CRISPR Genome Editing: Considerations for Therapeutic ......CRISPR Flexibility Addresses Diverse Mutations Non-homologous end joining typically disrupts a gene or eliminates a disease-causing

1© 2017 Editas Medicine© 2017 Editas Medicineeditasmedicine.com

CRISPR Genome Editing:

Considerations for Therapeutic Applications

November 9, 2017Cecilia Fernandez

2© 2017 Editas Medicine

Medicines that Aim to Repair Any Broken Gene

Potential to create the next major category of transformative medicines


CRISPR Provides Versatile Genome Editing Systems

Guide Sequence

Guide RNA

DNA

PAM

CutSites

Nuclease

HiFi

§ Complex of nuclease and guide RNA precisely locates and cuts genomic sites§ Ability to target several sites simultaneously using multiple guide RNAs§ Nuclease can be engineered to reach more sites and to modulate cutting

eS


CRISPR Flexibility Addresses Diverse Mutations

Non-homologous end joining typically disrupts a gene or eliminates

a disease-causing mutation

Homology-directed repair and targeted insertion aim to promote expression of

correct DNA sequences

Cut and Disrupt

Insertion orDeletion

e.g., Engineered T cells

PAM

Mutation

Cut and Replace

Copy

e.g., Hemoglobin Beta

Mutation

Cut and Remove

Deletion

e.g., LCA10

Mutation

e.g., Safe harbor

Insertion

Cut and Insert

5© 2017 Editas MedicineAsCpf1: Acidaminococcus species Cpf1; LbCpf1: Lachnospiraceae bacterium Cpf1; PAM: Protospacer Adjacent Motif; HiFi: High Fidelity; eS: enhanced Specificity

Broad Toolkit of CRISPR Nucleases

SaCas9SpCas9

AsCpf1 LbCpf1

PAM

SaCas9

PAM

SpCas9PAM

AsCpf1 LbCpf1

PAM

HiFi

SpCas9 SaCas9eS

HiFi

eS

Cas9

Cpf1

MULTIPLE

EDITING

SYSTEMS

ADVANCED

FORMS FOR

FLEXIBLE

TARGETING

ADVANCED

FORMS WITH

INCREASED

SPECIFICITY

6© 2017 Editas MedicineSpCas9: Streptococcus pyogenes Cas9; SaCas9: Staphylococcus aureus Cas9

Platform Enables Broad Product Opportunities

ReplaceDisrupt Remove

Lipid Nanoparticle ElectroporationViral Vector

Broad

Range of

Sites

Wide

Delivery

Options

Diverse

Spectrum

of Edits

~10x

SaCas9SpCas9 Editas

Platform

Cpf1 Variants

SaCas9 Variants

Cpf1SpCas9 Variants

Insert


Identify, Measure, Minimize

Lead Finding for Nuclease/gRNA and Specificity

In silico SelectionTesting of On-Target

Cutting(Cas9, Cpf1WT, nickase)

Targeted Panels for Detection of

Sites from Biased & Unbiased Screens

UnbiasedDetection of

Off-Target Cuts and Genomic Alterations

(e.g., GUIDE-Seq, UDiTaS™)

Proprietary in silico

Prediction of Cutting Sites


Identification of Robust gRNAs

§ S. pyogenes Cas9 RNPs in primary human T cells to knock-out PD-1

§ Several gRNAs performed well as assessed by FACS

§ Sequencing confirmed indels§ gRNAs analyzed by GUIDE-Seq to

identify off targets

U n trea te

d 4 9 5 1 5 2 5 3 5 5 5 6 9 7 9 8 9 91 0 0

1 0 11 0 2

1 0 31 0 4

0

2 0

4 0

6 0

8 0

1 0 0

g R N A ID

% P

D-1

Ne

ga

tiv

e


AsCpf1 emerging as the “go to” Cpf1 with Robust activity

Screening of multiple Cpf-1 orthologs and variants

0

10

20

30

40

50

60

70

80

MS1 MS5 MS11 MS18

% in

dels

by

T7E

1

% indels at four matched sites in U2OS

AsCpf1 FnCpf1 LbCpf1 Lb2Cpf1 SpCas9


Control and Specificity to Drive Precision

GUIDE-Seq Read Count

Guide RNAR

eads

On-Target Off-Target 1 Off-Target 2 Off-Target 3

§ GUIDE-Seq drives empirical demonstration of selectivity of product candidates

§ Off-targets identified by GUIDE-Seq would not be accurately predicted by in silico methods alone


A simple question with a complex answer

§ Sequence anchored detection approaches are limited to:– What is between the primers– Amplicon size

How Do You Best Measure Editing?

Intra-chromosomal Events Inter-chromosomal Events

Perfect repair

Insertion*^

Deletion*

Inversion

Duplication

Balanced Translocation

Unbalanced Translocation

+


A simple, robust method for capturing complex editing events in a single reaction

UDiTaS™ (Uni-Directional Targeted Sequencing)

Custom Transposon

ß UMI Barcodeß Pooling Barcodeß P5

5’

5’3’

3’ ddC

Custom Transposon Genomic DNA+

Tagmentation (~ 2Kb)

PCR Round 1 (hot start)

P5 primer

Sequence specific primer

PCR Round 2

UMI P7

P7BCBCBCP5

Sequencing

Picelli, S., et. al (2014). Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Research, 24(12), 2033–2040.


Measuring Structural Changes Using UDiTaS™

1

2

None0102030405060708090100

0 1 2 3 4 5 6 7 8

% In

dels

guide RNA #

AMP-seq

T7E1

UDiTaS

Targeted sequencingT7E1

UDiTaS§ Measurement of small Indelscorrelates well with targeted sequencing and T7E1 assays

§ Measurement of Inversions and Large Deletions





Broad

Range of

Sites

Wide

Delivery

Options

Diverse

Spectrum

of Edits

~10x

SaCas9SpCas9 Editas

Platform

Cpf1 Variants

SaCas9 Variants

Cpf1SpCas9 Variants

Insert


Scalable, Consistent Engineered Cell Therapies

EngineeredPatient Cell

PatientCell

RNA GuidedEndonuclease

RNAGuide

Ribonucleoprotein Particle (RNP)

+

Electroporation

Optimized Delivery of RNP to Primary T cells Via Electroporation


A completely non-enzymatic process for guide production

Generating Synthetic Covalently-Coupled Dual gRNA

Why make a synthetic guide?

• Targeted chemistries anywhere in the molecule

• Unhindered ends and modifications

• Scale up and purity are more compatible with CMC requirements

5’ 3’

5’

3’

5’

3’

+

covalently-coupled dual gRNA (dgRNA)

5’

3’


In vitro transcribed and synthetic covalently-coupled dgRNA are equivalent in cells

Cellular Editing Activity

-11 -10 -9 -8 -7 -60

20

40

60

80

Log concentation RNP (M)

% E

ditin

g (T

7E1)

IVT purified by vendorIVT purified by collaborator

unligated 2 part syntheticligated 2 part synthetic

IVT purified by vendor

covalently-coupled dgRNAIVT purified by collaborator

2-part synthetic


Development of an RNA-Seq based method for gRNA QC

Assessing gRNA purity and sequence fidelity

synthetic 100mer “A”

covalently-coupled dgRNA

synthetic 100mer “B”


Covalently-coupled dgRNA result in greater sequence fidelity in target region

gRNA purity and sequence fidelity

A

B

Covalently-Coupled dgRNA


Improved and Proprietary Guide RNA Structures

Single gRNA

Heterogeneous product(full-length, truncated, errors)

5’ 3’

Well-defined product(full-length)

5’ 3’

