Clark K Reed, Penaud-Budloo Magalie
Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
Methods Mol Biol. 2011;807:239-58. doi: 10.1007/978-1-61779-370-7_10.
Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression in a wide variety of animals, including primates. The rAAV genome is packaged into the virion as single-stranded DNA devoid of any viral genes. A proportion of the single-stranded genomes are converted into transcriptionally active double-stranded DNA (dsDNA) early after nuclear entry by second-strand synthesis mediated by host repair DNA polymerases or/and by annealing of the rAAV (-) and (+) strands. Second-generation, self-complementary vectors are packaged as single-strand hairpins and rapidly assume a dsDNA conformation independent of the action of polymerases. In both cases, linear dsDNA vector genomes circularize and can undergo concatemerization into higher order forms (McCarty et al. Annu Rev Genet 38: 819-845, 2004; Schultz and Chamberlain Mol Ther 16: 1189-1199, 2008; Duan et al. J Virol 72: 8568-8577, 1998). As a result, rAAV vector genomes are maintained mainly as circular monomeric and concatemeric episomal forms in skeletal muscle and liver (Schnepp et al. J Virol 77: 3495-3504, 2003; Penaud-Budloo et al. J Virol 82: 7875-7885, 2008; Nakai et al. J Virol 75: 6969-6976, 2001). Moreover, in nonhuman primate skeletal muscle, it has been shown that rAAV episomes assimilate into chromatin with a typical nucleosomal pattern that presumably is important for persistence and gene expression in quiescent tissues over a period of several years (Penaud-Budloo et al. J Virol 82: 7875-7885, 2008). Conversely, although rAAV is not considered as an integrative vector per se, introduction of exogenous DNA into the nuclear compartment can result in low-level vector assimilation into the host genome. One mechanism appears to involve vector insertion at sites of double-strand DNA breaks using cellular DNA repair enzymes. As rAAV gene transfer technology and applications mature, a better characterization of the genetic fate of the rAAV genome is critical to accurately evaluate the risk/benefit ratio for a particular disease indication. In this chapter, two complementary methods are detailed to enable characterization of rAAV molecular structure in a particular target tissue and estimation of its integration frequency.
重组腺相关病毒（rAAV）载体能够在包括灵长类动物在内的多种动物中介导长期基因表达。rAAV基因组以不含任何病毒基因的单链DNA形式包装进病毒粒子。一部分单链基因组在通过宿主修复DNA聚合酶介导的第二链合成或/和rAAV（-）链与（+）链退火进入细胞核后早期转化为转录活性双链DNA（dsDNA）。第二代自我互补载体以单链发夹形式包装，并迅速形成独立于聚合酶作用的dsDNA构象。在这两种情况下，线性dsDNA载体基因组都会环化，并可串联形成更高阶形式（麦卡蒂等人，《遗传学年度评论》38：819 - 845，2004；舒尔茨和张伯伦，《分子治疗》16：1189 - 1199，2008；段等人，《病毒学杂志》72：8568 - 8577，1998）。因此，rAAV载体基因组在骨骼肌和肝脏中主要以环状单体和串联附加体形式维持（施内普等人，《病毒学杂志》77：3495 - 3504，2003；佩诺 - 布德鲁等人，《病毒学杂志》82：7875 - 7885，2008；中井等人，《病毒学杂志》75：6969 - 6976，2001）。此外，在非人类灵长类动物骨骼肌中，已表明rAAV附加体以典型的核小体模式融入染色质，这可能对静止组织中数年的持久性和基因表达很重要（佩诺 - 布德鲁等人，《病毒学杂志》82：7875 - 7885，2008）。相反，尽管rAAV本身不被视为整合载体，但将外源DNA引入核区室可能导致低水平的载体整合到宿主基因组中。一种机制似乎涉及利用细胞DNA修复酶在双链DNA断裂位点插入载体。随着rAAV基因转移技术和应用的成熟，更好地表征rAAV基因组的遗传命运对于准确评估特定疾病适应症的风险/效益比至关重要。在本章中，详细介绍了两种互补方法，以能够表征特定靶组织中rAAV的分子结构并估计其整合频率。
