The potential of recombinant vesicular stomatitis virus-mediated virotherapy against metastatic colon cancer.

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related mortality in the United States. The liver and lung are the most common sites of distant metastasis of CRC. The approval of newer chemotherapeutic agents such as oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab has significantly improved survival, yet the majority of patients still succumb to the disease in less than 2 years. Novel therapeutic agents that can provide significant clinical benefit for metastatic CRC patients are needed. Oncolytic vesicular stomatitis virus (VSV) is a promising tool as a cancer therapeutic agent. In this study, we examined the feasibility of repeated intravenous infusions of rVSV in multiple CRC lung metastases, compared with repeated hepatic arterial administration in multifocal CRC liver metastasis in immune competent rats. We established a multifocal liver metastases model or the multiple lung metastases model using a CRC cell line, RCN-H4, implanted into syngeneic F344/DuCrj rats. 4.0x10(6) plaque-forming units (pfu) of recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein [rVSV-NDV/F(L289A)] were administered 3 times for 3 consecutive days locally via the hepatic artery for liver metastases or systemically via the penial vein for lung metastases. In the liver metastasis model, significantly enhanced survival was observed with rVSV-NDV/F(L289A)-treated rats (P=0.0196). Median survival was 110 and 25 days, respectively. In addition, 4 out of 7 of the rVSV-NDV/F(L289A)-treated rats demonstrated long-term survival exceeding 100 days. The long-term surviving rats were sacrificed to evaluate for residual malignancy. Liver tumors were not detected. In the lung metastasis model, median survival was 10 [VSV-NDV/F(L289A)-treated rats] and 7 days (control). Although survival was significantly prolonged (P<0.001), none of the rats achieved long-term survival. VSV virotherapy has potential for CRC liver and lung metastases, although systemic venous delivery is much less effective than locoregional delivery such as hepatic arterial infusion.

A versatile and tunable coating strategy allows control of nanocrystal delivery to cell types in the liver.

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.

Oncolytic vesicular stomatitis virus administered by isolated limb perfusion suppresses osteosarcoma growth.

A significant limitation to oncolytic virotherapy in vivo is the lack of a clinically relevant means of delivering the virus. We evaluated the oncolytic activity of vesicular stomatitis virus (VSV) in human osteosarcoma cells and explored isolated limb perfusion (ILP) as a novel oncolytic virus delivery system to extremity sarcoma in immune-competent rats. Human and rat osteosarcoma cells transduced with rVSV-lacZ uniformly expressed ß-gal. VSV was fully capable of replicating its RNA genome in all osteosarcoma cell lines, and efficiently killed them in time- and dose-dependent manners, whereas normal bone marrow stromal cells were refractory to the virus. VSV delivered by ILP inhibited growth of osteosarcoma xenografts more potently than that injected intravenously and intratumorally in the hind limb of immune-competent rats. Histopathological sections of tumor lesions treated by ILP-delivered VSV showed positive for VSV-G protein. There were no VSV-G expressions in perfused leg muscle, nonperfused leg muscle, brain, lung, and liver in VSV-treated rats. Our findings show efficient VSV gene expression and replication in osteosarcoma cells, suggesting that osteosarcoma may be a promising target for oncolytic virotherapy with VSV. Furthermore, we firstly showed that ILP of VSV against extremity sarcoma caused antitumor activity.

A functional recombinant human 4-1BB ligand for immune costimulatory therapy of cancer.

Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.

Mechanism of sustained release of vascular endothelial growth factor in accelerating experimental diabetic healing.

In this study, we hypothesize that local sustained release of vascular endothelial growth factor (VEGF), using adenovirus vector (ADV)-mediated gene transfer, accelerates experimental wound healing. This hypothesis was tested by determining the specific effects of VEGF(165) application on multiple aspects of the wound healing process, that is, time to complete wound closure and skin biomechanical properties. After showing accelerated wound healing in vivo, we studied the mechanism to explain the findings on multiple aspects of the wound healing cascade, including epithelialization, collagen deposition, and cell migration. Intradermal treatment of wounds in non-obese diabetic and db/db mice with ADV/VEGF(165) improves healing by enhancing tensile stiffness and/or increasing epithelialization and collagen deposition, as well as by decreasing time to wound closure. VEGF(165), in vitro, stimulates the migration of cultured human keratinocytes and fibroblasts, thus revealing a non-angiogenic effect of VEGF on wound closure. In conclusion, ADV/VEGF is effective in accelerating wound closure by stimulating angiogenesis, epithelialization, and collagen deposition. In the future, local administration and sustained, controlled release of VEGF(165) may decrease amputations in patients with diabetic foot ulcers and possibly accelerate closure of venous ulcers and pressure ulcers.