Covalently-Coupled Dual gRNA

5e−04

0.1502

0.0432

2e−04

0.5124

1.0384

7e−04

6e−04

1.2414

0.8864

7e−04

0.0011

0.0011

3e−04

0.0016

0.0055

1.0239

0.0192

0.0028

0.0033

3e−04

5e−04

1e−04

7e−04

7e−04

0.001

0.0015

0.0056

6e−04

0.0015

1e−04

0.011

0.133

0.0244

0.0066

1e−04

0.0072

0.0012

0.0298

1.0806

0.0914

1e−04

0.0055

5e−04

0.0102

2e−04

5e−04

0.003

0.016

0.0014

2e−04

0.0045

0.001

0.0123

0.0975

0.0532

1e−04

0.0172

0.0034

0.0014

0.3896

0.0961

0.0041

0.0528

1e−04

6e−04

3e−04

0.0038

0.3176

0.0413

0.0014

1e−04

1e−04

7e−04

6e−04

0.002

0.2954

0.0215

0.0015

0.0034

1e−04

9e−04

0.005

8e−04

0.0738

0.1507

0.0297

0.0056

0.0011

0.2056

6e−04

0.1205

0.0059

2e−04

2e−04

0.0042

0.001

9e−04

1e−04

0.0473

0.0139

7e−04

1e−04

0.0011

0.0749

0.0777

2e−04

2e−04

1e−04

0.002

0.1023

0.083

1e−04

0.0018

1e−04

0.001

0.001

0.1596

0.1036

1e−04

0.0067

5e−04

0.0293

6e−04

0.0015

0.0061

1e−04

2e−04

3e−04

0.0211

0.019

0.1122

0.0418

0.0129

1e−04

0.1447

3e−04

0.0853

0.1403

1e−04

6e−04

1e−04

0.0015

2e−04

0.004

0.0044

0.2824

0.048

0.0102

0.0107

3e−04

3e−04

0.0034

0.0027

0.002

0.3779

0.6421

0.0495

0.2605

0.0011

0.0079

0.001

2e−04

1e−04

3e−04

0.0015

0.0018

0.0015

0.0096

0.001

3e−04

0.0038

2e−04

0.0029

0.0018

0.002

9e−04

0.0165

1e−04

0.0016

1e−04

0.0037

0.0053

0.2478

0.1275

0.0076

0.089

6e−04

2e−04

3e−04

7e−04

0.0045

0.243

0.029

0.0025

5e−04

1e−04

0.0037

0.0029

0.0049

0.0272

0.213

0.1773

1e−04

5e−04

1e−04

6e−04

1e−04

0.0037

0.0023

0.3073

0.073

0.0038

0.0087

2e−04

1e−04

0.0016

0.001

0.0742

0.0772

0.016

0.0849

0.2282

0.0221

0.0382

5e−04

0.0058

7e−04

0.0037

2e−04

9e−04

2e−04

2e−04

2e−04

0.1642

0.0824

0.0056

9e−04

5e−04

0.0028

2e−04

0.0105

0.0279

0.0338

0.0405

5e−04

2e−04

0.0016

0.0016

0.0398

0.0813

0.0072

2e−04

2e−04

7e−04

2e−04

0.0289

0.0014

0.003

0.2042

0.1472

0.0049

0.0012

2e−04

0.0051

0.0028

0.1094

0.1565

0.0021

0.0037

0.0026

0.0058

0.004

0.0051

5e−04

5e−04

0.0047

0.0116

0.0326

0.115

0.0091

0.024

0.0014

9e−04

7e−04

0.0019

0.007

0.0088

0.1479

0.1092

0.0179

0.0289

2e−04

5e−04

0.0065

0.0403

0.0696

0.1195

9e−04

2e−04

2e−04

0.0035

0.0126

0.6543

0.3407

0.0021

0.0361

0.0016

5e−04

5e−04

0.0044

0.004

0.0028

0.0813

0.0263

2e−04

0.0366

0.0079

0.0226

0.0587

0.099

2e−04

0.0156

0.0082

0.0161

0.007

0.0917

0.1327

5e−04

0.1281

5e−04

0.0026

5e−04

7e−04

0.0077

0.3644

0.1726

0.024

0.0424

0.0014

2e−04

7e−04

0.0116

0.1693

0.2804

0.0238

2e−04

7e−04

