Knockdown of Gene Expression in Immune Cells in Humanized Mice

人源化小鼠免疫细胞基因表达的敲低

• 批准号: 8046953
• 负责人: Judy Lieberman
• 金额: $ 240.4万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8046953
• 关键词: Antibodies; Antibody Formation; Autoimmunity; BCL6 gene; Blocking Antibodies; Bone Marrow; CCR5 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell membrane; Cells; Chimera organism; Chronic; Clinical Trials; Communicable Diseases; Complex; Cytokine Gene; Cytomegalovirus; Dendritic Cells; Development; Disease; Double-Stranded RNA; Engineering; Fetal Liver; Gene Expression; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Polymorphism; Genetic Variation; Glutamate Carboxypeptidase II; Goals; Graft Rejection; HIV; HIV Envelope Protein gp120; HIV Infections; Hematopoietic; Hepatitis C; Human; Humoral Immunities; Immune; Immune System Diseases; Immune response; Immune system; Immunity; Immunodeficient Mouse; Immunologic Deficiency Syndromes; Immunology; In Vitro; Individual; Infection; Injection of therapeutic agent; Intravaginal Administration; Intravenous; Link; Lymphocyte; Lymphoid; Malaria; Methods; Molecular; Mus; Mycobacterium tuberculosis; Myeloid Cells; Natural Killer Cells; Outcome; Pathway interactions; Patients; Peripheral Blood Lymphocyte; Pharmaceutical Preparations; Process; Prostatic Neoplasms; Proteins; RNA Interference; Reagent; Recording of previous events; Regulatory Pathway; Regulatory T-Lymphocyte; Resistance; Role; Signaling Molecule; Small Interfering RNA; Small RNA; Subunit Vaccines; T cell differentiation; T cell response; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Testing; Thymus Gland; Time; Tissues; Transplantation; Vaccination; Vaccine Design; Vaccines; Viral; Viral Genes; Virus; aptamer; base; cell type; chemokine; cytokine; design; genetic manipulation; graft vs host disease; human disease; human tissue; immunopathology; in vivo; inhibiting antibody; killings; knock-down; knockout gene; macrophage; monocyte; peripheral blood; public health relevance; reconstitution; research study; response; targeted delivery; tool; transcription factor; transmission process; tumor; vaccine candidate

DESCRIPTION (provided by applicant): The immune response integrates interactions between diverse cell types, signaling molecules and pathways that can most effectively be studied in vivo. The immune response to infection or vaccination is the outcome of such a complex network of positive and negative regulatory pathways that it is often difficult to predict from in vitro experiments what will happen in vivo. Because immune genes are amongst the most highly evolving genes and the cytokine and chemokine responses in mice and humans often differ, mouse immune responses often differ qualitatively from human responses. Understanding and manipulating human immune responses is challenging, given the genetic diversity and unique history of environmental/infectious exposure of each individual. Experimental manipulations (i.e. gene knockout, blocking antibodies, and cytokines) that have been so powerful for dissecting the molecular basis of mouse immunology are rarely possible in humans. Two recent technologies, RNA interference (RNAi) and humanized mice with robust functional human immune responses, now provide an opportunity for genetic manipulation of the in vivo human immune response. The goal of this proposal is to join these two technologies to establish a method to knock down individual genes in CD4+ and CD8+ cells in humanized mice transplanted with human bone marrow, fetal liver and thymus (BLT mice) and use it to manipulate human immunity in vivo. BLT mice reconstitute an immune system that mimics human immune cell distribution in uninfected mice, as well as viral dynamics and B and T cell responses to HIV infection. RNAi is a ubiquitous mechanism for suppressing gene expression, which can be harnessed to knockdown individual genes by introducing exogenous small double-stranded RNAs with sequences complementary to a target gene (siRNAs) into a cell. The main obstacle to harnessing the power of RNAi for in vivo studies is delivering small RNAs across the plasma membrane, which is especially challenging for lymphocytes, which are both a moving target and resistant to transduction. We engineered chimeric small RNAs, composed of an aptamer that recognizes human CD4, linked at its 3'-end to a siRNA. CD4 aptamer- siRNA chimeras (CD4-AsiC) knockdown genes specifically in human CD4+ cells in peripheral blood, human tissue explants, and in BLT mice after intravaginal administration. Our first aim is to test and optimize CD4- AsiCs for systemic knockdown of gene expression in CD4 T cells, monocytes and macrophages. We will also engineer similar CD8 aptamer-siRNA chimeras for gene knockdown in CD8 T cells and NK cells. To test the utility of the AsiC method for studying human immunity in vivo, we will design CD4-AsiCs to knockdown FOXP3 and BCL6, transcription factors that direct CD4 T cell differentiation into suppressive Treg cells and antibody-promoting TFH cells, respectively. We will use them to test the hypothesis that enhancing TFH cells and suppressing Treg cells augments the humoral response to two HIV candidate vaccines, a gp120 subunit vaccine and whole killed virus. PUBLIC HEALTH RELEVANCE: Attempts to design effective vaccines for many of the world's serious chronic infectious diseases, including HIV, malaria and hepatitis C, have not been successful; vaccines that look promising in other species often fail in human clinical trials. This proposal will develop a powerful tool that will be useful for dissecting the contribution of individual immune cell proteins to human immune responses by knocking down individual genes one at a time in human CD4+ or CD8+ cells in vivo in humanized mice. These tools should not only provide a more scientific basis for vaccine design and understanding human immune protection and immunopathology, but the methods developed in this proposal could also be applied to knockdown gene expression in other cell types to study other complex human disease processes in vivo in human cells; this strategy potentially could also be adapted to design RNAi-based drugs suitable for systemic targeted delivery of small RNAs directed against disease-causing genes.

