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

描述（由申请人提供）：根据卫生和人类服务部的数据，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移植。目前用于控制移植排斥反应的药物是非特异性的，因此使患者面临感染疾病和恶性肿瘤的高风险。该项目的动机是移植的独特机会，即分离的器官可以使用离体方法进行治疗。外源性抗炎药可以在移植前引入移植物。白细胞介素-10是一种强大的免疫抑制细胞因子，已被证明可以减轻同种异体移植（或“同种异体移植”）的急性排斥反应，但白细胞介素-10蛋白的非特异性作用限制了其临床应用。该项目的目标是开发将IL-10基因体外导入移植组织内供体树突状细胞（dc）的方法。供体dc的表型决定了受体移植的命运。同种异体移植物中的供体dc在体外被迫表达IL-10时会产生抑制，但悬而未决的问题是供体dc在体内是否会保持抑制。在两种基因不同的小鼠品系之间进行皮肤移植将用于测试体外IL-10基因治疗的能力。小鼠（和人类）皮肤含有高密度的dc，因此很好地代表了通常移植的器官（如肾和肺），其中存在大量相同的细胞。IL-10基因颗粒将使用聚合物颗粒作为DNA载体应用于体外（移植前）异体皮肤移植。该系统需要在显示镍的PLGA颗粒表面使用低分子量多阳离子O10H6作为DNA凝聚剂。镍/his-tag相互作用提供了一种吸附O10H6的机制，释放质子化的鸟氨酸胺以最大限度地捕获DNA。通过对颗粒表面电荷的标定，可以优化不同剂量质粒DNA的负载。在项目第一阶段完成后（具体目标1），我们希望发现用IL-10基因颗粒处理的异体移植物在体内释放具有抑制表型的供体dc。排斥反应的评估将使用氟-19磁共振成像来测量同种异体移植物的局部炎症，这是排斥反应的早期迹象。该目标的关键里程碑是查明供体dc在体内与急性排斥反应延迟相关的表型。在第二阶段（具体目标2），我们希望记录IL-10修饰同种异体移植物对受体小鼠T细胞的反应变化。这一目标的关键里程碑是确定1)T细胞反应的性质和范围，以及2)与体外治疗相关的不良副作用。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。综上所述，我们希望本研究能为在富含dc的器官移植中测试IL-10基因治疗以实现特异性免疫抑制提供强有力的理论依据。

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.