Dataset Information

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies.

ABSTRACT: High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.

SUBMITTER: Bornert O

PROVIDER: S-EPMC7852329 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies.

Bornert Olivier O Kocher Thomas T Gretzmeier Christine C Liemberger Bernadette B Hainzl Stefan S Koller Ulrich U Nyström Alexander A

Matrix biology plus 20191120

High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies fo ...[more]

PMID: 33543014

Similar Datasets

Project description:BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients.MethodsA double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions.ResultsBoth topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin.ConclusionTopical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations.Trial registrationClinicalTrials.gov NCT02698735.FundingEpidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.

Project description:Dystrophic epidermolysis bullosa, a severely disabling hereditary skin fragility disorder, is caused by mutations in the gene coding for collagen VII, a specialized adhesion component of the dermal-epidermal junction zone. Both recessive and dominant forms are known; the latter account for about 40% of cases. Patients with dominant dystrophic epidermolysis bullosa exhibit a spectrum of symptoms ranging from mild localized to generalized skin manifestations. Individuals with the same mutation can display substantial phenotypic variance, emphasizing the role of modifying genes in this disorder. The etiology of dystrophic epidermolysis bullosa has been known for around two decades; however, important pathogenetic questions such as involvement of modifier genes remain unanswered and a causative therapy has yet to be developed. Much of the failure to make progress in these areas is due to the lack of suitable animal models that capture all aspects of this complex monogenetic disorder. Here, we report the first rat model of dominant dystrophic epidermolysis bullosa. Affected rats carry a spontaneous glycine to aspartic acid substitution, p.G1867D, within the main structural domain of collagen VII. This confers dominant-negative interference of protein folding and decreases the stability of mutant collagen VII molecules and their polymers, the anchoring fibrils. The phenotype comprises fragile and blister-prone skin, scarring and nail dystrophy. The model recapitulates all signs of the human disease with complete penetrance. Homozygous carriers of the mutation are more severely affected than heterozygous ones, demonstrating for the first time a gene-dosage effect of mutated alleles in dystrophic epidermolysis bullosa. This novel viable and workable animal model for dominant dystrophic epidermolysis bullosa will be valuable for addressing molecular disease mechanisms, effects of modifying genes, and development of novel molecular therapies for patients with dominantly transmitted skin disease.

Dataset Information

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies.

Publications

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets