Nanoparticle modified Human Fat Derived Mesenchymal Stem Cells for Brain Cancer

 描述(申请人提供)：胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤，占所有原发脑肿瘤的20%。尽管目前最好的治疗方法包括手术和化疗，但GBM的中位存活率仅为14.6个月。这种发病率和死亡率的一个重要原因是基底膜侵犯正常脑实质的能力，使局部治疗无效。越来越多的证据表明，脑肿瘤启动细胞(BTIC)是导致这种疾病治疗耐药性的一小部分细胞。为了使治疗有效，需要针对这些入侵细胞。一种有希望的方法涉及使用间充质干细胞(MSCs)，已发现它们优先迁移到癌细胞并在其上定居。此外，MSCs可以被改造成合成和释放抗肿瘤蛋白，如影响BTICs的骨形态发生蛋白4(BMP4)。MSCs可从骨髓(BM-MSC)和脂肪组织(AMSCs)获得。BM-MSCs获取困难，体外增殖能力有限，且随着供者年龄的增长，其效果下降。与BM-MSCs不同，AMSCs的来源更丰富，更容易从脂肪组织中获得，表达与细胞迁移有关的更高水平的表面标志，并已被证明具有抵抗肿瘤转化的作用。因此，资产证券化可能是一个更好的选择。虽然病毒基因传递方法通常用于修饰AMSCs，但它与插入突变和免疫原性有关，因此，在人类患者中使用的翻译能力可能有限。可生物降解的聚合物纳米颗粒能够有效地将非病毒基因传递到多种细胞类型，包括人类AMSCs(HAMSCs)，同时避免病毒的典型问题。在这项资助中，我们提出了一种新的技术来结合新鲜提取的脂肪组织(F.A.T.)以及纳米颗粒，以非病毒方式改造F.A.T内包含的原代hAMSCs，无需事先培养即可分泌抗癌蛋白，同时保持细胞向肿瘤细胞迁移的能力。我们的总体假设是，从F.A.T.获得的纳米颗粒修饰的hAMSCs在临床相关的活体人类GBM模型中保留了其肿瘤抑制特性。为了验证这一假设，我们将追求以下具体目标：(1)有效地将外源基因输送到新鲜提取的脂肪组织(F.A.T.)通过冷冻干燥的可生物降解纳米颗粒从患者那里获得。(2)检测纳米粒修饰的BMP4基因修饰的hAMSCs在体外是否具有抗胶质瘤作用。(3)以小鼠为动物模型，研究纳米粒修饰的BMP4基因修饰的hAMSC联合靶向放射治疗人GBM的安全性和有效性。目标1涉及研究和优化一种独特的技术，将纳米颗粒与我们患者的F.A.T.相结合。对于AIMS 2和AIMS 3，使用已经在商业hAMSCs中测试的纳米颗粒，我们现在将研究在添加纳米颗粒之前已经分离和培养的原代HAMSCs的修饰。我们的团队已经开发了在体外和体内使用的技术，并进一步确定了这些技术的特征。将利用微流体和纳米生物技术领域的新进展进行体外研究。活体研究将采用哺乳动物异种移植模型，植入人类GSC来源的GBM，这是最好的概括人类GBM。此外，在活体研究中，动物受试者将使用小动物辐射研究平台(SARRP)进行放射治疗，从而在小鼠的规模上重建传统的人类适形放射治疗。这项研究的结果将确定纳米颗粒修饰的hAMSCs是否可以提供一种安全有效的治疗方法，不仅适用于GBM患者，而且适用于多种类型的原发和转移性脑癌。为了将来的临床应用，纳米颗粒可以用于培养几天后从患者脂肪中获得的hAMSCs，或者作为治疗给予静脉注射，或者用于F.A.T.，并在手术期间重新给予所产生的hAMSCs。这可能会导致临床试验，为治疗脑癌患者提供一种革命性的新方法，并促进个性化药物的使用。

 DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor in adults, and accounts for 20% of all primary brain tumors. GBM has a median survival rate of only 14.6 months despite current best treatment practices including surgery and chemoradiation. A significant reason for this morbidity and mortality is the ability of GBM to invade normal brain parenchyma, making localized treatment ineffective. There is increasing evidence of a small subset of cells, brain tumor initiating cells (BTICs) that are responsible for the disease's treatment resistance. In order for treatment to be effective, these invading cells need to be targeted. One promising approach involves the use of mesenchymal stem cells (MSCs), which have been found to migrate preferentially to and home in on cancer cells. Moreover, MSCs can be engineered to synthesize and release anti-tumor proteins, like bone morphogenic protein 4 (BMP4), which affects BTICs. MSCs can be obtained from bone marrow (BM- MSC) and adipose tissue (AMSCs). BM-MSCs are difficult to obtain, have limited ex vivo proliferation capacity, and decrease in effectiveness with donor age. Unlike BM-MSCs, AMSCs are more abundant in supply, easier to obtain from fat tissue, express higher levels of surface markers implicated in cell migration, and have been shown to resist oncogenic transformation. AMSCs may therefore be a better option. The viral gene delivery method, though commonly used to modify AMSCs, is associated with insertional mutagenesis and immunogenicity, and, therefore, has potentially limited translational ability for use in human patients. Biodegradable, polymeric nanoparticles enable effective non-viral gene delivery to multiple cell types, including human AMSCs (hAMSCs), while avoiding the problems typical of viruses. In this grant, we propose a novel technology to combine Freshly-extracted Adipose Tissue (F.A.T.) and nanoparticles to non-virally engineer the primary hAMSCs contained within F.A.T without prior culture to secrete anti-cancer proteins while maintaining the cells' ability to migrate toward tumo cells. Our overall hypothesis is that nanoparticle-modified hAMSCs obtained from F.A.T. retain their tumor suppressive characteristics in a clinically relevant in vivo human GBM model. To test this hypothesis, we will pursue the following specific aims: (1) To effectively deliver exogenous genes of interest to Freshly-extracted Adipose Tissue (F.A.T.) from patients via lyophilized biodegradable nanoparticles. (2) To determine if nanoparticle-modified BMP4-secreting hAMSCs retain an anti-glioma effect in vitro. (3) To determine the safety and efficacy of nanoparticle-modified BMP4-secreting hAMSC treatment in combination with targeted radiation therapy on human GBM in an in vivo murine model. Aim 1 involves investigation and optimization of a unique technology of combining nanoparticles with F.A.T. from our patients. For aims 2 and 3, using nanoparticles already tested in commercial hAMSCs, we will now investigate the modification of primary hAMSCs that have been isolated and cultured prior to adding the nanoparticles. The techniques to be used in vitro and in vivo in this proposal have been developed and further characterized by our teams. In vitro studies will be conducted using new advancements in the fields of microfluidics and nanobiotechnology. In vivo studies will employ a mammalian xenograft model that engrafts human GSC-derived GBM, which bests recapitulates human GBM. Further, in the in vivo studies, animal subjects will be treated with radiation using Small Animal Radiation Research Platform (SARRP), thus recreating traditional conformal beam radiotherapy for humans on the scale of a mouse. The results of this study will determine whether nanoparticle-modified hAMSCs can provide a treatment that is safe and effective for not only patients with GBM, but many types of primary and metastatic brain cancers. For future clinical application, the nanoparticles could be administered either to hAMSCs obtained from patient fat after culturing for a few days or then given IV as a treatment or to F.A.T. with the resulting engineered hAMSCs re- administered during surgery. This may lead to clinical trials, with a revolutionary new way of treating patients with brain cancer and facilitating personalized medicine.