Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Abstract

22 Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools 23 for immunotherapy. However, their recalcitrance to standard transfection techniques has limited 24 both mechanistic studies and clinical applications. Current approaches for NK cell manipulation 25 rely on viral transduction or methods requiring NK cell activation, which can alter NK cell 26 function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) 27 efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. 28 Compared to electroporation, CARTs transfect NK cells two orders of magnitude more 29 efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype 30 and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric 31 antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our 32 knowledge, CARTs represent the first efficacious transfection technique for resting primary NK 33 cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical 34 applications of this understudied lymphocyte subset. 35 36 37 38 Natural killer (NK) cells are a group of innate lymphocytes that coordinate and execute 39 the rapid elimination of neoplastic and virus-infected cells 1,2 . Upon activation through a 40 combinatorial array of germline-encoded inhibitory and activating receptors, NK cells can 41 directly kill their targets via targeted release of perforin-and granzyme-containing granules, as 42 well as coordinate the downstream immune response by secreting pro-inflammatory cytokines 43 like IFNγ and TNFα 3,4 . The rapid and robust cytotoxicity of NK cells makes them excellent 44 assets for antiviral and anticancer immunotherapy, an enthusiasm most recently bolstered by 45 the use of chimeric antigen receptor (CAR) NK cells in treating CD19 + lymphoid cancers with 46 high efficacy and low toxicity 5-13 . Despite their clinical promise, several fundamental facets of 47 human NK cell biology are poorly understood. For example, the molecular underpinnings of 48 human NK cell memory 14-16 and the precise roles of various NK cell receptors in target cell 49 recognition remain unknown. 50 NK cells are notoriously difficult to manipulate, presenting challenges both for a deeper 51 understanding of fundamental NK cell biology and for the use of NK cells in clinical 52 applications 17-21 . Most clinical trials involving genetically-modified NK cells utilize viral 53 transduction (NCT02944162, NCT02892695, NCT03056339, NCT03579927) 5,22 . While viral 54 transduction enables stable transgene expression, it is costly, laborious, and induces robust NK 55 cell activation and apoptosis due to triggering of innate nucleic acid sensors, limiting its 56 practicality for mechanistic studies of NK cell biology 8,18,23-28 . Further, in clinical applications, 57 viral transduction carries the risks of sustained adverse effects and oncogenic potential ...

Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy

“…Currently, mRNA constructs have been employed to express tumor associated antigens (TAA) and neoantingens for the development of therapeutic and prophylactic vaccines, for the in-situ production of potent monoclonal antibodies and for the engineering of CAR T cells (Kranz et al, 2016;Rybakova et al, 2019;Wilk et al, 2020). The application of mRNA for these strategies will be discuss in the next sections.…”