259 Efficient In Vivo Delivery of Cholesterol Conjugated siRNA to the Liver Using Endogenous Chylomicron Remnant Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of[.]
OLIGONUCLEOTIDE & RNAI THERAPEUTICS III endosome escape via fusion with endosome membrane In the present study, lysophospholipid and GMO and Oleic acid (OA) was assessed for being helper lipids in siRNA delivery The lysophospholipid is a membrane fusion inhibitor as reported, and it makes the bilayers more stable This was veried by the cellular uptake result of siRNAFAM complexed with 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP)/DOPE and DOTAP/DOPE/LysoPC, determined by FACS or uorescence microscope After 24 h of post siRNA transfection, the cells were mainly shown green uorescence treated by FAMsiRNA-DOTAP/DOPE-Rhodamine-DOPE lipoplex, while the cells were mainly shown yellow (merge of green and red) treated by FAM-siRNA-DOTAP/DOPE/LysoPC-Rhodamine-DOPE This demonstrated that the formulations containing lysophospholipid were more stable than DOTAP/DOPE The stable complex might protect siRNA from degradation by the nuclease and followed by endocytosis as an intact particle But the siRNA effect by DOTAP/DOPE/LysoPC was not better or even less than that by DOTAP/DOPE This resulted from endosome entrapment due to the membrane fusion inhibition So to get efcient endosome escape, fusion promotor, like glycerol monoleate (GMO) or oleic acid (OA), was included into the lipid mixture This part of work is on-going It is expected that the combination of membrane fusion inhibitor and promoter will increase siRNA silencing effect by tailoring the structure of the lipoplex 257 c-Cbl shRNA-Expressing Adenovirus Functions as a Sensitizer of TRAIL-Induced Apoptosis Seeun Oh,1,2 So Y Kim,1,2 Bo K Sun,2 Dongho Kim,3 Suhung Lee,3 Yong J Lee,4 Joo-Hang Kim,1,2 Jae J Song.2 Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea; 2Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea; 3Genolution Pharmaceuticals, Inc., Seoul, Korea; Department of Surgery and Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA Recently we observed that c-Cbl-mediated degradation of TRAIL receptors is responsible for the development of the early phase of TRAIL resistance Our studies revealed a possible mechanism that Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is responsible for the rapid degradation of TRAIL receptors; Cbl binds to them and induces monoubiquitination of these receptors concurrent with their degeneration soon after TRAIL exposure, creating the early phase of acquired TRAIL resistance Therefore, we investigated the possibility of c-Cbl knock down as a sensitizer of TRAIL-based gene therapy First of all, several targets for efcient repression of c-Cbl proteins were screened by real time RT-PCR Selected target was designed for the shRNA and inserted into adenoviral shuttle vector for the production of adenovirus Then, we tested whether adenovirus-expressing si-Cbl RNA has downregulated Cbl expression and has functioned as a sensitizer of TRAIL-induced apoptosis in various tumor cell lines As a consequence, TRAIL enhanced apoptosis in Cbl-down regulated cells induced by preinfected c-Cbl shRNA-expressing adenovirus And this sensitizing effect of c-Cbl shRNA-expressing adenovirus was also conrmed in mouse model Based on the evidences, combined therapy using both TRAIL gene and c-Cbl shRNA is under investigation 258 ncRNAs Originating from the Dystrophin Gene as Biomarker for Assessing Antisense Therapy Matteo Bovolenta,1 Annarita Armaroli,1 Marcella Neri,1 Simona Brioschi,1 Marina Fabris,1 Chiara Scotton,1 Soa Falzarano,1 Paola Rimessi,1 Emanuele Valli,2 Giovanni Perini,2 Luciano Merlini,1 Francesca Gualandi,1 Alessandra Ferlini.1 Department of Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy; 2Department of Biology and Evolution, University of Bologna, Bologna, Italy The DMD gene is the largest in the human genome, it spans 2.2 Mb and is made of 79 exons and isoforms, all nely regulated and expressed in specic tissues Mutations in this gene lead to three distinct phenotypes: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy and X-linked dilated cardiomyopathy Therapeutic approaches are now becoming reality in DMD, nevertheless, clinical outcome measures may not always be sensitive enough to detect small changes in disease progression/regression and after short treatment periods It is therefore imperative to identify enrichment endpoints, as biomarkers, able to document benets of the treatment at early stages and at the individual level Recently, genome-wide approaches to monitor transcription have revealed a notable number of non coding RNAs (ncRNAs) with many regulatory functions To address this issue we designed a novel gene-specic Gene Expression tiling array covering the full DMD gene and used it to search for non-coding transcripts in polyA+ RNAs from human brain, heart, skeletal muscle and skin The search identied 13 sense and antisense oriented transcripts, originating from dystrophin introns/UTR regions Six of these ncRNAs (four sense polyA+ ncRNAs, located adjacent to dystrophin known promoter regions, and the antisense) were validated through Northern blotting and fully characterized by RACE PCR and sequencing Their length ranges from 1800 to 2800 bp, were unspliced, but one was spliced in several isoforms Compartmentalisation studies demonstrated that all the six ncRNAs are located in the nucleus None of these has an open reading frame, suggesting that they belong to the long non-coding RNA category These ncRNAs are highly represented in human heart, skeletal muscle and brain, known tissues of elective dystrophin expression CGH-DMD analysis in DMD patients with dystrophin exons 45-50 deletion (thus eligible for exon 51 skipping antisense therapy) allowed us to dene the deletion breakpoints therefore predicting the loss/maintenance at the genomic level of the ncRNAs region One sense oriented ncRNA, which localises within intron 44 and the corresponding genomic region was deleted in only one DMD boy, was consistently missing in his myogenic cells Further studies are in progress in order to prole the expression of these ncRNAs in patients’ myogenic cells before and after AONs treatment for exploring if these transcripts may be used as muscle transcriptomic biomarkers for monitoring the impact of novel treatment in dystrophinopathies 259 Efcient In Vivo Delivery of CholesterolConjugated siRNA to the Liver Using Endogenous Chylomicron Remnant Kazutaka Nishina,1 Kie Yoshida,1 Yoshitaka Uno,1 Hiroya Kuwahara,1 Wenying Piao,1 Hidehiro Mizusawa,1 Takanori Yokota.1 Neurology and Neurological Science, Tokyo Medical and Dental University, Tokyo, Japan RNA interference (RNAi) is a powerful tool of target-specic knockdown of gene expression For a therapeutic approach, however, efcient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ has not been established Intravenously administered cholesterol-conjugated siRNA (Chol-siRNA) was reported to reach S98 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy STEM CELL BIOLOGY liver by the interaction with serum high-density lipoprotein We here use endogenous chylomicron remnant (CMR) to deliver Chol-siRNA to liver specically We extracted endogenous chylomicron (CM) from rat lymph, and then we ex vivo bound Chol-siRNA with CMR which was made from CM by lipoprotein lipase Finally, we intraperitoneally administered Chol-siRNA with endogenous CMR to rat With this new method, intraperitoneal injection of Chol-siRNA targeting apolipoprotein B (apoB) could more efciently reduce endogenous apoB mRNA in the liver than intravenous injection of Chol-siRNA alone This delivery system is considered to use physiological pathway of CM and their receptors in the liver These ndings indicate that Chol-siRNA with endogenous CMR is effective and safe for RNAimediated gene silencing in the liver Stem Cell Biology 260 Controlled Nonviral Gene Delivery and Expression Using Stable Neural Stem Cell Line Transfected with a Hypoxia-Inducible Gene Expression System Yoon Ha,1 MengLu Liu,1 KeungNyun Kim,1 DoHeum Yoon.1 Neurosurgery, College of Medicine,Yonsei University, Seoul, Korea Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration We introduced a hypoxia-regulated plasmid-based system into mouse neural stem cells (NSCs) as an efcient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemicregulated gene expression in the spinal cord A synthetic hypoxia-responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed-expressing doublepromoter plasmid for cell