Efficient Targeting of Therapeutic Cells in Stroke and EAE

描述(申请人提供)：将干细胞用于治疗目的的前景是近年来科学和医学中最有前途的领域之一。在这一领域取得了重大进展，包括更好地了解干细胞生物学，确定新的干细胞来源，以及在各种疾病中取得令人鼓舞的治疗结果。神经性疾病仍然是医学上最大的挑战之一，几乎没有有效的治疗方法。使用干细胞的临床前研究令人鼓舞，并导致了一些针对帕金森氏症、多发性硬化症和中风的临床试验的启动。不幸的是，这些试验都没有显示出令人满意的治疗结果。这一失败的原因有很多种，其中一个主要原因是干细胞的生物分布和靶向效率低下。动脉内给药可能会绕过这一限制，已经有一些人尝试使用这种方法直接靶向大脑病变。限制这一方法的主要障碍是缺乏能够有效地将细胞与内皮结合的技术，以及由于细胞过度结合而导致微血栓的风险。我们的初步结果表明，过表达对接受体VLA-4大大提高了人神经胶质前体细胞对炎症区域的靶向效率。使用微流体体外黏附试验，细胞与激活的脑内皮细胞的结合比非VLA-4对照组显著增加(分别为71.511.7vs.36.43.3cell/FOV，p=0.045)。在脂多糖诱导的大鼠全身炎性脑模型中，动脉注射后，含有VLA-4转基因的细胞在体内表现出更强的归巢能力。实时、定量的全脑磁共振磁标记细胞成像显示，VLA-4+细胞仅停靠在同侧颈动脉的血管床内，表明存在首次通过的粘连机制。逐个像素的分析显示，注射Vla-4+细胞后，内毒素处理组的低信号像素数为3,979？705，而对照组为868？317(p=0.014)。有了这些令人鼓舞的结果，这项建议的总体目标是诱导多能干细胞来源的神经胶质前体细胞过度表达黏附分子VLA-4和LFA-1，以及趋化因子受体CXCR-4和CCR2。结合颈动脉内注射，我们假设在炎症性脑损伤内将发生高效和特异的植入。细胞与内皮细胞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上进行体内实验。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、脑血流和氧合情况。在我们的研究成功完成后，这种新的靶向方法可以显著提高基于细胞的治疗的疗效，并应用于许多医学领域。 与公共卫生相关：有效地将治疗细胞定位于脑损伤区域仍然是一项挑战。我们假设，细胞基因工程和对接受体(如VLA-4)的过度表达与动脉内方法相结合，将极大地提高靶向效率，并将为有效的细胞靶向创造一种新的范式。磁共振成像将被用来监测体内细胞结合以及脑血流和氧合，以确定这种新方法的非侵入性的有效性和安全性。

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.