Congenital erythropoietic porphyria (CEP) is a recessive autosomal disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. The severity of the disease, the lack of specific treatment except for allogeneic bone marrow transplantation, and the knowledge of the molecular lesions are strong arguments for gene therapy. An animal model of CEP has been designed to evaluate the feasibility of retroviral gene transfer in hematopoietic stem cells. We have previously demonstrated that the knockout of the Uros gene is lethal in mice (Uros(del) model). This work describes the achievement of a knock-in model, which reproduces a mutation of the UROS gene responsible for a severe UROS deficiency in humans (P248Q missense mutant). Homozygous mice display erythrodontia, moderate photosensitivity, hepatosplenomegaly, and hemolytic anemia. Uroporphyrin (99% type I isomer) accumulates in urine. Total porphyrins are increased in erythrocytes and feces, while Uros enzymatic activity is below 1% of the normal level in the different tissues analyzed. These pathological findings closely mimic the CEP disease in humans and demonstrate that the Uros(mut248) mouse represents a suitable model of the human disease for pathophysiological, pharmaceutical, and therapeutic purposes.
Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fech m1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fech m1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2-to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fech m1Pas was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIXliver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow. IntroductionErythropoietic protoporphyria (EPP; Mendelian Inheritance in Man [MIM] 177000) is an inherited disorder caused by partial deficiency of ferrochelatase (FECH; EC 4.99.1.1.), the last enzyme of the heme biosynthetic pathway. 1 FECH is an inner membrane mitochondrial enzyme catalyzing the insertion of ferrous iron into protoporphyrin IX (PPIX) to form heme. FECH deficiency in bone marrow erythroid cells is responsible for the primary overproduction of PPIX, leading to an accumulation of protoporphyrin in the bone marrow, plasma, erythrocytes, skin, bile, and feces. 2 Because of its hydrophobic nature, PPIX can be removed from the body only through the liver, where it is secreted into bile and then is excreted by fecal elimination. 3 More than100 mutations in the FECH gene, including missense, nonsense, splicing, deletions, and insertions, have been identified in EPP families (Human Gene Mutation Database, http:// archive.uwcm.ac.uk/uwcm/mg/hgmd0.html). Usually, EPP is inherited as an autosomal pseudodominant disorder, and clinical penetrance is mainly modulated by the presence of a common intronic single-nucleotide polymorphism (SNP), IVS3-48C, in trans to a dominant mutation. 4,5 The most common clinical manifestation of EPP is lifelong acute photosensitivity of sun-exposed skin appearing in early childhood. 6 Although EPP is generally a benign disease, hepatic complications such as cholelithiasis or, in rare cases (approximately 2%), rapid fatal liver disease with cirrhosis may occur. [7][8][9] Chronic liver disease i...
Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O 6 -methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O 6 -benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.
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