New advances and future outlook in the management and cure of hemoglobin disorders by Philip Leboulch

1. New advances and future outlook in the
management and cure of
c re
hemoglobin disorders
g
P. Leboulch – Bangkok TIF 2011

2. Advances in allogeneic hematopoietic
transplantation
Induction of γ-globin expression
γg p
Induced pluripotent stem (iPS) cells
Prospects for gene therapy

3. Mutations Causing -Thalassemia

4. Advances in allogeneic
hematopoietic transplantation

5. 2011

6. Overall Survival (OS) Disease Free Survival (DFS)

7. “SCURT”
SCURT
(Sickle Cell Unrelated Transplant Study)
Unrelated Donor Hematopoietic Cell Transplantation for
p p
children with severe SCD using a reduced intensity
regimen (BMT CTN 0601)
regimen (BMT‐CTN 0601)
A Multicenter Phase II Clinical Trial
A Multicenter Phase II Clinical Trial
Co‐PIs: Shalini Shenoy, MD
y,
Naynesh Kamani, MD

8. SCURT Trial: Current status
Data provided by DCC (Aug. 2011)
• I i i d i A il 2008
Initiated in April 2008
• Study now activated at 20 centers
• Actively enrolling eligible patients with 8/8 allele matched unrelated
Actively enrolling eligible patients with 8/8 allele matched unrelated
bone marrow donors
• Target accrual: 45 patients (incl. 8 who had UCBT)
Target accrual: 45 patients (incl. 8 who had UCBT)
• 21 have undergone BMT
– 8 males (median age: 13 1 y)
8 males (median age: 13.1 y)
– 13 females (median age: 13.6 y)
• Anticipated end date for enrollment: Nov 2011
Anticipated end date for enrollment: Nov 2011
Cord blood arm closed in January 2011 due to a high
incidence of graft rejection

9. Induction of γ-globin expression

10. SCIENCE VOL 322 19 DECEMBER 2008
SCIENCE VOL 334 18 NOVEMBER 2011

11. BLOOD, 10 MARCH 2011 – VOLUME 117, NUMBER 10

13. γ-globin expression levels obtained in postnatal mouse cells (wild-
type, SCD mice) and human thalassemic erythroid cells after
BCL11A KO (floxed) or knocked-down (shRNA) are inferieur or
equal to those achieved with γ- or β-globin lentiviral transfer,
even after administration of deacetylating agents (i.e., 5-azaD,
5 azaD,
SAHA)
If therapeutic strategy involves lentiviral transfer of BCL11A
shRNA ⇒ same i
hRNA issues as with globin l ti i l t
ith l bi lentiviral transfer
f
Small molecules inhibiting BCL11A (?)

14. Yearly lifelong costs of enzyme replacement for
Lysosome Storage Disorders
(average 50 kg adult)
• Gaucher disease (1991)
– $250 000 / year
$250,000
• Fabry disease (2004)
– $290 000 / year
$290,000
• MPS I (2004)
– $520 000 / year
$520,000
• MPS II (2007)
– $850 000 / year
$850,000
• MPS VI (2007)
– $800,000 / year
$ , y

15. Cost considerations for widespread application of
gene therapy vs. small molecule
The developing country of Thailand has decided to reimburse every allogenic CD34+
cell transplant for β th l
ll t l t f β-thalassemia because it is so much cheaper than lifelong bl d
i b i h h th lif l blood
transfusion and chelation.
Proposed pricing of one-time gene therapy for β-thalassemia < $200,000 and may drop
to < 50,000 with widespread diffusion.
Pricing of small molecule $1-$10 per day. When adjusted for inflation, this means
between $140,180 and $1,402,800 over an 80 year lifespan.

16. Induced pluripotent stem (iPS) cells

17. SCIENCE VOL 318 21 DECEMBER 2007
NATURE BIOTECHNOLOGY VOLUME 29 NUMBER 1 JANUARY 2011

18. Thalassemia patient ‐Induced pluripotent stem cells derivation (Thal‐iPS)
Why iPS cells?
1/ An alternative for gene therapy in the future ?
- Can grow in culture indefinitely
- Can differentiate into any cell types.
- Possible correction of any genetic defect by gene transfer or homologous recombination.
- Possible selection of safe corrected cells before engraftment to the patient.
2/ Our Thalassemia patient-Induced iPS cells is a good model to:
- Evaluate the hematopoietic potential of Thal-iPS cells.
- Study endogenous globin g
y g g gene expression in Thal-iPSC-derived erythroid cells in vitro and in vivo.
p y
- Study the βA(T87Q)-globin vector efficacy and properties.
- Investigate the oncogenic risks of a lentiviral βA(T87Q)-globin vector transferred into βE/β0- thalassaemia
patient.
- Compare lentiviral integration sites in corrected THAL-iPS cells with those of the patient.
Dr. Leila Maouche‐Chrétien (France) in
. e a aouc e C ét e ( a ce)
collaboration with Alisa Tubswan and
Prof. Suthat Fucharoen (Thailand)

