PATTERNS OF r AAV VECTOR INSERTION ASSOCIATED WITH LIVER TUMORS IN A MOUSE MODEL

小鼠模型中与肝肿瘤相关的 r AAV 载体插入模式

• 批准号: 9269161
• 负责人: HAIYAN FU
• 金额: $ 26.24万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269161
• 关键词: Address; Adopted; Affect; Animal Model; Back; Biological Assay; Blood - brain barrier anatomy; C3H/HeJ Mouse; Capsid; Cell Cycle; Clinical; DNA Integration; Development; Disclosure; Disease; Dose; Drug or chemical Tissue Distribution; Elements; Enhancers; Event; Family; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genetic Transcription; Genome; Healthcare Systems; Hereditary Disease; Human; Incidence; Infection; Injection of therapeutic agent; Investigation; Lead; Link; Liver; Liver neoplasms; Lysosomal Storage Diseases; Massive Parallel Sequencing; Measures; Mission; Mitotic; Modeling; Molecular Conformation; Mouse Strains; Mus; Muscle; Normal tissue morphology; Oncogene Activation; Oncogenes; Oncogenic; Patients; Pattern; Play; Positioning Attribute; Primary carcinoma of the liver cells; Public Health; Publications; Recombinant adeno-associated virus (rAAV); Recombinants; Reporting; Research; Resistance; Retina; Risk; Risk Assessment; Risk Factors; Risk Management; Role; Safety; Serotyping; Single-Stranded DNA; Site; Technology; Testing; Therapeutic; Tissue Model; Tissues; Toxic effect; Transgenes; Tumor Promotion; Tumor Suppressor Genes; Tumor Tissue; United States National Institutes of Health; Viral Genome; Viral Load result; Viral Vector; adeno-associated viral vector; animal tissue; cellular transduction; clinically relevant; comparative; cost; deep sequencing; design; disability; ds-DNA; exhaustion; experimental study; flexibility; gene therapy; genotoxicity; immunogenicity; integration site; mouse model; novel therapeutic intervention; preclinical study; public health relevance; success; therapy development; tool; transduction efficiency; tumor; tumor DNA; vector; vector genome

DESCRIPTION (provided by applicant): Recombinant AAV vectors are becoming an increasingly important gene transfer tool. Recent successes in clinical therapeutic applications suggest that the number of patients treated with these vectors is likely to increase rapidly in coming years. Preclinical studies have overwhelmingly supported the safety of rAAV gene therapy in numerous different tissues and animal models. However, a small number of studies have reported an excess incidence of hepatocellular carcinomas associated with rAAV treatment in mice, and in one case, this could be directly linked to vector DNA integration in a specific chromosomal locus. Other studies have found little evidence of such a link, despite exhaustive characterization of vector integration by massive parallel sequencing of DNA from tumors in rAAV infected mice. These inconsistent results leave the issue of rAAV genotoxicity in question, making it difficult to anticipate and disclose potential risks to patient groups. We have adopted a different strategy for the investigation of rAAV genotoxicity, essentially creating conditions where vector integration is likely to lead to tumor promotion in order to provide the sensitivity we need to detect oncogenic events, and then working back to dissect the mechanisms of genotoxicity as well as the vector elements that contribute to it. Using this strategy, we show in preliminary studies that we can readily measure an increase in tumor incidence associated with vector infection in a tumor-prone mouse strain (C3H/HeJ). This provides a valuable model for determining the comparative genotoxic potential of different rAAV vectors and, importantly, allows the characterization of the most likely mechanisms of oncogene activation. Using a rAAV vector designed to test the effects of strong read-through transcription on tumor promotion, our preliminary studies show tumor-associated vector genomes interacting with oncogenes through transcriptional read- through, transcriptional enhancer effects, and disruption of tumor suppressor genes. A second vector, of conventional design, was associated with excess tumors in this model through an apparently different mechanism, with little evidence for vector integration in tumors. Both of the vectors tested in our previous study were the self-complementary derivative of rAAV vector. In the proposed research, we will include three Aims designed to: 1. Directly compare self-complementary AAV and single-strand rAAV vectors for liver tumor promotion in the C3H/HeJ mouse model, and for differences in patterns of vector insertion in normal liver tissue from these animals. 2. Modify the scAAV vectors to determine what features contributed to tumor promotion, particularly from the vector that was not expected to activate oncogenes by transcriptional read- through. 3. Use deep sequencing technology to fully characterize rAAV integration sites in tumors and normal tissue with and without induction of cell-cycling. We will also relate the observed effects of vector infection in the tumor-prone mice to a tumor resistant mouse model to help extrapolate to liver tumor risk in humans. Together, these studies will greatly advance our understanding of the risk factors associated with rAAV vector gene therapy.

描述（由申请人提供）：重组 AAV 载体正在成为越来越重要的基因转移工具。最近在临床治疗应用中取得的成功表明，使用这些载体治疗的患者数量可能在未来几年迅速增加。临床前研究绝大多数支持 rAAV 基因治疗在许多不同组织和动物模型中的安全性。然而，少数研究报告了与 rAAV 治疗小鼠相关的肝细胞癌发病率过高，在一种情况下，这可能与载体 DNA 在特定染色体位点的整合直接相关。其他研究几乎没有发现这种联系的证据，尽管通过对感染 rAAV 的小鼠肿瘤 DNA 进行大规模平行测序，对载体整合进行了详尽的表征。这些不一致的结果使 rAAV 基因毒性问题成为疑问，从而难以预测和披露患者群体的潜在风险。我们有 采用了不同的策略来研究 rAAV 基因毒性，本质上是创造载体整合可能导致肿瘤促进的条件，以便提供我们检测致癌事件所需的灵敏度，然后回过头来剖析基因毒性的机制以及对其有贡献的向量元素。使用这种策略，我们在初步研究中表明，我们可以轻松测量与易患肿瘤小鼠品系（C3H/HeJ）中的载体感染相关的肿瘤发病率的增加。这为确定不同 rAAV 载体的相对基因毒性潜力提供了一个有价值的模型，并且重要的是，可以表征最可能的癌基因激活机制。使用旨在测试强通读转录对肿瘤促进的影响的rAAV载体，我们的初步研究表明肿瘤相关载体基因组通过转录通读、转录增强子效应和肿瘤抑制基因的破坏与癌基因相互作用。传统设计的第二种载体通过明显不同的机制与该模型中的过量肿瘤相关，几乎没有证据表明载体在肿瘤中整合。我们之前的研究中测试的两种载体都是rAAV载体的自互补衍生物。在拟议的研究中，我们将包括三个目标，旨在： 1. 直接比较自互补 AAV 和单链 rAAV 载体在 C3H/HeJ 小鼠模型中促进肝脏肿瘤的作用，以及正常肝脏中载体插入模式的差异来自这些动物的组织。 2. 修改 scAAV 载体以确定哪些特征有助于肿瘤促进，特别是来自预计不会通过转录通读激活癌基因的载体。 3. 使用深度测序技术全面表征肿瘤和正常组织中诱导和不诱导细胞周期的 rAAV 整合位点。我们还将在易患肿瘤的小鼠中观察到的载体感染的影响与抗肿瘤小鼠模型联系起来，以帮助推断人类肝脏肿瘤的风险。总之，这些研究将极大地增进我们对 rAAV 载体基因治疗相关危险因素的理解。