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。

• 批准号: 8497686
• 负责人: ARNON LAVIE
• 金额: $ 33.56万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 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/8497686
• 关键词: 2' -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

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.

描述（由申请人提供）：基于细胞的疗法有望彻底改变医学，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。限制细胞疗法翻译的一个主要因素是难以在体内追踪细胞的命运。正电子发射断层扫描（PET）是一种成像技术，可以实时、无创地监测体内情况。PET测量的信号来自放射性同位素，如氟-18，它经历了正电子发射。因此，含有这种同位素的分子——被称为PET探针——专门聚集在治疗细胞内，将揭示它们的位置、数量、半衰期等。限制治疗细胞应用于临床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。这项提议的长期目标是开发一种双重用途的系统，一是使用PET实时跟踪治疗细胞，二是在需要时消除细胞。当探针在治疗细胞中磷酸化时，在治疗细胞内实现PET探针的特异性积累。磷酸化将分子困在细胞内，因为带电分子不能穿过细胞膜。我们建议将人类核苷激酶基因插入治疗细胞，并要求该激酶能够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。该领域目前依赖于非人类酶，如HSV1-TK，以获得这种独特的酶活性。然而，作为一种PET报告酶，HSV1-TK有几个缺点，首先是免疫原性。为了避免这种情况，我们将采用改良版的人胸苷激酶2 （TK2）作为独特酶活性的来源。为了将这种酶与内源性TK2区分开来，我们将进行酶工程，为我们提供与野生型TK2活性谱不同的TK2变体，但仍然不会引起免疫反应。本提案的直接目标是通过解决与底物复合物中激酶的晶体结构来研究TK2底物特异性的决定因素（目标1），并利用这一理解来设计具有独特活性的TK2变体，用于新型PET探针（目标2）。我们将在小鼠模型中测试追踪表达我们的TK2变体的细胞的能力，并使用HSV1-TK将TK2系统与该领域的标准进行比较。这项研究的结果是一种非免疫原性PET报告酶，具有优化的PET探针活性，将允许对治疗细胞进行体内实时跟踪。这将对治疗细胞方法向临床的转变产生巨大影响。