Proteomic and Functional Analyses of Protein–DNA Complexes During Gene Transfer
Department of Pediatrics, Division of Neonatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.Molecular Therapy (Impact Factor: 6.23). 11/2012; 21(4). DOI: 10.1038/mt.2012.231
One of the barriers to successful nonviral gene delivery is the crowded cytoplasm, which plasmids need to actively traverse for gene expression. Relatively little is known about how this process occurs, but our lab and others have shown that the microtubule network and motors are required for plasmid movement to the nucleus. To further investigate how plasmids exploit normal physiological processes to transfect cells, we have taken a proteomics approach to identify the proteins that comprise the plasmid-trafficking complex. We have developed a live cell DNA-protein pull-down assay to isolate complexes at certain time points post-transfection (15 minutes to 4 hours) for analysis by mass spectrometry (MS). Plasmids containing promoter sequences bound hundreds of unique proteins as early as 15 minutes post-electroporation, while a plasmid lacking any eukaryotic sequences failed to bind many of the proteins. Specific proteins included microtubule-based motor proteins (e.g., kinesin and dynein), proteins involved in protein nuclear import (e.g., importin 1, 2, 4, and 7, Crm1, RAN, and several RAN-binding proteins), a number of heterogeneous nuclear ribonucleoprotein (hnRNP)- and mRNA-binding proteins, and transcription factors. The significance of several of the proteins involved in protein nuclear localization and plasmid trafficking was determined by monitoring movement of microinjected fluorescently labeled plasmids via live cell particle tracking in cells following protein knockdown by small-interfering RNA (siRNA) or through the use of specific inhibitors. While importin β1 was required for plasmid trafficking and subsequent nuclear import, importin α1 played no role in microtubule trafficking but was required for optimal plasmid nuclear import. Surprisingly, the nuclear export protein Crm1 also was found to complex with the transfected plasmids and was necessary for plasmid trafficking along microtubules and nuclear import. Our results show that various proteins involved in nuclear import and export influence intracellular trafficking of plasmids and subsequent nuclear accumulation.Molecular Therapy (2012); doi:10.1038/mt.2012.231.
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ABSTRACT: Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse though electrically permeabilized membrane. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, nuclear envelope) which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~ 4000 trajectories) reveals that DNA is actively transported via the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces active transport occurrence, velocities, and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.Molecular Therapy (2013); doi:10.1038/mt.2013.182.Molecular Therapy 08/2013; 12(12). DOI:10.1038/mt.2013.182 · 6.23 Impact Factor
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ABSTRACT: Over the last decade, the application of plasmid-based gene therapy has been widened to many disease models. This has been achieved primarily through advances in plasmid purification, delivery methods, and optimization of tissue-specific expression. Electroporation and hydrodynamic injections have been the most prevalent and successful modes of plasmid DNA delivery in animal models and clinical trials. Expression levels and longevity have been optimized through promoter selection, minimized immunogenicity against administered plasmid, and stable genomic integration. The requirement for short- or long-term transgene expression is primarily based on therapeutic need and desired outcome. Therapeutic applications of plasmid-based gene therapy are wide-ranging and include gene replacement, tumor targeting, neovascularization to treat ischemic and arteriosclerotic conditions, and ex vivo genetic modification for T cell based immunotherapy. The most successful clinical applications are most notably exemplified by successful outcomes of DNA vaccines against infectious diseases and effective treatment of critical limb ischemia. Safety assessments of short-term plasmid delivery-related effects on biological tissue and long-term potential risks associated with genomic integration have been extensively addressed paving the way for wider therapeutic application of plasmid-based gene therapy.Cancer Gene Therapy by Viral and Non-viral Vectors, First edited by Malcolm K. Brenner & Mien-Chie Hung, 03/2014: chapter Chapter 3. DNA Plasmids for Non-viral Gene Therapy of Cancer: pages 39-59; Wiley Blackwell., ISBN: 978-1-118-50162-7
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ABSTRACT: Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.Journal of Membrane Biology 08/2014; 247(12). DOI:10.1007/s00232-014-9720-6 · 2.46 Impact Factor
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