HSVtk Expression Research Articles

Effects of cancer-associated fibroblasts deletion using HSVtk suicide system in OSCC.

To evaluate the biological characteristics of oral cancer cells co-cultured with cancer-associated fibroblasts (CAFs)-HSVtk and to assess the reliability of the CAFs-HSVtk suicide system in a co-culture model. CAFs were lentivirus-transfected with PCDH-HSVtk. Ganciclovir (GCV) was added and the survival rates of the CAFs-HSVtk were measured. In parallel with the selective elimination of CAFs, comparison was made of the effects of CAF-HSVtk on tumor cell proliferation/migration in a CAFs-tumor co-cultural system. Cell death of co-cultured oral cancer cells was evaluated by flow cytometry. Q-PCR analysis showed that the expression of HSVtk in the CAFs-HSVtk group was significantly higher than in the control group (p < 0.01). The survival rates of CAFs-HSVtk with GCV were significantly reduced (p < 0.01). Following selective depletion of CAFs-HSVtk, the growth and migration rates of oral cancer cells co-cultured with CAFs-HSVtk were reduced in a mixture ratio of 1:2 (p < 0.01, p < 0.01). Enhanced proliferation and migration rates of oral cancer cells in co-culture were seriously impaired after deleting CAFs using the HSVtk suicide system, while oral tumor cell death was not affected. Therefore, CAFs-HSVtk can be utilized as a valid model for CAF signature identification.

Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma

Gene therapy has been introduced as an alternative to radiation and chemical therapy for glioblastoma. Biomimetic nanoparticles coated with cell membranes (CM) have advantages such as high biocompatibility and prolong half-life. To apply CM coated nanoparticles to gene delivery, the polyethylenimine (PEI25k)/plasmid DNA (pDNA) complexes were coated with CM from C6 rat glioblastoma cells. With the CM covering, the PEI25k/pDNA complexes formed stable nanoparticles with negative surface charge. The PEI25k/pDNA/CM nanoparticles had high colloidal stability and could be stored for approximately 20 days without aggregation. The transfection efficiency of the PEI25k/pDNA/CM nanoparticles was higher than that of the PEI25k/pDNA complex in serum-containing medium. This suggests that serum does not interfere with transfection efficiency of the nanoparticles. Moreover, the PEI25k/pDNA/CM nanoparticles had lower toxicity than the PEI25k/DNA complex in vitro and in vivo. The PEI25k/pDNA/CM nanoparticles prepared with CMs of different types of cells were transfected into cells. The results showed that the PEI25k/pDNA/CM nanoparticles with the C6 CM had the highest transfection efficiency to C6 cells, suggesting the homotypic targeting effect. The therapeutic effects of the nanoparticles were evaluated in intracranial C6 transplanted glioblastoma animal models. The PEI25k/pDNA/CM nanoparticles were prepared with herpes simplex virus thymidine kinase plasmid (pHSVtk) and injected into the tumor locally. The results showed that the PEI25k/pHSVtk/CM nanoparticles induced higher HSVtk expression compared with the PEI25k/pHSVtk complex. Furthermore, tumor size was reduced more efficiently by the PEI25k/pHSVtk/CM nanoparticles than by the PEI25k/pHSVtk complex. Overall results indicate that PEI25k/pDNA/CM nanoparticles are suitable for pDNA delivery to glioblastoma.

