Optimization and Validation of CAR Transduction into Human Primary NK Cells Using CRISPR and AAV

Abstract

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity, but genetic modification of primary human NK cells has been problematic compared to the ease in which primary human T cells are transduced. Despite some progress made in using lentiviral and retroviral vectors, the efficiency of viral transduction in NK cells remains relatively low. Additionally, using these viral vectors results in random integration events which can potentially affect the safety or efficacy of transduced immune cells. Recently, we described an efficient method for gene targeting in primary human NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined that approach with Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus, and optimized transduction efficiency using various AAV pseudotypes, single-stranded (ss) or self-complementary (sc) with a wide variety of homology arms, and homology repair (HR) andor non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For initial optimization we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms for HR resulted in optimal transduction efficiency. To validate the process for generating large numbers of CAR-NK cells needed for clinical application, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered by the transgene, and the NK cells maintained high CAR expression (mean 68% CAR+) during expansion. The expanded CD33-CAR-NK cells showed enhanced activation and antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML). Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome and low off-target insertion. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.