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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：
10047963
负责人：
Sheng Tong
金额：
$ 30.72万
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/10047963
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 Immunosuppressive Agents 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 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 patient subsets personalized strategies preclinical study programmed cell death ligand 1 response success therapeutic genome editing 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.

项目摘要免疫检查点通路的阻断已经成为多种疾病的有希望的治疗策略。癌症。然而，在临床前和临床研究中观察到的对免疫治疗的不同肿瘤反应，促进可针对复杂免疫环境定制的联合免疫疗法的开发个别患者。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。有鉴于此，我们的目标是开发一种用于CRISPR/Cas9的混合纳米颗粒-病毒载体系统。基于体内治疗性基因组编辑，其将用于免疫抑制的多重破坏。肿瘤微环境中的通路。CRISPR/Cas9系统在产生DNA双链方面非常有效。链断裂，从而通过非同源末端连接（NHEJ）途径破坏基因。然而，在这方面，在体内诱导不受控制的CRISPR/Cas9活性可能导致全身遗传毒性。我们将开发一个整合杆状病毒载体（BV）与磁性纳米颗粒的新型体内基因递送系统（MNP）。我们的研究表明，通过利用MNP介导的BV之间的相互作用，边集和内吞作用以及针对昆虫病毒的先天免疫，这种递送系统可以提供 CRISPR/Cas9活性的空间和时间控制。我们将使用MNP-BV系统来提供优化的 CRISPR/Cas9用于肿瘤组织中免疫抑制信号PD-L1和TGF-β的基因破坏。我们将使用两种良好建立的小鼠模型评估CRISPR/Cas9诱导的抗肿瘤免疫应答，免疫原性模型（MC 38），其中PD-1阻断的单药治疗是足够的，并且非免疫原性模型胰腺导管腺癌（PDAC），其描绘了大多数非免疫原性人类实体瘤，需要联合治疗的肿瘤在aim 1研究中，我们将设计和优化CRISPR/Cas9 用于靶向PD-L1和TGF-β的gRNA，将Cas9和gRNA包装到BV载体中，并构建MNP-1。 BV系统。在目标2研究中，我们将评估MNP-BV诱导的细胞培养物中的多重基因破坏，并确定在靶和脱靶插入缺失率。在aim 3研究中，我们将在体内测试对照 CRISPR/Cas9的递送，确定局部基因破坏与在小鼠肿瘤模型中用拮抗剂抗体进行全身阻断。拟议研究的成功将提供了一个多重的瘤内免疫工程平台，为临床转化铺平了道路。用于个性化癌症免疫治疗的高效体内基因组编辑策略。