inserm u876, université bordeaux ii victor segalen, bordeaux, france
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Inserm. Institut national de la santé et de la recherche médicale. Gene therapy of a mouse model of congenital erythropoietic porphyria improved by a selective advantage of corrected red blood cells. INSERM U876, Université Bordeaux II Victor Segalen, Bordeaux, France. - PowerPoint PPT PresentationTRANSCRIPT
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 RTRIPLEX
U3RU3
SASDRRE
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 RU3
RU3SASD
RREWPRE
Vectors TEEW or TEUW
Analyses
Transduced cells
CFC
5 weeks
2 week
s
Clonogenic tests
2 weeks
LTC-IC24hEGFP
CD34100 101 102 103 104
100
100
101
102
103
104
Cytometry
100 101 102 103 104
Fluorocytes
Num
ber
of c
ells
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 ce
llule
s EG
FP+
SPm CEP
020406080
100
6 11 18 25 32
% E
GFP
+ ce
lls
Time (days)
0
20
40
60
80
Metabolic Correction
Fluorescence des porphyrines
Num
ber
of c
ells
Fluorocytes
100 101 102 103 104
SPm controlTEEWTEUW
% transduction TEEW
fluorocytes (%)TEUWTEEW
SPm contrôle869.92.8
Porphyrin Fluorescence100 101 102 103 104
SPm CEP
Fluorocytes
SPm PEC73
69.917.3
Enzymatic CorrectionU
ROS
enzy
me
acti
vity
(nm
ol /
h / m
g)
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 kb2.5 kb
1.5 kb
4 kb
Copy/cell
5 kb
Size marker
Not transduced
PlasmidSPm control/TEUWSPm CEP/TEUW
Calculation of the Proviral Copy
Number
1,63,9
Transgene Expression after erythroid differentiation
020406080
100
EGFP
+ c
ells
(%
)0
100
200
300
400
URO
S Ac
t.
(nm
ol/h
/mg)100 101 102 103 10410
010
110
210
310
4
SPmcontrol
TEEW
GPA
EGFP
SPm control
TEEW
100 101 102 103 104100
101
102
103
104
SPmCEP
GPA
EGFP
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
URO
S ac
tivi
ty (
U/m
g of
pro
tein
s)
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)
Fluo
rocy
tes (
%)
II IV
SSC
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 por
phyr
ins
(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 anemiaRe
ticu
locy
tes
(%)
Hal
f-lif
e of
RBC
s
Hem
oglo
bin
(g/d
l)
024
68
1012
141618
+/+ 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
Sple
en/b
ody
wei
ght
(%)
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)
Fluo
rocy
tes (
%)
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
ivity
(U
/h/m
g of
pro
tein
s)
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
BCs
(%)
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
BCs
(%)
y = 2,34xR2 = 0,83
y = 0,36xR2 = 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 GFP
exp
ress
ion
betw
een
Gr-
1 +
cells
and
Ter
119+ c
ells
y = -0,29x + 31R2 = 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 (%)
Fluo
rocy
tes
(%)
Level of transduction necessary and efficient
05
1015202530354045
0 10 20 30 40 50 60 70 80 90 100
GFP+ RBCs (%)
Fluo
rocy
tes
(%)
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