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

• 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
• 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

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.

总结/摘要 免疫细胞疗法是治疗癌症和其他疾病的一种强大的新型“活药物”， 生产它们仍然是费力、低效和缓慢的。主要的瓶颈是如何准确地 在体外对极其大量的--通常是数十亿的--人类细胞的DNA进行改变。基因编辑， CRISPR-Cas9比慢病毒或逆转录病毒载体精确得多，但仍然难以提供受控的 在一些实施方案中，本发明提供了将大量的Cas9内切核酸酶导入人细胞，特别是免疫细胞中的方法。一个有希望的方法是 瞬时破坏质膜，允许DNA编辑蛋白直接转运到胞质溶胶中。 然而，目前和新兴的非病毒递送方法是不均匀的，对细胞有损伤，并且对于细胞来说太慢。 需要数十亿细胞的临床应用。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，使质膜透化，以促进DNA的有效递送， 编辑蛋白质。核心创新是使用粘弹性流体力来拉伸质膜， 细胞接触任何表面。因此，这种“非接触式”方法高效、温和、稳健， 在一个微通道中每分钟快速超过1亿个细胞。项目的K99阶段将侧重于 开发这项技术，用CRISPR对T细胞进行有效的基因编辑，以解决主要瓶颈。 在T细胞工程中。在目标1中，我们将开发用于核糖核蛋白递送和同种异体移植的粘弹性拉伸。 T细胞工程以每分钟10亿个细胞的速度进行，我们将描述细胞拉伸的生物学效应 在T细胞上。在目标2中，我们将使用这种方法从人外周血中产生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R 00阶段，粘弹性细胞拉伸将是 开发成一个高通量的“细胞手术”平台，用于直接移植外源蛋白和其他 纳米级货物进入胞质溶胶，朝着提高安全性，准确性和效率的长期目标 人类细胞中的基因编辑。基于K99期间获得的知识、技能和技术， Aim 3将专注于首次以蛋白质形式提供DNA修复因子，如Rad 52， 暂时增加同源性定向修复的频率，从而安全地提高修复的效率。 使用CRISPR进行精确的基因编辑。该项目的培训目标是为Sevenler博士提供 生物医学工程背景，并接受微流体领域领先专家的额外科学培训 (Dr.调色剂，首席导师，MGH/HMS），免疫学（Yarmush博士，共同导师，MGH/HMS），基因和药物输送 (Dr. Bhatia，MIT）和T细胞工程（Maus博士，MGH/HMS，Choi博士，MGH/HMS和Ritz博士，DFCI/HMS）。 这项额外的培训将使Sevenler博士做好准备，领导一个独立的生物医学研究项目。 工程学集中于改进阅读和写入生命分子信息的方法。

项目成果

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