Molecular Biology of Recombinant AAV Genomes

描述(由申请人提供)：AAV向量学的最新进展使重组AAV(RAAV)载体能够用于人体临床试验。然而，AAV载体的基本生物学特性仍未得到很好的了解。这一直是充分利用rAAV载体的有用性的主要限制。尽管许多研究集中在rAAV载体的组织亲和性和应用上，但rAAV基因组在体内的去向和AAV基因组转化的机制尚未得到很好的描述。临床前研究表明，对于人类受试者来说，令人不安的1x1014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它的效率。单链AAV基因组可以被宿主细胞识别为DNA损伤信号，导致AAV基因组在脱壳后立即发生级联丢失/降解。这种机制将不允许大量的自由SS AAV在较长时间内存在于主机中。因此，有效的rAAV转导的限制步骤更有可能是AAV病毒粒子的细胞内处理以及双链和双链AAV基因组的丢失/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)。研究甲型肝炎病毒基因组由单链DNA向双链DNA的转化。2)。研究双链AAV基因组的稳定性。对rAAV在体内整合的频率进行量化。这些特定目的的成功实现将有助于寻找新的策略来提高rAAV转导效率和安全利用rAAV载体。

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.