Gene therapy for osteoarthritis using AAV vector.

野生型AAV是一种直径20 nm、无复制能力、非致病性、稳定的细小病毒;我们试图使用基于腺相关病毒（AAV）的替代病毒载体来扩增这些“深层”软骨细胞。在AAV-GFP转导至原代人软骨细胞后，表达GFP的细胞的百分比在培养物中随时间增加。19岁患者的样本中荧光鳗的百分比为15.9%（转导后一天）、44.8%（第2天）、84.1%（第3天）和95.0%（第7天）;而来自84岁患者的样品中的那些分别为16.0%（第1天）、43.2%（第2天）、73.2%（第3天）和93.7%（第7天）。在本研究中，我们研究了AAV-GFP转导原代人软骨细胞以及软骨器官培养物的效率，并成功地获得了高效率的基因转导和持续的基因表达。结果，在原代软骨细胞中，高达95.0%或93.7%的细胞被转导;在软骨器官培养物中，45.3 ± 7.4%或46.0 ± 3.9%的不仅位于软骨内的浅层而且位于软骨内的深层的软骨细胞被原位转导，并且持续GFP表达长达28天。以前的研究使用腺病毒载体和血凝病毒的日本-脂质体（HVJ-脂质体）复合物导致效率低下的交付到软骨细胞位于软骨的深层。因此，AAV载体能够有效地将GFP基因递送到原代软骨细胞，并穿过致密的细胞外基质以覆盖驻留在软骨深层的软骨细胞。我们的研究结果表明，腺相关病毒载体是一种合适的基因转导软骨细胞的载体，并有望提供一种有前途的策略，使我们能够通过关节内注射直接在体内转导软骨细胞。

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.