Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo
Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo
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DOI：
10.1038/sj.gt.3302134
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发表时间：
2003-12-01
期刊：
影响因子：
5.1
通讯作者：
Samulski, RJ
中科院分区：
文献类型：
--
作者：
McCarty, DM;Fu, H;Samulski, RJ
重组腺相关病毒（rAAV）载体效率的一个重要限制是需要宿主细胞介导的从单链基因组合成双链DNA。我们已经在专门的自身互补rAAV（scAAV）载体中绕过了这一步骤，通过利用AAV在复制基因组是野生型（wt）长度的一半时包装DNA二聚体的倾向。为了有效地产生这些载体，我们已经从一个rAAV TR中删除了末端解析位点（trs），防止在突变末端开始复制。这些构建体产生单链反向重复基因组，在每个末端具有wt TR，并且在中间具有突变的TR。在解包后，病毒DNA通过突变TR内的分子内碱基配对折叠，然后通过基因组形成双链分子。我们已经使用scAAV来研究小鼠肝脏、肌肉和脑中rAAV转导的障碍。在每个组织中，scAAV的特征在于更快的基因表达起始和更高的转导效率。这项研究证实了早期的预测，互补链DNA合成是rAAV-2转导的主要障碍。scAAV不受这种屏障的影响，并提供了一种非常有效的载体，用于将基因转移到体内许多类型的细胞中。
An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of host-cell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.