307 Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S11[.]
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 STEM CELL THERAPIES I of cells in an inactive form Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time Since Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum, Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models demonstrated for the first time that nitrosative stress elicited by nNOSµ delocalization is an important mechanism underlying force loss in DMD Stem Cell Therapies I 310 Human Hematopoietic Stem Cells Can Be Functionally Isolated Based on Their Low Intracellular Reactive Oxygen Species (ROS) Activity Brian Ichwan,1 Cynthia E Dunbar,1 Andre Larochelle.1 National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 308 Prolonged Gene Silencing Effect Using Multilayered siRNA Coated Gold Nanoparticles Seung-Koo Lee,1 Myung-Shin Han,1 Ching-Hsuan Tung.1 Radiology, The Methodist Hospital Research Institute, Houston, TX Small interfering RNA (siRNA) has immense potential for the treatment of various diseases by silencing genes, but its delivery to the targeted cells and preservation of extended gene silencing remains challenging Recently we have developed a fabrication method to prepare multilayer siRNA delivery nano-vectors Up to layers of poly-L-lysine (PLL) and layers of siRNA were coated onto gold nanoparticles (AuNPs) by alternating the charged polyelectrolytes The formulated siRNA nanopaprticles were stable in physiological condition; however, the encapsulated siRNAs were release slowly when the shielding PLL layers were gradually degraded by intracellular proteases Cell culture results indicated that the siRNA effect correlated with the number of siRNA layers AuNPs with layers of siRNA (>80% inhibition) are much better than particles with 2-layer (72% inhibition) or 1-layer (57% inhibition) of siRNA in gene silencing Furthermore, the layering strategy could prolong inhibition effect With layers of siRNA, gene silencing effect sustained up to 10 days, while with Lipofectamin 2000 approach could only last for days after transfection 309 Implication of nNOSµ Delocalization on the Muscle Force in Dystrophin Deficient and δ-Sarcoglycan Knockout Mice Dejia Li,1 Yongping Yue,1 Yi Lai,1 Chady H Hakin,1 Dongsheng Duan.1 Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO The mechanism of force reduction is not completely understood in Duchenne muscular dystrophy (DMD), a dystrophin-deficient lethal disease Nitric oxide regulates muscle force Interestingly, neuronal nitric oxide synthase µ (nNOSµ), a major source of muscle nitric oxide, is lost from the sarcolemma in DMD muscle We hypothesize that nNOSµ delocalization contributes to force reduction in DMD To test this hypothesis, we generated dystrophin/nNOSµ double knockout mice Genetic elimination of nNOSµ significantly enhanced force in dystrophin-null mice Pharmacological inhibition of nNOS yielded similar results To further test our hypothesis, we studied δ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy These mice had minimal sarcolemmal nNOSµ delocalization and muscle force was less compromised Annihilation of nNOSµ did not improve their force either To determine whether nNOSµ delocalization itself inhibited force, we corrected muscle disease in dystrophin-null mice with micro-dystrophins that either restored or did not restore sarcolemmal nNOSµ Similar muscle force was obtained irrespective of nNOSµ localization Additional studies suggest that nNOSµ delocalization selectively inhibits muscle force in dystrophin-null mice via nitrosative stress In summary, we have S120 The search for novel approaches to isolate human hematopoietic stem cells (HSCs) has been a central question for functional stem cell studies and for the development of clinical applications, including gene therapy Various combinations of antibodies directed to cell surface markers have been used to isolate human HSCs but these protocols result in poor enrichment or require multiple complex steps Recently, a simple functional approach to enrich for murine HSCs has been described based on low levels of intracellular reactive oxygen species (ROS) (Blood 110: 3056, 2007) Long term HSCs are believed to reside predominantly in the osteoblastic niche, the lowest end of an oxygen gradient within the bone marrow (BM), to minimize the oxidative stress from ROS We hypothesized that a low intracellular level of ROS (ROSlow), in contrast to a high level of ROS (ROShigh), could select for human HSCs in G-CSF mobilized peripheral blood (MPB) and BM samples obtained from healthy volunteers Unfractionated MNCs or pre-enriched populations of CD34+ cells were incubated with the cell permeable fluorescent ROS indicator DCF-DA and total ROSlow/ROShigh populations as well as CD34+ or CD34+/CD38- ROSlow/ROShigh cells were isolated by FACS The ROSlow and ROShigh populations were compared in CFU assays, cell cycle analyses, flow studies and in vivo reconstitution in immune-deficient (NSG) mice Cell cycle analysis revealed a higher proportion of ROSlow BM MNC in the G0 phase of the cell cycle (9.6%) compared to the ROShigh fraction (0.8%), consistent with the more quiescent nature of the ROSlow BM cells In CFU assays, fewer ROSlow BM CD34+ cells-derived colonies (30 CFU/1000 cells plated) were generated compared to the ROShigh fraction (185 CFU/1000 cells plated), suggesting a depletion of more mature progenitors in the ROSlow population We estimated the frequency of CD34+CD38- ROSlow cells at in 3x106 BM cells Given a frequency of long-term HSCs in the BM of in 3x106 as previously calculated in limiting dilution transplantion studies, in CD34+CD38-ROSlow may have long-term repopulating capacity Transplantation of as few as 30 CD34+CD38-ROSlow BM cells resulted in 0.9% human cell engraftment in one NSG mouse two months after transplantation compared to less than 0.1% engraftment in a mouse transplanted with 30 CD34+ CD38-ROShigh BM cells In contrast to BM cells, cell cycle and CFU assays revealed no significant difference between the ROSlow and ROShigh MPB CD34+ cell population The frequency of CD34+CD38- cells was also comparable in the ROSlow (54%) and ROShigh (42%) MPB CD34+ cells When transplanted into NSG mice, ROSlow and ROShigh MPB CD34+ cells gave rise to similar human cell engraftment weeks after transplantation ROS levels in MPB samples were found to be increased compared to BM cells, suggesting that the five day period necessary for HSC mobilization by G-CSF may have led to significant intracellular ROS accumulation in HSCs, preventing their selection using this functional approach Our data indicate that primitive human HSCs that reside in a low-oxygen milieu of the bone marrow can be functionally enriched based on their lower levels of ROS Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... endoplasmic reticulum, Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging This reporter provides... provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models demonstrated... where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time Since Gluc is secreted from cells