PRECLINICAL DATA SUPPORTING THE INITIATION OF THE EDIT …

1© 2021 Editas Medicine© 2018 Editas Medicineeditasmedicine.com

PRECLINICAL DATA SUPPORTING THE INITIATION

OF THE EDIT-301 PHASE I/II RUBY CLINICAL

TRIAL FOR THE POTENTIAL TREATMENT OF

SICKLE CELL DISEASE

Edouard De Dreuzy1, Jack Heath1, Patricia Sousa1, Tusneem Janoudi1, Dawn Ciulla1,

Georgia Giannoukos1, Pavlina Wolf1, Harry An1, Meng Wu1, Tongyao Wang1, Eugenio

Marco1, Scott Hansen2, David K. Wood2, Sandra Teixeira1, Tamara T. Monesmith1, Kate

Zhang1, Kai-Hsin Chang1

1Editas Medicine, Inc., Cambridge, MA 2University of Minnesota, Minneapolis, MN

EHA2021

© 2021 Editas Medicine

2© 2021 Editas Medicine

Disclosures

Edouard De Dreuzy is a full-time employee

and shareholder in Editas Medicine

© 2021 Editas Medicine 2


aCD34+ hematopoietic stem and progenitor cell

HS: hypersensitive site; LCR: locus control region; TSS: transcriptional start site

Adapted from Higgs, Engel and Stamatoyannopoulos. Lancet 2012

β-globin

locus

Naturally occurring

HbF-inducing mutations

support clinical relevance

and safety of editing

Embryo Fetus Adult

Enhancer

(HS1-4)

LCR

Insulator

(5’HS)Insulator

(3’HS1)

HBG1HBG2HBE HBBHBD

Sickle hemoglobin (HbS) HbS HbF

α α

γ γ

HbF production

reduces RBC sickling

by inhibiting formation

of HbS fibers at low

oxygen levels

Unedited

CD34+ cellsa

from patients

with SCD

EDIT-301: Edited

CD34+ cellsa from

patients with SCD

CRISPR-Cas12a

editing at the

HBG1 and HBG2

promoter regions

induces

HbF expression

3

Naturally Occurring Mutations Support Clinical Relevance and Safety of

Editing at the HBG1/2 Promoter Region


Indel ProfileEditing Efficiency

Match target Site are 20mer protospacer sequence within the human genome with compatible PAM's for different nucleases (20-Ns)

EDIT-301 Editing using highly specific and potent CRISPR-Cas12a

enzyme

-20-1000

2

4

6

8

10

Indel Size

NH

EJ

In

de

ls (

%)

AsCas12a

SpCas9

AsCas12a staggered cut

generate larger deletions than

SpCas9, leading to higher HbF

induction at the HBG locus

De Dreuzy et al, ASH 2019

De Dreuzy et al, ASGCT 2018

Editas engineered AsCas12a

RNP demonstrates high editing

efficiency in CD34+ cells

De Dreuzy et al, ASH 2019

1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829303132333435363738390

20

40

60

80

100

RNP Version

Ind

els

(%

)

WT Cas12a

+ unmod

gRNA

Optimized

Conditions

Editing Specificity

AsCas12a is Highly Specific

primarily due to its DNA target

engagement mechanism that is

distinct from SpCas9

Gotta et al. Cold Spring Harbor 2019

0

20

40

Match target sites

Cu

t-s

ite

s (

Co

un

t)

Fig. to be

reformatted60

80

>100

AsCas12a

SpCas9

5© 2021 Editas MedicineUnedited cells did not undergo electroporation

High level of editing and robust HbF induction in edited CD34+ cells from

normal donors and patients with Sickle Cell Disease (SCD)

U n e d ite d E d ite d U n e d ite d E D IT -3 0 1

0

2 0

4 0

6 0

Hb

F (/

-lik

e)

(%)

Patients with SCDNormal donor

n=3 n=4

Robust ex vivo HbF expressionEfficient editing

U n e d ite d E d ite d U n e d ite d E D IT -3 0 1

0

2 0

4 0

6 0

8 0

1 0 0

Ind

els

(%

)

Patients with SCDNormal donor

n=3 n=4

6© 2021 Editas MedicineOxygen (%) 1% = 7.6mmHg

EDIT-301-derived RBCs have reduced sickling and improved rheological

properties versus unedited SCD-derived RBCs

Improved rheological behavior

Unedited SCD-derived RBCs EDIT-301 (edited SCD)-derived RBCsNormal donor-derived RBCs

No

rm

ali

ze

d v

elo

cit

y (

%)

O x y g e n (% )

0 2 4 6 8 1 0

4 0

6 0

8 0

1 0 0

Venous %O2

When placed in microfluidic channels, mimicking blood flow in microvasculature,

at a range of oxygen levelsWhen exposed to sodium metabisulfite

Oxygen level: 4%

HbF levels correlated with velocity

No

rm

ali

ze

d v

elo

cit

y (

%)

