Improving the Safety of Mesenchymal Stem Cell-Based Ex Vivo Therapy Using Herpes Simplex Virus Thymidine Kinase.

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC50) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.

Depletion of T cells via Inducible Caspase 9 Increases Safety of Adoptive T-Cell Therapy Against Chronic Hepatitis B.

T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo, induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity.

Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter.

Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones-N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk-were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and ßIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, ßIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers.

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here.

[Cys-Flanked Cationic Peptides For Cell Delivery of the Herpes Simplex Virus Thymidine Kinase Gene for Suicide Gene Therapy of Uterine Leiomyoma].

Uterine leiomyoma (UL) is the most common benign tumor in women of reproductive age. Gene therapy using suicidal genes appears to be a promising approach for UL treatment. One of key factors for success of gene therapy is the right choice of genetic construct carrier. A promising group of non-viral carriers for cell delivery of expression vectors is cationic Cys-flanked peptides which form tight complexes with DNA due to electrostatic interactions and the presence of interpeptide disulfide bonds. The paper reports a comparative study of the physico-chemical, toxic, and transfectional properties of the DNA-peptide complexes obtained by matrix polymerization or oxidative polycondensation of Cys-flanked peptides using the chain growth terminator 2-amino ethanethiol. We have demonstrated the therapeutic effect of the delivery of the pPTK-1 plasmid carrying the herpes simplex virus type 1 (HSV-1) thymidine kinase gene into PANC-1, and HEK-293T cell culture as well as into primary UL cells. It has been shown that the carriers obtained by oxidative polycondensation transform primary UL cells more efficiently than those produced by matrix polymerization. Treatment with ganciclovir resulted in the death of up to 40% of UL cells transfected with the pPTK-1 plasmid. The perspectives of use of the polyR6 carrier produced by oxidative polycondensation as a tool for the development of modular peptide carriers for the purposes of UL gene therapy were discussed.

Improvement of the dP-nucleoside-mediated herpes simplex virus thymidine kinase negative-selection system by manipulating dP metabolism genes.

A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(ß-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000 nM dP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E. coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E. coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100 nM dP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk.

Rapid Regulation of Human Mesenchymal Stem Cell Proliferation Using Inducible Caspase-9 Suicide Gene for Safe Cell-Based Therapy.

The regulation of transplanted cell proliferation and function is important to achieve safe cell-based therapies. We previously reported that the proliferation and function of transplanted cells, which expressed the herpes simplex virus thymidine kinase (HSVtk) suicide gene, could be controlled by ganciclovir (GCV) administration. However, there are some concerns regarding the use of GCV. It is reported that the inducible caspase-9 (iC9) gene, a human caspase-9-derived genetically engineered suicide gene, rapidly induces cell apoptosis in the presence of apoptosis inducers, such as AP20187. In this study, we used a combination of the iC9 gene and AP20187 to achieve rapid regulation of transplanted cell proliferation. Cells from the human mesenchymal stem cell line UE7T-13 were transfected with the iC9 gene to obtain UE7T-13/iC9 cells. AP20187 significantly reduced the number of UE7T-13/iC9 cells within 24 h in a concentration-dependent manner. This reduction was much faster than the reduction of HSVtk-expressing UE7T-13 cells induced by GCV addition. Subcutaneous AP20187 administration rapidly reduced the luminescence signal from NanoLuc luciferase (Nluc)-expressing UE7T-13/iC9 cells transplanted into mice. These results indicate that the combined use of the iC9 gene and AP20187 is effective in rapidly regulating transplanted cell proliferation.

Hinge Region in DNA Packaging Terminase pUL15 of Herpes Simplex Virus: A Potential Allosteric Target for Antiviral Drugs.

Approximately 80% of adults are infected with a member of the herpesviridae family. Herpesviruses establish life-long latent infections within neurons, which may reactivate into lytic infections due to stress or immune suppression. There are nine human herpesviruses (HHV) posing health concerns from benign conditions to life threatening encephalitis, including cancers associated with viral infections. The current treatment options for most HHV conditions mainly include several nucleoside and nucleotide analogs targeting viral DNA polymerase. Although these drugs help manage infections, their common mechanism of action may lead to the development of drug resistance, which is particularly devastating in immunocompromised patients. Therefore, new classes of drugs directed against novel targets in HHVs are necessary to alleviate this issue. We analyzed the conservation rates of all proteins in herpes simplex virus 1 (HHV-1), a representative of the HHV family and one of the most common viruses infecting the human population. Furthermore, we generated a full-length structure model of the most conserved HHV-1 protein, the DNA packaging terminase pUL15. A series of computational analyses were performed on the model to identify ATP and DNA binding sites and characterize the dynamics of the protein. Our study indicates that proteins involved in HHV-1 DNA packaging and cleavage are amongst the most conserved gene products of HHVs. Since the packaging protein pUL15 is the most conserved among all HHV-1 gene products, the virus will have a lower chance of developing resistance to small molecules targeting pUL15. A subsequent analysis of the structure of pUL15 revealed distinct ATP and DNA binding domains and the elastic network model identifies a functionally important hinge region between the two domains of pUL15. The atomic information on the active and allosteric sites in the ATP- and DNA-bound model of pUL15 presented in this study can inform the structure-based drug discovery of a new class of drugs to treat a wide range of HHVs.

