Publications by authors named "Matthias Titeux"

30 Publications

  • Page 1 of 1

Emerging drugs for the treatment of epidermolysis bullosa.

Expert Opin Emerg Drugs 2020 12 14;25(4):467-489. Epub 2020 Dec 14.

Imagine Institute, Laboratory of Genetic Skin Diseases, INSERM UMR 1163, Université de Paris , Paris, France.

Introduction: Epidermolysis Bullosa (EB) form a heterogeneous group of rare, sometimes life-threatening inherited skin diseases characterized by skin and mucosal blistering after mild trauma from birth. They display a wide range of disease severity, with multiple local and systemic complications with no satisfactory treatment.

Areas Covered: Approaches aiming to restore the functional expression of the defective protein such as and gene therapy, cell therapies, protein replacement and pharmacological approaches have shown promising results. In addition, improved knowledge of EB pathogenesis has open the way to symptom-relief therapies using repurposed drugs in some forms of EB.

Expert Opinion: A cure for all forms of EB will remain challenging, but it is anticipated that treatments for EB will rely on precision medicine, involving a combination of complementary approaches. Treatments aiming to restore the function of the defective genes will be combined with symptom-relief therapies to address the specific features of the different forms of EB and each patient complications. A growing number of biotech and pharmaceutical companies have shown an increasing interest in the treatment of EB and as a result, have implemented numerous clinical trials. Therefore, we anticipate the emergence of effective treatments in the near future.
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http://dx.doi.org/10.1080/14728214.2020.1839049DOI Listing
December 2020

Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa.

JCI Insight 2019 06 6;4(11). Epub 2019 Jun 6.

St John's Institute of Dermatology, School of Basic and Medical Biosciences, King's College London, London, United Kingdom.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.
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http://dx.doi.org/10.1172/jci.insight.126243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629162PMC
June 2019

Epidermolysis bullosa simplex-generalized severe type due to keratin 5 p.Glu477Lys mutation: Genotype-phenotype correlation and in silico modeling analysis.

Pediatr Dermatol 2019 Jan 4;36(1):132-138. Epub 2018 Dec 4.

Epidermolysis Bullosa Center, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Background/objectives: Epidermolysis bullosa is a group of diseases caused by mutations in skin structural proteins. Availability of genetic sequencing makes identification of causative mutations easier, and genotype-phenotype description and correlation are important. We describe six patients with a keratin 5 mutation resulting in a glutamic acid to lysine substitution at position 477 (p.Glu477Lys) who have a distinctive, severe and sometimes fatal phenotype. We also perform in silico modeling to show protein structural changes resulting in instability.

Methods: In this case series, we collected clinical data from six patients with this mutation identified from their national or local epidermolysis bullosa databases. We performed in silico modeling of the keratin 5-keratin 14 coil 2B complex using CCBuilder and rendered with Pymol (Schrodinger, LLC, New York, NY).

Results: Features include aplasia cutis congenita, generalized blistering, palmoplantar keratoderma, onychodystrophy, airway and developmental abnormalities, and a distinctive reticulated skin pattern. Our in silico model of the keratin 5 p.Glu477Lys mutation predicts conformational change and modification of the surface charge of the keratin heterodimer, severely impairing filament stability.

Conclusions: Early recognition of the features of this genotype will improve care. In silico analysis of mutated keratin structures provides useful insights into structural instability.
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http://dx.doi.org/10.1111/pde.13722DOI Listing
January 2019

Correction to: Antisense-Mediated Splice Modulation to Reframe Transcripts.

Methods Mol Biol 2018 ;1828:E1

INSERM UMR 1163, Paris, France.

The original version of this book was published with the following errors: "2'MOE" have been corrected into "2'MOEPS" in figure.6 - Chapter 35, multiple typo errors in page numbers: 532, 533, 534, 537, 542, 548 and 549. These errors has been updated.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_37DOI Listing
January 2018

Antisense-Mediated Splice Modulation to Reframe Transcripts.

Methods Mol Biol 2018 ;1828:531-552

INSERM UMR 1163, Paris, France.

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe skin genetic disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon skipping strategy to reframe transcripts.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_35DOI Listing
April 2019

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa.

Sci Transl Med 2018 08;10(455)

Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.
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http://dx.doi.org/10.1126/scitranslmed.aas9668DOI Listing
August 2018

A recessive form of hyper-IgE syndrome by disruption of ZNF341-dependent STAT3 transcription and activity.

