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Successful Therapeutic Effect in a Mouse Model of Erythropoietic Protoporphyria by Partial Genetic Correction and fluorescence-Based Selection of Hematopoietic Cells

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Successful Therapeutic Effect in a Mouse Model of Erythropoietic Protoporphyria by Partial Genetic Correction and fluorescence-Based Selection of Hematopoietic Cells Gene Therapy (2001) 8, 618–626 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells A Fontanellas1,2, M Mendez1, F Mazurier1, M Cario-Andre´1, S Navarro2, C Ged1, L Taine1,3, FGe´ronimi1, E Richard1, F Moreau-Gaudry1, R Enriquez de Salamanca2 and H de Verneuil1 1Laboratoire de Pathologie Mole´culaire et The´rapie Ge´nique, Universite´ Victor Segalen Bordeaux 2, France; 2Centro de Investigacio´n, Hospital 12 de Octubre, Madrid, Spain; and 3Laboratoire de Ge´ne´tique, CHU Pellegrin, Bordeaux, France Erythropoietic protoporphyria is characterized clinically by transplantation, the number of fluorescent erythrocytes skin photosensitivity and biochemically by a ferrochelatase decreased from 61% (EPP mice) to 19% for EPP mice deficiency resulting in an excessive accumulation of photore- engrafted with low fluorescent selected BM cells. Absence active protoporphyrin in erythrocytes, plasma and other of skin photosensitivity was observed in mice with less than organs. The availability of the Fechm1Pas/Fechm1Pas murine 20% of fluorescent RBC. A partial phenotypic correction was model allowed us to test a gene therapy protocol to correct found for animals with 20 to 40% of fluorescent RBC. In con- the porphyric phenotype. Gene therapy was performed by ex clusion, a partial correction of bone marrow cells is sufficient vivo transfer of human ferrochelatase cDNA with a retroviral to reverse the porphyric phenotype and restore normal hem- vector to deficient hematopoietic cells, followed by re-injec- atopoiesis. This selection system represents a rapid and tion of the transduced cells with or without selection in the efficient procedure and an excellent alternative to the use of porphyric mouse. Genetically corrected cells were separated potentially harmful gene markers in retroviral vectors. Gene by FACS from deficient ones by the absence of fluorescence Therapy (2001) 8, 618–626. when illuminated under ultraviolet light. Five months after Keywords: animal model; porphyria; cutaneous photosensitivity; genetic disease; bone marrow; gene transfer; retrovirus Introduction ure, which necessitates liver transplantation.1,2,4–7 The therapy of EPP is often limited to supportive care and is Erythropoietic protoporphyria (EPP) is a hereditary dis- only partially successful, especially in the severe forms. order caused by a decrease in ferrochelatase activity This monogenic disorder starts early in infancy and rep- (E.C.4.99.1.1). The enzyme is associated with the inner resents a good candidate for gene therapy in severe forms mitochondrial membrane and catalyzes the insertion of 1 since the genetic defect is well characterized at the mol- iron into protoporphyrin to form heme. This defect ecular level. Generally, EPP is an autosomal dominant results in a high accumulation of protoporphyrin in disease, but in some cases it is transmitted in a recessive erythrocytes, plasma, feces and other tissues such as the fashion.8–10 Following the cloning of the human ferrochel- liver and the skin.1,2 Clinically, the disease is charac- atase cDNA, several mutations have been identified terized by cutaneous photosensitivity due to the photore- showing a high molecular heterogeneity.8–13 active properties of protoporphyrin. Light between 360 and 450 nm initiates a chain of reactions in which proto- The pathophysiology of the human disease is still unclear, particularly with respect to the development of porphyrin acts as a photochemical radical. This photosen- 14 sitizing property induces an acute inflamation of the skin, liver failure. Acute hepatic failure is due to the accumu- characterized by burning pain, oedema and often pur- lation of protoporphyrin in the liver. Because the proto- pura.3 Occasionally, a mild microcytic, hypochromic ane- porphyrin mostly originates from the erythroid cells, mia may be observed. Signs of hepatic damage have also bone marrow (BM) transplantation can be a convenient been described, and can vary in severity from elevated therapy for severe cases of EPP. An EPP patient who serum transaminase activity to life-threatening liver fail- underwent allogeneic BM transplantation for leukemia had complete hematologic remission.15 A murine model of EPP, the Fechm1Pas/Fechm1Pas mouse16,17 was used for cell18,19 and gene therapy.18 The replacement of the major Correspondence: Fontanellas, Centro de Ingestigacio´n, Hospital part of the deficient BM cells by normal ones was able Universitario 12 de Octubre, Avenida de Cordoba, Km 5.400, 28041, to reduce protoporphyrin accumulation in erythrocytes. Madrid, Spain Received 27 October 2000; accepted 15 January 2001 Also, gene therapy to the bone marrow in the same Absence of porphyria phenotype following partial gene correction A Fontanellas et al 619 model can reverse the protoporphyrin