Kevin A. Goncalves2, John C. Davis2, Hans-Peter Kiem3, Andre Lieber11University of Washington, Department of Medicine, Division of Medical Genetics, Seattle, WA 98195

2Magenta Therapeutics, Cambridge, MA3Fred Hutchinson Cancer Research Center, Seattle, WA, USA

MGTA-145/plerixafor-mediated mobilization for in vivo HSC gene therapy

Presenter: Chang Li1, PhD

Disclosure of conflicts of interest

- Kevin A. Goncalves and John C. Davis are employees and shareholders of Magenta Therapeutics

- Hans-Peter Kiem is a consultant of Magenta Therapeutics and a co-founder of Ensoma Inc.

- Andre Lieber is a scientific co-founder of Ensoma Inc.

In vivo HSC gene therapy

Bonemarrow

Peripheralblood

Mobilization

of HSCs

Re-homing

HD-Ad5/35++

vector

• Avoids HSC collection and ex vivo manipulation

• No myeloablation; no conditioning; no transplantation

• Low-cost vector manufacturing

HDAd-mgmt/GFP

8 weeks

+ HDAd-SB

hCD46tgG-CSF/AMD3100

O6BG BCNU (7.5)

2 weeks

O6BG BCNU (10)

2 weeks

O6BG BCNU (10)

2 weeks

O6BG (30mg/kg) BCNU (10mg/kg)

26 weeks

EF1αmgmtP140K 2A GFP P

A

SB 100xPGK FlpEF1αHDAd-SB

HDAd-mgmt/GFP

%G

FP

+ce

lls in

PB

MC

t1 t2 t3 t4 t50

2 0

4 0

6 0

8 0

1 0 0

10 12 16 22 26 weeks p.t.

- High transgene marking following in vivo transduction and selection

Notes:AMD3100 = plerixaformgmtP140K provides a mechanism for drug resistance and the selective expansion of gene-modified cells.

- In vivo transduction

Limitations of current G-CSF-mediated mobilization

G-CSF G-CSF G-CSFG-CSF

Dex Dex PlerixaforHDAd HDAd

1 d1 d 16 h1 d 60 min 60 min 20 min

Several limitations exist for G-CSF:

• Requires 5-7 days of treatment

• Variable number of HSCs mobilized

• Unselective mobilization of committed cells leading to: 1) sequestration of HDAdand 2) high peripheral cytokine levels

• Contra-indicated for sickle cell disease

Aims and study design

Dex Dex PlerixaforHDAd HDAd

16 h 60 min 45 min 20 min

MGTA145

15 min

Aims:To test a MGTA-145/plerixafor-mediated mobilization regimen for in vivo HSC transduction.

Study design:Single-day mobilization

Followed by in vivo transduction in both “healthy” (CD46tg) and thalassemia (Hbbth3/CD46+/+) murine models

MGTA-145 is a synthetic form of chemokine GRO-β. It activates CXCR2 and, with plerixafor, rapidly mobilizes HSCs without the need for G-CSF.

MGTA-145 + plerixafor rapidly mobilizes HSCs

Time after MGTA-145 injection

# of

LSK

cel

ls (x

103 )

/ml b

lood

0

10

20

30

40

- # of LSK cells in blood measured by flow cytometry(LSK: Lineage−cKit+Sca1+, murine HSCs)

- Minimal elevation of cytokinesafter in vivo transduction

0

200

400

600

seru

m IL

-6 (p

g/m

l)

1 hr 6 hrs

*

*

Efficient in vivo transduction of HSCs following mobilization by MGTA-145 + plerixafor

0

20

40

60

80

100

% G

FP+

cells

in P

BMC

s

3d 4w 6w 8w 10w 12w

5 7.5 10 10 mg/kg BCNU

G-CSF + PlerixaforMGTA-145 + Plerixafor

blood

% G

FP+

cells

in fr

actio

ns

0

20

40

60

80

100

spleen bone marrow

% G

FP+

cells

in P

BMC

s

0

20

40

60

80

100

4w 8w 12w 16w

- GFP marking in PBMCs - GFP marking in lineage and LSK cells - GFP expression in secondary recipients

CD46tgHDAd-mgmt/GFP

+ HDAd-SB (IV)

wk 16C57Bl/6

secondary recipients

week 16

MGTA-145 + Plerixafor

analysis

O6BG/BCNULin- cells

wk 4 wk 10

- Experiment procedure

Genome-wide transgene integration analyses- LAM-PCR + deep sequencing (BM MNCs)

Sample 1: G-CSF + PlerixaforSample 2: MGTA-145 + Plerixafor

Minutes5 10 15 20 25 30 35

mVo

lts

0

50

100

150

200

250

300

mVo

lts

0

50

100

150

200

250

300

8293

455

2132

742

2193

530

4106

630

7934

8 1623

341

SPD-10AV Ch1-220nm3NP_Mice NC_2.met

Area

γ-gl

obin

/m

ouse

glo

bin

prot

ein

0

1020

304050

0

5

10

15

# of

CFU

-C(×

103 )

/ml b

lood

0

20406080

100

% γ

-glo

bin+

cells

in p

erip

hera

l RBC

s

Efficient in vivo transduction of HSCs following mobilization by MGTA-145 + plerixafor in thalassemia mice

Hbbth3/CD46tgHDAd-γ-globin/mgmt

+ HDAd-SB (IV)

wk 16C57Bl/6

secondary recipients

week 16

MGTA-145 + Plerixafor

analysis

O6BG/BCNULin- cells

wk 4 wk 10

- Experiment procedure

- Efficient mobilization

5 7.5 10 10 mg/kg BCNU

- Over 99% RBCs express γ-globin(Flow cytometry)

20190725

Minutes5 10 15 20 25 30 35

mV

olts

0

50

100

150

200

250

300

mV

olts

0

50

100

150

200

250

300

6499

323

3334

469

7067

436

SPD-10AV Ch1-220nm6NP_Mice NC_2.met

Area

mαmβ

CD46+/+ ctrlHbbth3/CD46 #536, wk14

hγ

- Therapeutic γ-globin production (36% over mouse β-major by HPLC)

Phenotypic correction after MGTA-145 + plerixafor mobilization and in vivo HSPCs transduction

Hbbth3/C46+/+

untreated

Gie

msa

retic

uloc

ytes

Hbbth3/C46+/+

wk14

- Corrected RBC morphology and reticulocytes

- Normal spleen size of treated mice

Hbbth3/CD46

treated (wk 14)untr

CD

46+/

+

Scale bar = 20µm

Summary

1 2 3 4 5

G-CSF

1h

day

plerixafor

HDAd

plerixafor

45 min

MGTA-145

15 min

HDAd

periphery periphery

HSCleukocyte

- Robust HSC mobilization- Significantly Less leukocytosis than G-CSF + Plerixafor- No cytokine release

HSCleukocyte

phenotypic correction of murine thalassemia

G-CSF + Plerixafor5 Day Regimen

MGTA-145 + Plerixafor1 Hour Regimen

- Robust HSC mobilization- Leukocytosis- Significant cytokine release

Acknowledgements

PI: André LieberLab members: Sucheol Gil

Hongjie WangAlex SovaJiho KimChristina GiangEli ChinZhinan Liu

Collaborators: Hans-Peter Kiem, Fred HutchKevin A. Goncalves, Magenta TherapeuticsJohn C. Davis, Magenta TherapeuticsThalia Papayannopoulou, UWZsuzanna Izsvak, GermanyTamás Raskó, GermanyAmit Pande, GermanyEvangelia Yannaki, Greece