ENGINEERING MOTOR PROTEIN TO ENHANCE GENE DELIVERY TO THE NUCLEUS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。非病毒基因传递载体，如脂质体和聚阳离子聚合物，比病毒载体更安全，因为它们不会引发免疫反应，而且不太可能对人类造成其他不利的生物影响。然而，病毒载体在将遗传物质输送到细胞中的效率明显高于非病毒基因递送系统。通过利用非病毒载体进行靶向和传递，并采用参与遗传物质细胞内运输的病毒蛋白的关键成分，我们可能能够组装出潜在更安全和更有效的基因治疗杂交载体。为了加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外区连接(NCD)，这是一种在果蝇中发现的与激动素相关的微管运动蛋白。Ron Vale博士为我们提供了NCD质粒，我们对其进行了修饰，在其上增加了GAL4 DNA结合域，使之成为可以装载质粒DNA的NCD-GAL4融合蛋白。在使用荧光素酶报告质粒的初步研究中，我们能够显示，与不含GAL4结合域的融合蛋白和对照质粒DNA相比，携带GAL4结合域的融合蛋白和质粒DNA的细胞中的荧光素酶活性显著增加。我们正试图通过进一步修饰NCD-GAL4融合蛋白来改善DNA运输，而不会干扰运动活性。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，将病毒内容传递到细胞核。我们使用嵌合体来可视化NCD的功能结构域，以及来自RBVI资源中心的各种序列分析工具。我们还使用Patricia Babbitt博士团队的鸟枪程序来寻找功能相似的远亲蛋白，并确定与哺乳动物运动蛋白结合的病毒蛋白的重要残基或片段(S)。

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.