Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors

We have assessed the suitability of retroviral vectors for gene therapy of recessive dystrophic epidermolysis bullosa (RDEB) in dogs expressing a mutated collagen type VII. Isolation and analysis of the 9 kb dog collagen type VII cDNA identified the causative genetic mutation G1906S and disclosed the interspecies conservation of collagen type VII. Highly efficient transfer of the wild-type collagen type VII cDNA to both dog RDEB and human primary RDEB collagen type VII-null keratinocytes using recombinant vectors derived from LZRS-Ires-zeo and MSCV retroviruses achieved sustained and permanent expression of the transgene product. The expression and post-translational modification profile of the recombinant collagen type VII was comparable to that of the wild-type counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Hypermotility was fully reverted in human RDEB keratinocytes, and strongly reduced in the dog RDEB cells. This observation suggests that not only infection efficiency but also high expression levels are required to ensure therapeutic efficacy in the presence of mutated gene products. Our results set the basis for preclinical gene therapy assays in the first immune-competent large animal model for an inherited skin disease and broaden the spectrum of preclinical and clinical applications of retroviral vectors in the transfer of large recombinant genes in epithelial cells.     

Identification of a lethal form of epidermolysis bullosa simplex associated with a homozygous genetic mutation in plectin

Genetic mutations in plectin, a cytoskeleton linker protein expressed in a large variety of tissues including skin, muscle, and nerves, cause epidermolysis bullosa simplex with muscular dystrophy, a recessive inherited disease characterized by blistering of the skin and late onset of muscular dystrophy, and Ogna epidermolysis bullosa simplex, a rare dominant inherited form of epidermolysis bullosa simplex with no muscular involvement. Here we report a novel homozygous genetic mutation (2727del14) in the plectin gene (PLEC1) associated with a lethal form of recessive inherited epidermolysis bullosa in a consanguineous family with three affected offspring. This new clinical variant of epidermolysis bullosa is characterized by general skin blistering, aplasia cutis of the limbs, developmental complications, and rapid demise after birth. Mutation 2727del14 is the first genetic defect described in PLEC1 that disrupts the plakin domain of plectin. The severe phenotype of the patients may be linked to the role of the N-terminal domain in the function of plectin and develops the understanding of the genotype-phenotype correlations in the genodermatoses affecting the dermal-epidermal junction.     

Immunofluorescence analysis of villous trophoblasts: a tool for prenatal diagnosis of inherited epidermolysis bullosa with pyloric atresia

Genetic mutations invalidating the genes for integrin alpha6beta4 and, in some cases, plectin are associated with junctional and simplex epidermolysis bullosa with pyloric atresia (PA-JEB and PA-EBS), respectively. These recessive inherited conditions are characterized by pregnancies with fetal bullae, pyloric atresia, polyhydramnios, and neonatal mucocutaneous blistering, which often results in early postnatal demise. To date, first-trimester DNA-based prenatal diagnosis is not applicable to affected kindred carrying as yet unidentified genetic mutations. Here, we show that first-trimester chorionic villi strongly express both integrin alpha6beta4 and plectin, which persist throughout the pregnancy. Based on this observation, we implemented 25 prenatal diagnoses in kindred at risk for PA-EB by immunomapping, which identified three PA-JEB-affected fetuses and 22 healthy ones. In 19 cases, including the three PA-JEB pregnancies that were prematurely terminated, the results were confirmed by chorionic villous DNA-based tests, which also led to the identification of seven previously unreported mutations in the alpha6beta4 integrin genes. Our prediction was further sustained by the birth of 22 healthy babies. These results validate chorionic villi immunofluorescence examination as a tool for prenatal diagnosis of PA-JEB and PA-EBS and indicate that this procedure could be devised for EB with muscular dystrophy, which is also associated with genetic mutations in plectin.     

Prenatal diagnosis of inherited epidermolysis bullosa in a patient with no family history: a case report and literature review

OBJECTIVE: The junctional form of epidermolysis bullosa (EB) is a recessively inherited mechanobullous disease in which minimal trauma results in blister formation at the dermal-epidermal junction. A rare form associated with pyloric atresia (JEB-PA) is a severe clinical subtype leading to rapid demise after birth, thus justifying prenatal diagnosis. The case characterized by abnormal ultrasound findings at 35 weeks of gestation (gastric dilatation associated with polyhydramnios) of a patient with no family history is reported. METHOD: Postabortion skin biopsies were analyzed by immunofluorescence that revealed marked reduction of integrin alpha6beta4 in accordance with the diagnosis of JEB-PA. RESULTS: Amniotic fluid contained excess total protein (4 MoM), abnormally high AFP (20.4 MoM) related to skin lesions and abnormally elevated digestive enzyme suggestive of fetal vomiting of bile. The electrophoretic pattern of cholinesterases was unusual (additional slow band). Maternal serum AFP was 3.14 MoM and free beta-hCG 13.1 MoM. Because of these concomitant findings, JEB-PA was suspected. CONCLUSION: The case under study was atypical because of late clinical manifestations of the disease: polyhydramnios, gastric enlargement. As maternal serum AFP at 15 weeks may be normal, it was suggested that discovery of polyhydramnios during the second or the third trimester should prompt biochemical analysis of amniotic fluid, such as AFP and GGTP assay in all cases.     

Treatment of two patients with Herlitz junctional epidermolysis bullosa with artificial skin bioequivalents

OBJECTIVE: To evaluate the effects of a treatment with artificial skin bioequivalents in Herlitz junctional epidermolysis bullosa (H-JEB). METHODS: Two infants, both homozygous for the Herlitz mutation R635X in the LAMB3 gene, who had refractory anemia and hypoproteinemia as a result of a continuous loss of body fluids through multiple large erosions, were treated with artificial skin bioequivalents. RESULTS: In the first patient, 10 of 13 acute or chronic wounds were found healed 3 to 6 weeks after the treatment, and the protein, iron, and hemoglobin levels normalized. Normal weight gain and marked improvement of the quality of life for this patient's family have been evident since. Nine treated wounds remained healed for at least 18 weeks and appeared to be more resistant to trauma. In a skin biopsy from a treated site obtained after 9 weeks, DNA of the graft was still detectable by polymerase chain reaction. In the second patient, who was treated at a late stage of the disease, only 2 of 18 chronic wounds were found healed at 3 weeks after the treatment. One site remained healed completely, and some additional islets of the grafts persisted for longer. There were no adverse events. CONCLUSION: Early treatment with artificial skin substitutes may improve the clinical course of H-JEB. However, a true cure of the cutaneous manifestations of H-JEB would require gene therapy of autologous epidermal stem cells, which could then be transplanted by using this or a similar cultured skin bioequivalent.     

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell-free assays and showed increased ability to induce pH-dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild-type dianthin. Conjugates made by chemically cross-linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing efficiency than conjugates of Tfn and wild-type dianthin. As a consequence, by fusion of membrane-acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non-specific and specific toxicity) of Tfn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymatic cytotoxins results in the acquisition of new physico-chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell-intoxication mechanisms.