158 Pulmonary Gene Delivery of Hybrid Vector, Lipopolyplex Containing N Lauroylsarcosine, Via Systemic Route Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gen[.]
CHEMICAL AND MOLECULAR CONJUGATES I 155 Intracellular Fragmentation of Cationic Gene Carriers Leads to the Enhanced Transgene Expression Tetsuji Yamaoka,1 Tomoko Hashimoto,1 Yumi Kobayashi,1 Akira Murakami.2 Department of Biomedical Engineering, National Cardiovascular Center Research Institute, Suita, Japan; 2Department of Biomelecular Engineering, Kyoto Institute of Technology, Kyoto, Japan Non-viral gene carriers have been developed as an alternative to the viral carriers because of biological safety, low cost, and easiness in manufacturing but their transfection efficiency is still low Various strategies for improving the transfection efficiency including recepter-mediated endocytosys, endosomal disruption, and nuclear transportation have been proposed for novel non-viral carriermediated gene transfer We have been focusing on the intracellular transcription step and designing polymeric gene carriers that lead to higher transgene expression Polymeric carriers with wide molecular diversity were evaluated in vitro, and it was found that the transgene recognition by the transcription factors greatly depends on the carrier properties In this work, the effect of molecular weight (Mw) of carriers on transcription efficiency on gene expression was utilized Various polypeptide-type carriers were mixed with pEGFP-N1 plasmidDNA at a given cation/anion ratio and incubated at 37 oC for 30 to allow the polyplex formation The polyplexes were mixed with fluorescence dye solution and microinjected into the cytosolic or nuclear compartoment of the COS-1 cells By measuring the fluorescent intensity under the fluorescent microscope, the amount of injected pDNA into the single cell and the EGFP expression were quantified The transcription efficiencies were calculated by dividing the EGFP intensity (expressed protein) with the dye intensity (inejected pDNA) Obtained results indicated that carriers with lower Mw lead to the higher transcription efficiency We also found a good correlation between the transcription efficiency and the polyplex formation ability According to these results, we designed novel in vitro trasnfection system utilizing an intracellular activity of furin proprotein convertase (PC) that cleaved C terminal of ArgXaa-Xaa-Arg Repetitive polypeptides containing furin-recognition sites were chemically synthesized and evaluated as gene carriers Polypeptides of longer than 16 amino acids were found to form complex effectively, while model peptides with the amino acid length of 4, which would be produced by furin digestion of the polypeptides did not form polyplexes up to the charge ratio of 50 By treating the polyplexes composed of H2N-(Arg-Lys-Lys-Arg)4-Cys-CONH2 in cell free system, release of free plasmid DNA was confirmed on agarose gel electrophoresis The PC-sensitive carrier, which contains seven cleavage sites were found to lead to high gene expression in vitro but H2N-(Arg-Lys)8-Cys-CONH2 with a same amino acid composition but no cleavage site did not These results indicate that this intracellular signal-responsive gene transfer system is the useful to improve the trangene expression in mammalian cells 156 High Level Therapeutic Effects of Fine Plasmid/PEI/Hyaluronic Acid Ternary Complex Particles in Tumor Model Mouse Tomoko Ito,1,2 Katsuyuki Hamada,3 Chieko Yoshihara,1 Yoshiyuki Koyama.1 Otsuma Women’s University, Tokyo, Japan; 2Present Address; Musashino University, Tokyo, Japan; 3Ehime University, Toon, Ehime, Japan Ionic complexes of plasmid with polycations or cationic lipids have been extensively explored as nonviral vector systems High gene expression on cultured cells has already been realized, but it remains challenging to achieve efficient in vivo gene transfection S62 There should be two major obstacles; the adverse interactions of the complex with biocomponents, and too large size of the complex to be delivered to the target cells We have developed a protective anionic polymer-coating on the DNA complex particles which re-charged the particles to negative The polyanion-coated DNA complexes showed very little interaction with blood components The remaining problem about the particle size was also solved by the protective polyanion-coating on the plasmid complex We found that the DNA/PEI/Hyaluronic acid (HA) ternary complex could be freeze-dried without loss of gene activity Very fine complex particles could be prepared by mixing the highly diluted solution of DNA, PEI, and HA, followed by freeze-dried, and rehydrated with small amount of water There was thus obtained small DNA complex particles (< 70 nm) at high concentration (> 500 ug/ml), which is applicable to in vivo experiments Plasmid encoding GM-CSF gene was then complexed with PEI and HA, and the therapeutic effects in tumor model mouse was examined Male mice at weeks of age were subcutaneously inoculated with B16 melanoma cells When the tumor size increased to mm in diameter, the plasmid complex was injected into