Gene therapy of human β-thalassemias by Philip Leboulch

1. Gene therapy of human β
py β-thalassemias
P. Leboulch – Bangkok TIF 2011

2. The only curative treatment for the β-hemoglobinopathies
currently is allogeneic hematopoietic transplantation, although
fewer than 25% patients have an HLA-matched related donor, and
those who do still face the risks of engraftment failure and Graft-
Versus-Host
Versus Host Disease (GVHD)
The β-hemoglobinopathies is a paradigm of gene therapy with
no intrinsic in vivo advantage for transduced autologous
hematopoietic stem cells (HSCs)

3. Target: The
Hematopoietic
Stem Cell
Lentiviral
L ti i l
Vector
Sustained
Correction of
Red Blood Cells
http://mentor.lscf.ucsb.edu/mcdb133/chapter03/hematopoiesis.htm

4. Mutations Causing -Thalassemia

5. Lentiviral vector with tagged β-globin gene having
β globin
anti-sickling properties
2 x cHS4 2 x cHS4
Insulator cores Insulator cores
human -globin gene
cPPT/FLAP
 266bp 644bp
266b 644b 845bp
845b 1153bp
1153b ppt
t
III II I p HS2 HS3 HS4
3’ enhancer
RRE
SIN + Insulator SIN + Insulator
(Δ 1 core in ≈ 50%) A-T87Q (Δ 1 core in ≈ 50%)
Insulators have unpredictable activity and tendency to instability: Dispensable

6. Phase I/II clinical trial: Initial focus on severe E/0-thalassemia
Severe in ~ 50% cases (similar to 0 Thalassemia major)
Transfusion dependency, iron chelation therapy and often splenectomy.
Candidates for ll
C did t f allogenic transplant when HLA-matched sibling donor.
i t l t h HLA t h d ibli d
Narrow differential Hb levels between severe and well tolerated cases
Mean spontaneous Hb levels separated by > 2 g/dl.
g/dl
High worldwide prevalence
In Thailand alone, ~ 3,000 new cases born each y
, , year.
~ 4% of the 350 million Southern China population carry a E- or a 0- allele.
High prevalence among US immigrants.

7. Pre-transplant clinical history of the first gene therapy patient
with severe E/0-thalassemia (Patient "PLB”)
Then 18 year old male with severe E/0 thalassemia and no HPFH or α mutation.
E/0-thalassemia mutation
Transfusion dependent since age 3 (> 225 ml RBCs /kg/year for Hb > 10 g/dl).
Spontaneous Hb levels as low as 4.5 g/dl.
Major hepato-splenomegaly (splenectomy at age 6) and growth retardation
retardation.
Failure of Hydroxyurea therapy (between ages 5 and 17).
Desferoxamine (5 days/week) since age 8, and oral Exjade since age 18 (although nausea). No
liver fibrosis. Moderate iron overload by liver MRI (561 mol/g).
Only child. No related, genoidentical HLA-matched donor. Match strict inclusion and
exclusion criteria.
Transplantation at age 19 on June 7, 2007

8. Overview of the Clinical Protocol
Vector Maximize
+ % Transduced
CD34+ cells
Cytokines HSCs
Bone Marrow Testing
or PB Harvest and Release
While Frozen
Busulfex
(2x108 unsorted BM cells/Kg
kept for rescue)
Bone Marrow
Conditioning
Maximize IV Infusion
Myeloablation Transduced Cells
Without ( 4x106 CD34+/Kg)
(≈
Immunosuppression (Spontaneous Homing)

9. Percentages of vector bearing cells in blood and bone marrow
(qPCR
( C – 4 years post-transplant) )
BONE MARROW
All nucleated cells 21.0 %
CD34+ 25.0 % ≈ 20%
CD45+ 19.4 %
Erythroblastes (glycoA+ CD71+) 34.6 %
Correction
BLOOD dyserythropoiesis
Granulo-Monocytes (CD15+)
y ( ) 18.6 %
Erythroblastes (CD45- CD71+) 1.1 %

10. HPLC analysis of globin chains in whole blood
(
(3 to 33 months post-transplant)
p p )
3 months
10.0
α
7.5 βA 33 months
h
Absroban 220 nm
10.0
5.0 α
7.5
obance 220 nm
nce
2.5 βA-T87Q
Q
5.0
0.0
βE
γG γA 2.5 βE
-2.5
0.0
00 γG γA
Absro
-5.0
-2.5
12 months -5.0
10.0
10 0
α
7.5
Absrobance 220 nm
βA-T87Q
5.0 βA-T87Q
βE x 100 = 34 - 37 %
2
2.5 βA Σ (all β + γ)
0.0 γG γA
-2.5
-2 5
A
-5.0

11. Conversion to transfusion independence for > 3.5 years
at ≈ 9 g/dL Hb, > 4.5 years post-transplantation (I)
Transplantation on June 7, 2007 Phlebotomies (200 ml each)
Last RBC transfusion on June 6, 2008 to remove iron
15
14
13
12
11
Hb (g/dL)
10
9
(
8
appendicitis
7
6
5
0.0 10.0 20.0 30.0 40.0
RBC transfusions
Months post-transplantation
Phlebotomies

