John F. Tisdale, MD Senior Investigator Molecular and Clinical Hematology Branch

1. Hematopoietic Stem Cells as Vehicles for Therapeutic Gene Delivery in Individuals with Sickle Cell Disease John F. Tisdale, MD Senior Investigator Molecular and Clinical Hematology Branch

2. Hematopoietic stem cells as vehicles for therapeutic gene delivery Allogeneic stem cell transplantation • Transplantation using stem cells from a normal donor • Bone marrow stem cells from a matched brother or sister carrying the normal gene Autologous stem cell gene transfer • Transplantation using the patient’s own modified stem cells • Bone marrow stem cells exposed to viral vectors carrying a normal or therapeutic gene

3. Hematopoietic stem cells as vehicles for therapeutic gene delivery Autologous stem cell gene transfer • Murine • High gene transfer rates easily achieved in vivo • Early human clinical • Equally high gene transfer rates estimated by in vitro assays • In vivo levels of <1/100,000 cells • Too low to expect clinical benefit • Predictive human HSC assays needed • Nonhuman primate competitive repopulation model developed

4. Neo not toxic to differentiation (Human Gene Therapy, 1999) Immune reaction not limiting (Human Gene Therapy, 2001) Optimal cytokine support ( Blood, 1998) Clinically feasible methods (Molecular Therapy, 2000) True stem cell transduction (Blood, 2000) Rhesus competitive repopulation model

5. Southern blotting: RC501 BM Monos 40W G1Na 6.25% PB Monos 40W PB Grans 40W G1Na 50% G1Na 3.125% G1Na 12.5% G1Na 25% FN STR

6. Prior studies on in vivo hematopoiesis • Direct murine retroviral tagging studies • Oligoclonal hematopoiesis • Clonal succession • Deterministic model • Indirect large animal and human studies • Polyclonal hematopoiesis • Clones contribute randomly • Stochastic model

7. Retroviral integration site analysis

8. Lineage specific integration site analysis

9. Alignment of flanking genomic DNA sequences

10. Multiple clones contribute to hematopoietic reconstitution

11. Capture-recapture procedure estimates polycolonal hematopoiesis in large animals • N=nD/X (D=originally captured/tagged, n=newly captured total, X=newly captured tagged) • 5-44 marked clones animal 1, 8-60 marked clones animal 2 contributing over the first year • 1,000 total clones based upon 5% marking • 5 stem cells per 107 bone marrow mononuclear cells • Data support stochastic model of human hematopoiesis

12. Steady state bone marrow comparable to G-CSF or G-CSF/SCF mobilized peripheral blood as stem cell source (Stem Cells, 2004) Neo not toxic to differentiation (Human Gene Therapy, 1999) Immune reaction not limiting (Human Gene Therapy, 2001) Optimal cytokine support ( Blood, 1998) Clinically feasible methods (Molecular Therapy, 2000) True stem cell transduction (Blood, 2000) 100 cGy TBI sufficient in mice (Human Gene Therapy, 2001) Low level engraftment in rhesus (Molecular Therapy, 2001) Low-dose busulfan promising (Experimental Hematology, 2006) Rhesus competitive repopulation model Clinical success feasible in simple disorders?

14. Steady state bone marrow comparable to G-CSF or G-CSF/SCF mobilized peripheral blood as stem cell source (Stem Cells, 2004) Neo not toxic to differentiation (Human Gene Therapy, 1999) Immune reaction not limiting (Human Gene Therapy, 2001) Optimal cytokine support ( Blood, 1998) Clinically feasible methods (Molecular Therapy, 2000) True stem cell transduction (Blood, 2000) Retroviral globin vectors including LCR elements unstable and prone to rearrangement 100 cGy TBI sufficient in mice (Human Gene Therapy, 2001) Low level engraftment in rhesus (Molecular Therapy, 2001) Low-dose busulfan promising alternative Rhesus competitive repopulation model

