Suicide Gene Research Articles

Sex differences in brain gene expression among suicide completers

BackgroundSuicide rates vary substantially by sex. Suicides committed by males significantly outnumber female suicides. Disparities in community and social factors provide a partial explanation for this phenomenon. Thus, the evaluation of sex differences at a biological level might contribute to the elucidation of the factors involved in this imbalance. The aim of the present study was to evaluate sex-specific gene expression patterns in the suicidal brain. Methodspostmortem samples from the dorsolateral prefrontal cortex (DLPFC) of 75 Latino individuals were analyzed. We considered the following groups: i) male suicides (n = 38), ii) female suicides (n = 10), iii) male controls (n = 20), and iv) female controls (n = 7). Gene expression profiles were evaluated by microarrays. Differentially expressed genes among the groups were identified with a linear model. Similarities and differences in the gene sets between the sexes were identified. ResultsDifferentially expressed genes were identified between suicides and controls of each sex: 1,729 genes in females and 1,997 genes in males. Female-exclusive suicide genes were related to cell proliferation and immune response. Meanwhile, male-exclusive suicide genes were associated to DNA binding and ribonucleic protein complex. Sex-independent suicide genes showed enrichment in mitochondrial and vesicular functions. LimitationsRelatively small sample size. Our diagnosis approach was limited to information found on coroner's records. The analysis was limited to a single brain area (DLPFC) and we used microarrays. ConclusionPreviously unexplored sex differences in the brain gene expression of suicide completers were identified, providing valuable foundation for the evaluation of sex-specific factors in suicide.

Demonstration of anti-tumour bystander killing with prodrug-preloaded suicide gene-engineered tumour cells: a potential improvement for cancer therapeutics

BackgroundTherapeutic approaches for cancer rely on careful consideration of finding the optimal way of delivering the pro-drug for cellular-based cancer treatment. Cell lines and cell cultures have been used in these studies to compare the in vitro and in vivo efficacy of autologous vs. allogeneic tumour cellular gene therapy. Here we have investigated and are reporting for the first time the effect of prodrug ganciclovir (GCV)-preloading (pre-treatment) in suicide gene therapy of cancer.MethodsThis study examines the effect of GCV-preloading (pre-treatment) on a range of tumour cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumour cell lines.ResultsFollowing co-culture of herpes simplex virus thymidine kinase (HSV-TK) modified tumour cells and unmodified tumour cells both in vitro and in vivo, GCV-preloading (pre-treatment) of TK-modified human and mouse mesothelioma cells and ovarian tumour cells allowed them to mediate efficiently bystander killing of neighbouring unmodified tumour cells in vitro. In contrast, GCV-preloading of TK-modified human and mouse mesothelioma cells and ovarian tumour cells abolished their in vivo ability to induce bystander killing of unmodified tumour cells, although there was some tumour regression compared to control groups but this was not statistically significant. These results suggest that preloading TK modified tumour cells with GCV needs further study to define the most effective strategy for an in vivo application to retain their bystander killing potential after exposure to lethal doses of GCV in vitro.ConclusionsThis study highlights the promising possibility of improving the efficacy of pro-drug system to prevent any damage to the immune system and enhancing this type of suicide gene therapy of cancer, as well as the need for further studies to explore the discrepancies between in vitro and in vivo results.

Detection of lentiviral suicide gene therapy in C6 rat glioma using hyperpolarised [1-13 C]pyruvate.

Hyperpolarised [1-13 C]pyruvate MRI has shown promise in monitoring therapeutic efficacy in a number of cancers including glioma. In this study, we assessed the pyruvate response to the lentiviral suicide gene therapy of herpes simplex virus-1 thymidine kinase with the prodrug ganciclovir (HSV-TK/GCV) in C6 rat glioma and compared it with traditional MR therapy markers. Female Wistar rats were inoculated with 106 C6 glioma cells. Treated animals received intratumoural lentiviral HSV-TK gene transfers on days 7 and 8 followed by 2-week GCV therapy starting on day 10. Animals were repeatedly imaged during therapy using volumetric MRI, diffusion and relaxation mapping, as well as metabolic [1-13 C]pyruvate MRS imaging. Survival (measured as time before animals reached a humane endpoint and were euthanised) was assessed up to day 30 posttherapy. HSV-TK/GCV gene therapy lengthened the median survival time from 12 to 25 days. This was accompanied by an apparent tumour growth arrest, but no changes in diffusion or relaxation parameters in treated animals. The metabolic response was more evident in the case-by-case analysis than in the group-level analysis. Treated animals also showed a 37 ± 15% decrease (P < 0.05, n = 5) in lactate-to-pyruvate ratio between therapy weeks, whereas a 44 ± 18% increase (P < 0.05, n = 6) was observed in control animals. Hyperpolarised [1-13 C]pyruvate MRI can offer complementary metabolic information to traditional MR methods to give a more comprehensive picture of the slowly developing gene therapy response. This may benefit the detection of the successful therapy response in patients.

