ ✓ 0 58 92 0

490 ✓ ✓ ✓ 0 48 74 0

492 ✓ ✓ ✓ 1 56 95 1

496 ✓ ✓ ✓ 0 58 72 0

502 ✓ ✓ ✓ 8 12 12 6

504 ✓ ✓ ✓ 0 53 71 0Non-

Specific

Control✓ 59


Digenome Assay with Lead Guides (64 and 323)

CEP290 Guide 323

on-target example

+

genomic DNA16

hours WGS Identify overabundant start sitesAlign &

analyze

CAS9 + Guide

Count of sites

Includes

on-target site


Editing Corrects CEP290 Splicing and Restores

mRNA and Protein Expression

0

20

40

60

80

100

120

Ctrl gRNAs323+64

Ctrl gRNAs323+64

IVS26#36 IVS26#35

% C

EP2

90

Exp

ress

ion

26-X-27

26-27

P = 0.0005 P = 0.02

Mutant (26-X-27)WT (26-27)

U6 323

U6 64

Sa Cas9CMV

+

+

CEP290

GAPDH

CEP290 mRNA Expression

CEP290 Protein Expression


Editing Causes Inversions, Deletions, and Indels

Exon 26 Exon 27

gRNA1 gRNA2

Deletion

Inversion

IndelsIndels

17

40

18

0

10

20

30

40

50

60

70

80

1

Large Inversions

Large Deletions

Small Indels

% E

ditin

g o

f Tota

l Alle

les

*


LCA10-IVS26 Mutation Decreases Splicing in a

Reporter System

G

CEP290

ex26 SD

FPCMV

CEP290

ex27 SA

WT CEP290

intron 26

gRNA1 gRNA2

GFP

G

CEP290

ex26 SD

FPCMV

CEP290

ex27 SA

IVS26 CEP290

intron 26

gRNA1 gRNA2

G FP* 128bp

Correct Splicing as Determined by GFP Expression

Relative

GFP 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

Relative

GFP Expression

G

CEP290

ex26 SD

FPCMV

CEP290

ex27 SA

Inversion

gRNA1gRNA2 *

G

CEP290

ex26 SD

FPCMV

CEP290

ex27 SA

Deletion GFP

?

0

2

4

6

8

10

12

14

16

18

20

WT IVS26 Deletion Inversion


EDIT-101: gRNAs plus SaCas9 in AAV5

U6 gRNA U6 gRNA Sa Cas9AAV5


Efficient Transduction and Editing of Mouse Retina by

Subretinal Delivery of EDIT-101

Transduction

efficiency by SR in

mouse retina:

24% (FACs) –

30% (Histo)

0

20

40

60

% P

roductive E

ditin

g

Whole Retina GFP+ Retinal Cells

GFP


Efficient Transduction of Photoreceptor Cells with

EDIT-101 in HuCEP290 KI Mice

AAV

ISH of AAV Vector Genome

IHC of Cas9 Protein

Counter-stained with

rhodopsin


Rapid Onset and Stable CEP290 Gene Editing by

EDIT-101 in HuCEP290 IVS26 KI Mice

Stable CEP290 Gene Editing

Over Time

0.42 1 2 4 8 16 260

5000

10000

15000

20000

Time Post Dosing (Weeks)

gR

NA

(m

ole

cule

s/c

ell)

*

*

*

0.42 1 2 4 8 16 260

1000

2000

3000

4000

5000

Time Post Dosing (Week)

Cas9 m

RN

A (

mole

cule

s/c

ell)

*

*

CRISPR/Cas9 Expression

Decreased Over Time

0.42 1 2 4 8 16 260

4

8

12

16

20

Time Post Dosing (Week)

Pro

ductive E

ditin

g (

%)

- M

edia

n *


Dose Response of EDIT-101 in HuCEP290 KI Mice

1010 1011 1012 1013 10140

20

40

60

80

EDIT-101 Dose Concentration (vg/mL)

% P

roductive E

dit /T

ransduced R

etina

Minimal Therapeutic

Target


Correlation of Productive CEP290 Editing Efficiency

and Expression Levels of Cas9 mRNA

10 100 1000 10000 1000000

20

40

60

80

100

Cas9 mRNA (Mol/Cell)

