Combinatorial suicide gene strategies for the safety of cell therapies.

Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.

Suicide gene therapy in cancer and HIV-1 infection: An alternative to conventional treatments.

Suicide Gene Therapy (SGT) aims to introduce a gene encoding either a toxin or an enzyme making the targeted cell more sensitive to chemotherapy. SGT represents an alternative approach to combat pathologies where conventional treatments fail such as pancreatic cancer or the high-grade glioblastoma which are still desperately lethal. We review the possibility to use SGT to treat these cancers which have shown promising results in vitro and in preclinical trials. However, SGT has so far failed in phase III clinical trials thus further improvements are awaited. We can now take advantages of the many advances made in SGT for treating cancer to combat other pathologies such as HIV-1 infection. In the review we also discuss the feasibility to add SGT to the therapeutic arsenal used to cure HIV-1-infected patients. Indeed, preliminary results suggest that both productive and latently infected cells are targeted by the SGT. In the last section, we address the limitations of this approach and how we might improve it.

Insight into susceptibility genes associated with bipolar disorder: a systematic review.

OBJECTIVE: Bipolar disorder (BD) is a severe disorder, and it is associated with an increased risk of mortality. About 25% of patients with BD have attempted and 11% have died by suicide. All these characteristics suggest that the disorders within the bipolar spectrum are a crucial public health problem. With the development of molecular genetics in recent decades, it was possible to more easily detect risk genes associated with this disorder. This study aimed at summarizing the findings of systematic reviews and meta-analyses on the topic and assessing the quality of the available evidence. MATERIALS AND METHODS: PubMed/Medline and Web of Science were searched to identify systematic reviews and meta-analyses published during 2013-2019. Standard methodology was applied to synthesize and assess the retrieved literature. RESULTS: This systematic review identifies a number of potential risk genes associated with bipolar disorder whose mechanism of action has yet to be confirmed. They are divided into several groups: 1) a list of the most significant susceptibility genetic factors associated with BD; 2) the implication of the ZNF804A gene in BD; 3) the role of genes involved in calcium signaling in BD; 4) DNA methylation in BD; 5) BD and risk suicide genes; 6) susceptibility genes for early-onset BD; 7) candidate genes common to both BD and schizophrenia; 8) genes involved in cognitive status in BD cases; 9) genes involved in structural alteration in BD brain tissue; 10) genes involved in lithium response in BD. CONCLUSIONS: Future research should concentrate on molecular mechanisms by which genetic variants play a major role in BD. Supplemental research is needed to replicate the applicable results.

Enhancement of nanoparticle-mediated double suicide gene expression driven by 'E9-hTERT promoter' switch in dedifferentiated thyroid cancer cells.

Differentiated thyroid cancer (DTC), such as papillary thyroid cancer, has a good prognosis after routine treatment. However, in the course of treatment, 5% to 20% of cases may dedifferentiate and can be transformed into dedifferentiated DTC (deDTC) or anaplastic thyroid cancer, leading to treatment failure. To date, several drugs have been used effectively for dedifferentiated thyroid cancer, whereas gene therapy may be a potential method. Literature reported that double suicide genes driven by human telomerase reverse transcriptase promoter (hTERTp) can specifically express in cancer cells and kill them. However, the weak activity of hTERTp limits its further research. To overcome this weakness, we constructed a novel chitosan nanocarrier containing double suicide genes driven by a 'gene switch' (a cascade of radiation enhancer E9 and a hTERTp). The vector was labeled with iodine-131 (131I). On one hand, E9 can significantly enhance the activity of hTERTp under the weak radiation of 131I, thereby increasing the expression of double suicide genes in deDTC cells. On the other hand, 131I also plays a certain killing role when it enters host cells. The proposed nanocarrier has good specificity for deDTC cells and thus deserves further study.

Recent progress in the research of suicide gene therapy for malignant glioma.

