Immunoevasive Engineered Living Blood Vessels

免疫逃避工程活血管

• 批准号: 10420546
• 负责人: Elliot Chaikof
• 金额: $ 61.18万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-05 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10420546
• 关键词: Allogenic; Aneurysm; Arteries; Biochemical; Biological; Biomechanics; Blood Vessels; CD47 gene; Cell Wall; Cells; Chemicals; Collagen; Collagen Fiber; Collagen Fibril; Elastin; Elements; Endothelial Cells; Engineering; Extracellular Matrix; Failure; HLA G antigen; HLA-A gene; Homing; Human; Immune system; Immunoassay; Immunologics; Implant; In Vitro; Infiltration; Mechanics; Microfabrication; Morphology; Mus; Natural Killer Cells; Operative Surgical Procedures; Patients; Phase; Phenotype; Physiological; Process; Production; Property; Proteins; Protocols documentation; Rattus; Reporting; Research Personnel; Risk; Smooth Muscle Myocytes; Source; Stenosis; Structure; T-Lymphocyte; Techniques; Thick; Tissue Engineering; Vascular Smooth Muscle; biodegradable polymer; design; differentiation protocol; genome editing; human pluripotent stem cell; hydrodynamic flow; immunogenicity; immunoregulation; implantation; in vivo; innovation; large scale production; macrophage; materials science; programmed cell death ligand 1; protein expression; reconstitution; response; scaffold; stem cell biology; stem cells; tool; vascular tissue engineering

Project Summary Recent innovations by project investigators have established an important new framework for the rapid and scalable production of engineered living blood vessels. Notably, we have designed new protocols for multiplex genome editing to generate human pluripotent stem cells (hPSCs) in which HLA-A, -B, and -C were selectively ablated, HLA class II molecules eliminated, and multiple tolerogenic factors, including HLA-G, PD-L1, and CD47 expressed. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) derived from these PSCs, using our previously reported chemically defined differentiation protocols, were protected from alloimmune rejection in vitro and in vivo. Further, we have developed new engineering approaches for the fabrication of mechanically robust, free-standing, ultrathin collagen sheets and related manufacturing tools for the scalable production of engineered living blood vessels. In this proposal, we postulate that immunoevasive blood vessels can be efficiently and rapidly manufactured using ‘hypoimmunogenic’ cells and planar extracellular matrix (ECM) scaffolds of defined composition, content, and microarchitecture. In the process, the efficacy of a variety of tolerogenic strategies will be evaluated. In this proposal we intend to: (1) Define the morphological and structural remodeling responses of an engineered living blood vessel substitute designed to mimic the microstructure of the native vessel wall. Engineered vessels will be fabricated by seeding primary human vascular wall cells on ultrathin ECM sheets consisting of collagen fibers or a collagen-elastin multilamellar composite. Biomechanical properties will be tuned in response to microstructure, and both biochemical and functional responses defined under simulated physiological conditions. Vessels will be implanted into immunodeficient SRG rats and both phenotypic stability and remodeling responses defined. (2) Generate ‘hypoimmunogenic’ vascular smooth muscle cells and endothelial cells that evade immunological rejection. ECs and SMCs will be derived from hypoimmunogenic hPSCs generated by multiplex genome editing and biological properties determined, including differentiation efficiency, functionality, absence of HLA proteins, and expression of tolerogenic factors. Angiogenic potential and vessel network formation will be assessed in vitro and in vivo. Alloreactivity will be evaluated using an in vitro panel of T cell, NK cell, and macrophage immunoassays, as well as in mice containing human immune system components. (3) Characterize the phenotypic stability, immunogenicity, and remodeling responses of immunoevasive engineered living blood vessels. Engineered vessels comprised of hypoimmunogenic cells will be produced and related biomechanical and biochemical properties characterized. We will determine the capacity of these vessels to maintain phenotypic stability after in vivo implantation in immunodeficient SRG rats. In the final phase of these studies, we will determine the ability of vessels engineered from hypoimmunogenic SMCs and ECs to evade immunological rejection in SRG rats reconstituted with elements of a human immune system.

项目摘要 项目调查人员的最新创新已经为快速建立了一个重要的新框架 以及可扩展的工程活血管的产生。值得注意的是，我们为 多重基因组编辑以生成人类多能干细胞（HPSC），其中HLA -A，-b和-c为 有选择地消除的HLA II类分子消除了，以及包括HLA-G，PD-L1，包括多种耐受因素 和CD47表示。源自这些的血管平滑肌细胞（SMC）和内皮细胞（EC） PSC使用我们先前报道的明确定义的分化方案，受到保护，免受 体外和体内的同种异体免疫排斥。此外，我们开发了新的工程方法 制造机械强大的，独立的，超薄的胶原蛋白板和相关的制造工具 工程活血管的可扩展产生。在此提案中，我们假设免疫撤离 血管可以使用“低免疫原性”细胞和平面细胞外有效，快速生产 矩阵（ECM）定义成分，内容和微体系结构的支架。在此过程中， 将评估各种耐受性策略。在此提案中，我们打算： （1）定义工程活血管的形态和结构重塑反应 替代旨在模仿天然容器壁的微观结构。工程船将是 通过在超薄的ECM片上播种原代人血管壁细胞，由胶原蛋白纤维或 胶原蛋白多层复合材料。生物力学特性将根据微观结构进行调整， 以及在模拟生理条件下定义的生化和功能反应。船将是 植入免疫缺陷的SRG大鼠以及定义的表型稳定性和重塑反应。 （2）产生逃避的“低免疫原性”血管平滑肌细胞和内皮细胞 免疫拒绝。 ECS和SMC将源自由低肿瘤的HPSC。 多重基因组编辑和生物学特性，包括分化效率，功能， HLA蛋白的缺乏和耐受因子的表达。血管生成潜力和血管网络 形成将在体外和体内评估。同种异体反应性将使用体外面板NK评估 细胞和巨噬细胞免疫测定以及含有人类免疫系统成分的小鼠中。 （3）表征表型稳定性，免疫原性和免疫厌氧反应的重塑反应 工程活血管。将产生由低免疫原性细胞组成的工程血管 以及相关的生物力学和生化特性。我们将确定这些能力 免疫缺陷型SRG大鼠体内植入后保持表型稳定性的血管。在最后阶段 在这些研究中，我们将确定从低免疫原性SMC和ECS设计到的血管的能力 SRG大鼠的免疫排斥与人类免疫系统的元素重组。