Five year outcome of lentiviral gene
therapy for human beta-thalassemia,
lessons and prospects
Cyprus TIF 2012 - P. Leboulch
The only curative treatment for the β-thalassemia 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 Disease (GVHD)
Ex vivo gene therapy by transfer of a therapeutic β-globin gene into
the patient’s own hematopoietic stem cells (HSCs) would alleviate
both donor shortage and GVHD.
It is however especially challenging and a paradigm of gene therapy
with no intrinsic in vivo advantage for corrected HSCs.
Lentiviral vector design with marked β-globin gene
2 x cHS4
Insulator cores
2 x cHS4
Insulator cores

cPPT/FLAP
human -globin gene
266bp 644bp
III
RRE
SIN + Insulator
(Δ 1 core in ≈ 50%)
•
•
II
I p
HS2
845bp
1153bp
HS3
HS4
ppt
3’ enhancer
A-T87Q
SIN + Insulator
(Δ 1 core in ≈ 50%)
Why A-T87 marking / mutation?
Provides anti-sickling properties (Leboulch and coll. Science 2001)
In-vivo biomarker for detection therapeutic globin by HPLC
Overview of the clinical protocol
CD34+ cells
Bone Marrow
or PB Harvest
Vector
+
Cytokines
Maximize
% Transduced
HSCs
Testing
and Release
While Frozen
Busulfex
(2x108
unsorted BM cells/Kg
kept for rescue)
Bone Marrow
Conditioning
Maximize
Myeloablation
Without
Immunosuppression
IV Infusion
Transduced Cells
(≈ 4x106 CD34+/Kg)
(Spontaneous Homing)
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 allogenic transplant when HLA-matched sibling donor.
Narrow differential Hb levels between severe and well tolerated cases
Mean spontaneous Hb levels separated by > 2 g/dl.
High worldwide prevalence
In Thailand alone, ~ 3,000 new cases born each year.
~ 4% of the 350 million Southern China population carry a E- or a 0- allele.
High prevalence among US immigrants.
Pre-transplant clinical history of the first gene therapy patient
without injection of backup cells
18 year old male with severe E/0-thalassemia (major) and no HPFH or α 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.
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 5 years ago at age 19 on June 7, 2007
Vector copy / cell
Vector copy numbers per circulating cell subsets
(up to 5 years post-transplantation)
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Whole blood
CD15 (neutrophils)
CD19 (lymphocytes B)
CD45-CD71+ (erythroblasts)
CD3+ (lymphocytes T)
CD14 (monocytes)
0
10
20
30
40
50
60
Months post-transplantation
Conversion to transfusion independence for 4.4 years
(5.4 years post-transplantation)
Published
Sept 2010
Transfused
Blood
Lentiglobin
Treatment
Transplantation on June 7, 2007
Last RBC transfusion on June 6, 2008
Conversion to transfusion independence (II)
Last transfusion > 4.4 years ago
Phlebotomies (200 ml each)
12
Hemoglobin (g/dL)
10
Hb totale
8
≈ 3.5 g/dL vector-encoded AT87Q-globin
6
HbAT87Q
HbF
HbE
4
2
HbA
0
0
10
20
30
40
Months post-transplantation
50
60
30
25
20
15
10
5
10
20
30
40
50
60
Months post-transplantation
4,0
3,5
3,0
2,5
2,0
1,5
1,0
10
20
30
40
50
60
4,0
3,5
3,0
2,5
2,0
1,5
1,0
Months post-transplantation
Reticulocytes
(x105/µL)
35
Red blood cells
(x106/µL)
Blood erythroblasts
(x103/µL)
Partial correction of dyseythropoiesis
and red blood cell maturation/lifespan
Partial decrease in plasma ferritin levels
Phlebotomies
Ferritin (µg/L)
3500
3000
2500
2000
1500
1000
4
8
12
16
20
24
28
32
36
40
44
48
Months post-transplantation
52
56
60
Sustained amelioration of the thalassemic phenotype
Complete MCH correction (28.4 pg)
(Average MCH for βE/β0-Thal patients = 19 pg)
Calculated expression βAT87Q > 70% endogenous βA-globin
on a per gene basis
Calculated increased RBC lifespan > 8.5-fold
Decreased circulating erythroblasts > 3-fold (> 9-fold for vector+ cells)
Significant decrease (> 2-fold) in plasma ferritin levels
Excellent clinical status (full time job)
Integration site (IS) analysis by LM-PCR and DNA pyrosequencing
(whole nucleated blood cells and purified sub-populations)
Low total number of different IS (< 300)
In actively transcribed regions, similar to generic HIV vector
24 IS both myeloid and lymphoid
100%
80
HMGA2
60
40
20
0
3
5
9
13
16
18
19
20
24
Months post-transplantation (whole nucleated blood cells)
Relative dominance of IS at the HMGA2 locus
(dominance relative to other IS, but > 80 % cells remain untransduced)
HMGA2
FBXL11
TBC1D5
PILRB
MKLN1
IRAK1
ZZEF1
RFX3
NUP98
ATXN10
EPB41L2
EIF1
PHF16
SAE1
GNA12
POLA2
Aberrant splicing of the third intron and polyadenylation within the
globin lentiviral vector
Globin-LV
ATG
E1 E2
51516 51517
E3
TAG
E4
Let-7 miRNAs
E5
Intron 3 (~ 113 kb)
Deletion of 1 copy insulator core
Sequencing of the main HMGA2 transcript:
Aberrant splicing within the vector insulator + polyadenylation
ERYTHROID-SPECIFIC EXPRESSION +++++
Cell type distribution of HMGA2 IS (3 months post-transplant)
CFU-GM colonies
~
7 % positive
(~ 67% of vector+)
BFU-E colonies
~
15 % positive
(~ 78% of vector+)
Lymphocytes (thorough search)
~
undetectable
(fresh sorted CD3 / CD19 or
lectin expanded)
LTC-IC (7 weeks)
~
6 % positive
(~ 50% of vector+)
First evidence in humans for long-term hematopoietic lineage bias
(myleoid vs. lymphoid vs. totipotent)
Further evidence is accumulating in mice …
•
By limited dilution transplant analyses from C. Eaves lab (e.g., Cell Stem Cell 2012;10:273-83).