Malignant glioma, which is characterized by diffuse infiltration into the normal brain parenchyma, is the most aggressive primary brain tumor with dismal prognosis. Over the past 40 years, the median survival has only slightly improved. Therefore, new therapeutic modalities must be developed. In the 1990s, suicide gene therapy began attracting attention for the treatment of malignant glioma. Some clinical trials used a viral vector for suicide gene transduction; however, it was found that viral vectors cannot cover the large invaded area of glioma cells. Interest in this therapy was recently revived because some types of stem cells possess a tumor-tropic migratory capacity, which can be used as cellular delivery vehicles. Immortalized, clonal neural stem cell (NSC) line has been used for patients with recurrent high-grade glioma, which showed safety and efficacy. Embryonic and induced pluripotent stem cells may be considered as sources of NSC because NSC is difficult to harvest, and ethical issues have been raised. Mesenchymal stem cells are alternative candidates for cellular vehicle and are easily harvested from the bone marrow. In addition, a new type of nonlytic, amphotropic retroviral replicating vector encoding suicide gene has shown efficacy in patients with recurrent high-grade glioma in a clinical trial. This replicating viral capacity is another possible candidate as delivery vehicle to tackle gliomas. Herein, we review the concept of suicide gene therapy, as well as recent progress in preclinical and clinical studies in this field.

Potential Alternatives to Conventional Cancer Therapeutic Approaches: The Way Forward.

onventional cancer therapeutic approaches broadly include chemotherapy, radiation therapy and surgery. These established approaches have evolved over several decades of clinical experience. For a complex disease like cancer, satisfactory treatment remains an enigma for the simple fact that the causal factors for cancer are extremely diverse. In order to overcome existing therapeutic limitations, consistent scientific endeavors have evolved several potential therapeutic approaches, majority of which focuses essentially on targeted drug delivery, minimal concomitant ramification, and selective high cytotoxicity. The current review focuses on highlighting some of these potential alternatives that are currently in various stages of in vitro, in vivo, and clinical trials. These include physical, chemical and biological entities that are avidly being explored for therapeutic alternatives. Some of these entities include suicide gene, micro RNA, modulatory peptides, ultrasonic waves, free radicals, nanoparticles, phytochemicals, and gene knockout, and stem cells. Each of these techniques may be exploited exclusively and in combination with conventional therapeutic approaches thereby enhancing the therapeutic efficacy of the treatment. The review intends to briefly discuss the mechanism of action, pros, and cons of potential alternatives to conventional therapeutic approaches.

Development of CAR T Cells Expressing a Suicide Gene Plus a Chimeric Antigen Receptor Targeting Signaling Lymphocytic-Activation Molecule F7.

Chimeric antigen receptors (CARs) are fusion proteins that contain antigen-recognition domains and T cell signaling domains. Signaling lymphocytic-activation molecule F7 (SLAMF7) is a promising target for CAR T cell therapies of the plasma cell malignancy multiple myeloma (MM) because SLAMF7 is expressed by MM but not normal nonhematopoietic cells. We designed CARs targeting SLAMF7. We transduced human T cells with anti-SLAMF7 CARs containing either CD28 or 4-1BB costimulatory domains. T cells expressing CD28-containing CARs or 4-1BB-containing CARs recognized SLAMF7 in vitro. SLAMF7-specific cytokine release was higher for T cells expressing CARs with CD28 versus 4-1BB domains. In murine solid tumor and disseminated tumor models, anti-tumor activity of T cells was superior with CD28-containing CARs versus 4-1BB-containing CARs. Because of SLAMF7 expression on some normal leukocytes, especially natural killer cells that control certain viral infections, the inclusion of a suicide gene with an anti-SLAMF7 CAR is prudent. We designed a construct with a CD28-containing anti-SLAMF7 CAR and a suicide gene. The suicide gene encoded a dimerization domain fused to a caspase-9 domain. T cells expressing the anti-SLAMF7 CAR plus suicide-gene construct specifically recognized SLAMF7 in vitro and eliminated tumors from mice. T cells expressing this construct were eliminated on demand by administering the dimerizing agent AP1903 (rimiducid).

