Transcript PowerPoint Presentation - Hematopoietic Stem Cells as Vehicles for

Bone marrow transplantation in sickle cell
disease
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 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
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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
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Roger Kurlander
Greg Kato
Mark Gladwin
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Elizabeth Kang
Jonathan Powell
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5 Research Court
– Mark Metzger
– Allen Krouse
– Barrington Thompson
– Bob Donahue
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Cindy Dunbar
– Stephanie Sellers
– Tong Wu
– Hyeoung Joon Kim
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Martha Kirby
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Leszek Lisowski
Selda Samakoglu
Michel Sadelain
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Terri Wakefield
Beth Link
Nona Coles
Karen Kendrick
Griffin Rodgers