This thesis was undertaken to determine if there is any interrelationship between the two terminal enzymes of the haem biosynthetic pathway, protoporphyrinogen oxidase (PPO) and ferrochelatase, with particular reference to porphyria variegata (PV) and erythropoietic protoporphyria (EPP)• It has previously been found that both enzymes were deficient in PV and EPP, there being a qualitative difference in so far as ferrochelatase deficiency is concerned. / IT2018

Objective To describe off-label utilization of erythropoietic agents in ICU patients.Methods A retrospective, observational study design was used to describe off-label utilization of erythropoietin alfa and darbepoetin alfa by 91,357 patients in the ICU during January 2002- June 2004 at 433 U.S. hospitals. Results Approximately 62% of patients received erythropoietic agents off-label in the ICU compared to 49% in the non-ICU population. Off-label use in the ICU was more likely in teaching hospitals, in larger sized hospitals, in females, for certain physician specialties, and in different regions of the U.S., The longer the hospital stay, the less likely off-label prescribing occurred. ConclusionsOff-label use of erythropoietic agents was common in the ICU. Multiple factors affected off-label use in the ICU although no single one was dominant.

Les porphyries érythropoïétiques (PE) : Porphyrie Erythropoïétique Congénitale -PEC- et Protoporphyrie Erythropoïétique -PPE- sont caractérisées par le déficit d’une des enzymes de la voie de biosynthèse de l’hème. Le traitement curatif des formes sévères de PE est la transplantation de moelle osseuse allogénique (TMOA). La PPE est parfois compliquée d’une insuffisance hépatique majeure nécessitant une greffe hépatique. Dans un modèle murin de PPE (Fechm1Pas/Fechm1Pas), nous avons démontré l’apparition progressive de lésions hépatiques dès la 2ème semaine de vie. Une TMO précoce (nouveau-né) a permis de prévenir l’apparition de ces lésions hépatiques et de corriger la photosensibilité cutanée démontrant l’efficacité de cette approche thérapeutique pour les formes sévères de PPE. La thérapie génique par greffe de cellules souches hématopoïétiques autologues corrigées représente une alternative à la TMOA en l’absence de donneur HLA-compatible. Nous avons développé des cellules souches pluripotentes induites (iPS) à partir de cellules épidermiques issues de modèles murins de PE et d’un patient PEC. La correction génique a été obtenue par transfert du gène lentiviral (ferrochélatase ou uroporphyrinogène III synthase (UROS). La pluripotence des cellules iPS a été caractérisée in vitro par la formation de corps embryoïdes et in vivo par la formation de tératomes. In vitro, la correction métabolique a été obtenue après différenciation des cellules iPS humaines en progéniteurs hématopoïétiques. Enfin dans une dernière partie, nous nous sommes intéressés à une approche pharmacologique de la PEC. Nous avons montré que les mutations C73R et P248Q entraînaient une instabilité et une dégradation accélérée de l’UROS par la voie du protéasome. Le traitement de souris UrosP248Q par un inhibiteur du protéasome (Velcade®) a permis la correction de la photosensibilité cutanée. Ces travaux ouvrent de nouvelles perspectives pour le traitement des porphyries érythropoïétiques. / Erythropoietic porphyrias (EP) : Congenital Erythropoietic Porphyria -CEP- and Erythropoietic Protoporphyria -EPP-) are characterized by a deficit of one enzyme implicated in heme biosynthetic pathway. The curative therapy for severe cases of EP is an HLA-compatible Bone Marrow Transplantation (BMT). EPP is sometimes complicated by a major hepatic failure requiring hepatic graft. In a murine model of EPP (Fechm1Pas/Fechm1Pas), we have demonstrated that hepatic lesions progressively appear 2 weeks after birth. Early BMT (in neonates) has made it possible to prevent hepatic lesions and correct skin photosensitivity, demonstrating the efficiency of this therapeutic approach in severe cases of EPP. The gene therapy by graft of corrected autologous hematopoietic stem cells represents an alternative to BMT when HLA-compatible donors are lacking. We have developed induced pluripotent stem cells (iPSC) from epidermic cells of murine models of EP and of one PEC patient. The gene correction was obtained by lentiviral gene transfer (ferrochelatase and uroporphyrinogen III synthase -UROS). The pluripotency of iPSC was characterized in vitro by the formation of embryoid bodies and in vivo by the formation of teratomas. In vitro, the metabolic correction was obtained after differentiation of human IPSC into hematopoietic progenitors. In the last part of this thesis, we have focused on a pharmacological approach of CEP. We have shown that C73R and P248Q mutations lead to instability and accelerated degradation of the UROS protein via the proteasome. Treating UrosP248Q mice with a proteasome inhibitor (Velcade®) has allowed the correction of skin photosensitivity. These works offer new prospects for the treatment of erythropoietic porphyrias.

