Molecular Biology of Recombinant AAV Genomes

AAV载体学的最新进展使重组AAV(RAAV)载体能够用于人类 临床试验。然而，AAV载体的基本生物学特性仍未得到很好的了解。这是一次重大的 限制充分利用rAAV载体的有用性。尽管有大量的研究活动 重点介绍了rAAV载体的组织亲和性及其应用，rAAV基因组在体内的去向，以及rAAV在体内的应用 甲型肝炎病毒基因组转化的机制尚未得到很好的描述。临床前临床研究表明 对于人类受试者来说，令人不安的剂量的Ix1014载体基因组可能是必要的。我们的假设是 RAAV基因组的不稳定性严重降低了其效率。单人搁浅(Ss)。甲型肝炎病毒基因组可以 被宿主细胞识别为DNA损伤信号，导致AAV基因组丢失/退化的级联 在揭开涂层后立即涂布。这样的机制将不允许基本上自由的SS AAV存在于主机中 延长期限。因此，有效的rAAV转导的限制步骤更有可能是细胞内 处理AAV病毒粒子以及双链和双链AAV基因组丢失/降解。此外， AAV基因组的随机整合是AAV载体的一个主要问题。为了解决这个问题，我们计划 量化rAAV整合频率。因此，我们的具体目标是1)。关于AAV转换的研究 基因组从单链DNA到双链DNA。2)。双链AAV的稳定性研究 基因组3)。对rAAV在体内整合的频率进行量化。成功执行这些特定的 AIMS将有助于确定提高rAAV转导效率和利用rAAV载体的新策略 安全无恙。

Recent advances in AAV vectorology have allowed recombinant AAV (rAAV) vectors to be used for human clinical trials. However, the basic biology of AAV vectors is still not well understood. This has been a major limitation for the full exploitation of the usefulness of rAAV vectors. Despite numerous research activities focusing on the tissue tropism and applications for rAAV vectors, the fate of rAAV genomes in vivo and the mechanisms of AAV genome conversion have not been well characterized. Preclinical clinical studies suggest that a disturbing dose of IxlO14 vector genomes may be necessary for a human subject. Our hypothesis is that rAAV genome instability seriously reduces its efficiency. The single stranded (ss). AAV genome can be recognized by host cells as DNA damage signal which leads to a cascade of AAV genome loss/degradation immediately after uncoating. Such mechanism will not allow substantial free ss AAV to exist in the host for an extended period. The limiting step for efficient rAAV transduction is therefore more likely to be intracellular processing of AAV virions and both ss and double stranded AAV genome loss/degradation. In addition, the random integration of AAV genomes is a major concern for AAV vectors. To address this issue, we plan to quantify rAAV integration frequency. Hence, our specific aims are 1). To study the conversion of AAV genome from single stranded DNA to double stranded form. 2). To study the stability of double stranded AAV genomes 3). To quantify the frequency of rAAV integration in vivo. The successful execution of these specific aims will help identify new strategies for improving rAAV transductionefficiency and utilizingrAAV vectors safely.