The oncopathic potency of Clostridium perfringens is independent of its alpha-toxin gene.

Hypoxia in solid tumors is a major obstacle in conventional treatment because of inefficient delivery of therapeutic agents to the lesions, but offers the potential for anaerobic bacterial colonization that can lead to tumor destruction. We have previously reported a recombinant Clostridium perfringens (Cp) strain constructed by deletion of the superoxide dismutase (sod) gene and insertion of the Panton-Valentine leukocidin (PVL) gene, Cp/sod(-)/PVL, which showed elevated oxygen sensitivity, tumor selectivity, and oncopathic potency in an orthotopic model of pancreatic cancer in immune-competent and syngeneic mice, and that led to substantial prolongation of animal survival. A major limitation to Cp/sod(-)/PVL in clinical applications is that it expresses phospholipase C (plc), the alpha-toxin and the major virulence determinant in Cp that is causative in the development of gas gangrene. In this study, the plc gene in Cp/sod(-)/PVL was knocked out to create Cp/plc(-)/sod(-)/PVL, which was shown to be incapable of inducing gas gangrene in mice. Intravenous injection of Cp/plc(-)/sod(-)/PVL spores led to a significant survival advantage in tumor-bearing mice with the same efficacy as Cp/sod(-)/PVL, indicating that the oncopathic potency of Cp is independent of a functional plc gene. The treatment also did not lead to an attenuated immune response to a subsequent pathogen challenge, indicating that a systemic immune-suppressive effect in the host is absent. Consequently, Cp/plc(-)/sod(-)/PVL is a novel oncopathic bacterial agent for the effective treatment of pancreatic cancer and other poorly vascularized tumors, with a substantially enhanced safety profile, which is essential for the development of translational studies in the future.

A genetically enhanced anaerobic bacterium for oncopathic therapy of pancreatic cancer.

BACKGROUND: A major obstacle in treatment of solid tumors is the inefficient delivery of therapeutic agents to the hypoxic cores. Hypoxia offers the potential for anaerobic bacteria colonization and tumor destruction by the bacteria, and dormant spores of wild-type Clostridium perfringens (Cp) germinate and proliferate within the hypoxic cores of pancreatic tumors in mice. However, the oncopathic effects of Cp were limited by host inflammatory responses and by Cp's residual tolerance to oxygen, which caused toxic effects in animals. METHODS: Recombinant Cp strains in which superoxide dismutase, a major oxygen tolerance gene, was deleted (Cp/sod(-)) were constructed to enhance its selective growth in tumors. In addition, Panton-Valentine Leukocidin (PVL), an inflammation-suppressing gene from Staphylococcus aureus, was inserted into the Cp/sod(-) genome to enhance its oncopathic potency. The ability of the recombinant Cp strains to kill tumors was investigated in C57/BL6 mice bearing murine PANC02 tumors. Systemic and organ toxic effects were assessed by monitoring serum chemistries and histopathological examination. Statistical tests were two-sided. RESULTS: Cp/sod(-) showed reduced toxic effects compared with wild-type Cp when spores were administered intravenously into PANC02 tumor-bearing mice. Mice treated with Cp/sod(-)/PVL spores demonstrated a reduction in neutrophils and macrophages in tumors, logarithmically elevated growth of intratumoral bacteria, enhanced tumor necrosis, and substantially prolonged survival without apparent systemic and organ toxic effects, compared with mice treated with both wild-type Cp and Cp/sod(-) spores. Accordingly, 47% of Cp/sod(-)/PVL-treated mice (n = 15) achieved tumor-free survival for over 120 days, whereas all mice treated with Cp/sod(-) or phosphate-buffered saline (n = 10 per group) died within 50 days. The median survival for Cp/sod(-)/PVL-treated mice was 77 days (95% confidence interval [CI] = 45 to 120 days) and for Cp/sod(-)-treated mice was 30 days (95% CI = 23 to 36 days; P < .001). CONCLUSIONS: Cp/sod(-)/PVL provides a prototype for a novel class of oncopathic microbes that may have potential for the safe and effective treatment of pancreatic cancer and other poorly vascularized tumors.

rVSV(M Delta 51)-M3 is an effective and safe oncolytic virus for cancer therapy.

