ENGINEERING MOTOR PROTEIN TO ENHANCE GENE DELIVERY TO THE NUCLEUS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Non-viral gene delivery vectors, such as liposomes and polycationic polymers, are safer than viral vectors since they do not elicit immune response and are less likely to cause other adverse biological effects on humans. However, viral vectors are significantly more effective in transporting genetic materials into cells than non-viral gene delivery systems. By utilizing non-viral vector for targeting and delivery, and adopting crucial component from viral proteins that are involved in intracellular transport of genetic materials, we may be able to assemble a hybrid vector for gene therapy that is potentially more safe and effective. To enhance transport of genetic materials from plasma membrane to the nucleus, are utilizing a modified form of nonclaret disjuctional (Ncd), a kinesin-related microtubule motor protein that is found in Drosophila. Dr. Ron Vale provided us with the Ncd plasmid, and we modified the plasmid by adding GAL4 DNA binding domain to the plasmid to make Ncd-GAL4 fusion protein that can load plasmid DNA as a cargo. In our preliminary study using luciferase reporter plasmid, we were able to show significantly increased level of luciferase activity in the cells transfected with the fusion protein and plasmid DNA with the GAL4 binding domain over the cells transfected with fusion protein and control plasmid DNA that did not contain the GAL4 binding domain. We are attempting to improve DNA transport by further modification of the Ncd-GAL4 fusion protein without perturbing the motor activity. The second part of this project involves sequence analysis of viral proteins that are thought to be involved in hijacking motor proteins of the host cell to deliver viral contents to the nucleus. We use Chimera to visualize functional domains of Ncd, and various sequence analysis tools from the RBVI resource center. We also use the Shotgun program from Dr. Patricia Babbitt's group to find distantly related proteins that have similar functions, and identify important residues or segment(s) of viral proteins that bind to mammalian motor proteins. As another approach to use molecular motors for drug delivery we are also attempting to use small peptides to target novel cargoes to endogenous dynein in cells. We have termed these peptides biomolecular adaptors for retrograde transport, or BART. We have designed peptides that bind to LC8, a subunit of dynein, and can be conjugated to microspheres as experimental cargo. If these peptide-cargo conjugates hitch onto an active dynein motor in a cell, they could be carried towards the cell�s interior along microtubules.