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

描述(申请人提供)：重组AAV载体正在成为一种日益重要的基因转移工具。最近在临床治疗应用方面的成功表明，在未来几年，使用这些载体治疗的患者数量可能会迅速增加。临床前研究已经压倒性地支持了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治疗相关的小鼠肝细胞癌的过度发病率，在一个案例中，这可能与特定染色体位置上的载体DNA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的小鼠肿瘤的DNA进行大规模平行测序，对载体整合进行了详尽的表征。这些不一致的结果给rAAV的遗传毒性留下了疑问，使得很难预测和披露患者群体的潜在风险。我们有 采用了一种不同的策略来研究rAAV的遗传毒性，本质上是创造了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敏感性，然后回溯到剖析遗传毒性的机制以及促成它的载体元件。使用这一策略，我们在初步研究中表明，我们可以很容易地在易患肿瘤的小鼠品系(C3H/HeJ)中测量与媒介感染相关的肿瘤发病率的增加。这为确定不同rAAV载体的相对遗传毒性潜力提供了一个有价值的模型，重要的是，它允许表征最可能的癌基因激活机制。利用设计用于测试强读通式转录对肿瘤促进作用的rAAV载体，我们的初步研究显示，肿瘤相关载体基因组通过转录通读、转录增强效应和肿瘤抑制基因的干扰而与癌基因相互作用。传统设计的第二个载体在这个模型中通过一个明显不同的机制与过量的肿瘤相关，几乎没有证据表明载体整合在肿瘤中。我们先前研究的两个载体都是rAAV载体的自互补导数。在拟议的研究中，我们将包括三个目标：1.直接比较自补型AAV和单链rAAV载体在C3H/HeJ小鼠模型中促进肝肿瘤的作用，并比较这些动物在正常肝组织中插入载体的模式的差异。2.修改scAAV载体，以确定哪些特征对肿瘤的促进作用，特别是从不期望通过转录通读激活癌基因的载体。3.利用深度测序技术，在诱导和不诱导细胞周期的情况下，对肿瘤和正常组织中的rAAV整合位点进行了全面的表征。我们还将把在易患肿瘤的小鼠中观察到的媒介感染的影响与具有肿瘤抗性的小鼠模型联系起来，以帮助推断人类患肝癌的风险。总之，这些研究将极大地促进我们对与rAAV载体基因治疗相关的风险因素的理解。

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.