1Magenta Therapeutics

Expansion with E478 Significantly Increases the Rate of

CRISPR-Mediated Homology Directed Repair (HDR) and

Improves Engraftment of Human Hematopoietic Stem Cells

Kevin A. Goncalves, PhD

Magenta Therapeutics

2Magenta Therapeutics

Increased CD34+ Cell Dose Leads to Improved Survival and Engraftment

ALLOGENEIC HEME-ONC AUTOLOGOUS HEME-ONC

Lee SH et.al. Biol Blood Marrow Transplant. 2005 Feb;11(2):122-8 Siena S, et. al.; J Clin Oncol. 2000 Mar;18(6):1360-77.

3Magenta Therapeutics

Aryl Hydrocarbon Receptor (AHR) Antagonists Drive HSC Renewal By Blocking Differentiation via a Defined Mechanism

CD34+ Cell

Growth factor

receptors

Differentiation

AHR

AHR

antagonist

XRE

Nucleus

CYP1B1/AHRR

Cytosol

• Identified in a screen of compounds that promoted CD34/CD133 expansion: StemRegenin 1

• Acts via antagonism of AHR which plays a prominent role in HSC differentiation

• AHR antagonist-mediated AHR inhibition is reversible

AHR

AHR

L

L

Boitano et al., Science, 2010

4Magenta Therapeutics

CD34+ CELL EXPANSION OVER 15 DAYS

During Ph1/2 Trial, AHR Antagonist Used to Manufacture MGTA-456 Expanded CD34+ Cord Blood Cells More Than 300-Fold

1 0 6

1 0 7

1 0 8

1 0 9

1 0 1 0

CD

34

+ C

ell

Nu

mb

er

P re

E x p a n s io n

P o s t

E x p a n s io n

1.44 x 109

4.4 x 106

Wagner et al. Cell Stem Cell 2016; 18:144-155

MGTA-456: ALLOGENEIC STEM CELL THERAPY

36 Patients Treated in Phase I/II Heme-Onc Study

5 Patients Treated in Phase II Study in Inherited Metabolic Diseases (IMDs)

5Magenta Therapeutics

MGTA-456 Has Been Clinically-Validated in Hem/Onc Patients

Historical Control

83% Engraftment

Median 26.5 days

MGTA-456

100% Engraftment

n = 21/21 patients

Median: 12 days

p<0.001

MYELOABLATIVE CONDITIONING

Flu / Cy / TBI 1320

MGTA-456

100% Engraftment

n = 9/9 patients

Median: 8 days

Historical Control

94% engraftment

Median: 17.5 days

p<0.001

Days

NON-MYELOABLATIVE CONDITIONING

Flu / Cy / TBI 200

Inc

ide

nc

e

0.0

0.2

0.4

0.6

0.8

1.0

0 7 14 21 28 35 42

DaysIn

cid

en

ce

Wagner et al., ASH 2017

MGTA-456

ASGCT PRESENTATIONS

ABSTRACT #120

MGTA-456 Heme-Onc Ph2 Study

John Wagner, MD

May 12th 3:45-5:30 PM

ABSTRACT #1302

MGTA-456 IMD Ph2 Study

John Wagner, MD

May 15th 8-9:45 AM

ABSTRACT #248

MGTA-456 Non-Clinical IMD Study

Sharon Hyzy, MS

May 12th 5:30-6:30 PM

6Magenta Therapeutics

E478 Is a Novel, Potent AHR Antagonist

AHR ANTAGONIST ACTIVITY IN VITRO EXPANSION AFTER 10 DAYS

0.0

001

0.0

01

0.0

10.1 1

10

0

2 ,0 0 0

4 ,0 0 0

6 ,0 0 0

8 ,0 0 0

C o n c e n tra tio n ( M )

Lu

min

es

ce

nc

e

E 4 7 8

S R 1

Unexpanded

Vehic

le

E478

0

1 0

2 0

3 0

Nu

mb

er

(x1

04)

Unexpanded

Vehic

le

E478

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Nu

mb

er

(x1

04)

CD34+ Number CD34+CD90+ Number

Mean ± SD (n=2)Mean ± SD (n=2)

