305 Scavenging in Reactive Oxygen Species Improves Gene Expression in Polyplex Supported Gene Delivery Nitroxyl Radical Containing Nanoparticles Assists Non Viral Gene Delivery System Molecular Therap[.]
GENE REGULATION I characterization of a replicator-binding protein complex involved in the prevention of silencing that mediated the interactions between LCR and Rep-P The protein complex consisting of hnRNP C1/C2, SWI/SNF, and MeCP1 (the LCR-associated remodeling complex, LARC) interacted with an essential asymmetric region within the Rep-P A Rep-P variant that cannot bind the protein complex failed to prevent gene silencing Silenced transgene constructs with the mutant Rep-P variant could be re-activeated by a DNA methyltransferase inhibitor (zebularine) The re-activation was transient and exposure to zebularine induced distinct, stable, methylation-independent transcriptional silencing that was accompanied by changes in H3K27me3 and H4K20me2 patterns Our observations suggest that the interaction of LARC complex with replicators plays a role in preventing methylation-dependent gene silencing and propose a novel, epigenetic mechanism of resistance to methylation inhibitors 303 Multimodal Targeted In Vivo Imaging of Brain Tumors Johanna M Niers,1 Mariam Kerami,1 John W Chen,2 Ralph Weissleder,2 Bakhos A Tannous.1 Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA; 2Radiology, Harvard Medical School and Massachusetts General Hospital, Boston, MA We have developed a multifaceted highly specific technique for multimodal imaging of brain tumors The naturally secreted Gaussia luciferase (Gluc) was fused at the C-terminus with a biotin acceptor peptide (BAP) followed by the transmembrane domain of the plateletderived growth factor receptor (Gluc-BAPTM) Upon expression in mammalian cells, the BAP is biotinylated by biotin ligase with both biotin and Gluc displayed on the cell surface Gioma cells were engineered ex vivo to express Gluc-BAPTM reporter and were injected intracranially Brain tumors formation were imaged with Gluc bioluminescence imaging after i.v injection of its substrate, coelenterazine Further, brain tumors expressing this reporter had high sensitivity for magnetic resonance (MR) and fluorescence tomographic imaging upon injection of streptavidin conjugated to magnetic nanoparticles or Alexa750 fluorophore respectively Moreover, single photon emission computed tomography (SPECT) showed enhanced imaging of these tumors upon injection with streptavidin complexed to biotin-DTPA-67Ga This work show for the first time a single small reporter (∼20 kDa) which can be imaged with all available modalities including bioluminescence, fluorescence, MR and SPECT/PET allowing real-time tracking of any cells transduced to express it in vivo 304 Regulation of B23/Nucleophosmin Protein Expression and Function by HIV-1 Derived miRTAR-5p and miR-TAR-3p Dominique L Ouellet,1,2 John J Rossi,2 Patrick Provost.1 Microbiology-Infectiology and Immunology, Université Laval, Québec, QC, Canada; 2Molecular and Cellular Biology, Beckman Research Institute at City of Hope, Duarte, CA messenger RNA (mRNA) in immunoprecipitated Argonautes (Ago) loading complexes Reporter gene activity assays have shown the presence of elements responsive to miR-TAR-5p and miR-TAR-3p in the 3’ untranslated region (UTR), but not in the 5’ UTR or coding region, of NPM/B23 mRNA Transposed to the endogenous B23/ NPM protein, these data indicate that HIV-1-derived miR-TAR-3p can regulate endogenous B23/NPM expression without affecting the level of its mRNA, in agreement with a regulation based on translational repression Immunofluorescence and coimmunoprecipitation experiments performed in Tat and/or Rev-transfected cells revealed an interaction between them and B23/NPM, pointing to the nucleolus as a niche for these HIV-1 proteins Short hairpin RNA-inducing downregulation of B23/NPM expression delocalized Tat and Rev proteins from the nucleolus, suggesting a modulation of B23/NPM by HIV in TAR RNA-expressing cells like memory CD4+ T latently infected cells Our study may provide novel insights into the biological role and functional significance of TAR-containing transcripts and TAR-derived miRNAs in HIV-1 pathogenesis 305 Scavenging in Reactive Oxygen Species Improves Gene Expression in Polyplex Supported Gene Delivery: Nitroxyl-Radical Containing Nanoparticles Assists Non-Viral Gene Delivery System Yukio Nagasaki,1,2,3,4 Kazuko Toh,1 Toru Yoshitomi,1 Yutaka Ikeda.1,4 Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2Graduate School of Complehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 3Satellite Laboratory, International Center for Materials Nanoarchitectonics (MANA), National Institute of Materials Science (NIMS), University of Tsukuba, Tsukuba, Ibaraki, Japan; 4Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, Japan Non-viral gene vector is one of the promising candidates for suitable gene carrier to avoid issues of viral vectors such as carcinogenicity Because DNA possesses negative charge, polyion complex with polycation is one of the principles to improve resistance against enzymatic degradation and internalization in cell across the membrane Though polyion complex is low risk to carcinogenicity, cationic materials are known to show high cytotoxicity due to the strong interaction with anionic cell membrane For example, PEI induces inflammatory cytokines such as TNF-α and IL-6, which activate NF-κB, followed by an induction of ROS generation to result in amplification of inflammation reaction, when it is added to the cultured cells We hypothesized here that inflammation caused by the generated cytokines strongly related to gene expression To suppress the inflammation, we examined scavenging effect of ROS on gene expression Nitroxyl radical is known to catalytically