Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy.
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.
Li
Juan
J
Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, NC 27599, USA.
Sun
Wunchang
W
Wang
Bing
B
Xiao
Xiao
X
Liu
Xiang-Qin
XQ
D000595:Amino Acid Sequence; D000818:Animals; D000229:Dependovirus / Q000235:genetics; D016189:Dystrophin / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D047668:Inteins; D051379:Mice; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D009137:Muscular Dystrophy, Animal / Q000235:genetics / Q000473:pathology / Q000628:therapy; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy; D019154:Protein Splicing
D016189:Dystrophin
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