842. Cell Permeable Peptides: Potential Use To Enhance Intracellular Gene Delivery

MOLECULAR CONJUGATES 842. Cell Permeable Peptides: Potential Use To Enhance Intracellular Gene Delivery Miguel Mano,1,2 Paula Verissimo,2 Carlos Faro,2 Abraham Loyter,3 Sergio Simoes,1,4 Maria C. Pedroso de Lima.1,2 1 Department of Gene Therapy, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal; 3Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel; 4Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. Over the past few years, the discovery that a number of small peptides, defined as cell permeable peptides (CPPs), are able to very efficiently cross cell membranes through a mechanism that is independent of receptors or transporters and that avoids lysosomal enzymatic degradation, has been enthusiastically considered of key interest for the development of new therapeutic approaches. For gene therapy purposes, their capacity to efficiently penetrate cell membranes, and most importantly the ability shared by a number of cell permeable peptides to accumulate inside the nucleus of cells, render them particularly suited to act as gene delivery vectors per se, or in association with other existing non-viral systems. Despite the extensive research on the unusual ability of these peptides to translocate across cell membranes and to promote the intracellular uptake of various cargo molecules, especially proteins, the mechanism of their internalization remains poorly understood. Reports that peptide penetration is a consequence of artifactual observations caused by a redistribution of surface-bound peptides upon cell fixation, and results of criterious reevaluations of the cellular uptake of these peptides that are consistent with the involvement of an endocytic process, have recently added great controversy to this field. Therefore, a careful evaluation of the ability of a recently described cell permeable peptide to penetrate and accumulate inside the nucleus of cells constituted the first aim of the present work. Analysis of peptide internalization in live cells, and results addressing the effect of several drugs which selectively compromise different internalization pathways, clearly demonstrated that the penetration of the peptide used in this study occurs through a mechanism distinct from classic endocytosis. The main goal of the present work was, however, to explore the observed cell permeable properties of this peptide to enhance intracellular/nuclear gene delivery. To this end, the biological performance of different conjugates obtained by electrostatic association of the cell permeable peptide with plasmid DNA was evaluated in terms of their capacity to deliver DNA into the cell nucleus and of the subsequent transgene expression. Additionally, the peptide was tested for its ability to potentiate gene transfer when associated with other non-viral gene delivery systems.

843. Single-Chain Polypeptide: Universal Module for Targeted Gene Delivery Valentina A. Anisimova,1 Igor G. Shemyakin.1 Protein Engineering, State Reaearch Center for Applied Microbiology, Obolensk, Russian Federation.

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For the present viruses remain to be the most effective gene delivery vehicles in comparing with non-viral ones due to the last lack one or more properties necessary for successful overcoming three membrane barriers of target cells. We have constructed multifunctional protein consisting of three parts: human protamine PI, responsible for binding, condensing and nuclear targeting of DNA; translocation domain of diphtheria toxin (DT) facilitating endosome escape and streptavidine (SA) as a mediator between DNA-protein S320

complexes and biotinilated ligands/antibodies which interact with specific molecules on the surface of target cells. SA in this construction provides as well possibility for screening of the most effective for gene delivery surface molecules. Gene of PI has been assembled from synthetic oligonucleotides taking into account codon usage frequency by Escherichia coli cells. The fusion protein (PI∆DT388SA) has been expressed in soluble form in E. coli and in insect cells using baculoviral system and purified up to homogeneity. It interacts with anti-DT antibodies and biotinilated molecules. DNA binding studies by gel-retardation demonstrate highly efficient neutralization of negative charge of plasmid DNA. Cell targeting efficiency of the protein-DNA complexes is under investigation.

844. Novel Pathways of Cell Entry and Trafficking Utilized by Ad Capsid Proteins in the Absence of the Whole Virus Altan Rentsendorj,1 Hasmik Agadjanian,1 Maguire Meghan,4 Jiansong Xie,2 Xinhua Chen,3 Michelle MacVeigh,2 HammAlvarez Sarah,2 Lali K. Medina-Kauwe.1 1 Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA; 2Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA; 3 Beth Israel Deaconess Medical Center, GI Division, Harvard Medical School, Boston, MA; 4Dept of Biochemistry, University of Southern California Keck School of Medicine, Los Angeles, CA. Cellular infection by Adenovirus serotype 5 (Ad5) is mediated by the viral capsid fiber and penton proteins. Whereas the fiber initiates high affinity binding to cellular Coxsackievirus Adenovirus Receptor (CAR) proteins, the penton binds to secondary integrin receptors, triggering receptor-mediated endocytosis of the virus. We have previously demonstrated that recombinant soluble penton proteins can assemble with plasmid DNA and deliver a reporter gene into cultured cells in the absence of the whole virus. To enhance and re-target penton-based non-viral gene delivery, we have attempted to recapitulate the infection mechanism of the Ad5 capsid by combining the penton and fiber proteins, and have formed gene delivery complexes that target DNA to CAR. As CAR does not normally undergo receptor-mediated endocytosis, we were surprised to find that the fiber protein alone, in the absence of the penton, also enabled gene transfer by binding CAR, but entered cells through an unknown mechanism. We have found subsequently that fiber uptake by HeLa cells is temperature-independent, actin dependent, and requires the fiber tail/shaft region. Additionally, fiber uptake is largely inhibited by heparin, implicating heparan sulfate glycosaminoglycans (HS-GAGs) as contributing to its internalization. Moreover, the fiber appears to localize to the nucleus after uptake (Figure B). In contrast, the penton undergoes temperature-dependent cell entry and accumulates around the nucleus after uptake (Figure A). Penton internalization requires intact actin whereas its intracellular trafficking requires intact microtubules. Additionally, we have uncovered a potential sorting motif normally found in cellular proteins destined for endocytic sorting, which appears to greatly affect the intracellular trafficking of the penton. This study describes our findings and raises the possibility that alternative pathways exist that may be utilized for both viral and non-viral cell entry.

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy