Image-Guided Gene-Radiation Therapy Targeting Hypoxic Tumors

肿瘤缺氧常见于许多人类癌症中；它们的存在与放化疗耐药有关，这是一种更具侵袭性的表型，是影响治疗结果的重要预后因素。这一提议的长期目标是开发一种图像引导的治疗策略，将低氧成像、放射治疗和基因治疗结合起来，以克服低氧介导的放射抵抗，提高癌症的治愈率。该策略的主要方面是1)肿瘤缺氧的成像和定位，2)图像引导的表达放射增敏效应基因和缺氧诱导标记基因的载体，3)通过分子成像验证载体传递到缺氧细胞，以及4)通过放射治疗肿瘤。要评估和验证该方法，有三个具体目标： 在特定的目标I中，我们将设计和构建表达来自A 低氧诱导的启动子和对抗DNA断裂修复的效应基因，并稳定地使用它们 转染人肿瘤细胞系。肿瘤细胞结构性表达各种效应基因的放射增敏程度 将在体外和体内进行评估；病毒tk基因在低氧细胞中的特异性表达将通过 TK介导的~(124)I-FIAU在裸鼠移植瘤中的microPET显像 在特定的目的II中，我们将评价腺病毒介导的标记基因和 效应基因、标记tk基因的低氧诱导性和效应基因的辐射增敏作用 在活体内。为了改善病毒分布和感染效率，我们将开发和评估条件复制物的使用 腺病毒与表达效应器和标记基因的复制缺陷型腺病毒相结合。 在具体目标三中，我们将把啮齿动物和人类肿瘤移植到裸鼠体内，鉴定和定位低氧 使用microPET/18F-FMISO成像的子体积。在这些图像的引导下，腺病毒载体将被输送到 肿瘤缺氧区和基于TK介导的124I-FIAU捕获的microPET成像将被用于验证 优先将载体运送到低氧细胞。然后将评估肿瘤对辐射的反应。 在初步研究中，我们已经表明反义Ku70或显性负性Ku70片段显著增加辐射敏感性。 体外和体内低氧肿瘤细胞，并通过腺病毒成功地将其带入肿瘤 载体，用于肿瘤乏氧靶向，我们用18F-FMISO进行了microPET成像，并开发了一种 用于指导腺病毒载体注射的立体定位模板。此外，我们还表明，低氧诱导的标记物 用~(124)I-FIAU/microPET可在体内快速检测到TK基因。从拟议的临床前研究中获得的信息 研究将指导临床策略的设计，以改善癌症治疗的结果。

Tumor hypoxia is often found in many human cancers; their presence has been implicated in radio- and chemoresistance, a more aggressive phenotype, and is an important prognostic factor of treatment outcome. The long term goal of this proposal is to develop an image-guided therapeutic strategy, combining hypoxia imaging, radiotherapy and gene therapy, to overcome hypoxia-mediated radioresistance and improve cancer cure. The major facets of this strategy are 1) the imaging and localization of tumor hypoxia, 2) image-guided delivery of vectors that express a radiosensitizing effector gene as well as a hypoxia-induced marker gene, 3) verification of vector delivery to hypoxic cells by molecular imaging, and 4) tumor eradication by radiation. To evaluate and validate this approach, there are three Specific Aims: In Specific Aim I, we shall design and construct vectors that express both a marker gene HSV1-tk from a hypoxia-inducible promoter and an effector gene that antagonizes the repair of DNA breaks, and use them to stably transfect tumor cell lines. The degree of radiosensitization of tumor cells that constitutively express various effector genes will be assessed in vitro and in vivo; the specific expression of the viral tk gene in hypoxic cells will be evaluated by microPET imaging of TK-mediated trapping of 124I-FIAU in tumors transplanted in nude mice. In Specific Aim II, we shall evaluate the efficacy of adenovirus-mediated delivery of the marker gene and the effector gene, the hypoxia inducibility of the marker tk gene, and the radiosensitizing effect of the effector gene in vitro and in vivo. To improve viral distribution and infection efficiency, we shall develop and evaluate the use of conditionally-replicative adenovirus in combination with replication-defective adenovirus expressing the effector and marker genes. In Specific Aim III, we shall transplant rodent and human tumors into nude rats, identify and localize the hypoxic sub-volume using microPET/18F-FMISO imaging. Guided by these images, adenoviral vectors will be delivered to the hypoxic region of the tumor and microPET imaging based on TK-mediated trapping of 124I-FIAU will be used to verify the preferential delivery of the vectors to hypoxic cells. The tumor's response to radiation will then be evaluated. In pilot studies, we have shown that antisense Ku70 or a dominant negative Ku70 fragment significantly radiosensitizes hypoxic tumor cells in vitro and in vivo, and that they can be successfully delivered into tumors by adenoviral vectors, For tumor hypoxia targeting, we have implemented microPET imaging with 18F-FMISO and developed a stereotaxic template for guiding adenoviral vector injection. Also, we have shown that the hypoxia-induction of the marker tk gene can be readily detected using 124I-FIAU / microPET in vivo. The information gained from the proposed preclinical studies will serve as a guide in the design of clinical strategy to improve the outcome of cancer therapy.