A Novel Ex-Vivo Model of Prolonged Osteoblast Suppression in Multiple Myeloma

Multiple myeloma (MM) is the most frequently involves the skeleton, with over 80% of patients developing osteolytic bone disease (MMBD). Importantly, lytic bone lesions in MM patients rarely heal even during long-term complete remission without detectable myeloma cells. Mesenchymal stem cells from MM patients (MM-MSCs) display distinct gene profiles and impaired osteoblast (OB) differentiation compared with those of normal donors, even after many passages in vitro. We found that MM causes long-term inhibition of OB differentiation by inducing epigenetic changes in the Runx2 promoter in MSCs, which persist in the absence of myeloma cells. Importantly, MSCs from myeloma patients expressed elevated Gfi1, a factor that modifies the chromatin structure of Runx2. However, the molecular mechanisms for MM-induced long-term OB suppression remain poorly understood. Exploring these mechanisms in animal models is costly, slow and complex. Further, it is difficult to replicate the bone marrow microenvironment in vitro.We have developed an ex-vivo bone organ culture assay (EVOCA) that more closely replicates the marrow microenvironment, to further investigate the molecular mechanisms responsible for the protracted OB suppression in MM and to screen bone anabolic agents for MMBD. 5TGM1 murine MM cells expressing thymidine kinase and GFP (5TGM1-GFI-TK) were co-cultured in EVOCA with mouse long bones (femurs and tibias from 4 weeks old mice). Bones cut longitudinally with marrow in place were cultured in 35 mm dishes +/- 5TGM1-GFI-TK cells (2x105/ml) and 10 % FCS in a 1:1 mix of RPMI/αMEM for 4 days. Conditioned media were collected and the bone resorption marker, c-terminal telopeptide crosslink of type 1 collagen (CTX) was measured by ELISA. CTX levels were increased in 5TGM1-containing EVOCAs compared to cultures without 5TGM1 cells (5.5±0.8 ng/ml vs. 9.8±1.0 ng/ml, p<0.001). 5TGM1-GFI-TK cell can be visualized by florescence microscopy, and HSV-TK expression permits selective cell killing with ganciclovir (Gcv), we then treated EVOCAs +/- 20 µg/ml Gcv on days 4-7 of culture. Myeloma cell survival was monitored by florescence microscopy and assay of IgG2b in the media. Gcv killed 100% of the myeloma cells. EVOCA cultures were continued for 1- 2 weeks more, then bones and outgrowth cells were homogenized, and lysates and RNA assayed for Runx2 and Osterix expression by Western blot and qPCR. Both Runx 2 and Osterix levels were 25% lower in EVOCAs treated with Gcv, and 50% lower compared to EVOCas not cultured with 5TGM1 cells. Runx2 and Osterix levels in EVOCAs cultured with Gcv but no MM cells were similar to levels in vehicle-treated bones. NFATc-1, cathepsin K, RANKL and OPG levels and osteoclast numbers were then measured in EVOCAs at 2 weeks post Gcv treatment. 5TGM1-containing EVOCAs had increased osteoclast numbers (2X) and levels of cathepsin K and NFATc-1. Further, RANKL/OPG ratios were increased in bone extracts from EVOCAs co-cultured with 5TGM1 cells compared with controls, regardless of Gcv treatment.We next modified EVOCA to evaluate new bone formation. A 0.5mm diameter hole was initially made with a drill bit in intact bones (uncut) and the bones co-cultured +/- 5TGM1-GFP-TK cells (2x105 /ml) 10% FCS in a 1:1 mix of RPMI/αMEM for 4 days. The cultures were then treated +/- Gcv for 3 days. At day 7, no 5TGM1 cells were detectable. The media was then changed to αMEM, and the EVOCAs continued for 1 week. Then 50µg/ml ascorbic acid and 10 mM β-glycerophosphate were added and the cultures continued for 7 more days. Bones were stained with Alizarin Red S to detect new bone formation at the drill site. New bone formation was confirmed by µCT. No new bone formation was detected in bones from EVOCAs initiated with 5TGM1 cells regardless of 5TGM1 removal. In contrast, new bone formation was easily evident in bones not exposed to MM cells.The results show that increased bone resorption and OB suppression persist in MM-exposed bone even after myeloma cells are removed. The assay should allow us to follow the sequential development of lytic bone lesions, tumor growth, and protracted OB suppression, providing a useful tool for studies of MMBD, and evaluation of anabolic agents and anti-myeloma therapy in an intact marrow microenvironment. DisclosuresRoodman:PharmaMar: Research Funding.

ApoE-modified liposomes mediate the antitumour effect of survivin promoter-driven HSVtk in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumour with high global morbidity and mortality associated with its multiple aetiologies, poor prognosis, resistance to chemotherapeutic drugs and high rate of recurrence. Here, we evaluated a gene therapy strategy that targets 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. Apolipoprotein E (ApoE)-modified liposomes were used for targeted gene delivery to the tumour tissue, and the survivin promoter was used to drive HSVtk expression in HCC cells. Our results showed that the survivin promoter was specifically activated in tumour cells, and HSVtk was expressed selectively in tumour cells. In combination with GCV treatment, HSVtk expression resulted in the inhibition of HCC cell proliferation via enhanced apoptosis. In addition, tail vein injection of ApoE-HSVtk significantly suppressed the growth of xenograft tumours through an apoptosis-dependent pathway and extended the survival time of tumour-bearing mice. In summary, this study illustrates an effective cancer-specific gene therapy strategy for HCC that can be further developed for future clinical trials.