0.0019

0.0061

0.0012

0.0119

0.027

0.0019

0.0012

0.0033

9e−04

0.0026

0.0082

0.2387

0.1481

0.0112

0.2189

0.0014

0.0021

0.0061

0.0256

0.0321

0.0955

0.2361

0.0354

5e−04

0.0044

0.0016

0.2352

0.4466

0.0023

0.0016

7e−04

0.0033

0.0144

0.013

0.1982

0.6248

0.0414

0.1614

0.0028

2e−04

0.0026

0.024

0.0035

0.0014

7e−04

5e−04

0.0061

0.0056

7e−04

0.0109

0.0012

7e−04

0.0142

2e−04

0.0093

0.0063

0.0012

0.0263

2e−04

2e−04

9e−04

0.0077

0.0175

0.0608

0.2385

0.0196

0.1896

0.0016

2e−04

0.003

0.003

0.0033

0.2056

0.0685

0.0154

0.0075

2e−04

0.0037

0.0144

0.0424

0.0652

0.3991

0.0123

0.0026

0.0019

5e−04

0.0151

0.0033

0.2603

0.1325

0.0221

0.034

5e−04

2e−04

0.0047

0.0016

0.0349

0.1742

0.0366

0.064

0.0525

9e−04

1e−04

1e−04

1e−04

0.0982

0.0655

0.3007

0.0727

2e−04

1e−04

2e−04

0.0121

0.0086

3e−04

3e−04

1e−04

5e−04

3e−04

2e−04

0.0044

3e−04

1e−04

1e−04

1e−04

0.0049

0.0114

8e−04

1e−04

5e−04

4e−04

0.0019

3e−04

0.0058

0.0032

0.0015

1e−04

1e−04

0.0076

0.0117

0.0084

1e−04

0.0013

0.0029

1e−04

0.0054

0.0045

1e−04

4e−04

1e−04

0.0089

0.0074

0.0025

0.001

0.0148

0.0511

0.0036

1e−04

3e−04

0.0065

2e−04

0.0099

0.0082

5e−04

0.0054

0.0378

3e−04

0.0183

0.0192

3e−04

1e−04

0.0014

0.0108

1e−04

6e−04

0.0043

0.0058

0.0019

1e−04

1e−04

0.005

0.0108

0.0331

6e−04

0.001

5e−04

0.0092

0.0042

0.0079

1e−04

2e−04

0.0509

0.2056

0.0023

0.0453

5e−04

1e−04

5e−04

1e−04

1e−04

0.001

1e−04

6e−04

6e−04

1e−04

1e−04

1e−04

0.0044

0.0397

1e−04

0.0186

1e−04

0.0025

0.0034

1e−04

0.0436

0.0034

0.0172

6e−04

0.0021

0.0051

0.0022

1e−04

1e−04

0.0039

1e−04

0.0034

0.0015

0.0122

0.031

−5−4−3−2−1

12345

−5−4−3−2−1

12345

−5−4−3−2−1

12345

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Position

Leng

th c

hang

e

0.26

0.58

1

% error

Leng

th C

hang

e(B

ases

)

Position (5’ à 3’)

Targeting Sequence

Leng

th C

hang

e(B

ases

)

Position (5’ à 3’)

Targeting SequenceHigh Low

Frequency of Error

5’

3’

5’3’

5’5’

3’





Broad

Range of

Sites

Wide

Delivery

Options

Diverse

Spectrum

of Edits

~10x

SaCas9SpCas9 Editas

Platform

Cpf1 Variants

SaCas9 Variants

Cpf1SpCas9 Variants

Insert


Cas9 Stimulates the Endogenous Repair Pathways

C-NHEJResection

Locus Unaltered

Small Deletions

Small Insertions

3’

HRSSA

Large Deletions Correction

MMEJBlunt EJ SD-MMEJ

Alt-NHEJ

3’

Deletions InsertionsDeletions

HDR

DSB5’

WT Cas9

5’


Cas9 is a Flexible Tool

WT Cas9

Blunt

5’

• Could we engage different pathways by using

these different variants?

