Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy

Abstract

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell-based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor-initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1-eGSH). Chr1-eGSH permitted robust transgene expression, but a 2 Mb-distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1-eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV-TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. Stem Cells Translational Medicine 2019;8:627&638.

Keywords: Gene editing; Genomic safe harbor; Induced pluripotent stem cells; Regenerative medicine; Suicide gene; Teratoma.

Figure 1

Generation of knockin hiPSCs carrying the HSV‐TK transgene at the Chr1‐eGSH locus. (A): Topological architecture of an extragenic genomic safe harbor on chromosome 1 (Chr‐eGSH). Transcripts and putative topologically associated domains are shown. (B): Experimental flow to create knockin human induced pluripotent stem cells (hiPSCs). (C): Schematic illustration of the WT and knockin alleles. The left and right homologous arms are indicated as HA‐L and HA‐R, respectively. The PCR primers used are shown as magenta arrows. (D): Targeted knockin of the HSV‐TK cassette detected by genomic PCR. No KI is an iPSC clone electroporated with vectors but without HSV‐TK. The results for all isolated clones are shown in Supporting Information Figure S3. (E): Bright field and fluorescence images of the representative knockin iPSC line (TK#1). Scale bar: 100 μm. (F): HSV‐TK expression levels in WT and knockin iPSCs. Relative expression to GAPDH (delta CT value) is shown. Data are represented as mean ± SD (n = 3 for each line). (G): Relative expression levels of 5′ and 3′ proximate genes in WT and knockin iPSCs. Relative expression to GAPDH (delta CT value) is shown. Data are represented as the mean ± SD (n = 3 for each line). Abbreviations: EFp, human elongation factor‐1 α promoter; HSV‐TK, herpes simplex virus thymidine kinase; IRES2, internal ribosomal entry site 2; N/D, not detected; pA, poly A; puroR, puromycin resistant gene.

Figure 2

Herpes simplex virus thymidine kinase (HSV‐TK)/ganciclovir eliminates knockin human induced pluripotent stem cells (hiPSCs). (A): in vitro cytotoxicity assay of WT and knockin hiPSCs. Cell viability was evaluated 48 hours after ganciclovir treatment at the indicated concentration. LC₅₀ of 409B2, TK#1, TK#2, and TK#3 was 203 μM, 0.043 μM, 0.048 μM, and 0.074 μM, respectively. Data are represented as mean ± SD (n = 6 for each line). (B): Cytotoxic effect of ganciclovir on OCT3/4 positive cells upon in vitro differentiation. The upper scheme shows the experimental flow. OCT3/4 expression was analyzed by qPCR at day 9 and was normalized to GAPDH expression. Data are represented as the mean ± SD (n = 3 for each line). (C): Effect of ganciclovir on embryoid body (EB) formation. Images at day 7 are shown. Scale bar: 250 μm. (D): Cytotoxic effect of ganciclovir on EBs. The volume of EBs at day 7 was analyzed. Data are represented as the mean ± SD (n = 8 for each line). LC₅₀ of TK#1, TK#2, and TK#3 was 0.018 μM, 0.005 μM, and 0.01 μM, respectively. (E): Relative expression levels of lineage maker genes and HSV‐TK in EBs formed in the presence and absence of ganciclovir treatment. Marker gene expression of 0.1 μM GCV‐treated EBs was compared with that of untreated EBs. Relative expression levels were normalized to GAPDH expression levels. Data are represented as mean ± SD (n = 3 for each line).

Figure 3

In vivo teratoma assay. (A–C): Tumoricidal effects of herpes simplex virus thymidine kinase (HSV‐TK)/ganciclovir (GCV) on established teratomas formed by TK#1 (A), TK#2 (B), or TK#3 (C) knockin human induced pluripotent stem cells (hiPSCs). The size (relative area) of each tumor was compared with that before GCV or vehicle injection. Size relative to the average start volume is shown. GCV treated teratomas were divided into an effective group and a noneffective group. Statistical significance was determined using two‐way analysis of variance (ANOVA). *, p < .05; **, p < .01. (D–F): Relative expression levels of HSV‐TK in TK#1 (D), TK#2 (E), or TK#3 (F) teratomas. Relative expression levels were normalized to GAPDH expression levels. Statistical significance was determined using Kruskal–Wallis ANOVA. Abbreviations: E, effective; NE, noneffective; C, control.

Figure 4

DNA methylation analyses of the promoter. (A): CpG islands of the EF‐1 α promoter and HSV‐TK transgene. CpG islands are indicated by boxes, with a gray box indicating the CpGs analyzed. (B): The average DNA methylation level of each CpG site. (C): Mean DNA methylation levels of the analyzed CpGs of human induced pluripotent stem cells and teratomas. Statistical significance was determined using the Student's t test. *, p < .05; **, p < .01. (D): Correlation between the DNA methylation of the EF‐1 α promoter and HSV‐TK expression. Relative expression levels of HSV‐TK were normalized to GAPDH expression levels. The Pearson's correlation coefficient is shown. Abbreviations: C, control teratomas; E, effective teratomas; HA‐L, left homologous arm for knockin; NE, noneffective teratomas.

Figure 5

Histological analysis of teratomas. Histological analysis of the injection sites in ganciclovir or vehicle treated teratomas. Representative images are shown. Scale bar: 100 μm.