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.

WITHDRAWN: Golgi membrane protein GP73 modified-liposome mediates the antitumor effect of surviving promoter-driven HSVtk in hepatocellular carcinoma

// Fanxiu Meng 1, * , Lina Zhang 1, * , Chunyang Xu 2 , Qi Zhang 1 , Yongfang Li 1 , Yingmin Zhang 1 , Na Zhao 1 , Baofeng Yu 1 , Rui Guo 1 , Hailong Wang 1 , Jun Xie 1 , Gongqin Sun 1, 3 , Gaopeng Li 4 , Jun Xu 4 , Qin Qin 5 and Xiushan Dong 6 1 Department of Biochemistry and Molecular Biology, Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi, China 2 Department of Biological Sciences, The Dietrich School of Arts and Sciences, University Of Pittsburgh, Pittsburgh, PA, USA 3 Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, USA 4 Department of General Surgery, Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan, Shanxi, China 5 Central Laboratory, Shanxi Provincial People's Hospital, Shanxi Medical University, Taiyuan, Shanxi, China 6 Department of General Surgery, Shanxi Dayi Hospital, Taiyuan, Shanxi, China * These authors contributed equally to this work Correspondence to: Baofeng Yu, email: shanxiyangchengdg@163.com Jun Xu, email: junxuty@163.com Keywords: hepatocellular carcinoma; gene therapy; HSVtk/GCV; GP73; apoptosis Received: July 29, 2017 Accepted: October 28, 2017 Published: January 02, 2018 ABSTRACT 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 very importance. 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 for HCC that can be further developed for future clinical trials.

The future of mesenchymal stem cell-based therapeutic approaches for cancer – From cells to ghosts

Mesenchymal stem cells (MSCs) are multipotent stromal cells which can differentiate into a variety of cell types including osteoblasts, adipocytes and chondrocytes. They are normally resident in adipose tissue, bone marrow and the umbilical cord, but can also be found in other tissues and are known to be recruited to sites of wound healing as well as growing tumours. The therapeutic potential of MSCs has been explored in a number of phase I/II and III clinical trials, of which several were targeted against graft-versus-host disease and to support engraftment of haematopoietic stem cells (HSCs), but currently only very few in the oncology field. There are now three clinical trials either ongoing or recruiting patients that use MSCs to treat tumour disease. In these, MSCs target gastrointestinal, lung and ovarian cancer, respectively. The first study uses MSCs loaded with a HSV-TK expression construct under the control of the CCL5 promoter, and has recently reported successful completion of Phase I/II. While no adverse side effects were seen during this study, no outcomes with respect to therapeutic benefits have been published. The other clinical trials targeting lung and ovarian cancer will be using MSCs expressing cytokines as therapeutic payload. Despite these encouraging early steps towards their clinical use, many questions are still unanswered regarding the biology of MSCs in normal and pathophysiological settings. In this review, in addition to summarising the current state of MSC-based therapeutic approaches for cancer, we will describe the remaining questions, obstacles and risks, as well as novel developments such as MSC-derived nanoghosts.

Development of a genetic sensor that eliminates p53 deficient cells

The TP53 gene fulfills a central role in protecting cells from genetic insult. Given this crucial role it might be surprising that p53 itself is not essential for cell survival. Indeed, TP53 is the single most mutated gene across different cancer types. Thus, both a theoretical and a question of significant practical applicability arise: can cells be programmed to make TP53 an essential gene? Here we present a genetic p53 sensor, in which the loss of p53 is coupled to the rise of HSV-TK expression. We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components.

Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells.

Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

Lentiviral HSV-Tk.007-mediated suicide gene therapy is not toxic for normal brain cells.

Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd.

Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for hypoxia-specific gene therapy

Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.

