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Molecular imaging of cell-based therapeutics using an engineered human enzyme.

使用工程人类酶对基于细胞的疗法进行分子成像。

基本信息

批准号：
8161788
负责人：
ARNON LAVIE
金额：
$ 36.83万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8161788
关键词：
2&apos -fluoro-5-methylarabinosyluracil Address Adverse effects Affect Animal Model Carbon Cell membrane Cell physiology Cells Cellular Immunity Charge Clinic Clinical Complex Deoxycytidine Deoxyglucose Development Disease Engineered Gene Engineering Enzymes Feedback Fluorine Genes Goals Half-Life Health Herpesvirus 1 Human Human Engineering Image Imagery Imaging technology Immune response Isotopes Life Location Measures Medicine Methods Modification Molecular Monitor Names Nature Normal tissue morphology Nucleosides Patients Phosphorylation Phosphotransferases Physiological Positron Positron-Emission Tomography Radioisotopes Radiopharmaceuticals Regenerative Medicine Reporter Reporter Genes Research Safety Signal Transduction Source Specificity Structure Substrate Specificity System T-Lymphocyte Technology Testing Therapeutic Therapeutic Uses Thymidine Time Tissues Translations Variant base cancer cell catalyst cellular imaging clinical application design enzyme substrate gain of function glucose analog hexokinase human TK2 protein immunogenic immunogenicity improved in vivo induced pluripotent stem cell interest molecular imaging mouse model mutant novel novel strategies nucleoside analog nucleoside kinase tumor

项目摘要

DESCRIPTION (provided by applicant): Cell-based therapeutics hold promise to revolutionize medicine, being applicable to numerous disease states. Due to the complex nature of cell-based medicine, its transition from proof-of-concept to clinical use has been slow. A major factor limiting the translation of cell-based therapeutics is the difficulty in tracking the fate of the cells in vivo. Positron emission tomography (PET) is an imaging technology that allows in vivo monitoring in real time and in a non-invasive manner. The signal measured in PET comes from radioactive isotopes, such a fluorine-18, that undergoes positron emission. Thus, a molecule containing such an isotope - called a PET probe - that specifically accumulates inside the therapeutic cells would reveal their location, number, half-life, etc. An additional factor limiting the advancement of therapeutic cells to the clinic has been the concern of unforeseen side effects from this novel approach. A safety mechanism that would allow for the elimination of the cells would greatly diminish this concern. The long-term goal of this proposal is to develop a dual-purpose system that would allow, one, for the real-time tracking of therapeutic cells using PET, and two, for the elimination of the cells if needed. Specific accumulation of the PET probe inside the therapeutic cells is achieved when the probe is phosphorylated in such cells. Phosphorylation traps the molecule within the cell due since charged molecules cannot traverse cell membranes. We propose to insert the gene of a human nucleoside kinase to the therapeutic cells, with the requirement that the kinase would impart unique activity to cells that express it. Such a unique enzymatic activity of a PET reporter enzyme would allow to preferentially phosphorylate novel PET probes in the therapeutic cells. The field currently relies on non-human enzymes, such as HSV1-TK, for that unique enzymatic activity. However, as a PET reporter enzyme HSV1-TK has several drawbacks, foremost being immunogenic. To circumvent this, we will employ a modified version of human thymidine kinase 2 (TK2) as the source for the unique enzymatic activity. To differentiate this enzyme from endogenous TK2, we will perform enzyme engineering to supply us with TK2 variants with an activity profile different to that of wild type TK2, but that still do not elicit an immune response. The immediate goals of this proposal are to study the determinants of substrate specificity of TK2 by solving crystal structures of the kinase in complex with substrates (Aim 1), and to exploit this understanding for the design of TK2 variants with unique activity towards novel PET probes (Aim 2). We will test the ability to track cells expressing our TK2 variants in mouse models, and compare the TK2-system to the standard in the field using HSV1-TK. The results of this research, a non-immunogenic PET reporter enzyme with optimized activity with PET probes, will allow the in vivo real time tracking of therapeutic cells. This would have a dramatic impact on the transition of therapeutic cell approaches to the clinic. PUBLIC HEALTH RELEVANCE: The use of cells in therapy is hampered by the difficulty in tracking the location and fate of the cells given to the patient. For the in vivo real time imaging of therapeutic cells using positron emission tomography (PET), we will engineer a unique version of a human enzyme that will have the ability to activate novel PET probes. The human source of the enzyme will circumvent an immune response against the therapeutic cells. Thus, the PET probes activated by the engineered enzyme will reveal the fate of therapeutic cells, with minimal background signal from the patient's cells that do not contain this ability. This development will facilitate the transition of therapeutic cells to the clinic.

描述（由申请人提供）：基于细胞的治疗有望彻底改变医学，适用于许多疾病状态。由于细胞医学的复杂性，其从概念验证到临床应用的过渡一直很缓慢。限制基于细胞的治疗剂的转化的主要因素是难以追踪细胞在体内的命运。正电子发射断层扫描（PET）是一种成像技术，允许在体内监测在真实的时间和在一个非侵入性的方式。PET中测量的信号来自放射性同位素，例如氟-18，其经历正电子发射。因此，含有这种同位素的分子-称为PET探针-特异性地在治疗细胞内积累，将揭示它们的位置，数量，半衰期等。限制治疗细胞向临床发展的另一个因素是对这种新方法不可预见的副作用的担忧。一种允许消除细胞的安全机制将大大减少这种担忧。该提案的长期目标是开发一种双重用途的系统，该系统将允许使用PET实时跟踪治疗细胞，以及在需要时消除细胞。当探针在这些细胞中被磷酸化时，实现PET探针在治疗细胞内的特异性积累。磷酸化将分子捕获在细胞内，因为带电分子不能穿过细胞膜。我们建议将人核苷激酶的基因插入治疗细胞，要求激酶将赋予表达它的细胞独特的活性。PET报告酶的这种独特的酶活性将允许在治疗细胞中优先磷酸化新的PET探针。该领域目前依赖非人类酶（例如HSV 1-TK）来获得独特的酶活性。然而，作为PET报告酶，HSV 1-TK具有几个缺点，首先是免疫原性。为了避免这一点，我们将采用人胸苷激酶2（TK 2）的修饰版本作为独特酶活性的来源。为了将这种酶与内源性TK 2区分开来，我们将进行酶工程改造，以向我们提供具有与野生型TK 2不同的活性谱的TK 2变体，但这些变体仍然不会引发免疫应答。该提案的直接目标是通过解决与底物复合的激酶的晶体结构来研究TK 2的底物特异性的决定因素（目的1），并利用这种理解来设计对新型PET探针具有独特活性的TK 2变体（目的2）。我们将测试在小鼠模型中追踪表达我们的TK 2变体的细胞的能力，并使用HSV 1-TK将TK 2系统与现场标准进行比较。这项研究的结果，一种非免疫原性PET报告酶与PET探针的优化活性，将允许在体内真实的时间跟踪治疗细胞。这将对治疗性细胞方法向临床的过渡产生巨大影响。公共卫生关系：细胞在治疗中的使用受到难以追踪给予患者的细胞的位置和命运的阻碍。为了使用正电子发射断层扫描（PET）对治疗细胞进行体内真实的时间成像，我们将设计一种独特的人类酶，该酶将具有激活新型PET探针的能力。人类来源的酶将避免对治疗细胞的免疫反应。因此，由工程酶激活的PET探针将揭示治疗细胞的命运，而来自不包含这种能力的患者细胞的背景信号最小。这一发展将促进治疗细胞向临床的过渡。