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.

rAAV9-UPII-TK-EGFP can precisely transduce a suicide gene and inhibit the growth of bladder tumors.

Bladder cancer is a common and widespread cancer of the human urinary system, and its incidence is increasing. Gene therapy is a promising treatment of bladder cancer. In our study, a recombinant adeno-associated virus (rAAV9-UPII-TK-EGFP) driven by a UPII promoter was constructed. The efficacy and safety of infection of bladder cells was tested in vivo and in vitro. The ability of rAAV9-UPII-TK-EGFP to penetrate the glycosaminoglycan (GAG) layer on the surface of bladder cells and to transduce the bladder cells in vivo was very high. Additionally, we confirmed that the TK/GCV system has a powerful cytotoxic effect on bladder tumor cells in vitro and in vivo. Thus, our data indicate that rAAV9-UPII-TK-EGFP is a precise gene drug delivery system for the treatment of bladder cancer, and the TK/GCV therapeutic strategy has a powerful antitumor effect. These findings can be widely used in clinical and scientific studies.

Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells.

Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a "safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies.

Study on the targeted therapy of oral squamous cell carcinoma with a plasmid expressing PE38KDEL toxin under control of the SERPINB3 promoter.

Oral squamous cell carcinoma (OSCC) has a poor prognosis and a high risk of recurrence. To improve the efficacy of OSCC therapy, it is of great significance to explore gene therapy for OSCC. The use of specific genes to regulate the targeted expression of suicide genes is a hot topic in gene therapy for cancer. The SERPINB3 gene is highly active in squamous cell carcinoma, but nearly undetectable or present at a low level in normal tissues. This specificity suggests that the SERPINB3 promoter can be used for targeted OSCC therapy. Pseudomonas aeruginosa secretes PE38KDEL, an exotoxin derivative, as a suicide gene used in gene therapy. A SERPINB3 promoter-mediated PE38KDEL expression vector was created. The SERPINB3 gene expression was tested in different cell lines by RT-qPCR and Western blotting, and the SERPINB3 promoter activity was detected by luciferase assay. The SERPINB3 promoter was more active in the TCA8113 cell line than in the other cell lines. The target therapeutic potential of the toxin vector pSERPINB3-PE38KDEL was tested in the SERPINB3-positive TCA8113 cell line, the SERPINB3-negative MG63 cell line, and normal L02 cell line. The SERPINB3 gene was expressed at a high level in TCA8113 cells but a low level in MG63 and L02 cells. Transfection of the pSERPINB3-PE38KDEL plasmid effectively inhibited the proliferation and invasion of TCA8113 cells and induced cell apoptosis, but no significant damage to MG63 and L02 cells was observed. The results of in vitro experiments indicated that the pSERPINB3-PE38KDEL plasmid could be a promising strategy for targeted OSCC gene therapy.

Specific driving of the suicide E gene by the CEA promoter enhances the effects of paclitaxel in lung cancer.

