Bone marrow derived stem cells for therapy of eye disease

描述(申请人提供)：骨髓来源的造血干细胞(HSCs)已被证明是视网膜新生血管的靶点。这些干细胞在视网膜变性的小鼠模型中具有血管和神经营养挽救作用，具有巨大的临床应用潜力，可用于治疗视网膜退行性疾病。然而，这些细胞发挥营养活性的机制仍不清楚。因此，在努力确定这些细胞的活性时，需要在它们的提取/鉴定方面做出重大改进。这项研究将开发一种基于细胞的利用小分子的正选择试验。我们将通过三个特定的目标来实现这一点特定的目标1：建立一种高通量的筛选方法来测试优先与CD44HI表达的细胞结合的小分子。使用基于竞争前细胞的类ELISA法，我们将为有效结合CD44HI表达细胞的小分子筛选非肽文库。比色法将被用来检测小分子抑制CD44抗体与骨髓来源细胞结合的能力。具体目标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溶液相方法学，将基于对高通量分析中确定的有效线索的系统修改来制备二级文库，并针对结合亲和力抑制抗体与CD44Hi表达细胞的结合进行优化。具体目标3：在视网膜神经元/血管变性动物模型中，利用已鉴定的小分子阳性分选CD44Hi表达的细胞，并评估这些细胞的营养/生物活性。通过将整个骨髓与已鉴定的小分子(S)孵育，然后筛选与小分子结合的细胞，我们可以分析这些细胞，以确定它们是否表达CD44Hi和/或具有营养活性。这将通过将这一亚群细胞注入患有视网膜变性的小鼠的玻璃体内，并观察血管和神经元退化的挽救情况来实现，如前所述。在糖尿病视网膜病变和老年性黄斑变性等视网膜疾病中，血管异常生长是导致成年人视力丧失的主要原因。这项研究将有助于开发治疗视网膜疾病的治疗药物，这些疾病是由可能导致失明的血管畸形引起的。

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.