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Gene therapy of a mouse model of congenital erythropoietic porphyria improved by a selective advantage of corrected red blood cells INSERM U876, Université.

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Presentation on theme: "Gene therapy of a mouse model of congenital erythropoietic porphyria improved by a selective advantage of corrected red blood cells INSERM U876, Université."— Presentation transcript:

1 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 Institut national de la santé et de la recherche médicale Inserm

2 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

3 cPPT CTSEF-1  US U5 R TRIPLEX  U3 RU3 SA SD 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 Analyses Lentiviral supernatant TEEW or TEUW, MOI 30 T0 Prestimulation 18h EF-1  EGFP or UROS U5 R  U3 RU3 SA SD RRE WPRE Vectors TEEW or TEUW

4 Analyses Transduced cells CFC 5 weeks 2 weeks Clonogenic tests 2 weeks LTC-IC 24h EGFP CD34 10 0 1 2 3 4 0 0 1 2 3 4 Cytometry 10 0 1 2 3 4 Fluorocytes Number of cells Porphyrins UROS enz Activity 72h

5 SPm control Percentage of transduction (TEEW) Population cellulaire totale 0 20 40 60 80 100 Cellules CD34 + CFCLTC-IC % de cellules EGFP + SPm CEP 0 20 40 60 80 100 611182532 % EGFP + cells Time (days) 0 20 40 60 80

6 Metabolic Correction Fluorescence des porphyrines Number of cells Fluorocytes 10 0 1 2 3 4 SPm control TEEW TEUW % transduction TEEW fluorocytes (%) TEUW TEEW SPm contrôle 86 9.9 2.8 Porphyrin Fluorescence 10 0 1 2 3 4 SPm CEP Fluorocytes SPm PEC 73 69.9 17.3

7 Enzymatic Correction UROS enzyme activity (nmol / h / mg) 0 20 40 60 80 100 120 140 TEUW (lenti) SPm control MFG-EGFP or TEEW MFG-US (onco-retro) SPm CEP

8 1.8 kb 5210 3 kb 2.5 kb 1.5 kb 4 kb Copy/cell 5 kb Size marker Not transduced Plasmid SPm control/TEUW SPm CEP/TEUW Calculation of the Proviral Copy Number 1,6 3,9

9 Transgene Expression after erythroid differentiation 0 20 40 60 80 100 EGFP + cells (%) 0 100 200 300 400 UROS Act. (nmol/h/mg) 10 0 1 2 3 4 0 1 2 3 4 SPm control TEEW GPA EGFP SPm control TEEW 10 0 1 2 3 4 0 1 2 3 4 SPm CEP GPA EGFP SPm CEP TEEW TEUW TEEWTEUWTEEWTEUW

10 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

11  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)

12  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

13  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)

14  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

15  U3 HS-40Ank p LTR WPRE cDNA UROS cPPT ESp-UROS LTR Experimental design CEP donors 5-FU BM Sca-1 + Cells 5 days Analyses Enzymatic Metabolic and Phenotypic Corrections CEP recipients 20 wks Busulfan (2 x 25mg/kg) ESp-UROS (MOI 2-200) 36h Experimental protocol Secondary CEP recipients Busulfan (2 x 25mg/kg)

16 Experimental design Control groups: normal BALB/c and CEP mice

17 Enzymatic correction in bone marrow UROS activity (U/mg of proteins) n=5 <0.2 n=4 0 5 10 15 20 25 +/+CEPIIIIIIIV n=4 n=8

18 Metabolic correction in peripheral blood Time (weeks) Fluorocytes ( %) IIIV SSC Fluorocytes 0.1 0 5 10 15 20 25 30 35 40 05101520 +/+ 30.3 CEP III 3.4 III 25.2 IVI 0.1 II ICEP +/+

19 Metabolic correction : porphyrins in urines Total porphyrins (µmol/L) < 0.2 5.8 3.8

20 Correction of hemolytic anemia Reticulocytes (%) Half-life of RBCs Hemoglobin (g/dl)

21 Spleen/body weight (%) Correction of splenomegaly

22 Phenotypic correction CEPI-III +/+IV 50µm

23 Long term expression of the transgene : secondary mice Time (weeks) Fluorocytes ( %)

24 Erythroid-specific expression of the therapeutic gene CEPII +/+ CEP BM Ter119 + Ter119 - BM UROS Activity (U/h/mg of proteins)

25  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

26 EF1pGFP LTR  U3 WPRE cPPT EGFP EF1  Lp CEP-HSC RBCs Granulocytes Platelets Lymphocytes HS-40Ank p LTR  U3 WPRE UROS cPPT ESpUROS- EF1pGFP EGFP EF1  Lp Selective advantage of corrected erythroid cells ?

27 GFP+ WBCs(%) GFP+ RBCs (%) Selective advantage of corrected red blood cells 4 weeks 0 10 20 30 40 50 60 70 80 01020304050607080 y = 0,36x R 2 = 0,86 Control vector EF1pGFP y = 2,29x R 2 = 0,82 Therapeutic vector ESpUROS-EF1pGFP 12 weeks GFP+ WBCs (%) GFP+ RBCs (%)

28 Normal mice EFpGFP Deficient mice ESpUROS-EFpGFP CEP mice Normal mice EFpGFP Deficient mice ESpUROS-EFpGFP CEP mice MNDpGFPEFpGFP ESpUROS-EFpGFP MNDpGFPEFpGFP ESpUROS-EFpGFP MNDpGFPEFpGFP ESpUROS-EFpGFP MNDpGFPEFpGFP ESpUROS-EFpGFP Selective advantage in bone marrow Normal mice CEP mice Ratio of GFP expression between Gr-1 + cells and Ter119 + cells

29 GFP+ RBCs (%) Fluorocytes (%) Level of transduction necessary and efficient 0 5 10 15 20 25 30 35 40 45 010203040506070809010 0 GFP+ RBCs (%) Fluorocytes (%)

30  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

31 INSERM E217, Bordeaux, France Robert-Richard Elodie Cario-Andre Muriel Costet Pierre Ged Cecile Guyonnet-Dupeyrat Véronique Lalanne Magalie Lamrissi-Garcia Isabelle Moreau-Gaudry Francois De Verneuil Hubert Inserm Aknowledgments

32

33 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

34 * CEP patient with GATA1 mutation (Phillips JD et al, 2007) Patients with CEP treated with stem cell transplantation

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