880 Lipid Substituted Poly(L Lysine) Polymers for Effective Transfection of Human Skin Fibroblasts Structure Function Relationships Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The[.]
CHEMICAL AND MOLECULAR CONJUGATES III 878 siRNA Delivery for HIV Treatment Using Pseudodendritic Oligoamines Verena Russ,1 Alexander Philipp,2 Ernst Wagner,2 John J Rossi.1 Department of Molecular Biology, City of Hope - Beckman Research Institute, Duarte, CA; 2Pharmaceutical BiologyBiotechnology, Ludwig-Maximilians University Munich, Munich, Germany RNA interference (RNAi) is an endogenous process, which employs small interfering RNAs (siRNA) to suppress targetspecific gene expression by mRNA degradation Reduction of gene expression through RNAi has a great potential to create a novel class of therapeutics that address the treatment of several previously untreatable diseases and viral infections like e.g HIV Significant efforts in terms of non-viral siRNA delivery have been made by developing cationic lipid based carriers, but the establishment of safe and effective methods both in vitro and in vivo is still challenging Currently, there are no efficient synthetic vectors available for non-viral gene therapy of HIV infections using therapeutic siRNAs Pseudodendrimers, degradable hyperbranched polymers, have been shown to deliver pDNA efficiently [1] Here, we explore the potential of pseudodendrimers for siRNA delivery We formed the pseudodendritic cores by coupling an excess of 1,6-hexandioldiacrylate (HD) to non-toxic 800Da oligoethylenimine (OEI) OEI is responsible for mediating endosomal release via the “proton-sponge effect” The labile ester bonds in HD are hydrolysed by esterases, thus decreasing cytotoxicity and increasing the intracellular unpackaging of the carrier, which releases the siRNA We established a structure activity relationship by modifying the surface of the pseudodendritic cores with different LMW oligoamines including ethanolamine, ethylenediamine, spermidine, spermine, pentaethylenhexamine and OEI Screening studies revealed that siRNA delivered by the spermine surface-modified conjugate showed the best down regulation of gene expression (up to 80%) in stably luciferase transfected cells In comparison to standard siRNA transfection agents (e.g Lipofectamine 2000) this pseudodendrimer exhibits a similar cytotoxicity profile and its siRNA complex yields better target gene silencing in human cell lines such as HEK293 and CEM, as shown by qRT-PCR This polymer can be exploited to create a biocompatible, safe and efficient siRNA carrier for HIV treatment Reference: [1] Russ V, Elfberg H, Thoma C, Kloeckner J, Ogris M, Wagner E Gene Ther 2008 Jan;15(1):18-29 879 Novel Lysine-Based Bioreducible Nonviral Gene Delivery Carriers Malavosklish Bikram,1 Mohamed I Nounou,1 Vicky Mody,1 Kalyopy Emmanouil,1 Zhuoyuan Lu.1 Pharmacological & Pharmaceutical Sciences, University of Houston, Houston, TX The development of biodegradable gene delivery systems, which have the ability to effectively deliver therapeutic DNA to a target tissue, is paramount to the success of nonviral gene delivery One approach to developing biodegradable polymers is to introduce disulfide bonds along the backbone of the polymers to ensure release of the DNA in the reductive environment of the cytoplasm, whilst simultaneously reducing the molecular weight of the polymers.1 Tantamount to the degradability of the polymers is the need to develop biocompatible polymers, which have low cytotoxicities so as to maintain cell viability and hence increase transfection efficiencies Therefore, to produce a biocompatible gene delivery system, we have designed and synthesized novel reducible copolymers of the type (AB)n, which consist of repeating units of the natural amino acid, L-lysine and cystamine bisacrylamide (CBA) These novel lysine-based reducible copolymers (LRCs) were then modified with ethylenediamine so as to introduce primary amines for efficient DNA condensation The molecular weight (MW) of the copolymers Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy was found to be ∼3.2 kDa with a polydispersity index of ∼1.2 A gel retardation assay showed complete condensation of DNA at N/P ratios [nitrogens of polymer/phosphates of DNA] higher than 20/1 To investigate the mechanism of DNA release from the polymer/ DNA complexes, fluorescence spectroscopy studies were performed with 1,4-dithio-DL-threitol (DTT) These data showed a significant reduction in