HSV-TK Expressing Mesenchymal Stem Cells Exert Inhibitory Effect on Cervical Cancer Model.

A growing area of research is focused on cancer therapy, and new therapeutic approaches are welcomed. Mesenchymal stem cell (MSC)-based gene therapy is a promising strategy in oncology. Intrinsic tropism and migration to tumor microenvironment with off lights are attractive features of this type of cell carrier. In this way, suicide genes have also found a good platform for better performance and have shown a stronger anti-tumor mechanism by riding on mesenchymal cells. In this study, we investigated the anti-tumor activity of intratumoral injected MSCs transduced with a lentivector expressing the HSV/TK in a mouse cervical cancer model. Following the injection of MSCs transduced with lentivector carrying TK, MSCs alone or PBS into the mice tumor, ganciclovir was administered intraperitoneally during 14 days, and tumor size, survival time, natural killer (NK) cells and cytotoxic T lymphocyte (CTL) activities were assessed. We demonstrated that combination of suicide therapy and cell therapy leading m,to successful tumor inhibition. Significant reduction in tumor size was detected in test group in comparison with controls. Also, potent antitumor NK and CTL activity was seen in treatment group in comparison with controls. Our data demonstrated that the mesenchymal cells expressing TK had inhibitory effect on cervical cancer model.

Potential of Chimeric Antigen Receptor T-Cells in Cancer Therapy.

Novel approaches for targeted delivery like nanoparticles, liposomes, polymer conjugates, etc. with better safety profile needs to be developed for cancer treatment. Chimeric antigen receptors (CAR) with modified thymus cells (T-cells) showed greater potential as a therapy due to its direct effect on immune system responsible for destruction of pathogens and said equivalent to the living drug. On activation of T-cell, it binds to the antigen domains treating refractory or relapsed cancers. The receptors are termed chimeric as it consists of T-cells functioning as well as antigen-binding combined in sole receptor. This therapy showed positive success towards hematological cancers and engineered for specific protein targeting. Though the therapy is associated to several challenges like incompetence towards tumor lysis and cytokine release rate, termination of cytotoxic activity after completion of tumor eradication, etc. The control mechanisms used by CAR T-cells are apoptosis by suicide genes, dual-antigen receptor, ON-switch tumor attack and bispecific molecules as activation switch. In solid tumors, CAR T-cell therapy showed promising signs of efficacy becoming a game-changing cell therapy. CAR T-cells are optimized using different engineering resolutions and lead to broadways for therapy adoption to benefit the cancer patients.

Suicide gene therapy for the treatment of high-grade glioma: past lessons, present trends, and future prospects.

Suicide gene therapy has represented an experimental cancer treatment modality for nearly 40 years. Among the various cancers experimentally treated by suicide gene therapy, high-grade gliomas have been the most prominent both in preclinical and clinical settings. Failure of a number of promising suicide gene therapy strategies in the clinic pointed toward a bleak future of this approach for the treatment of high-grade gliomas. Nevertheless, the development of new vectors and suicide genes, better prodrugs, more efficient delivery systems, and new combinatorial strategies represent active research areas that may eventually lead to better efficacy of suicide gene therapy. These trends are evident by the current increasing focus on suicide gene therapy for high-grade glioma treatment both in the laboratory and in the clinic. In this review, we give an overview of different suicide gene therapy approaches for glioma treatment and discuss clinical trials, delivery issues, and immune responses.

Combination Therapy by Tissue-Specific Suicide Gene and Bevacizumab in Intramedullary Spinal Cord Tumor.

PurposeMalignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth.Materials and MethodsIn the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hind-limb strength.ResultsThe complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone.ConclusionThis study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.

Gene therapies for high-grade gliomas: from the bench to the bedside.

Background:Gene therapy is the most attractive therapeutic approach against high-grade gliomas (HGGs). This is because of its theoretical capability to rework gene makeup in order to yield oncolytic effects. However, some factors still limit the upgrade of these therapies at a clinical level of evidence. We report an overview of glioblastoma gene therapies, mainly focused on the rationale, classification, advances and translational challenges.Methods:An extensive review of the online literature on gene therapy for HGGs was carried out. The PubMed/MEDLINE and ClinicalTrials.gov websites were the main sources. Articles in English published in the last five years were sorted according to the best match with the multiple relevant keywords chosen. A descriptive analysis of the clinical trials was also reported.Results:A total of 85 articles and 45 clinical trials were selected. The main types of gene therapies are the suicide gene, tumor suppressor gene, immunomodulatory gene and oncolytic therapies (virotherapies). The transfer of genetic material entails replication-deficient and replication-competent oncolytic viruses and nanoparticles, such as liposomes and cationic polymers, each of them having advantages and drawbacks. Forty-eight clinical trials were collected, mostly phase I/II.Conclusion:Gene therapies constitute a promising approach against HGGs. The selection of new and more effective target genes, the implementation of gene-delivery vectors capable of greater and safer spreading capacity, and the optimization of the administration routes constitute the main translational challenges of this approach. (www.actabiomedica.it)

A doxycycline-inducible C17.2 neural stem cell-based combination of differentiation and suicide gene therapy for an in vitro tumorigenic C6 glioma model

Neural stem cell (NSC)-based gene therapies have been recently developed as effective strategies for treating brain tumors through inherent tumor tropism. However, this methodology has two considerable challenges: preventing NSCs from dying from the therapeutic agents encoded by their equipped genes before reaching tumor sites and the clearance of exogenous NSCs after therapeutic treatments. For these purposes, we established a novel doxycycline-inducible retroviral plasmid pTRE3G-TKGFP. A herpes simplex type 1 thymidine kinase (HSV1TK)-green fluorescent protein (GFP) fusion protein coding sequence was integrated into the multiple cloning site II (MCS II) of the pQcXIX vector, and the CMV IE promoter (PCMV IE) of pQcXIX was replaced with the doxycycline-inducible TRE3G promoter (PTRE3G). We then cloned the coding sequences of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and soluble interleukin 6 receptor (sIL-6R) into MCS I to combine HSV1TK (TK)/ganciclovir (GCV)-based suicide gene therapy against tumorigenic C6 glioma cells. TNF-α, IL-6 and sIL6R could efficiently induce tumorigenic C6 glioma cell differentiation, resulted in down-regulating the cell proliferation rate and the tumorigenicity of glioma cells and up-regulating the production of differentiation markers, such as connexin-43. Furthermore, gap junctions could enhance the bystander effect in suicide gene therapy. Consequently, we found that the retroviral plasmid transfected NSCs exerted stronger remedial effects on tumorigenic C6 glioma cells through the combination of differentiation and suicide gene therapy than by suicide gene therapy alone. This study is also the first case applying NSCs to conduct the combination of differentiation and TK/GCV-based suicide gene therapy on glioma cells.

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

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). 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. 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. 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.