Chimeric antigen receptor (CAR+) modified T cells targeting prostate specific membrane antigen (PSMA) in patients (pts) with castrate metastatic prostate cancer (CMPC).

TPS3115 Background: A phase I dose-escalating study to assess safety, dose and targeting efficiency of genetically modified autologous human T cells targeted to PSMA was initiated. Preclinical models demonstrated anti-tumor activity and accumulation, migration, and persistence of these cells to tumor. The autologous PSMA-targeted T cells utilizes the P28z second generation chimeric antigen receptor following iv cyclophosphamide (Cy). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, rendering T cells sensitive to ganciclovir for immediate T cell elimination. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize these T cells Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts were dosed from 107 to 3 x 107 CAR+ T cells/kg.All 7 pts received 300mg/m2 of Cy one day before infusion. Baseline and post treatment imaging included FDG, FDHT and 18F-FIAU PET scans. Results: Three pts in cohort 1 received 1 x 107 CAR+ T cells/kg safely. A fourth pt received the same dose with a modified vector with higher copy number. One pt had stable disease for &gt; 6 months; a second pt has stable scans for &gt; 20 months; the third and fourth patients progressed. Of 3 pts in cohort 2, one received 1.5 x 107 CAR+ T cells/kg and 2 received 3 x 107 CAR+ cell/kg. All 3 had intermittent fever spikes up to 39oC associated with increased levels of IL-4, IL-8, IP-10, sIL-2ra and IL-6 suggesting T cell activation. CAR+ cells persisted in the circulation for up to 2 weeks. Scans with 18F-FIAU labeling suggests that imaging may be cell dose dependent. Conclusions: We have shown that pts can be safely treated with an ex vivo transduction, expansion and therapeutic protocol for the generation of PSMA targeted T cells. Cytokine production suggests in vivo activation and persistence of T cells in blood for up to 2 weeks. Ongoing imaging with 18F may be suboptimal; a second cohort of pts will be studied with 124I-FIAU. Clinical trial information: NCTO1140373.

Ultrasound-targeted HSVtk and Timp3 gene delivery for synergistically enhanced antitumor effects in hepatoma

Cancer gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited by non-targeted and insufficient gene transfer. We evaluated gene therapy targeting hepatocellular carcinoma (HCC) using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system and the tissue inhibitor of metalloproteinase 3 (Timp3) gene. Ultrasound-targeted microbubble destruction (UTMD) targeted gene delivery to the tumor tissue, and the α-fetoprotein promoter targeted HSVtk expression to the HCC cells. Human HepG2 cells transfected with the HSVtk or Timp3 gene demonstrated a reduction in cell viability by >40% compared with the vector control. Cell viability was further inhibited by over 50% with co-transfection of the genes. HepG2 cells were inoculated subcutaneously into athymic mice to induce tumors. UTMD-mediated delivery of HSVtk or Timp3 suppressed tumor growth by >45% and increased survival of tumor-bearing animals (P<0.01 vs vector control). Co-delivery of the genes resulted in a further 30% improvement in tumor suppression and significant extension of animal survival (P<0.01 vs vector control). Targeted gene delivery increased the number of apoptotic cells and decreased the vascular density of the tumors. Targeted co-delivery of the genes synergistically improved the antitumor effects and may provide an effective therapy for HCC.

Chimeric antigen receptor (CAR+) modified T cells targeting prostate-specific membrane antigen (PSMA) in patients (pts) with castrate metastatic prostate cancer (CMPC).

