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Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice. Synopsis: Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. Development of an in vitro phagocytosis assay, exploiting fluorescent virions and advanced imaging techniques, suitable to assess the susceptibility to phagocytosis of different LV versions in different experimental settings. Efficient tropism switch of LV from hepatocytes to liver endothelial cells in vitro and in vivo and shielding from phagocytosis. Reconstitution of FVIII activity in the normal range by a single intravenous administration of GP64-LV equipped with the endogenous endothelial-specific FVIII promoter. Proof-of-concept of stable long-term gene therapy for hemophilia A in both adult and newborn mice. Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. [ABSTRACT FROM AUTHOR]

Liver gene therapy with adeno‐associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X‐linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti‐F8 antibodies which neutralise F8 activity. Taking advantage of split‐intein‐mediated protein trans‐splicing, we divided the coding sequence of the large and highly secreted F8‐N6 variant in two separate AAV‐intein vectors whose co‐administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti‐F8 antibodies unlike animals treated with the single oversized AAV‐F8 vector under clinical development. Therefore, liver gene therapy with AAV‐F8‐N6 intein should be considered as a potential therapeutic strategy for HemA. Synopsis: Liver directed AAV‐intein mediated protein trans‐splicing results in stable therapeutic levels of F8 in mice thus representing a novel therapeutic strategy for haemophilia A. Split‐intein mediated protein trans‐splicing (PTS) allows reconstitution of large proteins via adeno‐associated viral (AAV) vectors in mouse liver.The highly secreted and active F8‐N6 (N6) variant has been adapted to fit into AAV intein vectors.A single systemic injection of AAV‐N6 intein targets liver of hemophilic mice resulting in stable therapeutic levels of F8 without eliciting anti‐F8 antibodies at the vector doses used. [ABSTRACT FROM AUTHOR]

Background: SHOX enhancer CNVs, affecting one or more of the seven recognized evolutionary conserved non‐coding elements (CNEs) represent one of the most frequent cause of SHOX‐haploinsufficiency. During the diagnostic workflow deletions/duplications have been identified downstream SHOX not including any of the these CNEs. Methods: Fine tiling aCGH and breakpoint PCR were used to characterize the critical interval and to search for novel alterations in a cohort of selected patients. Results: Screening of 252 controls provided evidence that duplications in this area represent likely benign variants whereas none of the deletions were detected. These findings suggested that other alterations relevant for SHOX‐haploinsufficiency might be missed by the standard diagnostic methods. To identify such undisclosed elements, the aCGH was used to reanalyze 52 unresolved cases with clinical features strongly suggestive of SHOX‐haploinsufficiency. This analysis followed by the screening of 210 patients detected two partially overlapping small deletions of ~12 and ~8 kb in four unrelated individuals, approximately 15 kb downstream SHOX, that were absent in 720 normal stature individuals. Conclusion: Our results strengthen the hypothesis that alterations of yet unidentified cis‐regulatory elements residing outside those investigated through conventional methods, might explain the phenotype in ISS/LWD patients thus enlarging the spectrum of variants contributing to SHOX‐haploinsufficiency. [ABSTRACT FROM AUTHOR]

The aim of the study is to investigate the relationship between the physico-chemical properties of superparamagnetic iron oxide nanoparticles (SPIONs) and their cytotoxicity profile in light of their potential biomedical application as nanocarriers for pancreatic cancer treatment. Two types of SPIONs were tested: magnetite nanoparticles (Fe3O4 NPs) and silica-coated magnetite nanoparticles (SiO2-Fe3O4 NPs). The physico-chemical properties of the 2 SPIONs were characterized by means of Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectrometry (EDS), and Selected Area Electron Diffraction (SAED). Their magnetic properties were quantified as magnetization saturation (Ms) and Remanence. The colloidal stability was investigated by Isoelectric Point Measurements and sedimentation tests. Finally, in vitro characterizations were performed to quantify the half maximal lethal concentration (LC50), by means of High Content Screening Analysis (HCSA), Flow cytometry (FC), and Laser Scanning Confocal Microscopy (LSCM). The obtained NPs present a spherical shape and a dimension between 10 and 20 nm, a superparamagnetic behavior and surface charge in agreement with their surface chemistry. The in vitro tests demonstrate that both NPs induce similar levels of cytotoxicity in a PANC-1 cell model and were internalized, with SiO2-Fe3O4 NPs associated to a slightly higher cellular internalization, probably due to their higher dispersability. [ABSTRACT FROM AUTHOR]