tumor tissue, or tail vein Apparently reduced growth of tumor was observed in some cases after intravenous injection Intratumor administration of the fine plasmid particles resulted in slower tumor growth and complete inhibition of tumor growth was also seen in two of the nine mice 157 The Effect of Spacer Length, Rigidity and Hydrophilicity of Charge-Reversal Amphiphiles on DNA and siRNA Delivery Xiao-xiang Zhang,1 Carla A H Prata,1 Thomas J McIntosh,2 Mark W Grinstaff.1 Biomedical Engineering and Chemistry, Boston University, Boston, MA; 2Cell Biology, Duke University Medical Center, Durham, NC In previous research in our lab, we found that a charge-reversal amphiphile, whose total charge can be reversed from +1 to -1 in the presence of an esterase, displays high transfection activity This charge-reversal effect facilitates the release of DNA from the lipoplex We also found that spacing of the cationic charges within the headgroup in a series of peptide-based lipids can afford improved transfection performance To take advantage of the charge-reversal effect, as well as to further understand how spacer length and composition affect transfection efficacy, here we present a study on a series of charge-reversal amphiphiles with different spacers separating the head groups from the hydrophobic chains Among them, only the amphiphiles possessing rigid and hydrophilic glycine spacers (B-Gly and B-GlyGly) showed effective DNA transfection in CHO and NIH 3T3 cells as well as siRNA gene knockdown in HepG2 and UASMC cells Ethidium bromide displacement assay revealed that DNA was released the fastest from the lipoplex of B-GlyGly in the presence of esterase Also, X-ray diffraction results indicate that the DNA is located between the adjacent lipid bilayers in the lipoplex of B-GlyGly These distinct features appear to be required for high transfection activity 158 Pulmonary Gene Delivery of Hybrid Vector, Lipopolyplex Containing N-Lauroylsarcosine, Via Systemic Route Tomoaki Kurosaki,1 Takashi Kitahara,1 Hideto To,1 Tomoyuki Hamamoto,1 Hitoshi Sasaki.1 Department of Hospital Pharmacy, Nagasaki University Hospital of Medicine and Dentistry, Nagasaki, Japan Purpose: Gene therapy is expected as a drastic method to treat pulmonary diseases such as cystic fibrosis, emphysema, asthma, and certain types of lung cancer The success of the gene therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy CHEMICAL AND MOLECULAR CONJUGATES I highly depends on the development of effective, secure, and targeted delivery vector In this experiment, we developed a novel hybrid vector, lipopolyplex, for pulmonary gene delivery via systemic route Methods: We constructed the lipopolyplex with plasmid DNA (pDNA), polyethylenimine (PEI), N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethlylammonium chloride (DOTMA), and N-lauroylsarcosine (LS) The size, ζ-potential, in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of lipopolyplex were evaluated The contribution index was estimated using a simultaneous optimization technique in which a multivariate spline interpolation (MSI) was incorporated Results: The ζ-potential of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was markedly decreased by addition of LS, without much effect on the size The lipopolyplexes containing LS showed low cytotoxicity and little aggregation with erythrocytes On the other hand, the lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D After intravenous injection of the complexes into mice, high gene expressions were observed in the lung, liver, and spleen In particular, the lipopolyplexes containing LS showed extremely higher transgene efficiency than lipopolyplex 2P-2D in the lung As a result of the MSI analysis, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index Conclusion: In conclusion, lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity 159 QPSPSPT Heptapeptide-Assisted Nuclear Delivery by Nucleolin Shuttling Amanda R Lowery,1 Todd D Giorgio.1,2 Biomedical Engineering, Vanderbilt University, Nashville, TN; Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN A novel, 7-mer peptide (QPSPSPT) has been discovered and shown to associate with the nuclei of MCF7 human mammary epithelial cells Previously, our lab has described the discovery process, which is based on bacteriophage (phage) display modified for the identification of intracellular ligands Additional work has characterized the capacity of QPSPSPT to mediate nuclear localization of phage and fluorescent proteins The heptapeptide has been shown to associate with nuclear proteins using an affinity chromatography approach Based on the results of affinity chromatography using cell lysate, nucleolin is a strong candidate for QPSPSPT association Nucleolin is a 100kDa protein concentrated in the nucleolus It has been reported to shuttle between the cell membrane and the nucleus to transport materials required for ribosome biogenesis.1 Nucleolin protein, but not nucleolin mRNA, persists during periods of serum restriction in cultured mammalian cells, suggesting that the chaperone activity of nucleolin may be available during in vitro