403 chemical manipulation of AAV tissue tropism

403 Chemical Manipulation of AAV Tissue Tropism Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S154 CHEMICAL AND MOLECULAR CONJUGATES II pr[.]

CHEMICAL AND MOLECULAR CONJUGATES II 400 Histidine-Rich Amphipathic Peptides Promote Efcient Delivery of Nucleic Acids into Mammalian Cells Antoine Kichler,1,2 A James Mason,3,4 Christian Leborgne,1 Daniel Scherman,2 Burkhard Bechinger.3 Genethon, BP60, Evry Cedex, France; 2Unité de Pharmacologie Chimique et Génétique et d’Imagerie, CNRS UMR 8151 – U1022 Inserm, Université Paris Descartes, Chimie Paristech, Paris Cedex, France; 3Faculté de Chimie, Institut Le Bel, Strasbourg, France; 4Pharmaceutical Science Division, King’s College London, 150 Stamford Street, London, United Kingdom Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases However, in order to enable a gene to become a pharmaceutical, efcient and safe methods of delivery have to be developed We found that cationic amphipathic histidine-rich peptide antibiotics can efciently deliver DNA into mammalian cells Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efciencies comparable to those of commercially available reagents Synthesis and evaluation of LAH mutants provided evidence that the transfection efciency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place Our results also suggest a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection Further results indicate that acidication of the endosome results in high local concentrations of free peptide in this organelle These peptides become then available to interact with the endosomal membranes and thereby are responsible for the delivery of the plasmid DNA complex to the cytoplasm When these data are taken together, they indicate a dual role of the peptide during the transfection process, namely DNA complexation and membrane permeabilization Finally, we will report that peptides of the LAH family are efcient siRNA delivery vehicles 401 MHC Class-I Mediated Antigen Presentation by a Non-Viral T-MEND Vector Expressing a High Luciferase Activity in Murine Derived Dendritic Cells (JAWS-II) Sharif M Shaheen,1 Hidetaka Akita,1 Takashi Nakamura,1 Shiroh Futaki,2 Atsushi Yamashita,3 Ryo Katoono,3 Nobuhiko Yui,3 Hideyoshi Harashima.1 Molecular Design of Pharmaceutics, Hokkaido University, Sapporo, Hokkaido, Japan; 2Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan; 3School of Material Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa, Japan Cell mediated immunity CMI (due to Cytotoxic T Lymphocyte) is an essential component of immune surveillance In order to develop an effective DNA vaccination here we have developed a non-viral vector of tetra lamellar multi envelope type of nano device (T-MEND) We have modied our previously published T-MEND nano device with two kinds of peptides (Stearylated octa arginine and stearylated KALA) and evaluated its transfection activity in a model of DC, murine derived dendritic cell (JAWS-II) STR-R8 and STR-KALA were optimized in both the inner and outer lipid layers of T-MEND Every stage of lipid layers was incorporated one by another monitoring the electrical potential Our modied T-MEND showed luciferase activity (RLU/mg protein) more than 107 and encouraged the system for antigen presentation activity It showed 17 times LacZ activity when an epitope (SINFEKLE) encoded ovalbumin plasmid DNA was packaged in this system Our modied nano device has been able to deliver SINFEKLE epitope encoded plasmid DNA to the nucleus and thereby MHC-I mediated antigen presentation occurred Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy subsequently when the antigen loaded cells were exposed to the B3Z hybridoma type T-Cell It might be a milestone in developing an effective non-viral vector for CMI type of DNA vaccination 402 Evaluation of Polymer-DNA Nanoparticles for Gene Transfer to Cardiac Muscles Xiaohong Qiu, Lepeng Zeng, Jon Urban, Alena Nikolskaya, Sarah Audet New Therapies and Diagnostics, Medtronic Inc, Mounds View, MN Background: Polymer-DNA nanoparticles, as promising nonviral gene therapy modality, are non-immunogenic, able to deliver unlimited sizes of nucleic acid, and present relatively simple quality control and regulation compared to viral vectors Negatively charged, non-ionic amphiphilic copolymers were recently found to promote gene delivery in cardiac and skeletal muscle The aim of this study was to characterize the ability of polymer-DNA nanoparticles with varied charge to transfect cells of cardiac origin, as well as cardiac tissue and skeletal muscle Methods: pCMV-GFP plasmids were associated with three different polymers at various doses: the linear cationic polyethylenimine in vivojetPEI, and two amphiphilic copolymers displaying few (tetronic 304) or no charges (pluronic L64) In vitro experiments were performed in either cultured neonatal rat ventricular cardiomyocytes (NRVM) or murine atrial cardiomyocyte cell line HL-5 GFP expression levels were assessed through ow cytometry at day post transfection In vivo evaluation of pGL4 DNA complexed with 10% tetronic 304 was carried out through direct injection in the left ventricular free wall and the tibialis anterior muscles of Sprague Dawley rats Levels of luciferase expression were monitored via IVIS Imaging System (Xenogen) for up to weeks After termination, luciferase activities in rat hearts were measured by luciferase assay Results: We observed that jetPEI mediated the highest GFP expression (30%) in NRVM cells, followed by Tetronic 304 (15%) and Pluronic L64 (10%) at individual optimum dose ranges However, all three polymer/DNA complexes exhibited high toxicities toward HL-5 cells, thus hampering examination of their transfection efcacy In contrast, no toxicities were detected in NRVM cells In rats, DNA/Tetronic 304 nanoparticles exhibited luciferase expression as early as day two after administration, reached peak on day four, and diminished at week In general, skeletal muscle showed higher transfection efciency than heart muscles Luciferase analysis on heart tissues displayed residual transgene expression under the highest DNA doses Conclusion: Our results indicated that plasmid vectors with various polymers different in particular by their charge are capable of achieving efcient gene transfer in vitro cultured rat ventricular cardiomyocytes Moreover, negatively charged DNA/Tetronic 304 