Hypermethylation and reduced expression of Gtl2, Rian and Mirg at the Dlk1-Dio3 imprinted locus as a marker for poor developmental potential of mouse embryonic stem cells.

Mouse embryonic stem cells (ESCs) have played a crucial role in biomedical research where they can be used to elucidate gene function through the generation of genetically modified mice. A critical requirement for the success of this technology is the ability of ESCs to contribute to viable chimaeras with germ-line transmission of the genetically modified allele. We have identified several ESC clones that cause embryonic death of chimaeras at mid to late gestation stages. These clones had a normal karyotype, were pathogen free and their in vitro differentiation potential was not compromised. Chimaeric embryos developed normally up to E13.5 but showed a significant decrease in embryo survival by E17.5 with frequent haemorrhaging. We investigated the relationship between the ESCs transcriptional and epigenomic state and their ability to contribute to viable chimaeras. RNA sequencing identified four genes (Gtl2, Rian, Mirg and Rtl1as) located in the Dlk1-Dio3 imprinted locus that were expressed at lower levels in the compromised ESC clones and this was confirmed by qRT-PCR. Bisulphite sequencing analysis showed significant hypermethylation at the Dlk1-Dio3 imprinted locus with no consistent differences in methylation patterns at other imprinted loci. Treatment of the compromised ESCs with 5-azacytidine reactivated stable expression of Gtl2 and rescued the lethal phenotype but only gave low level chimaeras.

From fiction to science: clinical potentials and regulatory considerations of gene editing.

Gene editing technologies such as CRISPR/Cas9 have emerged as an attractive tool not only for scientific research but also for the development of medicinal products. Their ability to induce precise double strand breaks into DNA enables targeted modifications of the genome including selective knockout of genes, correction of mutations or precise insertion of new genetic material into specific loci. Gene editing-based therapies hold a great potential for the treatment of numerous diseases and the first products are already being tested in clinical trials. The treatment indications include oncological malignancies, HIV, diseases of the hematopoietic system and metabolic disorders. This article reviews ongoing preclinical and clinical studies and discusses how gene editing technologies are altering the gene therapy landscape. In addition, it focusses on the regulatory challenges associated with such therapies and how they can be tackled during the drug development process.