72 Background: A phase I dose-escalating study to assess safety, dose and targeting efficiency of genetically modified autologous human T cells targeted to PSMA was initiated. Preclinical models demonstrated anti-tumor activity and accumulation, migration, and persistence of these cells to tumor. The autologous PSMA-targeted T cells utilizes the P28z second generation chimeric antigen receptor following iv cyclophosphamide (Cy). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, rendering T cells sensitive to ganciclovir for immediate T cell elimination. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize these T cells. Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts were dosed from 107 to 3 x 107 CAR+ T cells/kg. All 7 pts received 300mg/m2 of Cy one day before infusion. Baseline and post treatment imaging included FDG, FDHT and 18F-FIAU PET scans. Results: Three pts in cohort 1 received 1 x 107 CAR+ T cells/kg safely. A fourth pt received the same dose with a modified vector with higher copy number. One pt had stable disease for &gt; 6 months; a second pt has stable scans for &gt; 16 months; the third and fourth patients progressed. Of 3 pts in cohort 2, one received 1.5 x 107 CAR+ T cells/kg and 2 received 3 x 107 CAR+ cell/kg. All 3 had intermittent fever spikes up to 39oC associated with increased levels of IL-4, IL-8, IP-10, sIL-2ra and IL-6 suggesting T cell activation. CAR+ cells persisted in the circulation for up to 2 weeks. Scans with 18F-FIAU labeling suggests that imaging may be cell dose dependent. Conclusions: We have shown that pts can be safetly treated with an ex vivo transduction, expansion and therapeutic protocol for the generation of PSMA targeted T cells. Cytokine production suggests activation of these T cells with their persistence in blood for up to 2 weeks. If imaging with FIAU is suboptimal, a second cohort of pts will be studied with 124I- FIAU. Clinical trial information: NCT01140373.

The therapeutic effect of PEI-Mn0.5Zn0.5Fe2O4nanoparticles/pEgr1-HSV-TK/GCV associated with radiation and magnet-induced heating on hepatoma

Comprehensive therapy based on the integration of hyperthermia, radiation, gene therapy and chemotherapy is a promising area of study in cancer treatment. Using PEI-Mn(0.5)Zn(0.5)Fe(2)O(4) nanoparticles (PEI-MZF-NPs) as a gene transfer vector, the authors transfected self-prepared pEgr1-HSV-TK into HepG2 cells and measured the expression of the exogenous gene HSV-TK by RT-PCR. The results showed that HSV-TK was successfully transfected into HepG2 cells and the expression levels of HSV-TK remained stable. Besides, PEI-MZF-NPs were used as magnetic media for thermotherapy to treat hepatoma by magnet-induced heating, combined with radiation-gene therapy. Both in vitro and in vivo results suggest that this combined treatment with gene, radiation and heating has a better therapeutic effect than any of them alone. The apoptotic rate and necrotic rate of the combined treatment group was 51.84% and 15.45%, respectively. In contrast, it was only 20.55% and 6.80% in the radiation-gene group, 7.49% and 3.62% in the radiation-alone group, 15.23% and 7.90% in the heating-alone group, and only 3.52% and 2.16% in the blank control group. The inhibition rate of cell proliferation (88.5%) of the combined treatment group was significantly higher than that of the radiation-gene group (59.5%), radiation-alone group (37.6%) and heating-alone group (60.6%). The tumor volume and mass inhibition rate of the combined treatment group was 94.45% and 93.38%, respectively, significantly higher than 41.28% and 33.58% of the radiation-alone group, 60.76% and 52.18% of the radiation-gene group, 79.91% and 77.40% of the heating-alone group. It is therefore concluded that this combined application of heating, radiation and gene therapy has a good synergistic and complementary effect and PEI-MZF-NPs can act as a novel non-viral gene vector and magnetic induction medium, which offers a viable approach for the treatment of cancer.

Oxidative Stress-Regulated Lentiviral TK/GCV Gene Therapy for Lung Cancer Treatment

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lens-specific promoter LEP503

To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503. Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk). The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80°C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA). Expression efficiency of EGFP in RPEC group was 62.3%, 68.3% in NIH3T3 group, 75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0.29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51%, and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD(1) = -0.047, P = 0.671; MD(2) = 0.027, P = 0.912). The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV, it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.

Targeting castration resistant prostate cancer (CRPC) with autologous PSMA-directed CAR+ T cells.

