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，作为基因治疗载体已显示出巨大的前景。已证明递送RPE 65基因的重组AAV载体可治愈Leber先天性黑蒙（LCA）疾病，并且患者在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。将rAAV全身递送到人类受试者中将需要更多的载体。我们预计对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的现有能力。为了面对这一挑战，我们提出利用新概念来改善rAAV载体生产，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在促进rAAV复制和包装中相对于胞质载体辅助的互补辅助功能。为了进一步提高用于人基因治疗的重组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区室中来消除不需要的有复制能力的AAV颗粒形成。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： 1.研究腺病毒在细胞质辅助包装系统中对腺相关病毒的增强作用; 2.开发利用胞质rep/cap载体的rAAV生产系统; 3.设计无污染rAAV载体可规模化生产的方案。这些特定目标的实现将极大地增强rAAV载体在人类基因治疗领域中的应用。

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.