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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 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 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 Hematopoietic stem cells 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 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? 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. 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 1.0 .9 .8 .7 .6 .5 21% (5) .4 .3 .2 .1 0.0 0 90 180 270 360 450 540 630 720 Days Post Transplant TRM in all patients 810 900 990 1080 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 6 Days G-CSF (200 ug/kg) – 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) Harvest mobilized stem cells F1-Hybrid C57Bl6 (Kb) X BalbC(Kd) Recipient C57Bl6 (Kb) 100x106 cells Day -1 Week Day 0 (300 cGy) RAPA (3mg/kg) or CSA (20mg/kg) Why Rapamycin?? CsA TcR-CD3 IL-2 Rapa CD28 Effector Function Proliferation Anergy Induction of tolerance Rapamycin but not Cyclosporine Maintains Chimerism in the Absence of Long-Term Immunosuppression 100 CSA Rapa % Donor 80 60 40 20 0 0 8 16 24 Weeks post transplant 32 Sickle Hemoglobin is Replaced by Donor Hemoglobin in Chimeric Homozygote Mice Powell, J, et al., Transplantation, 2005 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 Protocol 03-DK-0170: Nonmyeloablative Allogeneic PBSC Transplantation for Adults with Severe Congenital Anemias Accrual: Adults with Hb SS, SC, or Sb0-thal 120 screened 2 ineligible due to stringent selection criteria 59 lack sufficient severity 59 HLA-typed 1 awaiting HLA typing 45 lack HLA-matched sibling donor 13 donor/recipeint pairs identified 2 ABO incompatible 11 donor/recipient pairs eligible 1 sudden death prior 10 patients transplanted 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 100 15 80 13 60 11 40 Myeloid Lymphoid Hgb 20 0 0 200 400 Days 600 9 7 5 800 hgb g/dL % Donor Mixed hematopoietic chimerism results in full replacement by donor type hemoglobin: YM Patient status at most recent follow-up Pt 1 (%) Donor CD3 (%) Donor CD14/15 (%) Donor RBC Hgb kg of recipient wt) Months post BMT 5.72 x 106 / (3.21 x 51 11 52 100 12.9 CD34 and CD3 (per 108) 2 7.56 / (2.27) 30 64 35 100 10.8 3 10 / (3.42) 38 71 99 100 13.7 (postpartum) 4 8.3 / (5.35) 37 0 0 0 12.4 5 5.51 / (3.71) 28 81 98 100 14.4 6 23.8 / (2.81) 25 27 98 100 13.9 7 18.8 / (3.32) 24 81 97 100 12.4 8 20.1 / (3.04) 23 63 96 100 12.2 9 16.6 / (3.7) 8 0 96 100 13.2 10 15.1 / (3.64) 7 42 100 100 10.3 Transplant outcome: Chimerism 120 Lymphoid Myeloid % Donor Chimerism 100 80 60 40 20 0 0 4 8 12 16 Months post transplant **All patients remain on sirolimus 20 24 Transplant outcome: Hemolytic parameters 1250 750 450 Retic LDH 500 300 404 250 0 9 212 166 Pre Post Total bilirubin 6 150 113 0 Pre 15 12 Post 12.6 3.8 3 9 9.4 1.1 0 Pre Post Hemoglobin 6 Pre Post Improvement in pulmonary hypertension (PHT) TRV (m/s) 4 • The reduction in TRV was observed immediately peritransplant 3.5 3 2.5 2 pre BP (mmHg) 130 0 1 3 6 9 12 SBP 110 • These patients with PHT tolerated the transplant procedure well 90 70 DBP 50 pre • The reduction in TRV remained stable despite a small increase in systemic blood pressure 0 1 3 6 9 12 IV morphine equivalent (mg) Narcotic usage post transplant 2000 1600 1200 800 400 0 0 4 8 12 16 Weeks post BMT 20 24 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 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 Rhesus competitive repopulation model 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) Clinical success feasible in simple disorders? 100 cGy TBI sufficient in mice Optimal cytokine support (Human Gene Therapy, 2001) ( Blood, 1998) Low level engraftment in rhesus (Molecular Therapy, 2001) Low-dose busulfan promising Clinically feasible methods (Experimental Hematology, 2006) (Molecular Therapy, 2000) True stem cell transduction (Blood, 2000) Rhesus competitive repopulation model 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) 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 Optimal cytokine support ( Blood, 1998) Clinically feasible methods (Molecular Therapy, 2000) True stem cell transduction (Blood, 2000) NATURE |VOL 406 | 6 JULY 2000 |www.nature.com Development of a preclinical nonhuman primate model for therapeutic ß-globin gene transfer b-globin gene LTR y RRE SD SA e Locus Control Region p HS2 HS3 HS4 dLTR 4 bp Insertion (Xba1) • 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 High level in vitro expression of human globin by rhesus erythroid cells after TNS9 gene transfer Collect mobilized CD34+ cells Transduce with TNS9 Erythroid culture 57.4% M1 Assess human βglobin expression In vivo expression of human β-globin at day 30 after transplantation Collect mobilized CD34+ cells Transduce with TNS9 Infuse after lethal XRT Assess human βglobin expression In vivo expression of human β-globin at extended follow up in both animals Production of chimeric vectors to overcome restriction from TRIM5-alpha and APOBEC3G, respectively Dose escalation study of chimeric vectors of HIV1 and SIV LCL8664 cells (Rhesus Lymphoblast) Transduction rate (%) CEMx174 cells (Human Lymphoblast) 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. 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 Rhesus macaques Total Body Irradiation (2x5Gy) <competitive assay> Transduction rate (%) The chi-HIV vector achieves superior transduction rates in vivo Day after transplantation Transduction rate (%) The chi-HIV vector achieves multi-lineage marking Day after transplantation In vivo GFP among red blood cells Hematopoietic stem cells as vehicles for therapeutic gene delivery: Future efforts for human application Allogeneic stem cell transplantation Validate results with continued accrual (Trial plan for 25 subjects) 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 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 Crew • • 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