MAGIC - Magnetic Resonance Assessment of Gene Imaging Constructs

描述（由申请人提供）：我们将开发一种基于酶激活报告分子的质子MRI检测肿瘤中转基因活性的新方法。具体而言，我们将合成，评估，优化和应用新的代理检测β-半乳糖苷酶活性产生的lacZ基因。基因疗法对癌症的治疗有很大的希望。然而，主要问题涉及评估递送到靶组织、生物分布的均匀性（相对于异质性）和确定基因是否表达。我们提出了一种新的方法来评估基因表达MRI。这项研究借鉴了光学检测报告基因的基础，特别是来自lacZ基因的β-半乳糖苷酶。最近，在Fe 3+离子存在下，3，4-环己烯七叶皂苷-β-吡喃半乳糖苷（市售为S-Gal（tm））在被β-gal活化后，已显示出产生强烈的黑色沉淀。这促使我们考虑沉淀物的顺磁性作为潜在的1H MRI造影剂。初步研究已经证明了可行性，我们现在希望将这一概念转化并发展到肿瘤细胞中进行体内检测。具体目标1将探索S-gal的效用，并进行调查以优化和验证该技术。将改变MRI参数，以在适当的空间和时间分辨率下最大化对比度。将评价药物给药，以最大化对比度并最小化毒性。具体目标2将专注于在活体小鼠中转化为肿瘤。MRI将通过与体内光学方法以及组织学和RT-PCR等传统方法进行比较来验证。具体目标3侧重于替代优化底物的合成和开发。我们相信这种方法既可以成为一种有价值的工具，用于检测p-gal活性在体内，并作为一个新的平台技术，适用于其他报告基因和内源性酶，如蛋白酶的原理证明。

DESCRIPTION (provided by applicant): We will develop a novel approach to detecting transgene activity in tumors based on proton MRI of enzyme activated reporter molecules. Specifically, we will synthesize, evaluate, optimize, and apply novel agents to detect (3-galactosidase activity generated by the lacZ gene. Gene therapy holds great promise for treatment of cancer. However, major problems involve assessing delivery to target tissue, the uniformity (versus heterogeneity) of bio distribution and determining whether the genes are expressed. We propose a novel approach for evaluating gene expression using MRI. This research draws on foundations in optical detection of reporter genes, specifically P- galactosidase from the lacZ gene. Recently, 3,4-cyclohexenoesculetin-p-galactopyranoside, marketed commercially as S-Gal(tm), has been shown to generate an intense black color as a precipitate upon activation by p-gal in the presence of Fe3+ ions. This prompted us to consider the paramagnetic properties of the precipitate as a potential 1H MRI contrast agent. Preliminary studies have demonstrated feasibility and we now wish to translate and develop the concept to tumor cells for detection in vivo. Specific aim 1 will explore the utility of S-gal, with investigations to optimize and validate the technique. MRI parameters will be varied to maximize contrast with appropriate spatial and temporal resolution. Agent administration will be evaluated to maximize contrast and minimize toxicity. Specific aim 2 will focus on translation to tumors in living mice. MRI will be validated by comparison with optical methods in vivo and traditional methods such as histology and RT-PCR. Specific aim 3 focuses on the synthesis and development of alternate optimized substrates. We believe this approach can both become a valuable tool for detecting p-gal activity in vivo and serve as a proof of principle for a novel platform technology adaptable to other reporter genes and endogenous enzymes such as proteases.