Bone marrow derived stem cells for therapy of eye disease

描述（由申请人提供）：骨髓来源的造血干细胞（hsc）已被证明可以靶向视网膜新生血管的部位。这些造血干细胞在视网膜变性小鼠模型中具有血管和神经营养修复作用，在视网膜退行性疾病的治疗中具有巨大的临床应用潜力。然而，这些细胞发挥其营养活性的机制尚不清楚。因此，为了表征这些细胞的活性，需要对其提取/鉴定进行重大改进。本研究将开发一种基于细胞的小分子阳性选择试验。我们将通过三个特定目标来实现这一目标：开发一种高通量筛选试验，以测试优先结合cd44hi表达细胞的小分子。使用基于前竞争细胞的elisa样分析，我们将筛选非肽文库，以有效结合cd44hi表达细胞的小分子。比色法将用于检测CD44抗体抑制骨髓源性细胞结合的小分子能力。专项目标2：通过化学修饰优化小分子的结合效率。利用用于文库合成的液相法，二级文库将基于高通量试验中鉴定的有效导联的系统修改来制备，并优化为结合亲和力抑制抗体与表达cd44hi的细胞结合。特异性目的3：利用鉴定的小分子积极筛选表达cd44hi的细胞，并评估这些细胞在视网膜神经元/血管变性动物模型中的营养/生物活性。通过将整个骨髓与鉴定的小分子孵育，然后对与小分子结合的细胞进行分类，我们可以分析这些细胞，看看它们是否表达cd44hi和/或具有营养活性。这将通过将该细胞亚群注射到视网膜变性小鼠体内，观察血管和神经元变性的恢复，如前所述。在视网膜疾病中，如糖尿病视网膜病变和年龄相关性黄斑变性，这是成年人视力丧失的主要原因，会发生血管异常生长。这项研究将有助于开发治疗由血管畸形引起的视网膜疾病的治疗药物，这些疾病可能导致失明。

DESCRIPTION (provided by applicant): Bone marrow-derived hematopoietic stem cells (HSCs) have been shown to target sites of neovascularization in the retina. These HSCs have vasculo- and neurotrophic rescue in mouse models of retinal degeneration, and have enormous potential for clinical use as therapeutics for treating retinal degenerative diseases. However, the mechanism by which these cells exert their trophic activity remains unknown. Therefore, in an effort to characterize the activity of these cells, significant improvements need to be made in their extraction/identification. This research will develop a cell-based positive selection assay utilizing small molecules. We will accomplish this through three specific aims Specific Aim 1: Develop a high through-put screening assay to test for small molecules that preferentially bind to CD44HI-expressing cells. Using a pre-competitive cell based ELISA-like assay, we will screen a non-peptidic library for small molecules that bind efficiently to CD44HI-expressing cells. A colorimetric assay will be used to assay the small molecules ability to inhibit binding to bone marrow derived cells by an antibody to CD44. Specific Aim 2: Optimize the binding efficiency of the small molecule through chemical modifications. Enlisting the solution phase methodology used for the library synthesis, secondary libraries will be prepared based on systematic modifications of the validated leads identified in the high through-put assay and optimized for binding affinity inhibiting antibody binding with CD44Hi-expressing cells. Specific Aim 3: Utilize identified small molecules to positively sort out CD44Hi-expressing cells and assess trophic/ biological activity of these cells in animal models of retinal neuronal/vascular degeneration. By incubating whole bone marrow with the identified small molecule(s) and then sorting for cells bound to the small molecule, we can analyze these cells to see if they are CD44Hi-expressing and/or possess trophic activity. This will be accomplished by injecting this subpopulation of cells intravitreally into mice with retinal degeneration and observing rescue of vascular and neuronal degeneration as previously described. In retinal diseases such as diabetic retinopathy, and age-related macular degeneration, the leading causes of vision loss in adults, abnormal blood vessel growth occurs. This research will contribute to the development of therapeutic drugs aimed at treating retinal diseases caused by blood vessel malformation that can result in blindness.