Targeted genome editing restores T cell differentiation in a humanized X-SCID pluripotent stem cell disease model

Claudio Mussolino,Celeste Pallant,Steven J Howe,Jamal Alzubi,Adrian J Thrasher,Toni Cathomen

doi:10.1038/s41598-017-12750-4

Abstract

The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application.

Highlights

T cells are a key component of the adaptive immunity, which provides host protection against pathogens and cancer
X-linked severe combined immunodeficiency (X-severe combined immunodeficiency (SCID)) embryonic stem cell (ESC) were transfected with transcription activator-like effector nucleases (TALENs) expression plasmids and the gamma chain (GC) donor
The expected PCR amplicon of 1.1 kb was detected in all four clones, whereas the 1.9 kb product, indicative of the ‘SCID allele’, was detected only in non-transfected samples and cells transfected with the GC donor alone (Fig. 1B)

Summary

Introduction

T cells are a key component of the adaptive immunity, which provides host protection against pathogens and cancer. Retroviral IL2RG gene transfer in haematopoietic stem cells (HSCs) has been assessed in autologous settings in several clinical trials The outcome of these studies has shown near complete immune reconstitution, with similar or even better outcome to that of mismatched allogeneic HSC transplantation[12]. Patient-specific IL2RG, JAK3 and WAS-deficient iPSC lines were corrected by designer nucleases and combined successfully with NK and T cell differentiation protocols[34,35,36]. These studies did not focus on stage-specific T cell development, which is important when modelling X-SCID in order to demonstrate the role of GC in IL-7 and IL-2 dependent T cell development and T cell expansion[7,37]

Methods

Results

Conclusion