ex vivo applications of cas12a - editas medicine€¦ · adapted from canver and orkin, blood, 2016...

1© 2020 Editas Medicine© 2020 Editas Medicineeditasmedicine.com

Ex vivo applications of Cas12a

Richard A Morgan

Keystone Symposia,

Engineering the Genome/Emerging Cellular Therapies

Banff AB, Canada

February 10, 2020

2© 2020 Editas Medicine

Disclosures

▪ Rick Morgan is a stockowner of and employee at Editas Medicine, Inc.


Cas12a: The Other CRISPR Nuclease

Naturally occurring ~40 nt

single guide RNA

5’ staggered DNA cut with

4 nt overhangs

Predominantly blunt DNA cut or

1 nt overhang

Separate crRNA and trRNA that

can be linked (~100 nt)

Cas9 Cpf1 (Cas12a)

Targets G-rich PAMs Targets T- and C-rich PAMs

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

Cas12a is Highly Specific Primarily Due to Intrinsic DNA Target Engagement Mechanism that is Distinct from SpCas9

Editas general suite of nucleases

SpCas9 AsCas12a

Zuris et al. Molecular Therapy Vol 27 No 4S1 April 2019Strohkendl et al. Mol Cell 2018, vol 71, 816-824

Swarts et al. Biochem Soc Trans 2019, vol 47, 1499-1510

4© 2020 Editas Medicineeditasmedicine.com

Cas12a Applications for Engineered Cell Medicines:

Sickle Cell Disease

NK Cell Therapy for Cancer


Etiology of Sickle Cell Disease

•Sickle cell disease (SCD) is caused by a single mutation E6V of the b-globin chain,

leading to polymerization of hemoglobin (Hb) and formation of sickle hemoglobin

(HbS) fibers when deoxygenated.

•Symptoms include anemia, acute chest syndrome, pain crises, and an array of

other complications.

•Patients suffer significant morbidity and early mortality.

Amino

Acid

GLU

VAL

G A G

DNA

G T Gbs-globin

b-globinb

a

b

a

Hemoglobin

Tetramer

bs

a a

bs

HbS

HbA

b

a

b

a

Low

O2

bs

a a

bs bs

a a

bs

bs

a a

bs

HbS Fiber

Bender MA. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, Amemiya A, editors. Gene Reviews® [Internet].

Seattle (WA): University of Washington, Seattle; 1993-2018.


Harnessing Natural Anti-sickling Hemoglobin

to Treat Sickle Cell Disease

Embryo Fetus Adult

b-globin locus

Enhancer

(HS1-4)

LCR

Insulator

(5’HS)Insulator

(3’HS1)

HBG1HBG2HBE HBBHBD

HbF

a a

g g

HbA

HbS b

a

b

a

Adapted from Canver and Orkin, Blood, 2016

Globin switchOnset of

SCD

Symptoms

d

b g

e

a

0

25

50

75

100

0 3 6 9 12 15 18

% G

lob

in S

yn

thesis

Months Post-conception

b

a

b

a

g

a a

g


Genome Editing to Reverse Hemoglobin Switching

for Treating Sickle Cell Disease

CRISPR-based

disruption of

HbF-repressing

machinery

Patient

CD34+ cells

Engineered

patient

CD34+ cells

with switching

reversed

bs

a a

bs

HbS

bs

a a

bsg

a a

g

HbF

bs

a a

bs

HbS

bs

a a

bs

From - Chang et al. Annual Meeting of the American Society of Hematology, San Diego, CA., December 2, 2018


Optimized CRISPR Cas12a-based Editing at the HBG Distal

CCAAT box Region – EDIT-301

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


Cas12a Showed Improved Editing in HSCs with

Desired Indel Size and Specificity



HbF: fetal hemoglobin; HbA: adult hemoglobin

Editing HBG1/2 Induces Robust Fetal Hemoglobin In Vivo

0

20

40

60

80

100

Unedited HBG1/2

Hb

F+

Re

d B

loo

d C

ell

s

High pan-cellular human HbF in red blood cellsRobust HbF in human CD34+ cells in vivo

89%

4%

n = 5 healthy human donors in NBSGW mice at 16 weeks

4%

52%

0

20

40

60

80

100

Unedited EDIT-301

Hb

F/ (H

bF

+ H

bA

)

100% 100%

HBG1/2

EditedHBG1/2

Edited



Cas12a Editing for SCD

Cas12a produced more long term HbF inducing indels than SpCas9 at the

HBG distal CCAAT box region.

