Molecular Biology of Recombinant AAV Genomes

AAV载体学的最新进展已经允许重组AAV（rAAV）载体用于人类免疫。 临床试验然而，AAV载体的基础生物学仍然没有很好地理解。这是一个重大的 限制了rAAV载体的有效性的充分利用。尽管有许多研究活动， 重点介绍了rAAV载体的组织嗜性和应用，rAAV基因组在体内的命运和rAAV载体的组织嗜性。 AAV基因组转化的机制尚未得到很好的表征。临床前临床研究表明 干扰剂量的1 × 1014载体基因组对于人类受试者可能是必需的。我们的假设是 rAAV基因组的不稳定性严重降低了其效率。单链（SS）。AAV基因组可以是 被宿主细胞识别为DNA损伤信号，其导致AAV基因组损失/降解的级联反应 在未涂覆后立即进行。这种机制将不允许大量游离ss AAV在宿主中存在持续时间。 延长期限。因此，有效rAAV转导的限制步骤更可能是细胞内的 AAV病毒体的加工以及ss和双链AAV基因组丢失/降解。此外该 AAV基因组的随机整合是AAV载体的主要关注点。为了解决这个问题，我们计划 定量rAAV整合频率。因此，我们的具体目标是1）。为了研究AAV的转化 基因组从单链DNA到双链形式。2）。研究双链腺相关病毒的稳定性 基因组3）。定量rAAV体内整合的频率。成功执行这些具体的 目的将有助于确定提高rAAV转导效率和利用rAAV载体的新策略 平安

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.