Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

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.