Abstract 2699: Application of whole genome loss of function CRISPR screen in primary natural killer cells

Abstract Natural killer cells (NKs) are cytotoxic lymphocytes of the innate immune system, operating against infections and cancer. NKs act in an antigen independent manner, preventing graft versus host disease in allogeneic settings. NKs can be isolated from healthy donors, activated, expanded and safely administered to cancer patients. Although NKs are highly cytotoxic against cancer cells in vitro, they lose viability and functionality in the suppressive tumor microenvironment (TME). Until recently, viral transduction of primary NKs was considered a technical hurdle. The latest progress using Baboon Lentiviral (BaLV) pseudotyping substantially increased transduction rates and facilitated the use of chimeric antigen receptor (CAR) NKs for cancer immunotherapies. Due to this challenge, most genetic manipulations were performed in single genes with limited capabilities. Large-scale functional manipulation of NKs using CRISPR-cas9 editing was not implemented, leaving a gap in our understanding of NK suppression. We generated a whole genome library of 55,548 guide RNAs (gdRNAs, 4 per gene), targeting 13,749 genes that were determined to be expressed in NKs and 138 (10%) olfactory receptor genes that are not expressed in NKs as control gdRNAs. The gdRNA plasmid was optimized based on our CAR-NK backbone, to express the gdRNA scaffold under U6 promotor and GFP under EF1α promotor. The library plasmids were sequenced and their gaussian distribution was verified, with high representation of all the different gdRNAs. Primary NKs were isolated from 3 healthy donors and transduced at 25% transduction rates using our optimized BaLV system. After a week expansion in vitro, NKs were electroporated with protein cas9-RFP using the maxCyte electroporator. RFP levels at 6 hours after electroporation were at least 80%. The cells were profiled after electroporation for cytotoxicity and for functionality against the K562 target cell line. IFNγ production and degranulation (CD107a) were similar before and after electroporation. Genomic DNA was extracted from the transduced NKs and the gdRNA barcodes were amplified by PCR and sequenced. The distribution of the gdRNA barcodes after sequencing exhibited high representation of almost all gdRNAs, with minimal loss of gdRNAs. Several gene knockouts were identified as important for survival/proliferation of NKs and are being validated using single gdRNAs in vitro and in vivo. NK cell immunotherapy is a promising approach for cancer treatment. Although recent clinical trials with CAR-NK present exciting results, many solid tumors do not benefit from NK immunotherapies due to the inactivation of NKs in the immune-suppressive TME. Here, we successfully implemented an unbiased genome wide loss-of-function CRISPR screen as an effective and informative method to identify novel candidates for NK cell suppression. Citation Format: Michal Sheffer, Isabel E. Kaplan, Eden Bobilev, Yasmin Z. Abdulhamid, Rizwan Romee. Application of whole genome loss of function CRISPR screen in primary natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2699.