Stem Cells as Delivery Vehicles and Imaging Probes for Glioma Gene Therapy

描述（由申请人提供）：恶性胶质瘤是最具破坏性的肿瘤之一，即使采用最好的治疗方法，诊断后也只能存活一到三年。手术和放射治疗（随后是辅助化疗）是标准的治疗方法，但由于无法确定肿瘤的边缘，常常失败。此外，由于多形性胶质母细胞瘤（GBM）的浸润性，在手术中不可能100%切除肿瘤。基因治疗有望改善GBM的预后，然而，包括缺乏有效载体在内的几个因素限制了基因治疗产生预期成功的能力。此外，将所需基因成功递送到所需位点的载体仍然缺乏。近年来，不同的干细胞已被成功地用于携带基因靶向肿瘤部位。基因转移技术与细胞移植的结合是一种优雅而有前途的方法，可以将治疗分子输送到中枢神经系统的正常细胞或肿瘤细胞中。我们已经报道了内皮祖细胞（EPCs），一类造血干细胞，能够迁移并融入植入胶质瘤的血管生成。当静脉注射或局部注射EPCs并加入血管生成时，EPCs在肿瘤周围积极迁移。由于EPCs的磁性标记，可以检测迁移和掺入。这些EPCs既可以作为基因进入肿瘤的载体，也可以作为基因进入肿瘤的递送载体。此外，通过磁标记（使用FDA批准的阿魏氧化物和硫酸鱼精蛋白）；这些细胞可以作为核磁共振成像的细胞探针来追踪给药后细胞的运动。当需要系统基因载体时，易获取、容易收获和成熟的基因操作技术使EPCs成为有吸引力的细胞载体。在本研究中，我们的目的是研究EPCs携带基因到肿瘤部位的能力，并利用这些转基因细胞作为细胞探针，通过磁共振成像（MRI）跟踪肿瘤的迁移和整合。本研究的目标将通过在裸鼠身上建立胶质瘤模型来实现，并通过全身或局部给药磁标记或未标记的转基因（携带人碘化钠同体，hNIS） EPCs，并通过MRI和核医学成像技术（SPECT）检测这些细胞的迁移，归巢和合并到肿瘤新生血管中。如果这些细胞在肿瘤部位携带和表达hNIS（这将通过SPECT检测到），它将开辟一个具有临床适用性的新研究领域，EPCs可以用作基因载体或递送载体。该提案的长期目标是从患者外周血中收集干细胞，并将其作为全身和局部管理的基因传递载体，将研究结果扩展到临床应用，也可以用作MRI的成像探针。本项目的研究结果将从两个方面促进诊断和治疗方法的发展。磁标记细胞通过靶向血管生成活性部位，有助于体内MRI检测肿瘤，这可能有助于临床医生制定抗血管生成治疗策略。如果我们能够有效地转染和追踪这些转基因细胞在胶质瘤部位的归巢，将开辟一条利用EPCs将基因（针对不同因子）递送到肿瘤部位的新途径。将磁性标记的细胞（例如总给药细胞的10%）与转基因细胞混合，MRI也可用于确认转基因细胞在感兴趣位置的迁移和归巢。此外，在动脉内输注过程中，通过施加外部磁场，磁性标记的转基因细胞可以递送到感兴趣的部位。

DESCRIPTION (provided by applicant): Malignant gliomas are among the most devastating tumors, with survival only one to three years after diagnosis even with the best of treatments. Surgery and radiation therapy (followed by adjuvant chemotherapy), which form the standard practice, very often fail because of uncertainty in delineating the margin of the tumor. Moreover due to infiltrative nature of glioblastomamulteforme (GBM), it is not possible to resect 100% of tumor mass during surgery. Gene therapy promises to improve the prognosis of GBM, however, several factors including the lack of an efficient vector limit the ability of gene therapy to produce the desired success. Moreover, the vehicles for successful delivery of desired gene at desired site is still lacking. In recent years, different stem cells have been successfully employed to carry a gene to target tumor sites. The combination of gene transfer techniques with cellular transplantation is an elegant and promising approach to the delivery of therapeutic molecules to normal or neoplastic cells in the CNS. We have reported the ability of endothelial progenitor cells (EPCs), a class of hematopoietic stem cells, to migrate and incorporate into the angiogenesis of implanted glioma. EPCs have migrated actively at the periphery of the tumors when administered intravenously or locally and incorporated into angiogenesis. Detection of migration and incorporation was possible due to magnetic labeling of EPCs. These EPCs can be used as carrier as well as delivery vehicles for gene into tumors. Moreover, by magnetic labeling (using FDA approved ferumoxides and protamine sulfate); these cells can be used as cellular probes for MRI to track the movement of the cells after administration. Accessibility, easy harvesting and established techniques for genetic manipulation renders EPCs as attractive cellular vehicles when systemic gene carrier is required. In this proposed research, we aim to investigate the ability of EPCs to carry a gene to the tumor sites and use these transgenic cells as cellular probes to track the migration and incorporation in the tumors by magnetic resonance imaging (MRI). The goals of this research will be achieved by making glioma model in nude rats and magnetically labeled or unlabeled transgenic (carrying human sodium iodide symporter, hNIS) EPCs will be administered either systemically or locally, and the migration, homing and incorporation of these cells into tumor neovasculatures will be detected by MRI and nuclear medicine imaging technique (SPECT). If these cells carry and express hNIS at tumor sites (which will be detected by SPECT), it will open a new area of investigation with clinical applicability, where EPCs can be used as gene carrier or delivery vehicles. The long-term goal of this proposal is to extend the findings into clinical use by collecting stem cells from patients' peripheral blood and manipulate them as gene delivery vehicles for both systemic and local administrations, which can also be used as imaging probes for MRI.The results of this proposed project will advance the methods of diagnosis and treatment in two ways. Magnetically labeled cells will help detect the tumors using in vivo MRI by targeting active site of angiogenesis and this may help clinician to plan anti-angiogenic treatment strategy. If we are able to efficiently transfect and track the homing of these transgenic cells at the site of glioma, it will open a new way of delivering gene (for different factors) to the site of tumors using EPCs. Mixing magnetically labeled cells (for example 10% of total administered cells) with transgenic cells, MRI can also be used to confirm the migration and homing of transgenic cells at the sites of interest. Moreover, magnetically labeled transgenic cells can be delivered to a site of interest by applying external magnetic field during intra-arterial infusion.