1. AML in mice after retroviral cell marking Heinrich-Pette-Institute, Hamburg Bernd Schiedlmeier, Martin Forster, Carol Stocking, Anke Wahlers, Oliver Frank, Wolfram Ostertag University Hospital Eppendorf, Hamburg Jochen Duellmann, Axel Zander, Boris Fehse University Freiburg Manfred Schmidt, Christof von Kalle EUFETS AG Klaus Kuehlcke, Hans-Georg Eckert Hannover Medical School Zhixiong Li, Johann Meyer, Christopher Baum CB 02

2. Oncogenic progression related to insertional mutagenesis Risk ~ 10 -7 per insertion in human TF-1 leukemia cells (Stocking et al., 1993) Insertional mutagenesis promotes tumor formation in numerous animal models, but single insertion never sufficient to explain malignancy No disease induction reported using replication-defective vectors designed for gene therapy in numerous preclinical and clinical trials, probably involving manipulation of >10 12 hematopoietic or lymphoid cells Side effects of transgene or active replication required for pathogenesis CB 02

3. Toxicity Assessment of Gene Transfer Technologies Animal experiments with long-term follow-up 2d 7mo MACS unselected 5mo ana- lysis dLNGFR SF EGFP SF tCD34 SF flCD34 SF One group of 5 recipients for each vector At least 5 recipients for each condition CB 02

4. dLNGFR group, 2° recipients (n=10) – AML M5: n=6 – Overt dysplasia: n=3 – Microscopic lesions: n=1 CB 02

5. AML after Retroviral Gene Marking in Mice Long latency: No overt disease in first cohort (7 mo) 10/10 secondary recipients developed dysplasia or AML M5 (5 mo) Leukemia is transplantable to 3° cohort (lethal) Monoclonal origin, heterogenous kinetics, however identical entity with reproducible phenotype Aberrant clone has single vector integration Vector is intact and continues to express dLNGFR Insertional activation of Evi-1 RCR and activation of endogenous MLV excluded CB 02

6. Vector integration in Evi-1 SD 681 9551131132 U3 R U5 E1 LTR - dLNGFR - LTR E1 E2 E3 M P1 P2 P3 P4 P5 S6 S8 S9 S10 S3 S4 S5 S1 H M PCR confirms integration and origin in primary recipient P2 PCR A B AUG CB 02

7. Evi-1 Transcription factor, known oncogene Endogenous expression in primitive stem cells Ectopic expression blocks granulocytic and erythroid differentiation promotes megakaryocytic hematopoiesis Activation implicated in MDS and AML (usually immature phenotype) Tg mice at increased risk for leukemia (dysplastic hematopoiesis) Not sufficient to explain AML M5 CB 02

8. dLNGFR: variant of p75NTR p75NTR dLNGFR Differentiation Apoptosis Juxtamembrane domain Death domain Ligand binding domain CB 02

9. dLNGFR: structurally related to antiapoptotic decoy receptors Marsters et al., Curr Biol 1997 Differentiation Apoptosis p75NTR dLNGFR Juxtamembrane domain Death domain Ligand binding domain DcR1 TRAIL family DcR2 Shedding of dLNGFR may generate soluble decoy receptor (see osteoprotegerin, OPG) CB 02

10. p75NTR and Trk receptors: A two-receptor-system for neurotrophins Survival Proliferation Differentiation Apoptosis Trk p75NTR NT Balanced growth p75NTR NGF BDNF NT-4 NT-3 TrkA TrkB TrkC CB 02

11. The combination of dLNGFR, Trk and NT transforms fibroblasts Hantzopoulos et al., Neuron 1994, 13:187 Survival Proliferation Differentiation Apoptosis Trk p75NTR NT Balanced growth Survival Proliferation No signal (?) Trk dLNGFR NT Transformation CB 02

12. AML cells express dLNGFR and TrkA and proliferate in response to NGF N L K S TrkA 4.4 kb GAPDH Survival Proliferation Enhancement TrkA dLNGFR NGF Expansion or Transformation ? CD11b 77 % dLNGFR Loss of balance CB 02

13. Expression of Neurotrophins and their Receptors in Human Hematopoiesis (Labouyrie et al., AJP 1999, 154:411) p75NTRabsentB cells (mouse mast cells) TrkA erythroblastsmono, baso, mast, B cells TrkBeo TrkBi erythroblastsmeg TrkCmyeloblastseo, meg, granulo TrkCimyeloblastsgranulo NGF BDNF NT-3 NT-4/5 ProgenitorsMature Cells bone marrow stroma cells, monocytic cells osteoblasts, osteoclasts, mast cells, B cells (T cells ?) CB 02

14. Trk receptors and human leukemia TrkA was detected in some leukemic cell lines, such as UT-7(acute megakaryoblastic leukemia), K562 and TF1 (erythroleukemia), and myeloid cell lines HEL, HL60 and KG1, but not in myeloid cell lines U937 and THP-1 (Chevalier et al., 1994, Auffray et al.,1996, Kaebisch et al., 1996). So far, there are only 3 reports on expression of p75NTR and Trk receptors in primary leukemia: 44% TrkA gene expression in patients with AML (Kaebisch et al., 1996). A translocation t(12;15) (p13;q25) was found in an AML patient, which resulted in a fusion RNA ETV6-TrkC (Eguchi et al., 1999). A deleted form of TrkA,  TrkA, was identified in AML patients. 75-aa deletion in the extracellular domain resulted in constitutive tyrosine phosphorylation of the protein, which also transforms fibroblasts (Reuther et al., 2000). These data suggest a possible role of Trk receptors and their mutant forms in leukemia development (however, so far no evidence for transformation of lymphatic cells). CB 02

15. AML after Retroviral Gene Transfer into Murine HSC Integration site causal role likely, but not sufficient Role of transgene causal contribution suggested Role of vector architectureno splice acceptor 5-FU exposure of donornot a strong mutagen, common procedure Forced expansion in serial BMT possibly promoting, but not cause Difference rodent vs. human cells ? Implications for other cell types ? U3RU5U3RU5 SD  dLNGFR CB 02