Gene therapy of a mouse model of Gene therapy of a mouse model of
congenital erythropoietic porphyria congenital erythropoietic porphyria
improved by a selective advantage improved by a selective advantage
of corrected red blood cellsof corrected red blood cells
INSERM U876, Université Bordeaux II Victor Segalen, Bordeaux, France INSERM U876, Université Bordeaux II Victor Segalen, Bordeaux, France
Institut nationalde la santé et de la recherche médicale
Inserm
Experimental Gene therapy in CEP
Géronimi et al, J Mol Med 2003
-Tissu source: mPB CD34+ normal and deficient cells
- Gene transfer: retroviral and lentiviral vectors
In vitro studies
cPPT CTS EF-1 USU5 R
TRIPLEX
U3
RU3SASD
RRE
Vecteur Trip-EF1-US (TEU)
WPRE
Gene Transfer into CD34+ Cells with SIN Lentivectors
Medium: Il-3, TPO, Flt3-L, SCF
CD34+ SPm: control or CEP
24h 24hAnalyses
Lentiviral supernatantTEEW or TEUW, MOI 30
T0
Prestimulation
18h
EF-1 EGFP or UROSU5 R
U3
RU3SASD
RREWPRE
Vectors TEEW or TEUW
Analyses
Transduced cells
CFC
5 weeks
2 week
s
Clonogenic tests
2 weeks
LTC-IC24hEG
FP
CD34
100 101 102 103 104
100
100
101
102
103
10
4
Cytometry
100 101 102 103 104
Fluorocytes
Nu
mb
er
of
cells
Porphyrins
Porphyrins UROS enz Activity
72h
SPm control
Percentage of transduction (TEEW)
Populationcellulaire
totale
0
20
40
60
80
100
CellulesCD34+
CFC LTC-IC
% d
e c
ellu
les E
GFP
+
SPm CEP
0
20
40
60
80
100
6 11 18 25 32
% E
GFP
+ c
ells
Time (days)
0
20
40
60
80
Metabolic Correction
Fluorescence des porphyrines
Nu
mb
er
of
cells
Fluorocytes
100 101 102 103 104
SPm controlTEEWTEUW
% transduction TEEW
fluorocytes (%)TEUW
TEEW
SPm contrôle
86
9.9
2.8
Porphyrin Fluorescence
100 101 102 103 104
SPm CEP
Fluorocytes
SPm PEC
73
69.9
17.3
Enzymatic CorrectionU
RO
S e
nzy
me a
cti
vit
y (
nm
ol /
h /
mg
)
0
20
40
60
80
100
120
140
TEUW(lenti)
SPm control
MFG-EGFPor TEEW
MFG-US(onco-retro)
SPm CEP
1.8 kb
5 2 1 0
3 kb
2.5 kb
1.5 kb
4 kb
Copy/cell
5 kb
Size marker
Not transduced
PlasmidSPm control/TEUW
SPm CEP/TEUW
Calculation of the Proviral Copy
Number
1,63,9
Transgene Expression after erythroid differentiation
0
20
40
60
80
100
EG
FP
+ c
ells (
%)
0
100
200
300
400
UR
OS
Act.
(n
mol/
h/m
g)100 101 102 103 1041
00
101
102
103
104
SPmcontrol
TEEW
GPA
EG
FP
SPm control
TEEW
100 101 102 103 104
100
101
102
103
104
SPmCEP
GPA
EG
FP
SPm CEP
TEEW
TEUW
TEUW TEUW
TEEW TEUW TEEW TEUW
Conclusions
- Maintenance of the transgene expression
after erythroid differentiation
Ex vivo gene therapy of a murine animal model
- Efficient gene transfer with lentivectors into
total cells, CFCs and LTC-ICs
Inherited disease caused by a deficiency in uroporphyrinogene III synthase (UROS) activity
Accumulation of porphyrins in erythrocytes, bone marrow, spleen, urine and feces.