•
By barcoded transplant analysis from P. Leboulch lab (submitted).
•
CD86- marks myeloid restriction from W. Kincade lab (Blood 2012;119:4889-97).
Hematopoietic homeostasis is maintained
Normal blood and bone marrow cytology – Normal cytoflurometry analysis
Normal karyotype and high resolution CGH-array chromosomal analysis –
No Trisomy 8 with specific probe
Lack of cytokine-independence in CFU-C assays
Normal LTC-IC counts
No preferred engraftment in NSG mice (coll. C. Eaves)
No abnormality detected for MPL. JAK2 and TET2
Asymptotic stabilization of the clone relative dominance
Natural cases of PNH with HMGA2 dysregulation followed for > 19 years
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)
MGMT glioblastoma trial (γ-RV vector)
- Lack of detection in mouse studies
- Evidence for initial integration bias ?
- Evidence for homeostatic in vivo selection ?
- General principle seen with other key genes
The percentage of partially dominant HMGA2 site is decreasing over time in
whole nucleated blood cells (qPCR – 5 years post-transplant) …
18
16
Genetically modified cells in blood
LentiGlobin vector
14
LentiGlobin vector at HMGA2
12
10
8
6
4
2
0
0
10
20
30
Months post-BMT
40
50
Integration site (IS) analysis by LM-PCR and DNA pyrosequencing
4 and 5 years post-transplantation
4 years
5 years post-transplantation
Insersion sites (% of each)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Number unique IS
WBC
WBC
CD45
CD3
CD19
CD15
CD14
Erythro
38
27
20
10
22
19
23
9
SPATS2
POLA2
ZZEF1
HMGA2
RFX3
PMS2P1
Already the most common after 3 years
qPCR based kinetics of the four most common IS
up to 5 years post-transplantation
Vector copy number
0.14
copies
0.12
HMGA2
0.10
RFX3
0.08
FBXL11
0.06
ZZEF1
0.04
0.02
0.00
0
10
20
30
40
50
60
Months post-transplantation
4 years
5 years
ZZEF1 = 1/3
HMGA2
ZZEF1 =
HMGA2
Second E/0-thalassemia (major) gene therapy patient
transplanted on November 24, 2011
•
23 year old woman, βE/β0-Thal Major
•
Transfusion dependent since her 2nd month of life. No HLA matched sibling donor
•
The transplantation was uneventful. Engrafted neutrophils by day 20 and had
delayed platelet reconstitution (no related complications)
•
The patient has returned to full time work
•
Peripheral vector copy numbers and therapeutic βAT87Q-globin protein in reticulocytes
approximately 3-fold greater than in the previous patient at same time-points
•
Red blood cell transfusions are now being tapered down to stimulate erythropoiesis
and increase βAT87Q-globin production
Parallel evolution of therapeutic Hb in the second β-thalassemia
patient after lentiviral gene therapy
PATIENT 1 (specific chain / non-a
chain)
globins [%]
20
15
87
g
E
d
10
5
g E
hemoglobins [g/dL]
PATIENT 1
0
0
2
4
6
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
8
87
g
E
d
0
2
Months post transplant
87
g
E
d
10
5
0
4
6
Months post transplant
8
hemoglobins [g/dL]
globins [%]
15
2
8
PATIENT 2
20
0
6
Months post transplant
PATIENT 2 (specific chain / non-a
chain)
g E
4
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
87
g
E
d
0
2
4
6
Months post transplant
8
Comparative globin chain analysis by reverse phase HPLC
Patient 1 (transfusion independent since 4.5 years ago) and Patient 2
Patient 1 « PLB »
12
10
Hb (g/dL)
8
6
10.59
9.70
4
5.82
0.00
0.71
0.40
0.00
0.04
0.05
7.36
0.13
1.34
0.80
0.02
0.49
0.31
