ENGINEERING MOTOR PROTEIN TO ENHANCE GENE DELIVERY TO THE NUCLEUS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 非病毒基因递送载体，如脂质体和聚阳离子聚合物，比病毒载体更安全，因为它们不会引起免疫应答，并且不太可能对人类造成其他不良生物学效应。 然而，病毒载体在将遗传物质转运到细胞中比非病毒基因递送系统显著更有效。 通过利用非病毒载体进行靶向和递送，并采用参与遗传物质细胞内转运的病毒蛋白的关键组分，我们可能能够组装用于基因治疗的杂交载体，其潜在地更安全和有效。 为了增强遗传物质从质膜到细胞核的运输，正在利用一种修饰形式的非红细胞分裂蛋白（Ncd），一种在果蝇中发现的驱动蛋白相关的微管马达蛋白。 罗恩维尔博士为我们提供了Ncd质粒，我们通过在质粒上添加GAL 4 DNA结合结构域来修饰该质粒，以制备可以装载质粒DNA作为货物的Ncd-GAL 4融合蛋白。 在我们使用荧光素酶报告质粒的初步研究中，我们能够显示在用融合蛋白和具有GAL 4结合结构域的质粒DNA转染的细胞中荧光素酶活性水平显著增加，超过用融合蛋白和不含GAL 4结合结构域的对照质粒DNA转染的细胞。 我们正试图通过进一步修饰Ncd-GAL 4融合蛋白而不干扰运动活性来改善DNA转运。 该项目的第二部分涉及病毒蛋白的序列分析，这些蛋白被认为参与劫持宿主细胞的马达蛋白，将病毒内容物输送到细胞核。 我们使用Chimera来可视化Ncd的功能结构域，以及RBVI资源中心的各种序列分析工具。 我们还使用Patricia Babyson博士小组的Shotgun程序来寻找具有相似功能的远亲蛋白，并鉴定与哺乳动物马达蛋白结合的病毒蛋白的重要残基或片段。 作为使用分子马达进行药物递送的另一种方法，我们还尝试使用小肽将新货物靶向细胞中的内源性动力蛋白。 我们将这些肽称为逆行转运的生物分子衔接子，或BART。我们设计了与LC 8（动力蛋白的一个亚基）结合的肽，并可以与微球结合作为实验货物。 如果这些肽-货物结合物与细胞中活跃的动力蛋白马达相连，它们就可以沿着沿着被运送到细胞内部。

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.