Clinical manifestations Severe skin photosensitivity
Splenomegaly
Erythrodontia
Redish-coloured urine
Hematologic features Haemolytic anemia
Fluorescent blood cells
Congenital erythropoietic porphyria (CEP)
Knock-in mouse model obtained by homologous recombinaison
Profound deficiency in UROS activity
Accumulation of porphyrins in RBC, BM, liver and spleen
Haemolytic anemia
Moderate skin photosensitivity
Severe splenomegaly
Useful model to test a gene therapy protocol
Ged et al., Genomics 2006
Murine model of CEP
+/+
CEP
Symptomatic treatments are inefficient
Allogenic bone marrow transplantation is the unique curative treatment for this severe disease
However, in the absence of a suitable donor
Alternative approach : ex vivo
HSCs gene therapy
Congenital erythropoietic porphyria (CEP)
Whether a specific expression limited to erythroid progeny of HSCs is sufficient to reverse the clinical phenotype ?
Whether a spontaneous in vivo survival advantage for corrected red blood cells does exist ?
What is the level of HSCs transduction that allows a complete correction of the disease ?
Specific aims
U3
HS-40 Ank p LTR WPRE cDNA UROS cPPTESp-UROS LTR
Experimental design
CEP donors
5-FU
BM Sca-1+ Cells
5 days
AnalysesEnzymatic
Metabolic and PhenotypicCorrections
CEP recipients
20 wks
Busulfan (2x25mg/kg
)
ESp-UROS (MOI 2-200)
36h
Experimental protocol
Secondary CEP recipients
Busulfan (2x25mg/kg
)
Mice MOI % CFC integration Proviral copy #
Group I (n=4) 200 83.3 15.5 ± 1.3
Group II (n=8) 20-60 62.5 - 68.2 3.8 ± 1.4
Group III (n=8) 6-20 42.9 - 45.5 0.6 ± 0.2
Group IV (n=4) 2 25 0.2 ± 0.1
Experimental design
Control groups: normal BALB/c and CEP mice
Enzymatic correction in bone marrow
UR
OS
acti
vit
y (
U/m
g o
f p
rote
ins)
n=5
n=5
<0.2
n=4
n=4
0
5
10
15
20
25
+/+ CEP I II III IV
n=4
n=8
Metabolic correction in peripheral blood
Time (weeks)
Flu
oroc
ytes
( %
)
II IV
SS
C
Fluorocytes
0.1
0
5
10
15
20
25
30
35
40
0 5 10 15 20
+/+
30.3
CEP
III
3.4
III
25.2
IVI
0.1
0.1
IIICEP+/+
Metabolic correction : porphyrins in urines
Tota
l p
orp
hyri
ns (
µm
ol/
L)
0
100
200
300
400
500
600
700
800
900
1000
+/+ CEP I II III IV
< 0.2 5.83.8
Correction of hemolytic anemiaR
eti
cu
locyte
s (
%)
Half
-lif
e o
f R
BC
s
Hem
og
lob
in (
g/d
l)
0
2
4
6
8
10
12
14
16
18
+/+ CEP I II III IV
0
2
4
6
8
10
+/+ CEP I II III IV0
10
20
30
40
50
60
+/+ CEP I II III IV
Sp
leen
/bod
y w
eig
ht
(%)
Correction of splenomegaly
0
1
2
3
4
5
6
7
8
9
+/+ CEP I II III IV
Phenotypic correction
CEP I-III +/+ IV
50µm 50µm 50µm 50µm
Long term expression of the transgene : secondary
mice
Time (weeks)
Flu
oroc
ytes
( %
)
0
5
10
15
20
25
30
35
40
0 5 10 15 20
+/+ CEP CEPII
Erythroid-specific expression of the therapeutic gene
0
5
10
15
20
25
30
35
40
CEPII +/+ CEP
BM
BM
Ter11
9+
Ter11
9+
Ter11
9+
Ter11
9-
Ter11
9-
Ter11
9-
BM
UR
OS
Act
ivit
y (
U/h
/mg
of p
rote
ins)
Erythroid-specific expression of the therapeutic gene led to a full enzymatic, metabolic and phenotypic correction of CEP mice.
