Optimizing systemic stem/progenitor cell therapy for AMD

描述（由申请人提供）：视网膜相关性黄斑变性（AMD）是老年人视力丧失的主要原因。尽管了解所涉及的细胞类型，但治疗干预有限，目前没有治疗“非渗出性AMD”的方法。虽然将RPE细胞移植到患者的视网膜下腔中提供了一种有希望的治疗方法，但迄今为止的结果由于以下原因而受到限制：1）晚期疾病中的移植，2）侵入性给药途径，以及3）RPE细胞的不完全分化状态。 移植的细胞。为了解决这些局限性，我们已经取得了许多令人兴奋的发现：1）RPE 65基因的强制表达允许小鼠造血干细胞（mHSC）在注射回循环中时回到视网膜并在RPE损失的急性和慢性小鼠模型中更新RPE单层并重建视觉功能; 2）内源性HSC释放的昼夜节律模式影响骨髓移植后的重建; 3）AMD中的小胶质细胞活化将需要“调节”以确保HSC的有效RPE再生; 4）高效的非病毒蛋白递送机制（T3 SS）能够将靶蛋白递送到宿主HSC中并促进其分化; 5）当用RPE 65编程时，人造血祖细胞（hHPC）表达RPE细胞标志物。基于这些观察结果，我们假设：“人或鼠HSC的成功治疗利用需要在注射到体循环中之前对其进行编程，在最佳植入潜力时注射，并通过抑制驻留小胶质细胞活化和/或恢复外周促炎和稳态单核细胞的平衡来预处理视网膜。“这一假设在三个目标中得到解决。在目的1中，我们将确定在SOD 2 KD小鼠模型中，程序化mHSC募集和掺入到损伤的RPE中对注射日的时间和供体HSC的年龄以及受体的年龄的依赖性。目标2将研究操纵视网膜环境的重要性， 控制驻留小胶质细胞的活化状态或外周单核细胞的流入对系统施用的程序化mHSC修复SOD 2 KD模型中RPE层的效率的影响。Aim 3将把我们的小鼠发现转化为hHPC。我们将在人CD 133+、CD 34-、CD 38-细胞中表达RPE 65，以使这些细胞向RPE细胞分化，并允许经历SOD 2 KD模型的SCID小鼠中的RPE再生。将利用间充质干细胞的共注射来减少驻留小胶质细胞的激活。我们的方法克服了目前人类干细胞治疗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.