Intracellular delivery of DNA-editing proteins by viscoelastic cell stretching

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.