Sci Immunol 2018 06;3(24)

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, 75015 Paris, France.

Heterozygosity for human () dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with DN mutations, ZNF341-deficient patients lack T helper 17 (T17) cells, have an excess of T2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the transcription-dependent autoinduction and sustained activity of STAT3.
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http://dx.doi.org/10.1126/sciimmunol.aat4956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141026PMC
June 2018

Intradermal Injection of Bone Marrow Mesenchymal Stromal Cells Corrects Recessive Dystrophic Epidermolysis Bullosa in a Xenograft Model.

J Invest Dermatol 2018 11 12;138(11):2483-2486. Epub 2018 Jul 12.

Laboratory of Genetic Skin Diseases, INSERM UMR 1163 and Imagine Institute of Genetic Diseases, Paris, France; Paris Descartes University-Sorbonne Paris Cite, Paris, France; Department of Genetics, Necker Hospital for Sick Children, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2018.04.028DOI Listing
November 2018

The Molecular Revolution in Cutaneous Biology: Emerging Landscape in Genomic Dermatology: New Mechanistic Ideas, Gene Editing, and Therapeutic Breakthroughs.

J Invest Dermatol 2017 05;137(5):e123-e129

INSERM UMR 1163, Paris, France; Imagine Institute, Paris, France; University Paris Descartes Sorbonne Cité, Paris, France; Department of Genetics, Necker Hospital for Sick Children, Paris, France. Electronic address:

Stunning technological advances in genomics have led to spectacular breakthroughs in the understanding of the underlying defects, biological pathways and therapeutic targets of skin diseases leading to new therapeutic interventions. Next-generation sequencing has revolutionized the identification of disease-causing genes and has a profound impact in deciphering gene and protein signatures in rare and frequent skin diseases. Gene addition strategies have shown efficacy in junctional EB and in recessive dystrophic EB (RDEB). TALENs and Cripsr/Cas9 have emerged as highly efficient new tools to edit genomic sequences to creat new models and to correct or disrupt mutated genes to treat human diseases. Therapeutic approaches have not been limited to DNA modification and strategies at the mRNA, protein and cellular levels have also emerged, some of which have already proven clinical efficacy in RDEB. Improved understanding of the pathogenesis of skin disorders has led to the development of specific drugs or repurposing of existing medicines as in basal cell nevus syndrome, alopecia areata, melanoma and EB simplex. These discoveries pave the way for improved targeted personalized medicine for rare and frequent diseases. It is likely that a growing number of orphan skin diseases will benefit from combinatory new therapies in a near future.
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http://dx.doi.org/10.1016/j.jid.2016.08.038DOI Listing
May 2017

Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model.

J Invest Dermatol 2016 12 3;136(12):2387-2395. Epub 2016 Aug 3.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1163, Paris, France; Imagine Institute, Paris, France; Paris Descartes University, Sorbonne Cité, Paris, France; Department of Genetics, Necker Hospital for Sick Children, Paris, France. Electronic address:

Dystrophic epidermolysis bullosa is a group of orphan genetic skin diseases dominantly or recessively inherited, caused by mutations in COL7A1 encoding type VII collagen, which forms anchoring fibrils. Individuals with recessive dystrophic epidermolysis bullosa develop severe skin and mucosal blistering after mild trauma. The exon skipping strategy consists of modulating splicing of a pre-mRNA to induce skipping of a mutated exon. We have targeted COL7A1 exons 73 and 80, which carry recurrent mutations and whose excision preserves the open reading frame. We first showed the dispensability of these exons for type VII collagen function in vivo. We then showed that transfection of primary recessive dystrophic epidermolysis bullosa keratinocytes and fibroblasts carrying null mutations in exon 73 and/or 80, with 2'-O-methyl antisense oligoribonucleotides, led to efficient ex vivo skipping of these exons (50-95%) and resulted in a significant level (up to 36%) of type VII collagen re-expression. Finally, one or two subcutaneous injections of antisense oligoribonucleotides at doses ranging from 400 μg up to 1 mg restored type VII collagen expression and anchoring fibril formation in vivo in a xenograft model of recessive dystrophic epidermolysis bullosa skin equivalent. This work provides a proof of principle for the treatment of patients with recessive dystrophic epidermolysis bullosa by exon skipping using subcutaneous administration of antisense oligoribonucleotides.
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http://dx.doi.org/10.1016/j.jid.2016.07.029DOI Listing
December 2016

Gene-Corrected Fibroblast Therapy for Recessive Dystrophic Epidermolysis Bullosa using a Self-Inactivating COL7A1 Retroviral Vector.

