Kanako Ide

Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor β-Expressing Macrophages.

Background Folate receptor β (FRβ) is induced during macrophage activation. A recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to an anti-FRβ antibody (anti–FRβ-PE38) has been reported to kill activated macrophages in inflammatory diseases. To elucidate the effect of an immunotoxin targeting FRβ on atherosclerosis, we determined the presence of FRβ-expressing macrophages in atherosclerotic lesions and administered the FRβ immunotoxin in apolipoprotein E–deficient mice. Methods and Results The FRβ-expressing macrophages were observed in atherosclerotic lesions of apolipoprotein E–deficient mice. At 15 or 35 weeks of age, the apolipoprotein E–deficient mice were divided into 3 groups and were intravenously administered 0.1 mg/kg of anti–FRβ-PE38 (immunotoxin group), 0.1 mg/kg of PE38 (toxin group), or 0.1 mL of saline (control group) every 3 days, for a total of 5 times for each age group. The mice were analyzed at 21 or 41 weeks of age. Treatment with the immunotoxin resulted in 31% and 22% reductions in atherosclerotic lesions of the 21- and 41-week-old mice, respectively (P<0.05). Administration of immunotoxin reduced the numbers of FRβ- and tumor necrosis factor-α–expressing macrophages, reduced cell proliferation, and increased the number of apoptotic cells (P<0.05). Real-time polymerase chain reaction demonstrated that the expression of FRβ and tumor necrosis factor-α mRNA was significantly decreased in the immunotoxin group (P<0.05). Conclusions These results suggest that FRβ-expressing macrophages exist in the atherosclerotic lesions of apolipoprotein E–deficient mice and that FRβ immunotoxin administration reduces the progression of atherosclerotic lesions in younger and older individuals. The recombinant FRβ immunotoxin targeting activated macrophages could provide a novel therapeutic tool for atherosclerosis. (J Am Heart Assoc. 2012;1:e003079 doi: 10.1161/JAHA.112.003079.)

Conditionally replicating adenovirus prevents pluripotent stem cell–derived teratoma by specifically eliminating undifferentiated cells

Incomplete abolition of tumorigenicity creates potential safety concerns in clinical trials of regenerative medicine based on human pluripotent stem cells (hPSCs). Here, we demonstrate that conditionally replicating adenoviruses that specifically target cancers using multiple factors (m-CRAs), originally developed as anticancer drugs, may also be useful as novel antitumorigenic agents in hPSC-based therapy. The survivin promoter was more active in undifferentiated hPSCs than the telomerase reverse transcriptase (TERT) promoter, whereas both promoters were minimally active in differentiated normal cells. Accordingly, survivin-responsive m-CRA (Surv.m-CRA) killed undifferentiated hPSCs more efficiently than TERT-responsive m-CRAs (Tert.m-CRA); both m-CRAs exhibited efficient viral replication and cytotoxicity in undifferentiated hPSCs, but not in cocultured differentiated normal cells. Pre-infection of hPSCs with Surv.m-CRA or Tert.m-CRA abolished in vivo teratoma formation in a dose-dependent manner following hPSC implantation into mice. Thus, m-CRAs, and in particular Surv.m-CRAs, represent novel antitumorigenic agents that could facilitate safe clinical applications of hPSC-based regenerative medicine.

Viral Vector-Based Innovative Approaches to Directly Abolishing Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine

Human pluripotent stem cells (hPSCs) are a promising source of regenerative material for clinical applications. However, hPSC transplant therapies pose the risk of teratoma formation and malignant transformation of undifferentiated remnants. These problems underscore the importance of developing technologies that completely prevent tumorigenesis to ensure safe clinical application. Research to date has contributed to establishing safe hPSC lines, improving the efficiency of differentiation induction, and indirectly ensuring the safety of products. Despite such efforts, guaranteeing the clinical safety of regenerative medicine products remains a key challenge. Given the intrinsic genome instability of hPSCs, selective growth advantage of cancer cells, and lessons learned through failures in previous attempts at hematopoietic stem cell gene therapy, conventional strategies are unlikely to completely overcome issues related to hPSC tumorigenesis. Researchers have recently embarked on studies aimed at locating and directly treating hPSC-derived tumorigenic cells. In particular, novel approaches to directly killing tumorigenic cells by transduction of suicide genes and oncolytic viruses are expected to improve the safety of hPSC-based therapy. This article discusses the current status and future perspectives of methods aimed at directly eradicating undifferentiated tumorigenic hPSCs, with a focus on viral vector transduction.

A Novel Construction of Lentiviral Vectors for Eliminating Tumorigenic Cells from Pluripotent Stem Cells

The risk of tumor formation poses a challenge for human pluripotent stem cell (hPSC)‐based transplantation therapy. Specific and total elimination of tumorigenic hPSCs by suicide genes (SGs) has not been achieved because no methodology currently exists for testing multiple candidate transgene constructs. Here, we present a novel method for efficient generation of tumorigenic cell‐targeting lentiviral vectors (TC‐LVs) with diverse promoters upstream of a fluorescent protein and SGs. Our two‐plasmid system achieved rapid and simultaneous construction of different TC‐LVs with different promoters. Ganciclovir (GCV) exerted remarkable cytotoxicity in herpes simplex virus thymidine kinase‐transduced hPSCs, and high specificity for undifferentiated cells was achieved using the survivin promoter (TC‐LV.Surv). Moreover, GCV treatment completely abolished teratoma formation by TC‐LV.Surv‐infected hPSCs transplanted into mice, without harmful effects. Thus, TC‐LV can efficiently identify the best promoter and SG for specific and complete elimination of tumorigenic hPSCs, facilitating the development of safe regenerative medicine. Stem Cells 2018;36:230–239

241. An Efficient Construction of Lentiviral Vectors That Identify and Eliminate Tumorigenic Cells in Pluripotent Stem Cells

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.View Large Image | Download PowerPoint Slide