568 Tracking Single Liposomes for Delivery of siRNA Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S217 OLIGONUCLEOTIDE & RNAI THERAPEUTICS[.]
OLIGONUCLEOTIDE & RNAI THERAPEUTICS I Adeno-associated virus (AAV) was used for long-term transduction of murine liver with shApoB A strong dose-dependent knock-down of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity No evidence was found for oversaturation of the endogenous RNAi pathway, since liver-specific microRNAs mir-122, mir29-a, and let7a were not significantly downregulated In addition, mir-122 target genes AldoA, Bckdk, and Ndrg3 were unaffected AAV-shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, while high-density lipoprotein cholesterol (HDL-C) remained unaffected Finally, elevated lipid accumulation was shown in murine liver transduced with AAV-shApoB, a known phenotypic side effect of lowering ApoB levels These results demonstrate a robust dosedependent knock-down of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAibased gene therapy for the treatment of hypercholesterolemia 565 Assessment of Zwitterionic Lipids as Vectors for Nucleic Acid Delivery Juliane Nguyen,1 Colin L Walsh,1 Francis C Szoka.1 Depts of Bioengineering, Therapeutic Science & Pharmaceutical Chemistry, UCSF, San Francisco, CA Nonviral RNAi delivery technology provides great potential for the treatment of many human diseases which are not addressable by current medicine Existing methods to deliver siRNA using cationic liposomes show high delivery efficiency but often cause unwanted toxic effects Anionic liposomal systems display low cytotoxicity but their use in vivo is hampered by insufficient nucleic acid delivery, large sizes and low encapsulation efficiency The objective of this research is to design a new class of lipids, which combine the advantages of both carrier systems into one vector: low cytotoxicity of anionic liposomes and high delivery efficiency of cationic liposomes These lipids should display a neutral or negative surface charge at physiological pH and become positively charged at the lower pH of the endosomes For this purpose we have synthesized a library of zwitterionic lipids that contain a cationic amine group and an anionic carboxylate group with pKa values of ∼ 5-6 These zwitterionic lipids were characterized for encapsulation of nucleic acids (pDNA and siRNA), size, zeta potential, and ability to mediate gene silencing of FVII in vivo 566 Multidrug Resistance Transporters and miRNAs Expression: Changes in Hepatocellular Carcinoma Florie Borel,1,2 Harald Petry,1 Peter Jansen,2 Sander van Deventer,1,3 Pavlina Konstantinova.1 Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; Department of Hepatology and Gastroenterology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands; 3Department of Hepatology and Gastroenterology, Leiden University Medical Centre, Leiden, Netherlands ATP-binding cassette (ABC) transporters family is involved in multidrug resistance by decreasing the intracellular concentration of toxic compounds This is a phenotype caused by overexpression of these transporters in tumor cells Overcoming increased drug extrusion could lead to significant improvement of chemotherapy effectiveness for hepatocellular carcinoma (HCC) patients In the current study we determined changes in expression pattern of ABC genes in HCC We established the expression profile of 15 ABC transporters in 19 non-treated HCC patients obtained from Collection des Carcinomes Hepatocellulaires (France) We show that 12 ABC genes are significantly up-regulated in HCC compared to adjacent healthy liver We next wanted to determine if these changes in ABC expression are associated with miRNA dysregulation The miRNA Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy profile of a subset of 10 patients reveals significant changes between HCC and healthy liver, for instance the let-7 family is times downregulated in HCC We have chosen a few down-regulated miRNAs which have bioinformatically predicted target in up-regulated ABC genes in order to validate them in vitro ABC, sub-family C transporters Abcc1 and Abcc2, also known as multidrug resistance proteins MRP1 and MRP2, were up-regulated in HCC Downregulation of MRP1/MRP2 could result in improved response of the tumors to chemotherapy We designed short hairpin RNAs: targeting murine Abcc1 and targeting murine Abcc2 In vitro assays in murine HCC cells showed endogenous mRNA knock-down of more than 50% with shAbcc11, shAbcc17, and shAbcc22, shAbcc23, shAbcc27, shAbcc28 The knock-down effect was sequence-specific and not due to shRNA off-targeting or toxicity scAAV8 transduces almost completely the murine liver scAAV8 expressing shAbcc11, shAbcc17, shAbcc22, shAbcc28 were therefore produced and injected in BL/6 mice and knock-down of Abcc1/Abcc2 was determined weeks post-injection Knocking-down multidrug resistance proteins may improve the effectiveness of the current anti-cancer therapies 567 Antisense Drug Development for Skipping Human Dystrophin Exons In Vitro and In Vivo Systematically Bo Wu,1 Ehsan Benrashid,1 Peijuan Lu,1 Caryn Cloer,1 Allen Zillmer,1 Mona Shaban,1 QiLong Lu.1 McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC Antisense therapy has recently been demonstrated