Bone marrow transplantation in sickle cell disease John

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 Gv. HD 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

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 • F 1 -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 c. Gy and a 30 d course of either • Cyclosporine (CSA) • Rapamycin (RAPA) Harvest mobilized stem cells F 1 -Hybrid C 57 Bl 6 (Kb) X Balb. C(Kd) Recipient C 57 Bl 6 (Kb) 100 x 106 cells Day -1 Week Day 0 (300 c. Gy) RAPA (3 mg/kg) or CSA (20 mg/kg)

Why Rapamycin? ? Cs. A Tc. R-CD 3 IL-2 Rapa CD 28 Effector Function Proliferation Anergy Induction of tolerance

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

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

Protocol 03 -DK-0170: Nonmyeloablative Allogeneic PBSC � Transplantation for Adults with Severe Congenital Anemias Eligibility: Adults with Hb SS, SC, or Sb 0 -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 c. Gy, Rapamycin, Campath 1 H

Protocol 03 -DK-0170: Nonmyeloablative Allogeneic PBSC � Transplantation for Adults with Severe Congenital Anemias Accrual: Adults with Hb SS, SC, or Sb 0 -thal

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

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

Patient status at most recent follow-up Pt 1 (%) Donor CD 3 (%) Donor CD 14/15 (%) Donor RBC Hgb kg of recipient wt) Months post BMT 5. 72 x 106 / (3. 21 x 51 11 52 100 12. 9 CD 34 and CD 3 (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 10. 3

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

Transplant outcome: Hemolytic parameters 1250 404 212 166 Pre 113 Pre Post 12. 6 3. 8 9. 4 1. 1 Pre Post

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

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

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 Gv. HD 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 c. Gy 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 c. Gy 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 HS 2 HS 3 HS 4 d. LTR 4 bp Insertion (Xba 1) • 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 TNS 9 gene transfer Collect mobilized CD 34+ cells Transduce with TNS 9 Erythroid culture 57. 4% Assess human βglobin expression

In vivo expression of human β-globin at day 30 after transplantation Collect mobilized CD 34+ cells Transduce with TNS 9 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 TRIM 5 -alpha and APOBEC 3 G, respectively

Dose escalation study of chimeric vectors of HIV 1 and SIV LCL 8664 cells (Rhesus Lymphoblast) Transduction rate (%) CEMx 174 cells (Human Lymphoblast) MOI The HIV 1 vector with s. HIVgagpol 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 HIV 1 vector Transduction (MOI=50) Single 24 hr Chi-HIV-GFP vector Rhesus CD 34+ cells <mixture> HIV 1 -YFP vector Transplantation G-CSF/SCF mobilized PBSCH Rhesus macaques Total Body Irradiation (2 x 5 Gy) <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