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I-SceI-mediated Genome Editing in the Canine Model Kiem lab Applications Meeting Feb 23, 2009.
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I-SceI-mediated Genome Editing in the Canine Model Kiem lab Applications Meeting Feb 23, 2009
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The donor template IDLV and I-SceI IDLVs were delivered into Target D17 cells The volume for both IDLVs used is indicated above each graph. Gene Correction Efficiency in Canine D17 Cells: Dual IDLV System SSC-H GFP 8.3e SSC-H GFP SSC-H GFP SSC-H GFP l0l25 l50 l100 l
Transcript of I-SceI-mediated Genome Editing in the Canine Model Kiem lab Applications Meeting Feb 23, 2009.
I-SceI-mediated Genome Editing in the Canine Model
Kiem labApplications Meeting
Feb 23, 2009
GFP Targeting Strategy.Gene Correction With A Dual IDLV System
Vector Provirus Target
GGGATCCAC TAGGGATAACAGGGTAAT CGGTC GCCACC ATG GTG TGA TAG GGC GAG GAGI-SceI
5’ LTR RRE cPPT hPGK GFP’ WPRE 3’ LTRSFFV MGMTP140K
Repair Template
GTC CTG CTG GAG TTC GTG TAA TGT ACA AGT AA
5’ LTR RRE cPPT SFFV I-SceI WPRE 3’ LTRI-SceI IDLV HA tag
5’ LTR RRE cPPT hPGK WPRE 3’ LTR
Stop codon(14 a.a.)
GGGATCCAC CGGTC GCCACC ATG GTG AGC AAG GGC GAG GAG
14-GFP
The donor template IDLV and I-SceI IDLVs were delivered into Target D17 cells
The volume for both IDLVs used is indicated above each graph.
Gene Correction Efficiency in Canine D17 Cells: Dual IDLV System
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
8.3e-3
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
1.44
0 200 400 600 800 1000100
101
102
103
104
SSC-HG
FP
2.44
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
3.75
0l 25l 50l 100l
GFP and I-SceI Expression in D17 Cells treated with the Dual IDLV System
I-SceIGFP
D17 cells targets were transduced with the two IDLVs at different relative ratios as indicated (I-SceI:repair template).
I-SceI IDLV 5.16 10.33 20.66Repair IDLV 1.17 1.17 1.17
ug p24/ml IDLV
0
10
20
30
40
50
60
0 10 20 30 40Days after transduction
% o
f HA
Tag
+ c
ells
Mock50ul/100ul100ul/100ul200ul/100ul
0123456789
10
0 10 20 30 40Days after transduction
% o
f GFP
+ c
ells
Mock50ul/100ul100ul/100ul200ul/100ul
I-SceI:template I-SceI:template
Targeting with An All-In-One IDLV System
Gene Target
5’ LTR RRE cPPT hPGK GFP’ WPRE 3’ LTRSFFV MGMTP140K
I-SceI + Repair Template
5’ LTR RRE cPPT hPGK 14-GFP WPRE 3’ LTRSFFV I-SceI
HA tag
I-SceI
Analysis of Gene Correction in D17-GFP’ Cells.
All-in-one IDLV System
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
0.061
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
0.33
0 200 400 600 800 1000100
101
102
103
104
SSC-HG
FP
1.72
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
2.44
0l 1l 10l 50l
The donor template and I-SceI were delivered into D17 targets using the all-in-one IDLV.
The volume of IDLV used is indicated above each graph.
