Novel adeno-associated virus vector production system development

描述(申请人提供)：AAV是一种单链DNA病毒，已显示出作为基因治疗载体的巨大前景。携带RPE65基因的重组AAV载体已被证明可以治疗Leber‘s先天性黑色素病(LCA)，患者接受载体后能够恢复正常视力。在血友病B临床试验中，接受AAV载体的患者能够表达治疗水平的因子IX基因表达。系统地将rAAV运送到人类受试者需要更多的载体。我们预计，用于人类临床试验的高质量rAAV载体的需求将超过我们领域目前的能力。为了应对这一挑战，我们建议利用一种新的概念来改进rAAV载体的生产，以消除野生型复制rcAAV颗粒并设计可扩展的制造工艺。我们还将研究腺病毒相对于细胞质载体辅助分子在促进rAAV复制和包装方面的辅助作用。为了进一步提高用于人类基因治疗的重组AAV的生产质量，我们假设可以通过将辅助功能和载体序列隔离到不同的细胞室来消除不需要的复制能力AAV颗粒的形成。该生产和处理系统将消除不适合大规模生产的质粒法和其他程序。因此，我们的三个具体目标是： 1.研究腺病毒在细胞质辅助包装系统中对AAV包膜的促进作用； 2.建立利用胞质rep/帽载体的rAAV生产系统； 3.设计一种无污染的可规模化生产rAAV载体的方案。这些特定目的的完成将极大地促进rAAV载体在人类基因治疗领域的应用。

DESCRIPTION (provided by applicant): AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. Recombinant AAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) disease and patients are able to regain normal vision after receiving the vectors. In hemophilia B clinical trial, patient receiving AAV vectors were able to express therapeutic level of factor IX gene expression. Systemic delivery of rAAV into human subjects would require a lot more vectors. We anticipate the demand for high quality rAAV vector for human clinical trials will exceed the current capability in our field. To face this challenge, we propose to improve rAAV vector production utilizing a new concept to eliminate wild type replication rcAAV particles and engineer scalable manufacturing processes. We would also investigate the complimentary helper function of adenovirus over cytoplasmic carrier helper in facilitating rAAV replication and packaging. In order to further improve the quality of recombinant AAV production for human gene therapy, we hypothesize that unwanted replication competent AAV particle formation could be eliminated by sequestering the helper function and vector sequences into different cellular compartments. The production and process system will eliminate plasmid transfection and other procedures which are not feasible for large scale production. Hence, our three specific aims are: 1. To study enhancement of AAV encapsidation by adenovirus in a cytoplasmic helper packaging system; 2. To develop a rAAV production system utilizing a cytoplasmic rep/cap carrier; 3. To engineer a protocol for scalable production of rAAV vectors free of contaminants. Completion of these specific aims will greatly enhance the way of rAAV vectors to be used in the field of human gene therapy.