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I-SceI-mediated Genome Editing in the Canine Model

I-SceI-mediated Genome Editing in the Canine Model. Kiem lab Applications Meeting Feb 23, 2009. G GGATCC AC TAGGGATAACAGGGTAAT CGGTC GCCACC ATG GTG T G A T AG GGC GAG GAG. I-SceI. GFP Targeting Strategy. Gene Correction With A Dual IDLV System. Vector Provirus Target. 5’ LTR. RRE.

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I-SceI-mediated Genome Editing in the Canine Model

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

  2. GGGATCCAC TAGGGATAACAGGGTAAT CGGTC GCCACC ATGGTG TGA TAGGGC GAG GAG I-SceI GFP Targeting Strategy.Gene Correction With A Dual IDLV System Vector Provirus Target 5’ LTR RRE cPPT SFFV MGMTP140K hPGK GFP’ WPRE 3’ LTR GGGATCCAC CGGTC GCCACC ATGGTG AGC AAGGGC GAG GAG Repair Template 5’ LTR RRE cPPT hPGK 14-GFP WPRE 3’ LTR GTC CTG CTG GAG TTC GTG TAA TGT ACA AGT AA Stop codon (14 a.a.) HA tag I-SceI IDLV 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 Gene Correction Efficiency in Canine D17 Cells: Dual IDLV System 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.

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

  5. HA tag 5’ LTR RRE cPPT SFFV I-SceI hPGK 14-GFP WPRE 3’ LTR Targeting with An All-In-One IDLV System I-SceI Gene Target 5’ LTR RRE cPPT SFFV MGMTP140K hPGK GFP’ WPRE 3’ LTR I-SceI + Repair Template

  6. 1l 10l 50l 0l 4 10 4 4 4 10 10 10 2.44 0.061 0.33 1.72 3 10 3 3 3 10 10 10 GFP GFP GFP GFP 2 10 2 2 2 10 10 10 1 10 1 1 1 10 10 10 0 10 0 0 0 10 10 10 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC-H SSC-H SSC-H SSC-H Analysis of Gene Correction in D17-GFP’ Cells.All-in-one IDLV System 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. 1.45 4 10 hPGK GFP’ 4 10 91.8 GFP 3 10 3 10 FACS sort hPGK PCR amplification of the gene target 14-GFP GFP Gene target 2 10 2 10 SSC-H 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) Clone 2 (corrected) Clone 3 (original) Clone 4 (original) Clone 5 (corrected) Clone 6 (original) 73 clones sequenced 41% corrected target 59% original target Analysis of Targeted D17 cells

  8. 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 Canine CD34+ Cells with Target Vector (O/N tdn) CFU counts CFU PCR analysis MGMT intracellular staining on liquid cultures 14d after transduction

  9. 4 4 10 10 4 4 10 10 4 10 0.14 4 4 0.085 10 10 1.8 4 2.24 10 2.5 5.45 1.88 3 3 10 10 3 3 10 10 3 10 3 3 10 10 3 10 MOI=10 MOI=20 Mock MOI=1 2 2 10 10 2 2 10 10 2 10 2 2 10 10 2 10 MGMT-PE O/N MGMT-PE MGMT-PE MGMT-PE 1 1 10 10 1 1 10 10 1 10 1 1 10 10 1 10 SSC-H 0 0 SSC-H SSC-H 10 SSC-H 10 0 0 10 10 0 10 0 0 10 10 0 0 200 400 600 800 0 200 400 600 800 1000 1000 10 1000 200 0 400 600 800 1000 0 200 400 800 600 8.46 0 200 400 600 800 1000 0 200 400 600 800 0 200 400 600 800 1000 1000 0 200 400 600 800 1000 O/N+4h MGMT-PE MGMT-PE MGMT-PE MGMT-PE SSC-H SSC-H SSC-H SSC-H Transduction of Canine CD34+ Cells with The Target Vector (O/N v. O/N+4h tdn) MGMT intracellular staining on liquid cultures 10d after transduction

  10. 4 10 4 4 10 4 4 4 4 10 10 10 4 4 10 10 4 10 3 10 3 3 10 3 3 3 3 10 10 10 3 3 10 10 3 Isotype-PE 10 1.05 1.21 CD34-PE 2 10 0.61 0.44 0.22 2 2 10 0.34 0.84 2 0.78 2 2 2 SSC-H 10 10 10 2 2 IDLV 10 10 2 10 SSC-H 1 10 1 1 10 1 ML3-GFP’ MOI=1 GFP 1 1 1 GFP GFP 10 10 10 1 1 10 10 1 10 0 10 0 0 10 0 0 0 0 200 400 600 800 1000 0 SSC-H 10 10 10 SSC-H SSC-H 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 10 10 800 1000 0 0 200 400 600 10 GFP 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 ML3-GFP’ MOI=10 GFP GFP GFP GFP SSC-H SSC-H SSC-H SSC-H SSC-H Gene Conversion in The Canine CD34+ Cell Line ML3 0l 2l 10l 50l

  11. RSCSPGW2 Mock MOI=0.5 MOI=1 MOI=10 Efficient Lentiviral Transduction of ML3 Cells Relative Vector Provirus Copy Number ML3 and D17 target cells have comparable amounts of the target vector provirus ML3 cells can be efficiently transduced with an integrating lentivirus

  12. Dog DLA-identical transplantation setting I. Collection and transduction of CD34+ cells with LHE site-containing integrating lentiviral vector II. Infusion of cells after conditioning by irradiation IV. Investigate repair efficiency in canine progenitors DLA identical recipient Donor 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

  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

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