Base Editing of Gamma Promoters Generates Durable Expression of Fetal for the Treatment of

Adrian P. Rybak, Elsie Zahr Akrawi, Conrad Rinaldi, Scott J. Haskett, Ling Lin, Jeffrey Marshall, Alexander Liquori, Luis Barrera, Jenny Olins, S. Haihua Chu, Jeremy Decker, Minerva Sanchez, Yeh-Chuin Poh, Matt Humes, Michael S. Packer, Nicole M. Gaudelli, Sarah Smith, Adam Hartigan and Giuseppe Ciaramella.

May 13, 2020 DISCLOSURE

I am a Beam employee and shareholder Normal Erythropoiesis, Sickle Cell Disease and Hereditary Persistence of (HPFH)

β-like Globin Hemoglobin Red blood cells System

β α Normal Gγ Aγ δ β α β (Adult) HBG2 HBG1 After birth, γ-globin is repressed Normal β:α hemoglobin Red blood cells form and and wild-type β-globin is expressed tetramers form function normally

Pain crises Sickle Cell Gγ Aγ δ βS Organ damage Disease HBG2 HBG1 Hemolysis (Adult) After birth, γ-globin is repressed and βS-globin tetramers form Polymers cause red blood Anemia sickle-causing βS-globin is expressed polymers under hypoxia cells to sickle under hypoxia

Hereditary Persistence of Fetal Gγ Aγ δ βS Hemoglobin HBG2 HBG1 (HPFH) γ-globin expression persists after Normal γ:α hemoglobin Red blood cells form and display birth and through adulthood tetramers form decreased sickling phenotype Base Editing at HBG1 and HBG2 (HBG1/2) Gene Promoters

β-like Globin Genes Hemoglobin Proteins Red blood cells System

Sickle Cell Pain crises Disease Gγ Aγ δ βS Organ damage (Adult) HBG2 HBG1 Hemolysis After birth, γ-globin is repressed and βS-globin tetramers form Polymers cause red blood Anemia sickle-causing βS-globin is expressed polymers under hypoxia cells to sickle under hypoxia Base Editing at HBG1 and HBG2 (HBG1/2) Gene Promoters

β-like Globin Genes Hemoglobin Proteins Red blood cells System

Base Editing recreates Gγ Aγ δ βS γ α naturally HBG2 HBG1 α γ occurring HPFH Re-expression of γ-globin Red blood cells with increased T decreases βS-globin and inhibits γ-globin have decreased A hemoglobin polymer formation sickling phenotype

C G

Guide RNA-targeting ABE produces A-to-G edits in HBG1/2 promotor regions and derepresses γ-globin expression Adenine Base Editing Technology

• Adenine Base Editor (ABE) comprises a deaminase enzyme fused to catalytically impaired CRISPR . T ▲ • Guide RNA (gRNA) directs the ABE to a target genomic DNA sequence and A exposes the editing window.

A • Deaminase chemically converts target adenine (A) to inosine (I) via A I I deamination.

• Guanine (G) subsequently replaces inosine during DNA repair or replication.

Gaudelli NM et al., Nature (2017); Gaudelli NM et al., Nature Biotechnology (2020). Base Editing at HBG1 and HBG2 (HBG1/2) Gene Promoters

β-like Globin Genes Hemoglobin Proteins Red blood cells System

BaseSickle Editing cell Pain crises recreatesdisease Gγ Aγ δ βS γ α Organ damage naturally(Adult) γ HBG2HBG2 HBG1HBG1 α Hemolysis occurring HPFH After birth, γ-globin is repressed and βReS--globinexpression tetramers of γ- globinform RedPolymers blood cellscause with red increased blood Anemia T S decreasespolymers βS under-globin hypoxia and inhibits cellsγ-globin to sickle have under decreased hypoxia sickle causingA β -globin is expressed hemoglobin polymer formation sickling phenotype

C G

Guide RNA-targeting ABE produces A-to-G edits in HBG1/2 promotor regions and derepresses γ-globin expression