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor ( TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD- scid IL2rγ null ( NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease ( GVHD) that occurs soon after cell transfer. We modified human primary CD4+ T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA- DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA- DR4 and to be deficient for murine class II MHC molecules. CD4+ T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells. [ABSTRACT FROM AUTHOR]

Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with β-thalassemia intermedia ( th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in β-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in β-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries. [ABSTRACT FROM AUTHOR]

The c- MET proto-oncogene, encoding the p190 hepatocyte growth factor tyrosine kinase receptor, can acquire oncogenic potential by multiple mechanisms, such as gene rearrangement, amplification and overexpression, point mutation, and ectopic expression, all resulting in its constitutive activation. Hepatocyte growth factor receptor truncated forms are generated by post-translational cleavage: p140 and p130 lack the kinase domain and are inactive. Their C-terminal remnant fragments are generally undetectable in normal cells, but a membrane-associated truncated form is recognized by anti-C-terminus antibodies in some human tumors, suggesting that a hepatocyte growth factor receptor lacking the ectodomain, but retaining the transmembrane and intracellular domains (Met-EC−), could acquire oncogenic properties. Herein we show that NIH-3T3 cells transduced with MET-EC− expressed a membrane-associated constitutively tyrosine-phosphorylated 60-kDa protein and, similarly to NIH-3T3 cells expressing the cytosolic oncoprotein Tpr-Met, showed activated extracellular regulated kinase 1/2 mitogen-activated protein kinase and Akt downstream transducers. Compared to control NIH-3T3 cells, NIH-3T3-Met-EC− cells grew faster and showed anchorage-independent growth and invasive properties in all aspects similar to cells expressing the transforming TPR-MET. Nude female mice injected subcutaneously with NIH-3T3-Met-EC− cells developed visible tumors, displaying the typical morphology of carcinomas with polygonal cells, in contrast to sarcomas with spindle-shaped cells induced by the injection of NIH-3T3-Tpr-Met cells. It is suggested that the different subcellular localization of the oncoproteins, more than differences in signal transduction, could be responsible for the tumor phenotype. All together, these data show that deletion of the ectodomain activates the hepatocyte growth factor receptor and its downstream signaling pathways, unleashing its transforming, invasive, and tumorigenic potential. ( Cancer Sci 2009; 100: 633–638) [ABSTRACT FROM AUTHOR]

Background: The exposure of liver to hepatotoxins, and their subsequent metabolism, results in increased reactive oxygen species (ROS), one of the major culprits in causing both acute liver cell injury and chronic liver diseases. The aim of this present study is to investigate the protective effects of lentiviral vector-mediated copper–zinc superoxide dismutase (LV-SOD1) gene transfer against ROS-induced cytotoxicity in Hep G2 cells and liver injury in mice. Methods: In vitro SOD1 efficacy was tested against two ROS-generating systems: hypoxanthine/xanthine oxidase (HX/XO) and hydroxyethyl radicals (HER), whereas in vivo SOD1 efficacy was evaluated in carbon tetrachloride (CCl4)-induced liver injury in C57BL/6 mice. Results: LV-SOD1 transduction in Hep G2 cells resulted in a significant increase in SOD activity in cell lysates, and it significantly decreased the toxicity induced by HX/XO and HER. High SOD1 expression in the liver was achieved via portal vein injection of LV-SOD1 in mice and these high levels were observed for 30 days, the length of the experiment to date. SOD1 overexpression significantly decreased the toxicity and restored liver function in the CCl4-treated mice. Conclusions: These findings demonstrate for the first time that LV transduction led to the long-term expression of fully functional transgene expression in both in vitro and in vivo systems. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme α-l-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I. [ABSTRACT FROM AUTHOR]