Site-Specific Integration Of TRAIL In IPSC-Derived Mesenchymal Stem Cells For Targeted Cancer Therapy

Backgroud Based on the tropism to the tumor stroma and low immunogenicity,both natural and genetically modified mesenchymal stem cells(MSCs)have been widely used as cellular vehicles to deliver anti-cancer effectors/genes in cancer treatment researches.The modified MSCs are customizable,thus could be more effective than natural MSCs under the specific pathological conditions.Currently,MSCs are mainly derived from adult tissues.Given the differences of donors,tissue sources and culture or passage conditions,there are challenges such as poor homogeneity,quality and repeatability,as well as difficulties in engineering and standardization when MSCs applied.Viral vectors,the most used tools for gene transduction,whereas carry potential risks of immune responses,limited gene loading capacity and potential risk of unintended insertional mutagenesis.Induced pluripotent stem cells(iPSCs)can be harnessed to generate MSCs(induced MSCs,i MSCs),and i MSCs have emerged as excellent candidates with the advantages of homogeneity,unlimited yield and easily modified.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)have advantages of preferentially inducing apoptosis in various tumor cells but no toxic effect on normal cells.In this study,therefore,we try to target the TRAIL gene at the r DNA locus using non-viral targeting vectors and TALENickases that were developed in the previous studies by our group.And the homogeneous therapeutic TRAIL-i MSCs would be derived from gene-modified TRAIL-i PSCs for in vitro and in vivo anti-tumor studies.Objective Based on an allogeneic cell and gene therapy strategy for tumors based on site-specific genetically modified human i PSCs,we try to generate an unlimited number of homogeneous TRAIL-i MSCs with efficient and stable expression of the therapeutic gene,and the anti-tumor efficacy of TRAIL-i MSCs will be validated in vitro and in vivo in preclinical cancer models.Methods(1)A non-viral vector plasmid minip Hrneo-ILZ-s TRAIL was successfully constructed;(2)Therapeutic gene TRAIL was efficiently integrated into the r DNA locus of i PSCs utilizing minip Hrneo ILZ-s TRAIL and TALENickases;(3)The cell karyotypes,stem cell characteristics,genome off-target,and transcription or expression levels of therapeutic gene TRAIL in TRAIL-i PSCs were verified;(4)TRAIL-iPSCs were stably differentiated into TRAIL-i MSCs.TRAIL-i MSCs were characterized by the transcription or expression levels of TRAIL,cell surface markers,stability of cell passage,differentiation ability and immunomodulatory ability;(5)The pro-apoptotic effect and related molecular expression in apoptotic signaling pathways of TRAIL-i MSCs treated various tumor cell lines were verified in vitro co-culture system;(6)The tumor suppressive effect and related molecular expression in apoptotic signaling pathways in A375 and MCF-7 xenograft-bearing mouse models treated by TRAIL-i MSCs were verified.The potential toxic and side effects of TRAIL-i MSCs treatment were assessed.Results(1)Targeted TRAIL into the rDNA locus in iPSCs;(2)Karyotypes and stem cell markers were not affected by exogenous gene intergration in TRAIL-i PSCs.Gene editing tools TALENickases did not cause insertion or deletion mutations in TRAIL-i PSCs;(3)Acquirement of innumerable TRAIL-i MSCs by directional differentiation;(4)High expression of TRAIL was detected in TRAIL-iPSCs and TRAIL-iMSCs.Different generations of TRAIL-i MSCs maintained the ability to continuously express the transgene protein TRAIL;(5)TRAIL-i MSCs could expand more rapidly and efficiently than primary MSCs,and they maintained expression of typical cell surface markers,ability of differentiation and immunomodulatory;(6)TRAIL-i MSCs significantly induced apoptosis in A375,A549,Hep G2,and MCF-7 cells by activating apoptotic signaling pathway in vitro;(7)TRAIL-i MSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and expressed the transgene protein human TRAIL;(8)TRAIL-i MSCs significantly inhibited tumor growth through the activation of apoptotic signaling pathways,resulted in tumor tissue apoptosis and necrosis in A549 or MCF-7 xenografts;(9)No obvious side effects in tumor-bearing mice models.Conclusions This study provides the first proof for prominent tissue tropism to and anti-tumor properties on breast,liver and lung cancer of gene-modified TRAIL-i PSCs derived TRAIL-i MSCs,paving the way for the development of not only an unlimited number of homogenous therapeutic MSCs but also a universal off-the-shelf stem cell-based preparations with good potential for clinical applications and industrial production.