accumulation as well as the photosensitization associated with the por- phyria.18 Interestingly, BM transplantation performed in very young animals (3–4 weeks old) can prevent hepato- biliary complications as well as hepatocyte alterations and partially revert protoporphyrin accumulation in the liver.19 However, allogeneic BM transplantation is limited by the need for an HLA-matched donor. In the absence of a suitable donor, autografting of genetically modified hem- atological cells seems to be an appealing alternative. An important step in view of future gene therapy in humans is developing efficient selection procedures to increase the frequency of genetically corrected cells before auto- Figure 1 (a) Identification of LFSN proviruses by Southern blot analysis logous transplantation. One approach relies on the in FDCP1 transduced and selected cells. Genomic DNA was prepared expression of a cotransfected marker gene. In the recent from normal, untransduced and transduced G418 selected cells. The DNA work of Pawliuk et al,18 a high proportion of transduced (10 ␮g per lane) was digested with SacI. This enzyme cuts in both long BM cells carrying the human ferrochelatase cDNA were terminal repeat (LTR) regions in our construction and produces a 3.8 kb selected ex vivo on the basis of the co-expression of the from the LFSN vector. DNA was separated on a 1% agarose gel, trans- + green fluorescent protein (EGFP). The authors demon- ferred to HybondN membrane (Amersham), and hybridized with a ferro- chelatase cDNA 32P-radiolabeled probe. 2.5, 5, 25, 50 and 125 pg of strated a complete and long-term correction of photosen- pLFSN were used with 10 ␮g of normal DNA corresponding to 0.25, 0.5, sitivity in this murine EPP model. However, the 2.5, 5 and 12.5 copies per cell of the transgene. The 1.4 kb band corre- approach using vectors encoding gene markers is not sponds to the mouse endogenous ferrochelatase gene which cross- acceptable for use in humans. The aim of our study was hybridized with the human cDNA probe. (b) Ferrochelatase expression in to develop a method of preselecting genetically corrected FDCP1 cells transduced with viral particles from Gp+env86/LXSN or + cells before transfusion using a method based on the Gp env86/LFSN17 clones and selected by G418. expression of the therapeutic gene, rather than on the expression of a potentially toxic marker gene. observed after 24 h of ALA addition (data not shown). Flow cytometry analysis of normal and deficient BM cells is shown in Figure 2a and b, respectively. An analysis Results was also performed with transduced deficient BM cells (Figure 2c) where two distinct peaks appeared. We sorted In vitro transduction of interleukin-3-dependent 35% of the cells showing the lowest fluorescence (Figure hematopoietic precursor cell line FDCP1 2d, open population, denoted LFSN-LowF) and 30% of The Gp+env86/LFSN17 clone was selected for sub- the cells exhibiting the highest fluorescence (Figure 2d, sequent experiments because of its high retroviral pro- closed population, denoted LFSN-HighF). Each of these duction (1.3 × 106 c.f.u.). Supernatants from this clone and two sorted cell populations was then grafted into four the control Gp+env86/LXSN clone were used to trans- deficient recipient mice. duce the FDCP1 cells in Retronectin-coated wells. Two days after the infection, cells were selected in the pres- Chimerism of transplanted animals ence of 1 mg/ml G418 for 2 weeks. A Southern blot The chimerism was similar in the five groups of mice 20 analysis showed that transduced and selected FDCP1 weeks after transplantation: normal mice grafted with cells carried two to three copies of the vector per cell normal BM cells (group 1) 97% (95–99%); deficient mice (Figure 1a). Ferrochelatase activity was 5.4-fold higher in grafted with deficient BM cells (group 2) 98% (95–100%); these cells compared with the LXSN-transduced or non- deficient mice grafted with transduced and nonselected transduced FDCP1 cells (Figure 1b). This clone was there- BM cells (group 3) 99.8% (98–100%); deficient mice fore selected for the transduction of BM cells from EPP grafted with transduced low-fluorescent BM sorted cells mice. (group 4) 98% (95–100%); and deficient mice grafted with transduced high-fluorescent BM sorted cells (group 5) Transduction of BM cells, fluorescence-based selection 98.8% (98–99.5%). and reimplantation into mice Two transduction experiments were performed. The first Hematological, enzymatic and metabolic correction of experiment showed that 25% of colony-forming cells the engrafted mice (CFC) were resistant to G418; 3.5 × 105 of these cells were Hematological parameters (Table 1) observed in mice injected directly into two different EPP mice. The second grafted with transduced and nonselected BM cells (group experiment gave rise to a 33% transduction in CFC. 3) were close to those of EPP mice (group 2). By contrast, Again, 3.5 × 105 of the cells were injected into two mice red blood cell (RBC) count, hematocrit and hemoglobin to complete the group of EPP animals grafted with non- concentration were normalized in mice grafted with selected transduced BM cells.
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