Vascular networks genetically engineered for protein drug delivery

用于蛋白质药物输送的基因工程血管网络

• 批准号: 10457445
• 负责人: Minglin Ma
• 金额: $ 57.24万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10457445
• 关键词: 3-Dimensional; Activated Partial Thromboplastin Time measurement; Address; Alginates; Allogenic; Biological Assay; Biology; Bioluminescence; Blood; Blood Circulation; Blood Coagulation Disorders; Blood Vessels; Blood coagulation; Caliber; Canis familiaris; Cell Line; Cell Survival; Cell Transplantation; Cells; Clinical; Clone Cells; Coagulation Process; Code; Competence; DNA Transposons; Data; Dermal; Development; Devices; Disease; Drug Delivery Systems; Encapsulated; Endothelial Cells; Engineering; Engraftment; Enzyme-Linked Immunosorbent Assay; Enzymes; Evaluation; Excision; Exons; Extracellular Matrix; F8 gene; Factor VIII; Fibrinogen; Fibroblasts; Future; Genetic Engineering; Genome; Genomics; Germ Cells; Greater sac of peritoneum; Hemophilia A; Hemorrhage; Homologous Transplantation; Human; Hydrogels; Immune; Immunocompetent; In Vitro; Inflammation; Infusion procedures; Inherited; Internal Ribosome Entry Site; Length; Life; Liver; Methods; Morbidity - disease rate; Mus; Mutation; Organoids; Patients; Phenotype; Plasma; Pluripotent Stem Cells; Production; Productivity; Protein Engineering; Research; Risk; Safety; System; Tail; Technology; Teratoma; Testing; Therapeutic; Time; Titrations; Transgenes; Translations; Transplantation; Transposase; Tubular formation; United States National Institutes of Health; Vascularization; White Blood Cell Count procedure; Whole Blood; Work; base; clinically relevant; density; experience; gene therapy; human pluripotent stem cell; immunoreaction; implantation; in vivo; induced pluripotent stem cell; inhibitor; new technology; novel; organoid transplantation; preclinical efficacy; prevent; response; sensor; subcutaneous

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.

项目总结/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 VIII（FVIII）。目前的治疗涉及在患者的整个生命周期内重复静脉输注FVIII浓缩物。 病人，这造成巨大的不适和发病率。或者，我们试图开发一种新的 持续的FVIII递送技术。最近，我们开发了一种非病毒离体基因治疗方法， 血友病A我们使用piggyBac DNA转座子系统将70个拷贝的F8基因插入到人类中， 多能干细胞（PSC）。我们将这些修饰的F8-PSC分化为内皮细胞（iEC;天然的 FVIII的生产者），并证明了极高水平的FVIII的生产。皮下 将我们的人F8-iEC植入免疫缺陷血友病（SCID-f8 ko）小鼠中，我们实现了高达600%的移植。 FVIII的循环水平，有效地纠正凝血缺陷。尽管取得了这些进展，我们的开放- 移植方法对于转化具有一些固有的局限性：1）非自体细胞的免疫排斥，和2） 对细胞传播和安全性的担忧。为了解决这些局限性，我们与Minglin博士合作， 马（康奈尔大学），谁拥有丰富的经验与设备的封装和移植细胞在小鼠 还有狗我们提出了一种技术，需要一种新的可检索封装设备。我们将集合我们的 F8-iEC进入稳定的3D血管类器官，然后将多个类器官嵌入藻酸盐水凝胶中 在管状封装装置（1 mm直径;可变长度）内。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，并产生FVIII， 植入腹膜腔后，血流处于治疗水平。为了验证这些假设，我们 提出三个具体目标。在Aim-1中，我们将对血管类器官进行基因工程改造， FVIII的临床相关水平。我们将开发一种新的无启动子的外显子陷阱传感器盒，以避免内部- piggyBac转座子的外显子整合。然后，我们将多个F8拷贝插入NIH合格的PSC系中 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， 最佳FVIII生产的配置，并确定免疫活性的安全性和长期疗效 血友病小鼠我们将评价细胞存活率、血浆中BDD-FVIII活性、凝血缺陷的纠正， 畸胎瘤形成的风险和治疗的可逆性。在Aim-3中，我们将评估安全性和长期 我们的设备在狗身上的功效。我们将首先产生犬特异性分泌FVIII的血管类器官。我们将 然后将我们的设备（IP）在健康狗中长达6个月，并评价可扩展性、安全性、可回收性， 和FVIII生产。最后，我们将在血友病A犬中测试我们的同种异体器械，并确定其安全性和 有效期长达1年。总之，我们建议研究开发一种新的技术，以提供FVIII， 血友病A我们设想这项研究可以为未来的人类研究铺平道路。