80-fold

p<0.001 47-fold

p<0.001

7Magenta Therapeutics

WEEK 16 NSG ENGRAFTMENT

All Human Hematopoietic Stem Cell Sources are Expandable with E478

Vehic

le

E478

0

1 0

2 0

3 0

4 0

5 0

hC

D4

5 (

%)

p<0.001

Vehic

le

E478

0

1 0

2 0

3 0

4 0

hC

D4

5 (

%)

p<0.001

Vehic

le

E478

0

2

4

6

8

1 0

hC

D4

5 (

%)

p<0.001

Cord Blood Bone Marrow Mobilized

Peripheral Blood

10 day cultures were started with the following CD34+ cell numbers:

Cord Blood: 7,000, Bone Marrow: 200,000, Mobilized Peripheral Blood: 100,000 n=8 mice

8Magenta Therapeutics

Unexpanded

Vehic

le

E478

1 0

1 0 0

1 0 0 0

1 0 0 0 0

SR

C N

um

be

r

E478 Significantly Increases HSCs in a Limit-Dilution Study Using Mobilized Peripheral Blood

ns

10-fold

p<0.001

WEEK 16 NSG ENGRAFTMENT

n=8 mice

9Magenta Therapeutics

E478 Enables a Variety of Gene Therapy Applications

E478 expands number of gene-modified HSCs:

1) Lentiviral Transduction

2) CRISPR-Cas9 Knockout

3) Gene Correction / Insertion

10Magenta Therapeutics

E478 Provides Higher Dose of Lenti-Transduced HSCs

IN VITRO EXPANSION AFTER 7 DAYS WEEK 16 NSG ENGRAFTMENT

CD34+CD90+

Cell Number

Transduction

Rate

Peripheral Blood

Engraftment

Transduction

Rate

Vehic

le

E478

0

5

1 0

1 5

2 0

2 5

Nu

mb

er

(x1

04)

Vehic

le

E478

0

2 0

4 0

6 0

Tra

ns

du

cti

on

(%

)

Vehic

le

E478

0

2

4

6

8

1 0

En

gra

ftm

en

t (%

)

Vehic

le

E478

0

2 0

4 0

6 0

Tra

ns

du

cti

on

(%

)

7-fold

7-fold

p<0.001

ns

n=8 mice

11Magenta Therapeutics

Vehic

le

E478

0

2

4

6

Nu

mb

er

(x1

04)

Vehic

le

E478

0

2 0

4 0

6 0

8 0

1 0 0

Ed

itin

g R

ate

(%

)

Vehic

le

E478

0

1

2

3

4

5

En

gra

ftm

en

t (%

)

Vehic

le

E478

0

2 0

4 0

6 0

8 0

1 0 0

Ed

itin

g R

ate

(%

)

E478 Provides Higher Dose of CRISPR-Edited HSCs

IN VITRO EXPANSION AFTER 7 DAYS WEEK 16 NSG ENGRAFTMENT

CD34+CD90+

Cell Number

Editing

Rate

Peripheral Blood

Engraftment

Editing

Rate

12-fold

12-fold

p<0.01 ns

n=8 mice

12Magenta Therapeutics

E478 Results in Higher Engraftment at All Timepoints Evaluated

Vehic

le

E478

Vehic

le

E478

Vehic

le

E478

Vehic

le

E478

0 .0

0 .5

1 .0

1 .5

2 .0

En

gra

ftm

en

t (%

)

1 D a y 3 D a y 7 D a y 1 0 D a y

2-f

old

4-f

old

10

-fo

ld

5-f

old

2 -fo ld p < 0 .0 5

3 -fo ld p < 0 .0 5

p<

0.0

05

p<

0.0

1

p<

0.0

5

Vehic

le

E478

Vehic

le

E478

Vehic

le

E478

Vehic

le

E478

0

2 0

4 0

6 0

8 0

1 0 0

Ed

itin

g R

ate

(%

)

1 D a y 3 D a y 7 D a y 1 0 D a y

n s n s n s n s

Thaw CD34+ Cells & Culture ± E478

Edit with CRISPR-Cas9

-1 0

Culture ± E478

1, 3, 7, or 10 days

Prior to Transplant

Into NSG Mice

EXPERIMENTAL SCHEMA WEEK 16 NSG ENGRAFTMENT

n=8 mice

Peripheral Blood

Engraftment

Editing

Rate

13Magenta Therapeutics

Does E478 Expand The Number of Gene-Corrected HSCs?

