Molecular Biology of Recombinant AAV Genomes

描述（由申请人提供）：AAV是一种单链DNA病毒，作为基因治疗载体已显示出巨大的前景。在临床试验中，已经证明递送RPE 65基因的rAAV载体治疗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。然而，它在其他遗传疾病如血友病中的应用仍然面临相当大的挑战。将rAAV全身递送到人类受试者中将需要比靶向视网膜组织多得多的载体。初步研究表明，rAAV载体在转导的各个阶段有99.9%以上的浪费。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，我们假设它将通过将辅助功能和载体测序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颗粒形成并改善rAAV生产。 将引入先进的基因组学和蛋白质组学来研究rAAV的包装偏好和包装机制。最后，我们确定了细胞质是rAAV基因组丢失的主要位点。 将建立一种创新的检测方法来跟踪细胞质宿主中的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.为了表征rAAV基因组完整性和rAAV包装机制~ 3. 研究重组腺相关病毒基因组在细胞质中的分子状态。 本申请中特定目的的完成将显著增强rAAV载体在人类基因治疗领域中的应用方式。

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.