Optimizing systemic stem/progenitor cell therapy for AMD

老年性黄斑变性(AMD)是老年人视力丧失的主要原因。 尽管知道所涉及的细胞类型，但治疗干预一直是有限的，而且存在 目前还没有治疗“非渗出性AMD”的方法。当RPE细胞移植到视网膜下时 患者空间提供了一种有希望的治疗方法，到目前为止，结果有限。 由于：1)晚期疾病的移植，2)侵入性给药途径，3) 移植细胞分化不完全状态。为了解决这些限制，我们有 有了许多令人兴奋的发现：1)强制表达RPE65基因使小鼠 造血干细胞(MHSc)，当被注射回循环时，回到视网膜和 在急性和慢性小鼠RPE丢失模型中更新RPE单层并重建 视功能；2)内源性HSC释放的昼夜节律模式影响重建 骨髓移植后；3)AMD的小胶质细胞激活需要“调节” 确保HSCs高效再生RPE；4)高效的非病毒蛋白输送 机械(T3SS)能够将靶蛋白送入宿主HSCs并促进其分化； 5)人造血祖细胞(HHPC)经RPE65编程后，表达RPE 细胞标志物。基于这些观察，我们假设：“成功的治疗 利用人或小鼠的HSC需要在注射到 体循环，它们在最佳植入潜力时的注射和 通过抑制驻留的小胶质细胞激活和/或 恢复外周血促炎症和内稳态单核细胞的平衡。 假说有三个目的。在目标1中，我们将确定招募的依赖性 并在SOD2KD小鼠模型中将程序化的MHSc整合到受损的RPE中 一天的注射时间和供者的年龄以及受者的年龄。目标2 将调查操纵视网膜环境的重要性，通过控制 驻留小胶质细胞的激活状态或外周血单核细胞的流入对小胶质细胞杀伤效率的影响 系统地应用程序化MHSc修复SOD2KD模型中的RPE层。目标 3将把我们的老鼠发现转化为hHPC。我们将在人CD133+、CD34-中表达RPE65- ，CD38-细胞将这些细胞分化为RPE细胞，并允许SCID中RPE再生 接受SOD2KD模型的小鼠。联合注射间充质干细胞将用于 减少驻留小胶质细胞的激活。我们的方法克服了当前的许多限制 AMD的人类干细胞疗法。

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.