Scott Howard 2009

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Bone marrow transplantation in sickle celldisease

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 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



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

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





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 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.



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)



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



Why Rapamycin??

IL-2CsA

CD28

Rapa

Anergy

Induction of tolerance

Effector Function

Proliferation

TcR-CD3



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



Sickle Hemoglobin is Replaced by Donor

Hemoglobin in Chimeric Homozygote Mice

Powell, J, et al., Transplantation, 2005



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



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



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



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



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



**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



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



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



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



• 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






•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



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?



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



NATURE |VOL 406 | 6 JULY 2000 |www.nature.com



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



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



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



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

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

( % )



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)



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



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



In vivo GFP among red blood cells

H t i ti t ll hi l f th ti



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)


H t i ti t ll hi l f th ti



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


C



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

Scott Howard 2009

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