Classical chemotherapy for lung cancer needs new strategies to enhance its antitumor effect. The cytotoxicity, nonspecificity, and low bioavailability of paclitaxel (PTX) limits their use in this type of cancer. Suicide gene therapy using tumor-specific promoters may increase treatment effectiveness. We used carcinoembryonic antigen (CEA) as a tumor-specific promoter to drive the bacteriophage E gene (pCEA-E) towards lung cancer cells (A-549 human and LL2 mice cell lines) but not normal lung cells (L132 human embryonic lung cell line), in association with PTX as a combined treatment. The study was carried out using cell cultures, tumor spheroid models (MTS), subcutaneous induced tumors and lung cancer stem cells (CSCs). pCEA-E induced significant inhibition of A-549 and LL2 cell proliferation in comparison to L132 cells, which have lower CEA expression levels. Moreover, pCEA-E induced an important decrease in volume growth of A-549 and LL2 MTS producing intense apoptosis, in comparison to L132 MTS. In addition, pCEA-E enhanced the antitumor effects of PTX when combined, showing a synergistic effect. This effect was also observed in A-549 CSCs, which have been related to the recurrence of cancer. The in vivo study corroborated the effectiveness of the pCEA-E-PTX combined therapy, inducing a greater decrease in tumor volume compared to PTX and pCEA-E alone. Our results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically towards lung cancer cells, and may be used to enhance the effectiveness of PTX against this type of tumor.

Targeted AAVP-based therapy in a mouse model of human glioblastoma: a comparison of cytotoxic versus suicide gene delivery strategies.

Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.

A double safety lock tumor-specific device for suicide gene therapy in breast cancer.

The complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.

Suicide Gene Therapy Against Malignant Gliomas by the Local Delivery of Genetically Engineered Umbilical Cord Mesenchymal Stem Cells as Cellular Vehicles.

BACKGROUND: Glioblastoma (GBM) is a malignant tumor that is difficult to eliminate, and new therapies are thus strongly desired. Mesenchymal stem cells (MSCs) have the ability to locate to injured tissues, inflammation sites and tumors and are thus good candidates for carrying antitumor genes for the treatment of tumors. Treating GBM with MSCs that have been transduced with the herpes simplex virus thymidine kinase (HSV-TK) gene has brought significant advances because MSCs can exert a bystander effect on tumor cells upon treatment with the prodrug ganciclovir (GCV). OBJECTIVE: In this study, we aimed to determine whether HSV-TK-expressing umbilical cord mesenchymal stem cells (MSCTKs) together with prodrug GCV treatment could exert a bystander killing effect on GBM. METHODS AND RESULTS: Compared with MSCTK: U87 ratio at 1:10,1:100 and 1:100, GCV concentration at 2.5µM or 250µM, when MSCTKs were cocultured with U87 cells at a ratio of 1:1, 25 µM GCV exerted a more stable killing effect. Higher amounts of MSCTKs cocultured with U87 cells were correlated with a better bystander effect exerted by the MSCTK/GCV system. We built U87-driven subcutaneous tumor models and brain intracranial tumor models to evaluate the efficiency of the MSCTK/GCV system on subcutaneous and intracranial tumors and found that MSCTK/GCV was effective in both models. The ratio of MSCTKs and tumor cells played a critical role in this therapeutic effect, with a higher MSCTK/U87 ratio exerting a better effect. CONCLUSION: This research suggested that the MSCTK/GCV system exerts a strong bystander effect on GBM tumor cells, and this system may be a promising assistant method for GBM postoperative therapy.

A CD33 Antigen-Targeted AAV6 Vector Expressing an Inducible Caspase-9 Suicide Gene Is Therapeutic in a Xenotransplantation Model of Acute Myeloid Leukemia.

Current chemotherapeutic regimens for acute myeloid leukemia (AML) have been modestly effective in patients and are associated with poor long-term survival (<30% at 5 years). Viral vector-based suicide gene therapy is an attractive option, if these vectors can target the AML cells with high specificity and efficiency. In this study, we have developed a receptor-specific adeno-associated virus (AAV) based vector to target the CD33 antigen which is overexpressed in leukemic cells. A targeting peptide was rationally designed from the antigen-binding regions of a CD33 monoclonal antibody. This peptide was further expressed on the capsid of the AAV6 vector, since this serotype was most efficient among AAV1-rh10 vectors to infect the pro-monocytic, human myeloid leukemia cells (U937). AAV6-CD33 vectors expressing a suicide gene, the inducible caspase 9 (iCasp9), and its prodrug AP20187 significantly reduced (∼59%) the viability of U937 cells. To further test its efficacy and specificity in vivo, AAV6-CD33 vectors were administered into a xenotransplantation model of AML in zebrafish through systemic delivery. We observed a significant antileukemic effect with AAV6-CD33 vectors, with a markedly higher survival (100% for AAV6-CD33 vectors vs 15% for mock-treated) and a higher number of TUNEL positive apoptotic cells after systemic vector delivery. Taken together, our work demonstrates the efficacy and translational potential of CD33-targeted AAV6 vectors for cytotoxic gene therapy in AML.

