Effective melanoma immunotherapy with interleukin-2 delivered by a novel polymeric nanoparticle

Interleukin-2 (IL-2) has been shown to possess antitumor activity in numerous preclinical and clinical studies. However, the short half-life of recombinant IL-2 protein in serum requires repeated high-dose injections, resulting in severe side effects. Although adenovirus-mediated IL-2 gene therapy has shown antitumor efficacy, the host antibody response to adenoviral particles and potential biosafety concerns still obstruct its clinical applications. Here we report a novel nanopolymer for IL-2 delivery, consisting of low molecular weight polyethylenimine (600 Da) linked by β-cyclodextrin and conjugated with folate (named H1). H1 was mixed with IL-2 plasmid to form H1/pIL-2 polyplexes of around 100 nm in diameter. Peritumoral injection of these polyplexes suppressed the tumor growth and prolonged the survival of C57/BL6 mice bearing B16-F1 melanoma grafts. Importantly, the antitumor effects of H1/pIL-2 (50 μg DNA) were similar to those of recombinant adenoviruses expressing IL-2 (rAdv-IL-2; 2 × 10(8) pfu). Furthermore, we showed that H1/pIL-2 stimulated the activation and proliferation of CD8+, CD4+ T cell, and natural killer cells in peripheral blood and increased the infiltration of CD8+, CD4+ Tcells, and natural killer cells into the tumor environment. In conclusion, these results show that H1/pIL-2 is an effective and safe melanoma therapeutic with an efficacy comparable to that of rAdv-IL-2. This treatment represents an alternative gene therapy strategy for melanoma.     

A novel and effective hepatocyte growth factor kringle 1 domain and p53 cocktail viral gene therapy for the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Kringle 1 domain of HGF (HGFK1) has been demonstrated as a potent anti-tumor molecule and p53 is a well established tumor suppressor. Recently we developed AAV transducing HGFK1 (AAV-HGFK1) as a gene therapy for HCC. Here we investigated the possibility of enhancing the effect of AAV-HGFK1 by combining it with Adv transducing p53 (Adv-p53). In vitro expression experiments suggested a small amount of Adv-p53 could increase the expression of AAV transgenes. AAV-HGFK1+Adv-p53 cocktail strongly inhibited the proliferation of microvascular endothelial cell (MEC) and two HCC cell lines, Hepa1-6 and McA-RH7777. In two orthotopic mice and rat HCC models the cocktail gene therapy also significantly reduced the tumor burdens and prolonged the survival time by inhibiting tumor angiogenesis and inducing tumor cell death. Significantly, tumor metastasis was completely prevented. AAV-HGFK1+Adv-p53 viral cocktail may be a promising cancer therapy for the treatment of HCC.     

Ascorbic acid inhibits ROS production, NF-kappa B activation and prevents ethanol-induced growth retardation and microencephaly

In this study, we established an embryo model to study the effects of ethanol on fetal development. When embryos of Xenopus laevis (the African clawed frog) were exposed to ethanol, the resultant tadpoles had significantly reduced brain sizes (microencephaly) and retarded growth rates. These effects, similar to those observed in human fetal alcohol syndrome (FAS), were dose- and time-dependent. We further showed that the antioxidant ascorbic acid (vitamin C) could inhibit the ethanol-induced reactive oxygen species (ROS) production and NF-kappaB activation and protect the ethanol-treated embryos against microencephaly and growth retardation. These results suggest the involvement of NF-kappaB and oxidative stress in ethanol-mediated developmental defects, and the potential use of ascorbic acid as a new and effective protective agent for FAS.     

Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus encoded LMP-1

Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma characterized by consistent association with Epstein-Barr virus (EBV). Of the EBV-encoded product, latent membrane protein-1 (LMP-1) is considered to be an onco-protein playing an essential role in cell transformation and metastasis. In this study, we used a recombinant adeno-associated virus type 2 vector (rAAV-2) to deliver small hairpin RNA (shRNA) targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells and evaluated the effect of long-term suppression of LMP-1 on NPC growth and metastasis in vivo and in vitro. An NPC metastasis nude mouse model with NPC xenograft transplanted in liver was established. The NPC C666-1 cells infected with rAAV-shRNA-LMP-1 or rAAV-EGFP were inoculated in the livers of nude mice. Formation of liver and lung metastasis was evaluated at day 14 after tumor inoculation. Our results demonstrate that rAAV-shRNA-LMP-1 effectively infected C666-1 cells and suppressed LMP-1 expression. Such suppression, in turn, did not significantly inhibit tumor growth, but prevented NPC metastasis in the liver as well as in the lung. Consistent with in vivo data, the in vitro studies in NPC C666-1 cell cultures showed that suppression of LMP-1 by rAAV-shRNA-LMP-1 significantly reduced cell mobility and transmembrane invasion ability. These results demonstrated for the first time that long-term suppression of EBV-encoded LMP-1 in vivo is an effective means for preventing NPC metastasis.     

Recombinant adeno-associated virus-mediated TRAIL gene therapy suppresses liver metastatic tumors

To evaluate the tumoricidal activity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on disseminated liver metastatic tumors, we constructed a recombinant adeno-associated virus (rAAV) expressing the extracellular domain (95-281aa) of human TRAIL (TRAIL(95-281), and the recombinant virus was designated as rAAV-TRAIL) using the 3-plasmid, helper-virus-free, packaging system. Transduction of mouse lymphoma EL-4 cells and Jurkat T cells lead to the expression of TRAIL(95-281) protein in both virus-transduced cells and the culture media, along with apoptosis of these cells in vitro. The therapeutic potential of rAAV-TRAIL was then evaluated in an orthotopic transplanted mouse model mimicking liver cancer metastasis, which was established by injection of EL-4 cells into the liver of C57BL/6 mice via the hepatic portal veins. Subsequent intraportal vein injection of rAAV-TRAIL, not the control virus, into the liver of these mice resulted in significant suppression of tumor growth and prolonged survival, while normal hepatocyte toxicity is undetectable. Histological and biochemical analysis in tumor tissue and serum confirmed that TRAIL(95-281) was stably expressed in relatively high level in hepatocytes and was secreted into the serum in active trimeric form. Futhermore, the mechanism for rAAV-TRAIL to inhibit tumor growth was by inducing apoptosis of the tumor cells metastasizing to the livers. These results strongly suggest that the rAAV-TRAIL-mediated gene delivery could be a promising approach for the treatment of liver metastasis cancer.     

The virology and developments toward control of human enterovirus 71

Enterovirus 71 (EV71), a member of the Enterovirus genus in the Picornaviridae family, was first recognized as a dermotrophic virus that usually cause mild, self-limiting hand-foot-and-mouth disease (HFMD). However, EV71 infection can sometimes induce a variety of severe neurological complications and even death. Current large outbreaks of EV71 make this virus being a major public health issue. Intense effort has been made to address its underlying pathogenesis and to develop effective means for combating EV71 infections. Here, we aimed to provide an overview of cellular mechanisms underlying EV71 infection and to assess potential agents for prevention and treatment of EV71 infections.     

The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer

The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI₆₀₀–CyD (H₁) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H₁ were examined. We found that folate molecules were successfully grafted to PEI₆₀₀–CyD. At N/P ratios between 5 and 30, the resulting H₁/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H₁ was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H₁ was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H₁-mediated transfection (50 μg luciferase plasmid (pLuc), N/P ratio = 20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 × 10⁹ pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H₁ is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation.     

Postmortem MRI changes of the brains of the rats of different ages

Brain degenerative changes were studied in the rats of different ages after cervical dislocation by T(2) MRI and histology. Appearance of dark spots in the T(2) images increased with increased duration after death. Quantitative analysis of the density of these spots revealed that the neonatal (1 week) and the old (9 months) animals had accelerated degenerative changes when compared with the young adult (1 month). The degenerative changes correlated with the accumulation of vacuoles or spaces in the brain tissue histologically. This study pointed out not only brain degenerative changes after death were associated with age, it also revealed that the MRI T(2) evaluations could be used as a way in postmortem investigation.