描述（由申请人提供）：免疫应答整合了不同细胞类型、信号分子和途径之间的相互作用，可以最有效地在体内进行研究。对感染或疫苗接种的免疫应答是阳性和阴性调节途径的复杂网络的结果，因此通常难以从体外实验预测体内会发生什么。由于免疫基因是最高度进化的基因之一，并且小鼠和人类中的细胞因子和趋化因子应答通常不同，因此小鼠免疫应答通常与人类应答在性质上不同。鉴于每个人的遗传多样性和独特的环境/感染暴露史，理解和操纵人类免疫反应具有挑战性。实验操作（即基因敲除、阻断抗体和细胞因子）对于剖析小鼠免疫学的分子基础是如此强大，但在人类中却很少可能。两种最新的技术，RNA干扰（RNAi）和人源化小鼠与强大的功能性人类免疫反应，现在提供了一个机会，在体内人类免疫反应的遗传操作。该提案的目标是将这两种技术结合起来，建立一种方法来敲除移植人骨髓、胎肝和胸腺的人源化小鼠（BLT小鼠）中CD 4+和CD 8+细胞中的单个基因，并利用它来操纵人体免疫力体内。BLT小鼠重建免疫系统，其模拟未感染小鼠中的人类免疫细胞分布，以及病毒动力学和对HIV感染的B和T细胞应答。RNAi是一种普遍存在的抑制基因表达的机制，其可以通过将具有与靶基因互补的序列的外源性小双链RNA（siRNA）引入细胞中来敲低个体基因。利用RNAi的力量进行体内研究的主要障碍是将小RNA递送穿过质膜，这对于淋巴细胞来说尤其具有挑战性，淋巴细胞既是移动的靶标又对转导具有抗性。我们设计了嵌合小RNA，由识别人CD 4的适体组成，在其3 '端连接到siRNA。CD 4适体- siRNA嵌合体（CD 4-AsiC）在外周血、人组织外植体和BLT小鼠中的人CD 4+细胞中特异性敲低基因。我们的第一个目标是测试和优化CD 4-AsiC用于系统性敲低CD 4 T细胞、单核细胞和巨噬细胞中的基因表达。我们还将设计类似的CD 8适体-siRNA嵌合体，用于CD 8 T细胞和NK细胞中的基因敲减。为了测试AsiC方法在体内研究人类免疫的效用，我们将设计CD 4-AsiC以敲低FOXP 3和BCL 6，FOXP 3和BCL 6是分别指导CD 4 T细胞分化为抑制性Treg细胞和抗体促进性TFH细胞的转录因子。我们将使用它们来检验增强TFH细胞和抑制Treg细胞增强对两种HIV候选疫苗（gp 120亚单位疫苗和全灭活病毒）的体液应答的假设。 公共卫生相关性：为世界上许多严重的慢性传染病（包括艾滋病毒、疟疾和丙型肝炎）设计有效疫苗的尝试一直没有成功;在其他物种中看起来有希望的疫苗往往在人体临床试验中失败。该提案将开发一种强大的工具，该工具将有助于通过在人源化小鼠体内敲低人CD 4+或CD 8+细胞中的单个基因一次一个来剖析单个免疫细胞蛋白对人类免疫应答的贡献。这些工具不仅应该为疫苗设计和理解人类免疫保护和免疫病理学提供更科学的基础，而且该提案中开发的方法也可以应用于敲除其他细胞类型中的基因表达，以在人体细胞中研究其他复杂的人体疾病过程;该策略也可能适用于设计基于RNAi的药物，其适合于针对致病基因的小RNA的系统靶向递送。