重组腺相关病毒载体的包装机制- - 夭折信号假说研究

Recombinant adeno-associated virus (rAAV) vectors have shown great promise not only in numerous animal experiments but also in human clinical trials. Recently, it has been approved for clinical use in Europe for the treatment of lipoprotein lipase deficiency (LPLD). However, to achieve a therapeutic level of transgene expression generally requires a very high dose. For example, a dose of 1x10E(11-12) rAAV particles is typically necessary for administration to a 25 gram mouse, which is equivalent to a dose greater than 10E14 viral particles for a human subject. Currently, AAV vector production is far from optimal and varies greatly depending on the vector sequence composition. Although the wild type AAV genomes are generally similar in size at 4.7 kb, short defective interference (DI) particles could indeed be formed. Southern blot analysis of rAAV genomes extracted from capsids often suggests a heterogeneous population. Therefore, we hypothesize that there may exist a hidden packaging termination signal for rAAV packaging machinery. They differ from canonical termination signals, AAV ITRs. These unknown sequences serve as a cryptic termination signal and would cause prematurely packaged AAV genomes and form DI particles which would not contribute to transgene expression. We innovatively propose to exploit Helicos Biosciences' true single molecule sequencing (tSMS) technology to analyze the fine structure of packaged rAAV genomes. By comparing 5' ends from various rAAV preparations using tSMS technology, we may define such cryptic packaging termination sequences. We also analyze whether AAV serotype, AAV packaging method and AAV helper functions such as rep&cap or adenovirus genes can affect such undesirable secondary packaging terminal signal usage. Identifying such termination signals for rAAV packaging would allow us to optimize rAAV cis content by eliminating these sequences and their analogs without affecting the gene function and expression levels. This study would have the potential to give rise to new strategies that will enhance the applications of rAAV vectors, improve the rAAV safety profile and make AAV mediated gene therapy more cost-effective.

重组腺相关病毒(rAAV)是欧美地区唯一上市基因药物Glybera的载体。然而现有rAAV载体设计和制备方面存在诸多缺陷，制约了其在基础研究和临床应用等方面的推广,阐明其包装机制是开发和优化rAAV载体系统的关键。与目前研究主要集中在AAV本身基因及腺病毒基因等反式作用因子不同，本项目试图阐明载体基因组上的顺式作用元件在包装过程中的作用。根据大量含不同基因载体包装效率的巨大差异且存在大量不完整AAV颗粒这些现象，本项目提出了rAAV包装的"夭折信号假说"，即病毒载体基因组上可能存在未知特异性序列，意外成为包装终止信号，提前停止包装，从而降低包装效率和质量。本项目将通过高通量tSMS序列分析技术，在单分子/单病毒层次上通过对不同基因组、不同血清型、不同包装系统中rAAV载体的序列分析，总结夭折信号特性，建立夭折信号数据库,探明夭折信号产生的机制，并根据其机制消除夭折信号，优化载体设计和制备。

重组腺相关病毒载体(rAAV)在众多临床实验取得成功并在欧美地区有多项上市的基因药物。然而现有 rAAV 载体设计和制备方面存在诸多缺陷, 制约了其在基础研究和临床应用等方面的推广, 阐明其包装机制是开发和优化 rAAV 载体系统的关键。在过去四年中，本项目试图阐明载体基因组上的顺式作用元件在包装过程中的作用。根据大量含不同基因载体包装效率的巨大差异且存在大量不完整 AAV 颗粒这些现象, 通过高通量 tSMS 序列分析技术, 在单分子/单病毒层次上通过对不同基因组、不同血清型、不同包装系统中 rAAV 载体的序列分析，验证了原来提出的rAAV 包装的“夭折信号假说”, 发现了病毒载体基因组上存在的特异性序列， 比如与AAV terminal resolution 位点的同源序列和能形成强二级结构的序列，都可以成为非正常的包装终止信号, 导致载体提前停止包装, 从而降低包装效率和质量。 此外，AAV ITR是原经典的包装起始信号，也是终止信号，大量的终止也出现在包装起始位置，导致大量载体仅含ITR。 根据对夭折信号的发现和特性的总结对优化载体设计和制备提出新的建议，与AAV terminal resolution 位点的同源序列和能形成强二级结构的序列应该在AAV载体通过序列优化时消除。未来的研究方向应该开发新型包装起始序列，消除AAV ITR同时作为起始信号和终止信号而成为产生不完整 AAV 颗粒的主要源头。本项目为完善全新的、经济的、高效的、无rcAAV污染的rAAV载体规模化制备系统提供主要的理论依据和使用价值。