react with ROS by its redox characteristics Thus, we newly designed nitroxyl-radical containing nanoparticle (RNP, Fig 1) MicroRNAs (miRNAs) are short, 21- to 24-nucleotide (nt) endogenous RNA species generated by the ribonuclease III Dicer and recognized as key regulators of gene expression As determined previously by our team and others, the transactivating response (TAR) element, a structured RNA located at the 5’ end of all transcripts derived from human immunodeficiency virus type (HIV-1), represents a source of functional miRNAs, namely miR-TAR-5p and miR-TAR-3p Proteomic analysis of stable, TAR miRNAexpressing Jurkat T cells unveiled a two-fold downregulation of B23/ Nucleophosmin (NPM), a 37-kDa nucleolar protein known to interact with HIV-1 Tat and Rev proteins Moreover, RNase protection assay (RPA) and RT-qPCR shown presence of TAR miRNAs and B23/NPM S118 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy GENE REGULATION I expression (data not shown) On the basis of these obtained results, RNP treatment as an ROS scavenger is promising strategy as a new strategy for non-viral gene delivery system 306 Gaussia Luciferase: A Biomarker for In Vivo Evaluation of Systemic Protein Delivery through Circulation Salim S El-Amouri,1 Phoung Cao,2 Dao Pan.1,2 Molecular and Cell Therapy Program, Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Department of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH Due to this design, we improved blood circulation time, reduction resistance, lowered toxicity and accumulation in tumor due to the EPR effect If the ROS scavenging character of RNP is effective to improve in gene expression, feasibility of the non-viral gene therapy will be expanded significantly To examine the effects of RNP on the transfection efficiency, PEI/pDNA polyplex was added to HeLa cells with or without RNP The gene expression increased significantly with the increasing amount of RNP and to finally attain about 2.5-fold higher than that without RNP addition(Fig.2) There is a clear need to develop novel tools to monitor in real-time the bio-distribution of gene and protein therapy in vivo In this study we aimed to evaluate the potential application of Gaussia luciferase (Gluc) as a reporter for real-time monitoring of in vivo protein biodistribution in mice To examine the in vivo Gluc production in blood stream and its bio-distribution profile, we injected naked plasmid DNA containing the coding sequence for the secreted Gluc under the control of the CMV promoter or a human alpha-1 antitrypsinbased hybrid promoter (hAAT) into mice by hydrodynamic tail-vein injection Robust Gluc levels were achieved (500-5000 folds above the background) in blood of all mice 1-3 days after injection, and then decreased rapidly to ∼10-folds as compared to control levels on day and thereafter Whole body imaging analysis showed that localized Gluc distribution could be observed with signals significantly above the background levels in all organs tested except the brain Among four regions of interest (ROI) evaluated, imaging quantification obtained at paw area was directly correlated to blood luciferase activities as determined using luminometer Moreover, three-dimensional (3D) bioluminescent imaging (BLI) revealed that the internal luminescent sources were positioned mostly in the liver, heart/spleen, and rectum regions as determined by photon density mapping analyses These observations are consistent with the fact that the liver is the main target for hydrodynamic gene delivery, and that the metabolic degradation of Gluc involves rectum system In perfused phAAT-Gluc injected animals, Gluc enzymatic activity was detected at highest levels in liver, and followed by kidney, lung, spleen and heart, suggesting that Gluc has been uptaken by the cells of those organs However, no Gluc activity was detected in the brain samples of all animal tested, indicating the Gluc was not able to pass the blood brain barrier To define the reuptake capability of Gluc in various cell types, non-tumorigenic cell lines were exposed to different volume of Gluc-containing medium for different time-points, resulting in different luciferase activities with variable intracellular half-life These results suggest that BLI can provide a reliable mean for a real-time quantization of Gluc in the circulation and peripheral organs of the same animal at multiple times Thus, the Gluc-based system can be a valuable tool for monitoring in vivo tracking of protein bio-distribution if in-frame fused with Gluc 307 Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis Johanna M Niers,1 Mariam Kerami,1 Lisa Pike,1 Grant Lewandrowski,1 Bakhos A Tannous.1 Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, MA Note that no significant difference in protein content was observed regardless of RNP concentrations We also confirmed that i) cellular uptake of polyplex improved with RNP addition; ii) TNF-α addition decreased gene expression; and iii) RNP addition recovers the gene Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer We have developed an apoptosis biosensor by fusing GFP to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V) and aspartic acid (D; DEVD) We showed that this fusion is retained in the cytoplasm S119 .. .GENE REGULATION I expression (data not shown) On the basis of these obtained results, RNP treatment as an ROS scavenger is promising strategy as a new strategy for non- viral gene delivery system. .. toxicity and accumulation in tumor due to the EPR effect If the ROS scavenging character of RNP is effective to improve in gene expression, feasibility of the non- viral gene therapy will be expanded... Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Department of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH Due to this design, we improved blood circulation