VLPs are engineered particles that mimic key features of viruses without carrying a complete viral genome capable of producing infectious progeny. Their structure can be adapted to package and transport specific molecular cargo, allowing researchers to deliver genome-editing components directly into target cells. In the new work, the particles were used to deliver Cas9-based ribonucleoprotein complexes, in which the Cas9 nuclease is preassembled with a guide RNA. Once inside a cell, the guide RNA directs Cas9 to a matching genomic sequence, where the enzyme creates a targeted DNA break. Repair of that break can disrupt the gene, producing a knockout. Because the editing machinery is delivered as a transient protein-RNA complex rather than expressed continuously from a viral vector, the approach can limit the duration of Cas9 activity and reduce some of the complications associated with prolonged nuclease expression.

The researchers report that the VLP toolkit supports multiple CRISPR modalities in human monocytes, macrophages and dendritic cells. In addition to conventional gene knockout, the platform can deliver base-editing systems, which chemically convert individual DNA bases without generating the double-stranded breaks typically associated with standard Cas9 editing. Base editors can therefore create precise genetic substitutions, although their activity remains dependent on the location and sequence context of the target. The same delivery strategy was also used for epigenetic silencing. In this form of editing, CRISPR-guided regulatory proteins are directed to a gene’s control region and alter its transcriptional state without necessarily changing the underlying DNA sequence. Together, these capabilities give researchers a way to compare permanent gene disruption with more targeted nucleotide changes or reversible gene repression in the same broad class of immune cells.

The study further expands the system beyond editing existing genomic sequences. By combining VLP delivery with adeno-associated virus-mediated donor delivery, the researchers enabled site-specific insertion of large DNA sequences through homology-directed repair. In this configuration, the editing machinery creates a break at a chosen genomic site, while the AAV supplies a donor template containing the intended genetic payload and flanking regions that match the target locus. The cell can use these homologous sequences to copy the donor DNA into the genome. Such targeted integration is technically demanding in primary myeloid cells, which are generally resistant to manipulation and may respond strongly to DNA delivery. The combined platform could nonetheless support the introduction of larger genetic elements, a feature relevant to engineered-cell therapies that require the addition of receptors, regulatory circuits or other functional modules.

A central component of the work is SLICeVLP, a system developed to make pooled CRISPR screening possible in primary human macrophages. SLICeVLP separates two delivery tasks: guide RNA is supplied by a VPX-lentivirus, while Cas9 protein is delivered independently through engineered VLPs. This division allows the researchers to introduce guide sequences in a pooled format and then provide the nuclease as a controlled, transient editing reagent. In a pooled loss-of-function screen, thousands of cells can receive different guides targeting many genes, after which the population is exposed to a biological stimulus or sorted according to a measurable phenotype. Sequencing the guide identities in distinct cell populations reveals which genetic perturbations are associated with changes in that phenotype. The approach makes it possible to investigate gene function without having to create and maintain a separate engineered cell line for every candidate gene.

The researchers applied SLICeVLP to study the molecular controls governing tumor necrosis factor, or TNF, and CD80 expression in human macrophages. TNF is a potent inflammatory cytokine that helps coordinate immune responses but can also contribute to pathological inflammation when produced in excess. CD80 is a costimulatory molecule involved in communication between antigen-presenting cells and lymphocytes. By screening for genes that altered the levels of these markers, the team identified regulators of macrophage activation and inflammatory polarization. The work also incorporated Perturb-seq, a method that combines pooled genetic perturbations with single-cell RNA sequencing. Rather than measuring only one surface marker or secreted factor, Perturb-seq captures broad transcriptional changes in individual cells and links those expression profiles to the guide RNA that each cell received. This allows researchers to distinguish distinct cellular states and reconstruct how gene perturbations reshape immune programs.

The screen converged on TNFAIP3, which encodes the protein A20, as a central regulator of inflammatory polarization. A20 is widely recognized as a negative regulator of nuclear factor-κB-associated signaling, a pathway that controls the expression of many inflammatory genes. In the reported experiments, ablation of TNFAIP3 drove macrophages toward a strongly proinflammatory state. These cells were resistant to suppressive repolarization, suggesting that removing this regulatory brake did more than temporarily increase inflammatory gene expression. Instead, the perturbation appeared to stabilize a cellular program that could not readily be redirected by signals normally used to induce a less inflammatory phenotype. The result illustrates how pooled screening can expose control points that are difficult to identify through studies of individual genes or measurements limited to a single immune marker.