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.

Nanoscale bubble ultrasound contrast agents-mediated suicide gene therapy system, Nanoscale bubble-LV5-YCD-TK/GCV/5-FC, effectively inhibits bladder cancer cell growth.

OBJECTIVE: Bladder cancer is the 2nd most common reason for human genitourinary cancer-associated mortality. This study aimed to investigate the effects of Nanoscale bubble ultrasound contrast agents-mediated yeast-cytosine-deaminase-thymidine kinase/ganciclovir (YCD-TK/GCV) or YCD-TK/5-fluorocytosine (5-FC) suicide gene therapy system on BIU-87 cell growth. MATERIALS AND METHODS: Targeted nanoscale bubble ultrasound contrast agents were prepared by utilizing thin-film hydration-sonication approach. Nanoscale bubble-LV5-YCD-TK/GCV(5-FC) was constructed and transfected to BIU-87 cells. Hematoxylin and eosin (HE) staining was used to evaluate inflammation. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to examine cell viability. Cell-cycle distribution was analyzed with cell cycle assay. Flow cytometry assay was utilized to test apoptosis of BIU-87 cells. YCD-TK expression was examined using Western blot and quantitative Real Time-PCR (qRT-PCR), respectively. RESULTS: YCD-TK highly expressed in Nanoscale bubble mediated suicide gene therapy system. Nanoscale bubble-mediated suicide gene therapy system significantly induced inflammatory response and apoptosis compared to that of Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly reduced cell viability compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly inhibited cell cycle arrest compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble-LV5-YCD-TK/GCV/5-FC therapy system significantly reduced BIU-87 cell viability compared to that of the Nanoscale bubble-associated groups (p<0.05). CONCLUSIONS: Nanoscale bubble-mediated suicide gene therapy system, bubble-LV5-YCD-TK/GCV/5-FC, acts as a novel therapeutic strategy for bladder cancer treatment.

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes.

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

The effect of gene therapy on postoperative recurrence of small hepatocellular carcinoma (less than 5cm).

To observe the curative effect of surgery combined with gene therapy on small hepatocellular carcinoma. Seventy-seven patients with small hepatocellular carcinoma (diameter < 5 cm) underwent surgical resection. The tumor located at the edge of the liver was treated by local excision or irregular hepatectomy. The tumor in the center of the liver was resected by hepatic lobectomy in order to ensure at least a 2-cm safety margin. Fifty-four patients underwent gene therapy (gene group) one or two times before operation, whereas 23 patients underwent surgery alone (control group) selected by themselves. The injectable gene was made of ADV-TK (adenovirus containing thymidine kinase suicide gene, with a concentration of 5 × 1012/ml). The prognosis of patients was analyzed by imaging twice a year. In the gene group, the 1-, 3-, and 5-year survival rates were 91.4, 63.6, and 52.1%. In the control group, the survival rates were 84.3, 54.4, and 32.6%, respectively. There was a significant difference in the overall survival rates between two groups. Factors associated with overall survival in univariate analysis included bilirubin, prothrombin activity, cirrhosis, and gene therapy (P < 0.05). In the multivariate analysis, it included cirrhosis, gene therapy, and bilirubin. The gene therapy hepatocellular carcinoma patients with a diameter < 5 cm could significantly reduce recurrence after operation. It was worthy of being popularized.