Intracellular delivery of DNA-editing proteins by viscoelastic cell stretching

通过粘弹性细胞拉伸在细胞内递送 DNA 编辑蛋白

基本信息

批准号：
10675729
负责人：
Derin Sevenler
金额：
$ 13.64万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-02 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10675729
关键词：
Address Allogenic Biological Biological Assay Biomanufacturing Biomedical Engineering Biophysics Biopolymers CD8B1 gene CRISPR/Cas technology Cell Therapy Cell membrane Cells Cellular immunotherapy Charge Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Communicable Diseases Cytosol DNA DNA Repair DNA delivery Dana-Farber Cancer Institute Data Devices Dextrans Disease Drug Delivery Systems Electroporation Emerging Technologies Ensure Face Frequencies Future Gene Delivery General Hospitals Genes Goals Human Immune Immunology In Vitro Jurkat Cells Knock-out Knowledge Lead Lentivirus Vector Life Liquid substance Malignant Neoplasms Measures Medical Medicine Mentors Methods Microfluidics Molecular Mus Oncology Operative Surgical Procedures Permeability Phase Phenotype Predisposition Production Proteins RAD52 gene Reading Reagent Research Retroviral Vector Ribonucleoproteins Safety Stretching Surface Suspensions System Systems Biology T cell therapy T-Cell Activation T-Cell Receptor T-Lymphocyte Technology Time Touch sensation Training Transfection Translating Transplantation Viral Vector Writing cell injury cell killing chimeric antigen receptor chimeric antigen receptor T cells clinical application endonuclease engineered T cells exhaustion gene therapy genomic locus immunocytochemistry improved in vivo innovation interest knockout gene medical schools nanoscale neoplastic cell programs receptor expression repaired scale up skills synthetic biology technology platform tumor vector viscoelasticity

项目摘要

Summary/Abstract Immune cell therapies are a powerful new class of “living medicines” for treating cancer and other diseases, but producing them remains laborious, inefficient, and slow. The chief bottleneck is the challenge of making accurate changes to the DNA of extremely large numbers—often billions—of human cells ex vivo. Gene editing with CRISPR-Cas9 is much more precise than lentiviral or retroviral vectors, but it remains difficult to deliver controlled amounts of the Cas9 endonuclease into human cells, particularly immune cells. A promising approach is to momentarily disrupt the plasma membrane, allowing direct transport of DNA-editing proteins into the cytosol. However, current & emerging nonviral delivery methods are nonuniform, damaging to cells, and too slow for clinical applications that require billions of cells. Therefore, the research objective of this proposal is to develop a very fast microfluidic method of permeabilizing the plasma membrane to facilitate efficient delivery of DNA- editing proteins. The central innovation is to use viscoelastic fluid forces to stretch the plasma membrane without cells touching any surfaces. As a result, this “contactless” approach is efficient, gentle, robust, and extraordinarily fast—exceeding 100 million cells per minute in a single microchannel. The K99 phase of the project will focus on developing this technology for efficient gene editing of T cells with CRISPR, to address the main bottleneck in T cell engineering. In Aim 1, we will develop viscoelastic stretching for ribonucleoprotein delivery and allogenic T cell engineering at one billion cells per minute, and we will characterize the biological effects of cell stretching on T cells. In Aim 2, we will use this method to generate allogenic chimeric antigen receptor (CAR) T cells from primary T cells, and assay their anti-tumor potency in vitro. In the R00 phase, viscoelastic cell stretching will be developed into a high throughput “cell surgery” platform for directly transplanting exogenous proteins and other nanoscale cargoes into the cytosol, towards the long-term goal of increasing the safety, accuracy, and efficiency of gene editing in human cells. Building upon the knowledge, skills, and technologies gained during the K99 phase, Aim 3 will focus on delivering DNA repair factors such as Rad52 in protein form for the first time, to temporarily increase the frequency of homology-directed repair and thereby safely increase the efficiency of precision gene editing with CRISPR. The training objective of this project is to provide Dr. Sevenler—who has a background in biomedical engineering—with additional scientific training from leading experts in microfluidics (Dr. Toner, lead mentor, MGH/HMS), immunology (Dr. Yarmush, co-mentor, MGH/HMS), gene & drug delivery (Dr. Bhatia, MIT), and T cell engineering (Dr. Maus, MGH/HMS, Dr. Choi, MGH/HMS and Dr. Ritz, DFCI/HMS). This additional training will prepare Dr. Sevenler to lead an independent research program in biomedical engineering focused on improved methods of reading and writing the molecular information of life.

摘要/摘要免疫细胞疗法是治疗癌症和其他疾病的一类强大的新型“活药物”，但是生产它们仍然是费力、低效且缓慢的，主要瓶颈是精确制造的挑战。通过离体基因编辑对大量（通常是数十亿）人类细胞的 DNA 进行改变。 CRISPR-Cas9 比慢病毒或逆转录病毒载体精确得多，但仍难以提供受控的病毒将一定量的 Cas9 核酸内切酶引入人体细胞，特别是免疫细胞中，是一种有希望的方法。暂时破坏质膜，允许 DNA 编辑蛋白直接转运到细胞质中。然而，当前和新兴的非病毒传递方法不均匀，对细胞有损害，而且速度太慢。需要数十亿个细胞的临床应用因此，本提案的研究目标是开发。一种非常快速的微流体方法，可透化质膜，以促进 DNA 的有效传递编辑蛋白质的核心创新是利用粘弹性流体力来拉伸质膜，而无需因此，这种“非接触式”方法高效、温和、稳健且非凡。快速——单个微通道中每分钟超过 1 亿个细胞，该项目的 K99 阶段将重点关注。开发这项利用 CRISPR 对 T 细胞进行高效基因编辑的技术，以解决主要瓶颈在T细胞工程中，我们将开发用于核糖核蛋白递送和同种异体的粘弹性拉伸。 T 细胞工程以每分钟 10 亿个细胞的速度进行，我们将表征细胞拉伸的生物效应在目标 2 中，我们将使用这种方法从 T 细胞中生成同种异体嵌合抗原受体 (CAR) T 细胞。原代T细胞，并在体外测定其抗肿瘤效力。在R00期，粘弹性细胞拉伸。发展成为高通量“细胞手术”平台，可直接移植外源蛋白等将纳米级货物进入细胞质，以实现提高安全性、准确性和效率的长期目标基于 K99 期间获得的知识、技能和技术。在第一阶段，Aim 3 将专注于首次以蛋白质形式提供 DNA 修复因子，例如 Rad52，以暂时增加同源定向修复的频率，从而安全地提高修复的效率该项目的培训目标是为 Sevenler 博士提供使用 CRISPR 的精准基因编辑技术。具有生物医学工程背景——接受微流体领域领先专家的额外科学培训（Toner 博士，MGH/HMS 首席导师）、免疫学（Yarmush 博士，MGH/HMS 联合导师）、基因与药物输送（Bhatia 博士，麻省理工学院）和 T 细胞工程（Maus 博士，MGH/HMS、Choi 博士，MGH/HMS 和 Ritz 博士，DFCI/HMS）。这项额外的培训将为 Sevenler 博士领导生物医学领域的独立研究项目做好准备工程学专注于改进读写生命分子信息的方法。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Rapid prototyping for high-pressure microfluidics.

DOI：
10.1038/s41598-023-28495-2
发表时间：
2023-01-22
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Rein, Carlie;Toner, Mehmet;Sevenler, Derin
通讯作者：
Sevenler, Derin

Attomolar sensitivity microRNA detection using real-time digital microarrays.

DOI：
10.1038/s41598-022-19912-z
发表时间：
2022-09-28
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Kanik, Fulya Ekiz;Celebi, Iris;Sevenler, Derin;Tanriverdi, Kahraman;Unlu, Nese Lortlar;Freedman, Jane E.;Unlu, M. Selim
通讯作者：
Unlu, M. Selim