Molecular Biology of Recombinant AAV Genomes

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。在临床试验中，携带RPE65基因的rAAV载体已被证明可以治愈Leber’s congenital amaurosis （LCA）疾病，患者在接受载体后能够恢复正常视力。然而，它在血友病等其他遗传疾病中的应用仍面临相当大的挑战。与瞄准视网膜组织相比，将rAAV系统地输送到人体受试者体内需要更多的载体。初步研究表明，超过99.9%的rAAV载体在rAAV转导的各个阶段被浪费。此外，rAAV基因组包装偏好仍然是一个令人不安的问题。为了进一步开发用于人类基因治疗的重组AAV，我们假设通过将辅助功能和载体测序分离到不同的细胞室中，可以消除不必要的复制能力AAV颗粒的形成，并提高rAAV的产量。将引入先进的基因组学和蛋白质组学来研究rAAV的包装偏好和封装机制。最后，我们发现细胞质是rAAV基因组丢失的主要位点。建立一种新颖的宿主细胞质中rAAV基因组追踪方法，并研究宿主细胞质中rAAV基因组减少的机制。因此，我们的三个具体目标是：1。开发和优化新一代rAAV封装系统。研究rAAV基因组完整性和rAAV包装机制。目的：研究重组AAV基因组在细胞质中的分子状态。本研究特定目的的完成，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. In clinical trial, rAAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) diseases and patients are able to regain normal vision after receiving the vectors. However, its use in other genetic diseases such as hemophilia still face considerable challenge. Systemic delivery of rAAV into human subjects would require a lot more vectors than targeting the retina tissue. Prelimary studies have demonstrated more than 99.9% of rAAV vectors were wasted in the varous stages of rAAV transduction. In addition, rAAV genomes packaging preference remains an unsettling issues. In order to further develop recombinant AAV for human gene therapy, we hypothesize that it would eliminate unwanted replication competent AAV particle formation and improve rAAV production by sequetering the helper function and vector sequencing into different cellular compartment. Advanced genomics and proteomics will be introduced to study rAAV packaging preference and encapsidation mechanism. Finally, we identified that cytoplasm is major site for rAAV genome loss. An innovative assay will be established to track rAAV genomes in cytoplasm host and mechanism will be studied to reduce rAAV genomes in cytoplasm of the host cells. Hence, our three specific aims are 1. To develop and optimize the next generation of rAAV packaging system~ 2. To characterize rAAV genome integrity and rAAV packaging mechanisms~ 3. To study the molecular status of recombinant AAV genomes in the cytoplasm. Completion of specific aims in this application will markedly enhance the way of rAAV vectors to be used in the human gene therapy field.