Chimeric antigen receptor macrophages (CAR-Ms) therapy has shown great promise in liver fibrosis, however limited anti-inflammatory capacity of CAR-Ms in the fibrotic foci compromises their anti-fibrotic potency. We here report tripartite motif containing 13 (TRIM13) engineered CAR-Ms for effectively manipulating the anti-inflammatory phenotype of CAR-Ms, augmenting their anti-fibrosis efficacy. Specifically, our efferocytosis-sparked lipid nanoparticles (ESLNPs) efficiently engineered fibrosis-associated macrophages to anti-inflammatory CAR-Ms by co-delivering mRNA encoding TRIM13 and anti-fibroblast activation protein (FAP) CAR respectively. Our data demonstrated these reprogrammed CAR-Ms exhibited a sustained anti-inflammatory phenotype via blocking the mitochondrial DNA (mtDNA)-STING pathway through the overexpression of TRIM13, and showed notable FAP-targeted phagocytosis. Treatment with ESLNPs in male mice with liver fibrosis obviously ameliorated fibrosis through synergizing anti-fibrotic and inflammation-resolution activities, ultimately prompting substantial hepatic function restoration. In sum, our findings established that remodeling and sustaining the anti-inflammatory phenotype of CAR-Ms markedly elevated their therapeutic efficacy in liver fibrosis, benefiting CAR-Ms therapy with broad application in other fibrotic diseases.

The antitumor efficacy of immune cell engagers that bind two targets on the same immune cell is limited by structural constraints, leading to incomplete coengagement and uncoordinated signaling. Here, we develop a trispecific macrophage engager (TrME) that both activates the prophagocytic receptor lipoprotein receptor-related protein 1 (LRP1) and blocks the antiphagocytic receptor signal regulatory protein alpha (SIRPα). This 'activate and block' AND logic gate, when coupled to a tumor-targeting moiety, enables coordinated signaling that enhances macrophage cytotoxicity against solid tumors. The TrME tandemly links monovalent LRP1 activator calreticulin, anti-SIRPα scFv and a tumor-associated antigen (TAA)-targeting arm through flexible linkers. Computational modeling and screening of tandem constructs revealed an optimal conformation for robust cis-targeting, allowing logic-gated control of ratiometric prophagocytic and antiphagocytic signaling. In situ generation of TrME by delivering mRNA encoding TAA-targeting TrME through an optimized lipid nanoparticle system activates macrophages and induces antitumor responses, significantly inhibiting tumor growth and prolonging survival in multiple solid tumor mouse models.

Triggering receptor expressed on myeloid cells 2 (TREM2), a critical sensor of cell debris, regulates macrophage efferocytosis to maintain tissue immune homeostasis. However, inflammatory mediators upregulate the sheddase ADAM17, leading to TREM2 cleavage, which impairs apoptotic cell clearance and exacerbates inflammation. We here report a synthetic cleavage-resistant TREM2 (CRT) to boost TREM2-dependent efferocytosis and alleviate inflammation associated with aberrantly accumulated apoptotic cells. CRT integrates the ligand-binding domain of TREM2 with its intracellular signaling adaptor DAP12 via a custom-engineered stalk and transmembrane segment. Our data demonstrate that CRT amplifies TREM2 signaling even in the presence of ADAM17. Customized lipid nanoparticles efficiently introduce CRT mRNA into macrophages, generating CRT-engineered macrophages (CRT-Ms) in situ. CRT-Ms effectively reduce apoptotic cell burden and alleviate inflammation in mouse models of metabolic-dysfunction-associated steatohepatitis and atherosclerosis. In sum, our findings establish that CRT strengthens TREM2-mediated macrophage efferocytosis and mitigates inflammation, with broad potential for apoptotic-cell-associated diseases.

Adoptive immune cell therapies, exemplified by chimeric antigen receptor T cells, have transformed the treatment of hematological malignancies. However, their broader clinical application is limited by complex ex vivo manufacturing, high cost, and safety concerns. In vivo immune cell engineering has emerged as an alternative strategy that delivers genetic instructions directly to immune cells, thereby generating or modulating therapeutic immune cells within the body and reducing the reliance on individualized in vitro operations. These advances underscore the need for a systematic evaluation of this emerging field. Therefore, this review systematically summarizes the mechanistic principles and delivery strategies underlying in vivo immune cell engineering, with an emphasis on in vivo CAR-T cell generation and the engineering of other immune cells. We then discuss major viral and non-viral delivery platforms and clarify how these platforms influence cargo delivery, cell specificity, and functional immune-cell programming. We further discuss recent preclinical and emerging clinical advances across cancer, autoimmune diseases, and degenerative diseases, while examining key translational challenges, including delivery specificity, off-target effects, controllability, persistence, and manufacturing standardization. Overall, although the field of in vivo immune cell engineering is advancing rapidly, its clinical success will depend on coordinated improvements in delivery precision, therapeutic efficacy, safety, and controllable immune-cell programming.