J Invest Dermatol 2016 07 16;136(7):1346-1354. Epub 2016 Mar 16.

Laboratory of Genetic Skin Diseases, Inserm UMR1163 and Imagine Institute of Genetic Diseases, Paris, France; University Paris Descartes-Sorbonne Paris Cite, Paris, France; Department of Genetics, Necker Hospital, Paris, France. Electronic address:

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack type VII collagen and therefore have severely impaired dermal-epidermal stability causing recurrent skin and mucosal blistering. There is currently no specific approved treatment for RDEB. We present preclinical data showing that intradermal injections of genetically corrected patient-derived RDEB fibroblasts using a Good Manufacturing Practices grade self-inactivating COL7A1 retroviral vector reverse the disease phenotype in a xenograft model in nude mice. We obtained 50% transduction efficiency in primary human RDEB fibroblasts with an average low copy number (range = 1-2) of integrated provirus. Transduced fibroblasts showed strong type VII collagen re-expression, improved adhesion properties, normal proliferative capabilities, and viability in vitro. We show that a single intradermal injection of 3 × 10(6) genetically corrected RDEB fibroblasts beneath RDEB skin equivalents grafted onto mice allows type VII collagen deposition, anchoring fibril formation at the dermal-epidermal junction, and improved dermal-epidermal adherence 2 months after treatment, supporting functional correction in vivo. Gene-corrected fibroblasts previously showed no tumorigenicity. These data show the efficacy and safety of gene-corrected fibroblast therapy using a self-inactivating vector that has now been good manufacturing grade-certified and pave the way for clinical translation to treat nonhealing wounds in RDEB patients.
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http://dx.doi.org/10.1016/j.jid.2016.02.811DOI Listing
July 2016

Comparison of 3 type VII collagen (C7) assays for serologic diagnosis of epidermolysis bullosa acquisita (EBA).

J Am Acad Dermatol 2016 Jun 3;74(6):1166-72. Epub 2016 Mar 3.

Department of Dermatology, APHP, Avicenne Hospital, Bobigny, France; Department of Histology, Unité de Formation et de Recherche (UFR) Léonard de Vinci, University Paris 13, Bobigny, France; Department of Pathology, APHP, Avicenne Hospital, Bobigny, France. Electronic address:

Background: Serologic diagnosis of epidermolysis bullosa acquisita (EBA) relies on the detection of circulating autoantibodies to type VII collagen (C7).

Objective: We sought to compare the diagnostic performances of a commercialized enzyme-linked immunosorbent assay (ELISA) using C7 noncollagenous (NC) domains (C7-NC1/NC2 ELISA) and indirect immunofluorescence (IIF) biochip test on NC1-C7-expressing transfected cells (IIFT), with a full-length-C7 ELISA developed in our laboratory.

Methods: C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were run on 77 nonselected consecutive EBA sera.

Results: C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were positive, respectively, for: 30%, 27%, and 65% of the 77 sera; 43%, 32%, and 80% of 44 sera labeling the salt-split-skin (SSS) floor (F) by IIF (SSS/F(+)); 9%, 22%, and 47% of 32 SSS/F(-) sera; 28%, 28%, and 58% of classic EBA; 41%, 41%, and 82% of inflammatory EBA; and 18%, 0%, and 55% of mucous-membrane-predominant EBA. Significant differences for all sera were found between: the 2 ELISAs for the 77 sera, SSS/F(+) and SSS/F(-) sera, and IIFT versus full-length-C7 ELISA.

Limitations: The retrospective design was a limitation.

Conclusion: C7-NC1/NC2 ELISA and IIFT sensitivities for serologic diagnoses of EBA were low. Full-length-C7 ELISA was significantly more sensitive and could serve as a reference test.
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http://dx.doi.org/10.1016/j.jaad.2016.01.005DOI Listing
June 2016

HEK293-based production platform for γ-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA.

Hum Gene Ther Clin Dev 2014 Dec;25(4):218-28

1 EUFETS GmbH , 55743 Idar-Oberstein, Germany .

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.
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http://dx.doi.org/10.1089/humc.2014.083DOI Listing
December 2014

Antisense-mediated exon skipping to reframe transcripts.

