Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo

被引:19
作者
Nakai, H
Fuess, S
Storm, TA
Meuse, LA
Kay, MA [1 ]
机构
[1] Stanford Univ, Sch Med, Dept Pediat, Stanford, CA 94305 USA
[2] Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA
关键词
adeno-associated virus; gene therapy; mouse; liver; inverted terminal repeat; recombination; integration; double-stranded DNA; naked DNA; Factor IX;
D O I
10.1016/S1525-0016(02)00034-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C - and L-, which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.
引用
收藏
页码:112 / 121
页数:10
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