Abstract
Membrane-disruption-based methods that enable intracellular delivery of various cargo types for clinical applications have been proposed as attractive candidates as next-generation delivery modalities because of potential benefits including safety, regulation and production [1]. Electroporation is the most widely used such method, but while it can be efficient for delivery of nucleic acids to some cell types, toxicity can be high, particularly in primary cells. We recently reported a novel vector-free technology, Solupore™ that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying compositions, properties and sizes [2]. A permeabilizing delivery solution that contains a low level of ethanol is used as the permeabilizing agent. The technology achieves delivery and reversal of cell permeabilization by precisely controlling the contact of the target cells with this solution. The procedure is rapid and is completed in less than 5 min. We have demonstrated that delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner and we have termed the method ‘soluporation’.In this study, we evaluated the ability of Solupore™ to deliver mRNA to primary human T cells and compared this with electroporation. GFP mRNA was delivered to T cells. GFP expression and cell viability were measured at 24 hr post-transfection. Cell proliferation was assessed at days 1-7 using an automated cell counter and functionality was examined by measuring IFNγ secretion.GFP expression in ‘soluporated’ cells ranged between 30-40% while expression in electroporated cells was variable and ranged between 60 and 98%. The viability of soluporated cells was similar to control untreated cells whereas viability in electroporated cells was reduced by 40-50%. The proliferation of soluporated cells over 7 days following transfection was similar to control cells while the proliferation of electroporated cells was significantly reduced. IFNγ secretion by soluporated cells was also unchanged compared to control while that of electroporated cells was significantly reduced.This study demonstrates that Solupore™ can achieve mRNA delivery to T cells with retention of cell viability and retention of proliferation and IFNγ capacity. The technology is gentle yet highly reproducible, compatible with high throughput and automated cell processing and has the potential to enable a broad range of T cell engineering applications.
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