Gene-edited cells: novel allogeneic gene/cell therapy for epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of rare genetic skin fragility disorders, which are hereditary. These disorders are associated with mutations in at least 16 genes that encode components of the epidermal adhesion complex. Currently, there are no effective treatments for this disorder. All current treatment approaches focus on topical treatments to prevent complications and infections. In recent years, significant progress has been achieved in the treatment of the severe genetic skin blistering condition known as EB through preclinical and clinical advancements. Promising developments have emerged in the areas of protein and cell therapies, such as allogeneic stem cell transplantation; in addition, RNA-based therapies and gene therapy approaches have also become a reality. Stem cells obtained from embryonic or adult tissues, including the skin, are undifferentiated cells with the ability to generate, maintain, and replace fully developed cells and tissues. Recent advancements in preclinical and clinical research have significantly enhanced stem cell therapy, presenting a promising treatment option for various diseases that are not effectively addressed by current medical treatments. Different types of stem cells such as primarily hematopoietic and mesenchymal, obtained from the patient or from a donor, have been utilized to treat severe forms of diseases, each with some beneficial effects. In addition, extensive research has shown that gene transfer methods targeting allogeneic and autologous epidermal stem cells to replace or correct the defective gene are promising. These methods can regenerate and restore the adhesion of primary keratinocytes in EB patients. The long-term treatment of skin lesions in a small number of patients has shown promising results through the transplantation of skin grafts produced from gene-corrected autologous epidermal stem cells. This article attempts to summarize the current situation, potential development prospects, and some of the challenges related to the cell therapy approach for EB treatment.

CRISPR/Cas9 knock-in toward creating a Rett syndrome cell model with a synonymous mutation in the MECP2 gene.

BACKGROUND: Rett syndrome is an X-linked dominant neurodevelopmental disease caused by mutation in the methyl-CpG-binding protein 2 (MECP2) gene. This gene encodes a methylated DNA-binding protein, which acts as a transcriptional regulatory factor. The present study aimed to establish a cell model of Rett syndrome with the MECP2 synonymous mutation c.354G>T (p.Gly118Gly). In addition, the molecular mechanism of pathogenesis of this mutation was also investigated. METHODS: To create a cell line containing the synonymous variant in MECP2 locus, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed repair precise gene editing method was used. In addition, employing the synthesis of cDNA, the effect of this variant on splicing was investigated. RESULTS: Using this model and molecular analysis, we found that the c.354G>T synonymous variant created a novel 5' cryptic splice donor site within the exon 3 of MECP2 gene, which resulted in the deletion of 25 nucleotides at the 3' end of exon 3 and presumably protein truncation. CONCLUSIONS: The results of the present study show that an apparently neutral synonymous polymorphism, which may be commonly classified as non-pathogenic, may indeed lead to the creation of an aberrant splice site, thereby resulting in disease.

Two novel mutations in the MECP2 gene in patients with Rett syndrome.

Rett syndrome (RTT) is an X-linked severe neurological disorder. Mutations in Methyl-CpG-Binding Protein2 (MECP2) gene are the main cause of RTT disease. In this study, we report the results of screening the MECP2 gene for mutations in 7 Iranian patients with RTT syndrome. MECP2 sequencing identified two novel mutations in the heterozygous state, a splice mutation, c.354G>T, p.Gly119Gly, resulting in a premature splice-donor site and a 20-bp deletion, c.1167-1186del20 (p.P390Rfs), leading to modifying the c-terminal parts of the protein and it also changes the reading frames of all coding sequence downstream of the mutation. Multiple sequence alignment showed that amino acid changes occurred in the well conserved protein regions across species. Based on the results of this study and literature reviews, about 70% of mutations are found in exon 3 and 4 of the MECP2 gene, and mutations in exon 4 are more common than other exons. Therefore, it is recommended that exon 4 to be a priority for screening the genetic analysis of RTT patients.