15. NATURE |VOL 406 | 6 JULY 2000 |www.nature.com

16. Locus Control Region b-globin gene dLTR LTR HS2 HS3 HS4 e p RRE y SD SA 4 bp Insertion (Xba1) Preclinical testing needed to improve the odds of successful clinical applicationNonhuman primate model for therapeutic ß-globin gene transfer • Modified vector developed to facilitate analysis and improve transduction rate in nonhuman primates • Vector produced at preclinical scale Both SIV and HIV (with alternate cyclophillin binding domain) backbone compared • Developed human ß-globin specific detection assays • Optimized lentiviral transduction procedures • Initiated in vivo non-human primate studies

17. Collect mobilized CD34+ cells Transduce with TNS9 Assess human -globin expression Infuse after lethal XRT In vivo expression of human -globin at day 30 after transplantation Confirmed by RNAse protection assay 32% and 13% of BM and PB cells positive, respectively, by genomic Southern blotting Clonogenic bone marrow progenitors positive for vector at 54%

18. In vivo expression of human β-globin at extended follow up in both animals Hayakawa et al., Hum Gene Ther., 20(6):563-72, 2009.

19. In vivo persistence of genetically modified cells limited by poor HSC transduction Hayakawa et al., Hum Gene Ther., 20(6):563-72, 2009.

20. In vivo persistence of genetically modified rhesus cells limited by block to HSC transduction Production of chimeric vectors to overcome restriction from TRIM5-alpha and APOBEC3G HIV1-Gag/Pol + SIV-CA plasmid Gag Pol Promoter PolyA CA SIV-Capsid (CA) HIV1- Rev/Tat + SIV-Vif plasmid Rev Vif Promoter PolyA Promoter Tat SIV-Vif & promoter

21. Screening of chimeric vector production by viral titer titers 5x106 1x107 * * * * * * * * * p<0.01

22. Dose escalation of chimeric vectors on human and rhesus cell lines CEMx174 cells (Human Lymphoblast) LCL8664 cells (Rhesus Lymphoblast) Transduction rate (%) MOI MOI The HIV1 vector with sCA (HIV) allowed efficient transduction of human and rhesus blood cell lines. Addition of sVif reduced transduction efficiency for the human blood cell line.

23. Competitive repopulation assay to compare HIV with HIV1 vectors Transduction (MOI=50) Single 24 hr Chi-HIV-GFP vector Rhesus CD34+ cells mixture HIV1-YFP vector Transplantation G-CSF/SCF mobilized PBSCH Total Body Irradiation (2x5Gy) Rhesus macaques competitive assay

24. The HIV vector achieves superior expression rates in vivo animal 1 % GFP / YFP HIV Days after transplantation Uchida, et al., J. Virol, 83(19):9854-62, 2009 HIV1

25. Competitive repopulation assay to compare HIV with SIV vectors Transduction (MOI=50) Single 24 hr Chi-HIV-GFP vector Rhesus CD34+ cells mixture SIV-YFP vector Transplantation G-CSF/SCF mobilized PBSCH Total Body Irradiation (2x5Gy) Rhesus macaques competitive assay

26. The HIV vector achieves equivalent expression rates in vivo animal 4 % GFP / YFP HIV Days after transplantation

27. The HIV vector achieves multilineage expression in vivo day 138 HIV SIV % GFP / YFP CD33+ Myeloid cells CD20+ B cells CD3+ T cells CD4+ T cells CD8+ T cells CD56+ NK cells CD71+ Reticulo cytes RBC+ cells CD41a+ Platelets

28. In vivo GFP among red blood cells

29. Autologous stem cell gene transfer Optimize lentiviral vectors for use in non-human primate (Modified HIV or SIV) Determine stem cell transduction efficiency (Test in myeloablated nonhuman primates) Determine vector directed globin expression (Compare vector designs to maximize expression) Determine integration pattern of optimized vector/transduction (Assess effects of additional safety measures including insulators) Initiate human clinical trials (Testing in thalassemia followed by SCD) Hematopoietic stem cells as vehicles for therapeutic gene delivery: Future efforts for human application

30. Tisdale lab Matt Hsieh Courtney Fitzhugh Pat Weitzel Naoya Uchida O.J. Phang Kareem Washington Department of Transfusion Medicine Susan Leitman Dave Stoncek Roger Kurlander Elizabeth Kang Bob Donahue Mark Metzger Barrington Thompson Allen Krouse Michel Sadelain Beth Link Terri Wakefield Nona Coles Karen Kendrick Griffin Rodgers Crew