highly efficient multi-gene knockout and transgene knock ... · © 2020 editas medicine 2...

1© 2020 Editas Medicine© 2020 Editas Medicineeditasmedicine.com

Highly Efficient Multi-Gene Knockout and Transgene Knock-in

using CRISPR-Cas12a in Induced Pluripotent Stem Cells for

the Generation of Engineered Cell Immunotherapies

JA Zuris, CM Margulies, R Viswanathan, JN Edelstein, R Pattali, KW Gareau, SN

Scott, S Lele, AC Wilson, JI Moon, N Dilmac, AE Friedland, RA Morgan

John Zuris, Ph.D.

May 15th, 2020

2© 2020 Editas Medicine

Disclosure

▪ John Zuris is an employee and shareholder at Editas Medicine


Develop best-in-class medicines for

hemoglobinopathies using Cas12a and solid

tumors using iPSC-derived cells

Leverage AAV-mediated editing with SaCas9

into additional therapeutic areas

CRISPR: clustered regularly interspaced short palindromic repeat; SaCas9: Staphylococcus aureus CRISPR-associated protein 9; Cas12a: CRISPR-associated protein 12a; iPSC: induced pluripotent stem cell

Building a Genomic Medicine Leader

CRISPR Gene Editing to Develop Differentiated, Transformational Medicines for High Unmet Need

Maintain Best-in-class Platform & Intellectual Property, and Advance Organizational Excellence

In Vivo CRISPR Medicines Engineered Cell Medicines


First in human CRISPR-Cas Gene Editing: EDIT-101 for LCA10

Mutated CEP290 Gene

Repaired CEP290 Gene

Genome Editing

Restored CEP290 Protein

Transcription and normal mRNA splicingProtein translation

EDIT-101

Cut and Remove

Deletion

MutationPAM

Chromosome 12 position q21.32


AGN-151587 (EDIT-101): SaCas9 and 2 gRNAs in AAV to

Correct LCA10-IVS26 Mutation

U6 U6323 64 hGRK1SV40

SD/SASa Cas9

NLS

pA

NLS

Kozak-ATG

Exon 26 Exon 27

323 64

Deletion

Inversion

IVS26

First patient dosed at Oregon Health & Science University (OHSU)

Casey Eye Institute, in February 2020


Develop best-in-class medicines for

hemoglobinopathies using Cas12a and solid

tumors using iPSC-derived cells

Leverage AAV-mediated editing with SaCas9

into additional therapeutic areas

CRISPR: clustered regularly interspaced short palindromic repeat; SaCas9: Staphylococcus aureus CRISPR-associated protein 9; Cas12a: CRISPR-associated protein 12a; iPSC: induced pluripotent stem cell

Building a Genomic Medicine Leader

CRISPR Gene Editing to Develop Differentiated, Transformational Medicines for High Unmet Need

Maintain Best-in-class Platform & Intellectual Property, and Advance Organizational Excellence

In Vivo CRISPR Medicines Engineered Cell Medicines


There Are Many Nucleases to Choose from but for Ex Vivo Gene Editing

Using AsCas12a Offers Several Potential Advantages to SpCas9

Variant PAM Frequency (bp)

SpCas9 NGG 1 in 8

SaCas9 NNGRRT 1 in 32

SaCas9 KKH NNNRRT 1 in 8

AsCas12a TTTV 1 in 43

AsCas12a RR TYCV/CCCC 1 in 18

AsCas12a RVR TATV 1 in 43

LbCas12a TTTV 1 in 43

Editas general suite of nucleases AsCas12a is a more specific nuclease compared to SpCas9

References on AsCas12a target specificity:

Kim et al. Nat Biotech 2016

Strohkendl et al. Mol Cell 2018

Swarts et al. Biochem Soc Trans 2019

PAM sites are not limiting for general KO

and large transgene KI applications

Small ~40 nt Cas12a guide is advantageous

for manufacturing and sequence fidelityMatched Target

Sites 1→25

From - Gotta et al. Genome Engineering: Frontiers of CRISPR/Cas Cold Spring Harbor, NY October 10, 2019

