UCLA

Cost-effective, efficient, vector-free, and nontoxic intracellular delivery technologies are required for a broad range of clinical and laboratory applications. In the context of the recent revolution in precise genomic editing enabled by the discovery and engineering of editing machinery (e.g., CRISPR/Cas9, base editors, etc.), introducing biomolecular payloads into target cells with high efficiency and minimal disruption to cell homeostasis has been become paramount. Methods for cell permeabilization that employ mechanical deformation have gained popularity as they may be less disruptive to cell biology than (traditional) electroporation. However, these methods require acquisition of specialized equipment and extensive training, which can be a hurdle for laboratories, especially those located in low resource areas of the world. Here, we report the development of a table-top “filtroporation” cell-processing technology that is efficient, non-toxic, scalable, and that does not require additional costly specialized materials or equipment. This platform utilizes commercially available poly(ethylene terephthalate) cell culture inserts and laboratory house vacuum. As cells are passed through the inserts’ pores, they become transiently permeabilized. Our studies show that this platform can deliver CRISPR/Cas9 ribonucleoproteins (RNPs) to effect gene knockout in CD34+ hematopoietic stem and progenitor cells (HSPCs) with high efficiency and minimal toxicity targeting the genomic locus relevant for treatment of β hemoglobinopathies, beta globin (HBB). We performed RNA-Seq and compared our results to electroporation, showing that filtroporation induces less apoptosis and inflammation, and preserves stem cell self-renewal potential. Additionally, we studied the membrane repair kinetics following filtroporation-induced permeabilization and found that the repair process occurs within 30 s of treatment. Our knockout studies revealed that membrane repair is, at least partly, mediated by inward scission of the membrane, suggesting that pores formed on the surface of treated cells are in the 50 – 100 nm range. The commercially available membranes employed for filtroporation are limited by low porosity (<1%) and the paucity of pore diameters available from commerical sources. We expand the reach of filtroporation by manufacturing custom membranes by microfabrication. We retain full control over pore diameter, distribution, and membrane thickness, enabling customization to apply filtration-mediated cell permeabilization to any cell type, as well as to scale up for clinical applications. We report our efforts to improve upon existing protocols for manufacturing of low- and high-porosity parylene C films.