Methods Mol Biol 2012 ;867:221-38

INSERM, U781, Paris, France.

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.
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http://dx.doi.org/10.1007/978-1-61779-767-5_15DOI Listing
July 2012

Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa.

J Invest Dermatol 2011 Apr 2;131(4):848-56. Epub 2010 Dec 2.

Division of Hematology-Oncology, Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.
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http://dx.doi.org/10.1038/jid.2010.346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151825PMC
April 2011

SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa.

Mol Ther 2010 Aug 18;18(8):1509-18. Epub 2010 May 18.

Inserm, U563, Toulouse, France.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.
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http://dx.doi.org/10.1038/mt.2010.91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927071PMC
August 2010

Gene therapy for recessive dystrophic epidermolysis bullosa.

Dermatol Clin 2010 Apr;28(2):361-6, xii

Institut National de la santé et de la recherche médicale, U563, Toulouse France.

Among the severe genetic disorders of the skin that are suitable for gene and cell therapy, most efforts have been made in the treatment of blistering diseases including dystrophic epidermolysis bullosa. This condition can be recessively or dominantly inherited, depending on the nature and position of the mutation or mutations in the gene encoding type VII collagen. At present, there is no specific treatment for recessive dystrophic epidermolysis bullosa, and gene and cell therapy approaches hold great promise. This article discusses the different gene therapy approaches that have been used for the treatment of this disease and the new perspectives that they open.
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http://dx.doi.org/10.1016/j.det.2010.02.003DOI Listing
April 2010

Human fibroblasts share immunosuppressive properties with bone marrow mesenchymal stem cells.

J Clin Immunol 2010 Jul 20;30(4):607-19. Epub 2010 Apr 20.

Etablissement Français du sang Pyrénées-Méditerranée, Toulouse, France.

Introduction: Bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells share immunosuppressive capacities, suggesting that the latter could be a general property of stromal cells.

Methods: To check this hypothesis, we compared human BM-MSC and fibroblasts for their in vitro multi-potentiality, expandability and their immunomodulatory properties under normalized optimized culture conditions.

Results: We report that, unlike BM-MSCs, fibroblasts cannot differentiate in vitro into adipocytes and osteoblasts and differ from BM-MSCs by the expression of membrane CD106, CD10 and CD26 and by the expression of collagen VII mRNA. Like BM-MSCs, fibroblasts are unable to provoke in vitro allogeneic reactions, but strongly suppress lymphocyte proliferation induced by allogeneic mixed lymphocyte culture (MLC) or mitogens. We show that fibroblasts' immunosuppressive capacity is independent from prostaglandin E2, IL-10 and the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase and is not abrogated after the depletion of CD8+ T lymphocytes, NK cells and monocytes.

Conclusion: Finally, fibroblasts and BM-MSCs act at an early stage through blockage of lymphocyte activation, as demonstrated by down-regulation of GZMB (granzyme B) and IL2RA (CD25) expression.
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http://dx.doi.org/10.1007/s10875-010-9415-4DOI Listing
July 2010

A frequent functional SNP in the MMP1 promoter is associated with higher disease severity in recessive dystrophic epidermolysis bullosa.

Hum Mutat 2008 Feb;29(2):267-76

INSERM, U563, Toulouse, France.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the COL7A1 gene encoding type VII collagen. Variations in severity between the different clinical forms of RDEB likely depend on the nature and location of COL7A1 mutations, but observed intrafamilial phenotypic variations suggest additional genetic and/or environmental factors. Candidate modifier genes include MMP1, encoding matrix metalloproteinase 1, the first gene implicated in RDEB before its primary role in the disease was excluded. Type VII collagen is a substrate of MMP1 and an imbalance between its synthesis and degradation could conceivably worsen the RDEB phenotype. Here, we studied a previously described family with three affected siblings of identical COL7A1 genotype but displaying great sibling-to-sibling variations in disease severity. RDEB severity did not correlate with type VII collagen synthesis levels, but with protein levels at the dermal-epidermal junction, suggesting increased degradation by metalloproteinases. This was supported by the presence of increased transcript and active MMP1 levels in the most severely affected children, who carried a known SNP (1G/2G) in the MMP1 promoter. This SNP creates a functional Ets binding site resulting in transcriptional upregulation. We next studied a French cohort of 31 unrelated RDEB patients harboring at least one in-frame COL7A1 mutation, ranging from mild localized RDEB to the severe Hallopeau-Siemens form. We found a strong genetic association between the 2G variant and the Hallopeau-Siemens disease type (odds ratio: 73.6). This is the first example of a modifier gene in RDEB and has implications for its prognosis and possible new treatments.
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http://dx.doi.org/10.1002/humu.20647DOI Listing
February 2008

TLR3 deficiency in patients with herpes simplex encephalitis.