% P

roductive E

dit /T

ransduced R

etina

Minimal Therapeutic Target


Retinal Structural Differences Between Mice and

NHPs and Adjusted Productive Editing

Monkey Retina Mouse Retina

▪ No Macula

▪ Photoreceptors: 85-90% cells

▪ 97% of photoreceptors are rods

▪ Entire retina collected for analysis but

only 30% neural retina transduced with

1ml AAV5 vector

In both species, quantified productive edits (inversions and deletions) must be

multiplied by 3.5x to determine total productive editing in photoreceptors

▪ Macula

▪ Photoreceptors: 25-30% cells

▪ Most foveal photoreceptors are cones

▪ 8mm retinal punch covering most of the

bleb collected for analysis but only

photoreceptors (25-30%) express GRK1

AAV5-GRK1-GFP

~30%

GFP+

AAV5-NHPCEP290gRNAs-GRK1-Cas9

RPE

RPE

POS

ONL

OPL

INL

IPL

GCL

Cas9

40X

40X

AAV5-NHPCEP290gRNAs-GRK1-Cas9

RPE

RPE

POS

ONL

OPL

INL

IPL

GCL

Cas9

40X

40X

AAV5-cynoCEPgRNAs-GRK1-SaCas9

Cas9 IHC ISH of AAV vector genome

AAV

GCL

INL

ONL

Cas9 IF

Cas9

GCL

ONL

INL

6 months

EDIT-101

IPL

OPL


Productive Editing Exceeds Therapeutic Threshold in

NHPs

“Productive Editing” = Editing in photoreceptors

that will restore wildtype CEP290 splicing

Based on demonstration that Cas9 expression is limited to photoreceptors cells,

level of productive editing in photoreceptors may be as high as 50%

I164

64

I164

65

I164

66

I164

67

I164

68

I164

690

20

40

60

Adju

ste

d P

roductive E

dits (

%)

1E+11 vg/mL 1E+12 vg/mL

6 wk

13 wk


Editing Efficiency Comparison: EDIT-101 in Mice vs

Surrogate NHP Vectors in NHPs

1010 1011 1012 1013 10140

20

40

60

80

Vector Dose Concentration (vg/mL)

% A

dju

ste

d P

roductive E

dit

Minimal

Therapeutic

Target

Sirion NHP

Sirion NHP in NHP

NW EDIT-101

Sirion EDIT-101


Immunogenicity Analysis

▪ What is the prevalence of pre-existing humoral or cellular

immunity to Cas9 proteins in humans and preclinical species?

▪ Does in vivo delivered CRISPR/Cas9 system induce a humoral

or cellular immune response to Cas9 proteins in humans and

preclinical species?

▪ Will the pre-existing or induced humoral or cellular responses

affect efficacy or safety?

▪ Scientists at Editas and FDA are collaborating to address

these questions.


Assay to Detect Anti-Cas9 Antibodies in Human

Serum in Accordance with FDA Guidance

Training set: 48 human

donors

• Validate assay (Sensitivity,

Selectivity and Precision)

• Set cut point for screening

assay

• Set cut point for confirmatory

assay

Sample set: 200 human donors

• Determine the prevalence

of anti-Cas9 antibodies in a

sample of US population

• SAMPLES TESTED

POSITIVE IN SCREENING

ASSAY ARE SUBJECTED

TO THE CONFIRMATORY

ASSAY

Anti-Cas9 Ab [ng/mL]

OD

Cas9 protein [µg]

OD

SCREENINGCUT-POINT

Determine [Cas9] for the Confirmatory Competition

Assay

Determine ScreeningCut-point

Determine normality of

distribution

Outlier evaluation

-B-B -B -B

Cas9 proteinAnti-Cas9 Ab

Biotin-Protein G

An ELISA assay was developed using anti-SaCas9 and anti-SpCas9 antibodies

Zuben Sauna www.fda.gov

27© 2018 Editas MedicineCollaboration between Zuben Sauna (OTAT/CBER/FDA) and Editas Medicine

Pre-existing human anti-Cas9 antibodies

Human anti-Cas9 antibodies SaCas9 SpCas9

Screening assay 19%

(38/200)

4.5%

(9/200)

Confirmatory assay 5.5%

(11/200)

1.5%

(1/200)

These results show low levels of pre-existing anti-Cas9 antibodies in humans

28© 2018 Editas MedicineCollaboration between Zuben Sauna (OTAT/CBER/FDA) and Editas Medicine

Humoral Response to Cas9 and AAV5 Capsid

SaCas9 SpCas9 AAV5 Capsid

Pre-existing human anti-Cas9 5.5%

(11/200)

1.5%

(1/200)

Pre-existing NHP anti-Cas9 10/20

Induced NHP anti-Cas9 1/6

Pre-existing NHP anti-AAV5 5/20

Induced NHP anti-AAV5 5/6


▪ Editing corrects splicing defect in cells from

LCA10-IVS26 patients and restores full-length

mRNA and protein

▪ EDIT-101 gRNAs are highly specific

▪ EDIT-101 and NHP-specific construct

efficiently edit photoreceptors in transgenic

mice and NHPs, respectively

▪ Immunogenicity studies suggest low levels of

anti-Cas9 antibodies in humans

Towards a CRISPR Medicine for LCA10

CEP290 gene editing medicine has the potential to have a

major impact on vision in LCA10 patients

Discovery of EDIT-101 for the Treatment of Leber’s ...€¦ · presentation represent the...

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