CBMS-08 INVESTIGATION FOR NICOTINIC EFFECTS ON STEM CELL’S PROPERTY IN HSV-TK/GCV GENE THERAPY

BACKGROUNDHerpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) system is one of feasible therapeutic strategies for defeating malignant gliomas. Stem cells with intrinsic tumor tropism are used for suicide gene vehicles, which make this therapy further realistic. Nicotine is known to affect cellular migration capacity in variety types of cells but whether nicotine impacts on stem cells’ migration capacity to gliomas is not scrutinized. In this research, we investigated nicotinic impact on stem cells’ properties including tumor tropism and gap junctional intercellular communication (GJIC), which is crucial to this therapeutic strategy.METHODSMouse induced pluripotent stem cell (iPSC)-derived neural stem cells (miPS-NSCs) and human dental pulp mesenchymal stem cells (hDPSCs) were used. Nicotine cytotoxicity for 24 hours was evaluated by MTT assay for stem cells and glioma cells; GS-9L and C6 (rat), GL261 (mouse), U251 and U87 (human). Tumor tropism to glioma-conditioned medium (CM) with or without non-toxic nicotine concentrations was assessed using Matrigel Invasion Chamber. Nicotine effect on GJIC was assessed with scrape loading/dye transfer assay (SL/DT assay) for co-culture of stem cells and glioma cells (stem cell/glioma cell) or parachute assay for glioma cells alone using high-content analysis.RESULTSMTT assay revealed 1 μM of nicotine, equivalent to serum nicotine concentration in habitual smoking, is the maximum safe concentration for stem cells and glioma cells. Tumor tropism (miPS-NSCs to GL261-CM, hDPSCs to U251- or U87-CM) and GJIC of co-culture of stem cells and glioma cells (miPS-NSC/GL261, hDPSC/U251) or glioma cells alone (GS-9L, C6, GL261 and U251) were not affected by 1 μM of nicotine.CONCLUSIONSPhysiological nicotine presence did not affect (1) stem cell’s tumor tropism to gliomas and (2) GJIC between stem cells and glioma cells or within glioma cells. HSV-tk/GCV therapy may retain its therapeutic efficacy against gliomas even under physiological nicotine concentrations.

Spatiotemporally controllable diphtheria toxin expression using a light-switchable transgene system combining multifunctional nanoparticle delivery system for targeted melanoma therapy

Gene therapy with external gene insertion (e. g. a suicide gene) and expression specifically in mutated tumor cells has shown to be a promising strategy in treatment of tumors. However, current tumor gene therapy often suffered from low efficiency in gene expression and off-target effects which may cause damage to normal tissues. To address these issues, in this study, a light-switchable transgene nanoparticle delivery system loaded with a diphtheria toxin A (DTA) segment encoded gene, a suicide gene for tumor cells, was developed. The nanoparticles contained vitamin E succinate-grafted polyethyleneimine core and arginylglycylaspartic acid (RGD)-modified pegylated hyaluronic acid shell for targeted delivery of the loaded gene to tumor cells via receptor-mediated (CD44 and αvβ3) endocytosis. Notably, the expression of target proteins in tumor cells could be conveniently regulated by adjusting the blue light intensity in the Light-On system. In in-vitro studies in cultured B16-F10 cells, the pG-DTA-loaded nano-micelles showed greatly improved inhibitory rate compared with the pG-DTA group. Moreover, in the tumor-bearing C57BL/6 mice model, the pG-DTA-loaded nanoparticle exhibited greatly improved efficacy and reduced systemic toxicity with significantly increased survival rate after 21 days. Significantly suppressed tumor angiogenesis was also identified in the nanoparticle-treated group likely due to the targeting ability of the RGD-modified nanoparticle. All the above results indicated that the combination of a light-switchable transgene system with a nanoparticle-based targeted delivery system have great potentials in gene therapy of malignant tumors with improved precision and efficacy.

Prodrugs for nitroreductase-based cancer therapy-3: Antitumor activity of the novel dinitroaniline prodrugs/Ssap-NtrB enzyme suicide gene system: Synthesis, in vitro and in silico evaluation in prostate cancer

Prodrugs for targeted tumor therapies have been extensively studied in recent years due to not only maximising therapeutic effects on tumor cells but also reducing or eliminating serious side effects on healthy cells. This strategy uses prodrugs which are safe for normal cells and form toxic metabolites (drugs) after selective reduction by enzymes in tumor tissues.In this study, prodrug candidates (1-36) containing nitro were designed, synthesized and characterized within the scope of chemical experiments. Drug-likeness properties of prodrug candidates were analyzed using DS 2018 to investigate undesired toxicity effects. In vitro cytotoxic effects of prodrug canditates were performed with MTT assay for human hepatoma cells (Hep3B) and prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) as healthy control. Non-toxic compounds (3, 5, 7, 10, 12, 15, 17, 19 and 21–23), and also compounds (1, 2, 5, 6, 9, 11, 14, 16, 20 and 24) which had low toxic effects, were selected to examine their suitability as prodrug canditates. The reduction profiles and kinetic studies of prodrug/Ssap-NtrB combinations were performed with biochemical analyses. Then, selected prodrug/Ssap-NtrB combinations were applied to prostate cancer cells to determine toxicity. The results of theoretical, in vitro cytotoxic and biochemical studies suggest 14/Ssap-NtrB, 22/Ssap-NtrB and 24/Ssap-NtrB may be potential prodrug/enzyme combinations for nitroreductase (Ntr)-based prostate cancer therapy.

