798 Biallelic COL7A1 editing in iPSCs via CRISPR/Cas9 for recessive dystrophic epidermolysis bullosa mutations

Abstract

The routine diagnosis of heritable skin and connective tissue disorders is complicated by the fact that in this group of disorders, clinical manifestations may result from genetic or phenotypic heterogeneity and the existence of new genes and/or novel disease subtypes. Autozygosity mapping (AM) has been proven to be a useful adjunct in the molecular diagnosis of homozygous autosomal recessive (AR) diseases. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using genomewide single nucleotide polymorphism (SNP) array-based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases (84.7%) were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analyzed with an EB-targeted next-generation sequencing (NGS) panel. This approach identified mutations in five additional cases, initially undiagnosed due to presence of compound heterozygosity, deep intronic mutations, or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 out of 46 cases (95.7%) diagnosed genetically. The yield of 84.7% using AM-guided sequencing was remarkably similar to that of the independent use of our previously reported NGS targeted panel, in which potential causative variants were identified in 76 of 91 (83.5%) families with EB. Thus, AM is an expedient and cost efficient approach to identify mutated genes consanguineous families.

New Frontier in the Management of Corneal Dystrophies: Basics, Development, and Challenges in Corneal Gene Therapy and Gene Editing

“…Followed by this, from gene-corrected iPSCs, 3-dimensional human skin equivalents (HSEs) were generated and differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted into immunodeficient mice, which showed wild-type expression of COL7A1 and restored anchoring fibrils with no evident off-targets activity or other safety concern. 51 Similar approaches can be implemented in corneal dystrophies as well, where patient-derived iPSC could be generated and corrected for its defective mutation using CRISPR tool followed by its differentiation into endothelial cells, which can be surgically grafted in patients cornea for the rescue of disease phenotype. Interestingly, in 1 study, it was shown that dental pulp contains a population of adult stem cells known as DPSc, that can be harvested from third molars with the capability to differentiate into corneal keratocytes.…”

Generation of Skin Organoids: Potential Opportunities and Challenges

“…For example, genetically modified autologous epidermal cells could provide a lasting treatment for vesicular skin diseases. Several studies have shown that it is possible to correct epidermolysis bullosa, a genetic skin disorder, by CRISPR/Cas9-based targeted genome editing (Itoh et al, 2011;Hirsch et al, 2017;Jacków et al, 2019). hiPSCs can become ideal sources of cells for generating skin organoids.…”

Therapy Development for Epidermolysis Bullosa

“…Additional efficiency can be gained by optimizing both, the format and the delivery of the gene-editing molecules. Currently, electroporation of COL7A1-specific RNPs together with single-stranded oligonucleotide HDR templates have resulted in the highest repair efficiencies [34].…”

“…The application of HDR-based approaches to the correction of patient-derived induced pluripotent stem cells (iPSCs) further increases the range of therapeutic options for patients, especially as the isolation of epidermal holoclones can be a limiting factor [14,34]. Pre-clinical studies using corrected iPSCs, that were then differentiated into keratinocytes and fibroblasts, and used to generate three-dimensional skin equivalents (HSEs) on the backs of immunodeficient mice, showed normal type VII collagen expression and restored anchoring fibrils [34]. Alternative strategies, not based on homology-directed repair, comprise base editing, which has proven to be a suitable option for correcting pathogenic mutations in RDEB [36].…”