Transfer and expression of the genes of Bacillus branched chain alpha-oxo acid decarboxylase in Lycopersicun esculentum

Engineering of higher plants for increased cold tolerance requires a chemical modification of membrane fluidity in both organelles and cytoplasm of plant cells. A small number of microorganisms use branched chain fatty acids as their membrane constituents to maintain membrane fluidity, instead of unsaturated fatty acids. One of the key enzymes facilitating synthesis of branched chain fatty acids in Bacillus subtilis 168s is the branched chain alpha-oxo acid decarboxylase. To examine the role of branched chain fatty acids in plants and the potential for low temperature tolerance, the A and B genes encoding the alpha and beta polypeptides, respectively, of the branched chain alpha-oxo acid decarboxylase were introduced into the genome of tomato plants. The mas promoters in the plant expression vector system facilitated the expression of the A and B genes. Transgenic plants were regenerated and shown to produce both the alpha and beta polypeptides. Comparative analysis of a small number of transgenic tomato plants and non-transformed control plants grown at 4ºC showed enhanced cold tolerance in the transformed plants. These findings, if confirmed by a larger scale analysis, suggest a potential role for branched chain fatty acids as a protective mechanism for growth of plants under sub optimal temperatures

Transfer and expression of the genes of Bacillus branched chain alpha-oxo acid decarboxylase in Lycopersicun esculentum

Engineering of higher plants for increased cold tolerance requires a chemical modification of membrane fluidity in both organelles and cytoplasm of plant cells. A small number of microorganisms use branched chain fatty acids as their membrane constituents to maintain membrane fluidity, instead of unsaturated fatty acids. One of the key enzymes facilitating synthesis of branched chain fatty acids in Bacillus subtilis 168s is the branched chain alpha-oxo acid decarboxylase. To examine the role of branched chain fatty acids in plants and the potential for low temperature tolerance, the A and B genes encoding the alpha and beta polypeptides, respectively, of the branched chain alpha-oxo acid decarboxylase were introduced into the genome of tomato plants. The mas promoters in the plant expression vector system facilitated the expression of the A and B genes. Transgenic plants were regenerated and shown to produce both the alpha and beta polypeptides. Comparative analysis of a small number of transgenic tomato plants and non-transformed control plants grown at 4\ubaC showed enhanced cold tolerance in the transformed plants. These findings, if confirmed by a larger scale analysis, suggest a potential role for branched chain fatty acids as a protective mechanism for growth of plants under sub optimal temperatures

Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

<p>Abstract</p> <p>Background</p> <p>We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding <it>Renilla </it>luciferase-<it>Aequorea </it>GFP fusion protein, β-galactosidase, and β-glucuronidase into the <it>F14.5L</it>, <it>J2R</it>, and <it>A56R </it>loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice</p> <p>Methods</p> <p>A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.</p> <p>Results</p> <p>we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.</p> <p>Conclusions</p> <p>These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.</p

Oncolytic vaccinia therapy of squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Novel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC) of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) as an oncolytic agent against a panel of six human head and neck SCC cell lines.</p> <p>Results</p> <p>All six cell lines supported viral transgene expression (β-galactosidase, green fluorescent protein, and luciferase) as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150-fold titer increase over 72 hrs) were observed in four of the cell lines. At a multiplicity of infection (MOI) of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in ≥ 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 × 10⁶pfu) intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2–4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression.</p> <p>Conclusion</p> <p>These results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both <it>in vitro </it>and <it>in vivo</it>, and support its continued investigation in future clinical trials.</p

Regression of Human Prostate Tumors and Metastases in Nude Mice following Treatment with the Recombinant Oncolytic Vaccinia Virus GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma

Significant Growth Inhibition of Canine Mammary Carcinoma Xenografts following Treatment with Oncolytic Vaccinia Virus GLV-1h68

Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma

Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli

Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast change

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

<p>Abstract</p> <p>Background</p> <p>Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic.</p> <p>Methods</p> <p>Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects.</p> <p>Results</p> <p>We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia.</p> <p>Conclusion</p> <p>Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.</p

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer. Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET. Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy