DNA Trafficking in Non-viral Gene Therapy: The Role of the CytoskeletonPublic Deposited
A primary limitation of non-viral gene therapy is the low level of expression achieved. One of the reasons that higher expression is not achieved is that there are many physical obstacles that prevent the DNA from reaching the nucleus to be expressed. A plasmid must cross the cell membrane, traverse the cytoplasm, and pass through the nuclear envelope in order to be transcribed. Recently, much work has focused on overcoming the barriers presented by the cell and nuclear membranes; however how plasmids navigate through the dense meshwork of the cytoplasm has remained unknown. Elucidating how DNA reaches the nucleus could greatly enhance gene therapy as increasing the efficiency of delivery and amount of plasmid that reaches the nucleus could significantly improve expression to more clinically relevant levels. This study shows that when cells are transfected by microinjection or electroporation in the presence of cytoskeleton altering drugs, the microtubule network is required for trafficking of plasmids to the nucleus. Further, the interaction between microtubules and plasmids was confirmed using an in vitro microtubule-binding assay. The ability of plasmids to traffic towards the nucleus is dependent upon the presence of specific sequences and is not a general phenomenon common to all DNA. Plasmids that contain binding sites for the cyclic AMP response element-binding protein (CREB) were shown, in vitro, to demonstrate an ability to interact with microtubules while plasmids lacking this sequence were unable to bind to microtubules. Further, it was noted that even in the presence of the microtubule-depolymerizing drug, nocodazole, some plasmids were able to traffic towards the nucleus. These studies show that a subset of nocodazole-resistant microtubules known to be acetylated, may not only provide a means for plasmid trafficking to the nucleus but may actually serve as a more efficient means for trafficking of plasmids to the nucleus. Taken together, the data presented could aid the design of a non-viral vector that can traffic to the nucleus more efficiently and thereby aid in increasing expression levels.