Oncolytic vesicular stomatitis virus (VSV) is being developed as a novel therapeutic agent for cancer treatment, although it is toxic in animals when administered systemically at high doses. Its safety can be substantively improved by an M Delta 51 deletion in the viral genome, and yet VSV(M Delta 51) induces a much greater, robust cellular inflammatory response in the host than wild-type VSV, which severely attenuates its oncolytic potency. We have reported that the oncolytic potency of wild-type VSV can be enhanced by vector-mediated expression of a heterologous viral gene that suppresses cellular inflammatory responses in the lesions. To develop an effective and safe VSV vector for cancer treatment, we tested the hypothesis that the oncolytic potency of VSV(M Delta 51) can be substantively elevated by vector-mediated expression of M3, a broad-spectrum and high-affinity chemokine-binding protein from murine gammaherpesvirus-68. The recombinant vector rVSV(M Delta 51)-M3 was used to treat rats bearing multifocal lesions (1-10 mm in diameter) of hepatocellular carcinoma (HCC) in their liver by hepatic artery infusion. Treatment led to a significant reduction of neutrophil and natural killer cell accumulation in the lesions, a 2-log elevation of intratumoral viral titer, substantively enhanced tumor necrosis, and prolonged animal survival with a 50% cure rate. Importantly, there were no apparent systemic and organ toxicities in the treated animals. These results indicate that the robust cellular inflammatory responses induced by VSV(M Delta 51) in HCC lesions can be overcome by vector-mediated intratumoral M3 expression, and that rVSV(M Delta 51)-M3 can be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future.

Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo.

Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

Site-specific transgene integration in the human genome catalyzed by phiBT1 phage integrase.

The Streptomyces phage phiBT1 integrase catalyzes recombination between phage attP and bacterial attB sites (att, attachment), resulting in phage DNA integration into the bacterial host genome in a unidirectional manner. Multiple pseudo-attB and -attP sites are present serendipitously in mammalian genomes and can recombine with wild-type attP and attB sequences. The phiBT1 system has been used previously to achieve site-specific integration of murine phenylalanine hydroxylase cDNA into hepatocytes of mice with phenylketonuria, which led to the complete and permanent correction of the disease phenotypes without apparent toxicities. Here we report the identification of three pseudo-attP and two pseudo-attB sites in human cells, which are located in intergenic regions of five different chromosomes. There are no microdeletions of human genomic sequences at the insertional junctions and the integrated transgenes are expressed. Human cells expressing phiBT1 integrase showed normal karyotypes without chromosomal translocations between the pseudo-attB and -attP sites. Polymerase chain reaction analyses were performed on genomic DNA isolated from various human cell types expressing phiBT1 integrase, using primers flanking the pseudo-attB and -attP sites from mismatched human chromosomes. No chromosomal translocation events were detected in normal human hepatocytes, peripheral blood mononuclear cells, vascular microendothelial cells, and two other transformed human cell lines, although one such event was observed in a human melanoma cell line. The results suggest that the occurrence of chromosomal translocations is human cell type dependent, and that the phiBT1 system for site-specific integration of transgenes into the human genome can be used in selected applications.

Combined VSV oncolytic virus and chemotherapy for squamous cell carcinoma.

OBJECTIVES: Vesicular stomatitis virus (VSV) is a negative-strand ribonucleic acid (RNA) virus that replicates specifically in tumor cells and has oncolytic effects in a variety of malignant tumors. We previously demonstrated recombinant VSV vectors incorporating viral fusion protein (rVSV-F) and interleukin 12 (rVSV-IL12) to have significant antitumor effects against squamous cell carcinoma (SCC) in a murine model. Here we evaluate the potential to combine a potent chemotherapeutic agent for SCC (cisplatin) with rVSV-F and rVSV-IL12 to improve efficacy. STUDY DESIGN: In vitro, three SCC cell lines were tested using rVSV-F and rVSV-IL12 with cisplatin, monitoring viral replication and cell survival. In an orthotopic floor of mouth murine SCC model, intratumoral injections of virus combined with systemic cisplatin were tested for tumor control and animal survival. RESULTS: In vitro, virus and cisplatin combination demonstrated rapid replication and enhanced tumor cell kill. Human keratinocytes were unaffected by virus and cisplatin. In vivo, combined rVSV-F with cisplatin reduced tumor burden and improved survival (P = .2 for both), while rVSV-IL12 monotherapy had better tumor control (P = .06) and survival (P = .024) than combination therapy. CONCLUSIONS: Addition of cisplatin did not affect the ability of either virus to replicate in or kill murine SCC cells in vitro. In vivo, combination therapy enhancedrVSV-F antitumor activity, but diminished rVSV-IL12 antitumor activity. Combination therapy may provide useful treatment for SCC with the development of more efficient viral vectors in combination with different chemotherapy agents or immunostimulatory agents.