The researchers also examined the consequences of TNFAIP3 loss in chimeric antigen receptor macrophages, or CAR macrophages. These engineered cells are designed to recognize defined molecular targets through a synthetic receptor and then use macrophage effector functions to attack or process target material. In the study, removal of TNFAIP3 enhanced the cells’ cytotoxicity and reinforced their inflammatory behavior. The finding suggests that manipulating intrinsic signaling regulators could alter the activity of CAR macrophages after they encounter their targets. At the same time, the results highlight the balance required in therapeutic cell design: a stronger inflammatory and cytotoxic program may improve target-cell destruction, but persistent inflammatory activation could also affect safety, durability and interactions with surrounding tissues. The VLP platform provides a way to investigate these trade-offs systematically in primary human cells.

An important feature of the technology is that it was developed with the physiological properties of myeloid cells in mind. These cells are specialized to detect pathogens and foreign material, so delivery systems that work efficiently in more permissive cell types can provoke innate immune responses or impair survival in monocytes and macrophages. The study reports that VLP-mediated delivery maintained viability and preserved innate immune responsiveness, allowing the engineered cells to remain suitable for downstream functional testing. This distinction is essential for immunotherapy research because an editing method that changes a cell’s baseline activation state may produce misleading conclusions about gene function or therapeutic performance. By combining transient protein delivery, programmable guide RNAs and modular donor systems, the toolkit is intended to provide editing while retaining the biological behavior that makes primary myeloid cells valuable experimental and therapeutic models.

The platform does not eliminate the broader challenges of myeloid-cell engineering, including donor-to-donor variation, delivery efficiency across cell states and the need to assess unintended genomic or transcriptional effects. Nevertheless, the study establishes VLPs as a flexible delivery framework for knockout, base editing, epigenetic regulation and targeted DNA integration in primary human myeloid cells. Its use in pooled loss-of-function and Perturb-seq screens demonstrates how the same technology can move from molecular engineering to large-scale discovery. By connecting genetic perturbations with inflammatory phenotypes and therapeutic functions, the work offers a route to designing macrophage and other myeloid-cell therapies on the basis of systematic functional evidence rather than trial and error. The identification of TNFAIP3 as a key regulator of macrophage polarization further shows how these screens may reveal genetic interventions capable of reshaping the behavior of engineered immune cells.

Subject of Research: Virus-like particle-based CRISPR gene engineering and pooled functional-genomics screening in primary human myeloid cells.

Article Title: Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.

Article References: Jung, H., Devant, P., Ching, C. et al. “Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.” Nature Biotechnology (2026). https://doi.org/10.1038/s41587-026-03258-2

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41587-026-03258-2

Keywords: Virus-like particles, CRISPR, primary human myeloid cells, monocytes, macrophages, dendritic cells, VLP-mediated delivery, base editing, epigenetic silencing, homology-directed repair, AAV, SLICeVLP, Perturb-seq, TNFAIP3, CAR macrophages, immunotherapy.

The Notch signaling pathway, a highly conserved cell communication system, is well-known for its pivotal role in determining cell differentiation and fate across various tissues. Its involvement in hematopoiesis, particularly in the regulation of progenitor cells that give rise to diverse myeloid lineages, has garnered increasing interest. However, a detailed exploration of its influence on monocyte progenitors, especially under inflammatory stimuli, had remained elusive until now.

Researchers focused on trilineage progenitors derived from human peripheral blood monocytes, which possess the remarkable capacity to differentiate into three distinct effector cell types: macrophages, dendritic cells, and osteoclasts. These cell types are integral players in immune defense, antigen presentation, and bone remodeling, respectively. Understanding the cues that drive progenitors toward one lineage or another under inflammatory conditions is critical for manipulating immune responses and attenuating pathological processes.

Employing a suite of sophisticated molecular and cellular techniques, the study meticulously dissected the role Notch signaling exerts when progenitors encounter inflammatory cytokines and environmental stressors. The researchers activated and inhibited components of the Notch pathway, observing consequent changes in gene expression, surface marker profiles, and functional capacities of differentiating cells. This comprehensive approach shed light on the dynamic interplay between external inflammatory cues and intrinsic Notch-mediated regulatory mechanisms.

A salient discovery of this investigation was the identification of distinct Notch-dependent transcriptional signatures that bias progenitor commitment towards macrophage or dendritic cell lineages. Under inflammatory conditions, heightened Notch activity preferentially steered progenitors to adopt macrophage phenotypes characterized by enhanced phagocytic and pro-inflammatory functions. Conversely, attenuation of Notch signaling skewed differentiation in favor of dendritic cells, which are vital for antigen presentation and activation of adaptive immunity.