19. Study Design
‐thal/HbE patient
Measurement of globin
expression
Pyrosequencing
Lentiviral vector
carrying A‐T87Q gene
Hematopoietic
reconstitution
Oct4, Sox2, Klf4, CFC assay in NOD/SCID mice
cMyc retroviruses
reprogramming ‐globin transfer differentiation
MOI=30
Thal‐iPSTG+
CD34‐ cells THAL‐iPS cells Hematopoietic cells
iPS cells
S ce s FACs analysis
characterization of hematopoietic markers

20. Characterization of Thal‐iPS cells
TRA1 60
TRA1-60 SSEA 3
SSEA-3 SSEA 1
SSEA-1
NANOG OCT 4
OCT‐4 SSEA‐4
Intestine DAPI DAPI
CNS Bronchea DAPI
Epithelium
Cartilage
CNS,
Muscle
M l Retina
R ti Adipose
Analysis of pluripotent genes Analysis of exogenous genes Analysis of transgene integration by
expression by RT-PCR silencing by RT-PCR PCR
endo OCT4
endo SOX2 pMig OCT4 OCT 4
endo KLF4 pMig SOX2
SOX 2
endo c-MYC
c MYC pMig KLF4
Mi KLF 4
NANOG pMig c-MYC c-MYC
β-ACTIN

21. In vitro hematopoietic differentiation of Thal iPS cells before (TG ) and after (TG+)
In vitro hematopoietic differentiation of Thal‐iPS cells before (TG‐) and after (TG+)
transduction with the lentiviral vector A‐T87Q gene.
Thal-iPSTG-
Thal-iPS
Thal iPSTG+
Mature BFU-E CFU-MIX CFU-G CFU-M

22. HPLC analysis of hemoglobin in BFU‐Es derived from Thal iPS and Thal iPSTG+
no adult Hb E
BFU-E patient Blood BFU-E.1 iPS-THAL TG-
0 5 10 20 25 30
CFU-GM. PS-THAL BFU-E 2 iPS-THAL TG+
HbAT87Q (%) = HbAT87Q /(HbAT87Q + HbF+ embryonic Hb) x 100
• BFU-Es from Thal-iPS produce mainly Hb F and embryonic Hb, with clear trend to switching in vivo
• Hb A (T87Q): highly expressed in BFU-E containing Lentiviral βA(T87Q)-globin vector
(level ranged from 40-85%) - no expression in non-erythroid cells

23. Hematopoietic engraftment of hematopoietic cells derived
from Thal‐iPS in immunodeficient‐mice
8 of 26 transplant mice showed engraftment with human cells (low levels)
10 5
10 4
Human CD45
3 weeks post transplantation 10 3
GpA+ cells sorted
(1 mouse)
10 2 Late erythroblast
0
0 10 2 10 3 10 4 10 5
Human glycophorin A
10 5 10 5
20
Human CD19/2
Human CD45
10 4 10 4
3 weeks post transplantation Myeloid cells
(1 mouse) 10 3 10 3
10 2 10 2
H
0 0
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Human glycophorin A Human CD15/33/66b
250K
10 5
Hum CD19/20
200K
10 4
FSC
150K
10 3
8 weeks post transplantation
man
100K
( 6 mice) 50K 10 2
B-lineage cells
0
0
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Human CD45 Human CD15/33/66b

24. Globin switching in vivo
?/(?+?)
1.000
1.20
1.00
0.100 0.80
β/(β+)
β/(β+)
0.633 0.60
0.010 n=2 0.96
0.40 n=22
0.011 0.57
0.20 0.42 n=1
0.004 n=2 n=3
0.001 n=2
0.00
Thal‐iPS BFU‐E
Thal iPS in vivo GPA+ THAL THAL patient blood
In vivo Thal patient CB
CB In vivo GPA+
in vivo GPA+ Adult normal
adult normal blood
iPS
BFU-E GPA+ blood CD 34+ CB
34 blood
Thal-iPS
• The ε to (γ + β) switch was ≈ complete after in vivo passage of iPS derived cells
• The β / (β + γ) ratio was ≈ 3 times higher after in vivo passage of iPS derived cells

25. Screening for clones with ‟safe” integrants in BFU‐Es from iPS THALTG+
g g
Criteria for iPS cells clones harboring safe genomic integration sites.
Criteria for iPS cells clones harboring safe genomic integration sites.
‐ distance of at least 50 kb from any genes
‐ distance of at least 300 kb from cancer‐related gene
‐ distance of at least 300 kb from any micro RNA
distance of at least 300 kb from any micro RNA
‐ location outside a transcription unit and ultraconserved regions (UCRs)
Total 24 individual BFU-Es
12 BFU-Es containing single copy number of the vector
2 of 12 (16%) BFU-Es containing safe integration site

26. Common Integration Sites (CIS) in ALD, β0/βE‐Thal patient and iPS THAL
Total of 4859 IS ( 2146 IS ALD P1, 1282 IS ALD P2, 357 IS Thal patient, and 1074 iPS THAL)
• located in CIS of the 3th order or higher
1698 IS • Most of CIS are shared integration in 3 studies
Most important CISs
50 IS • IS found from at least 2 studies
• should at least be of 6th order
# CIS Cluster
# CIS Cluster Shared Data set
Shared Data set
30 Shared in all data set
7 ALD & β‐Thal
13 ALD & iPS
0 iPS & β‐Thal
* considered 2, 3 or 4 insertions as CIS of 2nd, of 3rd or 4th order if they fell within a 30 kb, 50 kb or 100 kb window
of genomic sequence from each other, respectively.