Matched Target

Sites 1→25

Matched Target

Sites 1→25

Matched Target Site (20-Ns):

TTTVNNNNNNNNNNNNNNNNNNNNNGGRRT


Engineered Cas12a (EngCas12a) Robustly Edits at Higher Efficiency than

WT Cas12a and is Capable of Efficient Knock-in in T Cells

EngCas12a has superior activity High AAV6-mediated knock-inHigh knock-out across loci

Based on AsCas12a Ultra nuclease from IDT

Up to 40% double knock-in

Knock-in of a Single Transgene CargoEditing at Multiple Sites in B2M Exon Efficient Editing at Key Targets

TRAC B2M CIITA PD1

0

20

40

60

80

100

T cells

% E

dit

ing

by N

GS

RNP +

AAV

6

RNP o

nly

Alt

RNP +

AAV

6

Cel

ls o

nly

0

10

20

30

40

50

60

% K

no

ck-i

n

TCR– GFP+

B2M– mCherry+

*****


EngCas12a is Used in EDIT-301, an Experimental

Medicine for Sickle Cell Disease

>90% editing in HSCs with EngCas12a

>50% HbF levels that persist after engraftment

High specificity of WT nuclease retained in EngCas12a

Have since verified this attribute in many other cell types

From – De Dreuzy et al. Annual Meeting of the American Society of Hematology, Orlando. FL, December 9, 2019

Robust HbF in human CD34+ cells in vivo

4%

52%

0

20

40

60

80

100

Unedited EDIT-301

Hb

F/

(Hb

F+

Hb

A)

100%

HBG1/2Edited


The Case for Developing an Allogeneic Medicine for Oncology

$$$

Start Patient

Journey Hospital Apheresis

Centralized

Manufacturing

Production

Time - WeeksHospital

Patient

Treatment

Apheresis/gene editHealthy

DonorExpand Cells Hospital

Multiple Patient

Treatments

Autologous

Allogeneic

COGs Scope

$$


ab T cells gd T cells NK cells

Immune System Adaptive Innate

Tumor Recognition • ab T cell receptor

• CAR

• gd T cell receptor

• Innate receptors

• CAR

• Innate receptors

• Antibody-directed

• CAR

Graft-vs-Host Risk Higher Lower

CAR: chimeric antigen receptor

Potential Cell Types for Allogeneic Cell Medicines for Cancer

Pluripotent

Stem Cell

CD34+ Cells

INNATEADAPTIVE


Potential Advantages of NK Cell Therapy for Oncology

NK cells induce adaptive immune

responses against tumorsMultiple tumor-recognition mechanisms; potential to

combine with therapeutic antibodies to enhance ADCC

To date, no evidence of GvHD in patients treated with allogeneic NK cells, unlike off-the-shelf T cells

• Loss of MHC

• NKG2D

• NCRs

• CD16, etc…

Souza-Fonseca-Guimaraes, et al., Trends in Immunology, 2019, Vol. 40, 142-158

From - Morgan et al. Keystone Emerging Cellular Therapies: Cancer and Beyond/Engineering the Genome February 10 Banff, Alberta 2020

Rezvani et al. Mol Therapy 2017


TG

FB

1 L

og

2F

c

(mR

NA

expre

ssio

n)

Matched TCGA normal and GTExdatabase

Stomach cancer n=408 (pt)

n=211 (normal)

HNSCCn=529 (pt)

n=44 (normal)

TGFβ is an immunosuppressive factor

abundant in the solid tumor microenvironment

Case for Knocking out the TGFBR2 Protein on the Surface of NK Cells:

TGF-β is a Major NK Cell Suppressive Cytokine

TGF-b is Overexpressed in Many Solid Tumors

Souza-Fonseca-Guimaraes, et al., Trends in Immunol, 2019, Vol. 40, 142-158


Knockout TGFBR2

Modified from Saetersmoen et al. Seminars Immunopathology 2019

NK cell


Rationale Editing Biology

Efficient KO of TGFBR2 in Healthy Donor NK Cells with EngCas12a

Leads to Expected Loss of TGF-b Signaling

Cell cycle

progression

Anti-tumor

function

NK cell

0 2 0 6 0 1 8 0

0

2

4

6

D a ta 1

T im e (m in u te s )

