Nina M. Muñoz Kiem lab May 18 th , 2009

1. 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. GGGATCCAC TAGGGATAACAGGGTAAT CGGTC GCCACC ATGGTG TGA TAGGGC GAG GAG I-SceI GGGATCCAC CGGTC GCCACC ATGGTG AGC AAGGGC GAG GAG 5’ LTR RRE cPPT hPGK 14-GFP WPRE 3’ LTR Stop codon (14 a.a.) GFP Targeting Strategy.Gene Correction With A Two-IDLV System Gene Target 2 stop codons 5’ LTR RRE cPPT SFFV MGMTP140K hPGK GFP’ WPRE 3’ LTR Repair Template 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. 4 10 1.44 4 10 4 10 3 10 2.44 4 10 0l 25l 50l 100l 3.75 3 GFP 10 GFP 3 10 2 10 3 10 8.3e-3 GFP 2 10 GFP 2 10 1 10 2 10 1 10 1 10 0 10 1 10 0 200 400 600 800 1000 SSC-H 0 10 SSC-H 0 0 200 400 600 800 1000 10 SSC-H 0 0 200 400 600 800 1000 10 SSC-H 0 200 400 600 800 1000 Analysis of Gene Correction in GFP’-expressing D17 Cells. 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. 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. 4 10 hPGK GFP’ 4 10 91.8 3 10 3 10 FACS sort hPGK PCR amplification of the gene target 14-GFP GFP Gene target 2 10 2 10 Reverse primer Target-specific! Forward primer 1 10 1 10 SSC-H Repair template Sequence analysis 0 10 0 10 800 0 200 400 600 1000 0 200 400 600 800 1000 I-SceI site Stop codons Original target Clone 1 (+1bp, NHEJ) Clone 2 (corrected) Clone 3 (original) Clone 4 (original) Clone 5 (corrected) Clone 6 (original) 73 clones sequenced 41% corrected target 59% original target Sequencing Analysis of Gene Correction in D17-GFP’ Cells 1.45 GFP SSC-H

6. 4 10 4 4 10 10 4 10 3.4 7.1 2.8 0.49 3 10 3 3 10 10 3 10 2 10 2 2 10 10 2 10 1 10 1 1 10 10 1 10 0 10 0 0 10 10 0 10 0 200 400 600 800 1000 0 0 200 200 400 400 600 600 800 800 1000 1000 0 200 400 600 800 1000 MGMT-PE MGMT-PE MGMT-PE MGMT-PE SSC-H SSC-H SSC-H SSC-H Mock MOI=0.5 MOI=1 MOI=10 Transduction of Primary CD34+ Cells with The Gene Target CFU counts CFU PCR analysis MGMT intracellular staining on liquid cultures 14d after transduction

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.