Valproic acid enhances gene expression from viral gene transfer vectors.
ABSTRACT Viral vectors represent an efficient delivery method for in vitro and in vivo gene transfer, and their utility may be further enhanced through the use of pharmacologic agents that increase gene expression. Here, we demonstrate that valproic acid (VPA), a drug which is widely used for the treatment of epilepsy and mood disorders, enhances and prolongs expression of exogenous genes in cells transduced with various gene transfer agents, including adenovirus, adeno-associated virus and herpesvirus vectors. This effect occurs in a wide range of cell types, including both primary cells and cell lines, and appears to be associated with VPA's ability to function as a histone deacetylase inhibitor (HDACi). VPA treatment also enhanced adenovirally-vectored expression of a luciferase reporter gene in mice, as demonstrated by in vivo imaging. VPA was also less cytotoxic than a commonly used HDAC inhibitor, TSA, suggesting its use as a safer alternative. Taken together, these results suggest that VPA treatment may represent a useful approach to various gene transfer approaches in which enhanced transgene expression is desirable.
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ABSTRACT: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.Journal of Applied Microbiology 06/2007; 102(5):1337-49. DOI:10.1111/j.1365-2672.2006.03182.x · 2.39 Impact Factor
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ABSTRACT: Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2007. The central goal of the studies presented in this Thesis was to explore Fc receptor targeting as an approach to enhancing virally-vectored gene transfer in mammalian cells. To approach this question, two sets of experiments were performed. First, studies were conducted to test the hypothesis that the efficiency of gene transfer to dendritic cells (DC) by recombinant adenoviral (Ad) vectors could be improved by targeting the vector to the high affinity Fc-gamma receptor I (FcγRI). The long-term goal of these experiments was to improve Ad-based vaccine delivery. To ‘retarget’ Ad vectors to FcγRI, a targeting complex consisting of a trimeric adenovirus fiber-binding moiety fused to a single-chain antibody specific for FcγRI was generated. Transduction studies revealed that FcγRI-targeted Ad transduced primary monocyte-derived DC with greater efficiency than unmodified Ad vectors. In addition, FcγRI-targeted Ad elicited more efficient activation of antigen-specific autologous memory CD8+ T-cells than unmodified Ad vectors. Thus, FcγRI-targeted Ad demonstrated enhanced antigen delivery to DC. A second experimental model system was also used to study effects of Fc receptor targeting on virally-vectors gene transfer. In this case, a bacterial virus vector (recombinant bacteriophage λ) was decorated with intact antibody molecules, and the resulting immune complexes were then used to study the molecular mechanisms involved in FcγRI internalization and intracellular routing following receptor ligation. Bacteriophage λ immune complexes were found to transduce FcγRI expressing mammalian cells with approximately 20-50 fold greater efficiency than unmodified vii bacteriophage λ particles. The ability of other Fc receptor subtypes to support gene transfer by bacteriophage λ immune complexes was also assessed, and FcγRI was found to confer the greatest permissivity to phage-mediated gene expression. Pharmacologic studies revealed that actin microfilaments, which are important for clathrin-mediated endocytosis, are essential for efficient gene expression, following addition of bacteriophage λ immune complexes to FcγRI positive cells. In contrast, endosomotropic and microtubule-disrupting agents increased bacteriophage λ- mediated gene expression. Overall, these studies suggest that bacteriophage λ immune complexes bind to FcγRI and then enter mammalian cells through clathrincoated pits; most of the incoming bacteriophage particles then appear to enter endosomes, with the majority of bacteriophage λ particles presumably becoming degraded in lysosomes. Overall, these studies show that Fc receptor targeting can enhance gene transfer efficiency in a wide range of mammalian cell types, by both bacterial and eukaryotic virus vectors. This suggests that Fc receptor targeting may be a broadly useful approach to improve virally-vectored gene transfer to FcR-positive host cells such as dendritic cells.
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ABSTRACT: Virus vector-mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the cystic fibrosis transmembrane conductance regulator gene. The pathological proinflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limits the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (Treg) and to improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway.Gene Therapy advance online publication, 2 January 2014; doi:10.1038/gt.2013.78.Gene therapy 01/2014; 21(2). DOI:10.1038/gt.2013.78 · 4.20 Impact Factor