PMID- 20463753
OWN - NLM
STAT- MEDLINE
DCOM- 20110119
LR  - 20211020
IS  - 1476-5462 (Electronic)
IS  - 0969-7128 (Print)
IS  - 0969-7128 (Linking)
VI  - 17
IP  - 9
DP  - 2010 Sep
TI  - Self-complementary AAV mediates gene targeting and enhances endonuclease delivery 
      for double-strand break repair.
PG  - 1175-80
LID - 10.1038/gt.2010.65 [doi]
AB  - 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.
FAU - Hirsch, M L
AU  - Hirsch ML
AD  - UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel 
      Hill, NC 27599-7352, USA.
FAU - Green, L
AU  - Green L
FAU - Porteus, M H
AU  - Porteus MH
FAU - Samulski, R J
AU  - Samulski RJ
LA  - eng
GR  - PN2 EY018244-06/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20100513
PL  - England
TA  - Gene Ther
JT  - Gene therapy
JID - 9421525
RN  - 0 (DNA, Single-Stranded)
RN  - 9007-49-2 (DNA)
RN  - EC 3.1.- (Endodeoxyribonucleases)
SB  - IM
MH  - Cells, Cultured
MH  - DNA/metabolism
MH  - DNA Breaks, Double-Stranded
MH  - DNA Repair
MH  - DNA, Single-Stranded/metabolism
MH  - Dependovirus/*genetics
MH  - Endodeoxyribonucleases/*genetics/metabolism
MH  - *Gene Targeting
MH  - Genetic Vectors/*genetics
MH  - Humans
MH  - Transfection
PMC - PMC3152950
MID - NIHMS303716
COIS- Conflict of interest The authors declare no conflict of interest.
EDAT- 2010/05/14 06:00
MHDA- 2011/01/20 06:00
PMCR- 2011/09/01
CRDT- 2010/05/14 06:00
PHST- 2010/05/14 06:00 [entrez]
PHST- 2010/05/14 06:00 [pubmed]
PHST- 2011/01/20 06:00 [medline]
PHST- 2011/09/01 00:00 [pmc-release]
AID - gt201065 [pii]
AID - 10.1038/gt.2010.65 [doi]
PST - ppublish
SO  - Gene Ther. 2010 Sep;17(9):1175-80. doi: 10.1038/gt.2010.65. Epub 2010 May 13.