Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations

Tockner, B.; Köcher, Thomas; Hainzl, Stefan; Reichelt, Julia; Bauer, Johann; Koller, Ulrich; Murauer, Eva M.

doi:10.1038/gt.2016.57

Cited by 33 publications

(22 citation statements)

References 39 publications

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“…The procedure of western blotting was performed as previously described. 13 , 43 Ponceau red (Sigma-Aldrich) staining of the membrane was performed after electro-blotting. For detection of type VII collagen a rabbit anti-type VII collagen antibody (kindly provided by Dr. Alexander Nyström, Freiburg, Germany) was used at a dilution of 1:3,000 in Tris-buffered saline with 0.2% Tween (TBS-T) at 4°C overnight.…”

Section: Methodsmentioning

confidence: 99%

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

et al. 2017

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Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

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Section: Methodsmentioning

confidence: 99%

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

et al. 2017

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“…The procedure of western blotting was performed as previously described. 40 , 41 Ponceau red (Sigma-Aldrich, St. Louis, MO, USA) staining of the nitrocellulose membrane was performed after electro-blotting. Nitrocellulose membrane was blocked with blocking reagent (Roche Diagnostics, Mannheim, Germany) diluted 1:10 in Tris-buffered saline with 0.2% Tween (TBS-T) for 1 hr at room temperature (RT).…”

Section: Methodsmentioning

confidence: 99%

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

et al. 2017

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With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

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“…Preclinical studies have shown remarkable results in terms of gene correction/excision using both in vivo and ex vivo approaches. [64][65][66][67][68][69][70] In addition, a previous study of ex vivo gene therapy in an individual with junctional EB demonstrated restoration of laminin 332 expression following retroviralmediated transfection of epidermal stem cells with the LAMB3 gene, with long-lasting phenotypic correction in the grafted skin. 71 A US trial is currently evaluating the safety and efficacy of a retroviral vector and genetically engineered epithelial graft cultured from the skin of patients with RDEB (ClinicalTrials.gov identifier: NCT01263379).…”

Section: Gene Therapymentioning

confidence: 96%

Management of chronic wounds in patients with dystrophic epidermolysis bullosa: challenges and solutions

Rashidghamat¹,

Mellerio²

2017

CWCMR

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Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of severe inherited blistering diseases that affects 500,000 individuals worldwide. Clinically, individuals with EB have fragile skin and are susceptible to blistering following minimal trauma and show involvement of mucus membrane and other organs in some subtypes. Dystrophic EB (DEB) is divided into 2 major types depending on the inheritance pattern: recessive DEB (RDEB) and dominant DEB (DDEB). RDEB tends to be at the more severe end of the clinical spectrum and has a prevalence of 8 per 1 million of the population, accounting for approximately 5% of all cases of EB. RDEB is caused by loss-of-function mutations in the type VII collagen gene, COL7A1, which leads to reduced or absent type VII collagen (C7) and structurally defective anchoring fibrils at the dermal-epidermal junction. In this review, we will discuss the management of chronic wounds in individuals with DEB, highlighting the changes to practice and the novel therapies that may offer a solution to this debilitating and complex problem which is one of the greatest sources of morbidity in this disease.

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Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations

Cited by 33 publications

References 39 publications

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

Management of chronic wounds in patients with dystrophic epidermolysis bullosa: challenges and solutions

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