Engineering of Human Corneal Endothelial Grafts

描述（由申请人提供）：角膜内皮在维持角膜透明度方面起着关键作用。与其他物种不同，人类角膜内皮在疾病、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导致威胁视力的大泡性角膜病变。目前，恢复患有大泡性角膜病变的眼睛的视力的唯一解决方案依赖于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，加上在统称为“内皮角膜移植术”的手术中仅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的手术移植物是及时且至关重要的。（HCEC）。使用我们报道的体外模型系统，其中有丝分裂阻滞是由细胞连接成熟时的接触抑制介导的，我们已经表明，使用EDTA/bFGF产生单个HCECs的常规工程方法激活-连环蛋白/Wnt信号传导和正常HCEC表型向内皮-间充质转化（EMT）的丧失。相比之下，我们的新的工程方法的基础上瞬时敲低p120连环蛋白（p120）和Kaiso siRNA解锁有丝分裂阻滞激活p120/Kaiso信号，但不-连环蛋白/Wnt信号。我们通过切换到含有bFGF和LIF的无血清培养基进一步优化了这种p120-Kaiso敲低方案，并发现我们的方法进一步激活RohA-ROCK-经典BMP信号传导以将HCEC重编程为神经嵴样祖细胞，其增殖以维持正常的HCEC表型而没有EMT。因此，我们的新型组织工程技术可以成功地从从1/8的角巩膜缘剥离的后弹力膜（通常在常规角膜移植后丢弃）产生一个具有六边形形状的HCEC单层，其具有可比的体内细胞密度，并且直径的平均尺寸为11.0 ± 0.6 mm。也就是说，该技术将为每个供体角膜增加至少8个额外的可移植物。在该II期申请中，我们建议在可植入胶原膜上建立HCEC移植物的可再现GMP工程，使用新的包装系统运输这些移植物（目的1），并通过在NIH内皮功能障碍的体内小型猪模型中进行DMEK手术检查这些工程化HCEC移植物的安全性和有效性（目的2）。完成这两个目标将使公司能够收集向FDA提交IND所需的足够临床前数据。最终，公司可以通过满足未满足的全球需求来抓住独特的市场机遇。有一天，这种新的组织工程技术也可以被部署到工程其他类似的单层组织，如视网膜色素上皮（RPE），用于黄斑下移植治疗以RPE功能障碍为特征的视网膜致盲疾病。此外，这项技术的成功商业化将刺激科学界重新思考如何安全地扰乱“接触抑制”以使我们受益，即，通过维持正常的表型，以及这种新的再生方法是否可以避免直接从胚胎干细胞或诱导多能干细胞重新编程的需要。

DESCRIPTION (provided by applicant): The corneal endothelium plays a pivotal role in maintaining corneal transparency. Unlike in other species, the human corneal endothelium is notorious for its limited proliferative capacity in vivo after diseases, injury, aging, and surgery Persistent 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. Due to a severe global shortage of donor corneas, in conjunction with an increasing trend toward transplanting only the corneal endothelium in procedures collectively termed "endothelial keratoplasties," it is timely and paramount to develop a tissue engineering strategy to produce surgical grafts containing human corneal endothelial cells (HCECs). Using our reported in vitro model system, in which the mitotic block is mediated by contact inhibition when cell junctions mature, we have shown that the conventional engineering methods using EDTA/bFGF to generate single HCECs activates -catenin/Wnt signaling and the loss of the normal HCEC phenotype to endothelial-mesenchymal transition (EMT). In contrast, our novel engineering method based on transient knockdown by p120 catenin (p120) and Kaiso siRNAs unlocks the mitotic block by activating p120/Kaiso signaling but not -catenin/Wnt signaling. We have further optimized this p120-Kaiso knockdown regimen by switching to a serum-free medium containing bFGF and LIF and discovered that our method further activates RohA-ROCK-canonical BMP signaling to reprogram HCECs to neural-crest like progenitors which proliferates to maintain the normal HCEC phenotype without EMT. Consequently, our novel tissue engineering technology can successfully produce from Descemet membrane stripped from 1/8 of the corneoscleral rim (normally discarded after conventional corneal transplantation) one HCEC monolayer with a hexagonal shape, comparable in vivo cell density, and an average size of 11.0 ¿ 0.6 mm in diameter. That is, the technology will add at least an additional 8 transplantable grafts per one donor cornea. In this Phase II application, we propose to establish reproducible GMP engineering of HCEC grafts on an implantable collagen membrane, with a new packing system to transport these grafts (Aim 1), and to examine the safety and efficacy of these engineered HCEC grafts through the surgical procedure of DMEK in an in vivo NIH mini pig model of endothelial dysfunction (Aim 2). Completion of these two Aims will allow the Company to gather sufficient pre-clinical data needed for an IND submission to the FDA. Ultimately, the Company can capture a unique market opportunity by fulfilling an unmet global need. One day, this new tissue engineering technology can also be deployed to engineer other similar monolayer tissues such as retinal pigment epithelium (RPE) for submacular transplantation in treating retinal blinding diseases characterized by dysfunctional RPE. Furthermore, successful commercialization of this technology will stimulate the scientific community to re-think how "contact inhibition" can safely be perturbed to our benefit, i.e., by maintaining the normal phenotype, and whether this new regenerative approach can circumvent the need to reprogramming directly from embryonic stem cells or induced pluripotent stem cells.