Mechanisms of Nonviral Cardiovascular Gene Transfer Achieving the full potential of intraarterial or intravenous nonviral gene therapy will require quantitative bioengineering analysis of intracellular transport mechanisms. While adenovirus transduces nondividing cells, it is an expensive approach with known side effects. In contrast, nonviral gene therapy would be considerably more economical and potentially safer. However, transport of plasmids into the nucleus of cardiovascular cells is extremely inefficient in nondividing cell populations. Unfortunately, cells are not actively dividing in many in vivo tissues that are potential clinical targets for gene therapy. To overcome this intracellular barrier to plasmid transfer, we will conduct a bioengineering analysis of intracellular transport processes in an effort to catalyze rate limiting transport steps. We propose the following specific aims:
Aim 1 We seek to exploit endogenous cellular transport mechanisms involving nonclassical nuclear localization sequences such as the M9 sequence of heteronuclear ribonuclear protein (hnRNP) A1 that binds karyopherin-,B2 (transportin-1). We hypothesize that the M9 sequence results in specific plasmid import through the nuclear pore via karyopherin-,B2 mediated import. We provide data that the M9 sequence results in an unprecedented 83 percent transfection of confluent nondividing endothelium and a 63-fold improvement in total expresssion relative to current lipofection protocols. We present preliminary data that two mutants of the M9 sequence fail to provide this enhancement of gene transfer.
Aim 2 Based on preliminary studies of saturating the cytoplasmic compartment with plasmid, we hypothesize that endosome enhancement provides little benefit to nonviral gene transfer unless nuclear importation is catalyzed. We predict that enhancement of cytoplasmic levels of plasmid using engineered peptide sequences will facilitate increased M9-mediated import of plasmid and increased total expression.
Aim 3 We hypothesize that nuclear-targeting peptides can amplify in vivo expression obtained with lipid-based delivery systems. We predict that the mixture of cationic lipids with plasmids complexed with the peptides optimized in Aim 1 and 2 can maximize the transfection efficiency in vivo to achieve high expression levels in a rat femoral artery (noninjury) model. Overall, these studies will investigate the delivery of plasmids to the nucleus of nondividing endothelium or smooth muscle cells in order to improve nonviral gene transfer.
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