fluorescence intensity following the addition of LRCs to DNA After the addition of DTT, there was a 95 % increase in fluorescence intensity, which indicated the reduction of the disulfide bonds and the release of the DNA from the complexes The particle sizes of the LRC/DNA complexes were found to be between 100 to 231 nm with surface charges of 0.8 to 20 mV The transfection efficiencies of the complexes as determined with a luciferase assay showed that LRC complexes produced ∼7-8 times higher transfection efficiencies in human dermal fibroblasts (HDFs), ∼3 times higher transfection efficiencies in human breast adenocarcinoma cells (MCF-7s), and ∼4 times higher transfection efficiencies in metastatic mouse breast cancer cells (4T1s) as compared to the control In addition, confocal studies showed the association of released DNA with the nuclei of transfected cells Finally, the cytotoxicities of the LRCs showed significantly lower cytotoxicities as compared to the control Therefore, these results suggest that these novel LRCs are very efficient and biocompatible polymers for nonviral gene delivery References L V Christensen, C W Chang, J W Yockman, R Conners, H Jackson, Z Zhong, J Feijen, D A Bull, and S W Kim Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery J Control Release 118: 254-61 (2007) 880 Lipid-Substituted Poly(L-Lysine) Polymers for Effective Transfection of Human Skin Fibroblasts: Structure-Function Relationships Meysam Abbasi,1 Hasan Uludag,1,2,3 Vanessa Incani,3 Charlie Yu Ming Hsu,1 Andrea Jeffery.2 Biomedical Engineering, University of Alberta, Edmonton, AB, Canada; 2Chemical & Materials Engineering, University of Alberta, Edmonton, AB, Canada; 3Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Purpose: Enabling gene expression in skin fibroblasts using safe nonviral delivery systems has the potential to stimulate wound healing and aid in skin tissue engineering efforts In this study, several lipid-substituted poly(L-Lysines) (PLL) were investigated for their ability to deliver a plasmid DNA (pEGFP) to human skin fibroblasts Methods: Human CRL fibroblasts were used in this study and pEGFP-N2 plasmid DNA was used as a reporter gene Lipid-substituted polymers were synthesized from caprylic, myristic, palmitic, oleic and linoleic acids, and characterized by NMR Gel electrophoresis was performed for assessment of (i) pEGFP binding efficiency of polymers, (ii) pEGFP/polymer complex dissociation upon incubation with heparin, and (iii) pEGFP/polymer degradation by DNases Cellular uptake of pEGFP was assessed by fluorescent microscopy and flow cytometry Analysis of EGFP Expression was assessed by flow cytometry and PCR Results: While native and lipid-substituted PLLs showed complete complexation with pEGFP, polymers with higher lipid substitution were more resilient to dissociation after heparin treatment All polymers showed good protection of pEGFP against DNase I and DNase II digestion in vitro DNA delivery studies using fluorescently labeled pEGFP showed that native PLL lacked the ability to deliver pEGFP into cells, whereas most of the lipid-substituted PLLs gave efficient pEGFP delivery into the cells Extent of lipid substitution was an important factor in DNA delivery efficiency The intracellular pEGFP was intact after delivery with lipid-substituted polymers up to days An RT-PCR methodology indicated successful transcription of the EGFP gene, which was not the case when the cells were transfected with a blank plasmid lacking a EGFP gene Further studies with flow cytometry S335 CHEMICAL AND MOLECULAR CONJUGATES III showed that successful protein expression was obtained with PLLs substituted with myristic and stearic acid, the latter displaying a relatively lower toxicity Conclusion: We conclude that substituting lipids on PLL results in effective gene carriers and the extent of substitution, rather than the individual lipid, appeared to be critical for effective plasmid delivery 0.9 to 4.5 mg/kg/week, and delivered by weekly intraperitoneal injection Transcription, western and immunohistochemical analysis showed increased levels of dystrophin transcript, protein and correct localisation at the sarcolemma We characterised the physical properties, the interaction between nanoparticles and AON, and their diffusion pathways both by fluorescence and electron microscopy analysis We therefore showed that cationic nanoparticles have the capacity to both deliver antisense oligoribonucleotides in body-wide muscles and reduce the dose required for dystrophin rescue This non-viral approach may improve the therapeutic usage of antisense oligoribonucleotides in Duchenne muscular dystrophy as well as the delivery of RNA molecules with many implications in both basic research and medicine 881 Nanoparticle-Mediated Delivery of Antisense Oligoribonucleotides Allows Restoration of Dystrophin Expression in the mdx Mouse 1a: T1 nanoparticle scanning electron microscope 1b: Interaction between AON and T1 nanoparticles 1c: Biodistribution in the mdx mouse of fluorescent T1 A: diaphragm; B: gastrocnemius; C: heart 1d: Biodistribution of T1 by electron microscope analysis Paola Rimessi,1 Patrizia Sabatelli,1,2 Marina Fabris,1 Paola Braghetta,3 Elena Bassi,1 Pietro Spitali,1 Gaetano A Vattemi,4 Giuliano Tomelleri,4 Lara Mari,5 Daniela Perrone,6 Alessandro Medici,6 Marcella Neri,1 Matteo Bovolenta,1 Elena Martoni,1 Nadir M Maraldi,7 Francesca Gualandi,1 Luciano Merlini,1 Marco Ballestri,8 Luisa Tondelli,8 Katia Sparnacci,9 Paolo Bonaldo,3 Antonella Caputo,3 Michele Laus,9 Alessandra Ferlini.1 Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, FE, Italy; IGM-CNR, CNR Bologna, Bologna, BO, Italy; 3Department of Histology, Microbiology, and Medical Biotechnology, University of Padova, Padova, PD, Italy; 4Department of Neurological Sciences and Vision, Section of Clinical Neurology, University of Verona, Verona, VR, Italy; 5Department of Chemistry, University of Ferrara, Ferrara, FE, Italy; 6Department of Biology and Evolution, University of Ferrara, Ferrara, FE, Italy; 7Department of Human Anatomical Sciences, University of Bologna, Bologna, BO, Italy; 8ISOF, Consiglio Nazionale delle Ricerche, CNR Bologna, Bologna, BO, Italy; 9Department of Environmental and Life Sciences INSTM, University of Piemonte Orientale, Alessandria, AL, Italy For subsets of Duchenne muscular dystrophy mutations, antisense oligoribonucleotide mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein In the mdx murine model systemic delivery of antisense oligoribonucleotide, recognising the splice donor of dystrophin exon 23, has shown proof of concept We have been able to restore dystrophin expression in body-wide striated muscles of mdx animal model using different formulations of cationic nanoparticles These were loaded with low doses of 2’OMePS antisense oligoribonucleotide, ranging from S336 882 Synthesis and Evaluation of Amphiphilic Poly(tetrahydrofuran-b-Ethylene Oxide) Copolymers for DNA Delivery into Skeletal Muscle Catherine Pomel,2 Christian Leborgne,1 Hervé Cheradame,2 Daniel Scherman,1 Antoine Kichler,1 Philippe Guegan.2 Généthon - CNRS FRE 3087 - Université d’Evry, Centre de Recherche et d’Applications sur les Thérapies Géniques, Evry Cedex, France; 2CNRS UMR 7581, Université d’Evry, Laboratoire Matériaux Polymères aux Interfaces, Evry, France Purpose Amphiphilic triblock copolymers such as the pluronic poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) L64 (pEO13-pPO30-pEO13) have been shown to mediate more efficient gene transfer in muscle as compared to naked DNA We were interested in studying the effect of a chemical change of the central block of pluronic polymers on the transfection activity Methods We synthesized new amphiphilic copolymers in which the hydrophobic pPO block was replaced by poly(tetrahydrofuran) (pTHF) chains The resulting triblock pEO–pTHF–pEO polymers have been characterized by NMR and SEC and assayed for in vitro and in vivo gene transfer Results The animal experiments showed that the new copolymers are able to significantly increase the transfection efficiency of plasmid DNA after intramuscular injection Conclusions These results indicate that the capacity to enhance plasmid DNA transfection in skeletal muscle is not restricted to pEO–pPO–pEO arrangements Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... University of Padova, Padova, PD, Italy; 4Department of Neurological Sciences and Vision, Section of Clinical Neurology, University of Verona, Verona, VR, Italy; 5Department of Chemistry, University of. .. Italy; 6Department of Biology and Evolution, University of Ferrara, Ferrara, FE, Italy; 7Department of Human Anatomical Sciences, University of Bologna, Bologna, BO, Italy; 8ISOF, Consiglio Nazionale... interested in studying the effect of a chemical change of the central block of pluronic polymers on the transfection activity Methods We synthesized new amphiphilic copolymers in which the hydrophobic