A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging

被引:150
作者
Parks, RJ
Graham, FL
机构
[1] MCMASTER UNIV,DEPT BIOL,HAMILTON,ON L8S 4K1,CANADA
[2] MCMASTER UNIV,DEPT PATHOL,HAMILTON,ON L8S 4K1,CANADA
关键词
RECOMBINANT ADENOVIRUSES; GENE-TRANSFER; IN-VIVO; NONHUMAN-PRIMATES; CFTR GENE; TYPE-5; THERAPY; EXPRESSION; DELETION; IMPROVES;
D O I
10.1128/JVI.71.4.3293-3298.1997
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Adenoviruses (Ads) are intel mediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb, The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement, Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length, We have constructed a series of vectors with sizes ranging from 15.1 to; 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M., Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc, Natl. Acad. Sci, USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged, A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors, Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 lib, We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than similar to 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.
引用
收藏
页码:3293 / 3298
页数:6
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