241. An Efficient Construction of Lentiviral Vectors That Identify and Eliminate Tumorigenic Cells in Pluripotent Stem Cells
Authors: Kanako Ide, Kaoru Mitsui, Ken-ichiro Kosai
Journal: Molecular Therapy (2016)

Human pluripotent stem cells (hPSCs) are promising sources for cell transplantation therapy. However, incomplete abolition of tumorigenicity, including teratomas and cancers, causes potential safety concerns in their clinical trials. Importantly, most previous approaches focused on "INDIRECT" inhibition of tumorigenicity by reducing the reprogramming-associated oncogenic potential of hiPSCs. Because they cannot completely eliminate tumorigenic potentials due to the intrinsic characteristics of hPSCs, innovative safety approaches should be developed. In this regard, we first developed "adenoviral conditional targeting", which securely isolated target cells (Mol Ther. 14: 673-683. 2006), can increase the efficacy and safety by decreasing tumorigenicity. Second, we have recently developed a novel "oncolytic virus" strategy that specifically and efficiently eliminates undifferentiated cells, thereby inhibiting in vivo teratoma formation after hPSC transplantation (Mol Ther Methods Clin Dev. 2, 15026, 2015). Here we present the third antitumorigenic strategy by a novel methodology that can efficiently generate diverse "Tumorigenic Cell-targeting Lentiviral Vectors (TC-LVs). Tumorigenic cells in the transduced hPSCs can be specifically identified and efficiently killed by fluorescent genes and suicide genes, respectively, under the candidate promoters, which should be specifically and strongly activated in hPCSs in the undifferentiated and/or the transformed status, but not in the differentiated one. This system consists of two plasmids. One is the "LV-acceptor plasmid" (pLVA) that has a recombination cassette, upstream to the unit consisting of one of two fluorescence genes, 2A sequence and one of candidate suicide genes. The other is the "promoter-subcloning plasmid" (pPS) that has multicloning sites flanked by recombination sequences. We tested the feasibility and the utility of this construction system using the five candidate promoters. First, the promoters cloned into the pPS were surely transferred to pLVA having one of three candidate suicide genes and one of two fluorescent genes by using recombinase, resulting in the feasible and rapid generation of the different types of TC-LVs. To test the efficacy of this system, human and mouse PSCs were transduced with TC-LVs composing Venus (EGFP) and Herpes Simplex Virus Thymidine Kinase (HSV-tk) genes driven by either of the ubiquitously active CA or the cancer-specific survivin promoter, and the visualized PSCs were subsequently purified by a cell sorter. All of the PSCs in the undifferentiated status were almost perfectly killed by an addition of ganciclovir (GCV) in the GCV dose-dependent and the promoter activity-dependent manners, whereas they were viable in the differentiated status. The results importantly suggest the necessity of the best combination of the promoter (activity and specificity properties) and the suicide and the fluorescent genes. In conclusion, we have developed the novel method for a rapid generation of TC-LVs that can systematically identify the best promoter and suicide gene to surely eliminate tumorigenic cells, without harmful effect to the targeting differentiated cell. This methodology may facilitate the safe clinical application of PSCs-based cell therapy.