Vascular networks genetically engineered for protein drug delivery

项目摘要/摘要 血友病A是一种遗传性出血性疾病，由编码凝血因子的F8基因突变引起 (FVIII)。目前的治疗包括重复静脉注射。在生命的整个过程中，FVIII的输注浓缩 患者，这造成了极大的不适和发病率。或者，我们寻求开发一部小说 可持续交付FVIII的技术。最近，我们开发了一种非病毒的体外基因治疗方法 血友病A。我们使用了一种猪Bac DNA转座子系统，将70个拷贝的F8基因插入到人类体内。 多能干细胞(PSC)。我们将这些经过修饰的F8-PSCs分化为内皮细胞(IECs；Natural FVIII的生产商)，并展示了极高水平的FVIII的生产。皮下注射后 将我们的人F8-IECs植入免疫缺陷血友病(SCID-f8ko)小鼠，我们获得了高达600%的 循环中的FVIII水平，有效纠正凝血不足。尽管取得了这些进展，但我们的开放- 移植方法对于翻译有一些固有的局限性：1)非自体细胞的免疫排斥；2) 对细胞传播和安全的担忧。为了解决这些限制，我们与明林博士合作 Ma(康奈尔大学)，他在小鼠细胞的封装和移植设备方面拥有丰富的经验 还有狗。我们提出了一种新的可回收封装设备的技术。我们将把我们的 F8-IECS进入稳定的3D血管有机体，然后将多种有机类物质嵌入到海藻酸水凝胶中 管状封装装置内(直径1 mm；长度可变)。根据我们的初步数据， 我们假设我们的设备将保护细胞免受免疫排斥反应，并产生FVIII，将到达 植入腹膜后达到治疗水平的血流。为了检验这些假设，我们 提出三个具体目标。在AIM-1中，我们将遗传工程血管有机化合物来生产 临床相关水平的FVIII。我们将开发一种新的无启动子外显子陷阱传感器盒，以避免内部 我们的iggyBac转座子的外显子整合。然后我们将把多个F8拷贝插入到符合NIH标准的PSC系列中 以产生用于高FVIII生产的通用克隆。在AIM-2中，我们将建立一个封装设备 优化FVIII生产的配置并确定免疫活性的安全性和长期有效性 血友病小鼠。我们将评估细胞存活率，血浆中BDD-FVIII的活性，凝血缺陷的纠正， 畸胎瘤形成的风险，以及治疗的可逆性。在AIM-3中，我们将对其安全性和长期安全性进行评估 我们的设备在狗身上的功效。我们将首先产生犬特有的分泌FVIII的血管器官。我们会 然后移植我们的设备(I.P.)在健康狗身上持续6个月，并评估可扩展性、安全性、可恢复性、 和FVIII制作。最后，我们将在血友病A犬身上测试我们的同种异体设备，并建立安全和 有效时间长达1年。综上所述，我们建议开展研究，以开发一种新的技术来在 血友病答：我们预计这项研究将为未来的人类研究铺平道路。

PROJECT SUMMARY/ABSTRACT Hemophilia A is an inherited bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatment involves repeated i.v. infusions of FVIII concentrates throughout the life of the patient, which creates tremendous discomfort and morbidity. Alternatively, we seek to develop a novel technology for sustained FVIII delivery. Recently, we developed a non-viral ex vivo gene therapy approach for hemophilia A. We used a piggyBac DNA transposon system to insert 70 copies of the F8 gene into human pluripotent stem cells (PSCs). We differentiated these modified F8-PSCs into endothelial cells (iECs; natural producers of FVIII) and demonstrated the production of exceedingly high levels of FVIII. After subcutaneous engraftment of our human F8-iECs into immunodeficient hemophilic (SCID-f8ko) mice, we achieved up to 600% circulating levels of FVIII, effectively correcting the clotting deficiency. Notwithstanding this progress, our open- graft approach has some inherent limitations for translation: 1) immune rejection of non-autologous cells, and 2) concerns over cell dissemination and safety. To address these limitations, we have teamed up with Dr. Minglin Ma (Cornell), who has extensive experience with devices for encapsulation and transplantation of cells in mice and dogs. We propose a technology entailing a novel retrievable encapsulation device. We will assemble our F8-iECs into stable 3D vascular organoids and will then embed multiple organoids into an alginate hydrogel inside a tubular encapsulation device (1-mm diameter; variable length). Based on our preliminary data, we hypothesize that our device will protect the cells from immune rejection and produce FVIII that will reach the bloodstream at therapeutic levels upon implantation into the peritoneal cavity. To test these hypotheses, we propose three Specific Aims. In Aim-1, we will genetically engineer vascular organoids for the production of clinically relevant levels of FVIII. We will develop a new promoterless exon-trap sensor cassette to avoid intra- exon integration of our piggyBac transposon. We will then insert multiple F8 copies into NIH-eligible PSC lines to generate universal clones for high FVIII production. In Aim-2, we will establish an encapsulation device configuration for optimal FVIII production and determine the safety and long-term efficacy in immunocompetent hemophilic mice. We will evaluate cell survival, BDD-FVIII activity in plasma, correction of coagulation deficiency, risk of teratoma formation, and reversibility of the treatment. In Aim-3, we will evaluate the safety and long-term efficacy of our devices in dogs. We will first generate canine-specific FVIII-secreting vascular organoids. We will then transplant our devices (I.P.) in healthy dogs for up to 6 months and evaluate scalability, safety, retrievability, and FVIII production. Lastly, we will test our allogeneic devices in hemophilia A dogs and establish safety and efficacy for up to 1 year. In summary, we propose studies to develop a novel technology to deliver FVIII in hemophilia A. We envision this research could pave the way for future studies in humans.