McCarty D M, Fu H, Monahan P E, Toulson C E, Naik P, Samulski R J
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7352, USA.
Gene Ther. 2003 Dec;10(26):2112-8. doi: 10.1038/sj.gt.3302134.
An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-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.
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从单链基因组合成双链DNA。我们通过利用腺相关病毒（AAV）在复制基因组长度为野生型（wt）一半时包装DNA二聚体的趋势，在一种特殊的自我互补rAAV（scAAV）载体中绕过了这一步骤。为了高效生产这些载体，我们从一个rAAV TR中删除了末端分辨率位点（trs），阻止在突变末端起始复制。这些构建体产生单链、反向重复基因组，两端各有一个wt TR，中间有一个突变的TR。脱壳后，病毒DNA通过突变TR内的分子内碱基配对折叠，然后穿过基因组形成双链分子。我们使用scAAV研究了rAAV在小鼠肝脏、肌肉和大脑中转导的障碍。在每个组织中，scAAV的特点是基因表达起始更快且转导效率更高。这项研究证实了早期的预测，即互补链DNA合成是rAAV-2转导的主要障碍。scAAV不受此障碍影响，并为体内多种类型细胞的基因转移提供了一种极其有效的载体。
