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.

项目概要 项目研究人员最近的创新为快速研究建立了一个重要的新框架 值得注意的是，我们设计了新的方案。 多重基因组编辑生成人类多能干细胞 (hPSC)，其中 HLA-A、-B 和 -C 选择性消融、HLA II 类分子消除以及多种耐受因子，包括 HLA-G、PD-L1、 和 CD47 表达于这些细胞中。 PSCs，使用我们之前报道的化学定义的分化方案，受到保护 此外，我们还开发了新的工程方法。 制造机械坚固、独立式、超薄胶原蛋白片和相关制造工具 在这项提案中，我们假设免疫逃避。 使用“低免疫原性”细胞和平面细胞外可以有效、快速地制造血管 具有明确成分、内容和微结构的基质（ECM）支架在此过程中具有功效。 在本提案中，我们打算： (1) 定义工程活血管的形态和结构重塑反应 旨在模仿天然血管壁微观结构的替代品将是。 通过将原代人血管壁细胞接种在由胶原纤维或组成的超薄 ECM 片上制成 胶原蛋白-弹性蛋白多层复合材料的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。 植入免疫缺陷 SRG 大鼠并确定表型稳定性和重塑反应。 (2) 产生“低免疫原性”的血管平滑肌细胞和内皮细胞来逃避 EC 和 SMC 来源于免疫原性低的 hPSC。 确定多重基因组编辑和生物学特性，包括分化效率、功能、 缺乏 HLA 蛋白以及血管生成潜力和血管网络的表达。 将在体外评估形成，并使用体外 T 细胞、NK 组评估同种异体反应性。 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。 (3) 表征免疫逃避的表型稳定性、免疫原性和重塑反应 将产生由低免疫原性细胞组成的工程血管。 我们将确定这些特征的相关生物力学和生化特性。 在免疫缺陷 SRG 大鼠体内植入后，血管维持表型稳定性。 在这些研究中，我们将确定由低免疫原性 SMC 和 EC 改造而成的血管的能力 用人类免疫系统的成分重建的 SRG 大鼠可避免免疫排斥。