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Protein Trans -Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

2008; Mary Ann Liebert, Inc.; Volume: 19; Issue: 9 Linguagem: Inglês

10.1089/hum.2008.009

1557-7422

Juan Li, Wenchang Sun, Bing Wang, Xiao Xiao, Xiang-Qin Liu,

Muscle Physiology and Disorders

Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Split-inteins 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 split-genes 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.