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INSERM U876, Université Bordeaux II Victor Segalen, Bordeaux, France

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.

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INSERM U876, Université Bordeaux II Victor Segalen, Bordeaux, France

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  1. 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

  2. Experimental Gene therapy in CEP In vitro studies • Tissu source: mPB CD34+ normal and deficient cells • Gene transfer: retroviral and lentiviral vectors Géronimi et al, J Mol Med 2003

  3. Vecteur Trip-EF1a-US (TEU) DU3 RRE U5 cPPT CTS EF-1a R U3 R US SA SD TRIPLEX WPRE WPRE CD34+ SPm: control or CEP Prestimulation 24h 24h Analyses T0 18h Medium: Il-3, TPO, Flt3-L, SCF Gene Transfer into CD34+ Cells with SIN Lentivectors Vectors TEEW or TEUW DU3 RRE EF-1a U5 R U3 R EGFP or UROS SD SA Lentiviral supernatant TEEW or TEUW, MOI 30

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

  5. SPm control SPm CEP 100 100 80 80 80 60 60 60 % EGFP+ cells % de cellules EGFP+ 40 20 40 40 0 6 11 18 25 32 20 20 Time (days) 0 0 Population cellulaire totale Cellules CD34+ CFC LTC-IC Percentage of transduction (TEEW)

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

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

  8. 3,9 1,6 Calculation of the Proviral Copy Number Not transduced SPm CEP/TEUW SPm control/TEUW Plasmid Size marker 5 kb 4 kb 3 kb 2.5 kb 1.8 kb 1.5 kb 5 2 1 0 Copy/cell

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

  10. Conclusions • Efficient gene transfer with lentivectors into • total cells, CFCs and LTC-ICs • Maintenance of the transgene expression after erythroid differentiation Ex vivo gene therapy of a murine animal model

  11. Clinical manifestations • Severe skin photosensitivity • Splenomegaly • Erythrodontia • Redish-coloured urine • Hematologic features • Haemolytic anemia • Fluorescent blood cells Congenital erythropoietic porphyria (CEP) • Inherited disease caused by a deficiency in uroporphyrinogene III synthase (UROS) activity • Accumulation of porphyrins in erythrocytes, bone marrow, spleen, urine and feces.

  12. Murine model of 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 +/+ CEP Ged et al., Genomics 2006

  13. Congenital erythropoietic porphyria (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

  14. Specific aims • 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 ?

  15. Experimental design D U3 ESp-UROS cDNA UROS HS-40 Ank p WPRE LTR LTR cPPT Analyses Experimental protocol Enzymatic Metabolic and Phenotypic Corrections ESp-UROS (MOI 2-200) Busulfan (2x25mg/kg) 5-FU 5 days 36h Busulfan (2x25mg/kg) 20 wks BM Sca-1+ Cells CEP donors CEP recipients Secondary CEP recipients

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

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

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

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

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

  21. Correction of splenomegaly Spleen/body weight (%)

  22. Phenotypic correction +/+ CEP I-III IV 50µm 50µm 50µm 50µm

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

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

  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. Selective advantage of corrected erythroid cells ? CEP-HSC D U3 EF1pGFP EF1aLp WPRE EGFP cPPT LTR D U3 ESpUROS-EF1pGFP HS-40 Ank p WPRE EF1aLp EGFP UROS cPPT LTR RBCs Granulocytes Lymphocytes Platelets

  27. Selective advantage of corrected red blood cells 12 weeks Therapeutic vector ESpUROS-EF1pGFP y = 2,29x GFP+ RBCs (%) R2 = 0,82 Control vector EF1pGFP y = 0,36x R2 = 0,86 GFP+ WBCs (%) 4 weeks 80 70 60 50 40 GFP+ RBCs (%) 30 20 10 0 0 10 20 30 40 50 60 70 80 GFP+ WBCs(%)

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

  29. Level of transduction necessary and efficient 45 40 35 30 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 10 0 Fluorocytes (%) Fluorocytes (%) GFP+ RBCs (%) GFP+ RBCs (%)

  30. Conclusion • 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

  31. Inserm Aknowledgments 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

  32. Congenital Erythropoietic Porphyria Treatment of CEP 1. Symptomatic treatment - sunscreen lotions - b-carotene - oral charcoal - hydroxyurea - splenectomy - repeated transfusions 2. Curative treatment - Stem cell transplantation (compatibility) - Gene therapy in the future ?

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

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