Novel adeno-associated virus vector production system development

描述（由申请人提供）：AAV是一种单链DNA病毒，作为基因治疗载体具有很大的前景。携带RPE65基因的重组AAV载体已被证明可以治愈Leber's congenital amaurosis （LCA）病，患者在接受载体后能够恢复正常视力。在B型血友病临床试验中，接受AAV载体的患者能够表达治疗水平的因子IX基因表达。将rAAV系统地输送到人体需要更多的载体。我们预计对用于人体临床试验的高质量rAAV载体的需求将超过我们目前领域的能力。为了应对这一挑战，我们提出利用一种新的概念来改善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对细胞质载体辅助物的辅助功能，以促进rAAV的复制和包装。为了进一步提高用于人类基因治疗的重组AAV生产的质量，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中来消除不必要的复制能力AAV颗粒的形成。生产和工艺系统将消除质粒转染和其他不适合大规模生产的程序。因此，我们的三个具体目标是：

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.