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HLA DR Genome Editing with TALENs in Human iPSCs Produced Immune-Tolerant Dendritic Cells.

ABSTRACT: Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4⁺ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.

SUBMITTER: Kwon YW

PROVIDER: S-EPMC8163544 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome. Methods:HPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole. Results:Although ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity. Conclusions:Our study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.

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HLA DR Genome Editing with TALENs in Human iPSCs Produced Immune-Tolerant Dendritic Cells.

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