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Non-viral Genetic Modification of Antigen-presenting Cells in Allografts

同种异体移植物中抗原呈递细胞的非病毒遗传修饰

基本信息

批准号：
8035128
负责人：
WILSON S MENG
金额：
$ 35.71万
依托单位：
DUQUESNE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035128
关键词：
Acute Adjuvant Adsorption Adverse effects Allogenic Allografting Amines Anti-Inflammatory Agents Anti-inflammatory Antigen-Presenting Cells Attenuated Back Biomedical Research C57BL/6 Mouse Cells Charge Clinic Clinical Collaborations Communicable Diseases Complex Cytotoxic T-Lymphocytes DNA DNA Binding Data Dendritic Cells Development Dose Drug usage Frequencies Gene Transfer Gene-Modified Generations Genes Genetic Goals Graft Rejection Health Human IL10 gene Immune system Immunosuppression Immunosuppressive Agents In Vitro Inbred BALB C Mice Inflammation Inflammatory Interleukin-10 Kidney Kidney Transplantation Lead Leukocytes Longevity Lung Lung Transplantation Lymphocyte Suppression Magnetic Resonance Magnetic Resonance Imaging Malignant Neoplasms Measures Mediating Methods Modeling Modification Molecular Weight Morbidity - disease rate Mouse Strains Mus Nature Nickel Organ Organ Transplantation Ornithine Outcome Patients Pediatric Hospitals Pharmaceutical Preparations Phase Phenotype Proteins Recombinant Interleukin-10 Regulatory T-Lymphocyte Research Rodent Role Scientist Skin Skin Transplantation Skin graft Solid Specificity Surface System T cell response T-Lymphocyte Testing Therapeutic Therapeutic Uses Transfection Translations Transplant Recipients Transplantation Transplanted tissue United States United States Dept. of Health and Human Services Viral Antigens allograft rejection cytokine density design functional outcomes gene therapy high risk immunogenicity implantation improved in vivo lymph nodes molecular marker mortality particle plasmid DNA polycation research study skin allograft success vector virus genetics

项目摘要

DESCRIPTION (provided by applicant): According to the Department of Health and Human Services, more than 25,000 patients undergo solid organ transplantation every year in the U.S. Approximately 100,000 patients are currently waiting for compatible transplants. The current drugs used in controlling transplant rejection are non-specific, thereby subjecting patients to high risks of infectious diseases and malignancies. This project is motivated by the unique opportunity in transplantation in that isolated organs can be treated using ex vivo methods. Exogenous anti-inflammatory agents can be introduced into transplants prior to implantation. Interleukin-10 is a powerful immunosuppressive cytokine that has been shown to attenuate acute rejection of allogeneic transplant (or "allografts"), but the non-specific effects of IL-10 protein limit its clinical use. The goal of this project is to develop methods to introduce the IL-10 gene ex vivo into donor dendritic cells (DCs) reside within transplant tissues. The phenotype of donor DCs dictates the fate of transplants in recipients. While donor DCs in allografts become inhibitory when forced to express IL-10 in vitro, the outstanding question is whether the donor DCs will remain inhibitory in vivo. Skin grafting between two genetically distinct mouse strains will be used to test the ability of the ex vivo IL-10 gene treatment. The mouse (and human) skin contains high density of DCs, therefore a good representation of commonly transplanted organs (e.g. kidney and lung) in which a significant number of the same cells resides. IL-10 gene particles will be applied to skin allografts ex vivo (prior to transplantation) using polymeric particles as a DNA carrier. The system entails using the low molecular weight polycation O10H6 as a DNA condensing agent on the surface of nickel-displaying PLGA particles. The nickel/his-tag interaction provides a mechanism for adsorbing O10H6, freeing the protonated ornithine amines to maximize DNA capture. By calibrating the particles' surface charge loading of different doses of plasmid DNA can be optimized. Upon completion of the first phase of the project (specific aim 1), we expect to find that allografts treated ex vivo with the IL-10 gene particles to release in vivo donor DCs with inhibitory phenotypes. Rejection will be assessed using florine-19 magnetic resonance imaging to measure in allografts localized inflammation, an early sign of rejection. The key milestone of this aim is to pinpoint the phenotype of donor DCs in vivo associated with a delay in acute rejection. In the second phase (specific aim 2), we expect to document changes in the recipient mice's T cells in respond to IL-10 modified allografts. Key milestones of this aim are to determine 1) the nature and scope of the T cell response, and 2) unwanted side effects associated with the ex vivo treatment. The proposed experiments will be carried out in collaborations with scientists at the Pittsburgh NMR Center for Biomedical Research and Children's Hospital of Pittsburgh. In summary, we expect the research to provide strong rationale for testing ex vivo IL-10 gene therapy in transplantation of DC-rich organs to achieve specific immunosuppression. PUBLIC HEALTH RELEVANCE: This project emphasizes the development of an ex vivo gene therapy approach to attenuate acute transplant rejection. It aims to test if the longevity of transplants can be extended by forcing white blood cells associated with transplant organs to produce interleukin-10. A polymeric system with low inflammatory potential will be used in ex vivo IL-10 gene transfection of skin transplants in mice. The research entails characterization of the donor's white blood cells in the recipient, how the recipient's immune system responds toward the modified transplants, and the extent to which the transplant remains viable in recipient mice. Successful completion of the study will lead to improved health outcome of a significant number of patients by decreasing the need for indefinite use of non-specific immunosuppressants in transplant recipients.