Greater than 90% indels were achieved after optimization of electroporation

conditions and selection of best performing Cas12a variant and gRNA

modifications

EDIT-301 had no detectable off target editing and contained highly edited

long term HSCs (> 90 % indels) that engrafted mice with high polyclonality

and no lineage skewing.

50% HbF levels were observed in vivo with pancellular distribution.

EDIT-301 program, heading toward IND in 2020



Oncology Cell Therapy Development

$$$

Start Patient

Journey Hospital Apheresis

Centralized

Manufacturing

Production

Time - WeeksHospital

Patient

Treatment

Apheresis/gene edit

iPSC

Healthy

DonorExpand Cells Hospital

Multiple Patient

Treatments

Multiple gene

edited MCBScalable

ManufacturingHospital

Off-the-shelf

Patient Treatments

Autologous

Off-the-shelf

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


• Antibody-directed

• CAR

Graft-vs-Host Risk Higher Lower

CAR: chimeric antigen receptor

Potential Cell Types for Allogeneic Cell Medicines

Pluripotent

Stem Cell

CD34+ Cells

INNATEADAPTIVE


Multiple Gene Edits Required for the Generation of an

“Off-the-shelf” Allogeneic T Cell ProductsMultiple Gene Edits Allow for the Generation

of an “Off-the-shelf” Allogeneic T Cell Product

Host CD4T Cell

TCR

EngineeredT Cell

MHC Class I

Host CD8T Cell

Host Epithelial

Cell

MHC Class II

CIITA

B2M

Response: (+)

Multi-geneEdit

EngineeredT Cell

Host Epithelial

Cell

Host CD8T CellTCR

MHC Class II

CIITA

Host CD4T Cell

B2M

MHC Class I

Response: (–)

GvHR, HvGR

Zuris et al. Molecular Therapy Vol 27 No 4S1 April 2019


Making an Allo T Cell: Cas12a Comparable to SpCas9

0

20

40

60

80

100

TRAC B2M CIITA

% E

dit

ing

by

NG

S

Cpf1

Cas9

Zuris et al. Molecular Therapy Vol 27 No 4S1 April 2019

0

20

40

60

80

100

TRAC B2M CIITA

% E

dit

ing

by N

GS

Efficient Multiplexing by Cas12a

Cas12a

Cas9


ab T cells gd T cells NK cells

Immune System Adaptive Innate

Tumor Recognition • ab T cell receptor

• CAR

• gd T cell receptor


• CAR


• Antibody-directed

• CAR

Graft-vs-Host Risk Higher Lower

CAR: chimeric antigen receptor

Potential Cell Types for Allogeneic Cell Medicines

Pluripotent

Stem Cell

CD34+ Cells

INNATEADAPTIVE


NK Cells Can Both Directly Kill Tumors and

Stimulate the Endogenous Immune System

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


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

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


Potency Comparison of Engineered Cas12a and SpCas9

in NK Cells at the Same Target Site in the Genome

Control target site for comparing SpCas9 and AsCas12a

MS5

MS5 chosen from Kleinstiver et al. Nat Biotech 2016

n = 2

0

20

40

60

80

100

0.01 0.1 1 10

% E

ditin

g b

y N

GS

RNP concentration (µM)

Potency of Cas12a vs. Cas9 in NK cells

Cas12a

Cas9

vol 34, 869-874


0

20

40

60

80

100

1 10 100 1000 10000

% E

ditin

g b

y N

GS

RNP concentration (nM)

Editing of TGFBR2 in three different donors

Donor 1

Donor 2

Donor 3

Rationale Editing Biology

Robust Gene Editing at TGFBR2 in Healthy Donor

NK Cells with an Engineered Cas12a

Cell cycle

progression

Anti-tumor

function

NK cell

P

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


TGFBR2 Knockout (KO) NK Cells Showed Superior Effector

Function than Unedited Control NK Cells

Attached 2D tumor cell monolayer

NK cell addition

24hr0hr

Assay readouts

• % Cytolysis by xCELLigence

• IFNγ production by FACS

xCELLigence assay

Target - SKOV3

OvCa cells

Control

NK

%C

yto

lysis

at

14hr

(E:T

=4:1

, n

o c

yto

kin

e trt)

