| Project/Area Number |
12470203
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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 |
Hematology
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| Research Institution | Jichi Medical School |
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Principal Investigator |
OZAWA Keiya Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUKAMI Hiroaki Jichi Med. Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (20311938)
KUME Akihiro Jichi Med. Sch., Faculty of Medicine, Assistant Professor, 医学部, 助教授 (10264293)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Gene therapy / Gene transfer / AAV vector / packaging cell line / AAVS1 locus / Rep / ITR / TVI method |
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| Research Abstract |
We studied the gene transfer methods using adeno-associated virus (AAV).
1. Development of the method for AAV vector production: We developed novel packaging cell lines for AAV vector production by regulating the expression of cytotoxic AAV proteins through Cre/loxP system. First, we applied combined Cre/loxP system using variant loxP and wild-type loxP to the simultaneous regulation of Rep and Cap expressions. Second, we developed a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap through the introduction of a Cre recombinase gene.
2. Establishment and application of highly sensitive detection method for AAV vector-mediated transgenes : Long PCR (sometimes combined with nested PCR) was conducted with appropriate primer sets located on the D region of ITR. We also collected many samples from the experimental animals which received intramuscular injections of AAV vectors.
3. Development of the method for targeted vector integration (TVI) into a defined locus on chromosome 19 using AAV-derived components (ITR and Rep gene) : 293 and K562 cells were transfected with the neoγ gene using the TVI method. We amplified junctional regions between cellular and transgene sequences by Alu-PCR. As a result, no cellular sequences regarded as a common recognition motif of the Rep proteins was found. We also developed mutant Rep-expression vectors with reduced cytotoxicity.
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