Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

Hu, Youjin; Liu, Xionghao; Long, Panpan; Xiao, Di; Cun, Jintao; Li, Zhuo; Xue, Jinfeng; Wu, Yong; Luo, Sha; Wu, Lingqian

doi:10.1155/2013/135189

Cited by 10 publications

(4 citation statements)

References 45 publications

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“…These properties indicate that the rDNA locus may be an ideal safe locus for transgene integration [ 7 , 8 ]. In our previous studies, exogenous genes have been targeted to the rDNA region and stably expressed in various cell lines by non-viral plasmids, including immortal adult cells and stem cells [ 9 , 10 , 11 , 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Wang

Zhao

Duan

et al. 2018

IJMS

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Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites. After co-transfection into mESCs with sgRNA-Cas9n and a non-viral rDNA targeting vector pMrnF9, harboring the homology donor template and the human F9 expression cassette, a recombination efficiency of 66.7% was achieved and all targeted clones were confirmed to be site-specific integration of F9 in the rDNA locus by PCR and southern blotting. Targeted mESCs retained the main pluripotent properties and were then differentiated into hepatic progenitor like cells (HPLCs) and mature hepatocytes, which were characterized by hepatic markers and functional assays. Importantly, the differentiated cells could transcribe exogenous F9 and secrete coagulation factor IX (FIX) proteins, suggesting active transcription and stable inheritance of transgenes in the rDNA locus. After intrasplenical transplantation in severe combined immune deficiency (SCID) mice, targeted HPLCs could survive and migrate from spleen to liver, resulting in secretion of exogenous FIX into blood. In summary, we demonstrate an efficient and site-specific gene targeting strategy in rDNA locus for stem cell-based gene therapy for hemophilia B.

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Section: Introductionmentioning

confidence: 99%

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Wang

Zhao

Duan

et al. 2018

IJMS

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“…In this context, iPSCs can be genetically modified to express anti-cancer factors, from which a large number of therapeutic MSCs with relative uniform quality can be derived. We have previously constructed a novel non-viral targeting vector, the ribosomal DNA targeting vector (pHrneo), which has targeted therapeutic genes into the rDNA locus of different human cell types including hepatocyte cell lines [35], MSCs [36], and embryonic stem cells [37]. More importantly, we have used this vector to target IL-24 into the ribosomal DNA locus of human iPSCs and obtained an iPSC cell line with the expression of exogenous gene IL-24 (IL-24-iP-SCs).…”

Section: Discussionmentioning

confidence: 99%

Mesenchymal stem cells derived from iPSCs expressing interleukin-24 inhibit the growth of melanoma in the tumor-bearing mouse model

Liu

Wang

et al. 2020

Cancer Cell Int

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Background: Interleukin-24 (IL-24) is a therapeutic gene for melanoma, which can induce melanoma cell apoptosis. Mesenchymal stem cells (MSCs) show promise as a carrier to delivery anti-cancer factors to tumor tissues. Induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells (MSCs). We previously developed a novel non-viral gene targeting vector to target IL-24 to human iPSCs. This study aims to investigate whether MSCs derived from the iPSCs with the site-specific integration of IL-24 can inhibit the growth of melanoma in a tumor-bearing mouse model via retro-orbital injection.Methods: IL-24-iPSCs were differentiated into IL-24-iMSCs in vitro, of which cellular properties and potential of differentiation were characterized. The expression of IL-24 in the IL-24-iMSCs was measured by qRT-PCR, Western Blotting, and ELISA analysis. IL-24-iMSCs were transplanted into the melanoma-bearing mice by retro-orbital intravenous injection. The inhibitory effect of IL-24-iMSCs on the melanoma cells was investigated in a co-culture system and tumorbearing mice. The molecular mechanisms underlying IL-24-iMSCs in exerting anti-tumor effect were also explored.Results: iPSCs-derived iMSCs have the typical profile of cell surface markers of MSCs and have the ability to differentiate into osteoblasts, adipocytes, and chondroblasts. The expression level of IL-24 in IL-24-iMSCs reached 95.39 ng/10 6 cells/24 h, which is significantly higher than that in iMSCs, inducing melanoma cells apoptosis more effectively in vitro compared with iMSCs. IL-24-iMSCs exerted a significant inhibitory effect on the growth of melanoma in subcutaneous mouse models, in which the migration of IL-24-iMSCs to tumor tissue was confirmed. Additionally, increased expression of Bax and Cleaved caspase-3 and down-regulation of Bcl-2 were observed in the mice treated with IL-24-iMSCs. Conclusion: MSCs derived from iPSCs with the integration of IL-24 at rDNA locus can inhibit the growth of melanoma in tumor-bearing mouse models when administrated via retro-orbital injection.

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“…Many studies have demonstrated that genetically engineered MSCs inhibit tumor growth in lung cancer, melanoma, glioma, liver cancer and breast cancer [ 41 , 42 , 45 , 46 ]. In a previous study we targeted neomycin gene into the rDNA locus of hBM-MSCs and generated stably transformed hBM-MSCs with G418 selection [ 17 ]. We also demonstrated proliferation of transformed hBM-MSCs upon culturing [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study we targeted neomycin gene into the rDNA locus of hBM-MSCs and generated stably transformed hBM-MSCs with G418 selection [ 17 ]. We also demonstrated proliferation of transformed hBM-MSCs upon culturing [ 17 ]. Therefore, a new strategy was necessary to rapidly obtain abundant transformed MSCs for clinical applications, whenever necessary.…”

Section: Discussionmentioning

confidence: 99%

Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci

Liu

Chen

et al. 2017

Oncotarget

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Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than control iMSCs. IL24-iMSCs efficiently differentiated into osteoblasts, chondrocytes and adipocytes. Functionally, IL24-iMSCs induced in vitro apoptosis in B16-F10 melanoma cells more efficiently than control iMSCs when co-cultured in Transwell assays. In vivo tumor xenograft studies in mice demonstrated that IL24-iMSCs inhibited melanoma growth more than control iMSCs did. Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with IL24-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of iPSCs through gene targeting with non-viral vectors into a rDNA locus. The ability of these genetically modified MSCs to inhibit in vivo melanoma growth is suggestive of the clinical potential of autologous cell therapy in cancer.

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Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

Cited by 10 publications

References 45 publications

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Mesenchymal stem cells derived from iPSCs expressing interleukin-24 inhibit the growth of melanoma in the tumor-bearing mouse model

Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci

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