scott howard 2009
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Bone marrow transplantation in sickle celldisease
John F. Tisdale, MD
Senior Investigator
Molecular and Clinical Hematology Branch
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• 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 wil l have discovered the molecular basis of diseases, and wi l l have discovered
why molecules of certain drugs are effective in treatment, and others are not effective.”
Linus Pauling 1952
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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
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Hematopoietic stem cells
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Hematopoietic stem cells as vehicles for
therapeutic gene delivery
Allogeneic stem cell transplantation
Autologous stem cell gene transfer
– Transplantation using autologous stem
cells which have been corrected by
transfer of a normal or therapeutic gene
•Retroviral vectors
– Transplantation using allogeneic
stem cells from a normal donor
•HLA-matched sibling
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Hematopoietic stem cells as vehicles for
therapeutic gene delivery
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?
Allogeneic stem cell transplantation
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Nonmyeloablative conditioning sufficient for
reliable allogeneic PBSC engraftment
• Cytoxan/fludarabine based immune ablativeconditioning piloted in patients with metastaticcancer – Chi lds, R.W., et al ., JCO, 17, 2044, 1999.
– Chi lds, 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 H ematother and Stem Cell Res, 11, 809-816, 2002.
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Application to sickle cell disease?
• NIH experience overall (n>100) – Engraftment through donor
alloimmune response
– GVHD common
• T cell alloreactivity notnecessary in nonmalignant
disorders
– Treatment related mortality 21%
• GVHD principal cause• Prohibitive in nonmalignant
disorders
TRM in all patients
Days Post Transplant
1080990900810720630540450360270180900
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0.0
21% (5)
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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 with300 cGy and a 30d course of either
• Cyclosporine (CSA)
• Rapamycin (RAPA)
F1-Hybrid
C57Bl6 (K b) X BalbC(K d)
6 DaysG-CSF
(200 ug/kg)
Harvest mobilized
stem cells
100x106
cells
RAPA (3mg/kg)
or CSA (20mg/kg)
Recipient
C57Bl6 (K b)
Day 0
(300 cGy)
Week
Day -1
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Why Rapamycin??
IL-2CsA
CD28
Rapa
Anergy
Induction of tolerance
Effector Function
Proliferation
TcR-CD3
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Rapamycin but not Cyclosporine Maintains
Chimerism in the Absence of Long-Term
Immunosuppression
0
20
40
60
80
100
0 8 16 24 32Weeks post transplan
CSA
Rapa
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Sickle Hemoglobin is Replaced by Donor
Hemoglobin in Chimeric Homozygote Mice
Powell, J, et al., Transplantation, 2005
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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
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2 ineligible due to stringent selection criteria 59 lack sufficient severity
1 awaiting HLA typing 45 lack HLA-matched sibling donor
2 ABO incompatible
1 sudden death prior
10 patients transplanted
11 donor/recipient pairs eligible
13 donor/recipeint pairs identified
59 HLA-typed
120 screened
Protocol 03-DK-0170: Nonmyeloablative
Allogeneic PBSC Transplantation for Adults
with Severe Congenital Anemias
Accrual: Adults with Hb SS, SC, or Sb0-thal
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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
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0
20
40
60
80
100
0 200 400 600 800
Days
5
7
9
11
13
15
h g b g / d L
MyeloidLymphoidHgb
Mixed hematopoietic chimerism results in full
replacement by donor type hemoglobin: YM
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Patient status at most recent follow-up Pt CD34 and CD3 (per
kg of recipient wt)
Months
post BMT
(%) Donor
CD3
(%) Donor
CD14/15
(%)
Donor
RBC
Hgb
1 5.72 x 106 / (3.21 x
108)
51 11 52 100 12.9
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
T l
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**All patients remain on sirolimus
Months post transplant
% D o n o r C h
i m e r i s m
0
20
40
60
80
100
120
0 4 8 12 16 20 24
Lymphoid Myeloid
Transplant outcome:Chimerism
T l
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LDH
0
250
500
7501250
Total
bilirubin
0
3
6
9
Hemoglobin6
9
12
15
Retic
0
150
300
450
1.1
3.8
166
404
113
212
9.4
12.6
Pre Post Pre Post
PostPrePostPre
Transplant outcome:Hemolytic parameters
I t i l
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Improvement in pulmonary
hypertension (PHT)
2
2.5
3
3.5
4
pre 0 1 3 6 9 12
T R V ( m / s )
B P ( m
m H g )
50
70
90
110
130
pre 0 1 3 6 9 12
• The reduction in TRVwas observedimmediately peri-transplant
• The reduction in TRVremained stable despite asmall increase insystemic blood pressure
• These patients with PHTtolerated the transplant
procedure well
SBP
DBP
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Narcotic usage post transplant