lipofection and Zeocin-selection to establish a hypoxia-regulated stable NSC line (NSC-Epo-SV-Luc) A non-hypoxia-regulated stable NSC line (NSCSV-Luc) was also established as a control Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC-Epo-SV-Luc, but not in NSC-SV-Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down-regulated to a low basal level after reoxygenation of the hypoxic cells Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again After transplantation into a rat spinal cord injury (SCI) model, only NSC-Epo-SV-Luc showed ischemic injury-specic luciferase expression and achieved a maximal induction of approx 2.5 to 4.3-fold above the basal expression in uninjured rats Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia-regulated luciferase expression after differentiation We propose that NSCs engineered with the EpoSV-therapeutic gene will be valuable for developing a controllable stem cell-mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses 261 Non-Viral Carriers Deliver mRNA Encoding Marker Proteins in HeLa cells and mRNA Encoding CXCR4 in Mesenchymal Stem Cells Joanna Rejman, Geertrui Tavernier, Joseph Demeester, Stefaan S D Desmedt Research Group on Nanomedicines, Faculty of Pharmacy, Ghent, Belgium Introduction The use of mRNA as a therapeutic agent has several advantages when compared to pDNA; there are no immunogenic CpG motifs, there is no risk for insertional mutagenesis and mRNA does not need to cross the nuclear barrier to bring forth protein expression mRNA has the ideal properties to be used in particular applications, mainly when transient protein expression is required, e.g vaccination strategies or temporal change of properties of (stem) cells Efcient packaging of nucleic acids is a necessity to accomplish their therapeutic effect Viral carriers have already shown their capacity to transfect cells very efciently but their toxicity is a serious concern Alternatively, the ability of the less toxic cationic lipids and polymers to form complexes with nucleic acids and to deliver them into cells, is currently widely investigated Objectives It was our aim to evaluate transfection efficiency of mRNA complexed with one of four non-viral carriers; two cationic lipids (Lipofectamine and DOTAP/DOPE) and two cationic polymers (linear poly(ethyleneimine and poly-beta-aminoester) Therefore, mRNA encoding marker proteins (Luciferase or GFP) was used to transfect Hela cells and mRNA encoding CXCR4 to transfect mesenchymal stem cells (MSCs) Our rationale for the latter approach is to improve migration capacity of MSCs towards damaged tissue which excretes, among other cytokines, SDF-1, a protein which interacts specically with the CXCR4 receptor MSCs have already been proven to migrate to injuries and to ameliorate the nal outcome, however their migration capacity is insufcient Transfection of HeLa cells with mRNA encoding marker proteins mRNA encoding luciferase was complexed to Lipofectamine, DOTAP/DOPE, linear PEI or poly-beta-aminoester The complexes were incubated with HeLa cells and luciferase expression was assessed by a bioluminescence assay Although all four cationic carriers resulted in signicant luciferase expression, Lipofectamine was by far the most efcient, yielding a luciferase expression lasting for up to days We also assessed the proportion of cells that become transfected by means of transfection with mRNA encoding GFP and compared it with transfection by the pDNA analogue For both cationic lipids as well as for the polymer PbAE, transfection with mRNA yielded a substantially larger fraction of transfected cells than transfection with pDNA Transfection of MSCs with mRNA encoding CXCR4 S99 ... intraperitoneal injection of Chol -siRNA targeting apolipoprotein B (apoB) could more efciently reduce endogenous apoB mRNA in the liver than intravenous injection of Chol -siRNA alone This delivery. .. considered to use physiological pathway of CM and their receptors in the liver These ndings indicate that Chol -siRNA with endogenous CMR is effective and safe for RNAimediated gene silencing in the liver. .. BIOLOGY liver by the interaction with serum high-density lipoprotein We here use endogenous chylomicron remnant (CMR) to deliver Chol -siRNA to liver specically We extracted endogenous chylomicron