Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells

Fontanellas, Antonio; Méndez, Manuel; Mazurier, Frédéric; Cario‐André, Muriel; Navarro, Susana; Ged, Cécile; Taine, Laurence; Geronimi, Fabien; Richard, Élodie; Moreau‐Gaudry, François; Salamanca, Rafael Enrı́quez de; Verneuil, Hubert de

doi:10.1038/sj.gt.3301427

Cited by 30 publications

(17 citation statements)

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“…BG/BCNU injection was able to cure the pathologic phenotype, mice were subjected to a photosensitivity assay at week 20 after BMT as described. 30,32 Normal mice did not demonstrate macroscopic nor microscopic skin lesions 4 days after UV exposure, whereas severe macroscopic lesions and immune cell infiltrates were observed in nontransplanted EPP mice. In the untreated nonmyeloablated group, severe macroscopic and microscopic skin lesions were still observed.…”

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confidence: 99%

“…28 Gene therapy of the EPP mouse model with retroviral vectors was performed by us and others, but needed preselection of transduced cells for transplantation to be efficient. 30,31 We then demonstrated that preselection can be avoided by using SIN-erythroidspecific lentiviral vectors, but a lethal preconditioning regimen of recipient mice was still required to obtain efficient engraftment, avoiding dilution with endogenous deficient HSCs. 32 We have recently demonstrated that the coexpression of the selective G156A-MGMT and the therapeutic FECH genes under the control of EF1-a promoter in the context of a bicistronic SIN-lentiviral construct can selectively protect and correct human EPP lymphoblastoid cell lines in vitro after O 6 BG and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) administration.…”

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Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

et al. 2004

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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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Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

et al. 2004

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“…29 Bone marrow transplants and gene therapies using recombinant viruses to express therapeutic levels of ferrochelatase in erythroid-lineage cells have been successfully accomplished in a mouse model of recessive protoporphyria. [30][31][32] Mutations affecting ferrochelatase activity are heterogeneous. Missense and nonsense mutations have been reported in several protoporphyric patients 33,36,37,[39][40][41][42][43] ; however, more common are splice-donor and acceptor-site mutations, which lead to exon Reprints: David A. Brenner, University of North Carolina at Chapel Hill, CB#7038, Department of Medicine, Chapel Hill, NC 27599; e-mail: dab@med.unc.edu.…”

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An exon 10 deletion in the mouse ferrochelatase gene has a dominant-negative effect and causes mild protoporphyria

Magness

Maeda

Brenner

2002

Blood

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Protoporphyria is generally inherited as an autosomal dominant disorder. The enzymatic defect of protoporphyria is a deficiency in ferrochelatase, which chelates iron and protoporphyrin IX to form heme. Patients with protoporphyria have decreased ferrochelatase activities that range from 5% to 30% of normal caused by heterogeneous mutations in the ferrochelatase gene. The molecular mechanism by which the ferrochelatase activity is decreased to less than an expected 50% is unresolved. In this study, we assessed the effect of a ferrochelatase exon 10 deletion, a common mutation in human protoporphyria, introduced into the mouse by gene targeting. F1 crosses produced (؉/؉), (؉/؊), and (؊/؊) mice at a ratio of 1:2:0; (؊/؊) embryos were detected at 3.5 days postcoitus, consistent with embryonic lethality for the homozygous mutant genotype. Heterozygotes demonstrated equivalent levels of wildtype and mutant ferrochelatase messenger RNAs and 2 immunoreactive proteins that corresponded to the full-length and an exon 10-deleted ferrochelatase protein. Ferrochelatase activities in the heterozygotes were an average of 37% of normal, and protoporphyrin levels were elevated in erythrocytes and bile. Heterozygous mice exhibited skin photosensitivity but no liver disease. These results lend support for a dominant-negative effect of a mutant allele on ferrochelatase activity in patients with protoporphyria. IntroductionFerrochelatase catalyzes the formation of heme by chelating ferrous iron to protoporphyrin IX. High ferrochelatase activity is required during erythropoiesis to provide heme for hemoglobin, but low constitutive activity is required in all cell types to provide heme for cytochromes and other essential heme-containing proteins. The ferrochelatase gene is located on chromosome 18 and consists of 11 exons spanning about 40 to 50 kilobases (kb) in both mice and humans. [1][2][3][4] Mammalian ferrochelatase is a metalloenzyme that possess a 2-iron/2-sulfur cluster, a structural component of the protein that is located near the carboxyl terminus and is required for enzymatic activity. 5 The ferrochelatase protein is translated with a mitochondrial targeting peptide, which is cleaved off after translocation of the enzyme to its active site in the inner mitochondrial membrane. 6,7 The mammalian enzyme is active in situ as an 80-to 82-kd homodimer as suggested by radiation inactivation studies 8 and confirmed by x-ray crystallography. 9,10 Protoporphyria is a disease caused by a deficiency in ferrochelatase activity. 11 The principal clinical manifestation of protoporphyria is severe photosensitivity and in rare cases hepatobiliary disease, which can advance to hepatic failure and require liver transplantation. 12 The symptoms of protoporphyria are due to the partial enzymatic block, which results in excess protoporphyrin that accumulates in tissues, serum, and bile. 13,14 Approximately 80% of the excess protoporphyrin originates from developing erythroid cells and 20% from the liver. 15 Cutaneous photosensitivity occurs ...