with great potential for frame-restoring of dystrophin mRNA in human muscle cells and in local muscles of Duchenne muscular dystrophy (DMD) patients Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs), for targeted skipping of human dystrophin exons Identification of AOs with highest efficiency and specificity is therefore crucial for AO drug development In this study, we applied three cell culture systems, C2C12 myoblasts containing a GFP-reporter gene expressing human dystrophin exon 50, normal human myoblasts and a culture of DMD patient-derived skin fibroblasts, to screen AOs targeting human dystrophin exon 50 The GFP reporter system was most effective and sensitive for quantitative measurement of exon skipping efficiency The efficacy of selected AOs was further investigated with Vivo-Morpholino chemistry in the hDMD transgenic mouse carrying the full-length human dystrophin gene Our results suggest that a combination of in vitro cell culture based selection systems and a Vivo-Morpholino based evaluation in vivo systemically, provides stringent screening to identify effective AOs targeting human dystrophin exon for drug development to treat DMD patients 568 Tracking Single Liposomes for Delivery of siRNA Matthew R Tiffany,1 Francis C Szoka.1 Bioengineering, Therapeutic Science & Pharmaceutical Chemistry, University of California, San Francisco, San Francsico, CA The impetus for this work is based upon the observation that liposomes, and non-viral vectors as a whole, are inefficient at delivering their contents to the cytosol For example, a single liposome encapsulates at least ∼10^2 siRNA molecules, and at the very least 10^3 liposomes are bound per cell in a typical transfection Thus the potential for delivery starts at 10^5 siRNAs per cell In contrast, current estimates indicate that the cell needs ∼10^3 siRNAs for effective silencing The two log discrepancy between the amount dosed and the amount for a biological outcome suggests that either only a small fraction of molecules get delivered to the cytoplasm in S217 OLIGONUCLEOTIDE & RNAI THERAPEUTICS I an active form or only a small fraction of liposomes deliver their contents Internalization of liposomes is poorly understood at the single particle scale First the liposome must interact with the cell at the plasma membrane where it can be endocytosed by a number of different mechanisms, potentially down a pathway resulting in nonproductive delivery Along this pathway the liposome experiences a drop in pH as the endosome transitions into a lysosome It is believed that if the cargo does not escape from the liposome before the lysosome then delivery has failed There are a variety of lipids that have been made to tune endosomal escape to a pH typically between and The most accepted mechanism of delivery involves mixing of cationic or zwitterionic vector lipids with the anionic lipids of the endosome The oppositely charged lipid molecules undergo an electrostatic interaction resulting in charge neutralization and a bilayer phase change from lamellar to inverted hexagonal This process allows the contents to release not only from the vector due to lipid mixing but also into the cytoplasm because of the phase inversion We are studying the endocytosis and release of siRNA from liposomes using biochemical and optical techniques To provide a consistent liposomecell interaction we will use a melanoma cell adhesion molecule (MCAM) targeting scFv antibody Our first aim is to determine the uptake kinetics of a control formulation (POPC:Cholesterol) We will then move into single particle tracking of control liposomes and determine endosomal pH change, modes of motion (active, simple, or constrained diffusion), fraction of successful delivery events, and contents release Finally, we will repeat the studies with liposome formulations that are pH responsive (e.g DOPE:Cholesterol hemisuccinate), including a new class of zwitterionic lipids There is evidence demonstrating that pH responsive liposomes are better vectors than non-pH responsive liposomes on a population scale, however, what is lacking is a fundamental understanding of how, when and to what extent delivery occurs at the individual liposome level This work is funded by the National Institute of Biomedical Imaging and Bioengineering project number 2R01EB003008-06 569 RNAi-Mediated Gene Therapy of Human Diseases Annemart Koornneef,1 Piotr Maczuga,1,2 Florie Borel,1,3 Angelina Huseinovic,1 Peter Jansen,1,3 Harald Petry,1 Pavlina Konstantinova.1 Amsterdam Molecular Therapeutics B.V., Amsterdam, Netherlands; 2Department of Gastroenterology, Leiden University Medical Center, Leiden, Netherlands; 3Department of Hepatology, Academic Medical Center, Amsterdam, Netherlands AAV vectors allow efficient, safe, long-term gene delivery in a wide range of tissues and therefore are excellent vectors for gene therapy AAV vectors can efficiently deliver short hairpin RNAs (shRNAs) and microRNAs (miRNAs) that inhibit the production of a disease-related mRNA by RNA interference (RNAi) Our research is focused on treatment of two liver diseases: hypercholesterolemia and hepatocellular carcinoma Additionally, we are developing RNAi-based gene therapy for Huntington’s disease by AAV-delivered miRNA in the brain