title: Improved splicing of adeno-associated viral (AAV) capsid protein-supplying pre-mRNAs leads to increased recombinant AAV vector production.
abstract: Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Missouri-Columbia School of Medicine, Life Sciences Center, Columbia, MO 65211, USA.
authors.forename: K David
authors.initials: KD
authors.lastname: Farris
authors.forename: David J
authors.initials: DJ
authors.lastname: Pintel
mesh_terms: D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D012333:RNA, Messenger
references.citation: J Virol Methods. 2003 Dec;114(1):1-10
references.pmid: 14599673
references.citation: J Virol. 2000 Dec;74(24):11456-63
references.pmid: 11090141
references.citation: J Virol. 1988 Sep;62(9):3356-63
references.pmid: 2841488
references.citation: Mol Cell Biol. 1998 Oct;18(10):5921-9
references.pmid: 9742109
references.citation: Mol Cell Biol. 2002 Jun;22(11):3639-52
references.pmid: 11997501
references.citation: Virology. 1991 Mar;181(1):22-34
references.pmid: 1825251
references.citation: J Virol. 1988 Aug;62(8):2745-54
references.pmid: 2839699
references.citation: Hum Gene Ther. 2002 Nov 1;13(16):1935-43
references.pmid: 12427305
references.citation: EMBO J. 2003 Apr 1;22(7):1716-24
references.pmid: 12660177
references.citation: J Virol. 1997 Jul;71(7):5236-43
references.pmid: 9188591
references.citation: Virology. 2002 Feb 15;293(2):345-55
references.pmid: 11886255
references.citation: J Virol. 1997 Sep;71(9):6816-22
references.pmid: 9261406
references.citation: J Virol. 1998 Mar;72(3):2224-32
references.pmid: 9499080
references.citation: Genes Dev. 1992 Jun;6(6):1107-19
references.pmid: 1592259
references.citation: J Virol. 2006 Jul;80(13):6559-67
references.pmid: 16775342
references.citation: Virology. 2001 Dec 5;291(1):140-51
references.pmid: 11878883
references.citation: Brief Funct Genomic Proteomic. 2008 Jul;7(4):303-11
references.pmid: 18632744
references.citation: Virology. 1966 Jun;29(2):353-7
references.pmid: 5943543
references.citation: Proc Natl Acad Sci U S A. 1966 Jun;55(6):1467-74
references.pmid: 5227666
references.citation: Proc Natl Acad Sci U S A. 1967 Oct;58(4):1410-5
references.pmid: 4295829
references.citation: J Virol. 1997 Mar;71(3):1897-905
references.pmid: 9032320
references.citation: EMBO J. 1991 Dec;10(12):3941-50
references.pmid: 1657596
references.citation: J Virol Methods. 2006 Dec;138(1-2):85-98
references.pmid: 16950522
references.citation: Gene Ther. 2002 Sep;9(18):1199-206
references.pmid: 12215886
references.citation: J Virol Methods. 2006 Nov;137(2):193-204
references.pmid: 16860883
references.citation: Biochem Biophys Res Commun. 2000 Sep 24;276(2):559-63
references.pmid: 11027513
references.citation: J Virol. 2002 Dec;76(24):12435-47
references.pmid: 12438569
references.citation: J Virol. 1998 Jul;72(7):5472-80
references.pmid: 9621003
references.citation: Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211-5
references.pmid: 2156265
references.citation: J Virol. 2006 Aug;80(16):7807-15
references.pmid: 16873238
references.citation: Virology. 2005 Apr 25;335(1):10-21
references.pmid: 15823602
references.citation: J Virol. 2000 Nov;74(21):9878-88
references.pmid: 11024114