Modified neural stem cells for slycosylation-independent treatment of MPS IIIB

 描述(申请者提供)：世界梦想用干细胞治愈每一种疾病。我们都知道这不太可能。但使用干细胞再生组织的主要障碍是 治疗疾病是指与受者细胞在基因上不相同的干细胞容易发生免疫排斥反应。一种克服这一障碍的创新解决方案使用患者自己的体细胞来生成称为IPSCs(诱导多能干细胞)的干细胞。一旦生成，这些ipscs就可以在没有排斥反应的情况下应用于患者，因为ipscs与患者中的所有其他细胞在基因上是相同的。然而，对于大多数遗传性疾病来说，管理IPSCs并不是一个解决方案，因为产生的IPSCs也包含致病的基因缺陷。我们已经组建了一个拥有干细胞生物学、神经治疗学、转化科学和神经药理学专业知识的团队来解决这个问题。我们建议使用一种名为CRISPR-Cas9(簇状规则间隔短回文重复序列-CRISPR相关蛋白9)的新技术来修复受者干细胞中的缺陷基因。CRISPR-CAS9是迄今为止最有效的基因校正系统。我们的方法将使用CRISPR-Cas9来纠正从患者分离的IPSCs中的遗传缺陷。矫正后的干细胞将被移植回患者体内，以提供具有该基因功能副本的细胞。我们建议使用神经干细胞治疗遗传性儿童神经疾病粘多糖病IIIB(MPS IIIB)来测试这一概念。经过基因矫正的自体干细胞疗法非常适合于像MPS IIIB这样的疾病，这些疾病是由于缺乏分泌蛋白(在这种情况下是NAGLU[α-N-乙酰氨基葡萄糖苷酶])，因为非常少量的经过矫正的干细胞可以产生很大的治疗效果。我们还将使用糖基化非依赖的溶酶体靶向，使用一种源自IGF2的多肽来改善分泌的NAGLU在大脑中的摄取。在这项计划的R21阶段，我们将进行体外研究，以确定我们矫正的干细胞如何有效地分泌可被MPS IIIB细胞摄取的正常NAGLU和NAGLU-IGF2。我们还将进行活体研究，以确定三种不同剂量的神经干细胞在新生动物和成年动物大脑中的分布情况。在R33阶段的提案中，我们将把这些概念放在一起，用于体内研究矫正后的神经干细胞对MPS IIIB疾病的影响。我们将评估生化、神经病理和神经行为结果。其目标是为这种毁灭性的、无法治愈的疾病创造一种永久的治疗方法，并建立一个治疗其他遗传性疾病的平台，这些疾病是由缺乏功能的、分泌的蛋白质引起的。

 DESCRIPTION (provided by applicant): The world dreams of curing every disease with stem cells. We all know this is unlikely. But the main barrier to using stem cells to regenerate tissues and treat diseases is that stem cells that are not genetically identical to the recipient's cells ae prone to immune rejection. An innovative solution to overcome this barrier uses the patient's own somatic cells to generate stem cells called iPSCs (induced pluripotent stem cells). Once generated, these iPSCs can be administered to the patient without fear of rejection because iPSCs are genetically identical to all other cells in the patient. However, administering iPSCs is not a solution for most inherited diseases because the generated iPSCs also contain the disease-causing genetic defect. We have assembled a team with expertise in stem cell biology, neurotherapeutics, translational science, and neuropharmacology to solve this problem. We propose to use a new technology called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) to repair the faulty gene in the recipient's stem cells. CRISPR-Cas9 is the most efficient gene correction system to date. Our approach will use CRISPR-Cas9 to correct the genetic defect in iPSCs isolated from patients. The corrected stem cells will be transplanted back to the patient to provide cells that have a functioning copy of the gene. We propose to test this concept using neural stem cells to treat the inherited childhood neurological disease mucopolysaccharidosis type IIIB (MPS IIIB). Gene-corrected, autologous stem cell therapy is ideally suited to diseases like MPS IIIB, which are due to lack of a secreted protein (in this case, NAGLU [alpha-N-acetylglucosaminidase]), because a very small number of corrected stem cells can have a large therapeutic effect. We will also employ glycosylation-independent lysosomal targeting using a peptide derived from IGF2 that improves the uptake of secreted NAGLU into the brain. In the R21 phase of this proposal, we will perform in vitro studies to determine how effectively our corrected stem cells secrete normal NAGLU and NAGLU-IGF2 that can be taken up by MPS IIIB cells. We will also perform in vivo studies to determine how well the neural stem cells distribute throughout the brain at three different doses, in neonatal and adult animals. In the R33 phase of the proposal, we will put these concepts together for in vivo studies of the effect of corrected neural stem cell on MPS IIIB disease. We will assess biochemical, neuropathological, and neurobehavioral outcomes. The goal is to create a permanent treatment for this devastating, untreatable disease, and to build a platform for treating other genetic diseases that are caused by the lack of a functioning, secreted protein.