Golgi membrane protein GP73 modified-liposome mediates the antitumor effect of survivin promoter-driven HSVtk in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide, and there is currently no effective therapeutic strategy in clinical practice. Gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited because of the lack of tumor-specific targets and insufficient gene transfer. The study of targeted transport of therapeutic genes in HCC treatment seems to be very important. In this study, we evaluated a gene therapy approach targeting HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. GP73-modified liposomes targeted gene delivery to the tumor tissue, and the survivin promoter drove HSVtk expression in the HCC cells. Our results showed that the survivin promoter was specifically activated in tumor cells and HSVtk was expressed selectively in tumor cells. Combined with GCV treatment, HSVtk expression resulted in suppression of HCC cell proliferation via enhancing apoptosis. Moreover, tail vein injection of GP73-HSVtk significantly suppressed the growth of xenograft tumors through an apoptosis-dependent pathway and extended the survival of tumor-bearing mice without damaging the mice liver functions. Taken together, this study demonstrates an effective cancer-specific gene therapy strategy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system for HCC that can be further developed for future clinical trials.

Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

Long-term treatment with valganciclovir improves lentiviral suicide gene therapy of glioblastoma.

BACKGROUND: Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. METHODS: Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. RESULTS: TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. CONCLUSION: Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy.

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell-based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor-initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1-eGSH). Chr1-eGSH permitted robust transgene expression, but a 2 Mb-distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1-eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV-TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. Stem Cells Translational Medicine 2019;8:627&638.

Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.

Origins of Suicide Gene Therapy.

"Tumor chemosensitivity" can be achieved by the expression of the herpes simplex virus thymidine kinase gene in cells, followed by the conversion of the "prodrug" ganciclovir into the therapeutic drug inside the cells. This system presaged other combinations of suicide genes and prodrugs, including cytosine deaminase/5-fluorocytosine, purine nucleoside phosphorylase/6-methylpurine deoxyriboside, and horseradish peroxidase/indole-3-acetic acid.

Cancer Suicide Gene Therapy with TK.007.

Cancer is a devastating disease characterized by uncontrolled and aggressive cell growth. Suicide gene therapy (SGT) facilitating induction of malignancy-specific cell death represents a novel therapeutic approach to treat cancer, which has been investigated in several cancer types with very promising results. In addition, SGT has been suggested as a safeguard in adoptive immunotherapy and regenerative-medicine settings. Generally, SGT consists of two steps-vector-mediated delivery of suicide genes into tumors and subsequent activation of the suicide mechanism, e.g., by administration of a specific prodrug. This chapter provides a framework of protocols for basic and translational research using the Herpes-simplex-virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system, the most widely used suicide gene approach. The protocols provide standard guidelines for the preparation of high-titer third-generation lentiviral vectors encoding a genetically improved HSV-TK version known as TK.007 and its application in in vitro and in vivo treatment setups.

Osteonectin Promoter-Mediated Suicide Gene Therapy of Prostate Cancer.

Suicide gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene, combined with the prodrug ganciclovir (GCV) medication, is a promising approach for the treatment of malignant tumors, including prostate cancer. The success of this therapeutic strategy requires tissue- or tumor-specific gene expression and efficient gene delivery. In this chapter, we describe the experimental protocols of key methodologies, including promoter construction, reporter assay, adenoviral vector construction and preparation, HSV-tk enzymatic assay and cytotoxicity assay to evaluate the specificity and efficacy of osteonectin promoter-mediated HSV-tk/GCV suicide gene therapy of prostate cancer.

Suicide Gene Therapy for Oral Squamous Cell Carcinoma.

Suicide gene therapy has been tested for the treatment of a variety of cancers, including oral cancer. Among the various suicide gene therapy approaches that have been reported, the Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system is one of the most extensively studied systems, holding great promise in cancer therapy. In this chapter, we describe methods to use the HSV-tk/GCV system to achieve antitumor activity, both in cultured oral cancer cells and in orthotopic and subcutaneous murine models of oral squamous cell carcinoma, using ligand-associated lipoplexes for enhancing therapeutic delivery.

Suicide Gene Therapy of Oral Squamous Cell Carcinoma and Cervical Carcinoma In Vitro.

Suicide gene therapy induced by the Herpes Simplex Virus thymidine kinase/ganciclovir (HSV-tk/GCV) system has been utilized to successfully treat various cancers. We describe TransfeX-mediated transfection of pCMV.Luc into HeLa cervical carcinoma and HSC-3, FaDu, and H357 oral squamous cell carcinoma (OSCC) cell lines in the presence of 10% serum. This method has proved to be highly efficient, with low nonspecific cytotoxicity. The plasmid pNGVL1-tk encoding HSV-tk under the control of the CMV promoter was delivered to the cells in vitro via TransfeX, a cationic liposomal reagent, followed by treatment with ganciclovir. The Alamar Blue cell viability assay was used to determine levels of the suicide effect.

Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.

Graphene oxide-hydroxyapatite nanocomposites effectively deliver HSV-TK suicide gene to inhibit human breast cancer growth.

Gene therapy with herpes simplex virus thymidine kinase gene (HSV-TK), which is also known as "suicide" gene therapy, is effective in various tumor models. The lack of a safe and efficient gene delivery system has become a major obstacle to "suicide" gene therapy. In this study, the cytotoxicity and transfection efficiency of graphene oxide-hydroxyapatite (GO-Hap) were analyzed by MTS and flow cytometry, respectively. A series of assays were performed to evaluate the effects of GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment on growth of human breast normal and cancer cells. The results showed that GO-HAp nanocomposites effectively transfected cells with minimum toxicity. GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment inhibited the proliferation and induced cell apoptosis in cancer cells, while the cytotoxic effects are tolerable in normal breast cells. We conclude that the GO-HAp nanocomposites have significant potential as a gene delivery vector for cancer therapy.