Immune cell engineering has emerged as a transformative frontier in medicine, reshaping therapeutic strategies for cancer, autoimmunity and infectious diseases. Advances in delivery technologies such as viral vectors, lipid nanoparticles and polymer-based systems, together with precise gene editing tools including mRNA platforms and CRISPR, have enabled the efficient programming of immune cells with enhanced specificity and potency. Chimeric antigen receptor (CAR) designs exemplify how synthetic receptors can redirect immune recognition and function, and are now being applied not only to T cells but also to macrophages, natural killer cells and dendritic cells. These innovations are expanding therapeutic opportunities from hematologic malignancies to solid tumors and chronic inflammatory disorders. In parallel, the transition from ex vivo manipulation to in vivo reprogramming is beginning to address manufacturing bottlenecks and improve clinical accessibility. Moreover, artificial intelligence is increasingly driving rational design of vectors, CAR structures, and signaling networks for next-generation therapies. In this Review, we summarize current approaches to immune cell engineering, compare the characteristics of delivery systems and the design of engineered regulatory elements, and highlight therapeutic applications, while also discussing the outstanding challenges of safety, persistence and microenvironmental barriers that must be overcome to achieve durable and widely applicable therapies.

Chimeric antigen receptor microglia (CAR-M)-mediated amyloid-β oligomers (AβO) phagocytosis shows great promise in Alzheimer's disease (AD) treatment, however, the limited AβO degradation of CAR-M compromises their anti-AβO potency. This work here reports an in situ engineered agonistic anti-C-type lectin domain containing 7 A (CLEC7A) nanobody to accelerate AβO degradation of CAR-M, augmenting their anti-AβO efficacy. Specifically, with the intranasal-delivered microglia-targeting lipid nanoparticles (LNP), this work generates an AβO-specific degradation-potentiated CAR-M by introducing dual mRNAs encoding AβO-specific CAR and anti-CLEC7A nanobody into the cerebral microglia. These data show that these engineered CAR-M exhibited superior phagocytic function and promoted intracellular AβO degradation via activating CLEC7A-spleen tyrosine kinase (SYK) signaling pathway through the local secretion of anti-CLEC7A nanobody. In the APP/PS1 mouse model of AD, these in situ reprogrammed CAR-M significantly reduced cerebral Aβ levels, suppressed neuroinflammation, and restored cognitive function. In sum, these findings demonstrate that potentiating AβO degradation within CAR-M effectively alleviates AD pathology, providing a promising therapeutic strategy for AD with broad application in other neurodegenerative diseases.

Chimeric antigen receptor macrophage (CAR-M) therapy represents a promising therapeutic approach for treating glioblastoma multiforme (GBM). However, durable antitumorigenic macrophage phenotype of CAR-Ms is limited by the highly immunosuppressive tumor microenvironment (TME), wherein Siglec-sialic acid signaling directly drives macrophage polarization toward a protumorigenic phenotype. We here report an in situ synthetic SIGLEC9-based chimeric switch receptor (CSR) for diverting the inhibitory signal into positive ones, augmenting the sustained proinflammatory phenotype and tumoricidal immunity of CAR-Ms in the GBM niche. Specifically, our results showed that macrophage-targeted ionizable lipid nanoparticles efficiently introduce dual circRNAs into macrophages to generate CSR functionalized CAR-Ms in vitro and in vivo. The modified macrophages maintained a proinflammatory state, exhibited superior phagocytic activity, resulting in rapid and efficient eradication of IL13Rα2-positive tumor cells. Moreover, an injectable nanoparticle-hydrogel system for reprogramming macrophages surrounding the glioma resection cavity initiated a locoregional antitumor immune response and elicited robust long-term immunological memory, inhibiting tumor relapse in the postoperative GBM model. In sum, our findings establish that the engineered SIGLEC9-based CSR significantly promotes the maintenance of an antitumoral phenotype of CAR-Ms in the hypersialylated acidic TME, contributing to the improvement of engineered macrophage-based immunotherapy against GBM.