Rejection of metastatic 4T1 breast cancer by attenuation of Treg cells in combination with immune stimulation.

4T1 breast carcinoma is a highly malignant and poorly immunogenic murine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments. We hypothesize that the ineffectiveness of immune stimulatory treatment is mediated by the suppressive effects of CD4(+)CD25(+) regulatory T (Treg) cells, which can be attenuated by engaging the glucocorticoid-induced tumor necrosis factor receptor family-related protein with its natural ligand (GITRL); further, combination treatment with existing immune stimulation regimens will augment anti-tumor immunity and eradicate metastatic 4T1 tumors in mice.A soluble homodimeric form of mouse GITRL (mIg-mGITRLs) was molecularly constructed and used to treat orthotopic 4T1 tumors established in immune-competent, syngeneic Balb/c mice. When applied in combination with adenovirus-mediated intratumoral murine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) gene delivery plus systemic 4-1BB activation, mIg-mGITRLs attenuated the immune-suppressive function of splenic Treg cells, which led to elevated interferon-gamma (IFN-gamma) production, tumor-specific cytolytic T-cell activities, tumor rejection and long-term survival in 65% of the animals without apparent toxicities. The results demonstrate that addition of mIg-mGITRLs to an immune-stimulatory treatment regimen significantly improved long-term survival without apparent toxicity, and could potentially be clinically translated into an effective and safe treatment modality for metastatic breast cancer in patients.

Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus.

Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-gamma, interleukin 2 (IL-2), or tumor necrosis factor alpha, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment.

Fusogenic vesicular stomatitis virus for the treatment of head and neck squamous carcinomas.

OBJECTIVES: This study investigates the efficacy of recombinant fusogenic VSV [rVSV-NDV/F(L289A) or rVSV-F] in the treatment of head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN AND SETTING: The in vitro replication and cytotoxicity of rVSV-F were studied in two human SCC cell lines, in one murine SCC cell line, and in human keratinocytes. The effects on tumor size and animal survival were investigated following in vivo rVSV-F treatment of floor-of-mouth tumor model C3H/HeJ mice. RESULTS: Recombinant VSV-F preferentially induced rapid syncytia formation, and replicated in (P < 0.04) and killed (P < 1 x 10(-13)) all three SCC lines tested. The virus had no observable effect on human keratinocytes. Tumor size was smaller (P < 0.03) and overall survival was better (P < 0.001) for treated animals than for control animals. CONCLUSION/SIGNIFICANCE: Recombinant VSV-F confers a modest survival benefit for HNSCC in this orthotopic murine model. This oncolytic virus holds promise as a novel cancer treatment for recurrent HNSCC.

Metabolic basis of sexual dimorphism in PKU mice after genome-targeted PAH gene therapy.

We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that results in targeted insertion of transgenes into mammalian genomes, and its use in the delivery of murine phenylalanine hydroxylase (PAH) complementary DNA (cDNA) into the hepatocytes of male phenylketonuria (PKU) mice, leading to a complete and permanent correction of their hyperphenylalaninemic phenotype. In this study, we report only partial phenotypic correction in female PKU mice, even though hepatic PAH activities in both sexes after gene treatment were similar. Daily injections of tetrahydrobiopterin (BH4), an essential co-factor for phenylalanine hydroxylation, in the gene-treated females led to complete correction of their PKU phenotype. After gonadectomy, serum phenylalanine levels in the gene-treated females were reduced to normal, whereas those in the gene-treated males remained unchanged. The sterile gene-treated PKU mice were subjected to daily sex hormone injections. Whereas the estradiol-treated sterile males developed hyperphenylalaninemia, the dihydrotestosterone-treated sterile females remained normal phenylalaninemic. The results indicate that it is estrogen that suppresses the steady-state levels of BH4 in mouse hepatocytes that became limiting, which is the underlying mechanism for the observed sexual dimorphism in PKU mice after PAH gene treatment. Livers of the PAH gene-corrected PKU mice also appeared normal and without apparent pathologies.