Intriguingly, the study revealed that Notch signaling also inhibits osteoclastogenesis from monocyte progenitors in inflamed environments, suggesting a protective mechanism against pathological bone resorption commonly observed in chronic inflammatory diseases such as rheumatoid arthritis. This nuanced regulation underscores Notch’s role as a multifunctional gatekeeper balancing immune defense and tissue homeostasis.

Delving deeper into molecular pathways, the team elucidated that Notch signaling modulates key transcription factors including NF-κB, IRF8, and PU.1, which are instrumental in lineage specification. These factors orchestrate gene networks that define terminal differentiation programs and functional phenotypes. The crosstalk between Notch and these transcriptional regulators represents a sophisticated regulatory nexus modulating progenitor plasticity.

Another pivotal facet of the study involved the temporal dynamics of Notch activation. Researchers demonstrated that early versus late activation of Notch signals yields divergent differentiation outcomes, emphasizing the importance of signal timing in hematopoietic programming. Such temporal control mechanisms could be exploited to fine-tune immune responses for therapeutic benefit.

Furthermore, the findings implicate inflammatory cytokines such as TNF-α and IL-6 as modulators of Notch receptor and ligand expression on progenitor cells, thereby integrating extrinsic inflammatory signals with intrinsic differentiation programs. This interface constitutes an adaptive regulatory loop whereby systemic inflammation directly influences progenitor cell fate via Notch pathways.

Implications of these insights are profound, especially for designing targeted immunotherapies. By manipulating Notch signaling components within monocyte progenitors, it may be possible to recalibrate immune responses in diseases characterized by dysregulated inflammation and aberrant myeloid cell function, including autoimmune disorders, chronic infections, and cancer.

Moreover, the selective inhibition of Notch pathways to prevent excess osteoclast formation could herald new treatments for inflammatory bone loss, offering a dual benefit of immune modulation and preservation of skeletal integrity. The translational potential of these findings positions Notch signaling as a promising target in the development of next-generation immunomodulators.

This study also underscores the critical importance of studying human cells within physiologically relevant inflammatory milieus, moving beyond animal models to capture the complexity and heterogeneity of human immune regulation. Such approaches are essential for bridging the gap between bench research and clinical application.

While the results provide compelling evidence for Notch’s multifaceted roles, the authors acknowledge limitations, including the need for in vivo validation and exploration of Notch interactions with other signaling pathways such as Wnt and Hedgehog. Future research is poised to untangle these complex networks, offering richer insights into immune progenitor biology.

In conclusion, this groundbreaking investigation delineates how Notch signaling dynamically governs the fate of human peripheral blood monocyte trilineage progenitors under inflammatory conditions, finely tuning the balance between macrophage, dendritic cell, and osteoclast lineages. These findings invigorate the field with fresh mechanistic understanding and lay a robust foundation for harnessing Notch pathways in therapeutic innovation.

As chronic inflammatory conditions continue to impose significant health burdens worldwide, the modulation of progenitor cell fate through Notch offers a beacon of hope. The ability to direct immune cell differentiation with precision could revolutionize treatment paradigms, enabling tailored interventions that restore immune equilibrium without broad immunosuppression.

The research community and clinical practitioners alike will keenly watch forthcoming studies that build upon these seminal discoveries. By integrating molecular insights with clinical needs, the path toward transformative immune therapies may be rapidly accelerated, fulfilling the promise of precision medicine.

Continued investment in decoding cell signaling mechanisms and their contextual dependencies remains paramount. The elucidation of Notch’s role herein exemplifies the power of fundamental research to illuminate complex biological systems and inspire novel therapeutic strategies.

This study handles complexities of immune differentiation with elegant experimental strategies, offering clarity into a previously obscure regulatory axis. Its publication marks a significant milestone in both immunology and cell biology, likely to galvanize further inquiries and technological advancements.

The intersection of Notch signaling and inflammatory microenvironments unveiled by this research reflects the evolving landscape of hematopoietic science, one where signaling pathways are viewed not in isolation but as integrated systems influencing disease outcomes and clinical opportunities alike.

Subject of Research: The influence of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitors under inflammatory conditions.

Article Title: Effects of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitor under inflammatory conditions.

Article References:
Aničić, S., Filipović, M., Krešić, I. et al. Effects of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitor under inflammatory conditions. Cell Death Discov. 11, 519 (2025). https://doi.org/10.1038/s41420-025-02807-z

Image Credits: AI Generated

DOI: 10 November 2025