H. de Verneuil

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

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 O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-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.

Gene transfer of the uroporphyrinogen III synthase cDNA into haematopoietic progenitor cells in view of a future gene therapy in congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder characterized by an overproduction and accumulation of porphyrins in bone marrow. This autosomal recessive disease results from a deficiency of uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the haem biosynthetic pathway. It is phenotypically heterogeneous: patients with mild disease have cutaneous involvement, while more severely affected patients are transfusion dependent. The cloning of UROIIIS cDNA and genomic DNA has allowed the molecular characterization of the genetic defect in a number of families. To date, 22 different mutations have been characterized. Allogeneic bone marrow transplantation is the only curative treatment available for the severe, transfusion-dependent, cases. When bone marrow transplantation cannot be performed owing to the absence of a suitable donor, the autografting of genetically modified cells is an appealing alternative. The best approach to somatic gene therapy in this disease involves the use of recombinant retroviral vectors to transduce cells ex vivo, followed by autologous transplantation of the genetically modified cells. We investigated retroviral transfer in deficient human fibroblasts, immortalized lymphoblasts as well as bone marrow cells, and obtained a complete restoration of the enzymatic activity and full metabolic correction.Using K562 cells, an erythroleukaemic cell line, the expression of the transgene remained stable during 3 months and during erythroid differentiation of the cells. Finally, a 1.6- to 1.9-fold increase in enzyme activity compared to the endogenous level was found in normal CD34+ cells, a population of heterogeneous cells known to contain the progenitor/stem cells for long-term expression. The future availability of a mouse model of the disease will permit ex vivo gene therapy experiments on the entire animal.

Prenatal diagnosis in congenital erythropoietic porphyria by metabolic measurement and DNA mutation analysis

Identification of uroporphyrinogen III synthase (UROIIIS) gene mutations in patients with congenital erythropoietic porphyria (CEP) allows fast and reliable carrier detection and prenatal diagnosis. We describe here the first case of prenatal diagnosis by concomitant measurement of uroporphyrin I in amniotic fluid and direct detection of the gene mutation. A French couple, whose first child was diagnosed with CEP, requested prenatal diagnosis at 16 weeks of gestation. Uroporphyrin I was dramatically increased in amniotic fluid and the fetus was homozygous for the C73R mutation, the most common mutation in this disease. The pregnancy was then terminated.

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

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fechm1Pas/Fechm1Pas murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34+-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34+-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP+ cells led to 83–100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in long-term culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

A Prospective Study of Filaggrin Null Mutations in Keratoconus Patients with or without Atopic Disorders

Background: Atopic dermatitis (AD) is significantly associated with keratoconus (KC). An inherited component for KC has been suggested. Filaggrin (FLG) mutations are a strong genetic risk factor for AD. Since filaggrin is also expressed in the corneal epithelium, we hypothesized a common aetiology for ichthyosis vulgaris (IV), AD and KC. Objectives: We examined the prevalence of AD and IV in a KC population. We also studied the expression of filaggrin in normal and KC cornea and analysed 2 prevalent loss-of-function FLG alleles (R501X and 2282del4) in a KC population. Finally we examined whether the population with KC and FLG mutations had specific clinical characteristics. Results: Of 89 KC patients, 38 had current or a history of AD and/or IV. Five patients were carriers of at least 1 FLG mutant allele and had a clinical diagnosis of AD and IV with a severer KC. Conclusion: The low frequency of FLG mutations is surprising since 42.7% of our KC population had AD associated or not with IV; the expected frequency would have been 12–15%, based on our previous studies. Further studies are required to look at other possible FLG mutations or other candidate genes.

Usefulness of a global clinical ichthyosis vulgaris scoring system for predicting common FLG null mutations in an adult caucasian population

Background  Loss of function FLG alleles were first identified as causative of ichthyosis vulgaris (IV) and were subsequently found to be major predisposing factors for atopic dermatitis (AD) and atopic disorders.

Is genotyping useful for the screening of medium-chain acyl-CoA dehydrogenase deficiency in France?

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (McKusick 201450) is the most common enzyme deficiency in mitochondrial fatty acid oxidation. Typical clinical manifestations of the disease are fasting intolerance and acute episodes of hypoglycaemic coma, which can be fatal if the patient is not treated (Roe and Coates 1989). The disease can manifest as sudden unexpected death as well as remain asymptomatic for years (Duran et al 1986). The diagnosis relies on biochemical tests: hypoketotic hypoglycaemia and dicarboxylic aciduria with specific glycine and carnitine conjugates. In asymptomatic cases or between acute episodes, these abnormalities may be observed after a phenylpropionate loading test. The enzyme defect is assessed by measuring MCAD enzyme activity in cultured fibroblasts (Roe and Coates 1989). The mode of inheritance is autosomal recessive and a single base substitution A985G (resulting in the missense mutation K329E) accounts for approximately 90% of the disease-causing alleles in diagnosed patients (Gregersen et al 1993). A simple dietary treatment can prevent acute illness. The aim of this study was the determination of the frequency of G985 mutated allele of MCAD gene in order to evaluate the usefulness of a systematic screening programme based on a genotyping method.

