OPTIMIZING SYSTEMIC STEM/PROGENITOR CELL THERAPY FOR AMD

描述(申请人提供)：老年性黄斑变性(AMD)是导致老年人视力丧失的主要原因。尽管对所涉及的细胞类型有所了解，但治疗干预一直是有限的，目前还没有治疗“非渗出性AMD”的方法。虽然RPE细胞移植到患者的视网膜下腔提供了一种有希望的治疗方法，但由于以下原因，迄今为止的结果有限：1)晚期疾病的移植，2)侵入性给药途径，3)不完全分化状态。 移植的细胞。为了解决这些局限性，我们取得了一些令人兴奋的发现：1)强制表达RPE65基因使小鼠造血干细胞(MHSc)在回注到循环中时能够回到视网膜，并在急性和慢性RPE丧失的小鼠模型中更新RPE单层并重建视功能；2)内源性HSC释放的昼夜规律影响骨髓移植后的重建；3)AMD中的小胶质细胞激活将需要“调节”以确保HSC高效地再生RPE；4)高效的非病毒蛋白输送机制(T3SS)能够将靶蛋白输送到宿主HSC并促进其分化；5)人造血祖细胞(HHPC)在RPE65诱导下表达RPE细胞标志。基于这些观察，我们假设：“人类或小鼠HSC的成功治疗需要在注射到体循环之前编程，在最佳植入潜力时注射，并通过抑制驻留的小胶质细胞激活和/或恢复外周促炎症和内稳态单核细胞的平衡来对视网膜进行预适应。”这一假说有三个目的。在目标1中，我们将确定SOD2KD小鼠模型中程序化MHSc的募集和整合到受损的RPE中的依赖关系，包括注射时间、供体HSC的年龄以及受体的年龄。目标2将通过以下方式调查操纵视网膜环境的重要性 在SOD2KD模型中，控制驻留的小胶质细胞的激活状态或外周血单核细胞的流入对系统给药程序化MHSc修复RPE层的效率有影响。AIM 3将把我们的小鼠发现转化为hHPC。我们将在人CD133+、CD34-、CD38-细胞中表达RPE65，以使这些细胞分化为RPE细胞，并允许经历SOD2KD模型的SCID小鼠再生RPE。联合注射间充质干细胞将被用来减少常驻小胶质细胞的激活。我们的方法克服了目前人类干细胞疗法治疗AMD的许多局限性。

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the leading cause of visual loss in the elderly. Despite knowledge of the cell types involved, therapeutic intervention has been limited and there is currently no treatment for "nonexudative AMD". While RPE cell transplantation into the subretinal space of patients offered a promising therapeutic approach, outcomes to date have been limited due to: 1) transplantation in late stage disease, 2) the invasive route of administration, and 3) incomplete differentiation status of the transplanted cells. To address these limitations, we have made a number of exciting discoveries: 1) forced expression of the RPE65 gene allows mouse hematopoietic stem cells (mHSC), when injected back into the circulation, to home to the retina and renew the RPE monolayer in both acute and chronic mouse models of RPE loss and re-establish visual function; 2) the circadian pattern to endogenous HSC release impacts reconstitution following bone marrow transplantation; 3) microglial activation in AMD will require "modulation" to ensure efficient RPE regeneration by HSCs; 4) a highly effective non-viral protein delivery machinery (T3SS) is able to deliver target proteins into host HSCs and promote their differentiation; 5) human hematopoietic progenitor cells (hHPCs), when programmed with RPE65, express RPE cell markers. Based on these observations we hypothesize that: "Successful therapeutic utilization of human or murine HSC requires their programming prior to injection into the systemic circulation, their injection at the time of optimal engraftment potential and preconditioning of the retina by either suppression of resident microglia activation and/or restoring the balance of peripheral pro-inflammatory and homeostatic monocytes." The hypothesis is addressed in three Aims. In Aim 1 we will determine the dependence of recruitment and incorporation of programmed mHSC into the injured RPE in the SOD2 KD mouse model upon the time of day of injection and the age of the donor HSC as well as the age of the recipient. Aim 2 will investigate the importance of manipulating the retinal environment, by controlling either the activation state of the resident microglia or the influx of peripheral monocytes on the efficiency of systemically administered programmed mHSC to repair the RPE layer in the SOD2 KD model. Aim 3 will translate our mouse findings into hHPCs. We will express RPE65, in human CD133+, CD 34- , CD38- cells to differentiate these cells toward RPE cells and allow RPE regeneration in SCID mice undergoing the SOD2 KD model. Co-injection of mesenchymal stem cells will be utilized to reduce activation of resident microglia. Our approach overcomes many of the current limitations of human stem cell therapies for AMD.