Science 2007 Sep;317(5844):1522-7

Human Genetics of Infectious Diseases, Institut National de la Santé et de la Recherche Médicale (INSERM), U550, Faculty Necker, Paris 75015, France.

Some Toll and Toll-like receptors (TLRs) provide immunity to experimental infections in animal models, but their contribution to host defense in natural ecosystems is unknown. We report a dominant-negative TLR3 allele in otherwise healthy children with herpes simplex virus 1 (HSV-1) encephalitis. TLR3 is expressed in the central nervous system (CNS), where it is required to control HSV-1, which spreads from the epithelium to the CNS via cranial nerves. TLR3 is also expressed in epithelial and dendritic cells, which apparently use TLR3-independent pathways to prevent further dissemination of HSV-1 and to provide resistance to other pathogens in TLR3-deficient patients. Human TLR3 appears to be redundant in host defense to most microbes but is vital for natural immunity to HSV-1 in the CNS, which suggests that neurotropic viruses have contributed to the evolutionary maintenance of TLR3.
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http://dx.doi.org/10.1126/science.1139522DOI Listing
September 2007

DNA-based prenatal diagnosis of harlequin ichthyosis and characterization of ABCA12 mutation consequences.

J Invest Dermatol 2007 Mar 2;127(3):568-73. Epub 2006 Nov 2.

Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Until the identification of ABCA12 as the causative gene, prenatal diagnosis (PD) for harlequin ichthyosis (HI) had been performed by electron microscopic observation of fetal skin biopsy samples. We report the first case of HI DNA-based PD. Direct sequence analysis of ABCA12 revealed that the deceased proband was a compound heterozygote for two novel mutations. The maternal nonsense mutation p.Ser1249Term likely leads to nonsense-mediated messenger RNA decay. The paternal mutation c.7436G>A affects the last codon of exon 50 and was expected to be a splice site mutation. For their third pregnancy, the parents requested PD. Direct sequence analysis of fetal genomic DNA from amniotic fluid cells at 17 weeks gestation revealed the fetus was a compound heterozygote for both mutations. The parents requested the pregnancy to be terminated. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products from the allele carrying the splice site mutation. Four of them lead to premature termination codons whereas the two others produced shortened proteins missing 21 and 31 amino acids from the second ATP-binding cassette. This report provides evidence for residual ABCA12 expression in HI, and demonstrates the efficiency of early DNA-based PD of HI.
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http://dx.doi.org/10.1038/sj.jid.5700617DOI Listing
March 2007

Human keratinocytes acquire cellular cytotoxicity under UV-B irradiation. Implication of granzyme B and perforin.

J Biol Chem 2006 May 8;281(19):13525-13532. Epub 2006 Mar 8.

INSERM U563, CPTP, Bat B, Pavillon Lefebvre, Place du Dr. Baylac, Centre Hospitalier Universitaire Purpan, BP 3028, 31024 Toulouse cedex 3, France; Service d'Hématologie, Centre Hospitalier Universitaire Purpan, 31059 Toulouse, France.

Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.
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http://dx.doi.org/10.1074/jbc.M512694200DOI Listing
May 2006

Recessive dystrophic epidermolysis bullosa caused by COL7A1 hemizygosity and a missense mutation with complex effects on splicing.

Hum Mutat 2006 Mar;27(3):291-2

Inserm, U563, Toulouse, F-31059, France.

Loss-of-function mutations in the gene encoding type VII collagen, COL7A1, are the molecular basis of the blistering skin disorder, recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 maps to a region of the short arm of chromosome 3 that has been found to be deleted in many types of malignancies. We have characterized the first case of a large genomic deletion in chromosome 3p21.31 that removes COL7A1 entirely in an RDEB patient. This interstitial deletion spans 255 to 520 kb and encompasses 9 to 15 genes, but seems to have no pathological consequences other than RDEB. We show that the second, hemizygous allele of COL7A1 in this patient bears a base substitution within exon 94, c.7245G>A. This translates into an amino acid substitution, p.M2415I, and leads to a complex splicing abnormality that allows marginal levels of functional mRNA and protein to be synthesized. We propose that the leakiness of the splicing defect enables the partial rescue of collagen VII deficiency. This is consistent with the diagnosis of the moderately severe form of RDEB in the proband, at variance with the most severe form, RDEB Hallopeau-Siemens, that would arise from complete collagen VII deficiency.
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http://dx.doi.org/10.1002/humu.9406DOI Listing
March 2006

Three severe cases of EBS Dowling-Meara caused by missense and frameshift mutations in the keratin 14 gene.