The functional reconstruction of bone defects caused by trauma, infection, surgical resection and degenerative diseases poses substantial clinical challenges. Bone tissue engineering (BTE) holds immense potential for treating bone defects while avoiding complications commonly associated with conventional autografts, allografts and internal fixation. Bioactive hydrogels with exceptional drug delivery capabilities, excellent biocompatibility and tunable physicochemical properties have emerged as promising biomaterial scaffolds for BTE. This review provides a comprehensive overview of recent advancements in bioactive hydrogel-based strategies for BTE applications. An initial introduction to bone physiology is followed by a critical discussion of the design considerations for hydrogel platforms, specifically biomaterial selection, innovative crosslinking mechanisms and bioactive functionalization. Furthermore, hydrogel engineering for the controlled delivery of bioactive cargo is critically examined, with an emphasis on spatiotemporally programmable release behaviors that coordinate osteogenesis, angiogenesis and immunomodulation. Finally, key translational challenges are discussed, and the emerging role of artificial intelligence-assisted design is explored as a transformative approach for facilitating the clinical translation of next-generation bioactive hydrogels for BTE.

The clinical significance of circulating tumor DNA (ctDNA) mutation profiling is extensive, involving the guidance of targeted therapies and the monitoring of cancer recurrence. However, the identification of ctDNA mutations is challenging due to the presence of abundant wildtype DNA and the severe fragmentation of ctDNA in blood or urine. Certain PCR methods utilize additional oligonucleotides to suppress wildtype DNA amplification, leading to increased costs, potential cross-reactions, and longer amplicons. To tackle these issues, a novel PCR strategy has been developed by introducing dual-function probes, which enhance allelic discrimination and generate fluorescence signals simultaneously. Both the dual-function Taqman probe and MGB probe, with optimized length and quantity, enhance discrimination between mutant and wild-type DNA. Lower Mg2+ concentrations and higher temperatures were found to enhance discrimination in this study. Exceptional sensitivity, reaching 0.01 % or 0.03 % variant allele frequencies (VAFs), was achieved for six mutations with amplicon lengths ranging from 62 to 83 bp. Additionally, a remarkable sensitivity of 0.03 % VAFs was demonstrated in two multiplexed assays. Clinical validation using 17 ctDNA samples revealed complete concordance with digital droplet PCR (ddPCR) targeting the EGFR L858R mutation. This new technique, offering benefits such as heightened sensitivity, simplified reactions, cost-effectiveness, and shorter amplicons, has the potential for widespread utilization in ctDNA mutation profiling.

Immunotherapy provides a new alternative treatment for patients with malignant tumors, offering them renewed hope. However, many patients experience limited efficacy with immunotherapy, largely due to the tumor microenvironment (TME), which often inhibits the immune response. This reduced efficacy may be attributed to several factors, including hypoxia, immune escape, off-target toxicity, and insufficient drug accumulation. It is widely thought that ultrasound-mediated microbubbles can be used as effective carriers to enhance the effectiveness of immunotherapy by leveraging their active targeting and/or passive targeting of the TME. In this paper, we expound on the targeting effects of ultrasound combined with drug-loaded microbubbles on various components of the TME. Besides, we review how ultrasound-mediated microbubble targeting can refine tumor immunotherapy.

The development of proteolysis-targeting chimeras (PROTACs) represents a promising strategy for targeted protein degradation in cancer therapy. However, the limited tumor-specific targeting and the inherent unfavorable physicochemical properties of PROTACs lead to insufficient cellular uptake and suboptimal antitumor immune responses. Herein, as a proof of concept, we developed an oncolytic virus-PROTAC conjugate (BPAD) by efficiently coupling bromodomain-containing protein 4 (BRD4)-targeting PROTACs with oncolytic viruses (OVs). In BPAD, the potent and highly selective infection of OVs to tumor cells enhances both cellular uptake and tumor-selective delivery of PROTACs, resulting in a 640-fold increase in the protein degradation efficiency. Moreover, prior to OV-induced tumor lysis, the preferential replication of OVs within tumor cells, combined with BRD4 degradation, promotes the secretion of type I interferons and facilitates dendritic cell maturation. Overall, the BPAD strategy enables the development of biologically derived macromolecular PROTAC conjugates, thereby enhancing the clinical translation potential of diverse PROTACs.