No

rm

ali

ze

d p

SM

AD

2/3

N o E P

T G F B R 2 K O

0 2 0 6 0 1 8 0

0

2

4

6

D a ta 1


No

rm

ali

ze

d p

SM

AD

2/3

N o E P

T G F B R 2 K O

0 2 0 6 0 1 8 0

0

2

4

6

D a ta 1


No

rm

ali

ze

d p

SM

AD

2/3

N o E P

T G F B R 2 K O

pSMAD2/33 technical replicates

Stimulation with 10ng/mL TGF-β

Cont

KO

KO NK cells, minimal pSMAD2/3

in presence of TGF-β


0.001 0.01 0.1 1 10

0

20

40

60

80

100

RNP concentration (µM)

% E

dit

ing

by N

GS

Editing at TGFBR2 in Three Different Donors

Donor 1

Donor 2

Donor 3P


Efficient Transgene Knock-in with EngCas12a in Healthy

Donor NK Cells Enables Testing of New Biology

50% knock-in with EngCas12a Tumor-targeting CAR knock-in enhances killing

0 25 50 75 100 125

0.5

0.6

0.7

0.8

0.9

1.0

Time (h)

Sp

hero

id S

ize

2:1 E:T ratio

CAR Knock-in

GFP Knock-in

Enhanced tumor killing with CAR

1 10

0

10

20

30

40

50

AAV6 MOI x 1e5 (vg/cell)

% K

no

ck-i

n

TRAC site 1

TRAC site 2


EngCas12a Can Overcome the Editing Challenges in Creating Highly

Engineered Cells for Targeting Solid Tumors

Modified from Saetersmoen et al. Seminars Immunopathology 2019

Knockout TGFBR2

Summary of what we achieved with EngCas12a platform for potentially making medicines for oncology:

- EngCas12a retains intrinsic AsCas12a specificity advantage over SpCas9

- EngCas12a can robustly edit targets across cell types, including functional KO of TGFBR2 in NK cells

- EngCas12a can efficiently knock-in transgene cargos into T cells and NK cells

Potential

Knock-ins

NK cell


The Case for Developing an iPSC-Derived Medicine for Oncology

iPSC Multiple gene

edited MCBScalable

ManufacturingHospital

Off-the-shelf

Patient Treatments

Off-the-shelf

$


Advancing Universal Allogeneic Cell Medicines to Treat Cancer

Differentiated

somatic celliPSC Edited clonal iPSC line iNK cells

Technology from

BlueRock Therapeutics

High editing efficiency

with engineered Cas12a

Genome

characterization builds

on in vivo editing

capabilities

Developing a

proprietary, scalable

differentiation method

Highly engineered line

Low COGs

Off-the-shelf

Infinitely renewable

Defined genome

0

0

DifferentiateDedifferentiateEditing +

clonal identification

Our process:

iPSC platform allows for highly edited cells


Knock-in of Many Transgenes Poses a Major Manufacturing Challenge

Saetersmoen et al. Seminars Immunopathology 2019

Potential

Knock-ins

Potential Knock-out: TGFBR2

First key step in our iPSC editing platform:

Demonstrate highly efficient dsDNA break

occurrence (i.e. knock-out) in order to

maximize opportunity knock-in efficiency


Highly Efficient Gene Editing in iPSCs Using EngCas12a

Recommended Lonza conditions for SpCas9

Additional advances to our iPSC editing platform:

- Made up to four edits at once and developed assays to assess which edit combinations show most promise

- Developed method to edit cells in a consistent manner so that editing and differentiation results are consistent

- Developed method to detect the possible infrequent occurrence of a translocation between two on-target edits