J Invest Dermatol 2006 Apr;126(4):773-6

INSERM, U563, Toulouse, France.

We report three unrelated patients affected at birth with an unusually severe form of epidermolysis bullosa simplex Dowling-Meara type (EBS-DM) because of mutations in KRT14 encoding keratin 14. Two patients were heterozygous for the previously described p.M119T mutation. The third patient was heterozygous for a novel c.1246delC mutation predicting the replacement of the helix termination peptide and the tail domain by a 25 amino-acid aberrant carboxyterminal sequence. At age 2 years, patients carrying the p.M119T mutation still suffered from severe EBS-DM, whereas the patient harboring the c.1246delC mutation has improved over time. These cases illustrate genotype-phenotype correlations and have implications for genetic counselling of EBS.
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http://dx.doi.org/10.1038/sj.jid.5700154DOI Listing
April 2006

Synemin expression in developing normal and pathological human retina and lens.

Exp Neurol 2003 Oct;183(2):499-507

Biologie moléculaire de la différenciation, Université Paris 7, case postale 7136, 2 place Jussieu, 75005, Paris, France.

Synemin (Syn) is an intermediate filament (IF) protein. To gain insight into a morphogenetic role of Syn, we have studied its expression patterns in the developing human retina and lens and compared it with those of other IF proteins. In addition, we have tested Syn expression in fetuses (23 and 28 weeks) affected by Walker-Warburg syndrome (WWS), Meckel syndrome, and trisomy 13. In the retina, Syn expression starts in the nerve fiber and ganglion cell layers (NFL and GCL) at 15 weeks, remains there in up to 20 weeks, and spreads to other layers and may be colocalized with vimentin, GFA, or neurofilaments in the subsequent 16 weeks. This expansion of Synemin expression from 20 to 28 weeks is not observed in WWS in which Syn immunoreactivity in NFL is reduced and Vim expression is increased. Changes are seen in Syn or vimentin expressions in the retinae of 23-week-old Meckel syndrome or 28-week-old trisomy 13 fetuses. Syn expression in the lens is, at first (16 weeks), uniformly distributed, becoming stronger in the epithelium of the anterior part at 25 weeks and later. As in the retina, Syn expression in lens is also selectively affected in WWS. The colocalization of Synemin with vimentin, GFA, or NF supports the idea that Syn is a key cross-linking protein that connects different cytoskeletal structures. Moreover, stagnant Syn expression in WWS retina and lens reinforces the notion of a significant role of this protein in morphogenesis.
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http://dx.doi.org/10.1016/s0014-4886(03)00240-1DOI Listing
October 2003

Differences in the activation of the GFAP gene promoter by prion and viral infections.

Brain Res Mol Brain Res 2002 Dec;109(1-2):119-27

Biologie Moléculaire de la Différenciation, Université Paris-7, Case Postale 7136, 2 Place Jussieu, 75005 Paris, France.

The expression of glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. After surgical injury or viral infections, an increase in this protein reflects reactive gliosis in the brain. We analyzed the activation of the GFAP gene in transgenic mice using a prion and two different viruses (rabies and Theiler viruses). Inoculation of the transgenic mice with the C506M3 mouse prion strain resulted in activation of the GFAP-lacZ transgene. Expression of the GFAP transgene increased concomitantly with the expression of GFAP in astrocytes from the infected mice. In contrast, infection with rabies or Theiler's virus had no effect on the expression of the GFAP transgene, showing that the glial reactions to these infectious agents involved different mechanisms. These findings indicate that the activation of the endogenous GFAP gene as a consequence of viral infection could involve different regulatory pathways than activation as a result of prion infection. The first 2 kb upstream from the start codon of the GFAP gene seems to provide enough activation domains to produce efficient activation of the reporter gene in prion-infected mice.
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http://dx.doi.org/10.1016/s0169-328x(02)00547-8DOI Listing
December 2002