title: Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy.
abstract: 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.
authors.affiliation: Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, NC 27599, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Wunchang
authors.initials: W
authors.lastname: Sun
authors.forename: Bing
authors.initials: B
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
mesh_terms: 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
chemical_list: D016189:Dystrophin
references.citation: Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7851-6
references.pmid: 9223276
references.citation: Mol Ther. 2007 Oct;15(10):1856-62
references.pmid: 17653101
references.citation: Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9226-31
references.pmid: 9689062
references.citation: Mol Ther. 2001 Oct;4(4):383-91
references.pmid: 11592843
references.citation: Hum Mol Genet. 1995 Aug;4(8):1251-8
references.pmid: 7581361
references.citation: J Virol. 2000 Feb;74(3):1436-42
references.pmid: 10627554
references.citation: Hum Gene Ther. 2002 Aug 10;13(12):1451-60
references.pmid: 12215266
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):11999-2004
references.pmid: 16103356
references.citation: Mol Ther. 2002 Jun;5(6):716-22
references.pmid: 12027555
references.citation: Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13714-9
references.pmid: 11095710
references.citation: Gene Ther. 1999 Jan;6(1):74-82
references.pmid: 10341878
references.citation: J Thromb Haemost. 2003 Feb;1(2):220-6
references.pmid: 12871492
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6716-21
references.pmid: 10841568
references.citation: Hum Gene Ther. 1997 Nov 1;8(16):1891-900
references.pmid: 9382955
references.citation: Nat Med. 1997 Mar;3(3):306-12
references.pmid: 9055858
references.citation: Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10158-63
references.pmid: 9707617
references.citation: Hum Gene Ther. 2003 Jan 20;14(2):143-52
references.pmid: 12614565
references.citation: Biochim Biophys Acta. 1998 Sep 8;1387(1-2):422-32
references.pmid: 9748659
references.citation: J Virol. 1996 Nov;70(11):8098-108
references.pmid: 8892935
references.citation: J Biol Chem. 1990 Mar 15;265(8):4560-6
references.pmid: 2407739
references.citation: Nature. 1990 Jan 11;343(6254):180-2
references.pmid: 2404210
references.citation: Nat Biotechnol. 2000 May;18(5):527-32
references.pmid: 10802620
references.citation: Nat Med. 2000 May;6(5):599-602
references.pmid: 10802720
references.citation: Microsc Res Tech. 2000 Feb 1-15;48(3-4):223-38
references.pmid: 10679969
references.citation: Nat Med. 2000 May;6(5):595-8
references.pmid: 10802719
references.citation: Cell. 1987 Jul 31;50(3):509-17
references.pmid: 3607877
references.citation: J Biol Chem. 1993 Feb 15;268(5):3781-90
references.pmid: 7679117
references.citation: Nat Med. 2002 Mar;8(3):253-61
references.pmid: 11875496