27. Conclusions from our Thal‐iPS study from β0/βE‐thalassemia gene therapy patient
Thal-iPS cells are able to differentiate into multiple blood cell types both in vitro and in
vivo in immunodeficient-mice but level of multilineage engraftment very low in NSG mice
g g y
16% of transduced Thal-iPS cells with βA(T87Q)-globin lentiviral vector satisfied the
stringent criteria for “safe” areas of vector integration
g g
BFU-Es derived from genetically corrected Thal-iPS cells show high levels of Lentivector-
derived βA(T87Q)-globin expression - Clear trend to globin class switching in vivo
g p g g
Comparative analysis combining IS data show shared CIS between Thal-iPS, Thal
gene therapy patient and 2 ALD gene therapy patients with no bearing of the
therapeutic DNA insert (lentivector-backbone only)
CIS of 3rd order or higher occur at least 100 times more often than expected under the
random lentiviral distribution (evidence of non-random integration rather than in vivo
selection?)
Multiple and formidable hurdles remain for the use of iPS cells (both safety and efficacy)

28. Prospects for gene therapy

29. Conversion to transfusion independence of the first βE/β0-thalassemia
thalassemia
(major) patient for > 3.5 years at ≈ 9 g/dL Hb, > 4.5 years post-gene therapy

30. Intrinsic integration bias independent from DNA inserts
BLOOD, 3 JUNE 2010 – VOLUME 115, NUMBER 22
October 2011 / Volume 6 / Issue 10 / e24247
October 2010 / Volume 6 / Issue 11 / e1001008

31. Second E/0-thalassemia (major) gene therapy patient transplanted on
November 24 2011
24,
Patient 1 = PLB Patient 2 = MHV
Globin chains in PLB reticulocytes Globin chains in MHV reticulocytes 26 days
32 days post‐transplantation
y p p p
post‐transplantation
p
87Q)/(+) 4.4% 8.9%
87Q)/() 3.6% 8.2%
(87Q)/(E) 9.5%
9 5% 20.7%
20 7%
(87Q)/() 9.0% 18.5%
(E)/() 46.4% 43.0%
()/() 49.2% 48.1%

32. PROSPECTS
Optimized βA(T87Q)-globin lentiviral vector validated (higher transduction
globin
potency) – New or amended trial to be filed in France and US (early 2012)
Continuation Phase I/II for 0-thalassemia (major Cooley) and sickle cell
(major,
disease
Pilot Phase IIb/III for E/0-thalassemia (major)
thalassemia
Ex vivo or In vivo selection for transduced HSCs
Conditional suicide for enhanced safety
HSC and progenitor expansion
Decreased α-globin (β-thalassemia) by shRNAs
(coll.
(coll with Dr. Jim VADOLAS, Australia)
Dr VADOLAS

33. SCIENCE VOL 329 10 SEPTEMBER 2010

34. BLOOD, 19 MAY 2011 – VOLUME 117, NUMBER 20

35. Naturally occuring truncated (t)Epo-R variants in humans
Truncation of the C-terminal moiety of Epo-R is responsible for « dominant benign hereditary
erythrocytosis » (de la Chapelle et al. PNAS 1993)
« … cross-country skier having won three Olympic
gold medals and two world championships »
Hypersensitivity of erythroid progenitors to Epo (Juvonen et al. Blood 1991; Prchal et al. Eur J
Haematol 1996)
Epo Epo Epo
Jak2(P) Jak2(P) Jak2(P)
Y343 STAT5(P) STAT5(P)
-
Y401 Y1
Y429 SHP-1(P)
Y12
Y18

36. Co-expression of truncated Epo-R results in cure of thalassemia mice
at very low vector copy levels
A B
100
40
80
30
human Hb (%)
huRBC (%)
60
20
40 LG P= LG/HA-Y1
LG P= LG/HA-Y1 0,006 r2 = P = 0,079 r2
0,005 r2 = P = 0,144 r2 0,690 = 0,336
10 20
0,696 = 0,247
0 0
0 0,2 0,4 0,6 0,8 0 0,2 0,4 0,6 0,8
Copy per WBC Copy per WBC
C 1,9
1,7
- Phenotypic correction with
1,5
< 0.01vector copy in WBCs
log Epo
1,3
1,1
- No long-term leukemogenesis
0,9 P = 0,0000007
r2 = 0,857
and organ pathology (>10 months
0,7
and secondary transplants)
0,5
0 20 40 60 80 100
huRBC (%)

37. Thank you for your attention
37