• Could we selectively stimulate HDR?

D10A Nickases

5’ Overhang

5’

5’

N863A Nickases

3’ Overhang

5’

3’


DSBs Generated by D10A are Predominantly

Repaired by HDR

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

WT N863A D10A

Insertions

Deletions

HDR

N863A D10AWT

Mod

ifica

tion

(%)

3’ 5’

Bothmer et al., Nat Comm 2017


Do Gene Conversion and Gene Correction have the

same Genetic Requirement?

Gene Conversion

HBB HBD

Do they both dependent on the HR pathway?

Gene Correction

ssODN


Gene Conversion and Gene Correction have Different

Genetic Requirements

Neg.

Cont.

K.D.

Rad51FF

Rad51

Brca2

0

10

20

30

Gen

e C

orre

ctio

n

1000 bp donor

1000 bp donor0

10

20

FFRad51

Brca2

0

10

20

30

Neg.

Cont.

K.D.

Rad51

Gen

e C

onve

rsio

n

FFRad51

Brca2

0

10

20

30

Neg.

Cont.

K.D.

Rad51

Gen

e C

onve

rsio

n

SS ODN donor Endogenous HBD Plasmid donor

HR is required for repair from double stranded donors (endogenous homology tracks or plasmids) but not single stranded donors


Conclusions from the Dual Nick Analysis

• Different ends activate different DNA repair pathways

• Different donors stimulate different pathways

Gene Correction mediated by ssODN is not HR dependent

C-NHEJ

Alt-NHEJ HDR

D10A Nickases

5’

N863A Nickases

5’

WT Cas9

5’

Bothmer et al., Nat Comm 2017

28© 2017 Editas MedicineLCA10: Leber Congenital Amaurosis type 10; HSV: Herpes Simplex Virus

Pipeline Strategy to Enable Successful Medicines

Medical Need

§ Severe diseases where current treatments, if any, are poor

§ Potential for durable therapies to provide unique benefit

Biology & Clinical

§ Clear biological hypothesis for genomic intervention

§ Favorable clinical and regulatory path

Technical

§ Validated delivery approaches

§ Mutation feasibly corrected

Eye

§ LCA10 (EDIT-101)§ Ocular HSV§ Additional ocular indications

Lung

§ Cystic Fibrosis

Muscle

§ Duchenne Muscular Dystrophy

Bone Marrow & Blood

§ Hemoglobinopathies§ Engineered T cells for cancer§ Additional bone marrow and blood

indications

Liver

§ Alpha-1 Antitrypsin Deficiency§ Infectious diseases of liver

Prioritization Principles Product Pipeline

29© 2017 Editas Medicine 29© Editas Medicine

Thank You

§ Hayat Abdulkerim

§ Luis Barrera

§ Anne Bothmer

§ Frank Buquicchio

§ Dawn Ciulla

§ Cecilia Cotta-Ramusino

§ Georgia Giannoukos

§ Kiran Gogi

§ Jennifer Gori

§ Fred Harbinski

§ Hari Jayaram

§ Eugenio Marco

§ Carrie Margulies

§ Tanushree Phadke

§ Terence Ta

§ Grant Welstead

§ Chris Wilson

§ Vic Myer

§ I2 Pharmaceutical Team

CRISPR Genome Editing: Considerations for Therapeutic ......CRISPR Flexibility Addresses Diverse Mutations Non-homologous end joining typically disrupts a gene or eliminates a disease-causing

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