Magnetic nanoparticle mediated delivery of neurotherapeutic genes to multipotent neural stem cell transplant populations

成人的大脑和脊髓（合称为中枢神经系统或CNS）在受伤和疾病后修复不良。这导致患有严重脑/脊髓损伤和疾病的患者恢复有限，并对其生活质量产生深远影响。在神经系统中发现的干细胞被称为神经干细胞或NSCs，它们具有巨大的潜力，可以增加中枢神经系统损伤/疾病部位的修复，因为它们可以产生新的干细胞，取代中枢神经系统丢失的细胞类型，还可以抑制损伤反应。这些细胞可以在中枢神经系统中长距离迁移，特别是被吸引到神经系统受损的区域。因此，这些工程干细胞的一个重要用途是充当运输“载体”，将治疗分子运送到中枢神经系统的损伤/疾病区域。在神经学实验研究中，将基因传递给干细胞最常见的方法是使用修饰过的病毒作为载体。用这些病毒感染细胞可以将感兴趣的基因转移到被感染的细胞中。然而，使用病毒的实验方法可能在技术上困难，耗时和昂贵，并且可能改变神经干细胞的基本细胞特性，例如它们的分裂能力和新细胞类型的发生。这种方法也有其他缺点，包括主要的安全问题（如免疫系统的反应和致癌效应），以及难以实现人类和兽医临床治疗所需的大规模生产。最先进的递送系统使用具有铁芯的小颗粒，称为磁性纳米颗粒（MNPs），在这方面有许多好处，可以有效地用于基因的递送。初步临床试验表明，向体内注射MNPs似乎是安全的。MNPs可以与基因连接，并被吸收到细胞中介导基因传递。尽管基于MNP的系统被认为在传递基因方面不如病毒有效，但我们已经证明，施加静态或振荡磁场（一种称为“磁效应”的方法）可以显著增加MNP的基因传递。DMC的研究小组最近证明，MNPs可以用于安全地将基因传递到用于神经细胞移植治疗的几种主要细胞类型（包括啮齿动物和犬类）。所使用的方法与安全问题无关，也不会改变干细胞的基本特性，如它们的分裂或迁移能力，或它们产生的子细胞的类型/数量。我们开发的基于MNP的基因转移方法非常安全，技术简单，快速且相对便宜，因此它们的使用将为基因传递提供一种有效和方便的方法，并可为实验室和资助机构节省大量成本。这项研究将以最近的发现为基础，开发利用MNPs将治疗分子的基因编码传递给干细胞（然后用于移植）的方法。我们将评估不同的策略，以优化被基因包裹的MNPs进入干细胞的摄取，并确定该过程是否对细胞的存活和发育有不利影响。进一步的目标是开发一种新的，技术上简单和廉价的方法，使用特殊的“电子”显微镜在高倍倍率下检查干细胞和MNPs的相互作用。我们还将研究振荡磁场增加MNPs基因传递的潜在机制。工程干细胞的修复增强潜力将在脊髓的“活体”3D切片模型中进行测试，该模型可以作为移植细胞的受伤“宿主”组织发挥良好的功能。这种方法为活体动物干细胞移植治疗的研究提供了一种替代手术移植的方法，从而大大减少了实验研究中动物的使用和痛苦。

The adult brain and spinal cord (together called the central nervous system or CNS) repair poorly after injury and disease. This leads to limited recovery for patients with severe brain/spinal cord injury and disease with profound consequences for their quality of life. Stem cells found in the nervous system called neural stem cells or NSCs have enormous potential for increasing repair at injury/disease sites in the CNS as they can give rise to new stem cells, replace lost cell types of the CNS and can also suppress injury responses. These cells can migrate long distances in the CNS and are particularly attracted into areas of damage in the nervous system. Therefore, an important use for these engineered stem cells is to function as as transport 'vehicles' to deliver therapeutic molecules to injury/disease areas in the CNS. In experimental neurology research, the most common way of delivering genes to stem cells is to use modified viruses as vehicles. Infection of cells with these viruses enables the transfer of genes of interest into the infected cells. However, experimental methods using viruses can be be technically difficult, time consuming and expensive and can alter the basic cell properties of neural stem cells such as their division capability and their genesis of new cell types. This method also has other drawbacks including major safety issues (such as reactions from the immune system and cancer causing effects) and difficulties in achieving the large scale production that is required for human and veterinary clinical therapies. State-of-the-art delivery systems using small particles with an iron core called magnetic nanoparticles (MNPs) have many benefits in this regard and can be used effectively for delivery of genes. Preliminary clinical trials have shown that injecting MNPs into the body appears to be safe. MNPs can be linked with genes and taken up into cells to mediate gene delivery. Although MNP based systems are thought to be less effective than viruses for delivering genes, we have shown that applying static or oscillating magnetic fields (a method called 'magnetofection') can dramatically increase gene delivery by MNPs. DMC's group proved recently that MNPs can be used to safely deliver genes to several major cell types (of both rodent and canine origin) that are used in neural cell transplantation therapies. The methods used were not associated with safety issues and did not alter basic stem cell properties like their division or migration capability or the types/numbers of daughter cells they give rise to. The methods we have developed for MNP based gene transfer are very safe, technically simple, quick and relatively inexpensive, therefore their use will provide an effective and convenient method for delivering genes and can result in significant cost savings to laboratories and funding agencies.This study will build on recent findings to develop methods to use MNPs to deliver genes coding for therapeutic molecules to stem cells (that will then be used for transplantation). We will assess different strategies to optimise uptake of MNPs coated with genes into stem cells and establish if this procedure has adverse effects on the survival and development of the cells. A further goal is to develop a new, technically easy and cheap method to examine the interactions of stem cells and MNPs at high magnification using a special 'electron' microscope. We will also investigate the underlying mechanisms by which oscillating magnetic fields increase gene delivery by MNPs. The repair enhancing potential of the engineered stem cells will be tested in a 'living' 3D slice model of the spinal cord, that can function well as injured 'host' tissue for transplant cells. This method provides a robust alternative to the use of surgical transplantation to study stem cell transplantation therapies in living animals, thereby significantly reducing animal usage and suffering in experimental research.