High Aspect-Ratio Nanostructures for Genetic Engineering

Background: Chimeric antigen receptor T cell (CAR-T) therapy is a treatment using genetically reprogrammed T cells for cancer immunotherapy. However, it is challenging to transfect T cells with high efficiency while preserving critical biological polyfunctionalities. Viruses and bulk electroporation offer low transfection efficiency, induce aberrant cytokine productions, and delay cell proliferation, leading to less efficacious and more costly treatments. Emerging methods such as nanoparticles suffer from poorly controllable intracellular release of cargo while microfluidics run into high operating costs from use of biomolecule concentrates in continuous flow.<br/><br/>Methods: Here, we describe a high aspect-ratio nanostructure-based method named magnetic nano-electro-injection (MagNEI) platform for T cell immuno-engineering. During MagNEI transfection, localized electric fields transiently open membrane pores of cells magnetically stabilized onto hollow nanochannels. The electric fields then electrophoretically inject DNA into T cells. Once DNA enters the cells, magnetic forces are applied <i>via</i> FDA-approved Dynabeads to preferentially transport them into the nuclei.<br/><br/>Results: MagNEI provided up to 90/80/50% delivery efficiency for high molecular weight proteins, mRNA, and DNA. The long-term, stable expression of DNA plasmids was 3-4 folds better than gold standard AAVs and Lonza bulk electroporation. While viruses and Lonza bulk electroporation adversely reduced T cell proliferation by 30% and T cell migration by 80%, MagNEI did not. MagNEI also did not cause significant increase in IL-6 cytokine productions or changes in gene expression relative to untreated control T cells.<br/><br/>Conclusions: Our results demonstrate the technical and biological superiority of MagNEI as a transfection method. We envision that the use of MagNEI platform can overcome transfection difficulties for genetic engineering of primary immune cells to advance CAR-T therapy.