H b F ( / - lik e ) (% )

0 2 0 4 0 6 0 8 0

4 0

6 0

8 0

1 0 0

R2=0.9321

Reduced sickling

U n e d ite d E D IT -3 0 1

0

1 0

2 0

3 0

4 0

5 0

Sic

kle

d R

BC

s (

%)

Mean HbF (%): 53.819.9


Consistent and robust large-scale manufacturing of edited CD34+ cells

from normal donors

Robust large-scale manufacturing

n=8

CD34 Purity Editing

0

20

40

60

80

100

n=8

Recovery

0

20

40

60

80

100

n=8 n=8

%%

Viability

0

20

40

60

80

100

%

Consistent editing profile

Indel Length

0

20

10

Ind

el

%

10 20 30 40-10-20-30-40-50

0

Deletions are represented as negative values ;

Insertions as positive values

Large scale

Edited batch (n=5)


Cas12a RNP is highly specific and no off-target editing was detected in

large scale manufacturing batches

Discovery

Phase

In silico modeling

Predicted sites base

on sequence

Digenome-Seq

Cleaved sites on

naked DNA

GUIDE-Seq

Cleaved sites in

cellular context

Verification

Phase

Targeted Sequencing

in CD34+ cells

Editing activity at

“discovered”

candidate sites

No off-target editing

observed in Cas12a

treated CD34+ cells

9© 2021 Editas MedicineDirect RNA-seq analysis conducted using the Oxford Nanopore System on erythroid cells RNA harvested after 7 days of erythroid differentiation

No detectable unintended globin transcript variants in edited CD34-

derived erythroid precursors (Large scale batches)

Large-scale manufacturing –

Unedited CD34 derived Erythroid Precursor


Edited CD34 derived Erythroid Precursors

Unedited Erythroid Precursors

Edited Erythroid Precursors

N=3 CD34 normal donors

Donor1

Donor2

Donor3

Donor1

Donor3

Donor2

10© 2021 Editas MedicineUHPLC-MS analysis of erythroid cells lysates harvested after 18 days of erythroid differentiation

No detectable unintended globin protein variants in edited CD34-derived

RBCs (Large scale batches)


Unedited CD34 derived erythroid cells


Edited CD34 derived erythroid cells

Donor1

Donor3

Donor2

Donor1

Donor3

Donor2

Heme β A AγT

N=3 CD34 normal donors

Gγ Aγ Wash Heme β A AγT Gγ Aγ Wash

Time (min)Time (min)


Infusion of edited CD34+ cells manufactured on a large scale to

NSG mice leads to polyclonal engraftment with no lineage skewing

Efficient editing

maintained in vivo

Bone Marrow

at 20 weeks

n=32 mice/donor

1 2 3

0

2 0

4 0

6 0

8 0

1 0 0

Ed

itin

g %

0

100

80

60

40

20

Ed

itin

g (

%)

No lineage skewing after engraftment

Blood

Stable polyclonal engraftment

Bone marrow at 20 weeks post-infusion (Female NSG mice) Blood draws over 20 weeks and BM

Ind

els

(%

)

Time (weeks)

0

100

80

60

40

20

Ind

els

(%

)

0

100

80

60

40

20

124 8 16 20C D 4 5

l e u k o c y t e s

C D 1 9

B c e l l s

C D 3 3

m y e lo id c e l l s

C D 3

T c e l l s

C D 2 3 5

e r y t h r o id s

0

2 0

4 0

6 0

8 0

1 0 0

Po

pu

lat

ion

(%

)

U n e d i t e d C D 3 4 + c e l l s

E d i t e d C D 3 4 + c e l l s

n=46–48 mice/treatment (3 donors) Representative data from one NSG mouse.

Each color or color shade represents an individual

indel signature.

Blood Bone marrow

Unedited CD34+ cells

Edited CD34+ cells

Donor


Conclusions

High levels of editing were achieved in CD34+ cells using highly specific Cas12a enzyme,

leading to potentially therapeutically relevant levels of HbF expression

EDIT-301(edited SCD)-derived RBCs demonstrated a significant reduction in sickling and

improved rheological properties

Large-scale process suitable for use in clinical manufacturing led to consistent editing

without off-target and unintended HBG variants. Infusion of the edited cells in mice gave rise to

multilineage and polyclonal engraftment with persistence of high levels of editing.

These results support the initiation of the RUBY clinical trial, a phase 1/2 study of EDIT-301

to treat patients with severe SCD (NCT#04853576).

13© 2021 Editas Medicine 13© Editas Medicine

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Genomic Medicine

Company

Developing

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