gene delivery studies.2 At this conference in 2006, nucleolin was reported to associate with DNA compacted with PEG-substituted polylysine and to modulate transgene expression, among other characteristics.3, The nucleolinspecific recognition element or mechanism, however, was not identified The intentional use of nucleolin to modulate intracellular transport of chemical gene therapies has not been described due, in part, to a lack of appropriate, nucleolin-specific ligands Nucleolin expression has recently been reported to be inversely correlated with gene expression mediated by AAV vectors.5 Through additional studies regulating the expression of nucleolin, this work aims to link nucleolin shuttling to the mechanism of QPSPSPT-mediated nuclear delivery The degree to which QPSPSPT can modulate nucleolinspecific cellular entry and nuclear delivery of chemically formulated pDNA will be reported The extent of transgene expression in these in vitro systems will also be characterized This work was funded by the US Army Medical Research and Materiel Command BCRPCDMRP under awards BC023276, ‘Nuclear Targeting of Drug and Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy Gene Therapies to Breast Carcinoma’ and BC044010, ‘Discovery and Assessment of Novel Therapeutic and Imaging Agents for Breast Cancer: Differential Nuclear Targeting Peptides’ Tuteja, R.; Tuteja, N., Nucleolin: a multifunctional major nucleolar phosphoprotein Crit Rev Biochem Mol Biol 1998, 33, (6), 407-36 Kim, S K.; Srivastava, M., Stability of Nucleolin protein as the basis for the differential expression of Nucleolin mRNA and protein during serum starvation DNA Cell Biol 2003, 22, (3), 171-8 Davis, P B.; Cooper, M J., Vectors for airway gene delivery AAPS J 2007, 9, (1), E11-7 Chen, X.; Davis, P B., Compacted DNA Nanoparticles Transfect Cells by Binding to Cell Surface Nucleolin Molecular Therapy 2006, 13, S152 Johnson, J S.; Samulski, R J., Enhancement of AAV infection by mobilizing capsids into and out of the nucleolus Journal of Virology 2008, preprint 160 Chitosan-PEG-FA-DNA Nanoparticules for Non-Viral Gene Therapy and Expression of the IL1Ra Gene in an Experimental Model of Rheumatoid Arthritis Christian Jreyssaty,1,2 Qin Shi,1 Patrick Lavigne,1 Mohammed Benderdour,1 Franỗoise M Winnik,3 Kerong Dai,4 Julio Fernandes.1,2 Orthopaedics Research Laboratory, Research Center, SacréCoeur Hospital, Montreal, QC, Canada; 2Biomedical Engineering Institute, Faculty of Medicine, University of Montreal, Montreal, QC, Canada; 3Department of Physical Chemistry and Polymer Science, Faculty of Pharmacy, University of Montreal, Montreal, QC, Canada; 4Department of Orthopaedics, School of Medicine, Ninth People’s Hospital - Jiao Tong University, Shanghai, China Gene therapy is considered to be one of the medicine challenges of the coming decade, whose success depends on the ability to deliver therapeutic DNA into target cells Non-viral polymers such as chitosan, a cationic polymer can be easily combined with DNA Once the complex formed, the DNA is protected from the nucleases degradation The first objective of this study was to define the characteristics of the best suited chitosan nanoparticle for a maximum selective in vitro transfection in KB cells The nanoparticles vary by the presence or absence of folic acid (FA), and the molecular weight (MW) of chitosan (5, 25 and 50 KDa) The nanoparticle was then selected and combined with the IL-1Ra gene, a natural blocker of the inflammatory cytokine interleukin-1 The second objective was to inject these carriers using the hydrodynamic method in a rat model of adjuvant induced arthritis (AIA) and evaluate the protective effects of IL-1Ra against inflammation in vivo The in vitro study showed that Chitosan-DNA nanoparticles in the presence of folic acid and a chitosan Mw of 25 KDa demonstrated a selective transfection in KB cells The in vivo study showed the presence of recombinant IL-1Ra protein in rat’s serum The protective effects of nanoparticles were evident by lower levels of inflammatory factors (Interleukin1-β and prostaglandins E2) expression and macroscopic decrease of limb inflammation Non-viral gene therapy with Chitosan-PEG-FA-DNA nanoparticles containing the IL-1Ra gene appears to significantly decrease inflammation in this experimental model of arthritis In our future studies, we seek to improve the characteristics of Chitosan nanoparticles to achieve a better in vivo transfection and thereafter analyze the gene expression in different rat’s organs to determine the distribution and toxicity of nanoparticles S63 ... inflammation Non-viral gene therapy with Chitosan-PEG-FA-DNA nanoparticles containing the IL-1Ra gene appears to significantly decrease inflammation in this experimental model of arthritis In... into and out of the nucleolus Journal of Virology 2008, preprint 160 Chitosan-PEG-FA-DNA Nanoparticules for Non-Viral Gene Therapy and Expression of the IL1Ra Gene in an Experimental Model of Rheumatoid... analysis, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index Conclusion: In conclusion, lipopolyplexes containing LS are safe and