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Bone marrow transplantation in sickle cell disease

Bone marrow transplantation in sickle cell disease . John F. Tisdale, MD Senior Investigator Molecular and Clinical Hematology Branch. First disease for which molecular defect identified Single substitution at position 6 of ß-globin chain Abnormal Hb polymerization upon deoxygenation

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Bone marrow transplantation in sickle cell disease

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  1. Bone marrow transplantation in sickle cell disease John F. Tisdale, MD Senior Investigator Molecular and Clinical Hematology Branch

  2. First disease for which molecular defect identified • Single substitution at position 6 of ß-globin chain • Abnormal Hb polymerization upon deoxygenation • Ideal for hematopoietic stem cell based approach “I believe medicine is just now entering into a new era when progress will be much more rapid than before, when scientists will have discovered the molecular basis of diseases, and will have discovered why molecules of certain drugs are effective in treatment, and others are not effective.”Linus Pauling 1952

  3. Conventional Sources of Stem Cells • Somatic stem cells • Harvested from mature organs or tissues (bone marrow) • Multipotent, may be tissue specific, pluripotent? • Many established clinical uses • Embryonic stem cells • Derived from ICM of blastocyst • Pluripotent, differentiate to all cell lineages • Encumbered by technical and ethical issues • May be induced from adult tissues

  4. Hematopoietic stem cells

  5. Hematopoietic stem cells as vehicles for therapeutic gene delivery Allogeneic stem cell transplantation • Transplantation using allogeneic stem cells from a normal donor • HLA-matched sibling Autologous stem cell gene transfer • Transplantation using autologous stem cells which have been corrected by transfer of a normal or therapeutic gene • Retroviral vectors

  6. Hematopoietic stem cells as vehicles for therapeutic gene delivery Allogeneic stem cell transplantation • Myeloablative transplantation curative in children with sickle cell disease • Cumulative experience with over 200 children • Survival 82-86% • Rejection 7-10% • Acute GvHD 15-20% • Stable mixed chimerism sufficient • 13/50 surviving patients 11-99% donor chimerism (Walters et al., BBMT, 7, 665, 2001) • Toxic conditioning and GVHD limit application to children • Engraftment without ablation?

  7. Nonmyeloablative conditioning sufficient for reliable allogeneic PBSC engraftment • Cytoxan/fludarabine based immune ablative conditioning piloted in patients with metastatic cancer • Childs, R.W., et al., JCO, 17, 2044, 1999. • Childs, R., et al., NEJM, 343: 750-758, 2000. • Extended to high-risk patients ineligible for conventional myeloablative conditioning • Kang, E.M., et al., Blood, 99, 698-701, 2002. • Kang, E.M., et al., J Hematother and Stem Cell Res, 11, 809-816, 2002.

  8. Application to sickle cell disease? • NIH experience overall (n>100) • Engraftment through donor alloimmune response • GVHD common • T cell alloreactivity not necessary in nonmalignant disorders • Treatment related mortality 21% • GVHD principal cause • Prohibitive in nonmalignant disorders

  9. F1-Hybrid C57Bl6 (Kb) X BalbC(Kd) Harvest mobilized stem cells 6 Days G-CSF (200 ug/kg) 100x106 cells Recipient C57Bl6 (Kb) Day -1 Week Day 0 (300 cGy) RAPA (3mg/kg) or CSA (20mg/kg) A Murine Model of Nonmyeloablative Stem Cell Transplantation for the Treatment of Sickle Cell Disease • Develop regimen that: • Promotes tolerance without need for long term immunosuppression • Allow for stable mixed chimerism • F1-Hybrid donor mice • Myeloid-flow cytometry • Erythroid-Hb electrophoresis • Donors mobilized with G-CSF • Mobilized cells collected day 6 • Recipient mice conditioned with 300 cGy and a 30d course of either • Cyclosporine (CSA) • Rapamycin (RAPA)

  10. CsA Rapa IL-2 TcR-CD3 CD28 Effector Function Proliferation Anergy Induction of tolerance Why Rapamycin??

  11. Rapamycin but not Cyclosporine Maintains Chimerism in the Absence of Long-Term Immunosuppression

  12. Donor Recipient 1 Recipient 2 Recipient 3 SS Control ASC Sickle Hemoglobin is Replaced by Donor Hemoglobin in Chimeric Homozygote Mice Powell, J, et al., Transplantation, 2005

  13. Protocol 03-DK-0170: Nonmyeloablative Allogeneic PBSC Transplantation for Adults with Severe Congenital Anemias Eligibility: Adults with Hb SS, SC, or Sb0-thal Severe end-organ damage • stroke or abnormal CNS vessel • pulmonary hypertension (TRV ≥2.5 m/s) • renal damage • Or modifiable complication(s), not ameliorated by hydroxyurea • > 2 hospital admissions per year for pain crises (VOC) • previous acute chest syndromes (ACS) • red cell alloimmunization • osteonecrosis of multiple joints • Conditioning • 300 cGy, Rapamycin, Campath 1H

  14. Protocol 03-DK-0170: Nonmyeloablative Allogeneic PBSC Transplantation for Adults with Severe Congenital Anemias Accrual: Adults with Hb SS, SC, or Sb0-thal

  15. Transplant course • All patients tolerated conditioning without serious adverse events • No need for nutritional support • No acute or chronic GVHD • No sickle cell anemia related events • All experienced normalization of Hb with donor type

  16. Mixed hematopoietic chimerism results in full replacement by donor type hemoglobin: YM

  17. Patient status at most recent follow-up

  18. % Donor Chimerism Months post transplant Transplant outcome:Chimerism **All patients remain on sirolimus

  19. 1250 404 212 166 113 Pre Pre Post Post 12.6 3.8 9.4 1.1 Pre Pre Post Post Transplant outcome:Hemolytic parameters

  20. Improvement in pulmonary hypertension (PHT) • The reduction in TRV was observed immediately peri-transplant • The reduction in TRV remained stable despite a small increase in systemic blood pressure • These patients with PHT tolerated the transplant procedure well TRV (m/s) SBP BP (mmHg) DBP

  21. Narcotic usage post transplant IV morphine equivalent (mg) Weeks post BMT

  22. Conclusions • Allogeneic PBSC transplantation after low dose TBI, campath, rapamycin conditioning sufficient to revert the sickle phenotype • Reversal of end organ damage • Low toxicity allows application in adults with severe disease • ‘Split’ or mixed chimerism and absence of acute or chronic GvHD suggests operational tolerance • Longer follow-up and further accrual necessary • Alternative strategies need exploration

  23. 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

  24. 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?

  25. 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 unstable Lentiviral vectors appear promising 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

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

  27. Locus Control Region b-globin gene dLTR LTR HS2 HS3 HS4 e p RRE y SD SA 4 bp Insertion (Xba1) Development of a preclinical nonhuman 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 backbone compared • Developed human ß-globin specific detection assays • Optimized lentiviral transduction procedures • Initiated in vivo non-human primate studies

  28. Collect mobilized CD34+ cells Transduce with TNS9 Erythroid culture Assess human β-globin expression High level in vitro expression of human globin by rhesus erythroid cells after TNS9 gene transfer 57.4%

  29. Collect mobilized CD34+ cells Transduce with TNS9 Infuse after lethal XRT Assess human β-globin expression In vivo expression of human β-globin at day 30 after transplantation

  30. In vivo expression of human β-globin at extended follow up in both animals

  31. Production of chimeric vectors to overcome restriction from TRIM5-alpha and APOBEC3G, respectively

  32. Dose escalation study of chimeric vectors of HIV1 and SIV LCL8664 cells (Rhesus Lymphoblast) CEMx174 cells (Human Lymphoblast) Transduction rate (%) MOI MOI The HIV1 vector with sHIVgagpol allowed good transduction of human and rhesus blood cell lines. Addition of simian Vif reduced transduction efficiency for the human blood cell line.

  33. In vivo rhesus study to compare chi-HIV vector with HIV1 vector 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>

  34. The chi-HIV vector achieves superior transduction rates in vivo Transduction rate (%) Day after transplantation

  35. The chi-HIV vector achieves multi-lineage marking Transduction rate (%) Day after transplantation

  36. In vivo GFP among red blood cells

  37. Validate results with continued accrual (Trial plan for 25 subjects) Allogeneic stem cell transplantation Expand to multicenter trial design (Facilitate recruitment) Determine engraftment level sufficient to revert phenotype (Compare marrow progenitor chimerism with peripheral blood) Utilize animal model to address additional questions (Compare degree of host conditioning required) Tolerance for alternative donor transplantation (Haploidentical or cord blood-01-DK-0122) Hematopoietic stem cells as vehicles for therapeutic gene delivery: Future efforts for human application

  38. 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) Determine degree of host conditioning required (Test safety and efficacy of in vivo selection strategies) • Persons and Tisdale, Semin Hematol. 2004, 41(4):279-86 Hematopoietic stem cells as vehicles for therapeutic gene delivery: Future efforts for human application

  39. Tisdale lab Jun Hayakawa Naoya Uchida Courtney Fitzhugh O.J. Phang Kareem Washington Matt Hsieh Coen Lap Camille Madison Department of Transfusion Medicine Charley Carter E.J. Read Susan Leitman Dave Stoncek Roger Kurlander Greg Kato Mark Gladwin Elizabeth Kang Jonathan Powell 5 Research Court Mark Metzger Allen Krouse Barrington Thompson Bob Donahue Cindy Dunbar Stephanie Sellers Tong Wu Hyeoung Joon Kim Martha Kirby Leszek Lisowski Selda Samakoglu Michel Sadelain Terri Wakefield Beth Link Nona Coles Karen Kendrick Griffin Rodgers Crew

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