Development of a gene delivery vector based on a novel concept: RNP vector derived from Sendai virus
| Project/Area Number |
18390169
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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 |
Applied pharmacology
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| Research Institution | DNAVEC Corporation |
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Principal Investigator |
HIRONAKA Takashi DNAVEC Corporation, Production Center, Depertment of Science & Technology Development, Chief (50373543)
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| Co-Investigator(Kenkyū-buntansha) |
BAN Hiroshi DNAVEC Corporation, Depertment of Science & Technology Development, Researcher (60373544)
INOUE Makoto DNAVEC Corporation, Depertment of Science & Technology Development, Director (30373541)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,880,000 (Direct Cost: ¥14,800,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2007: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2006: ¥11,200,000 (Direct Cost: ¥11,200,000)
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| Keywords | Drug transport |
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| Research Abstract |
We examined the possible use of ribonucleoprotein (RNP) derived from Sendai virus (SeV) as a novel RNA replicon vector for gene delivery. For use in this study, we reconstructed non-transmissible SeV vectors carrying the luciferase, GFP or LacZ gene and purified RNP from cells infected with these vectors. First, we examined RNP production and processing conditions for the preparation of highly concentrated RNP from SeV vector-infected cells and established a method of large-scale RNP preparation. Also, we found that addition of a stabilizer increased the preservation stability of RNP during storage. In addition, lyophilization was found to be an effective method of preservation, offering the possibility of long-term storage of RNP. The result of the comparative evaluation study of several transfection reagents showed that cationic lipids such as DOTAP and DOSPER enabled efficient RNP transfection in vitro. The transport pathway of RNP into cells seemed to be via endocytosis from the re
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sult of the experiment with chloroquine or cytochalasin B added to the system. On the other hand, we were able to transduce RNP to the muscle in vivo and the transduction efficiency of RNP alone without transfection reagent was higher than that of RNP/DOTAP complex. We detected an efficient transgene expression when a second administration of RNP vector alone was performed after the primary administration of SeV or RNP vector in the tibialis anterior muscle of rats. This result suggested that multiple dosing with RNP could be possible. In addition, the presence of antibodies against SeV vector in the serum of the animals strongly indicated that RNP was successfully transduced and the gene carried by the RNP was expressed in the cells. These results, which indicated that multiple dosing with RNP was possible in the presence of serum neutralizing antibodies, satisfied the concept of RNP vector that we had initially set as the objective of this investigation. Furthermore, bupivacain treatment of the muscle vigorously enhanced the transduction efficiency, and gene expression was confirmed in the muscle fiber cells. In addition, we found that sonoporation, a physical gene transfer method, allowed gene transduction into tissues other than muscle such as hypodermis, auricle, and footpad. In sum, this investigation showed the usefulness of SeV-derived RNP as a novel gene delivery vector. Less
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Report
(3 results)
Research Products
(2 results)
