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Controllable In Vivo Genome Editing for Immune-Checkpoint Blockade in Solid Tumors

用于实体瘤免疫检查点封锁的可控体内基因组编辑

基本信息

批准号：
9939589
负责人：
Sheng Tong
金额：
$ 49.44万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-21 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9939589
关键词：
Address Advanced Malignant Neoplasm Adverse effects Agonist Allografting Antibodies Antigen-Antibody Complex Antitumor Response Biodistribution CRISPR/Cas technology Cancer Patient Cell Culture Techniques Clinical Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Colon Adenocarcinoma Combination immunotherapy Combined Modality Therapy Complex Coupled DNA Double Strand Break DNA Sequence Data Development Disease remission Drug Kinetics Endocytosis Engineering Gene Deletion Genes Genetic Genome Guide RNA Human Hybrids Immune Immune checkpoint inhibitor Immune response Immune system Immunity Immunologics Immunooncology Immunotherapeutic agent Immunotherapy In Vitro Individual Insect Viruses Lead Light MC38 Magnetic nanoparticles Magnetism Malignant Neoplasms Mediating Modeling Modification Mus Natural Immunity Nonhomologous DNA End Joining Normal tissue morphology OX40 Organ PD-1 blockade PD-L1 blockade Pancreatic Ductal Adenocarcinoma Pathologic Processes Pathway interactions Phenotype Proteins Signal Transduction Solid Neoplasm Specificity System Testing Therapeutic Therapeutic Effect Tissues Treatment Efficacy Tumor Immunity Tumor Tissue Viral Viral Vector anti-cancer anti-tumor immune response base cancer heterogeneity cancer immunotherapy cancer type clinical application clinical translation design engineered nucleases gene delivery system genome editing genotoxicity human disease image guided immune checkpoint immune checkpoint blockade immunoengineering immunogenic in vivo individual patient insertion/deletion mutation iron oxide nanoparticle magnetic field mouse model multiplexed imaging novel nuclease pancreatic ductal adenocarcinoma model patient subsets personalized strategies preclinical study programmed cell death ligand 1 response success therapeutic genome editing therapeutically effective transduction efficiency tumor tumor microenvironment vector vector-induced viral nanoparticle

项目摘要

Project Summary The blockade of immune-checkpoint pathways has emerged as a promising therapeutic strategy for a variety of cancers. However, the diverse tumor responses to immunotherapy seen in preclinical and clinical studies prompt the development of combination immunotherapies that can be tailored to the complex immune milieu of individual patients. On the other hand, the severe adverse effects associated with the combination therapies with multiple antagonist antibodies address the necessity for alternative safe and effective therapeutic approaches. In light of this, we aim to develop a hybrid nanoparticle-viral vector system for CRISPR/Cas9- based in vivo therapeutic genome editing, which will be used for multiplexed disruption of immune suppressive pathways in the tumor microenvironment. CRISPR/Cas9 systems are very efficient in generating DNA double- strand breaks, thus disrupting genes through the non-homologous end-joining (NHEJ) pathway. However, inducing uncontrolled CRISPR/Cas9 activities in vivo may lead to systemic genotoxicity. We will develop a novel in vivo gene delivery system that integrates a baculoviral vector (BV) with magnetic nanoparticles (MNPs). Our studies have shown that by taking advantage of the interplay between the MNP-mediated BV margination and endocytosis and the innate immunity against insect viruses, this delivery system can provide spatial and temporal control of CRISPR/Cas9 activity. We will use the MNP-BV system to deliver optimized CRISPR/Cas9 for gene disruption of immune suppressive signals PD-L1 and TGF- in the tumor tissue. We will evaluate CRISPR/Cas9 induced anti-tumor immune responses using two well-established mouse models, an immunogenic model (MC38) where monotherapy with PD-1 blockade is sufficient, and non-immunogenic models pancreatic ductal adenocarcinoma (PDAC) which portrays most non-immunogenic human solid tumors that require combination strategies. In aim 1 studies, we will design and optimize CRISPR/Cas9 gRNAs for targeting PD-L1 and TGF-, package Cas9 and gRNAs into a BV vector, and construct the MNP- BV system. In aim 2 studies, we will evaluate MNP-BV-induced multiplexed gene disruption in cell culture, and determine the on-target and off-target indel rates. In aim 3 studies, we will test the controlled in vivo delivery of CRISPR/Cas9, determine the immunological and therapeutic effects of local gene disruption vs. systemic blockade with antagonist antibody in mouse tumor models. The success of the proposed studies will provide a multiplexed intratumoral immunoengineering platform and pave the way for the clinical translation of a highly efficient in vivo genome editing strategy for personalized cancer immunotherapy.

项目摘要阻断免疫检查点通路已成为治疗多种疾病的一种有希望的策略。癌症。然而，在临床前和临床研究中看到的对免疫治疗的不同肿瘤反应促进针对复杂免疫环境量身定做的联合免疫疗法的发展个别病人的情况。另一方面，与联合治疗相关的严重不良反应使用多种拮抗剂抗体解决了替代安全有效治疗的必要性接近了。有鉴于此，我们的目标是开发一种针对CRISPR/Cas9的纳米颗粒-病毒混合载体系统。基于体内治疗性基因组编辑，将用于免疫抑制的多路中断肿瘤微环境中的通路。CRISPR/Cas9系统在产生DNA双基因的过程中非常有效链断裂，从而通过非同源末端连接(NHEJ)途径扰乱基因。然而，在体内诱导不受控制的CRISPR/Cas9活性可能导致全身遗传毒性。我们将开发一种整合杆状病毒载体和磁性纳米颗粒的新型体内基因递送系统 (MNPs)。我们的研究表明，通过利用MNP介导的BV之间的相互作用边际作用和内吞作用以及对昆虫病毒的天然免疫力，这种递送系统可以提供 CRISPR/Cas9活性的时空控制。我们将使用MNP-BV系统提供优化的 CRISPR/CAS9用于阻断肿瘤组织中免疫抑制信号PD-L1和转化生长因子-的基因。我们将使用两个成熟的小鼠模型评估CRISPR/Cas9诱导的抗肿瘤免疫反应，一种免疫原性模型(MC38)，其中PD-1阻断的单一治疗就足够了，并且是非免疫原性的模型胰腺导管腺癌(PDAC)，它描绘了大多数非免疫原性的人类实体需要联合治疗的肿瘤。在目标1研究中，我们将设计和优化CRISPR/CAS9 靶向PD-L1和转化生长因子-的GRNAs，将CAS9和GRNAs包装到BV载体中，构建MNP- BV系统。在Aim 2研究中，我们将评估MNP-BV在细胞培养中诱导的多重基因破坏，并确定目标上和目标外的插入率。在Aim 3研究中，我们将在体内测试对照 CRISPR/Cas9的交付，确定局部基因破坏的免疫和治疗效果与拮抗抗体对小鼠肿瘤模型的全身阻断作用。拟议研究的成功将提供多元化的肿瘤内免疫工程平台，为临床翻译铺平道路一种用于个性化癌症免疫治疗的高效体内基因组编辑策略。