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方面起着关键作用。与其他物种不同，人的角膜内皮细胞在疾病、损伤、衰老和手术后体内增殖能力有限而臭名昭著。持续性的角膜内皮功能障碍会导致威胁视力的大泡性角膜病变。目前，恢复大泡性角膜病变患者视力的唯一方法是移植含有健康角膜内皮的身体供体角膜。由于全球供体角膜严重短缺，加上在统称为“内皮角膜成形术”的手术中仅移植角膜内皮细胞的趋势日益增加，开发一种组织工程策略来生产含有人角膜内皮细胞(HCECs)的外科移植物是及时且至关重要的。利用我们报道的体外模型系统，当细胞连接成熟时，有丝分裂抑制由接触抑制介导，我们已经表明，使用EDTA/bFGF产生单个HCEC的传统工程方法激活了-catenin/Wnt信号，并失去了正常的HCEC表型向内皮-间充质转化(EMT)。相反，我们的新的工程方法基于p120连环蛋白(P120)和Kaiso siRNAs的瞬时击倒，通过激活p120/Kaiso信号而不是非连环蛋白/Wnt信号来解锁有丝分裂阻断。我们进一步优化了这种p120-Kaiso基因敲除方案，改用含有bFGF和LIF的无血清培养液，并发现我们的方法进一步激活Roha-Rock-Canonical BMP信号，将HCEC重新编程为神经峰样祖细胞，在没有EMT的情况下增殖以维持正常的HCEC表型。因此，我们新的组织工程技术可以成功地从1/8的角巩膜(通常在常规角膜移植后丢弃)中剥离出一层HCEC单层，其形状为六角形，体内细胞密度与之相当，平均直径为11.0±0.6 mm。也就是说，该技术将为每个捐赠者的角膜增加至少8个可移植的移植物。在这个第二阶段的应用中，我们建议在可植入的胶原膜上建立可重复使用的HCEC移植物的GMP工程，并使用新的包装系统来运输这些移植物(目标1)，并通过在活体NIH小型猪的内皮功能障碍模型中通过DMEK的手术过程来检验这些工程的HCEC移植物的安全性和有效性(目标2)。完成这两个目标将使该公司能够收集向FDA提交IND所需的足够的临床前数据。最终，该公司可以通过满足未得到满足的全球需求来抓住一个独特的市场机会。有朝一日，这项新的组织工程技术也可以应用于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失明疾病。此外，这项技术的成功商业化将促使科学界重新思考如何安全地干扰“接触抑制”，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的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.