EC50 < 100 nM

AAV

S1

B2M

Targ

et 3

Targ

et 4

Targ

et 5

Targ

et 6

0

20

40

60

80

100

Potential target sites for iNK

% E

dit

ing

by N

GS

0.01 0.1 1 10

0

20

40

60

80

100

RNP Concentration (µM)%

Ed

itin

g b

y N

GS

Potent RNP targeting AAVS1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

20

40

60

80

100

Condition

% E

dit

ing

by N

GS

Electroporation pulse code and buffer screen


Transgene Knock-in in an iPSC Requires Additional Considerations

Attributes

Donor type

AAV Plasmid ssODN

Efficient transgene insertion High Low Moderate

Toxicity when delivered to cells Moderate High Moderate

Off-target integration events Low Moderate Low

Delivery of large cargos Moderate HIGH Low

Manufacturing challenge High LOW High

Donor Nature

Plasmid

Linear plasmid

AAV

Long ssDNA

RNP Dose

Electroporation

Pulse Code

Buffer

Additives

Rock/CloneR

BCL-XL

NHEJ Inhibitors

p53 Inhibitors

Donor Composition

Homology arm length

Optimization is needed for plasmid-based

transgene knock-in in iPSCs

Factors to consider while making a

highly-edited iPSC-derived NK cell

Several parameters that could be tested:


Transgene Knock-in Efficiency Using Plasmid Approaches 20% in iPSCs

mCherry plasmid onlyRNP + mCherry plasmid

MH

C I

-

mCherry+

MH

C I

-

mCherry+

Efficient Editing at Potential Targets

This knock-in efficiency should greatly reduce the number of iPSC clones that need to be analyzed

AAVS1 B2M0

20

40

60

80

100

iPSCs

% E

dit

ing

by N

GS

Editing with EngCas12a

18.4

1.9 0.07

0.034.1172.1

7.7 95.8

Efficient Knock-in of Transgene Cargo


We Demonstrate that Edited iPSCs Can be Differentiated into Functional

iPSC-Derived NK cells (iNKs) with Enhanced Tumor Killing Activity


Advancing Universal Allogeneic Cell Medicines to Treat Cancer

Differentiated

somatic celliPSC Edited clonal iPSC line iNK cells

Technology from


High editing efficiency

with engineered Cas12a

Genome

characterization builds

on in vivo editing

capabilities

Developing a

proprietary, scalable

differentiation method

Highly engineered line

Low COGs

Off-the-shelf

Infinitely renewable

Defined genome

0

0

DifferentiateDedifferentiateEditing +

clonal identification

Process of going from an edited iPSC to a differentiated iNK cell iNK killing of SKOV3 cells

1:4 1:2 1:1 2:1 4:1 8:1

0

20

40

60

80

100

E:T Ratio

% C

yto

lysis

WT

KO


▪ EngCas12a is more potent than Wild Type Cas12a but retains its strongly superior

specificity compared to SpCas9

▪ EngCas12a is capable of highly efficient KO and transgene KI across cell types

ENGCAS12A AS A POWERFUL

GENE EDITING PLATFORM

▪ Allogeneic NK cells are an effective cancer cell therapy without evidence of Graft

vs Host Disease

▪ Using EngCas12a we obtain robust editing to reduce TGF-β impact on NK cells

WHY NK CELLS FOR CANCER?

▪ iPSCs offer the potential to create Off-the-Shelf therapies at low costs

▪ Using the best nuclease possible can increase chances of finding clones with the

desired edit and inserted construct

▪ Edited iPSCs can differentiate into iNKs with enhanced tumor killing ability

BUILDING A PLATFORM TO

MAKE HIGHLY ENGINEERED

IPSC-DERIVED NK CELLS

Summary


Acknowledgements

Partners

Allergan

Bristol-Myers Squibb


Sandhill Therapeutics

Integrated DNA Technologies

GenEdit

Editas Colleagues

iNK Program Team

Sickle Program Team

Healthy Donor NK Program Team

Discovery Biology Team

Lead Discovery Team

Biological Development Team

Boulder Chemistry Team

highly efficient multi-gene knockout and transgene knock ... · © 2020 editas medicine 2...

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