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