Missense UROS mutations causing congenital erythropoietic porphyria reduce UROS homeostasis that can be rescued by proteasome inhibition.

Congenital erythropoietic porphyria (CEP) is an inborn error of heme biosynthesis characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in deleterious porphyrin accumulation in blood cells responsible for hemolytic anemia and cutaneous photosensitivity. We analyzed here the molecular basis of UROS impairment associated with twenty nine UROS missense mutations actually described in CEP patients. Using a computational and biophysical joint approach we predicted that most disease-causing mutations would affect UROS folding and stability. Through the analysis of enhanced green fluorescent protein-tagged versions of UROS enzyme we experimentally confirmed these data and showed that thermodynamic instability and premature protein degradation is a major mechanism accounting for the enzymatic deficiency associated with twenty UROS mutants in human cells. Since the intracellular loss in protein homeostasis is in excellent agreement with the in vitro destabilization, we used molecular dynamic simulation to rely structural 3D modification with UROS disability. We found that destabilizing mutations could be clustered within three types of mechanism according to side chain rearrangements or contact alterations within the pathogenic UROS enzyme so that the severity degree correlated with cellular protein instability. Furthermore, proteasome inhibition using bortezomib, a clinically available drug, significantly enhanced proteostasis of each unstable UROS mutant. Finally, we show evidence that abnormal protein homeostasis is a prevalent mechanism responsible for UROS deficiency and that modulators of UROS proteolysis such as proteasome inhibitors or chemical chaperones may represent an attractive therapeutic option to reduce porphyrin accumulation and prevent skin photosensitivity in CEP patients when the genotype includes a missense variant.

Genetic background influences hepcidin response to iron imbalance in a mouse model of hemolytic anemia (Congenital erythropoietic porphyria).

Clinical severity is heterogeneous among patients suffering from congenital erythropoietic porphyria (CEP) suggesting a modulation of the disease (UROS deficiency) by environmental factors and modifier genes. A KI model of CEP due to a missense mutation of UROS gene present in human has been developed on 3 congenic mouse strains (BALB/c, C57BL/6, and 129/Sv) in order to study the impact of genetic background on disease severity. To detect putative modifiers of disease expression in congenic mice, hematologic data, iron parameters, porphyrin content and tissue samples were collected. Regenerative hemolytic anemia, a consequence of porphyrin excess in RBCs, had various expressions: 129/Sv mice were more hemolytic, BALB/c had more regenerative response to anemia, C57BL/6 were less affected. Iron status and hemolysis level were directly related: C57BL/6 and BALB/c had moderate hemolysis and active erythropoiesis able to reduce iron overload in the liver, while, 129/Sv showed an imbalance between iron release due to hemolysis and erythroid use. The negative control of hepcidin on the ferroportin iron exporter appeared strain specific in the CEP mice models tested. Full repression of hepcidin was observed in BALB/c and 129/Sv mice, favoring parenchymal iron overload in the liver. Unchanged hepcidin levels in C57BL/6 resulted in retention of iron predominantly in reticuloendothelial tissues. These findings open the field for potential therapeutic applications in the human disease, of hepcidin agonists and iron depletion in chronic hemolytic anemia.

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients.

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.

Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones.

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors.

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.

FGFR3 has tumor suppressor properties in cells with epithelial phenotype.

BACKGROUND: Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical. RESULTS: FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors. CONCLUSION: In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.

Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse.

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and ß1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria.

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.

XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.

Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.

In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens.

BACKGROUND: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. RESULTS: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. CONCLUSION: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells.

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut248) mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.

Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients. METHODS: RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose. RESULTS: We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo. CONCLUSIONS: The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation.

The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity.

The JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.

Erythropoietic porphyrias: animal models and update in gene-based therapies.

The inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided into erythropoietic and hepatic according to the predominant porphyrin-accumulating tissue. Three different erythropoietic porphyrias (EP) have been described: erythropoietic protoporphyria (EPP, MIM 177000) the most frequent, congenital erythropoietic porphyria (CEP, MIM 263700), and the very rare hepatoerythropoietic porphyria (HEP, MIM 176100). Bone marrow transplantation is considered as the only curative treatment for severe cases of erythropoietic porphyria (especially CEP), if donors are available. Some EPP patients who undergo liver failure may require hepatic transplantation. Murine models of EPP and CEP have been developed and mimic most of the human disease features. These models allow a better understanding of the pathophysiological mechanisms involved in EP as well as the development of new therapeutic strategies. The restoration of deficient enzymatic activity in the bone marrow compartment following gene therapy has been extensively studied. Murine oncoretroviral, and recently, lentiviral vectors have been successfully used to transduce hematopoietic stem cells, allowing full metabolic and phenotypic correction of both EPP and CEP mice. In CEP, a selective survival advantage of corrected cells was demonstrated in mice, reinforcing the arguments for a gene therapy approach in the human disease. These successful results form the basis for gene therapy clinical trials in severe forms of erythropoietic porphyrias.

Murine retroviral but not human cellular promoters induce in vivo erythroid-specific deregulation that can be partially prevented by insulators.

We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.

[Successful gene therapy of mice with congenital erythropoietic porphyria]. / Succès de la thérapie génique d'un modèle murin de porphyrie érythropoïétique congénitale.

Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut 248) mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs.

Hepcidin: immunoanalytic characteristics.

Hepcidin has progressively become essential in clinical practice for the diagnosis and follow-up of a large spectrum of diseases. Anyway, its own biochemical and structural characteristics have complicated and delayed the acquisition of a standardized quantifying tool of the peptide.