Experimental Design with

Mobilized Peripheral Blood CD34+ Cells

2 Day Pre-Stimulation

± E478

Edit with CRISPR/Cas9

and AAV.GFP Donor

1 Day Expansion

± E478

4 Day Pre-Stimulation

± E478

Edit with CRISPR/Cas9

and AAV.GFP Donor

4 Day Expansion

± E478

2+1 4+4

14Magenta Therapeutics

All CD34+CD90+ Cells Are Actively Cycling After Three Days in Culture

0 1 2 3 4 5

0

2 5

5 0

7 5

1 0 0

D a y s in c u ltu re

Fre

qu

en

cy

(%

) G 0

G 1

S -G 2 -M

CELL CYCLE PROFILE OF CD34+CD90+ MOBILIZED BLOOD CELLS

DMSO Vehicle: dashed line

E478: solid linen=2 cell donors

15Magenta Therapeutics

Expansion with E478 Leads to Higher Rates of HDR and Numbers of HDR+ CD34+CD90+ Cells Compared to Conventional Approaches

IN VITRO EXPANSION OF CD34+CD90+ CELLS

n=2

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

5

1 0

1 5

Nu

mb

er

(x1

05)

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

1 0

2 0

3 0

4 0

HD

R (

%)

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

1

2

3

4

Nu

mb

er

(x1

05)

Cell Number Rate of HDR HDR+ Cell Number

21-fold

p<0.01

3-fold

p<0.01

6-fold

p<0.01

ns

134-fold

p<0.01

3-fold

p<0.01

2+1 4+42+1 4+4 2+1 4+4

16Magenta Therapeutics

Expansion with E478 Leads to Higher Rates of HDR and Numbers of HDR+ NSG-Engrafting Cells Compared to Conventional Approaches

WEEK 16 NSG ENGRAFTMENT

n=8 mice

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

2

4

6

8

1 0

En

gra

ftm

en

t (%

)

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

5

1 0

1 5

2 0

2 5

HD

R (

%)

AA

V O

nly

Vehic

le

E478

Vehic

le

E478

0

4 0 0 0

8 0 0 0

1 2 0 0 0

Nu

mb

er

En

gra

fta

ble

HD

R+

ce

lls

pe

r m

L

Engraftment Rate of HDR Engraftable HDR+

Cell Number10-fold

p<0.001

4-fold

p<0.001

18-fold

p<0.001

ns

169-fold

p<0.001

12-fold

p<0.001

2+1 4+42+1 4+4 2+1 4+4

BLQ

17Magenta Therapeutics

Expansion with E478 Leads to High Rates of HDR and Numbers of HDR+ NSG-Engrafting Cells in Secondary Recipients

Vehic

le (

2+1)

E478 (

4+4)

0

5

1 0

1 5

2 0

2 5

HD

R (

%)

Vehic

le (

2+1)

E478 (

4+4)

0

2 0 0 0

4 0 0 0

6 0 0 0

Nu

mb

er

En

gra

fta

ble

HD

R+

ce

lls

pe

r m

L

Vehic

le (

2+1)

E478 (

4+4)

0

2

4

6

En

gra

ftm

en

t (%

)

WEEK 16 NSG ENGRAFTMENT

n=8 mice

Engraftment Rate of HDR Engraftable HDR+

Cell Number

12-fold

p<0.00117-fold

p<0.001216-fold

p<0.001

18Magenta Therapeutics

E478 Increases the Dose of Gene-Modified HSCs

• Increased CD34+ cell dose leads to improved engraftment and gene therapy outcomes

• AHR antagonism allows for robust expansion of HSCs by blocking differentiation

• MGTA-456 provides clinical validation for expansion of HSCs via AHR antagonism

• E478 is a novel, potent AHR antagonist capable of expanding all human HSC sources: mobilized

blood, bone marrow, and cord blood

• Compared to conventional approaches, ex vivo expansion with E478:

1. Results in significantly higher engraftment of lentiviral vector-transduced cells by 7-fold

2. Results in higher engraftment of CRISPR-Cas9 knockout cells by 12-fold

3. Enables 18-fold higher rates of HDR, a high dose of HDR+ HSCs, and a ~200-fold increase inthe engraftment of HDR+ HSCs

19Magenta Therapeutics

Acknowledgments

Magenta R&D Team

Megan Hoban

Sharon Hyzy

Katia George

Anthony Boitano

Michael Cooke

John Davis