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.

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.

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.

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.

An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.

Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2(50-100)) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2(50-100) to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.

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.

Syncytia induction enhances the oncolytic potential of vesicular stomatitis virus in virotherapy for cancer.

Vesicular stomatitis virus (VSV) selectively replicates in tumor but not in normal cells and is being developed as an oncolytic agent for cancer therapy. Here we report the construction of a recombinant VSV capable of inducing syncytia formation between tumor cells through membrane fusion at neutral pH, which led to enhanced oncolytic properties against multifocal hepatocellular carcinoma (HCC) in the livers of immunocompetent rats. Recombinant VSV vectors were constructed by insertion into their genome a transcription unit expressing a control or fusion protein derived from Newcastle disease virus. In vitro characterization of the recombinant fusogenic VSV vector on human and rat HCC cells showed extensive syncytia formation and significantly enhanced cytotoxic effects. In vivo, administration of fusogenic VSV into the hepatic artery of Buffalo rats bearing syngeneic multifocal HCC lesions in their livers resulted in syncytia formation exclusively within the tumors, and there was no collateral damage to the neighboring hepatic parenchyma. The fusogenic VSV also conferred a significant survival advantage over a nonfusogenic control virus in the treated animals (P = 0.0078, log-rank test). The results suggest that fusogenic VSV can be developed into an effective and safe therapeutic agent for cancer treatment in patients, including those with multifocal HCC in the liver.

Oncolytic vesicular stomatitis virus for treatment of orthotopic hepatocellular carcinoma in immune-competent rats.

Tumor-targeted replicating viruses are being developed as a novel class of oncolytic agents. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with inherent specificity for replication in tumor cells due to their attenuated antiviral responses. VSV as an oncolytic virus is particularly appealing for its exceptionally rapid replication rate in tumor cells, such that the oncolytic effects could be maximally manifested before the onset of potentially neutralizing antiviral immune responses in the host. To easily monitor VSV replication, we have rescued a recombinant VSV (rVSV) vector expressing the green fluorescent protein (GFP) gene (rVSV-GFP). Using this GFP-expressing virus, we have demonstrated the oncolytic potential of VSV against human and rat hepatocellular carcinoma (HCC). We found that rVSV-GFP replicated efficiently in cultured human and rat HCC cells, whereas normal human and rat hepatocytes were refractory. When a single dose of the vector was injected intratumorally into large orthotopically implanted HCC in immune-competent rats, rVSV-GFP effectively and selectively replicated throughout the solid tumor mass without apparent hepatotoxicity, caused tumor destruction, and inhibited tumor growth, which led to significant prolongation of animal survival. Our results show that VSV is an effective oncolytic agent against HCC in immune-competent hosts and warrants further development for future therapy in patients with HCC.

Basal insulin gene expression significantly improves conventional insulin therapy in type 1 diabetic rats.

Although a conventional insulin regimen for type 1 diabetes with twice-daily insulin injections is effective in preventing postprandial blood glucose excursions, this treatment is limited by its inadequate control of fasting hyperglycemia. Alternatively, sustained basal hepatic insulin gene expression has been shown to result in fasting normoglycemia in type 1 diabetic rats, although the treated animals still exhibited moderate postprandial hyperglycemia. To test the hypothesis that basal hepatic insulin production can be used as an auxiliary treatment to conventional insulin therapy for achieving better glycemic control, streptozotocin-induced diabetic rats were treated with twice-daily insulin injections, basal hepatic insulin production, or both in combination. Diabetic rats treated by conventional insulin therapy still suffered from fasting hyperglycemia, but when complemented with basal hepatic insulin production, near-normoglycemia under both fed and fasting conditions was achieved without fasting hypoglycemia. In addition, the combination-treated animals showed significantly enhanced glucose tolerance and markedly improved profiles in lipid metabolism. Furthermore, the combination treatment reduced the elevated fructosamine, glycated hemoglobin, and advanced glycation end products concentrations to normal. These results provide a proof of concept for basal hepatic insulin production as an adjuvant treatment to conventional insulin therapy in type 1 diabetes.

Systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immune-competent mice.

In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.

Adenovirus-mediated expression of glucokinase in the liver as an adjuvant treatment for type 1 diabetes.

Glucokinase (GK) plays a crucial role in hepatic glucose disposal. Its activity is decreased in patients with maturity-onset diabetes of the young and in some animal models of diabetes. We investigated the feasibility of manipulating GK expression as an adjuvant treatment for type 1 diabetes, using an E1/E3-deleted adenoviral vector (Ad.EF1(alpha)GK) delivered to the liver of streptozotocin-induced type 1 diabetic rats. First, we studied the metabolic impact of constitutive glucokinase expression in the absence of insulin. Normal blood glucose levels were observed after gene transfer, and glucose tolerance was substantially enhanced compared with diabetic control animals, suggesting that hepatic GK expression is a feasible mechanism to enhance glucose disposal. In a second study we administered Ad.EF1(alpha)GK together with subcutaneous insulin injections to determine whether the combined action of insulin plus GK activity would provide better glucose homeostasis than insulin treatment alone. This combination approach resulted in constant, near-normal glucose values under fed conditions. Furthermore, the animals stayed in the normoglycemic range after an overnight fast, indicating that the risk to develop hypoglycemia is not increased by expression of GK. Alterations of other metabolic routes were observed, suggesting that insulin-regulated expression of GK may be necessary to use the strategy as a treatment of type 1 diabetes.

Intratumoral delivery of adenovirus-mediated interleukin-12 gene in mice with metastatic cancer in the liver.

Clinical trials of recombinant human interleukin-12 (rhIL-12) delivered by intravenous administration have shown dose-limiting toxicities with limited tumor responses at the doses tested. We have previously reported that intratumoral injection of an adenovirus vector expressing murine interleukin-12 (Adv.RSV-mIL-12) was effective in inducing antitumor immune responses, tumor regression, and long-term survival in mice with established metastatic cancer in the liver. We now report additional studies in the same murine tumor model to assess the safety of intratumoral Adv.RSV-mIL-12 injection. At vector doses that were previously shown to be therapeutically effective, no inflammation in the liver or lungs, and no significant elevations in serum creatinine and aminotransferases were seen after vector injection. Serum elevations of IL-12 and interferon-gamma (IFN-gamma) were 17- and 19-fold lower than peak levels after intravenous recombinant IL-12 at the maximal tolerated dose in clinical trials. No elevations in serum proinflammatory cytokines (interleukin-6, tumor necrosis factor-alpha) were noted up to 2 weeks after vector injection. No systemic dissemination of the vector was detected on polymerase chain reaction (PCR) assays at therapeutically effective vector doses. At higher supratherapeutic vector doses of Adv.RSV-mIL-12, however, inflammation in the liver and lungs with elevation in serum aminotransferases were seen, but not in controls injected with the equivalent particle number of an empty adenoviral vector. These results support the cautious testing in patients with hepatic metastases of adenovirus mediated IL-12 gene delivery by intratumoral injection.

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 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.

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.