1. I-SceI-mediated Genome Editing in the Canine Model Kiem lab Applications Meeting Feb 23, 2009

2. GFP Targeting Strategy. Gene Correction With A Dual IDLV System Vector Provirus Target GGGATCCAC TAGGGATAACAGGGTAAT CGGTC GCCACC ATG GTG TGA TAG GGC GAG GAG I-SceI 5’ LTRRREcPPThPGKGFP’WPRE3’ LTRSFFVMGMT P140K Repair Template GTC CTG CTG GAG TTC GTG TAA TGT ACA AGT AA 5’ LTRRREcPPTSFFVI-SceIWPRE3’ LTR I-SceI IDLV HA tag 5’ LTRRREcPPThPGKWPRE3’ LTR Stop codon (  14 a.a.) GGGATCCAC CGGTC GCCACC ATG GTG AGC AAG GGC GAG GAG  14-GFP

3. 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 02004006008001000 10 0 1 2 3 4 SSC-H GFP 8.3e-3 02004006008001000 10 0 1 2 3 4 SSC-H GFP 1.44 02004006008001000 10 0 1 2 3 4 SSC-H GFP 2.44 02004006008001000 10 0 1 2 3 4 SSC-H GFP 3.75 0l0l25  l50  l100  l

4. GFP and I-SceI Expression in D17 Cells treated with the Dual IDLV System I-SceI GFP D17 cells targets were transduced with the two IDLVs at different relative ratios as indicated (I-SceI:repair template). I-SceI:template

5. Targeting with An All-In-One IDLV System Gene Target 5’ LTRRREcPPThPGKGFP’WPRE3’ LTRSFFVMGMT P140K I-SceI + Repair Template 5’ LTRRREcPPThPGK  14-GFP WPRE3’ LTRSFFVI-SceI HA tag I-SceI

6. Analysis of Gene Correction in D17-GFP’ Cells. All-in-one IDLV System 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.061 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.33 02004006008001000 10 0 1 2 3 4 SSC-H GFP 1.72 02004006008001000 10 0 1 2 3 4 SSC-H GFP 2.44 0  l 1l1l10  l50  l 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.

7. Analysis of Targeted D17 cells 0200400600 800 1000 10 0 1 2 3 4 SSC-H GFP 1.45 02004006008001000 10 0 1 2 3 4 SSC-H GFP 91.8 FACS sort PCR amplification of the gene target Reverse primer Target-specific! hPGKGFP’ Forward primer Gene target hPGK  14-GFP Repair template Sequence analysis 73 clones sequenced 41% corrected target 59% original target Original target Clone 1 (+1bp) Clone 2 (corrected) Clone 3 (original) Clone 4 (original) Clone 5 (corrected) Clone 6 (original) I-SceI site Stop codons

8. Transduction of Canine CD34 + Cells with Target Vector (O/N tdn) CFU countsCFU PCR analysis 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 0.49 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 2.8 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 3.4 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 7.1 MockMOI=0.5MOI=1MOI=10 MGMT intracellular staining on liquid cultures 14d after transduction

9. Transduction of Canine CD34 + Cells with The Target Vector (O/N v. O/N+4h tdn) 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 0.14 0200400 600 800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 1.8 0 200 400600800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 2.24 0200400600800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 2.5 0200400600800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 0.085 0200400600800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 1.88 0200400600800 1000 10 0 1 2 3 4 SSC-H MGMT-PE 5.45 02004006008001000 10 0 1 2 3 4 SSC-H MGMT-PE 8.46 Mock MOI=1 MOI=10MOI=20 O/N O/N+4h MGMT intracellular staining on liquid cultures 10d after transduction

10. 02004006008001000 0 1 2 3 4 SSC-H Isotype-PE 0200400600 800 1000 0 1 2 3 4 SSC-H CD34-PE 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.22 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.44 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.61 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.84 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.34 02004006008001000 10 0 1 2 3 4 SSC-H GFP 0.78 02004006008001000 10 0 1 2 3 4 SSC-H GFP 1.05 02004006008001000 10 0 1 2 3 4 SSC-H GFP 1.21 0  l2l2l10  l50  l ML3-GFP’ MOI=1 ML3-GFP’ MOI=10 IDLV Gene Conversion in The Canine CD34 + Cell Line ML3

11. Efficient Lentiviral Transduction of ML3 Cells MockMOI=0.5 MOI=1MOI=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 Relative Vector Provirus Copy Number

12. Dog DLA-identical transplantation setting Donor DLA 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 O 6 BG 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

13. In Vivo Selection to Increase the Percentage of Canine CD34 Cells with I-SceI Targets Days after Transplantation

14. 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

15. 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