Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.
Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders.
Hirsch
M L
ML
UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7352, USA.
Green
L
L
Porteus
M H
MH
Samulski
R J
RJ
D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D014162:Transfection
D004277:DNA, Single-Stranded; D004247:DNA; D004706:Endodeoxyribonucleases
Nature. 2005 Jun 2;435(7042):646-51
15806097
Mol Ther. 2003 Jan;7(1):101-11
12573623
Nat Biotechnol. 2007 Jul;25(7):778-85
17603475
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6064-8
8016116
J Virol. 1997 Feb;71(2):941-8
8995611
Mol Cell Biol. 1997 Nov;17(11):6386-93
9343400
Mol Cell Biol. 1995 Apr;15(4):1968-73
7891691
Nucleic Acids Res. 1995 Dec 25;23(24):5012-9
8559659
Mol Cell Biol. 2001 Jan;21(1):289-97
11113203
Science. 2003 May 2;300(5620):763
12730593
Nucleic Acids Res. 1993 Feb 11;21(3):407-12
8441653
Mol Ther. 2008 Feb;16(2):280-9
18059373
Nat Protoc. 2006;1(3):1412-28
17406430
Hum Gene Ther. 2005 Apr;16(4):522-6
15871683
Nature. 1986 Nov 6-12;324(6092):34-8
3785372
Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077-81
6281795
J Virol. 1987 Oct;61(10):3096-101
3041032
Cell. 1987 Nov 6;51(3):503-12
2822260
Mol Ther. 2008 Jan;16(1):187-93
17955022
J Virol. 2000 Oct;74(20):9451-63
11000214
Nat Biotechnol. 2007 Nov;25(11):1298-306
17965707
J Virol. 1996 May;70(5):3227-34
8627803
Mol Ther. 2001 Apr;3(4):526-30
11319913
Gene Ther. 2001 Aug;8(16):1248-54
11509958
Mol Cell Biol. 2003 May;23(10):3550-7
12724413
J Virol. 2007 Dec;81(23):12936-45
17898048
Nat Biotechnol. 2000 Apr;18(4):399-404
10748519
Mol Cell Biol. 1989 Apr;9(4):1621-7
2725520
J Virol. 2000 May;74(10):4612-20
10775597
Nat Genet. 1998 Apr;18(4):325-30
9537413
Mol Cell Biol. 2003 May;23(10):3558-65
12724414
Nature. 1987 Dec 10-16;330(6148):576-8
3683574
Cell. 1986 Feb 14;44(3):419-28
3002636
J Virol. 2003 Dec;77(23):12881-5
14610209
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3608-12
8622983
Mol Cell Biol. 1993 Feb;13(2):918-27
8423811
J Virol. 1999 Sep;73(9):7376-80
10438827
Science. 2004 Feb 20;303(5661):1198-201
14976317
J Mol Biol. 2007 Sep 14;372(2):397-406
17663993