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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
3© 2020 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
4© 2020 Editas Medicine
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
5© 2020 Editas Medicine
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
6© 2020 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
7© 2020 Editas Medicine
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
8© 2020 Editas Medicine
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+
*****
9© 2020 Editas Medicine
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
10© 2020 Editas Medicine
The Case for Developing an Allogeneic Medicine for Oncology
$$$
Start Patient
Journey Hospital Apheresis
Centralized
Manufacturing
Production
Time - WeeksHospital
Patient
Treatment
Apheresis/gene editHealthy
DonorExpand Cells Hospital
Multiple Patient
Treatments
Autologous
Allogeneic
COGs Scope
$$
11© 2020 Editas Medicine
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
12© 2020 Editas Medicine
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
13© 2020 Editas Medicine
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
From - Morgan et al. Keystone Emerging Cellular Therapies: Cancer and Beyond/Engineering the Genome February 10 Banff, Alberta 2020
Knockout TGFBR2
Modified from Saetersmoen et al. Seminars Immunopathology 2019
NK cell
14© 2020 Editas Medicine
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
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
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
pSMAD2/33 technical replicates
Stimulation with 10ng/mL TGF-β
Cont
KO
KO NK cells, minimal pSMAD2/3
in presence of TGF-β
From - Morgan et al. Keystone Emerging Cellular Therapies: Cancer and Beyond/Engineering the Genome February 10 Banff, Alberta 2020
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
15© 2020 Editas Medicine
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
16© 2020 Editas Medicine
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
17© 2020 Editas Medicine
The Case for Developing an iPSC-Derived Medicine for Oncology
iPSC Multiple gene
edited MCBScalable
ManufacturingHospital
Off-the-shelf
Patient Treatments
Off-the-shelf
$
26© 2020 Editas Medicine
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
18© 2020 Editas Medicine
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
19© 2020 Editas Medicine
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
20© 2020 Editas Medicine
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:
21© 2020 Editas Medicine
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
22© 2020 Editas Medicine
We Demonstrate that Edited iPSCs Can be Differentiated into Functional
iPSC-Derived NK cells (iNKs) with Enhanced Tumor Killing Activity
26© 2020 Editas Medicine
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
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
23© 2020 Editas Medicine
▪ 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
24© 2020 Editas Medicine
Acknowledgements
Partners
Allergan
Bristol-Myers Squibb
BlueRock Therapeutics
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