AAV2 and hepatocellular carcinoma

项目摘要 该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）基因组在致癌插入诱变中，并最终在 诱发肝细胞癌（HCC）。在过去的半个世纪，AAV 2被认为是一种非致病性病毒 因为超过90%的人类群体是AAV 2血清阳性的，但是还没有已知的疾病被发现。 与病毒感染有关。此外，自20世纪90年代后期以来，重组AAV载体已经被用于治疗癌症。 作为一种基因递送系统，它被广泛研究和开发，以治疗许多遗传疾病， 实验室和诊所，到目前为止，还没有不良事件的报告。但上述 众所周知的知识最近受到临床观察的质疑。在193例人HCC中的11例中， 样品，仅含有3 '-非翻译区的部分野生型（WT）AAV2基因组的克隆整合 观察了我们对AAV生物学和3 '-非翻译区有着长期的兴趣， 特别是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的细胞周期，包括基因组拯救、复制和胞苷化。对rAAV的影响 转基因表达也进行了检查，这最终导致了D-序列取代的发展。 单链rAAV载体以及D序列缺失的单链DNA微型载体。年初 年，我们开发了polyA缺失的WT AAV和rAAV载体，并为生产性生命周期提供了证据 在完全不存在常规polyA信号的情况下，最近，我们把整个 ITR 2与来自其他AAV血清型的ITR的重组以产生高滴度、高效力的rAAV载体。虽然我们 广泛研究了polyA信号、D序列和各种ITR的作用及其在病毒基因组中的功能 整合，或在HCC的诱导尚未得到严格的审查。此外，AAV的模式 在体外人肝细胞或原代人肝细胞中， 肝细胞异种移植小鼠体内，一个有价值的，临床相关的肝脏模型。我们和其他人 出版物指出，该模型具有基因治疗应用的显著潜力。我们也有 设计了额外的策略来克服这种新动物模型的变量， 实验结果。 因此，在本提案中，我们将追求以下具体目标和相关假设： 具体目的1：3 '-非翻译区在体外AAV病毒基因组整合中的作用。 具体目标2：3 '-非翻译区在AAV介导的人HCC体内启动中的作用。 这些研究不仅为研究病毒致癌插入基因的表达建立了一个模板动物模型， 因此，这些研究不仅可以避免突变，而且从这些研究中获得的知识也将适用于设计更安全的AAV。 为将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.