La protoporphyrie érythropoïétique (PPE) est une maladie héréditaire rare caractérisée par un déficit en activité FECH responsable d’une accumulation de PPIX. Elle se manifeste par une photosensibilité très invalidante. Il n’existe pas de traitement efficace pour la PPE. 95 % des malades présentent un allèle FECH hypomorphe (c.315-48C) en trans d'une mutation FECH délétère, ce qui entraine une diminution de l'activité FECH résiduelle dans les érythroblastes en dessous d'un seuil critique d'environ 35 % de l'activité normale. L’allèle hypomorphe (c.315-48C) favorise l'utilisation d'un site cryptique d'épissage situé en -63 de l’intron 3 générant un ARNm FECH incluant une partie de l’intron 3 et possédant un codon stop prématuré. L’ARN est alors dégradé par NMD pendant sa maturation. Nous avons déjà identifié un oligonucléotide antisens (ASO-V1) qui redirige l'épissage vers le site accepteur physiologique de l’intron 3 et augmente la production d’ARN FECH WT. Nous avons développé par ce travail une nouvelle stratégie d’adressage d’ASO-V1 en utilisant des peptides ciblant le récepteur de la transferrine (RTf1) qui est exprimé à un niveau très élevé dans les progéniteurs érythroïdes en différenciation concomitamment à la FECH. Nous avons développé des peptides bifonctionnels à partir des séquences peptidiques ciblant le RTf1 tout en les couplant à des séquences Cell Penetrating Peptide (CPP) qui facilitent la sortie de l’ASO-V1 de la vésicule endosomale. Après la transfection des lignées lymphoblastoïdes de malades PPE par différents nanocomplexes RTf1-CPP/ASO-V1, nous avons pu montrer que plusieurs des peptides bifonctionnels utilisés permettaient une redirection efficace et prolongée de l’épissage cryptique vers l’épissage physiologique exon3-exon4 et que cela permettait une correction des taux d’ARN FECH WT. Nous avons ensuite testé l’effet des nanocomplexes RTf1-CPP/ASO-V1, ex vivo, dans les progéniteurs érythroïdes en différenciation de différents sujets atteints de PPE et nous sommes arrivés à augmenter l’ARN FECH WT et diminuer significativement l’accumulation de la PPIX dans ces cellules par rapport à celles transfectées par des nanocomplexes RTf1-CPP/ASO-Mock. La prochaine étape de notre étude serait d’apporter la preuve de concept, in vivo, dans un modèle murin humanisé de PPE après l'administration de nanocomplexes RTf1-CPP/ASOV1 / Erythropoietic protoporphyria (EPP) is a rare hereditary disease characterized by a deficiency in FECH activity responsible for the accumulation of PPIX. EPP is manifested by a very disabling photosensitivity. There is no effective treatment for EPP. 95% of the patients present a hypomorphic FECH allele (c.315-48C) in trans of a deleterious FECH mutation, resulting in a decrease in residual FECH activity in erythroblasts below a critical threshold of about 35% of normal activity. The hypomorphic allele (c.315-48C) promotes the use of a cryptic splicing site located at -63 of the intron 3 generating a FECH mRNA including a part of the intron 3 and possessing a premature stop codon. The RNA is then degraded by NMD during its maturation. We have previously identified an antisense oligonucleotide (ASO-V1) that redirects splicing to the physiological acceptor site of intron 3 and increases the production of WT FECH mRNA. Here, we developed a new ASO-V1 addressing strategy using transferrin receptor (TRf1) targeted peptides. TfR1 is expressed at a very high level in differentiating erythroid progenitors concomitantly with FECH. We developed bifunctional peptides from peptide sequences targeting TfR1 while coupling them to Cell Penetrating Peptide (CPP) sequences that facilitate the release of ASO-V1 from the endosomal vesicle. We transfected the lymphoblastoid cell lines from EPP patients by different TfR1-CPP/ASO-V1 nanocomplexes and we demonstated that several of the bifunctional peptides allowed an efficient and prolonged redirection of the cryptic splicing towards the exon3-exon4 physiological splicing and the correction of the WT FECH mRNA levels. Then, we tested the effect of TfR1-CPP/ASO-V1 nanocomplexes, ex vivo, in differentiating erythroid progenitors of different EPP subjects and we were able to increase WT FECH mRNA and decrease significantly the accumulation of the PPIX in these cells compared to those transfected by TfR1-CPP/ASO-Scr nanocomplexes. The next step of our study would be to provide a proof of concept, in vivo, in a humanized murine model of EPP after the administration of TfR1-CPP/ASOV-1 nanocomplexes

