AAV2 and hepatocellular carcinoma

项目摘要 这项建议的主要目的是充分评估腺病毒3‘-非翻译区的潜在作用。 相关病毒血清型2(AAV2)基因组中的致癌插入突变并最终在 诱发肝细胞癌。在过去的半个世纪里，aav2被认为是一种非致病性病毒。 因为超过90%的人是AAV2血清阳性的，但还没有已知的疾病 与病毒感染有关。此外，自20世纪90年代末以来，重组AAV载体已经 作为一种基因传递系统被广泛研究和开发，用于治疗许多遗传性疾病 在实验室和临床上，到目前为止，还没有不良事件的报道。然而，上述情况 最近，一项临床观察对众所周知的知识提出了质疑。193例人肝细胞癌中有11例 仅含3‘-非翻译区的野生型(WT)AAV2部分基因组的克隆整合 都观察到了。我们对AAV生物学和3‘非翻译区有长期的兴趣 尤其是AAV基因组。二十多年前，我们阐明了D序列在生命中的作用 WT AAV2的周期，包括基因组拯救、复制和封装。它对重组腺病毒的影响 还检测了转基因表达，这最终导致了D-序列替代的发展 单链rAAV载体以及D序列缺失的单链DNA微型载体。今年早些时候 年，我们开发了Polya缺失的WT AAV和rAAV载体，并为生产生命周期提供了证据 在完全没有常规POLYA信号的情况下的WT AAV2。最近，我们用整个 ITR2与来自其他AAV血清型的ITR产生高滴度、高效力的rAAV载体。尽管我们有 广泛研究了PolyA信号、D序列和各种ITR的作用，以及它们在病毒基因组中的功能 整合，还是在诱导肝细胞癌方面还没有得到严格的研究。此外，甲型肝炎病毒的流行模式 高通量方法尚未在体外或原代人肝细胞中发现整合。 肝细胞异种移植小鼠体内，一种有价值的，临床相关的肝脏模型。我们的人和其他人 文献表明，该模型在基因治疗应用中具有巨大的潜力。我们还有 设计了额外的策略来克服这种新动物模型的变量，这些变量可能会影响 实验结果。 因此，在这项建议中，我们将追求以下具体目标和相关假设： 具体目的1：3‘非翻译区在甲型肝炎病毒基因组整合中的作用。 特定目的2：3‘-非翻译区在AAV介导的体内人肝癌启动中的作用。 这些研究不仅将建立研究病毒致癌插入的模板动物模型 突变，但从这些研究中获得的知识也将适用于更安全的AAV的设计 为未来的基因治疗研究提供载体。

Project Abstract The main aim of this proposal is to fully evaluate the potential role of 3'-untranslated region of the adeno- associated virus serotype 2 (AAV2) genome in the oncogenic insertional mutagenesis and eventually in inducing hepatocellular carcinoma (HCC). AAV2 is considered a non-pathogenic virus for the past half century because more than 90% of the human population is AAV2 seropositive, and yet no known-disease has been associated with the viral infection. In addition, since the late 1990s, recombinant AAV vectors have been extensively studied and developed as a system of gene delivery to treat a number of genetic diseases both in the laboratory and in the clinic, and thus far, no adverse event has ever been reported. However, the above well-known knowledge has recently been questioned by a clinical observation. In 11 of 193 human HCC samples, clonal integrations of partial wild-type (WT) AAV2 genome containing only the 3'-untranslated region were observed. We have had a long-term interest in AAV biology in general, and in the 3'-untranslated region of AAV genome in particular. More than two decades ago, we elucidated the role of the D-sequence in the life cycle of the WT AAV2, including genome rescue, replication and encapsidation. Its influence on rAAV transgene expression was also examined, which eventually led to the development of D-sequence-substituted single-stranded rAAV vectors as well as D-sequence-deleted single-stranded DNA mini-vectors. Earlier this year, we developed polyA-deleted WT AAV and rAAV vectors and provided evidence for a productive life cycle of WT AAV2 in the complete absence of a conventional polyA signal. More recently, we substituted the entire ITR2 with ITRs from other AAV serotypes to produce high-titer, high-potency rAAV vectors. Although we have extensively studied the role of polyA signal, D-sequence and various ITRs, their functions in the viral genome integration, or in the induction of HCC has not been rigorously examined. In addition, the patterns of AAV integration has not been revealed by high-throughput methods in human liver cells in vitro, or in primary human hepatocytes xenografted mice in vivo, a valuable, clinically relevant liver model. Both our and others publications indicated that this model has significant potential for gene therapy applications. We also have designed additional strategies to overcome the variables of this new animal model that may influence experimental outcomes. Thus, in this proposal, we will pursue the following Specific Aims and related hypotheses: Specific Aim 1: Role of the 3'-untranslated region in AAV viral genome integration in vitro. Specific Aim 2: Role of the 3'-untranslated region in AAV-mediated of human HCC initiation in vivo. These studies will not only establish a template animal model for studying viral oncogenic insertional mutagenesis, but also the knowledge gained from these studies will be applicable in the design of safer AAV vectors for gene therapy studies in the future.