Mason Jeffrey B, Gurda Brittney L, Engiles Julie B, Hankenson Kurt D, Wilson James M, Richardson Dean W
Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348-1692, USA.
Hum Gene Ther Methods. 2013 Jun;24(3):185-94. doi: 10.1089/hgtb.2012.199.
Our aim was to investigate serotype-specific cell and tissue-transduction tropisms, transgene expression levels and longevity, and immunogenicity of candidate rAAV serotypes in rat osteochondral cells, tissues, and stifle joints. In vitro, we used six rAAV serotypes and two promoters to transduce synoviocytes and chondrocytes. Serotypes rAAV2/5 and 2/2 yielded the highest transduction efficiency 4 days after transduction. No differences were detected between cytomegalovirus and chicken β-actin promoters. In vivo, intra-articular injection was used to introduce four rAAV serotypes into 4-month-old rats in the left stifle joint. Eleven months later, serotype 2/5 vector, diluted with saline or surfactant, was injected into the right stifle joint of the same rats. Rats were analyzed up to 12 months after initial injection. Bioluminescence was detected at 7 days and all serotypes tested displayed bioluminescence above controls after 1 year in the left stifle. Gene expression was detected in the right stifle joints of all rats with the exception of rats previously injected with serotype 2/5. We observed no difference irrespective of whether the luciferin was injected subcutaneously or intraperitoneally. However, surfactant-diluted vectors led to increased gene expression compared with saline-diluted vectors. Cell- and tissue-specific transduction was observed in rat stifles injected with an nLacZ-containing rAAV. Transduction was greatest in stromal tissues and mesenchymal cell types. Exposure to a specific serotype did not inhibit subsequent transduction with a different serotype at a second vector injection. Including surfactant as a vector diluent increased gene expression within the stifle joint and should be considered for in vivo gene therapy applications.
我们的目的是研究候选重组腺相关病毒（rAAV）血清型在大鼠骨软骨细胞、组织和膝关节中的血清型特异性细胞和组织转导趋向性、转基因表达水平和持久性以及免疫原性。在体外，我们使用六种rAAV血清型和两种启动子转导滑膜细胞和软骨细胞。转导后4天，rAAV2/5和2/2血清型产生的转导效率最高。巨细胞病毒启动子和鸡β-肌动蛋白启动子之间未检测到差异。在体内，通过关节内注射将四种rAAV血清型引入4月龄大鼠的左膝关节。11个月后，将用盐水或表面活性剂稀释的2/5血清型载体注射到同一只大鼠的右膝关节。在初次注射后长达12个月对大鼠进行分析。在7天时检测到生物发光，并且在左膝关节中，所有测试的血清型在1年后的生物发光均高于对照。除先前注射2/5血清型的大鼠外，在所有大鼠的右膝关节中均检测到基因表达。无论荧光素是皮下注射还是腹腔注射，我们均未观察到差异。然而，与盐水稀释的载体相比，表面活性剂稀释的载体导致基因表达增加。在用含nLacZ的rAAV注射的大鼠膝关节中观察到细胞和组织特异性转导。在基质组织和间充质细胞类型中转导最大。在第二次载体注射时，暴露于特定血清型并不抑制随后用不同血清型进行的转导。将表面活性剂作为载体稀释剂可增加膝关节内的基因表达，在体内基因治疗应用中应予以考虑。
