technologyIn vitro maturation of BMP-7-responsive pancraeatic beta cell progenitors by oxygen modulation

技术通过氧调节使 BMP-7 反应性胰腺 β 细胞祖细胞体外成熟

• 批准号: 9344589
• 负责人: Juan Dominguez-Bendala
• 金额: $ 71.09万
• 依托单位: OPHYSIO, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9344589
• 关键词: Academia; Administrator; Adult; Alpha Cell; Area; Autoimmune Diabetes; Automobile Driving; Award; BMP7 gene; Beta Cell; Biology; Biotechnology; Cell Differentiation process; Cell Lineage; Cell Maturation; Cell Therapy; Cells; Clinical; Clinical Trials; Collaborations; Contracts; Coupled; Cyclic GMP; Data; Development; Device Designs; Devices; Diabetes Mellitus; Embryo; Endocrine; Enhancement Technology; Evolution; Exhibits; Exposure to; FDA approved; Funding; Generations; Glucose; Growth; Human; Immunity; In Situ; In Vitro; Industry; Insulin; Insulin-Dependent Diabetes Mellitus; Islets of Langerhans Transplantation; Knowledge; Lead; Legal patent; Length; Licensing; Mediating; Methods; Mission; Mus; Natural regeneration; Nature; Non-Insulin-Dependent Diabetes Mellitus; Organ; Organ Donor; Oxygen; Pancreas; Pathway interactions; Patients; Phase; Phenotype; Physiological; Pluripotent Stem Cells; Population; Preparation; Procedures; Process; Production; Progress Reports; Protocols documentation; Publications; Publishing; Reporting; Research; Research Institute; Safety; Source; Stem cells; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Traction; Transplantation; United States National Institutes of Health; Universities; Washington; Work; base; cell type; clinical application; design; drug discovery; effective therapy; experience; experimental study; falls; human embryonic stem cell; human pluripotent stem cell; improved; in vivo; islet; mouse model; novel; phase 2 study; progenitor; prospective; receptor; response; scale up; success; tissue preparation

PROJECT SUMMARY Islet transplantation represents the current cell therapy standard for type 1 diabetes (T1D). However, the gap between the availability of donor organs and the clinical demand for them calls for the development of alternative/renewable sources of insulin-producing cells. In addition to this therapeutic need, a steady supply of islets is also needed for research and drug discovery purposes. Human embryonic stem cells (hESc) differentiated into pancreatic -cell precursors are presently the subject of Phase I/II clinical trials. However, the success of this approach hinges on the assumption that the microenvironment that leads to effective maturation in a mouse model will be the same in human patients with autoimmune diabetes. The safety of partially differentiated hESc-derived products, efficacy of the macro- encapsulation devices used to shield them from allo- and auto-immunity, and lag time to functional maturation remain open questions. The use of insulin-producing cells that are mature and functional at the time of transplantation may circumvent some of these problems. However, despite claims to the contrary, there is no current protocol to date that yields -like cells capable of reversing diabetes right after transplantation. In collaboration with our partners at the University of Miami, Ophysio, Inc. has successfully developed a platform to aid in the terminal in vitro differentiation of pancreatic progenitors (PPs) of different origins (hESc and native murine pancreas). This patented technology is based on the accurate targeting of physiological oxygenation throughout cell aggregates in culture –which conventional means of culture fail to achieve. Oxygen tension lies at the crossroads of key pancreatic differentiation pathways, and its evolution throughout development has been conclusively shown to drive cell fate. Here we seek to extend these principles to the terminal maturation of a novel sub-population of PPs that our collaborators have described in human non-endocrine pancreatic tissue (hNEPT), which comprises 98% of the pancreas and is routinely discarded after islet isolation. This sub-population, identified through in vitro lineage-tracing techniques, is characterized by its responsiveness to the FDA- approved bone morphogenetic protein 7 (BMP-7). hNEPT exposure to BMP-7 results in the efficient (up to 15% in preliminary data) generation of endocrine cells that secrete insulin at levels that fall right within the range published for human isolated islets and exhibit robust glucose responsiveness in vitro and in vivo. Our Phase II studies aim at capitalizing on our Phase I data. These include not only the proof of principle that oxygen modulation improves BMP-7-mediated conversion of hNEPT, but also new findings on the phenotype of BMP-7 responsive cells that will allow for their prospective isolation from raw hNEPT preparations. Our specific aims are: (1) To determine whether in vitro targeting of physiological pO2 in PDX1 (P2RY1)+/ALK3+-sorted hNEPT subpopulations results in functional -like cells capable of reversing diabetes in mice; and (2) To scale up the process using an entire organ (10-12 ml of hNEPT pellet after islet isolation) using Ophysio’s new T75 oxygen-modulating devices (designed in the context of our previous award 2R44DK083832-02). In addition to the optimization and scale up of the process, we will simultaneously establish cGMP manufacturing protocols, file for IP protection of the final method and begin licensing contracts with parties for use of the process to obtain the cells for research purposes. We contend that BMP-7-responsive PPs from hNEPT represent a valid alternative to hESc for clinical applications, as this technology capitalizes on current clinical strategies (islet isolation and transplantation) for which there are already well established networks; increased safety of adult cell products vs. hESc-derived ones; and ease of in vitro expansion/differentiation using a single, FDA-approved clinical product. Coupled with Ophysio’s technology for enhanced in vitro maturation, this approach has rapid translational potential for the treatment of diabetes mellitus.