Today’s 1 In vitro optimization 2 γ-globin upregulation 3 In vivo performance Agenda: Optimize A-to-G base editing in Maximize γ-globin protein Long-term engraftment, mobilized human CD34+ levels produced in erythroid retention of editing, hematopoietic stem/progenitor cells. γ-globin protein upregulation, cells (HSPCs) by titrating ABE and multi-lineage mRNA and guide RNA. hematopoietic reconstitution. Experimental Outline

Seed Electroporation Cell Viability and Mobilized (EP) + NGS Analysis CD34 HSPCs (108 cells/mL) HBG1/2 Gene Promoter Base Editing (ABE + guide RNA) -48 h 0 h 48 h NGS Globin Protein Analysis Analysis (UPLC) Erythroid Differentiation (EDIFF) (In Vitro) Day 14 Day 18 Cell Expansion + Erythroid Differentiation

CD34+ Enucleated Erythroid cells Maximizing A-to-G Base Editing at HBG1/2 Gene Promoters

HBG1/2 Promoter HBG1/2 Promoter Gamma Globin Base Editing (%) Base Editing (%) Protein Levels (%) (48 h Post-EP) (Day 14 EDIFF) (Day 18 EDIFF)

100 100 ) 100

%

(

s

n

i )

80 ) 80 80

b

% %

o

( (

l

g

g g

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60 n 60 60

i i

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40 40 a 40

t

e e

o

s s

T

a a

/

n

B B

20 20 i 20

b

o

l g

0 0 - 0 g *Next Generation Variant

• Base editing at HBG1/2 promoters in CD34+ HSPCs is dependent on ABE mRNA and guide RNA concentration.

• Base editing levels increase following in vitro-mediated erythroid differentiation (EDIFF).

• Increased gamma globin protein levels with increasing A-to-G base editing at HBG1/2 promoters in erythroid cells. HBG1/2 Gene Promoter Base Editing is Tightly Correlated with Gamma Globin Protein Induction In Vitro MC028 MC033

● ● ● ● 60 ●●

60 ● ●● 2 ● R = 0.993 ● ● ● ● ●

● %

50

● ● e

k ●

i ● l ● − ● 40

b ●

l 40

a

t o

like (%) likeglobins ●

T

–

/

n

훽

i b

o 30

l ● g

− ●

g ● ● 20 ● ● ● 20 ● ●

globin/Total globin/Total ● -

훾 ● ● ● ● ● ● 10 0 25 50 75 0 25 50 75 A-to-G editing at target base in HBG1/2 promotersPosition 5 (%)A−>G%

• Linear regression analysis demonstrates that HBG1/2 gene promoter base editing and gamma globin protein levels in human erythroid cells in vitro are tightly correlated.

• Analysis is consistent with achieving >60% gamma globin protein levels at high base editing levels. Base Editing at HBG1/2 Gene Promoters in SCD Patient Cells Increases Gamma Globin Levels in Erythroid Cells In Vitro

HBG1/2 Promoter Gamma Globin Sickle β-Globin

Base Editing (%) Protein Levels (%) Protein Levels (%)

) )

100 100 % 100

%

(

(

s

s

n

n

i

i ) 80 80 b

b 80

o

%

o

l

(

l

g

g

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60 60 k 60

i

i

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40 a 40

t 40

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a

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n

i

n B

20 i 20 20

b

b

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l

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l

g

g

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0 0 S g 0 β *Next Generation Variant

• >80% base editing at HBG1/2 gene promoters achieved in in vitro-derived erythroid cells from a homozygous sickle cell disease (SCD) donor.

• >60% gamma globin protein levels (relative to total β-like globins) with a concomitant decrease in sickle β-globin. Experimental Outline

Seed Electroporation Cell Viability and Mobilized (EP) + NGS Analysis CD34 HSPCs (108 cells/mL) HBG1/2 Gene Promoter Base Editing (ABE + guide RNA) -48 h 0 h 48 h

Bone Marrow Bone Marrow Analysis Analysis

NBSGW Mouse Transplantation (In Vivo) 8 weeks 16 weeks Long-Term Engraftment Human CD34+ HSPCs Retain Long-term Engraftment and HBG1/2 Gene Promoter Base Editing In Vivo

Human Chimerism (%) HBG1/2 Promoter Base Editing (%) )

% 100

( 100

) Unedited

+

5 )

