New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors
New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors
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DOI：
10.1038/nm1197-1295
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发表时间：
1997-11-01
期刊：
影响因子：
82.9
通讯作者：
Samulski, RJ
中科院分区：
文献类型：
--
作者：
Ferrari, FK;Xiao, X;Samulski, RJ
腺相关病毒（AAV）载体是目前病毒传递系统的一个有希望的替代方案。基于aav的基因治疗研究领域是由我们对这种独特的细小病毒的生物学和生命周期的理解所驱动的。AAV是一种有缺陷的人细小病毒，具有非致病性、靶向整合能力和广泛的宿主范围（人、猿、鼠、犬和禽）等许多自然特征，对人类基因治疗载体具有吸引力。然而，重组AAV （rAAV）载体的应用仍存在一些问题。这些障碍的例子包括载体随机整合，无法包装大基因（bbb5 kbp），以及生成高滴度载体所需的繁琐协议。鉴于这种病毒的隐蔽性，只有在对更容易检测的rAAV载体进行广泛研究后，对其生物学的新见解才浮出水面，这并不奇怪。再加上rAAV具有转导分裂细胞和非分裂细胞的能力，最近的体内研究已经在多种组织中实现了高效、长期的基因转移，包括肺、肌肉、大脑和肝脏。最近，直接将rAAV注射到小鼠的大脑和肌肉中导致转基因表达超过1.5年”。这些观察结果的独特之处在于，转导的细胞不会引起细胞毒性t淋巴细胞（CTL）反应，这表明这些载体与目前的重组腺病毒载体之间存在重大差异。此外，人促红细胞生成素和因子IX rAAV载体的递送已导致两种蛋白的系统性生产，分别长达40周和26周。”在一个大型动物模型中，用rAAV载体表达因子IX感染血友病犬已导致转基因表达长达4个月”。此外，rAAV基因治疗|囊肿纤维化目前正在临床环境中进行人体I期毒性试验。这些最近的rAAV体内实验结果与产生高滴度重组AAV制剂的能力直接相关。重组AAV的生产现有的rAAV包装系统是利用辅助腺病毒（Ad）瞬时转染293细胞。所有AAV载体均来源于质粒底物，该质粒底物仅包含所选转基因侧翼的145 bp AAV反向末端重复序列（ITR）。这些ITR序列是AAV复制、包装和整合所必需的唯一顺式作用元件。”所有的病毒编码序列（96%的基因组）被移除，并从辅助质粒中以反式形式提供（图1）”。由于AAV辅助质粒与AAV载体结构不存在重叠，因此野生型AAV的产生和对残留病毒基因产生免疫应答的可能性都不大
Adeno-associated virus (AAV) vectors represent a promising alternative to current viral delivery systems". The field of AAV-based gene therapy research is driven by our understanding of the biology and life cycle of this unique parvovirus. Classified as a defective human parvovirus, AAV has many natural features that are attractive for a human gene therapy vector, including its non-pathogenicity, targeted integrating capability and broad host range (human, simian, murine, canine and avian). However, some problems with the application of recombinant AAV (rAAV) vectors are still unresolved. Examples of these obstacles include vectors integrating randomly, inability to package large genes (> 5 kbp), and the tedious protocols required to generate high-titer vector. It is not surprising, given the cryptic nature of this virus, that new insights into its biology have surfaced only after extensive research with the more easily assayed rAAV vectors. Coupled with the ability to transduce both dividing and nondividing cells, recent studies with rAAV in vivo have resulted in efficient, long-term gene transfer in a variety of tissues, including lung", muscle “, brain'and liver “. Recently, direct injection of rAAV into the brain and muscle of mice has resulted in transgene expression for over 1.5 years". These observations were unique in that the transduced cells did not elicit a cytotoxic T-lymphocyte (CTL) response, demonstrating a major difference between these vectors and the current recombinant adenovirus vectors. In addition, delivery of human erythropoietin and factor IX rAAV vectors has resulted in systemic production of therapeutic levels of both proteins for up to 40 and 26 weeks, respectively". In a large animal model, infection of hemophilic dogs with a rAAV vector expressing factor IX has resulted in transgene expression now up to four months". Furthermore, rAAV gene therapy for| cystī fibrosis is now being tested in the clinical setting with phase I toxicity trials in humans. These recent in vivo results with rAAV have been directly correlated with the ability to generate high-titer recombinant AAV preparations.Production of recombinant AAV The existing rAAV packaging systems use transient transfection of 293 cells infected with helper adenovirus (Ad). All AAV vectors are derived from a plasmid substrate that contains only the 145-bp AAV inverted terminal repeats (ITR) flanking the transgene of choice. These ITR sequences are the only cis-acting elements necessary for AAV replication, packaging and integration". All viral coding sequences (96% of the genome) are re-moved and provided in trans from a helper plasmid (Fig. 1)". As there is no overlap between the AAV helper plasmid and the AAV vector construct, both the generation of wild-type AAV and the possibility of immune response to residual viral gene