0
40 0
80 0
1200
1600
2000
0 4 8 12 16 20 24
I V m o r p h i n e e q u i v a l e n t ( m g )
Weeks post BMT
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• Allogeneic PBSC transplantation after low doseTBI, campath, rapamycin conditioning sufficientto revert the sickle phenotype
– Reversal of end organ damage
• Low toxicity allows application in adults withsevere disease
• ‘Split’ or mixed chimerism and absence of acuteor chronic GvHD suggests operational tolerance
• Longer follow-up and further accrual necessary
• Alternative strategies need exploration
Conclusions
i i ll hi l f
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Hematopoietic stem cells as vehicles for
therapeutic gene delivery
Autologous stem cell gene transfer
•Murine
– High gene transfer rates easilyachieved in vivo
•Early human clinical
– Equally high gene transfer ratesestimated 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 competitiverepopulation model developed
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Rhesus competitive repopulation model
Optimal cytokine support( Blood, 1998)
Clinically feasible methods(Molecular Therapy, 2000)
True stem cell transduction( Blood, 2000)
Neo not toxic to differentiation(Human Gene Therapy, 1999)
Immune reaction not limiting
(Human Gene Therapy, 2001)
Steady state bone marrow comparable
to G-CSF or G-CSF/SCF mobilized
peripheral blood as stem cell source
(Stem Cells, 2004)
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)
Clinical
success
feasible insimple
disorders?
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Rhesus competitive repopulation model
Optimal cytokine support( Blood, 1998)
Clinically feasible methods(Molecular Therapy, 2000)
True stem cell transduction( Blood, 2000)
Neo not toxic to differentiation(Human Gene Therapy, 1999)
Immune reaction not limiting
(Human Gene Therapy, 2001)
Steady state bone marrow comparable
to G-CSF or G-CSF/SCF mobilized
peripheral blood as stem cell source
(Stem Cells, 2004)
100 cGy TBI sufficient in mice(Human Gene Therapy, 2001)
Low level engraftment in rhesus
( Molecular Therapy, 2001)
Low-dose busulfan promising
alternative
Retroviral globin vectors unstable
Lentiviral vectors appear promising
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NATURE |VOL 406 | 6 JULY 2000 |www.nature.com
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Development of a preclinical nonhuman primate model for
therapeutic ß-globin gene transfer
• Modified vector developed to facilitate analysis andimprove 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
SA
RRE
SD
pe
Locus Control Region-globin gene
dLTR LTR HS2 HS3 HS4
y
4 bp Insertion (Xba1)
Hi h l l i it i f h l bi b
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High level in vitro expression of human globin by
rhesus erythroid cells after TNS9 gene transfer
M1
57.4%
Collect mobilized
CD34+ cells
Transduce
with TNS9
Assess human β-
globin expression
Erythroid
culture
I i i f h β l bi t d 30 ft
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In vivo expression of human β-globin at day 30 after
transplantation
Collect mobilized
CD34+ cells
Transduce
with TNS9
Assess human β-
globin expression
Infuse after
lethal XRT
In vivo expression of human β globin at extended
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In vivo expression of human β-globin at extended
follow up in both animals
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Production of chimeric vectors to overcome restriction from TRIM5-alpha
and APOBEC3G, respectively
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Dose escalation study of chimeric vectors of HIV1 and SIV
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.
CEMx174 cells
(Human Lymphoblast)
LCL8664 cells
(Rhesus Lymphoblast)
MOI
MOI
T r a n s d u c t i o n r a t e
( % )
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In vivo rhesus study to compare chi-HIV vector with HIV1 vector
Rhesus macaques
Rhesus CD34+ cells
Transplantation
Transduction
(MOI=50)
Single 24 hr
Chi-HIV-GFP vector
<competitive assay>
<mixture>
HIV1-YFP vector
G-CSF/SCF mobilized
PBSCH
Total Body Irradiation
(2x5Gy)
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T r a n s d
u c t i o n r a t e
( % )
The chi-HIV vector achieves superior transduction rates in vivo
Day after transplantation
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The chi-HIV vector achieves multi-lineage marking
T r a n sd
u c t i o n
r a t e
( %)
Day after transplantation
In vivo GFP among red blood cells
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In vivo GFP among red blood cells
H t i ti t ll hi l f th ti
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Hematopoietic stem cells as vehicles for therapeutic
gene delivery: Future efforts for human application
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)
Allogeneic stem cell transplantation
H t i ti t ll hi l f th ti
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Hematopoietic stem cells as vehicles for therapeutic
gene delivery: Future efforts for human application
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
Autologous stem cell gene transfer
C
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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
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