title: Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity.
abstract: Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.Molecular Therapy - Nucleic Acids (2012) 1, e54; doi:10.1038/mtna.2012.46; published online 13 November 2012.
authors: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors: Justin
authors: J
authors: Judd
authors: Fang
authors: F
authors: Wei
authors: Peter Q
authors: PQ
authors: Nguyen
authors: Lawrence J
authors: LJ
authors: Tartaglia
authors: Mavis
authors: M
authors: Agbandje-McKenna
authors: Jonathan J
authors: JJ
authors: Silberg
authors: Junghae
authors: J
authors: Suh
references: J Virol. 2002 May;76(9):4559-66
references: 11932421
references: Nat Genet. 1994 Oct;8(2):148-54
references: 7842013
references: J Virol. 1998 Feb;72(2):1438-45
references: 9445046
references: Nat Med. 1999 Jan;5(1):71-7
references: 9883842
references: Virology. 1999 Dec 20;265(2):274-85
references: 10600599
references: ACS Nano. 2011 May 24;5(5):3523-35
references: 21473596
references: Science. 2001 Nov 30;294(5548):1929-32
references: 11729319
references: Biochemistry. 2006 Aug 15;45(32):9639-47
references: 16893165
references: J Virol. 2002 May;76(9):4580-90
references: 11932423
references: Proc Natl Acad Sci U S A. 2003 May 13;100(10):6081-6
references: 12716974
references: Methods Mol Biol. 2008;434:161-70
references: 18470644
references: Nat Biotechnol. 2003 Sep;21(9):1040-6
references: 12897791
references: J Virol. 2007 Aug;81(15):7833-43
references: 17507473
references: J Mol Biol. 2004 Feb 6;336(1):263-73
references: 14741221
references: J Virol. 2004 Jun;78(12):6595-609
references: 15163751
references: Hum Gene Ther. 2001 Sep 20;12(14):1697-711
references: 11560765
references: J Virol. 2004 May;78(9):4421-32
references: 15078923
references: Protein Eng Des Sel. 2004 Jul;17(7):595-602
references: 15331775
references: Biotechnol Bioeng. 2005 Oct 5;92(1):24-34
references: 15937953
references: J Virol. 2005 Sep;79(18):11776-87
references: 16140755
references: Hum Gene Ther. 2007 Apr;18(4):367-78
references: 17437357
references: Eur J Pharm Sci. 2012 Jun 14;46(3):167-72
references: 22406091
references: Gene. 1989 Apr 15;77(1):51-9
references: 2744487
references: Nat Med. 1999 Dec;5(12):1438
references: 10581091
references: Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10220-5
references: 20479244
references: Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10405-10
references: 12136130
references: EMBO J. 1991 Dec;10(12):3941-50
references: 1657596
references: Mol Ther. 2010 Jan;18(1):80-6
references: 19904234
references: J Virol. 2005 May;79(9):5296-303
references: 15827144
references: Mol Ther. 2005 Jun;11(6):856-65
references: 15922956
references: J Control Release. 2005 Nov 2;108(1):161-77
references: 16125817
references: J Struct Biol. 2009 Mar;165(3):146-56
references: 19121398
references: J Virol. 2000 Sep;74(18):8635-47
references: 10954565
references: Mol Ther. 2003 Jul;8(1):151-7
references: 12842438
references: Nat Biotechnol. 1999 Feb;17(2):181-6
references: 10052356
references: Arch Biochem Biophys. 2010 Apr 1;496(1):1-8
references: 20102698
references: Mol Ther. 2006 Apr;13(4):671-82
references: 16442847
references: Nat Biotechnol. 2006 Feb;24(2):198-204
references: 16429148
references: Nat Biotechnol. 2004 Dec;22(12):1567-72
references: 15558047
references: Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211-5
references: 2156265
references: Mol Imaging. 2010 Apr;9(2):59-75
references: 20236604
references: J Gene Med. 2009 Dec;11(12):1103-13
references: 19777441
references: J Virol. 1994 Jan;68(1):170-6
references: 8254726
references: Gene Ther. 2008 Dec;15(24):1618-22
references: 18668144
references: Mol Ther. 2008 Jul;16(7):1252-1260
references: 28178482
references: J Virol. 2006 Dec;80(23):11556-70
references: 16971437
references: Hum Gene Ther. 1998 Sep 1;9(13):1929-37
references: 9741431