Bone marrow transplantation in sickle cell disease by djh75337

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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                     1.0


   – GVHD common                              .9

                                              .8

                                              .7


      • T cell alloreactivity not             .6
                                              .5
                                                                                                   21% (5)
         necessary in nonmalignant            .4

                                              .3


         disorders                            .2

                                              .1
                                             0.0


   – Treatment related mortality 21%
                                                   0   90    180   270   360   450   540   630   720   810   900   990 1080

                                                                   Days Post Transplant
                                       TRM in all patients

      • 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:                        F1-Hybrid
                                         C57Bl6 (Kb) X BalbC(Kd)
                                                                                  Harvest mobilized
                                                                                     stem cells
 – 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                                                              100x106
                                                                                        cells

 – Erythroid-Hb electrophoresis
                                              Recipient
•Donors mobilized with G-CSF                 C57Bl6 (Kb)                 Day -1


•Mobilized cells collected day 6
•Recipient mice conditioned with                                    Week


 300 cGy and a 30d course of either                Day 0
                                                                             RAPA (3mg/kg)
                                                  (300 cGy)                  or CSA (20mg/kg)
       • Cyclosporine (CSA)
       • Rapamycin (RAPA)
          Why Rapamycin??
           CsA                 IL-2   Rapa

TcR-CD3               CD28




                                             Effector Function
                                             Proliferation




      Anergy
      Induction of tolerance
     Rapamycin but not Cyclosporine Maintains
      Chimerism in the Absence of Long-Term
               Immunosuppression
          100
                    CSA
                    Rapa
          80

          60
% Donor




          40

          20

           0
                0   8           16             24   32
                           Weeks post transplant
 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
Mixed hematopoietic chimerism results in full
replacement by donor type hemoglobin: YM

            100                                   15

            80                                    13




                                                       hgb g/dL
  % Donor




            60                                    11

            40                       Myeloid      9
                                     Lymphoid
            20                       Hgb          7

             0                                     5
                  0   200   400    600          800
                            Days
Patient status at most recent follow-up
Pt   CD34 and CD3 (per      Months     (%) Donor   (%) Donor   (%)     Hgb
     kg of recipient wt)    post BMT   CD3         CD14/15     Donor
                                                               RBC

1    5.72 x 106 / (3.21 x   51         11          52          100     12.9
     108)

2    7.56 / (2.27)          30         64          35          100     10.8
3    10 / (3.42)            38         71          99          100     13.7 (post-
                                                                       partum)
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
                    100
% Donor Chimerism




                    80

                    60

                    40

                    20

                     0
                          0     4        8        12        16       20        24
                                     Months post transplant

                                **All patients remain on sirolimus
              Transplant outcome:
                   Hemolytic parameters
 750
1250                              450
                            LDH                             Retic
500                               300
       404
                                        212
250                  166
                                  150
                                                     113

  0      Pre            Post
                                    0         Pre       Post
  9                               15
                       Total
  6                  bilirubin    12                 12.6
       3.8
  3                                9    9.4
                      1.1
                                                    Hemoglobin
  0                                6
             Pre       Post                   Pre       Post
                         Improvement in pulmonary
                            hypertension (PHT)
              4
                                                    • The reduction in TRV
             3.5
TRV (m/s)




                                                      was observed
              3                                       immediately peri-
             2.5
                                                      transplant
              2                                     • The reduction in TRV
                   pre   0     1   3   6   9   12
                                                      remained stable despite a
             130     SBP                              small increase in
                                                      systemic blood pressure
 BP (mmHg)




             110

             90                                     • These patients with PHT
                     DBP                              tolerated the transplant
             70

             50
                                                      procedure well
                   pre     0   1   3   6   9   12
                      Narcotic usage post transplant
IV morphine equivalent (mg)



                              2000

                              1600

                              1200

                              800

                              400

                                 0
                                     0   4   8    12     16   20   24
                                             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 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      Neo not toxic to differentiation
 to G-CSF or G-CSF/SCF mobilized           (Human Gene Therapy, 1999)
 peripheral blood as stem cell source    Immune reaction not limiting
           (Stem Cells, 2004)              (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           Clinically feasible methods
   (Molecular Therapy, 2001)                (Molecular Therapy, 2000)
 Low-dose busulfan promising              True stem cell transduction
 (Experimental Hematology, 2006)                  (Blood, 2000)
            Rhesus competitive repopulation model
Steady state bone marrow comparable         Neo not toxic to differentiation
 to G-CSF or G-CSF/SCF mobilized               (Human Gene Therapy, 1999)
 peripheral blood as stem cell source        Immune reaction not limiting
           (Stem Cells, 2004)                  (Human Gene Therapy, 2001)




                                         Retroviral globin vectors unstable
                                        Lentiviral vectors appear promising


100 cGy TBI sufficient in mice
  (Human Gene Therapy, 2001)
                                               Optimal cytokine support
                                                     ( Blood, 1998)
Low level engraftment in rhesus
                                              Clinically feasible methods
   (Molecular Therapy, 2001)                    (Molecular Therapy, 2000)
 Low-dose busulfan promising                  True stem cell transduction
          alternative                                 (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                  Locus Control Region


    LTR                                              HS2            HS3         HS4   dLTR
                      e                        p
            y RRE
          SD     SA


                                 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   Transduce   Erythroid   Assess human β-
  CD34+ cells       with TNS9    culture    globin expression




                                   57.4%
                                     M1
In vivo expression of human β-globin at day 30 after
                   transplantation
Collect mobilized   Transduce   Infuse after   Assess human β-
  CD34+ cells       with TNS9   lethal XRT     globin 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

                               CEMx174 cells                        LCL8664 cells
                            (Human Lymphoblast)                 (Rhesus 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.
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>
                        The chi-HIV vector achieves superior transduction rates in vivo
Transduction rate (%)




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




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

								
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