Hypercholesterolemia: As a first proof-ofconcept in vivo, the Apolipoprotein B (ApoB) gene expression was decreased in murine livers transduced with AAV-shApoB and AAVmiApoB Transduction of murine livers with AAV-miApoB resulted in a reduction of murine plasma cholesterol and ApoB expression for up to months while the inhibitory effect of AAV-shApoB started to wear off at weeks Ongoing research aims to determine the mechanism underlying the differences seen between long-term AAVshApoB and AAV-miApoB efficacy in murine livers We hypothesize that the long-term stability of the miApoB is due to its lower toxicity and off-target properties compared to shApoB because expression of miApoB is specifically limited to hepatocytes by a liver-specific promoter Our results demonstrate a robust long-term knock-down of ApoB by AAV-delivered miApoB in murine liver, thus providing an S218 excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia and associated cardiovascular disease Hepatocellular carcinoma (HCC): We are aiming to discover new targets for RNAi-mediated gene therapy of this devastating disease The ATP-binding cassette transporters (ABC) are involved in tumor resistance by decreasing the intracellular concentration of toxic compounds Therefore, we determined changes in expression of 15 ABC transporters in HCC by screening their profile in 19 paired HCC patient samples Furthermore, we identified changes in expression of 79 cellular miRNAs between HCC and healthy liver We are currently verifying the bioinformatically predicted miRNA targets in the ABC genes in vitro The ABCC1 and ABCC2 genes were significantly up-regulated in the HCC patient samples We designed shRNAs targeting ABCC1/2 and demonstrated a strong knock-down in vitro and we are currently trying to determine their efficacy in vivo using AAV-shRNA vectors Huntington’s disease (HD): HD is a dominant neurodegenerative disorder caused by poly-CAG expansions in the Huntingtin (Htt) gene We are currently using artificial miRNAs to silence several target genes involved in HD in vitro We are optimizing the miRNA molecules in order to obtain best efficacy and specificity against the target genes For clinical applications, the miRNA expression should be switched on and off at desired points Therefore, we are currently developing inducible miRNA expression system that can be used as an additional safety measure in the future RNAi-based gene therapy of genetic diseases 570 Silencing the Human Eag1 Potassium Channel in Glioma Cells by Using Short Interfering RNA Ludmylla C Cunha,1 Ana Paula P Faria,1 Bruno C Gonzaga,1 Elaine Del Bel,2 Luis A Pardo,3 Walter Stühmer,3 Ricardo Titzede-Almeida.1 Lab Tecnologias para Terapia Gênica, FAV, UnB, University of Brasília - UnB, Brasília, Distrito Federal, Brazil; 2Lab Neurofisiologia e Biologia Molecular, Dept Morfologia Estomatologia e Fisiologia, FORP, University of São Paulo - USP, Ribeirão Preto, São Paulo, Brazil; 3Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Göttingen, Germany Ether a go-go (Eag) potassium channels are natively expressed in mammalian brain regions Most cancer cell lines and tumor tissues also express Eag1, the prototypic Eag potassium channel In the present study we set up a protocol to silence Eag1 from glioma cells in culture For that, we used dsRNA sequences synthetized by Qiagen targeted to the previously described mRNA Eag1 interfering sequence GTCCACTTGGTCCATGTCCAG The dsRNA was transfected (150pmol) into glioma cells (U251MG) in culture using Lipofectamine® (Invitrogen) The negative control dsRNA was the commercial scramble All-Star® (Qiagen) The cell culture media used in our study were Optimen® (Gibco) and DMEM® (Gibco), for the first hs and for the remaining 24h of incubation, respectively We used commercial kits for RNA extraction (RNeasy Plus MiniKit®, Qiagen) and reverse transcription (SuperScript™ First-Strand Synthesis System for RT-PCR, Invitrogen) The relative expression (2-∆∆CT) of Eag1 was determined by using a quantitative PCR protocol (qPCR) with SYBR Green® (Applied Biosystem 7500 Fast Real-Time PCR System) The forward and reverse primers for Eag1 were 5’- TCTGTCCTGTTTGCCATATGATGT-3’ and 5’-CGGAGCAGCCGGACAA-3’ For the housekeeping gene transferrin, we used the primers 5’- GACTTTGGATCGGTTGGTGC-3’, and 5’-CCAAGAACCGCTTTATCCAGAT-3’.The PCR mixture contained the following reagents: SYBR Green mix, 5,0 µL; cDNA, 250ng; 0,4 µL of each primer 10µM/µL; milliQ water up to 10µL The qPCR cycles used in our study were: initial denaturation step at 95ºC for 5min, followed by 40 cycles of amplification (denaturation Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... the uptake kinetics of a control formulation (POPC:Cholesterol) We will then move into single particle tracking of control liposomes and determine endosomal pH change, modes of motion (active,...OLIGONUCLEOTIDE & RNAI THERAPEUTICS I an active form or only a small fraction of liposomes deliver their contents Internalization of liposomes is poorly understood at the single particle scale First the liposome... liposome before the lysosome then delivery has failed There are a variety of lipids that have been made to tune endosomal escape to a pH typically between and The most accepted mechanism of delivery