Molecular Biology of Recombinant AAV Genomes

描述（由申请人提供）：AAV载体学的最新进展允许重组AAV（rAAV）载体用于人体临床试验。然而，AAV载体的基础生物学仍然没有很好地理解。这是充分利用rAAV载体有用性的主要限制。尽管许多研究活动集中在rAAV载体的组织嗜性和应用上，但rAAV基因组在体内的命运和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，1 × 1014个载体基因组的干扰剂量对于人类受试者可能是必要的。我们的假设是rAAV基因组的不稳定性严重降低了其效率。单链（ss）AAV基因组可以被宿主细胞识别为DNA损伤信号，其导致在未包被后立即发生AAV基因组损失/降解的级联。这种机制将不允许大量游离ss AAV在宿主中存在延长的时间。因此，有效rAAV转导的限制步骤更可能是AAV病毒体的细胞内加工以及ss和双链AAV基因组丢失/降解。此外，AAV基因组的随机整合是AAV载体的主要关注点。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）。目的研究腺相关病毒（AAV）基因组由单链DNA向双链DNA的转化。2）。研究双链AAV基因组的稳定性3）。定量rAAV体内整合的频率。这些特定目标的成功执行将有助于确定提高rAAV转导效率和安全利用rAAV载体的新策略。

DESCRIPTION (provided by applicant): 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 1x1014 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 transduction efficiency and utilizing rAAV vectors safely.