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed that mALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. Generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. The delivery of stable and highly active mALAS2 variants has the potential to expand and improve upon current PDT regimes. Mutations in the C-terminus of human ALAS2 (hALAS2) can increase hALAS2 activity and are associated with X-linked erythropoietic protoporphyria (XLEPP), a disease phenotypically characterized by elevated levels or PPIX and zinc protoporphyrin in erythroblasts. This is apparently due to enhanced cellular hALAS2 activity, but the biochemical relationship between these C-terminal mutations and increased hALAS2 activity is not well understood. HALAS2 and three XLEPP variants were studied both in vitro to compare kinetic and structural parameters and ex vivo in HeLa and K562 cells. Two XLEPP variants, delAGTG, and Q548X, exhibited higher catalytic rates and affinity for succinyl-CoA than wild-type hALAS2, had increased transition temperatures, and caused porphyrin accumulation in HeLa and K562 cells. Another XLEPP mutation, delAT, had an increased transition temperature and caused porphyrin accumulation in mammalian cells, but exhibited a reduced catalytic rate at 37[deg]C in comparison to wild-type hALAS2. The XLEPP variants, unlike wild-type hALAS2, were more structurally responsive upon binding of succinyl-CoA, and adopted distinct features in tertiary and PLP cofactor-binding site. These results imply that the C-terminus of hALAS2 is important for regulating its structural integrity, which affects kinetic activity and stability. XLEPP has only recently been identified as a blood disorder, and thus there are no specific treatments. One potential treatment involves the use of the antibiotic isonicotinic acid hydrazide (isoniazid, INH), commonly used to treat tuberculosis. INH can cause sideroblastic anemia as a side-effect and has traditionally been thought to do so by limiting PLP availability to hALAS2 via direct inhibition of pyridoxal kinase, and reacting with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We postulated that in addition to PLP-dependent inhibition of hALAS2, INH directly acts on hALAS2. Using FACS and confocal microscopy, we show here that INH reduces protoporphyrin IX accumulation in HeLa cells expressing either wild-type human hALAS2 or XLEPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) restored cellular hALAS2 activity in the presence of INH. Kinetic analyses with purified hALAS2 demonstrated non-competitive or uncompetitive inhibition with an apparent Ki of 1.5 uM. Circular dichroism studies revealed that INH triggers structural changes in hALAS2 that interfere with the association of hALAS2 with its PLP cofactor. These studies demonstrate that hALAS2 can be directly inhibited by INH, provide insight into the mechanism of inhibition, and support the prospective use of INH in treating patients with XLEPP and potentially other cutaneous porphyrias.

No description available.

ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms.