Engineering of Human Corneal Endothelial Grafts

描述（申请人提供）：角膜内皮在维持角膜透明度中起着关键作用。与其他物种不同，人类角膜内皮在损伤、衰老和手术后的体内增殖能力有限。持续的角膜内皮功能障碍可导致威胁视力的大疱性角膜病变。目前，恢复大疱性角膜病变视力的唯一方法是移植含有健康角膜内皮的尸体供体角膜。由于全球供体角膜严重短缺，加上在“内皮角膜移植术”中只移植角膜内皮的趋势日益增加，因此开发一种组织工程策略来生产含有人角膜内皮细胞（HCEC）的外科移植物是及时和至关重要的。使用我们报道的体外模型系统，当细胞连接成熟时，有丝分裂阻断是通过接触抑制介导的，我们的初步研究表明，基于EDTA/bFGF的传统工程方法解锁的有丝分裂阻断激活了¿-catenin/Wnt信号，并且由于内皮-间充质转化（EMT）而有失去正常表型的风险。我们进一步发现，这种有丝分裂阻断也可以通过敲低p120-catenin来选择性地激活p120-catenin /Kaiso而不是¿-catenin/Wnt信号来解锁。因此，我们的新型组织工程技术成功地从常规角膜移植后通常丢弃的角膜巩膜边缘的1/8剥离的Descemet膜中制备出具有六边形形状和高细胞密度的HCEC单层，平均尺寸为3.7 ~ 0.7 mm2 （2.1 ~ 0.4 mm直径）。因此，在这个I期应用中，我们希望通过优化p120-catenin siRNA敲除方案，然后再敲除Kaiso siRNA (Aim 1)，以及通过添加nocodazole来增强p120-catenin核转运（Aim 2），来证明HCEC单层细胞的大小可以进一步扩大。这两个目标的完成将使我们能够进一步在上皮脱落的羊膜上制造扩大的HCEC单层，最终从一个供体边缘生产8个HCEC移植物，并在II期进行临床前动物实验。我们设想，这种基于siRNA的新型组织工程技术也可以应用于体内和体外的增殖开关，而不会冒着在其他接触抑制组织中EMT失去正常功能的风险。进一步的探索

DESCRIPTION (provided by applicant): The corneal endothelium plays a pivotal role in maintaining corneal transparency. Unlike other species, the human corneal endothelium is notorious for its limited proliferative capacity in vivo after injury, aging, and surgery. Persistet corneal endothelial dysfunction leads to sight- threatening bullous keratopathy. Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy relies upon transplantation of a cadaver donor cornea containing a healthy corneal endothelium. Because of a severe global shortage of donor corneas in conjunction with an increasing trend toward transplanting only the corneal endothelium in procedures collectively termed "endothelial keratoplasty", it is timely and paramount to develop a tissue engineering strategy to produce surgical grafts containing human corneal endothelial cells (HCEC). Using our reported in vitro model system, in which the mitotic block is mediated by contact inhibition when cell junctions mature, our preliminary studies showed that such mitotic block unlocked by the conventional engineering method based on EDTA/bFGF activates ¿-catenin/Wnt signaling and runs the risk of losing the normal phenotype to fibrous metaplasia because of endothelial-mesenchymal transition (EMT). We have further discovered such mitotic block can also uniquely be unlocked by knockdown of p120-catenin to selectively activate p120- catenin/Kaiso but not ¿-catenin/Wnt signaling. Consequently, our novel tissue engineering technology has successfully produced HCEC monolayers with a hexagonal shape and high cell density and an average size of 3.7 ¿ 0.7 mm2 (2.1 ¿ 0.4 mm in diameter) from stripped Descemet membrane of 1/8 of the corneoscleral rim normally discarded after conventional corneal transplantation. Thus, in this Phase I application, we would like to prove the concept that the size of HCEC monolayers can further be enlarged by optimizing the regimen of p120- catenin siRNA knockdown followed by additional Kaiso siRNA knockdown (Aim 1), and by addition of nocodazole to enhance p120-catenin nuclear translocation (Aim 2). Completion of these two Aims will allow us further fabricate expanded HCEC monolayers on epithelially- denuded amniotic membrane to ultimately produce 8 HCEC grafts from one donor rim and to conduct pre-clinical animal experiments in Phase II. We envision that this novel tissue engineering technology based on siRNA can also be applied to switch on and off proliferation both in vivo and ex vivo without risking the loss of normal function to EMT in other contact- inhibited tissues. Further exploration of how contact inhibition is controlled by p120- catenin/Kaiso signaling may unravel other therapeutic potentials in burgeoning regenerative medicine for treating a number of diseases characterized by the lack of regeneration due to aging, surgery, or degeneration. PUBLIC HEALTH RELEVANCE: This Phase I application proposes to develop a novel strategy of engineering human corneal endothelium based on selective activation of p120ctn/Kaiso signaling using interference RNA technology targeted at p120-catenin. Using our reported in vitro model system, we have provided strong preliminary data supporting the plausibility of further expanding human corneal endothelial monolayers by additional Kaiso siRNA knockdown with or without nocodazole. By switching on and off p120/Kaiso signaling, our novel engineering strategy may control cellular proliferation without disrupting their intercellular junctions, hence avoiding both the use of enzymatically dissociated single cells and the risk of losing the normal cell phenotype to fibrous metaplasia. Such engineered grafts may one day be used to improve the surgical procedure of endothelial keratoplasty for restoring sight in patients suffering from bullous keratopathy due to dysfunctional human corneal endothelium. Furthermore, the said technology may also be applied to engineer other similar tissues, such as the retinal pigment epithelium, in the future.