Analysis of Targeted D17 cells
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
1.45
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP
91.8
FACS sort PCR amplification of the gene target
Reverse primerTarget-specific!
hPGK GFP’Forward primer
Gene target
hPGK 14-GFP
Repair template
Sequence analysis
73 clones sequenced 41% corrected target59% original target
Original targetClone 1 (+1bp)Clone 2 (corrected)Clone 3 (original)Clone 4 (original)Clone 5 (corrected)Clone 6 (original)
I-SceI site Stop codons
Transduction of Canine CD34+ Cells with Target Vector (O/N tdn)
0
5
10
15
20
25
30
35
40
0 0.5 1 10MOI
Tota
l # o
f col
onie
s
0
5
10
15
20
25
30
35
0 0.5 1 10MOI
% o
f pos
itiv
e co
loni
es
CFU counts CFU PCR analysis
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
0.49
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
2.8
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
3.4
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
7.1
Mock MOI=0.5 MOI=1 MOI=10
MGMT intracellular staining on liquid cultures 14d after transduction
Transduction of Canine CD34+ Cells with The Target Vector (O/N v. O/N+4h tdn)
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
0.14
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE1.8
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
2.24
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
2.5
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
0.085
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
1.88
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
5.45
0 200 400 600 800 1000100
101
102
103
104
SSC-H
MG
MT-
PE
8.46
Mock MOI=1 MOI=10 MOI=20
O/N
O/N+4h
MGMT intracellular staining on liquid cultures 10d after transduction
0 200 400 600 800 10000
1
2
3
4
SSC-H
Isot
ype-
PE
0 200 400 600 800 10000
1
2
3
4
SSC-H
CD34
-PE
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.22
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.44
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.61
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.84
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.34
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 0.78
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 1.05
0 200 400 600 800 1000100
101
102
103
104
SSC-H
GFP 1.21
0l 2l 10l 50l
ML3-GFP’MOI=1
ML3-GFP’MOI=10
IDLV
Gene Conversion in The Canine CD34+ Cell Line ML3
Efficient Lentiviral Transduction of ML3 Cells
0 50K 100K 150K 200K 250KSSC-A
0
102
103
104
105
FITC
-A
0.03
0 50K 100K 150K 200K 250KSSC-A
0102
103
104
105FI
TC-A
2.36
0 50K 100K 150K 200K 250KSSC-A
0102
103
104
105
FITC
-A
4.97
0 50K 100K 150K 200K 250KSSC-A
0102
103
104
105
FITC
-A
19.2
Mock MOI=0.5
MOI=1 MOI=10
RSCSPGW2
ML3 cells can be efficiently transduced with an integrating lentivirus
ML3 and D17 target cells have comparable amounts of the target vector provirus
0
50
100
150
200
250
ML3 parent ML3-GFP' moi1
ML3-GFP' moi10
D17 parent D17-GFP' moi0.9
D17-GFP' moi4.5
Cell Line
Rela
tive
am
ount
of l
enti
viru
sR
elat
ive
Vec
tor P
rovi
rus
Cop
y N
umbe
r
Dog DLA-identical transplantation setting
DonorDLA identical recipient
I. Collection and transduction of CD34+
cells with LHE site-containing integrating
lentiviral vector
II. Infusion of cells after conditioning by
irradiation
III. Iterative treatments with O6BG and BCNU or
temozolomide followed by collection of CD34+ cells
with stably integrated LHE site-containing target.
VI. Infusion of cells after myeloablative
conditioning V. Transduction with IDLV encoding
repair template and I-SceI
IV. Investigate repair efficiency in canine progenitors
In Vivo Selection to Increase the Percentage of Canine CD34 Cells with I-SceI Targets
Days after Transplantation
Summary• Efficient IDLV targeting using an EGFP reporter system
• A ratio of I-SceI IDLV: Donor IDLV 4.4:1 gave efficient targeting (lowest ratio tried to date)
• Similar efficiency with all-in one IDLV vector in D17 cells
• Demonstrated Gene Correction at the Molecular level
• Established conditions for efficient introduction of target vector in canine CD34+ cells
Future Experiments• Investigate the effect of the repair template : I-SceI ratio on
gene repair efficiency and toxicity
• Test a negative selection marker (e.g. Cytosine Deaminase) to eliminate background random integrants from the IDLV.
• Evaluate LHE-mediated genome editing of canine hematopoietic progenitors and repopulating cells.
• Generate an I-AniI-mediated reporter system
