1 / 34

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.

loan
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related