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

基本信息

批准号：
10617773
负责人：
Minglin Ma
金额：
$ 66.71万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
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/10617773
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 Canis familiaris Cell Line Cell Survival Cell Transplantation Cells Clinical Clone Cells Coagulation Process Code Competence Cytoprotection DNA Transposons Data Dermal Development Devices Diameter Disease Drug Delivery Systems Eligibility Determination 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 Immunologic Deficiency Syndromes In Vitro Inflammation Infusion procedures Inherited Internal Ribosome Entry Site Length Life Liver Maps 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 clinically relevant density experience gene therapy human pluripotent stem cell immunoreaction implantation in vivo induced pluripotent stem cell inhibitor new technology novel preclinical efficacy prevent programs response sensor subcutaneous

项目摘要

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.

项目摘要/摘要血友病A是由F8基因编码凝结因子突变引起的遗传出血障碍 VIII（FVIII）。当前的治疗涉及重复的静脉FVIII的输注集中在整个生命中患者，会产生极大的不适和发病率。另外，我们试图发展小说持续FVIII交付的技术。最近，我们开发了一种非病毒外基因治疗方法血友病A.我们使用Piggybac DNA转座子系统将F8基因的70份插入人多能干细胞（PSC）。我们将这些改良的F8-PSC区分为内皮细胞（IEC；天然 FVIII）的生产者，并证明了FVIII水平极高的产生。皮下植入人类F8-eics融入免疫缺陷的血友病（SCID-F8KO）小鼠中，我们达到了600％ FVIII的循环水平，有效地纠正了凝结缺乏症。尽管取得了进展，我们的开放移植方法对翻译具有一定的固有局限性：1）非自动细胞的免疫排斥和2）关注细胞传播和安全性。为了解决这些限制，我们与Minglin博士合作 MA（Cornell），他在封装和移植小鼠的设备方面拥有丰富的经验和狗。我们提出了一项需要新颖的可检索封装装置的技术。我们将组装我们的 F8-EECS进入稳定的3D血管器官，然后将多个类正骨嵌入藻酸盐水凝胶中在管状封装装置（直径1毫米；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的影响，并产生将到达的FVIII 植入腹膜腔后治疗水平的血液。为了检验这些假设，我们提出三个具体目标。在AIM-1中，我们将在基因上进行遗传设计血管器官，以生产 FVIII的临床相关水平。我们将开发一个新的无启动子外显子陷阱传感器盒，以避免内部我们的Piggybac转座子的外显子整合。然后，我们将插入多个F8副本中的NIH资格PSC线生成用于高FVIII生产的通用克隆。在AIM-2中，我们将建立一个封装设备配置最佳FVIII生产并确定免疫能力的安全性和长期功效血友病小鼠。我们将评估细胞存活，血浆中的BDD-FVIII活性，校正凝结缺乏症，形成畸胎瘤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的安全性我们在狗中设备的功效。我们将首先生成犬类特异性的FVIII分泌血管器官。我们将然后将我们的设备（i.p.）移植到健康的狗中长达6个月，并评估可伸缩性，安全性，可检索性，和FVIII生产。最后，我们将测试我们在血友病A狗中的同种异体设备，并建立安全性和最多1年的功效。总而言之，我们建议研究开发一种新型技术，以提供FVIII 血友病A.我们设想这项研究可能为未来在人类研究的方式铺平道路。