Construction and characterization of a new TRAIL soluble form, active at picomolar concentrations.

Apoptosis induction has emerged as a treatment option for anticancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein, is a potent and specific pro-apoptotic protein ligand, which activates the extrinsic apoptosis pathway of the cell death receptors. Here we describe the construction and characterization of a new soluble TRAIL, sfTRAIL, stabilized with the trimerization Foldon domain from the Fibritin protein of the bacteriophage T4. Supernatants of 0.22 µM-filtered supernatants were produced in Vero-transduced cells with HSV1-derived viral amplicon vectors. Experiments were undertaken in two known TRAIL-sensitive (U373 and MDA.MB.231) and two TRAIL-resistant (MCF7 and A549) cell lines, to determine (i) whether the sfTRAIL protein is synthetized and, (ii) whether sfTRAIL could induce receptor-mediated apoptosis. Our results showed that sfTRAIL was able to induce apoptosis at concentrations as low as 1899.29 pg/mL (27.71 pM), independently of caspase-9 activation, and reduction in cell viability at 998.73 fM.

Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures of neuronal cells and into the brain of living animals.

HSV-1-derived amplicon vectors: recent technological improvements and remaining difficulties--a review.

Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. As the vector genome carries no virus genes, amplicons are both non-toxic for the infected cells and non-pathogenic for the inoculated organisms. In addition, the large transgenic capacity of amplicons, which allow delivery of up to 150 Kbp of foreign DNA, makes these vectors one of the most powerful, interesting and versatile gene delivery platforms. We present here recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review also discusses the many difficulties still pending to be solved, in order to achieve stable and physiologically regulated transgene expression.

HSV-1-derived amplicon vectors: recent technological improvements and remaining difficulties: a review

Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. As the vector genome carries no virus genes, amplicons are both non-toxic for the infected cells and non-pathogenic for the inoculated organisms. In addition, the large transgenic capacity of amplicons, which allow delivery of up to 150 Kbp of foreign DNA, makes these vectors one of the most powerful, interesting and versatile gene delivery platforms. We present here recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review also discusses the many difficulties still pending to be solved, in order to achieve stable and physiologically regulated transgene expression.