Download Highly Efficient CRISPR/Cas9 Gene Editing and

Highly Efficient CRISPR/Cas9 Gene Editing
and Long-Term Engraftment of Human
Hematopoietic Stem and Progenitor Cells
J. M. Heath, A. Chalishazar, C.S. Lee, W. Selleck, C. Cotta-Ramusino, D. Bumcrot, J.L. Gori
Disclosure:
Jennifer Gori and Co-Authors are Full-time Employees of Editas Medicine
Gene-Modified Autologous Hematopoietic Stem and
Progenitor Cell Therapy
ex vivo approach to gene correction of hematopoietic diseases
CD34+ HSC
erythroid myeloid lymphoid
© 2016 Editas Medicine
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Rationale for Delivery of Cas9 RNP for Gene
Editing in HSCs
Effective gene editing and transient nuclease expression
 Hypothesis
- Cas9 RNP would support gene editing in HSCs without impacting
viability or functionality in vivo
 Electroporation of Cas9/gRNA ribonucleoprotein (RNP)
- High efficiency
- Limited exposure
WT Cas9
Cas9/gRNA treated
Human CD34+ cells
D10A Nickase
24h 48h 72h
5’
PAM
Blunt
Effective for NHEJ
© 2016 Editas Medicine
PAM
PAM
5’ Overhang
Effective for HDR
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a-Cas9
a-Actin
Efficient and Reproducible Editing in HSCs
Fold change in live CD34+ cells
(7-AAD-AnnexinV-)
Comparison of Wild-Type and D10A SpCas9 at b-hemoglobin locus (HBB)
% Gene editing
100
75
50
25
0
CB-HSC mPB-HSC CB-HSC mPB-HSC
WT Cas9
4
3
2
1
0
RNP
D10A Nickase
 Reproducible gene editing across 20 donors
 Maintenance of viability of RNP treated HSCs
© 2016 Editas Medicine
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Control
Gene-Edited HSCs Maintain Erythroid and Myeloid
Multipotency ex vivo
Analysis of gene editing in clonal derivatives of edited HSCs
GEMM
E
GM
M
100
G
% Gene edited colonies
# Hematopoietic colonies
200
150
100
50
Erythroid
Myeloid
75
50
25
0
0
RNP
control
Monoallelic
Biallelic
 RNP treated HSCs
- Differentiate into erythroid and myeloid colonies
- Monoallelic and biallelic gene disruption detected in HSC clones
© 2016 Editas Medicine
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Long-Term Engraftment of Cas9/gRNA RNP
Treated Human HSCs
Compare engraftment of RNP treated and control human CD34+ cells in mouse
xenograft model
Busulfan
± D10A RNP
targeting HBB
-4d
CD34+ cells
-2d
Prestimulation
-1d
0d
Conditioning Infusion
Parameter
n
Busulfan (mg/kg)
25
Control HSC mice
6
RNP HSC mice
7
CD34+ cell dose
570,000
4m
Endpoint
 Reconstitution of human hematopoiesis in vivo (4+ months)
 Gene editing in marrow, spleen, blood (human subsets)
© 2016 Editas Medicine
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Gene Edited Cells Reconstitute Peripheral Blood
Human CD45+ lymphoid and myeloid cells at 4 months
85% human CD45+
Mouse CD45
Subset within human CD45+ gate
Human CD45
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Gene Edited Human Blood Cells and HSCs
Repopulate the Bone Marrow and Spleen
5 million HSCs recovered from bone marrow of each recipient
Bone Marrow
100
70% human CD45+
Spleen
100
RNP
Control
50
18% HSC
25
50
13% HSC
25
0
0
Subset within human CD45+ gate
© 2016 Editas Medicine
RNP
Control
75
% Positive
% Positive
75
73% human CD45+
Subset within human CD45+ gate
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Efficient Gene Editing Detected in Human Blood
Cells in the Bone Marrow and Spleen
Gene editing in vivo equal to editing in pre-infusion product
Bone Marrow
75
50
25
100
RNP
Control
% Gene editing
% Gene editing
100
Spleen
0
RNP
Control
75
50
25
0
 50% gene editing HSC before transplantation
 50% gene editing in engrafted cells in the blood, marrow,
and spleen 4 months after transplantation
© 2016 Editas Medicine
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Gene Editing is Maintained in HSC Progeny
Differentiated in vivo
Gene editing in engrafted HSCs is maintained in progeny in vivo
Sorted Bone Marrow Fractions
Erythroid (CD235)
% Gene editing
100
10
75
50
25
0
HSC
Myeloid
10
10
5
11%
4
3
12%
0
Erythroid
-10
3
-10
3
0
10
3
10
HSCs (CD34)
© 2016 Editas Medicine
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4
10
5
Summary and Conclusions
 Cas9/gRNA RNP supports efficient and reproducible gene
editing in human HSCs across donors (57% ± 8)
 Gene edited HSCs retain phenotype, viability, and
differentiation potential ex vivo
 Gene edited human HSCs retain long-term engraftment and
multipotency in vivo (50% editing and 85% human blood
reconstitution)
© 2016 Editas Medicine
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Electroporation of D10A RNP with Donor Supports
Homology Directed Repair in HSCs
80
% Gene editing
NHEJ
60
40
HDR
20
0
 12% homology directed repair achieved after co-delivery of
D10A RNP and single strand oligonucleotide donor
© 2016 Editas Medicine
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Jack Heath
Aditi Chalishazar
Christina Lee
Will Selleck
Tanushree Phadke
Erik Corcoran
Cecilia Cotta-Ramusino
David Bumcrot