TPS4700 Background: Based on our preclinical animal models, we initiated a phase I dose-escalating study to assess safety, dose requirement and targeting efficiency of genetically directed autologous human T cells targeted to Prostate Specific Membrane Antigen (PSMA). Our approach is based on the infusion of autologous PSMA-targeted T cells utilizing the P28z second generation chimeric antigen receptor (CAR) in patients (pts) with metastatic CRPC, following iv cyclophosphamide (Cy) (trial NCT01140373). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, and renders T cells sensitive to ganciclovir for immediate T cell elimination if needed. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize adoptively transferred T cells. The aims of the trial are to assess: (1) safety of PSMA-targeted T cells; (2) biologic and anti-tumor effects; (3) T cell persistence at tumor site; and (4) immune response. Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts will be treated at 3 dose levels from 107 to 108 CAR+ T cells/kg. Four patients have been enrolled; 3 have been treated with 300mg/m2 of Cy one day before infusion of 107 CAR+ T cells/kg. Pts underwent baseline and post treatment CT, bone and PET scans. Pts are followed weekly, then monthly with blood work including immune and vector sequence monitoring. Results: The first 3 pts within the first cohort were successfully treated without toxicity. Two had stable disease for greater than 6 months with the third patient having disease progression. There were no acute adverse events. Conclusion: We have established an ex vivo transduction, expansion and therapeutic protocol for the generation and testing of safe, clinical-grade, PSMA targeted T cells. Pts enrolled at the next dose level of 3 x 107 CAR+ T cells/kg will be assessed as described and by imaging the transduced T cell population using 18F-FIAU as a radiotracer. The data pertaining to the planned T cell imaging will be presented as well.

Over-expression of miR-145 enhances the effectiveness of HSVtk gene therapy for malignant glioma

This study attempts to combine two findings toward developing a rational strategy for improved therapy for glioma. One of the findings, made in this pre-clinical study, is that an hTERT-targeting ribozyme-controlled HSVtk gene (hTERT.Rz.HSVtk) exerts anti-tumor effects. The second observation is that the over-expression of a small noncoding RNA, miR-145, causes down-regulation of metastasis-related genes, such as PLAUR, SPOCK3, ADAM22, SLC7A5 and FASCN1. While blocking in vivo tumor growth only slightly, over-expression of miR-145 significantly inhibits both the migration and invasion of U87MG/U373MG glioma cells. We hypothesized that a simultaneous adenoviral-mediated over-expression of miR-145 might enhance the anti-tumor effects of hTERT.Rz.HSVtk and that a combination therapy with miR-145 and the HSVtk gene would be an effective approach for treating glioma. We tested this by developing adenoviral vectors that over-express miR-145 under the CMV promoter and employing them in combination with hTERT.Rz.HSVtk expression, both in vitro and in vivo in animal studies. We found that the adenovirus Ad5CMV.Rz.HSVtk.miR145 harboring an HSVtk expression cassette plus miR-145 produced prolonged survival benefits compared to administration of Ad5CMV.Rz.HSVtk or Ad5CMV.miR-145 alone. This study demonstrates that combination therapy using the hTERT.Rz.HSVtk gene together with miR-145 over-expression produces enhanced anti-tumor effects compared to that resulting from hTERT.Rz.HSVtk gene therapy alone.

In vitro and in vivo double-enhanced suicide gene therapy mediated by generation 5 polyamidoamine dendrimers for PC-3 cell line

BackgroundOne of the most frequently used and efficient suicide gene therapies for prostate cancer is HSV-TK/GCV system, but its application has been limited due to lack of favorable gene vector and the reduction of "bystander effect". We investigated the effect of a novel combination of HSV-TK/GCV fused with Cx43 and gemcitabine using non-viral vector generation 5 polyamidoamine dendrimers (G5-PAMAM-D) on PC-3 cells.MethodsRT-PCR and Western blot were used to detect TK and Cx43 expression. Cell viability and proliferation were measured by using MTT assay. Cell apoptosis was detected with double-staining of Annexin V-FITC and propidium iodide (PI) by flow cytometry. Nude mice models were established to evaluate the therapeutic effect in vivo.ResultsG5-PAMAM-D efficiently delivered recombinant plasmids into PC-3 cells and HSV-TK and Cx43 could be expressed successfully. With gemcitabine, G5-PAMAM-D mediated HSV-TK and Cx43 expression effectively inhibited prostate cancer PC-3 cell proliferation, leading to more cellular apoptosis and inhibiting PC-3 tumor growth in nude mice models.ConclusionsThis study illustrates that this new suicide gene system mediated by G5-PAMAM-D is effective in decreasing PC-3 cell proliferation and inducing cell apoptosis, and inhibiting tumor growth in vivo. In a word, our study could provide a potential approach for gene therapy of prostate cancer.