Suprisingly, this full phenotypic correction of the disease was obtained with only 45% of transduction of CFCs suggesting a selective advantage of corrected cells
EF1pGFP LTR
U3
WPRE cPPT EGFPEF1Lp
CEP-HSC
RBCs
Granulocytes Platelets Lymphocytes
HS-40 Ank p LTR
U3
WPRE UROScPPTESpUROS-EF1pGFP
EGFPEF1Lp
Selective advantage of corrected erythroid cells ?
GFP+ WBCs(%)
GFP
+ R
BC
s (
%)
Selective advantage of corrected red blood cells
4 weeks
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60 70 80
y = 0,36xR2 = 0,86
Control vectorEF1pGFP
y = 2,29xR2 = 0,82
Therapeutic vector ESpUROS-EF1pGFP
12 weeks
GFP+ WBCs (%)
GFP
+ R
BC
s (
%)
y = 2,34x
R2 = 0,83
y = 0,36x
R2 = 0,80
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60 70 80
0
0,5
1
1,5
2
2,5
3Gr-1 positive cellsearly normoblasts (III)intermediate normoblats (II)RBCs in BM (I)RBCs in peripheral blood
MNDpGFP EFpGFP EFpGFP ESpUROS-EFpGFP
Selective advantage in bone marrow
Normal mice CEP mice
Rat
io o
f G
FP
exp
ress
ion
bet
wee
n G
r-1 +
ce
lls
and
Ter
119+
cel
ls
y = -0,29x + 31
R2 = 0,80
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40 50 60 70 80 90 100
GFP+ RBCs (%)
Flu
oro
cyte
s (
%)
Level of transduction necessary and efficient
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40 50 60 70 80 90 100
GFP+ RBCs (%)
Flu
oro
cyte
s (
%)
A specific expression limited to erythroid progeny of HSCs is sufficient to reverse the phenotype.
A survival advantage of corrected RBCs has been demonstrated.
The level of transduction of HSCs necessary to obtain a complete correction of the disease is about 30-40%.
A long term correction was also observed in secondary mice
This study forms the basis of a gene therapy clinical trial for the patients suffering this severe porphyria disease
Conclusion
INSERM E217, Bordeaux, France
Robert-Richard ElodieCario-Andre Muriel
Costet PierreGed Cecile
Guyonnet-Dupeyrat Véronique
Lalanne MagalieLamrissi-Garcia IsabelleMoreau-Gaudry Francois
De Verneuil Hubert
Inserm
Aknowledgments
Congenital Erythropoietic Porphyria
2. Curative treatment- Stem cell transplantation (compatibility)- Gene therapy in the future ?
Treatment of CEP
1. Symptomatic treatment- sunscreen lotions- -carotene - oral charcoal- hydroxyurea- splenectomy- repeated transfusions
Case number
Age at transplant
Sex Conditioning Stem cell source Clinical out come Follow-up
1
10 years
F
Busulfan/cyclo
BM sibling
Died CMV infection
11 months
2 4 years F Busulfan/cyclo
Anti-thymocyte
UCB sibling Alive Complete recovery
10 months
3 22 months F Busulfan/cyclo
X 2
BM X2 sibling Alive Complete recovery
12 years
4 18 months F Busulfan/cyclo BM sibling Alive Complete recovery
35 months
5 11 years M BM BM Died sepsis 10 days
6 23 months M Busulfan/cyclo BM sibling Alive Complete recovery
15 months
7 23 months F ? CD34+ BM Alive Complete recovery
16 months
8 22 months
24 months
F Busulfan/cyclo
X 2
CD34+ BM X 2
Haplo-identical sibling
Alive Complete recovery
10 years
9 4 years M Busulfan/cyclo
Anti-thymocyte
BM not related
Alive CMV infection Complete recovery
6 years
10 2 years F Busulfan/cyclo
Anti-thymocyte
BM not related Alive Complete recovery
4 years ½
11* 3 years M ? HSC from HLA unrelated donor
Alive Complete recovery
2 years
* CEP patient with GATA1 mutation (Phillips JD et al, 2007)
Patients with CEP treated with stem cell transplantationPatients with CEP treated with stem cell transplantation