Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6
Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6
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DOI：
10.1038/mt.2010.205
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发表时间：
2011-01-01
影响因子：
12.4
通讯作者：
Chamberlain, Jeffrey S.
中科院分区：
文献类型：
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作者：
Odom, Guy L.;Gregorevic, Paul;Chamberlain, Jeffrey S.
重组腺相关病毒（rAAV）载体介导的基因转移是治疗许多疾病的一种有前景的方法。然而，rAAV载体的实用性长期以来因DNA包装容量小（约4.8 kb）而受到阻碍。这种限制可能会阻碍关键调控元件和/或较大编码序列的包装和递送，例如类似于杜氏肌营养不良症 (DMD) 基因治疗感兴趣的 14-kb 肌营养不良蛋白互补 DNA (cDNA)。在这里，我们证明了在血管内共递送两个独立的 rAAV6 载体（共享 372 个核苷酸的中央同源重组区域）后，体内编码高功能“小型肌营养不良蛋白”蛋白（Delta H2-R19，222 kd）的表达盒（7.3 kb）的重建。与之前报道的反式剪接方法类似，一个rAAV载体向启动子提供类似于1/2的微型肌营养不良蛋白初始部分，而第二个载体提供剩余的微型肌营养不良蛋白cDNA，随后提供多聚腺苷酸化信号。值得注意的是，向营养不良小鼠施用适度剂量的两种编码小型肌营养不良蛋白的rAAV载体[2 x 10(12)载体基因组(vg)]可引起生理性能的改善，表明疾病状态的预防或改善。这些研究提供的证据表明，比单个rAAV基因组通常携带的功能性肌营养不良蛋白转基因更大的功能性肌营养不良蛋白转基因可以在与rAAV6血管内共递送后通过同源重组(HR)在体内重建。
Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (similar to 4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the similar to 14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (Delta H2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the similar to 1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 x 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.