Abstract 2751: ENIGMACTM discovery platform enables gene to function target validation for macrophage therapy

Abstract The advent of Single cell RNA sequencing (scRNA-seq) has improved our understanding of cellular heterogeneity by providing in-depth transcriptome information at the single cell level. Several scRNA-seq papers reported sequencing of the tumor CD45 positive fraction, or selected immune cells, giving access to hundreds of sequencing datasets using multiple platforms. Because of the difficulties in performing gene-to-function studies in the immuno-oncology and more precisely in the macrophage field, this “dataset infodemic” did not translate into an abundance of new validated targets. Human macrophages are studied in vitro mainly using cell lines (such as THP1) or monocyte-derived macrophages (MDM) differentiated from human monocytes obtained from blood of healthy donors. However, cell lines are limited by their lack of physiological relevance as in contrast to primary cells they are mutated and fail to respond to, or do not require, important immunological stimulations. MDM cultures are mainly limited by yield, as monocytes from blood do not proliferate in vitro. Moreover, human primary macrophages are not easily genetically modified due to their extreme resistance to transfection and transduction. This phenomenon can be partially explained by their expression of several restriction enzymes such as APOBEC, TRIM and SAMHD1 which hypermutate or degrade the transfected/transduced genetic payload. All these challenges represent a major barrier for target validation and drug screening in the macrophage field. The main goal of the ENIGMAC™ discovery platform is to support gene-to-function studies and to deliver new validated macrophage targets. The platform is designed to develop novel assay systems, unique genome editing methods, and supported by the multi-omics bioinformatics analyses. We use a human Induced Pluripotent Stem Cell (iPSC) line that yields macrophages phenotypically and functionally very similar to MDM. By employing this iPSC system we can produce millions of macrophages per week, which allows multiple high throughput assays to support target validation and drug discovery. Furthermore, we developed several technologies to allow fast and reliable gene editing of macrophages. Our proprietary toolbox allows us to perform gene Knock In (KI), Knock Out (KO) and Knock Down (KD) with high efficiency both at iPSC and macrophage level while maintaining expression/silencing during macrophage differentiation. KD can be performed using a pooled approach that enables screening by flow cytometry-based phenotypes at large scale. In conclusion, the ENIGMAC™ platform represents a unique tool for gene-to-function studies using human macrophages. Notably, it is disease agnostic and can be integrated with a variety of disease-specific conditions and phenotypic readouts. Citation Format: Martha Lopez-Yrigoyen, Thomas W. Monteiro Crozier, Helena Engman, Yuxin Cui, Moritz Haneklaus, Krzysztof B. Wicher, Stephen Myatt, Jeffrey W. Pollard, Luca Cassetta, Carola Ries. ENIGMACTM discovery platform enables gene to function target validation for macrophage therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2751.