% CytolysisAverage of 3 donors, 5 independent

experiments

TGFBR2

KO

***

Co

nt r

ol

TG

FB

R2

KO

0

2 0

4 0

6 0

8 0

1 0 0

D a t a 2

% C

yto

lys

is

%IFNγ+NK cells at 24hrAverage of 3 donors

IL-15 IL15+TGFβ

Treatment during assay

%IF

Nγ

+N

K c

ells

+I L

- 15

+I L

- 15

+T

GF

b

0

1 0

2 0

3 0

4 0

C o p y o f I L 1 5 v s I L 1 5 + T G F B

%IF

Ng

+C

D5

6+

NK

ce

lls U n e d i t e d

T G F B R 2 K O

*** **


TGFBR2 KO Exerted Superior Control of Tumor Spheroids

for >125hrs in the Presence of TGF-β

Time Post Effector Addition (hours)

No

rma

lize

d T

ota

l In

tegra

ted

Red

Ob

ject In

ten

sity

(RC

U x

mm

2/im

age

)

20:1 E:T(3 Donors, 5 Independent Experiments)

SK-OV-3 spheroid + 10ng/mL TGF-b

No

rma

lize

d T

ota

l In

tegra

ted

Red

Ob

ject In

ten

sity

(RC

U x

mm

2/im

age

)

Time Post Effector Addition (hours)

20:1 E:T(3 Donors, 5 Independent Experiments)

PC3 spheroid + 10ng/mL TGF-b Control NK

TGFBR2 KO

+ IL -1 5 + IL -1 5 + T G F b

0

2 0

4 0

6 0

8 0

IL 1 5 v s IL 1 5 + T G F B

%IF

Ng

+C

D5

6+

NK

ce

lls U n e d ite d

C IS H K O

T G F B R 2 K O

D K O

Tumor

spheroid

125hr0hr

NK cell addition Spheroid assayAssay readouts

• Changes in spheroid size over time

(killing by NK cells)

(measurement of red object intensity)

+ IL -1 5 + IL -1 5 + T G F b

0

2 0

4 0

6 0

8 0

IL 1 5 v s IL 1 5 + T G F B

%IF

Ng

+C

D5

6+

NK

ce

lls U n e d ite d

C IS H K O

T G F B R 2 K O

D K O

TGF-b


Multiple

innate receptors

Antibody-directed

cellular cytotoxicity

(ADCC)

NK CELL ADCC

NK CELL

Multiple

innate receptors CARs

ADCC

NK CELL

Multiple

innate receptors

CRISPR editing

Improved ADCC, persistence,

and tumor micro-environment

(TME) resistance

Improved ADCC, persistence, and

additional TME resistance

Improved recognition of tumor cells

lacking T cell antigens for PD-1

nonresponding tumors with innate

receptors and CARs

CRISPR editing

NK Therapeutic Strategy for Winning in Solid Tumors


Cas12a Editing for NK Cell-based Therapies

The next generation of allo and off-the-shelf cell therapies for cancer will

require robust and specific gene editing.

Cas12a produces efficient and specific gene editing, comparable or superior

to SpCas9 in both primary T cells and NK cells.

Greater than 90% editing obtained targeting the TGFBR2 gene in primary

human NK Cells.

TGFBR2 editing NK cells demonstrated superior effector cell function in both

short and long-term cell killing assay.

Gene edited healthy donor NK program progressing to IND-enabling studies


Acknowledgements

Sickle Cell Disease TeamKaiHsin Chang, Edouard de Dreuzy, Jack Heath

Healthy Donor NK Cell TeamKarrie Wong, Chris Borges, John Zuris

EditasDiscovery, Development, Chemistry, and Operations

PartnersBristol-Myers Squibb

BlueRock Therapeutics

Sandhill Therapeutics

Integrated DNA Technologies

GenEdit

ex vivo applications of cas12a - editas medicine€¦ · adapted from canver and orkin, blood, 2016...

Documents