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

描述(申请人提供)：恶性胶质瘤是最具破坏性的肿瘤之一，即使是最好的治疗方法，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)形成了标准的做法，但由于肿瘤边缘的不确定性，常常失败。此外，由于胶质母细胞瘤(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取得预期成功的能力。此外，还缺乏在理想位置成功传递所需基因的载体。近年来，不同的干细胞被成功地用来携带一种基因来靶向肿瘤部位。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或肿瘤细胞。我们已经报道了内皮祖细胞(EPC)，一类造血干细胞，迁移并整合到植入性胶质瘤的血管生成中的能力。在静脉或局部给药时，内皮祖细胞在肿瘤周围活跃迁移，并与血管生成结合。由于内皮祖细胞的磁性标记，可以检测到迁移和整合。这些内皮祖细胞既可以作为载体，也可以作为基因导入肿瘤的载体。此外，通过磁性标记(使用FDA批准的亚铁氧化物和鱼精蛋白硫酸盐)，这些细胞可以用作磁共振成像的细胞探针，以跟踪给药后细胞的运动。当需要系统性基因载体时，可获得性、易于收获和成熟的基因操作技术使内皮祖细胞成为有吸引力的细胞载体。在这项拟议的研究中，我们旨在研究内皮祖细胞将基因携带到肿瘤部位的能力，并使用这些转基因细胞作为细胞探针，通过磁共振成像(MRI)跟踪肿瘤中的迁移和掺入。本研究的目的是通过建立裸鼠脑胶质瘤模型，将磁性标记或未标记的转基因(携带人钠碘转运体，hNIS)内皮祖细胞进行全身或局部给药，并通过核磁共振成像和核医学成像技术(SPECT)检测这些细胞在肿瘤新生血管中的迁移、归巢和整合情况。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)，这将开辟一个具有临床应用价值的新的研究领域，其中内皮祖细胞可以用作基因载体或输送载体。这项提议的长期目标是通过收集患者外周血中的干细胞将这些发现扩展到临床应用，并将它们作为全身和局部给药的基因输送载体，这些载体也可以用作磁共振成像探针。这项提议的项目的结果将在两个方面促进诊断和治疗方法的进步。磁性标记细胞将通过靶向血管生成的活性部位，帮助体内MRI检测肿瘤，这可能有助于临床医生制定抗血管生成的治疗策略。如果我们能够有效地将这些转基因细胞导入并跟踪这些转基因细胞在胶质瘤部位的归巢，这将开辟一条利用内皮祖细胞将基因(针对不同因素)输送到肿瘤部位的新途径。将磁性标记的细胞(例如，总注射细胞的10%)与转基因细胞混合，MRI也可以用来确认转基因细胞在感兴趣的部位的迁移和归巢。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被输送到感兴趣的部位。

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.