项目概要 胰岛移植代表了当前 1 型糖尿病 (T1D) 的细胞治疗标准。 然而，供体器官的可用性与临床需求之间的差距 呼吁开发胰岛素产生细胞的替代/可再生来源。在 除了这种治疗需求外，还需要稳定的胰岛供应用于研究和 药物发现的目的。人胚胎干细胞（hESc）分化为胰腺细胞 -细胞前体目前是 I/II 期临床试验的主题。然而，成功的 这种方法取决于这样的假设：导致有效的微环境 小鼠模型中的成熟与患有自身免疫性糖尿病的人类患者相同。 部分分化 hESc 衍生产品的安全性、宏观功效 用于保护它们免受同种免疫和自身免疫的封装装置，以及延迟时间 功能成熟仍然是一个悬而未决的问题。使用产生胰岛素的细胞 移植时的成熟和功能可能会避免其中一些问题。 然而，尽管有相反的说法，但迄今为止还没有任何协议可以产生类似  的结果 移植后能够立即逆转糖尿病的细胞。 Ophyio, Inc. 与迈阿密大学的合作伙伴合作，成功 开发了一个平台来帮助胰腺祖细胞进行终末体外分化 (PP) 不同来源（hESc 和天然鼠胰腺）。这项专利技术是 基于整个细胞聚集体中生理氧合的准确靶向 文化——这是传统文化手段无法实现的。氧张力位于 关键胰腺分化途径的十字路口及其整个演变过程 发育已被最终证明可以驱动细胞的命运。在这里我们寻求扩展这些 我们的合作者提出的新的 PP 亚群最终成熟的原则 已在人类非内分泌胰腺组织 (hNEPT) 中进行了描述，该组织占 98% 胰岛并在胰岛分离后通常被丢弃。该亚群已确定 通过体外谱系追踪技术，其特点是对 FDA 的响应 批准的骨形态发生蛋白 7 (BMP-7)。 hNEPT 暴露于 BMP-7 导致 高效（初步数据高达 15%）产生分泌胰岛素的内分泌细胞 水平正好落在针对人类孤立胰岛公布的范围内，并且表现出稳健的 体外和体内的葡萄糖反应性。我们的第二阶段研究旨在利用我们的 第一阶段数据。这些不仅包括氧调节改善的原理证明 BMP-7 介导的 hNEPT 转化，以及 BMP-7 表型的新发现 反应细胞，这将允许它们从原始 hNEPT 制剂中进行前瞻性分离。 我们的具体目标是： (1) 确定 PDX1 中生理 pO2 的体外靶向是否有效 (P2RY1)+/ALK3+ 分选的 hNEPT 亚群产生功能性  样细胞，能够 逆转小鼠糖尿病； (2) 使用整个器官（10-12 ml 使用 Ophyio 的新型 T75 氧气调节装置（胰岛分离后的 hNEPT 颗粒） （根据我们之前的奖项 2R44DK083832-02 设计）。除了 工艺优化和放大，同步建立cGMP 制造协议，申请最终方法的知识产权保护并开始许可合同 与各方就使用该过程获取用于研究目的的细胞进行合作。 我们认为来自 hNEPT 的 BMP-7 响应 PP 是 hESc 的有效替代品 用于临床应用，因为该技术利用了当前的临床策略（胰岛 隔离和移植），已经有完善的网络；增加 成体细胞产品与 hESc 衍生产品的安全性；和易于体外 使用 FDA 批准的单一临床产品进行扩展/分化。加上 OPhysio 的增强体外成熟技术，该方法具有快速转化性 治疗糖尿病的潜力。