bA
bAT87Q
5.55
g
6.27
2
0
6.64
6.98
0.24
1.62
1.00
0.34
1.38
0.92
0.39
1.61
0.61
1.04
1.18
1.72
bE
Patient 2 « MHV »
12
Hb (g/dL)
10
8
bA
6
8.17
7.79
4
7.64
8.95
0
0.05
0.06
6.15
5.63
6.48
0.10
0.35
0.27
bAT87Q
g
2
0.00
0.42
0.60
6.40
0.14
0.35
0.35
0.13
0.31
0.41
0.21
0.65
0.93
0.32
0.99
1.33
0.33
0.82
1.11
bE
Integration site (IS) analysis by LM-PCR and DNA pyrosequencing
in Patient 2 “MHV” 6 months post-transplantation
100%
CD34
WBC
CD45
CD3
CD19
CD15
CD14
Erythro
218 unique sites
Insertion sites (% of
each)
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
JUP
C12ORF64
CIP29*
BST2
DHX8
CEP290
CARM1
PFAS
TNKS2
MPRIP
FBXL11(1)
C4ORF45
C19ORF47
SPOCK3
ACSL4
WDHD1
JAK3*
low
PLEKHA3
TCF12*
IMAA
BC031827 DHDDS
GUSBL2
ARID1A*
GUSBL2
LHX3
NBR1*
MAG
HORMAD2
CLN3
VWF
SAPS3
FLJ45340 TK1*
TNFSF12
FCHSD2
NCK1*
CEP110*
SAFB
ARIH1
AK090973 B7
CHD6,CHD5* C11ORF49
SETD2*
DKFZP686B0790
GATAD2B
TAFII105
EIF4G3
TXNRD3
RAB5C*
C6ORF106
SIRT3
IQGAP1
low
low
GTF2IRD2
VAPA
DKFZP434
N1217
FBXL11(3)
KIF1B
FBXL11(2)
RAB14
SUV420H1
KIAA0827
MALAT1
MAP1S
C17ORF26
DNAL1
RPS6KA1*
RBM5*
TBLR1
SPOP
NAV3
NIP30
low
SLC19A2
GTF3C2
IL6ST*
BTBD11
NR_002821
low
DKFZP4
34
C2019
ATP6V1A
TXNDC11
UBA2,SAE2
NUP88
RHOA*
CUTL1
MIER2
PLEKHA3
C9ORF86
VDP
MAP3K3
UNQ747
low
VPS16
ZNF318*
SF3A3
ZNF280B
NHN1
PTRF
CRAMP1L
PB1
HMGA2*
PMS2P1
SPATS2
MEX3C
CSNK1G1
low
Modified vector BB305 (G. Veres & M. Finer – bluebird bio) for
continuation French trial and for US trial
•
Removal insulator and TAT independent
together with novel purification system
•
Higher GMP production titers and >2-3-fold better
human CD34+ transduction efficiency
•
Greater vector copy number (VCN) per cell
(important for β0-thal)
•
High βAT87Q-globin protein expression even in nonthalassemic erythroid cells (e.g., βS/ βS)
•
Maintained low genotoxic potential in IVIM assays
(coll. C. Baum)
•
Vector substitution in French trial planned for
next patients
•
US trial with new vector filed with RAC
(unanimous approval) and with FDA in December
VCN ~ 3
Institute of Emerging Diseases and Innovative Therapies – INSERM U. 962 – University Paris 11
Bluebird bio – France and Harvard Medical School, Brigham and Women’s Hospital, Boston, MA
Emmanuel Payen
Olivier Negre
Leila Maouche-Chrétien
Alisa Tubsuwan
Soumeya Abed
Robert Pawliuk
Karen Westerman
Resy Cavallesco
Julian Down
Kathy Hehir
Philippe Leboulch
Hopital Necker (AP-HP), Paris
Marina Cavazzana-Calvo (current PI clinical trial)
Olivier Hermine
Felipe Suarez
Salima Hacein-Bey Abina
Antoine Ribeil
Bluebird bio, Inc, Cambridge, MA and bluebird bio - France
Olivier Negre
Gabor Veres
Béatrix Gillet-Legrand
Yves Beuzard
Robert Kutner
Dave Davidson
Maria Denaro
Kathleen Kirby
Mitchell Finer
Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de Paul
Eliane Gluckman
Françoise Bernaudin
Gérard Socié
University of Pennsylvania School of Medicine, Philadelphia, PA
Frederick Bushman
Gary Wang
Troy Brady
German Cancer Research Center (DKFZ), Heidelberg, Germany
Annette Deichmann
Manfred Schmidt
Kristof Von Kalle
Indiana Vector Production Facility, Indianapolis, IN
Ken Cornetta
Scott Cross
Chris Ballas
Terry Fox Laboratory, University of British Columbia, Vancouver, Canada
Melanie Kardel
Alice Cheung
Connie Eaves
Mahidol University and Ramathibodi Hospital, Bangkok, Thailand
Alisa Tubsuwan
Suradej Hongeng
Suthat Fucharoen