ILDS Newsletter No. 19

After having returned from an outstanding and stimulating world congress in Seoul, it is my pleasure to invite you to read the fi rst ILDS newsletter edited by the newly elected Board. The ILDS Board and I as its President will do our best to stay in touch with you, and to jointly work on many important issues to be tackled and solved during the forthcoming four years. Please see a photo of the new Board as well as a list of all Board members as well as their email addresses below. Please contact me as your President directly, or contact the league through Regional Board members, or simply those who you know best. The new Board of the ILDS has started its work for the ongoing term, and we are aiming to identify the areas that deserve most of our attention. To prepare the ground, the Board will hold a full day strategy retreat in October 2011, prior to the EADV meeting in Lisbon. We will have several committees within the ILDS Board, and the chairpersons will report in one of the forthcoming newsletters on their work in detail. Thus, you can follow and comment our activities. It is the predominant task of the ILDS to organize the World Congress of Dermatology every four years, and therefore the preparations for the next WCD have to be initiated right away. The Program Committee is already assigned and will, under the leadership of Prof Jean Bolognia, work hard to ensure a successful program. However, some framework discussions need to take place at the retreat meeting before we can go ahead. Questions pertinent to the WCD include: what are the unique strengths of a WCD, how can we improve further its attractiveness, should other professions – i.e. dermatological nurses – be involved as well, how can we best balance between the diff erent subspecialties of dermatology, and what is the ideal length of a WCD? Some of you have already come up with suggestions, and we ask you to contact us for criticisms or proposals. As you may know, there many other issues in which the ILDS is involved. In fact, the ILDS entertains strong philanthropic activities, such as the Regional Dermatology Training Center in Moshi, Tanzania. Here, in the Kilimanjaro region, many African dermatologists are trained, and we can be proud of our successful work, which – by the way – is supported by many of our member societies, either independently or via the International Foundation of Dermatology, a subcommittee of the ILDS. As it is often the case, such successful activities are seen by others, who then are keen to support and to help further. In one of the forthcoming issues of the ILDS newsletter, we will report on a new project in the context of the Moshi training center, called Hats on for Skin Health. I am sure you will be surprised and pleased. The ILDS is the league of dermatological societies around the globe, and we believe it is important that the leaders of these societies engage more frequently. Therefore, the ILDS will host receptions for the Presidents and selected offi cers of our member societies, in order to exchange thoughts, concerns and new concepts on a global scale. We will regularly hold such receptions at the annual meetings of AAD and EADV, with other possible locations. Next year, we will organize an ILDS Dermatology Summit, which will take place from June 4–5, in Berlin. Presidents and other representatives of all member societies are being invited to discuss opportunities and challenges in dermatology on a global scale. From these discussions, we hope to initiate programs and activities that will strengthen and further develop our discipline. At our retreat meeting in October this year, we will discuss a draft agenda which will be distributed to all member societies for further input. The scope of dermatology diff ers geographically, and we need to fi nd ways to strengthen areas of our discipline that are not represented globally, but which are of utmost importance where they belong to dermatology. Such fi elds include, among others, venereology (which belongs to dermatology is many parts of the world), allergy, dermatopathology, proctology, leprology, mycology, and even andrology. We will defi nitively involve you, and learn from you, on our way towards a successful future. Personally, I strongly believe that the ILDS, the International League of Dermatological Societies, is the organization to identify and to tackle the problems we are facing in dermatology, and to develop strategies to identify and overcome them. Without any doubt, the ILDS represents an enormous source of knowledge and experience and, together, we will be strong enough to bring dermatology and its subspecialities into an excellent and rewarding position, thus maintaining and improving the care for our patients. Please help us, invest time and support our projects.

Hepatic porphyrin concentration and uroporphyrinogen decarboxylase activity in hepatitis C virus infection: HPC and UROD in HCV infection

Previous studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with porphyria cutanea tarda (PCT). The aim of this study was to assess hepatic porphyrin concentrations (HPC) and hepatic uroporphyrinogen decarboxylase (UROD) activity in HCV‐infected patients free of PCT. Thirty‐two HCV‐infected patients (20 M, 12 F, mean age 51 years) and seven control patients (4 M, 3 F, mean age 59 years) free of liver disease, were studied. Knodell’s score was determined on liver biopsy by two independent anatomopathologists. Measurement of HPC and hepatic UROD activity levels were carried out on liver biopsy. Relative to controls, HCV‐infected patients had high HPC levels (mean ± SD: 47 ± 20 vs. 17 ± 6 pmol/mg protein, P < 0.001) and low hepatic UROD activity levels (514 ± 95 vs. 619 ± 125 pmol Copro/h/mg protein, P < 0.05). HPC was not correlated with hepatic UROD activity and the increase was due to coproporphyrin accumulation. No correlation was observed between HPC or hepatic UROD activity values and HCV‐RNA concentrations, Knodell’s score, hepatic fibrosis, periportal necrosis, periportal inflammation or hepatic iron content in HCV‐infected patients. Hepatocellular necrosis was significantly correlated with HPC value (P < 0.005). Hence, in HCV‐infected patients, HPC is significantly increased and hepatic UROD activity is very slightly decreased as compared to controls. HPC values and UROD activity are not correlated with HCV‐RNA concentrations, hepatic iron content and hepatic fibrosis. The small increase in HPC values in hepatitis C infection is linked with hepatic injury and not with a direct effect on hepatic UROD enzyme.