Mroske Cameron, Rivera Hector, Ul-Hasan Taihra, Chatterjee Saswati, Wong K K
Division of Hematology and Division of Stem Cell Transplantation, City of Hope, Duarte, CA 91010, USA.
Hum Gene Ther Methods. 2012 Apr;23(2):128-36. doi: 10.1089/hgtb.2011.231. Epub 2012 May 21.
Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5' ITR (T>G, nt 2) and the downstream MscI site of the 3' ITR (del. nt 4672-4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR-chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.
反向末端重复序列（ITR）的完整性对于重组腺相关病毒（rAAV）的复制、包装和转导至关重要，rAAV是一种很有前景的基因治疗载体。由于AAV ITRs的GC含量为70%且呈回文结构，其测序难度极大。本研究旨在开发一种可靠的ITR测序方法。对AAV2的两个分子克隆pTZAAV和pAV2的ITR进行了如下测序：（1）在变性剂存在的情况下直接从质粒DNA进行测序（直接测序法，DSM），或（2）首先在以7-脱氮-dGTP替代dGTP的反应中进行扩增，然后对所得扩增产物进行测序（扩增测序法，ASM）。DSM和ASM技术均产生了清晰的色谱图，能够读取ITR发夹结构，并揭示了每个ITR中迄今未报道的突变。pTZAAV和pAV2在5' ITR的上游MscI位点（T>G，第2位核苷酸）和3' ITR的下游MscI位点（缺失第4672 - 4679位核苷酸）具有相同的突变。pAV2的色谱图还显示该构建体的ITR以FLOP/FLOP方向排列。此外，DSM成功用于从多种rAAV转导的组织类型中回收ITR - 染色体连接序列。DSM和ASM均可用于对AAV ITR发夹结构进行测序，且两种技术都能可靠地检测ITR中的突变。由于DSM和ASM提供了一种验证ITR完整性的方法，它们构成了rAAV基因治疗工艺开发的有力工具。
