Montreal Neurological Institute, Montreal H3A 2B4, Canada.
J Gene Med. 2010 Mar;12(3):266-75. doi: 10.1002/jgm.1433.
Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle.
To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle.
Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer.
We have developed a method of increasing CAR levels in both normal and regenerating muscle.
高效的腺病毒（AdV）介导的基因转移仅在未成熟肌肉或再生肌肉中才有可能，这表明一种发育调控事件在限制 AdV 摄取成熟骨骼肌中起着重要作用。以前，我们发现主要柯萨奇病毒和腺病毒受体（CAR）的表达在肌肉成熟过程中受到严重下调，并且在肌肉特异性 CAR 转基因小鼠中，AdV 介导的基因转移到成熟骨骼肌中有显著增强。
为了评估增加 CAR 表达是否也可以增强对具有许多再生纤维的营养不良肌肉的基因转移，我们将 CAR 转基因小鼠与肌营养不良蛋白缺陷型小鼠（mdx/CAR）进行了杂交。我们还测试了一种两步方案，即在给予 AdV 之前，通过使用表达 CAR 的重组腺相关病毒（AAV2）在靶肌肉中增加 CAR 水平。最后，我们评估了组蛋白去乙酰化酶抑制剂对成肌细胞和 mdx 肌肉中 CAR 和 AdV 转导效率的影响。
尽管由于这些动物中持续的肌肉再生，成年 mdx 小鼠中可以实现比正常小鼠更高的转导率，但在 mdx 背景下（mdx/CAR 转基因小鼠）CAR 的表达仍然显著提高了成熟肌肉对 AdV 介导的肌营养不良蛋白基因转移的易感性。AAV2-CAR 预先给予正常肌肉会导致随后注射 AdV 时的转导明显增加。组蛋白去乙酰化酶抑制剂丙戊酸增加了成肌细胞和 mdx 肌肉中的 CAR 转录本和蛋白水平，并且也增加了 AdV 介导的基因转移。
我们已经开发出一种在正常和再生肌肉中增加 CAR 水平的方法。