Structure-Activity Relationships for CYP4B1 Bioactivation of 4-Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates.

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 μM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.

Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants.

CRISPR/Cas9 gene-editing technologies have been very effective in editing target genes in all major crop plants and offer unprecedented potentials in crop improvement. A major challenge in using CRISPR gene-editing technology for agricultural applications is that the target gene-edited crop plants need to be transgene free to maintain trait stability and to gain regulatory approval for commercial production. In this article, we present various strategies for generating transgene-free and target gene-edited crop plants. The CRISPR transgenes can be removed by genetic segregation if the crop plants are reproduced sexually. Marker-assisted tracking and eliminating transgenes greatly decrease the time and labor needed for identifying the ideal transgene-free plants. Transgenes can be programed to undergo self-elimination when CRISPR genes and suicide genes are sequentially activated, greatly accelerating the isolation of transgene-free and target gene-edited plants. Transgene-free plants can also be generated using approaches that are considered non-transgenic such as ribonucleoprotein transfection, transient expression of transgenes without DNA integration, and nano-biotechnology. Here, we discuss the advantages and disadvantages of the various strategies in generating transgene-free plants and provide guidance for adopting the best strategies in editing a crop plant.

High-Throughput Preparation of Antibacterial Polymers from Natural Product Derivatives via the Hantzsch Reaction.

SummaryThe Hantzsch and free-radical polymerization reactions were combined in a one-pot high-throughput (HTP) system to simultaneously prepare 30 unique polymers in parallel. Six aldehydes derived from natural products were used as the starting materials to rapidly prepare the library of 30 poly(1,4-dihydropyridines). From this library, HTP evaluation methods led to the identification of an antibacterial polymer. Mechanistic studies revealed that the dihydropyridine group in the polymer side-chain structure plays an important role in resisting bacterial attachment to the polymer surface, thus leading to the antibacterial function of this polymer. This research demonstrates the value of multicomponent reactions (MCRs) in interdisciplinary fields by discovering functional polymers for possible practical applications. It also provides insights to further developing new functional polymers using MCRs and HTP methods with important implications in organic chemistry, polymer chemistry, and materials science.

Cell-based interferon gene therapy using proliferation-controllable, interferon-releasing mesenchymal stem cells

An important safety concern on cell-based gene therapy is that few methods have been available to control the proliferation and functioning of therapeutic protein-expressing cells after transplantation. We previously reported that the proliferation and functioning of the cells transfected with herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, can be controlled by administration of ganciclovir. In this study, we tried to control the amount of murine interferon-γ (IFN-γ) secreted from transplanted murine mesenchymal stem cell line C3H10T1/2 cells to achieve safe cell-based IFN-γ gene therapy for cancer. C3H10T1/2 cells were transfected with HSVtk- and murine IFN-γ-expressing plasmid vectors to obtain C3H10T1/2/HSVtk/IFN-γ cells. C3H10T1/2/HSVtk/IFN-γ cells released IFN-γ and were sensitive to ganciclovir. C3H10T1/2/HSVtk/IFN-γ cells significantly suppressed the proliferation of murine adenocarcinoma cell line colon26 cells both in vitro and in vivo. Moreover, subcutaneous administration of ganciclovir to mice transplanted with NanoLuc luciferase-expressing C3H10T1/2/HSVtk cells for three consecutive days reduced the luminescence signals from the transplanted cells. These results indicate that the cell regulation system using HSVtk gene and ganciclovir can be useful for safe and efficient cell-based IFN-γ gene therapy for cancer.

Targeting the EGF-Receptor and CD38 in Solid and Haematological Malignancies with Nanobody-Based Heavy Chain Antibodies and AAV Vectors

Abstract Background Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells. Methods We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays. Results EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples. Conclusion Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors. Legal entity responsible for the study Friedrich Koch-Nolte. Funding DFG (German Research Foundation) and SFB (Collaborative Research Centres). Disclosure The author has declared no conflicts of interest.

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.

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.