Characterization of genome integrity for oversized recombinant AAV vector.
Characterization of genome integrity for oversized recombinant AAV vector.
复制标题
超大重组AAV载体的基因组完整性的表征。
DOI：
10.1038/mt.2009.258
复制
发表时间：
2010-01
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
重组腺相关病毒(RAAV)的包装容量限制了其在基因治疗中的应用。最近的研究表明，rAAV可以实现超过4.7kb包装限制的持续转基因表达。为了阐明过大的rAAV载体导致转基因表达的机制，我们构建了一系列基因组范围为2.9~7.2kb的rAAV载体。载体中含有一个复制起始点和一个氨苄西林抗性标记，以促进环状、转导后AAV基因组的恢复。Southern斑点杂交和银染证实在不同大小的载体上都能产生rAAVs。然而，与常规载体相比，超大载体的载体产量下降了大约10倍。碱性Southern杂交表明，超大载体的包装基因组被截断。在上述载体转导的细胞中，环状rAAV单体在感染后24小时即可被解救。无论初始载体大小如何，几乎没有恢复的AAV基因组大小为5kb。在接受上述载体的小鼠中，在载体注射后第21天，对于超大的载体，可以恢复更大的环状rAAV基因组。我们的研究表明，部分包装的rAAV序列可能相互补充，以恢复完整的表达盒。
Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.
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DOI：
10.1073/pnas.87.6.2211
发表时间：
1990-03-01
影响因子：
11.1
作者：
KOTIN, RM;SINISCALCO, M;BERNS, KI
通讯作者：
BERNS, KI
影响因子：
12.4
作者：
Chen, Lingxia;Zhu, Fuxiang;Xiao, Weidong
通讯作者：
Xiao, Weidong
影响因子：
82.9
作者：
Ferrari, FK;Xiao, X;Samulski, RJ
通讯作者：
Samulski, RJ
DOI：
10.1073/pnas.182412299
发表时间：
2002-09-03
影响因子：
11.1
作者：
Gao, GP;Alvira, MR;Wilson, JM
通讯作者：
Wilson, JM
影响因子：
3.7
作者：
Duan, DS;Yan, ZY;Engelhardt, JF
通讯作者：
Engelhardt, JF