In Vivo Image Guidance for Gene-Radiation Therapy

描述(由申请人提供)： 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。为了评估和验证这种方法，有三个特定的目标：在特定的目标I，我们将设计和构建表达来自低氧诱导启动子的标记基因HSV1-tk和拮抗DNA断裂修复的效应基因的载体，并使用它们稳定地转染肿瘤细胞系。将在体外和体内评估组成性或诱导性表达各种效应基因的肿瘤细胞的放射增敏程度；将通过TK介导的124I-FIAU在裸鼠移植瘤中的MicroPET成像来评估病毒tk基因在低氧细胞中的特异性表达。在特定的目的II中，将构建含有缺氧标记基因和效应基因的复制缺陷型腺病毒。然后，将在体外和体内研究腺病毒介导的标记基因和效应基因的有效性，标记基因tk的低氧诱导性，以及效应基因的辐射增敏效应。在特定目标III中，我们将把啮齿动物和人类肿瘤移植到裸鼠体内，利用microPET/18F-FMISO成像技术识别和定位缺氧亚体积。在这些图像的引导下，腺病毒载体将被输送到肿瘤的缺氧区，基于TK介导的124I-FIAU捕获的microPET成像将被用于验证载体被优先输送到缺氧细胞。然后将评估肿瘤对辐射的反应。在初步研究中，我们已经证明了反义Ku70或显性负性Ku70片段在体外和体内都能显著增敏低氧肿瘤细胞，并且它们可以通过腺病毒载体成功地输送到肿瘤中。对于肿瘤乏氧靶向，我们用18F-FMISO进行了microPET成像，并开发了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，利用124I-FIAU/microPET可以在体内很容易地检测到标记tk基因的缺氧诱导。从拟议的临床前研究中获得的信息将在设计临床策略以改善癌症治疗结果时起到指导作用。

DESCRIPTION (provided by applicant): Tumor hypoxia is often found in many human cancers; their presence has been implicated in radio- and chemo-resistance, 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 or inducibly 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, replication defective adenovirus containing the hypoxia marker gene and the effector gene will be constructed. 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 will then be studied in vitro and in vivo. 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.