In Vivo Delivery of siRNAs as a Novel Approach to Inducing Mixed Chimerism

siRNA 体内递送作为诱导混合嵌合状态的新方法

基本信息

批准号：
7753595
负责人：
Megan Sykes
金额：
$ 26.29万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-15 至 2010-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7753595
关键词：
Achievement Activated Lymphocyte Address Affinity Alloantigen Allogeneic Bone Marrow Transplantation Allogenic Allograft Tolerance Animals Antigens Autoimmune Diseases Binding Bone Marrow Transplantation Cell Culture Techniques Cells Chimeric Proteins Chimerism Chronic Clinic Clinical Complex Cultured Cells Cyclin D1 Data Development Disease Dose Engraftment Gene Expression Goals Hematopoiesis Hematopoietic Hematopoietic stem cells Hemoglobinopathies Human Immune Immune Tolerance Immunoglobulin Fragments Immunosuppression Immunosuppressive Agents Inborn Errors of Metabolism Incidence Individual Integrins Intercellular adhesion molecule 1 Kinetics Knowledge Laboratories Lead Leukocytes Life Ligands Link Lymphocyte Lymphocyte Function-Associated Antigen-1 Marrow Methods Modeling Molecular Conformation Mus Organ Transplantation Patients Peptides Pharmaceutical Preparations Pre-Clinical Model Procedures Protamines RNA Interference Reagent Regimen Small Interfering RNA Solid Source Specificity System T-Cell Activation T-Lymphocyte Testing Therapeutic Therapeutic immunosuppression Time Tissue Grafts Toxic effect Transcript Translations Virus Diseases Whole-Body Irradiation Work anergy base cell type central tolerance conditioning design drug development graft vs host disease in vivo interest novel novel strategies novel therapeutic intervention protein complex public health relevance success

项目摘要

DESCRIPTION (provided by applicant): The success of solid organ transplantation is limited by the toxicity of chronic immunosuppressive drugs and late graft loss due to chronic rejection. These problems would be overcome by induction of donor-specific tolerance. Establishment of mixed allogeneic chimerism using non-myeloablative conditioning achieves this goal in mice with minimal toxicity. Achievement of mixed chimerism requires a method of overcoming the pre- existing T cell barrier to donor marrow engraftment. This can be achieved by combining bone marrow transplantation (BMT) with costimulation blockade using anti-CD154 mAb. However, clinical use of anti-CD154 is associated with thromboembolic complications, and efficacy in large animals is limited. Thus, alternative approaches are needed. The goal of the studies proposed herein is to establish a system of in vivo delivery of small interfering (si)RNAs specifically into activated T cells as a novel therapeutic approach to inducing tolerance to allogeneic bone marrow grafts, thereby achieving durable mixed chimerism and subsequent central deletional tolerance. We hypothesize that in vivo delivery of siRNAs silencing transcripts that are critical for T cell activation, proliferation, and/or survival into T cells at the time of allogeneic BMT will result in anergy and deletion specifically of pre-existing donor-reactive T cells. Mixed allogeneic chimerism and central deletion of newly developing T cells will assure life-long donor-specific tolerance. Dr. Lieberman and colleagues have developed a method to introduce siRNAs specifically into activated lymphocytes using a fusion protein composed of domains 1 and 2 of murine ICAM-1 linked to an siRNA-binding protamine peptide. Since ICAM-1 encounters a high affinity form of its ligand LFA-1 only on activated lymphocytes, this construct will be used to target siRNAs to recipient T cells recognizing donor alloantigens in association with BMT. We aim to: 1) Demonstrate specific binding of the ICAM-1-protamine fusion protein and determine efficiency of delivery and knockdown in cultured cells; 2) Validate and characterize the specificity and efficiency of binding, as well as the level of knockdown induced by the ICAM-1 construct-siRNA complex in vivo; and 3) Test the hypothesis that specific silencing of RasGRP1, cyclin D1, and bcl-xL in activated T cells is sufficient to tolerize donor-reactive T cells, permitting achievement of durable mixed chimerism and subsequent central tolerance. These studies will lead us toward a novel, clinically applicable strategy to achieve durable mixed chimerism and tolerance. PUBLIC HEALTH RELEVANCE: The proposed studies will lead us toward a novel, clinically applicable strategy to achieve durable mixed chimerism and organ allograft tolerance, avoiding the need for chronic immunosuppressive therapy and its attendant toxicities.

描述（申请人提供）：实体器官移植的成功受到慢性免疫抑制药物的毒性和慢性排斥导致的晚期移植物损失的限制。这些问题可以通过诱导供体特异性耐受来克服。使用非清髓性条件建立混合同种异体嵌合体以最小的毒性在小鼠中实现了这一目标。混合嵌合的实现需要一种克服供体骨髓植入之前存在的 T 细胞障碍的方法。这可以通过将骨髓移植 (BMT) 与使用抗 CD154 mAb 的共刺激阻断相结合来实现。然而，抗CD154的临床使用与血栓栓塞并发症相关，并且在大型动物中的疗效有限。因此，需要替代方法。本文提出的研究的目标是建立一种体内递送小干扰（si）RNA特异性进入活化T细胞的系统，作为诱导对同种异体骨髓移植物耐受的新治疗方法，从而实现持久的混合嵌合状态和随后的中枢性嵌合状态。缺失耐受性。我们假设，在同种异体 BMT 时，体内递送对 T 细胞活化、增殖和/或存活至关重要的 siRNA 沉默转录物将导致先前存在的供体反应性 T 细胞的无反应性和特异性缺失。混合同种异体嵌合和新发育 T 细胞的中心缺失将确保终生供体特异性耐受。 Lieberman 博士及其同事开发了一种方法，使用由鼠 ICAM-1 的结构域 1 和 2 与 siRNA 结合鱼精蛋白肽连接组成的融合蛋白，将 siRNA 特异性引入激活的淋巴细胞中。由于 ICAM-1 仅在活化的淋巴细胞上遇到其配体 LFA-1 的高亲和力形式，因此该构建体将用于将 siRNA 靶向识别与 BMT 相关的供体同种抗原的受体 T 细胞。我们的目标是： 1) 展示 ICAM-1-鱼精蛋白融合蛋白的特异性结合，并确定培养细胞中的递送和敲低效率； 2) 验证和表征结合的特异性和效率，以及体内 ICAM-1 构建体-siRNA 复合物诱导的敲低水平； 3) 测试以下假设：活化 T 细胞中 RasGRP1、细胞周期蛋白 D1 和 bcl-xL 的特异性沉默足以耐受供体反应性 T 细胞，从而实现持久的混合嵌合状态和随后的中枢耐受。这些研究将引导我们找到一种新颖的、临床适用的策略来实现持久的混合嵌合和耐受。公共健康相关性：拟议的研究将引导我们找到一种新颖的、临床适用的策略，以实现持久的混合嵌合和器官同种异体移植耐受，避免长期免疫抑制治疗及其伴随的毒性。