1. Nina M. Muñoz Kiem lab May 18th, 2009
A Color Change Reporter System For I-SceI-mediated Genome Editing In Canine Hematopoietic Stem Cells
Nina M. Muñoz
Kiem lab
May 18th, 2009

2. GFP Targeting Strategy. Gene Correction With A Two-IDLV System
Gene Target
GGGATCCAC TAGGGATAACAGGGTAAT CGGTC GCCACC ATG GTG TGA TAG GGC GAG GAG
I-SceI
2 stop codons
5’ LTR
RRE
cPPT
SFFV
MGMTP140K
hPGK
GFP’
WPRE
3’ LTR
Repair Template
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
GTC CTG CTG GAG TTC GTG TAA TGT ACA AGT AA
I-SceI
HA tag
5’ LTR
RRE
cPPT
SFFV
I-SceI
WPRE
3’ LTR

3. Analysis of Gene Correction in GFP’-expressing D17 Cells.
200
400
600
800
1000 10 1 2 3 4
SSC-H
GFP
8.3e-3
1.44
2.44
3.75
0l
25l
50l
100l
The repair template and I-SceI were delivered into D17 cells stably expressing the GFP’ target
using the two IDLVs.
The volume of each IDLV used is indicated above each graph.

4. The expression of I-SceI (by HA Tag) and GFP was followed over time.
GFP and I-SceI Expression in D17-GFP’ Cells treated with The Two-IDLV System
GFP
I-SceI
D17 cells stably expressing the GFP’ target were transduced with the two IDLVs.
The expression of I-SceI (by HA Tag) and GFP was followed over time.

5. Sequencing Analysis of Gene Correction in D17-GFP’ Cells
200
400
600
800
1000 10 1 2 3 4
SSC-H
GFP
1.45
200
400
600
800
1000 10 1 2 3 4
SSC-H
GFP
91.8
FACS sort
PCR amplification of the gene target
Reverse primer
Target-specific!
hPGK
GFP’
Forward primer
Gene target
14-GFP
Repair template
Sequence analysis
73 clones sequenced
41% corrected target
59% original target
Original target
Clone 1 (+1bp, NHEJ)
Clone 2 (corrected)
Clone 3 (original)
Clone 4 (original)
Clone 5 (corrected)
Clone 6 (original)
I-SceI site
Stop codons

6. Transduction of Primary CD34+ Cells with The Gene Target
CFU counts
CFU PCR analysis
MGMT intracellular staining on liquid cultures 14d after transduction
200
400
600
800
1000 10 1 2 3 4
SSC-H
MGMT-PE
0.49
2.8
3.4
7.1
Mock
MOI=0.5
MOI=1
MOI=10

7. Canine Secondary Transplant for I-SceI-mediated Gene Correction
I. Collect and transduce CD34+ cells with lentiviral vector encoding target gene containing I-SceI restriction site and MGMTP140K
II. Infuse cells after conditioning by irradiation
Donor
DLA identical recipient
V. Infuse cells after myeloablative conditioning
III. Treat iteratively with O6BG and BCNU
IV. Collect CD34+ cells and
transduce with IDLVs encoding I-SceI and repair template

8. Canine Secondary Transplant for ISceI-mediated Gene Conversion: Initial Observations
Dog H084 was transplanted on 4/16/09
CD34+ cells product of apheresis from HLA-identical sibling (H089) were exposed twice (O/N+7h) to the lentiviral vector encoding the target gene (MOI=10).
H084 received 3.7x106 cells/kg.
After 3 weeks, VNTR analysis showed full chimerism (100% donor cells in peripheral blood).
PCR analysis on transduced CD34+ cells plated for CFUs before infusion demonstrated 6.8% transduced cells.

9. Future Experiments
Assess marking levels by TaqMan and MGMT intracellular staining on peripheral blood and bone marrow cells.
Initiate O6BG/BCNU treatments, once platelets levels are stable, in order to increase marking levels (if any).
Evaluate gene conversion in vitro using CD34+ cells from dog H084 and the two-IDLV system.