Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences
Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences
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DOI：
10.1089/hgtb.2011.034
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发表时间：
2012-02-01
影响因子：
--
通讯作者：
Baiker, Armin
中科院分区：
文献类型：
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作者：
Aurnhammer, Christine;Haase, Maren;Baiker, Armin
基于各种天然存在的腺相关病毒（AAV）血清型的病毒载体是人类基因治疗中最有前途的工具之一。对于重组AAV（rAAV）载体的生产，研究人员主要集中于将基于血清型2（AAV2）的人工AAV基因组交叉包装到来自其他血清型的衣壳中。在包装的基因组内，反向末端重复序列（ITR）是rAAV载体产生所需的唯一顺式作用病毒元件，并且描绘了所有AAV 2衍生的载体基因组的最小公分母。到目前为止，由于其广泛的二级发夹结构形成，无法建立用于检测和定量AAV2 ITR的定量PCR（qPCR）。因此，目前基于qPCR的方法靶向载体编码的转基因或调控元件。在此，我们建立了一种分子生物学方法，该方法允许基于AAV2 ITR序列特异性qPCR对AAV2基因组进行准确和可重复的定量。引物和标记的探针位于ITR序列内，并且被设计用于检测野生型AAV 2和基于AAV 2的载体。该方法适用于检测来自AAV2载体颗粒的单链DNA和来自载体质粒的双链DNA。通过与质粒标准品比较，检测限确定为每个反应50个ITR序列拷贝。总之，该方法描述了促进AAV2 ITR序列的检测和定量的第一个qPCR系统。由于该方法可普遍用于所有基于AAV2基因组的载体，因此它将在未来显著简化rAAV2载体滴定。
Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.