4 50 nM ABE* D

75 % 75

(

C 20 nM ABE*

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h

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t 50 nM ABE7.10 i

5 50 50

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+ *Next Generation 5

4 Variant D

C 0 0 h 8 16 8 16 8 16 8 16 0 8 16 0 8 16 0 8 16 0 8 16 Mean±SEM n=3 (8 weeks) Post-Transplant (weeks) Post-Transplant (weeks) n=6-7 (16 weeks)

• >90% human chimerism and >90% base editing at HBG1/2 gene promoters achieved in bone marrow samples at 16 weeks post-transplantation. HBG1/2 Gene Promoter Edited CD34+ HSPCs Display Long-Term Multi-Lineage Hematopoietic Reconstitution In Vivo

100

100 Human HSPCs ) 100 Human Erythroid Cells Unedited %

50 ( 50

) ) s 50 nM ABE*

30 l 20

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% 75

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A 5 *Next Generation

h y l Variant G 0 0 h 0 8 16 8 16 8 16 8 16 0 8 8 16 0 8 8 16 0 8 8 16 0 8 8 16 Post-Transplant (weeks) Post-Transplant (weeks)

100 Human Myeloid Cells 100 Human Lymphoid Cells )

) 60 60

% %

( (

+

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D Mean±SEM

C C h h 10 10 n=3 (8 weeks) n=6-7 (16 weeks) 0 0 8 16 8 16 8 16 8 16 8 16 8 16 8 16 8 16 Post-Transplant (weeks) Post-Transplant (weeks) HBG1/2 Gene Promoter Base Editing is Maintained Long-Term Post-Engraftment with Elevated Gamma Globin Levels In Vivo

HBG1/2 Promoter Base Editing (%) Gamma Globin Protein Levels (%) (Sorted Bone Marrow Cells) (Sorted Human Erythroid Cells)

100 ) 100

% (

Unedited

s 65.4±0.9

n i ) 60.9±0.8

b 50 nM ABE* %

75 o 75

(

l

g

g 20 nM ABE*

e 43.4±0.6

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50 - 50 50 nM ABE7.10

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25 / 25 n B 1.1±0.5

i *Next Generation

b o

l Variant g

0 - 0 g + + + + + + + + + + + + + + + + M 5 9 4 A M 5 9 4 A M 5 19 4 yA BM 15 19 34 yA B 1 D1 3 ly B 1 D1 3 ly B D1 D 3 l k D D D l lk D C D G lk D C D G lk C C CD G ul C C C G u C -C u C -C Bu - B e- B ge B ge ge g a a a ea ne ne ne in Li Li Li L Mean±SEM Mean±SEM Human Cell Populations n=4-7 (16 weeks) n=5 (16 weeks)

• >90% base editing achieved in sorted human HSPCs, myeloid, lymphoid and erythroid cells at 16 weeks post-transplantation.

• >65% gamma globin protein levels expressed in sorted base edited erythroid cells compared to unedited cells.

• Similar human chimerism, HBG1/2 promoter base editing and gamma globin protein upregulation has been achieved in a second mobilized CD34+ HSPC donor at 18 weeks post-transplantation. Key Takeaways ✓ In vitro optimization • Increased base editing of the HBG1/2 gene promoters was achieved in mobilized CD34+ HSPCs in a dose-dependent manner using ABE mRNA and guide RNA.

✓ γ-globin upregulation • Base editing highly correlated with γ-globin production (R2=0.99) and suggests that >60% γ-globin protein induction could be achieved in vitro.

• SCD Patient Cells: >80% base editing was observed in erythroid cells in vitro, resulting in upregulation (>60%) of γ-globin protein levels with a concomitant decrease in sickle β-globin.

✓ In vivo performance • Human CD34+ HSPCs retained long-term engraftment and >90% human chimerism, maintained >90% base editing at HBG1/2 gene promoters, and displayed multi-lineage hematopoietic reconstitution.

• Base edited erythroid cell progeny produced high (>65%) γ-globin levels compared to unedited cells (<1.5%). Thank you!

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“A Novel Base Editing Approach to Directly Edit the Causative Mutation in Sickle Cell Disease”

▪ Session Date: Wednesday, May 13, 2020 ▪ Presentation Time: 5:30pm - 6:30pm ▪ Abstract number: 808

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