Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy
Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy
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DOI：
10.1089/hum.2008.009
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发表时间：
2008-09-01
影响因子：
4.2
通讯作者：
Liu, Xiang-Qin
中科院分区：
文献类型：
--
作者：
Li, Juan;Sun, Wunchang;Liu, Xiang-Qin
内含子催化蛋白质剪接的方式类似于自剪接内含子催化RNA剪接的方式。裂解素以高度特异性的方式通过反式剪接催化两个不同多肽的精确连接。在这里，我们报告了一种利用蛋白质反式剪接来规避基因治疗载体包装大小限制的方法。为了验证这种方法，我们选择了一个大的dystrophin基因和腺相关病毒(AAV)载体，它具有较小的包装尺寸。将一个高功能的6.3kb Becker形式的dystrophin基因分成两段，并通过添加适当的分裂内含子编码序列进行修饰，使分裂基因足够小，可以包装在AAV载体中。这两个分裂基因在共传递到靶细胞后，产生两个多肽，在体外细胞培养中自发反式剪接形成预期的Becker-form dystrophin蛋白。通过AAV1载体将分裂基因导入Duchenne肌营养不良症小鼠模型的肌肉中，实现了治疗性基因表达和益处。
Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Splitinteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly functional 6.3-kb Becker-form dystrophin cDNA was broken into two pieces and modified by adding appropriate split-intein coding sequences, resulting in splitgenes sufficiently small for packaging in AAV vectors. The two split-genes, after codelivery into target cells, produced two polypeptides that spontaneously trans-spliced to form the expected Becker-form dystrophin protein in cell Culture in vitro. Delivering the split-genes by AAV1 vectors into the muscle of a mouse model of Duchenne muscular dystrophy rendered therapeutic gene expression and benefits.