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“…Les résultats d'une thé-rapie génique efficace dans un nouveau modèle murin de cette porphyrie sont exposés dans cet article. < modèle murin de protoporphyrie érythropoïétique ou PPE, maladie différente de la PEC [10][11][12]. Le vecteur lentiviral thérapeutique utilisé, dénommé Esp-UROS, est un vecteur dit « SIN » (self inactivation) ou autoinactivé, possédant un promoteur spécifiquement activé dans la lignée érythroïde (HS40/Ankyrine), la séquence ADNc UROS humaine, ainsi que l'élément de régulation post-transcriptionnel WPRE.…”

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“…Cet avantage sélectif des précurseurs des globules rouges corrigés constitue un argument important pour espérer un bénéfice théra-peutique chez l'homme, même si la totalité des cellules souches déficientes n'a pas été corrigée. Ces résultats sont différents de ceux qui sont obtenus dans la protoporphyrie érythropoïétique : il n'y a pas d'anémie hémolytique dans cette maladie, et l'avantage sélectif des progéniteurs érythroïdes corrigés n'y existe pas [10][11][12].…”

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Succès de la thérapie génique d’un modèle murin de porphyrie érythropoïétique congénitale

et al. 2008

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Une maladie sévère traitée par allogreffe de moelle osseuseParmi les porphyries héréditaires, la porphyrie érythro-poïétique congénitale (PEC) ou maladie de Günther, est la plus sévère [1]. C'est une maladie génétique rare, de transmission autosomique récessive, caractérisée par un déficit en uroporphyrinogène III synthase (UROS), la quatrième enzyme de la biosynthèse de l'hème [1][2][3]. L'augmentation très importante de synthèse des porphyrines dans la moelle osseuse du fait du déficit enzymatique entraîne leur accumulation dans le sang et les tissus, expliquant la photosensibilité cutanée caractéristique chez les malades. Les patients pré-sentent une excrétion massive de porphyrines dans les urines (majoritairement l'uroporphyrine I) et les selles. L'anémie hémolytique est due à la fragilité globulaire produite par l'accumulation de porphyrines dans ces cellules. Le traitement symptomatique de la maladie est déce-vant : utilisation de crèmes antisolaires et réduction de l'exposition aux rayonnements UV, prise de β-carotène qui peut limiter la photosensibilité, prise d'hydroxyurée qui diminue l'érythropoïèse et donc l'hyperhémolyse, réduisant ainsi les besoins transfusionnels. La splé-nectomie a une efficacité très variable sur le processus hémolytique et son efficacité à long terme est très discutée. La sévérité de la maladie chez un grand nombre de patients (photosensibilité extrême avec évolution sclérodermiforme et mutilante du visage et des mains, anémie hémolytique nécessitant des transfusions répétées) conduit à proposer une greffe de moelle osseuse dans l'enfance, à condition de disposer d'un donneur HLA compatible. Treize cas de transplantation médullaire ont été publiés chez des enfants ou adolescents de 18 mois à 15 ans [3,[4][5][6][7], avec un succès thérapeutique majeur, mais un risque non négli-geable en terme de mortalité (deux décès par compli-> Les porphyries héréditaires représentent un ensemble de maladies métaboliques caractéri-sées par une synthèse, une accumulation et une excrétion accrues de porphyrines et/ou de leurs précurseurs, l'acide delta aminolévulinique et le porphobilinogène. Chacune de ces maladies a pu être reliée à un déficit spécifique d'une des enzymes de la biosynthèse de l'hème, et nous avons précédemment publié dans Médecine/Sciences les progrès effectués dans la connaissance des gènes, la pathologie moléculaire des porphyries ainsi que des modèles animaux indispensables pour des études physiopathologiques et thérapeuti-ques. Parmi les porphyries érythropoïétiques, la porphyrie érythropoïétique congénitale (PEC), ou maladie de Günther, la plus sévère des porphyries, est une maladie génétique caractérisée par un déficit en uroporphyrinogène III synthase (UROS). Elle est actuellement traitée par greffe de moelle osseuse allogénique dans les formes graves ; elle pourrait bénéficier dans le futur d'une thérapie génique ciblée sur les cellules souches/progéni-trices hématopoïétiques. Les résultats d'une thé-rapie génique efficace dans un nouveau modèle murin de cette porphyri...

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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

Cited by 30 publications

References 33 publications

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

An exon 10 deletion in the mouse ferrochelatase gene has a dominant-negative effect and causes mild protoporphyria

Succès de la thérapie génique d’un modèle murin de porphyrie érythropoïétique congénitale

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