12. Conversion to transfusion independence (II)

13. Coordinated βE- and γ-globin expression
3
normalized HPLC Areas)
Ratios bloo globins / βE
2,5
A
s
2
E
1,5
od
A+G G
d
Gene th
therapy patient
ti t
1
87
0,5
,
R
(n
0
15
20
25
30
35
40
45
50
0
5
10
Months post transplantation
post-transplantation
Homogenous γ-globin expression by HPLC in single BFU-E colonies
regardless of vector integration sites (HMGA2 or not)
BLOOD, Vol 94, No ( (November 1), 1999:pp 3199-3204

14. Progressive decrease in plasma Ferritin concentration
g p
5000
4500
4000
Ferritin (µg/L)
3500
3000
2500
2000
1500
0.0 10.0 20.0 30.0 40.0 50.0
Months post BMT
transfusion

15. Integration site (IS) analysis by LM-PCR and DNA pyrosequencing
(whole nucleated blood cells and purified sub-populations)
sub populations)
Low total number of different IS (< 300)
In actively transcribed regions, similar to generic HIV vector
y g , g
24 IS both myeloid and lymphoid
HMGA2
FBXL11
10% TBC1D5
PILRB
8 MKLN1
IRAK1
6 HMGA2 ZZEF1
RFX3
NUP98
4 ATXN10
EPB41L2
2 EIF1
PHF16
0 SAE1
3 5 9 13 16 18 19 20 24 GNA12
POLA2
Months post-transplantation (whole nucleated blood cells)
Relative d i
R l ti dominance of IS at the HMGA2 l
f t th locus
(dominance relative to other IS, but > 85 % cells remain untransduced)

16. Vector integration in the third intron of the HMGA2 gene
Globin-LV Let-7 miRNAs
ATG TAG
51516 51517
E1 E2 E3 E4 E5
Intron 3 (~ 113 kb)
Deletion of 1 copy insulator core
py
Normal degradation of RNA by Let-7 miRNAs (multiple targets in E5)
Increased expression of truncated HMGA2 in benign
p g
proliferations (e.g., lipomas, HPN)

17. Kinetics of integration sites (IS) distribution in nucleated
blood cells quantified by qPCR:
Stability of HMGA2 partial dominance at 2-4 %

18. Proportion of vector bearing BFU-Es
40 months post-transplant
2%
10%
1
2
3
88%
1. Non modified BFU-Es
2. BFU-Es genetically modified at HMGA2 IS
3. BFU-Es genetically modified at other sites

19. HMGA2 IS is frequently retrieved by DNA pyrosequencing in vivo after
retroviral and lentiviral human CD34+ gene transfer
HMGA2 in X-SCID trial (γ-RV vector)
> 15 cluster IS in HMGA2 (aggregates of patients data):
- several in HMGA2 Intron 3
- several with tendency to increase with time and then stabilize
- 2 with truncated RNA by aberrant splicing Intron 3 into vector
HMGA2 in ALD trial (LV vector)
1 IS in HMGA2 Intron 3 in patient P1:
- only in B lymphocytes and 1 time-point
Wiskot Aldrich trial (γ-RV vector)
- Lack of detection in mouse studies
- Evidence for initial integration bias ?
- E id
Evidence for homeostatic i vivo selection ?
f h t ti in i l ti
- General principle seen with other key genes

20. 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

21. Second E/0-thalassemia (major) gene therapy patient transplanted on
November 24 2011
24,
Patient 1 = PLB Patient 2 = MHV
Globin chains in PLB Globin chains in MHV reticulocytes 26
reticulocytes 32 days post-
y y p days post-transplantation
y p p
transplantation
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%

22. 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

23. Institute of Emerging Diseases and Innovative Therapies – INSERM U. 962 – University Paris 11
Emmanuel Payen Olivier Negre Leila Maouche-Chrétien
Floriane Fusil Béatrix Gillet-Legrand Yves Beuzard
Céline Courne Philippe Leboulch
Harvard Medical School, Brigham and Women s Hospital Boston MA
School Women’s Hospital, Boston,
Robert Pawliuk Karen Westerman Resy cavalleso
Shamil Sunyaev Philippe Leboulch
Hopital Necker (AP-HP), Paris
p ( )
Marina Cavazzana-Calvo Salima Hacein-Bey Abina Laure Cacavelli
University of Pennsylvania School of Medicine, Philadelphia, PA
Frederick Bushman Gary Wang Troy Brady
Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de Paul
Eliane Gluckman Françoise Bernaudin Gérard Socié
Robert Girot Jean Soulier Nabil Kabbara
Bluebird bio, Inc, Cambridge, MA
Kathy Hehir Maria Denaro Julian Down
Gabor Veres Mitchell Finer
Indiana Vector Production Facility, Indianapolis, IN
Ken Cornetta Scott Cross Chris Ballas
Mahidol University and Ramathibodi Hospital, Bangkok, Thailand
Alisa Tubsuwan Suparek Bornwornpinyo Suradej Hongeng
Suthat Fucharoen