CRISPR-Cas9 Modified iPSC-CAR-NK Cell for Lymphoma Cancer Treatment

Abstract

The human immune system is composed of a network of interactions between physical barriers, cells, and proteins aiming to defend the body from foreign and internal threats. However, lymphoma occurs when T or B lymphocytes from the adaptive immune system begin behaving abnormally through unchecked growth or increased survival. Though there is no cure for lymphoma, many different prospective treatment methods are available for patients, including CAR therapy. CAR therapy employs genetically edited recombinant antigen receptors on immune effector cells to significantly enhance and redirect the specificity of the cells’ antitumor activity. Out of all immune system cells, NK cells remain the most prospective cells for CAR therapy due to their activity, superior safety relative to T cells, and their high potential for “off-the-shelf” production. In this paper, we will explore modifications that would theoretically maximize the activity and efficacy of NK cells in response to lymphoma. After considering various factors for cell design, I hypothesize that the most advantageous features for CAR-NK therapy are the addition of IL-15R alpha receptor, removal of NKG2A inhibitory receptor, and enhancement of HLA-E expression on iPSC-derived CAR-NK cells. Currently, with the simplicity and efficiency of CRISPR-Cas9 gene-editing technology, we will employ the Cas9 double nicking approach by electroporating plasmids into undifferentiated iPSCs to make desired changes to the expression of any gene in NK cells. Once we produce the modified NK cells, we will monitor their functionality and cytotoxicity through various experiments, including chromium release assay, flow cytometry, immunofluorescence microscopy, and enzyme-linked immunosorbent assay.