Molecular Biology of Recombinant AAV Genomes

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。在临床试验中，携带RPE65基因的重组腺相关病毒载体已被证明可以治疗Leber‘s先天性黑色素病，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性疾病中的应用仍然面临着相当大的挑战。系统地将rAAV输送到人类受试者体内需要比靶向视网膜组织多得多的载体。初步研究表明，在rAAV转导的不同阶段，超过99.9%的rAAV载体被浪费。此外，rAAV基因组的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，我们假设它将通过将辅助功能和载体测序隔离在不同的细胞室，从而消除不需要的复制能力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的包装偏好和包膜机制。最后，我们确定细胞质是rAAV基因组丢失的主要部位。将建立一种新的方法来追踪细胞质宿主中的rAAV基因组，并将研究减少宿主细胞细胞质中的rAAV基因组的机制。因此，我们的三个具体目标是：1.开发和优化下一代rAAV包装系统~2.研究rAAV基因组的完整性和包装机制~3.研究重组AAV基因组在细胞质中的分子状态。这一应用中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.