| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Wild-type AAV is a 20 nm diameter, replication incompetent, non-pathogenic, stable parvoviru ; We sought to transduce these "deep" chondrocytes using an alternative viral vector based on adeno-associated virus (AAV). Following AAV-GFP transduction to the primary human chondrocytes, the percentage of cells expressing GFP increased over time in culture. The percentages of fluorescent eel in the samples from the 19-year-old patient were 15.9 % (one day after transduction), 44.8 % (day 2), 84.1 % (day 3), and 95.0 % (day 7); and those in the samples from the 84-year-old patient were 16.0 % (day 1), 43.2 % (day 2), 73.2 % (day 3), and 93.7 % (day 7). In the present study, we examined the efficiency of AAV-GFP transduction of primary human chondrocytes as well as cartilage organ cultures, and succeeded in obtaining high efficiency of gene transduction and sustained gene expression. As a result, in the primary chondrocytes, up to 95.0 % or 93.7 % of cells were transduced ; in the cartilage organ cultures, 45.3 ± 7.4 % or 46.0 ± 3.9 % of chondrocytes that are located not only in superficial layer but also in deep layer within the cartilage were transduced in situ and lasted GFP expression for up to 28 days. Previous studies using adenovirus vectors and the hemagglutinating virus of Japan-liposome (HVJ-liposome) complex resulted in inefficient delivery to chondrocytes there located in the deep layer of the cartilage. Therefore, the AAV vector was able to deliver GFP gen to primary chondrocytes efficiently and pass through the dense extracellular matrix to transduce the chondrocytes that reside in the deep layer of the cartilage. The results of our study indicate that the AAV vector is an appropriate vector for gene transduction to chondrocytes and expected to provide a promising strategy that may allow us to directly transduce chondrocytes in vivo through intra articular injection.
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