AAV2 and hepatocellular carcinoma

AAV2 与肝细胞癌

• 批准号: 9528459
• 负责人: Arun Srivastava
• 金额: $ 18.47万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9528459
• 关键词: 3' Untranslated Regions; Adenoviruses; Adverse event; Animal Model; Biology; Capsid; Clinic; Clinical; Clinical Trials; DNA; DNA Viruses; Defective Viruses; Dependovirus; Development; Diploidy; Disease; Elements; Enhancers; Fibroblasts; Future; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genetic Diseases; Genome; Hela Cells; Helper Viruses; Hepatocyte; Human; In Vitro; Incidence; Infection; Injections; Insertional Mutagenesis; Inverted Terminal Repeat; Knowledge; Laboratories; Lead; Life Cycle Stages; Liver; Manuscripts; Mediating; Methods; Modeling; Mus; Muscle; Neonatal; Nucleotides; Oncogenic; Outcome; Parvovirus; Pattern; Peptide Signal Sequences; Polyadenylation; Population; Primary carcinoma of the liver cells; Proteins; Public Health; Publications; Publishing; Recombinant adeno-associated virus (rAAV); Recombinants; Reporting; Risk; Role; Sampling; Serotyping; Signal Transduction; Single-Stranded DNA; Skeletal Muscle; Structure; Study models; System; Techniques; Viral; Viral Genes; Viral Genome; Viral Vector; Virus; Virus Diseases; Xenograft procedure; adeno-associated viral vector; base; clinically relevant; co-infection; design; gene delivery system; gene therapy; gene therapy clinical trial; genotoxicity; humanized mouse; in vivo; integration site; interest; member; mouse model; nonhuman primate; novel; promoter; seropositive; transgene expression; tumor; vector

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.

项目摘要 该提案的主要目的是充分评估腺-的3'-非翻译区的潜在作用。 相关病毒血清型 2 (AAV2) 基因组在致癌插入突变中并最终在 诱发肝细胞癌（HCC）。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% 的人口 AAV2 血清呈阳性，但尚未发现任何已知疾病 与病毒感染有关。此外，自20世纪90年代末以来，重组AAV载体已被广泛应用。 作为基因传递系统进行了广泛的研究和开发，用于治疗多种遗传疾病 实验室和诊所，迄今为止，尚未报告任何不良事件。然而，上述 众所周知的知识最近受到临床观察的质疑。 193 例人类 HCC 中有 11 例 样品，仅包含 3'-非翻译区的部分野生型 (WT) AAV2 基因组的克隆整合 被观察到。我们对 AAV 生物学以及 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 的循环，包括基因组拯救、复制和衣壳化。对rAAV的影响 还检查了转基因表达，最终导致了 D 序列取代的开发 单链 rAAV 载体以及 D 序列缺失的单链 DNA 微型载体。早些时候这个 同年，我们开发了多聚腺苷酸缺失的 WT AAV 和 rAAV 载体，并为生产生命周期提供了证据 WT AAV2 在完全没有传统的多聚腺苷酸信号的情况下。最近，我们替换了整个 ITR2 与其他 AAV 血清型的 ITR 结合可产生高滴度、高效力的 rAAV 载体。虽然我们有 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 整合或诱导 HCC 的过程尚未经过严格检验。此外，AAV 的模式 体外人肝细胞或原代人肝细胞中的高通量方法尚未揭示整合 小鼠体内肝细胞异种移植，是一种有价值的、临床相关的肝脏模型。我们的和其他人 出版物表明该模型在基因治疗应用中具有巨大潜力。我们还有 设计了额外的策略来克服这种新动物模型可能影响的变量 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： 具体目标 1：3'-非翻译区在 AAV 病毒基因组体外整合中的作用。 具体目标 2：3'-非翻译区在 AAV 介导的人 HCC 体内起始中的作用。 这些研究不仅将建立用于研究病毒致癌插入的模板动物模型 诱变，而且从这些研究中获得的知识将适用于更安全的 AAV 的设计 未来基因治疗研究的载体。