RNAi Screening in Hematopoietic Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The discovery and development of RNAi and CRISPR/Cas9 genetic screening technologies have provided researchers with invaluable tools for wide-scale and rapid genetic screening. A theme of our research program has been to develop methodology for efficient application of these screening technologies in immune cell lineages, and to implement screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our efforts are generally focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to collaborative efforts in the LISB to generate integrated models of host-pathogen interactions. In the context of our screening projects, precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells, heating, and electrochemistry at the electrode-electrolyte interface. We previously described the use of microfabricated electrodes based on the conducting polymer PEDOT:PSS, which substantially increases cell viability and transfection efficiency. As a proof-of-concept, we demonstrated the enhanced delivery of Cas9 protein, gRNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enabled rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms. In FY25 we leveraged this platform, coined BADGER, to generate genetically engineered myeloid cells (GEMys), addressing key barriers to clinical translation of macrophage-based immunotherapies. Both therapeutic strategies, cytokine knock-in and immunosuppressive gene knock-out, were functionally validated in vitro, and in the case of STAT6 KO, also demonstrated therapeutic efficacy in vivo. The ability to improve transduction efficiency and insert large gene constructs enables more complex, programmable therapies. These findings support the use of BADGER as a platform for scalable and flexible cell therapy manufacturing. In collaboration with Rosie Kaplan’s NCI research group, the development of inducible IL-12 GEMys represent a promising avenue for reducing systemic toxicity associated with constitutive IL-12 delivery. However, engineering primary myeloid cells with large inducible constructs poses challenges for transduction efficiency. Our data show that BADGER-enhanced electrotransduction increases both transduction rates and inducible IL-12 secretion. Furthermore, we demonstrate that BADGER reduces the required viral titer by over an order of magnitude, which may lower manufacturing costs and variability. While current inducible GEMys produce less IL-12 than constitutive counterparts, BADGER allows further optimization of construct design and delivery for future clinical translation. BADGER-mediated gene knock-out of M2 polarization pathways also yielded functionally skewed macrophages with reduced immunosuppressive phenotypes. STAT6 KO cells displayed diminished IL-4 and tumor-induced M2 polarization in vitro, and modest tumor suppression in vivo. These results validate STAT6 as a therapeutic target and establish BADGER as a rapid method to generate potent engineered macrophages. The contrast between IL-4 and tumor-induced polarization phenotypes highlights the importance of using physiologically relevant models to assess therapeutic potential. Together, our data establish BADGER as a versatile tool for engineering low-proliferating immune cell populations with complex payloads. By improving both knock-in and knock-out strategies, BADGER has the potential to expand the scope and feasibility of next-generation cell therapies targeting the tumor microenvironment. Functional genomics and chemical screens are powerful for discovering cellular regulators and therapeutic targets, but traditionally rely on immortalized cell lines and protein-based imaging readouts, limiting physiological relevance. In FY25, in collaboration with NCI and NIAMS colleagues, we adapted the hybridization chain reaction (HCR), an isothermal RNA FISH amplification method, to an automated high-content imaging format we called high-content HCR (hcHCR). This protocol enables measurement of mRNA abundance at single-cell resolution in primary human cells and is adaptable to diverse perturbations. hcHCR offers a practical approach for low- to medium-throughput screens where transcript-level responses are the primary endpoint. In previous genome-scale siRNA screens of the LPS response in human macrophages, we identified the antiviral protein IFIT1 as an important regulator of inflammatory and antiviral gene expression. In FY25, we found that Ifit1-/- mice are protected against endotoxin shock and salmonella infection. Mass spectrometric analysis of Ifit1-/- macrophages showed reduced IL-1β protein levels, although the Il1b transcript levels were elevated in both RNAseq and qPCR analysis. Consistent with this, the pro-IL-1β protein levels were reduced when the inflammasome was activated in Ifit1-/- macrophages by respiratory syncytial virus (RSV) infection. Analysis with miRNA antagomirs identified a role for IFIT1-dependent miRNA-mediated suppression of IL-1β. Epigenetic analysis of H3K27Ac enrichment, a marker for open chromatin, showed enhanced signal at the miR-22 gene locus in Ifit1-/- macrophages. This suggests that IFIT1 promotes epigenetic modulation of the miR-22 gene locus to inhibit its expression, which limits miR-22 Il1b transcript targeting to support and enhance IL-1β protein translation. Our data extends the previously identified role of IFIT1 in inhibiting the translation of viral mRNAs to show that, through suppression of miR-22 expression, IFIT1 also supports translation of IL-1β, a critical protein in the host inflammatory response against microbial infections.