nanoparticles allow signicant improvement of gene transfer to myocardium, with reference to naked DNA, and therefore hold promise as valuable therapeutic gene carriers for in vivo heart gene delivery 403 Chemical Manipulation of AAV Tissue Tropism Eric D Horowitz,1 Jana L Phillips,1 Aravind Asokan.1,2 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Genetics, University of North Carolina, Chapel Hill, NC Rational and combinatorial mutagenesis of adeno-associated viral (AAV) capsid proteins has traditionally been used to create AAV vectors with novel tissue tropisms for gene therapy applications Development of methods to chemically modify naturally occurring amino acid residues in AAV capsid proteins would add another dimension to vector design Specically, chemoselective reactions S155 CHEMICAL AND MOLECULAR CONJUGATES II that can target specic amino acid side chains can be exploited to alter charge, polarity, hydrophobicity and hydrogen bonding potential within receptor binding domains on AAV capsids Such ability to alter specic receptor footprints on the AAV capsid surface enables generation of synthetic vectors with altered tissue tropism Such an approach could create a facile method for generating synthetic AAV vectors based on existing AAV templates Using a battery of biochemical and biological assays, we studied the effect of several bioconjugation reactions on receptor usage, antigenicity, transduction efciency and tissue tropism of chemically modied AAV vectors In particular, we report the discovery of new synthetic AAV vectors demonstrating expanded tissue tropism in mouse models in vivo biocompatible polymers, which have low cytotoxicities so as to maintain cell viability and hence increase transfection efciencies Therefore, to produce a biocompatible gene delivery system, we have designed and synthesized novel reducible linear L-lysine copolymers (LLCs) of the type (AB)n, which consist of repeating units of the natural amino acid, L-lysine grafted with ethylenediamine and cystamine bisacrylamide (CBA) (Figure 1) 404 Versatile Self-Assembling Nanoparticles for Enhanced siRNA Delivery into Mammalian Cells Gulam H Dar,1 Madhusudhana R Nalam,1 Vijaya Gopal.1 W212, West Wing First Floor, Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India Current nucleic acid delivery systems are still challenged by a requirement to enhance uptake both in vivo and in vitro Recent approaches for plasmid DNA delivery that include cationic lipids formulated with co-lipids, in addition to nucleic acid-binding designer cationic peptides and cationic polymers, are suitable for introducing siRNA into cells Although RNAi offers considerable potential as a therapeutic tool, obstacles in transporting siRNA into cells to achieve efcacious delivery need to be overcome siRNAs are also quickly degraded in circulation thereby impeding systemic delivery This serves as a starting ground for devising approaches to introduce siRNA into cells effectively and with minimal cytotoxicity We have recently developed delivery formulations composed of a designed synthetic 18-mer peptide P18 and versatile recombinant peptides TAT-Mu or Mu-Mu that are efcient for siRNA delivery Cationic lipid DHDEAC (N, N-Di-n-hexadecyl-N,N-dihydroxyethyl ammonium chloride, formulated with the co-lipid cholesterol, DHDEAC:Chol at 1:1 mole ratio) along with these peptides were tested for delivery of siRNA against an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293 and HeLa cells Fluorescence Activated Cell Sorting of cells transfected in the presence and absence of the peptides indicated similar levels of uptake of siRNA The formulations resulted in knockdown of GAPDH expression by >82% in HEK293/HeLa cells It appears that siRNA is efciently disassembled from the formulation containing the peptides enabling efcient targeting of mRNA Transfection efciency was also determined in PC-3 tumor derived cell line Our observations demonstrate the ability of peptide-mediated delivery to induce a specic RNAi response where optimal gene knockdown was observed at 1:16:1 (siRNA: peptide: lipid) The potential of these formulations for targeted delivery and therapeutic applications will be discussed The molecular weight (MW) of the copolymers was found to be ∼3.2 kDa with a polydispersity index of ∼1.2 Gel retardation assays showed complete condensation of DNA at N/P ratios greater than 20/1 and exceptional LLC/pDNA polyplex stability during incubation with DNase I To investigate the mechanism of DNA release from the polymer/pDNA complexes, uorescence spectroscopy studies were performed with 1,4-dithio-DL-threitol (DTT) These data showed a signicant reduction in uorescence intensity following the addition of LLCs to DNA After the addition of DTT, there was a 95 % increase in uorescence intensity, which indicated the reduction of the disulde bonds and the release of the DNA from the complexes The particle sizes of LLC/pDNA polyplexes were found to be between 100-231 nm with surface charges of 0.8-17 mV respectively The transfection efciencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced ve times higher transfection efciencies in HDF cells, three times higher transfection efciencies in MCF-7 cells, and four times higher transfection efciencies in MA cells as compared to the optimal PLL control (Figure 2) 405 Novel L-Lysine Reducible Copolymers for Efcient Nonviral Gene Delivery Mohamed M Ismail Nounou,1 Samuel Chung,1 Kalyopy Emmanouil,1 Thu Pham,1 Zhuoyuan Lu,1 Malavosklish Bikram.1 Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 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 disulde 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 Tantamount to the degradability of the polymers is the need to develop S156 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... transduction efciency and tissue tropism of chemically modied AAV vectors In particular, we report the discovery of new synthetic AAV vectors demonstrating expanded tissue tropism in mouse models... facile method for generating synthetic AAV vectors based on existing AAV templates Using a battery of biochemical and biological assays, we studied the effect of several bioconjugation reactions... receptor binding domains on AAV capsids Such ability to alter specic receptor footprints on the AAV capsid surface enables generation of synthetic vectors with altered tissue tropism Such an approach