MAGIC - Magnetic Resonance Assessment of Gene Imaging Constructs

描述(申请人提供)：我们将开发一种基于酶激活报告分子的质子磁共振成像来检测肿瘤中转基因活性的新方法。具体地说，我们将合成、评估、优化和应用新的试剂来检测由LacZ基因产生的3-半乳糖苷酶活性。基因疗法在治疗癌症方面大有可为。然而，主要的问题涉及评估对目标组织的传递，生物分布的一致性(与异质性)，以及确定基因是否表达。我们提出了一种利用核磁共振技术评估基因表达的新方法。这项研究的基础是光学检测报告基因，特别是来自LacZ基因的P-半乳糖苷酶。最近，商业上被称为S-半乳糖苷的3，4-环己烯七叶皂苷被证明在Fe3+存在下被p-半乳糖活化后产生强烈的黑色作为沉淀物。这促使我们考虑将沉淀物的顺磁性作为潜在的1H磁共振造影剂。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为肿瘤细胞，并在体内进行检测。具体目标1将探索S-GAL的用途，并进行调查以优化和验证该技术。MRI参数将变化，以最大限度地提高对比度，并具有适当的空间和时间分辨率。将对给药进行评估，以最大限度地提高对比度并将毒性降至最低。特定目标2将专注于将其转化为活体小鼠的肿瘤。MRI将通过与体内光学方法以及组织学和RT-PCR等传统方法的比较来验证。具体目标3侧重于交替优化底物的合成和开发。我们相信，这种方法既可以成为检测体内p-Gal活性的有价值的工具，也可以作为一种新的平台技术的原理证明，该平台技术适用于其他报告基因和内源性酶，如蛋白酶。

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.