Intracellular delivery of DNA-editing proteins by viscoelastic cell stretching

摘要/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的“活药”，但 生产它们仍然费力、低效且缓慢。主要的瓶颈是如何准确地 极大数量--通常是数十亿--体外培养的人类细胞的DNA变化。用来编辑基因 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 进入人体细胞，特别是免疫细胞的Cas9核酸内切酶的量。一个有希望的方法是 瞬间破坏质膜，允许DNA编辑蛋白直接运输到细胞质中。 然而，当前和新兴的非病毒传递方法不均匀，对细胞造成损害，并且速度太慢 需要数十亿个细胞的临床应用。因此，这项建议的研究目标是发展 一种非常快速的使质膜通透性的微流控方法，以促进DNA的有效传递- 编辑蛋白质。中心的创新是使用粘弹性流体力来拉伸质膜而不需要 接触任何表面的细胞。因此，这种非接触式方法是高效、温和、健壮的，而且出类拔萃 在单个微通道中快速超过每分钟1亿个细胞。项目的K99阶段将专注于 利用CRISPR开发这项高效T细胞基因编辑的技术，以解决主要瓶颈 在T细胞工程方面。在目标1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 以每分钟10亿个细胞的速度进行T细胞工程，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来产生同种异体嵌合抗原受体(CAR)T细胞 原代T细胞，并测定其体外抗肿瘤活性。在R00阶段，粘弹性细胞拉伸将 发展成为可直接移植外源蛋白等的高通量“细胞手术”平台 纳米级的货物进入胞浆，朝着提高安全性、准确性和效率的长期目标前进 人类细胞中的基因编辑。以K99期间获得的知识、技能和技术为基础 阶段，Aim 3将专注于首次以蛋白质形式提供DNA修复因子，如Rad52，以 暂时增加同源定向修复的频率，从而安全地提高 使用CRISPR进行精确的基因编辑。该项目的培训目标是为塞文勒博士提供 生物医学工程背景--来自微流体领域顶尖专家的额外科学培训 (托纳博士，MGH/HMS首席导师)，免疫学(Yarmush博士，MGH/HMS联合导师)，基因和药物输送 (麻省理工学院Bhatia博士)和T细胞工程(MGH/HMS Maus博士、MGH/HMS Choi博士和DFCI/HMS Ritz博士)。 这项额外的培训将为塞文勒博士领导一个独立的生物医学研究项目做好准备 工程学的重点是改进读取和写入生命分子信息的方法。

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.