Efficient Targeting of Therapeutic Cells in Stroke and EAE

中风和 EAE 治疗细胞的有效靶向

• 批准号: 8370236
• 负责人: Piotr Walczak
• 金额: $ 35.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370236
• 关键词: Adhesions; Adverse effects; Animals; Area; Binding; Biodistribution; Biological Assay; Blood - brain barrier anatomy; Blood flow; Brain; Brain Injuries; Bypass; CCR; CXCR4 gene; Carotid Arteries; Cell Adhesion; Cell Adhesion Molecules; Cell Respiration; Cell Therapy; Cell model; Cells; Cerebrovascular Circulation; Cerebrum; Clinical; Clinical Trials; Devices; Diffusion Magnetic Resonance Imaging; Disadvantaged; Disease; Docking; Endothelial Cells; Endothelium; Engraftment; Ensure; Exhibits; Experimental Autoimmune Encephalomyelitis; Extravasation; Failure; Genetic Engineering; Histology; Homing; Hot Spot; Human; Image; In Vitro; Inflammation; Inflammatory; Injection of therapeutic agent; Integrin alpha4beta1; Intra-Arterial Injections; Intracarotid; Intravenous; Ipsilateral; Ischemia; Label; Lesion; Ligands; Lipopolysaccharides; Magnetic Resonance Imaging; Mediating; Medicine; Metabolism; Microfluidics; Modeling; Molecular; Monitor; Multiple Sclerosis; Neurons; Outcome; Parkinson Disease; Pathology; Patients; Pilot Projects; Rattus; Relative (related person); Research Personnel; Risk; Route; Safety; Science; Source; Spin Labels; Stem cells; Stroke; Surface; Techniques; Testing; Therapeutic; Time; Transgenes; Vascular Cell Adhesion Molecule-1; Weight; base; brain tissue; cellular engineering; cellular targeting; chemokine receptor; clinical application; cytokine; design; effective therapy; improved; in vitro testing; in vivo; induced pluripotent stem cell; injection/infusion; nervous system disorder; novel strategies; overexpression; preclinical study; precursor cell; prevent; progenitor; receptor; research study; response; stem cell biology; targeted delivery; therapeutic target; time use; tissue oxygenation; vascular bed

DESCRIPTION (provided by applicant): The prospect of using stem cells for therapeutic purposes has been one of the most promising fields of science and medicine in recent years. Progress in this area has been substantial, including a better understanding of stem cell biology, the identification of new sources of stem cells, and encouraging therapeutic results in a variety of diseases. Neurological disorders remain one of the greatest challenges in medicine, with little or no effective treatments available. Preclinical studies using stem cells have been encouraging and have led to the initiation of a few clinical trials for Parkinson's disease, multiple sclerosis and stroke. Unfortunately, none of these trials demonstrated a satisfactory therapeutic outcome. There are many suggested reasons for that failure, with one of the primary reasons being an inefficient biodistribution and targeting of stem cells. Intraarterial delivery could potentially bypass this limitation, and a few attempts have been made to use this approach for direct targeting of brain lesions. The major obstacle limiting this approach is the lack of techniques tha enable efficient binding of cells to endothelium, as well as the risk of microembolism as a result of excessive cell binding. Our preliminary results indicate that overexpression of the docking receptor VLA-4 greatly improves the targeting efficiency of human, glial progenitors towards areas of inflammation. Using a microfluidics in vitro adhesion assay, cell binding to activated brain endothelial cells greatly increased as compared to non-VLA-4 controls (71.5¿11.7 vs. 36.4¿3.3 cells/FOV, respectively, p=0.045). In a LPS-induced rat global inflammatory brain model, cells containing the VLA-4 transgene demonstrated much enhanced homing in vivo following intraarterial injection. Real-time, quantitative serial whole brain MR imaging of magnetically labeled cells revealed that, VLA-4+ cells docked exclusively within the vascular bed of the ipsilateral carotid artery indicating a first pass adhesion mechanism. Pixel-by-pixel analysis revealed that injection of VLA-4+ cells in LPS-treated animals resulted in 3,979¿705 hypointense pixels as compared to 868¿317 in VLA-4- LPS-treated controls (p=0.014). With these encouraging results, the overall aim of this proposal is to induce pluripotent stem cells-derived glial precursors to overexpress the adhesion molecules VLA-4 and LFA-1 and the chemokine receptors CXCR-4 and CCR2. Combined with intracarotid delivery, we hypothesize that a highly efficient and specific engraftment within inflammatory brain lesions will occur. The ability of cells to bind to endothelium and extravasate into the brain parenchyma will be initially tested in vitro using a microfluidics model blood brain barrier. Experiments will then be performed in vivo in rat models of stroke and autoimmune encephalomyelitis. To ensure the safety of this approach, we will monitor cell delivery, cerebral blood flow, and oxygenation in real-time using MRI. Upon successful completion of our studies, this new targeting approach could significantly improve the efficacy of cell-based therapy with applications in many areas of medicine. PUBLIC HEALTH RELEVANCE: Efficient targeting of therapeutic cells to areas of brain damage continues to be a challenge. We hypothesized that genetic engineering of cells and overexpression of docking receptors such as VLA-4 combined with intraarterial approach would greatly improve targeting efficiency and would create a new paradigm for efficient cellular targeting. Magnetic resonance imaging will be used to monitor both in vivo cell binding and cerebral blood flow and oxygenation, in order to determine the efficiency and safety of this new approach non- invasively.

描述（由申请人提供）：利用干细胞进行治疗的前景是近年来最有前途的科学和医学领域之一。该领域取得了巨大进展，包括更好地了解干细胞生物学、鉴定干细胞的新来源以及在多种疾病中取得令人鼓舞的治疗结果。神经系统疾病仍然是医学上最大的挑战之一，有效的治疗方法很少或根本没有。使用干细胞的临床前研究令人鼓舞，并导致启动了一些针对帕金森病、多发性硬化症和中风的临床试验。不幸的是，这些试验均未显示出令人满意的治疗结果。造成这种失败的原因有很多，其中主要原因之一是干细胞的生物分布和靶向效率低下。动脉内递送可能会绕过这一限制，并且已经进行了一些尝试使用这种方法直接靶向脑部病变。限制这种方法的主要障碍是缺乏能够使细胞与内皮有效结合的技术，以及由于细胞过度结合而导致微栓塞的风险。我们的初步结果表明，对接受体 VLA-4 的过度表达极大地提高了人类神经胶质祖细胞对炎症区域的靶向效率。使用微流体体外粘附测定，与非 VLA-4 对照相比，细胞与活化脑内皮细胞的结合大大增加（分别为 71.5×11.7 与 36.4×3.3 细胞/FOV，p=0.045）。在 LPS 诱导的大鼠整体炎症脑模型中，含有 VLA-4 转基因的细胞在动脉注射后表现出体内归巢能力大大增强。磁性标记细胞的实时定量连续全脑 MR 成像显示，VLA-4+ 细胞仅停靠在同侧颈动脉的血管床内，表明首过粘附机制。逐像素分析显示，在 LPS 处理的动物中注射 VLA-4+ 细胞会产生 3,979×705 个低信号像素，而在 VLA-4-LPS 处理的对照组中则产生 868×317 个低信号像素（p=0.014）。有了这些令人鼓舞的结果，该提案的总体目标是诱导多能干细胞衍生的神经胶质前体过度表达粘附分子 VLA-4 和 LFA-1 以及趋化因子受体 CXCR-4 和 CCR2。与颈动脉内递送相结合，我们假设将在炎症性脑损伤内发生高效且特异性的植入。细胞与内皮结合并渗入脑实质的能力最初将是 使用微流体模型血脑屏障进行体外测试。然后将在中风和自身免疫性脑脊髓炎的大鼠模型中进行体内实验。为了确保这种方法的安全性，我们将使用 MRI 实时监测细胞输送、脑血流量和氧合。成功完成我们的研究后，这种新的靶向方法可以显着提高细胞疗法的功效，并在许多医学领域得到应用。 公共卫生相关性：有效地将治疗细胞靶向脑损伤区域仍然是一个挑战。我们假设细胞基因工程和对接受体（例如 VLA-4）的过度表达与动脉内方法相结合将大大提高靶向效率，并为有效的细胞靶向创建新的范例。磁共振成像将用于监测体内细胞结合以及脑血流和氧合，以便无创地确定这种新方法的效率和安全性。