描述（由申请人提供）：根据美国卫生与公众服务部的数据，美国每年有超过25，000名患者接受实体器官移植，目前约有100，000名患者正在等待相容的移植。目前用于控制移植排斥反应的药物是非特异性的，从而使患者面临感染性疾病和恶性肿瘤的高风险。该项目的动机是移植中的独特机会，因为离体器官可以使用离体方法进行治疗。外源性抗炎剂可以在植入前引入移植物中。白细胞介素-10是一种强大的免疫抑制细胞因子，已显示其减弱同种异体移植物（或“同种异体移植物”）的急性排斥，但IL-10蛋白的非特异性作用限制了其临床应用。本项目的目标是开发将IL-10基因离体导入移植组织内的供体树突状细胞（DC）的方法。供体DC的表型决定了受体中移植物的命运。尽管同种异体移植物中的供体DC在体外被迫表达IL-10时变得具有抑制性，但突出的问题是供体DC在体内是否仍具有抑制性。两种遗传上不同的小鼠品系之间的皮肤移植将用于测试离体IL-10基因治疗的能力。小鼠（和人）皮肤含有高密度的DC，因此是其中存在大量相同细胞的通常移植器官（例如肾和肺）的良好代表。使用聚合物颗粒作为DNA载体，将IL-10基因颗粒应用于离体皮肤同种异体移植物（移植前）。该系统需要使用低分子量聚阳离子O 10 H6作为镍显示PLGA颗粒表面上的DNA缩合剂。镍/组氨酸标签相互作用提供了一种吸附O 10 H6的机制，释放质子化的鸟氨酸胺以最大限度地捕获DNA。通过校准颗粒的表面电荷，可以优化不同剂量的质粒DNA的负载。在完成该项目的第一阶段（具体目标1）后，我们期望发现用IL-10基因颗粒离体处理的同种异体移植物在体内释放具有抑制表型的供体DC。排斥反应将通过florine-19磁共振成像来评估，以测量同种异体移植物中的局部炎症，这是排斥反应的早期迹象。这一目标的关键里程碑是确定与急性排斥反应延迟相关的体内供体DC的表型。在第二阶段（具体目标2），我们希望记录受体小鼠T细胞对IL-10修饰的同种异体移植物的反应变化。这一目标的关键里程碑是确定1）T细胞应答的性质和范围，以及2）与离体治疗相关的不必要的副作用。拟议的实验将与匹兹堡核磁共振生物医学研究中心和匹兹堡儿童医院的科学家合作进行。总之，我们期望该研究为在富含DC的器官移植中测试离体IL-10基因治疗以实现特异性免疫抑制提供强有力的理论基础。公共卫生相关性：该项目强调开发一种减轻急性移植排斥反应的离体基因治疗方法。它旨在测试是否可以通过迫使与移植器官相关的白色血细胞产生白细胞介素10来延长移植的寿命。具有低炎症潜能的聚合物系统将用于小鼠皮肤移植物的离体IL-10基因转染。这项研究需要对受体中供体的白色血细胞进行表征，受体的免疫系统如何对修饰的移植物做出反应，以及移植物在受体小鼠中保持活力的程度。本研究的成功完成将通过减少移植受者无限期使用非特异性免疫抑制剂的需求，改善大量患者的健康结局。