563. Rapid and Nuclease-Free Generation of Gene-Repaired Human iPS Cells from IL2RG-Defective Fibroblasts

Abstract

Recent developments of artificial nucleases, such as TALENs and CRISPR/Cas9, have made gene repair therapy of inherited diseases more realistic. However, off-target chromosomal breaks resulting in imprecise DNA repair by the nucleases are still significant concerns from safety aspects, which is the most critical issue of clinical gene therapy. Furthermore, prolonged culture during induction and manipulation of induced pluripotent stem cells (iPSCs) has been shown to increase point mutations and copy number variations in the iPSC genome, raising additional safety concerns for therapeutic applications of iPSCs. In order to minimize cell culture for iPSC induction and achieve efficient gene repair without using nucleases, we examined a strategy of consecutive iPSC induction and gene repair without cell cloning between the two processes. For this purpose, we utilized Sendai virus vector (SeV)-mediated iPSC induction and helper-dependent adenoviral vector (HDAdV)-mediated gene targeting.To mimic somatic cells from patients of X-linked severe combined immunodeficiency, we firstly knocked out exon 1 of the common gamma chain of human interleukin-2 receptor (IL2RG) locus by utilizing TALENs, and subsequently derived fibroblast-like cells. Next, these cells were subject to iPSC induction by using the SeV expressing Yamanaka factors, SeVdp(KOSM). Instead of isolating and characterizing individual SeV-iPS colonies before gene targeting, iPSC-like colonies were grown as a cell population. Twenty-two to 26 days after SeV infection, the cells were transduced with a HDAdV targeting the IL2RG locus. This HDAdV was designed to introduce the PGK-neo cassette into intron 4 and to repair the mutation of exon 1. Cells were subject to G418/FIAU selection to isolate gene-targeted SeV-iPS colonies. Correct targeting at the IL2RG locus by the vector in G418/FIAU double-resistant colonies was verified by PCR analyses. From two independent experiments, the gene targeting frequencies were similar to those in authentic human iPSCs at 1.6 – 1.8 × 10-5 per cell and 60 – 75% of chromosomal integration (G418/FIAU-resistance), among them 60 – 80% of gene-targeted colonies had the exon 1 mutation repaired. At the time of HDAdV infection, the SeV-iPSCs already showed characteristics of pluripotency, revealed by bisulfite sequencing of the NANOG locus and TRA-1-60 immunostaining. The pluripotency was also maintained in gene-targeted iPSC colonies. From the start of iPSC induction (infection with SeV) to isolation of gene-repaired iPSC colonies, it took only 40 days. We are currently analyzing the expression of IL2RG in differentiated T cells derived from the gene-repaired iPSCs.These results suggest that, by combining efficient methods for iPSC induction and gene targeting, gene repair therapy using patients’ somatic cells becomes more realistic.