Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.

No abstract

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.