| Project/Area Number |
16300166
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | National Institute of Advanced Industrial Science and Technology |
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Principal Investigator |
NAKANISHI Mahito National Institute of Advanced Industrial Science and Technology, Bioterapeutic Research Laboratory, Director (10172355)
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| Project Period (FY) |
2004 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,460,000 (Direct Cost: ¥14,800,000、Indirect Cost: ¥660,000)
Fiscal Year 2007: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2006: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥5,400,000 (Direct Cost: ¥5,400,000)
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| Keywords | Nuclear transport / Nano-size particle / Gene delivery / Drug delivery system / Nuclear transport signal / Gene expression / ラムダファージ / 遺伝子発現系 / 核転移シグナル / プロテオソーム / 核 / 標的化 / 遺伝子治療 / ポリオーマウイルス / 変異体 |
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| Research Abstract |
Gene transfer and expression in mammalian cells should become a technology required in advanced medical application in future. Virus vectors are favored in current medical application as they have their intrinsic machinery to deriver the genetic materials across two major barriers, the cell membrane and the nuclear membrane. However, safer non-viral vectors are desired in many clinical applications. Performance of non-viral vectors has been improved through enhancing the transport across the cell membrane. However, no technology realizing the transport of large DNA molecules across the nuclear membrane has been established yet. In this project, I investigate the conditions allowing the efficient nuclear transport of the DNA encapsulating nano-sized particles. For this purpose, I employ bacteriophage Lambda as a model. Various peptides with nuclear transport activity(nuclear transport signal, NLS) based on T antigen of SV40 were displayed on the surface of the head of the phage particle by fusing them to D protein, one of two major capsid proteins. All of these NLS peptide assisted the nuclear transport of the D proteins, but only a few could assist the transport of nano sized particles. By using the nuclear transport of the phage particles injecting into the cytoplasm by microinjection as an Index, I succeeded to optimize the signals so that up to 5% of the particles could be transported into the nucleus actively through the nuclear pore complex(NPC). On the other hand, I also found that the NLS-mediated particle disruption in the cytoplasm, possibly through the proteasome, limited the efficiency of the nuclear transport. This is the first evidence that the NLS peptide derived from SV40 T antigen, used most popularly in the research of nuclear transport, may not necessarily be suited for transport of large particles.
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