Improved Gene Delivery and In Vivo Imaging

目前，由于缺乏一种非侵入性和临床有用的手段来监测载体的持久性、体内的生物分布和转基因表达，基因治疗受到了限制。当前项目的目标是测试设计的策略，以改善在腺病毒介导的基因治疗的局部给药后基因表达的空间分布，从而使整个器官在尽可能少的病毒的情况下获得足够的临床疗效(以限制毒性)。一种基于钠碘转运体(NIS)的新报告基因将表征其监控治疗性基因表达的能力，并与正在开发的其他催化活性测量方法进行对比。该计划有三个具体目标 建议进行的研究。1.比较NIS报告基因和CD/HSV-1 TK治疗基因表达的分布。如果能够证明NIS活性是治疗性基因表达的替代标记，NIS成像可以消除目前用于评估转基因表达的活检，并允许对转基因表达进行动态和全身监测。2.评估新的载体配方和注射条件，旨在优化腺病毒在前列腺内的放置，以获得最大的器官覆盖率。这项工作将为项目3.3中描述的临床试验提供科学基础。将监测转基因表达的前药标记方法与NIS方法进行比较。这一目标 测试旁观者效应可以测量并超过基因表达区域的假设。旁观者效应将使用结合的放射性标记前药([~3H]-更昔洛韦-MP，[14C]-5-FdUMP)的放射自显影来测量，并与转导NIS后的碘定位进行比较。最后，测量将扩展到使用5-FU的[19F]-MR化学位移成像和使用NIS活性的SPECT成像的123(I)摄取的非侵入性技术。建议的研究需要一个大型动物模型。我们计划使用大型杂交狗的前列腺。所描述的研究代表了“原则证据”，并将产生重要的新知识，使基因治疗和医学界受益。

Gene therapy is currently limited by the lack of a non-invasive and clinically useful means to monitor vector persistence and biodistribution and transgene expression in vivo. The goal of the current project is to test strategies devised to improve the spatial distribution of gene expression following the local administration of adenovirus-mediated gene therapy so that the entire organ receives a viral dose sufficient for clinical efficacy with as little virus as possible (to limit toxicity). A new reporter gene based on the sodium iodide symporter (NIS) will be characterized with regard to its ability to monitor therapeutic gene expression and contrasted with other measurements of catalytic activity under development. There are three specific aims of the proposed studies. 1. Compare the distribution of NIS reporter gene expression to that of CD/HSV-1 TK therapeutic gene expression. If it can be shown that NIS activity is a surrogate marker of therapeutic gene expression, NIS imaging could eliminate the biopsy currently used to assess transgene expression and allow for dynamic and whole body monitoring of transgene expression. 2. Evaluate new vector formulations and injections conditions designed to optimize placement of the adenovirus within the prostate to obtain maximal organ coverage. This work will provide the scientific basis for a clinical trial described in Project 3. 3. Contrast the prodrug-labeled approach of monitoring transgene expression to the NIS approach. This aim tests the hypothesis that the bystander effect can be measured and exceeds the area of gene expression. Bystander effect will be measured using autoradiography of bound radiolabeled prodrugs ([3H]-ganciclovir-MP, [14C]-5-FdUMP) and compared to iodide localization following transduction of NIS. Finally the measurements will be extended to non-invasive techniques using [19F]-MR chemical shift imaging of 5-FU and 123(I)uptake using SPECT imaging of NIS activity. A large animal model is needed for the proposed studies. We plan to use the prostates of large, mongrel dogs. The studies described represent "proof of principle" and will generate significant new knowledge that will benefit the gene therapy and medical communities.