CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

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. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, 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 and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. 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. ### Competing Interest Statement Dr. Lee reports stock from Courier Therapeutics, personal fees and stock options from Caribou Biosciences, personal fees from Intellia Therapeutics, personal fees from Merck, Sharp, and Dohme, grants, stock, and personal fees from Kiadis Pharma, outside the submitted work; In addition, Dr. Lee has patents US62/825,007; US63/105,722; US62928,524; PCT-370 US201/032,670; WO-2019/222,503-A1; PCT-US2020/018,384; US62/805,394; US62/987,935; US62/900,245; US62/815,625; Self-driving CAR with royalties paid to Kiadis Pharma and Membership on the NIH Novel and Exception Therapies and Research Advisory Committee (NExTRAC). Dr. Naeimi Kararoudi reports personal fees from Kiadis Pharma; In addition, Dr. Naeimi Kararoudi has patents US62/825,007; WO2019222503A1; USPTO63/105,722; PCT/US2020/02545; US63/018,108; US62/928,524; US62/987,935; Self-driving CAR with royalties paid by Kiadis Pharma. Dr. Moriarity is founder of and has sponsor research with Catamaran Bio, he is an inventor of WO2017214569A1. Dr. Meyer and Dr. Likhite report a patent PCT/US2020/025454 with royalties paid by Kiadis. Dr. Chakravarti reports In addition, Dr. Chakravarti has a patent PM21 particles to improve bone marrow homin of NK cells licensed to Kiadis.