Pathogenic amyloid-β (Aβ) accumulation defines Alzheimer's disease (AD), directly inflicting neuronal damage and driving chronic neuroinflammation. While both central microglia and peripheral macrophages are critical for Aβ clearance, their functional impairment in AD inexorably leads to escalating Aβ burden and disease progression. We here report an in situ engineered synthetic Aβ disaggregator (SAD) delivered to macrophages via neuroprotective DHA-based lipid nanoparticles (DLNPs). This platform transcends current therapeutic limitations by not only potently dismantling neurotoxic Aβ aggregates but also by fundamentally reprogramming peripheral macrophages to enhance Aβ clearance. Specifically, our results demonstrate that DLNPs effectively reprogram peripheral macrophages to produce and secrete cerebral-penetrating SAD both in vitro and in vivo. The SAD can promote cerebral Aβ disaggregation, thereby inhibiting neuroinflammatory pathology progression. Moreover, the DLNPs efficiently reprogram the peripheral macrophages to enhance phagocytosis, further facilitating drainage of Aβ and reducing cerebral Aβ accumulation in mouse models. Collectively, these findings uncover a dual-action mechanism of SAD through the synergistic interplay of direct Aβ disaggregation and enhanced macrophage-mediated clearance. In sum, our findings establish that the central-peripheral targeting therapeutic strategy significantly reversed AD pathology, highlighting the therapeutic potential of mRNA-based in situ fusion protein in AD treatment.

Overwhelming cardiomyocyte death and excessive cardiac fibrosis post myocardial infarction (MI) collectively lead to heart failure and mortality. For treating this devastating disease, it is essential to eliminate fibrosis and reconstitute the damaged myocardium, yet effective strategies remain elusive. Here, we created pleiotropic chimeric antigen receptor-monocytes (pCAR-Mos), revitalizing the injured heart via synergistic fibrosis clearance and myocardial reconstitution. Specifically, we engineered monocytes to express fibroblast activation protein (FAP)-chimeric antigen receptor (CAR) and secrete the cardioregenerative protein Agrin. CAR-mediated phagocytosis of myofibroblasts, which was further enhanced by Agrin, significantly attenuated fibrotic scar formation. Moreover, Agrin secretion promoted cardiomyocyte regeneration, thereby facilitating replenishment of functional myocardium. Treatment with pCAR-Mos remodeled the cardiac fibrotic microenvironment and substantially restored cardiac function in MI mice. In sum, our findings confirmed that pCAR-Mos exerted potent phagocytic activity against profibrotic myofibroblasts while simultaneously enabling myocardial reconstitution, thereby providing a reversible treatment strategy for MI with broad application in other fibrotic diseases.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Epstein‒Barr virus (EBV)-induced B-cell overactivation could lead to inflammatory injury to the CNS, which is thought to underlie the initiation and progression of MS. To specifically eradicate these B cells, we report in situ EBNA1-specific chimeric antigen receptor (CAR)-T cells that were transiently programmed with circular RNA (circRNA)-laden CD7-targeted lipid nanoparticles (CD7-LNP). We demonstrate that systematic injection of CD7-LNP can efficiently introduce CAR circRNA to T lymphocytes and yield in vivo CAR-T cells. These in situ CAR-T cells were able to specifically clear EBNA1-specific B cells and significantly mitigate the progression of MS in a MS mouse model. Thus, in situ generation of EBNA1-specific CAR-T cells hold promise as a therapeutic strategy for MS that avoids the risks of general immunosuppression, and warrant further clinical trials.

Glioblastoma (GBM) remains a highly lethal form of cancer due to its molecular heterogeneity and the immunosuppressive microenvironment surrounding the tumor. Here, we report a modular immunotherapy platform characterized by its flexibility to simultaneously target multiple antigens. Specifically, we utilize engineered E. coli Nissle to colonize tumors and produce bispecific engagers that simultaneously target EGFRvIII and interleukin (IL)-13Rα2. These tags direct in situ-reprogrammed chimeric antigen receptor (CAR) macrophages, which are edited using nanoparticles and delivered within a shear-thinning hydrogel, to execute targeted phagocytosis. This probiotic-macrophage crosstalk eliminates tumor cells while converting protumor M2 macrophages into immunostimulatory M1 effectors. In aggressive orthotopic GBM mouse models, this strategy achieves 83% survival at the 120-day endpoint, representing a 5-fold improvement over single-target controls and establishing durable immunological memory that effectively combats recurrence. By functioning as multifunctional immune hubs, this platform offers a versatile framework designed to overcome the antigenic complexity of solid tumors.

Chimeric antigen receptor macrophage (CAR-M) therapy has shown great promise in solid malignancies; however, the phenotypic re-domestication of CAR-Ms in the immunosuppressive tumor niche restricts their antitumor immunity. We here report an in situ engineered chimeric interleukin (IL)-2 signaling receptor (CSR) for controllably manipulating the proinflammatory phenotype of CAR-Ms, augmenting their sustained tumoricidal immunity. Specifically, our in-house-customized lipid nanoparticles efficiently introduce dual circular RNAs into macrophages to generate CSR-functionalized CAR-Ms. The intracellular inflammatory signaling pathway of CAR-Ms can be stimulated with the IL-2 therapeutic via the synthetic IL-2 receptor, which induces the antitumor phenotype shifting of CAR-Ms. Moreover, hydrogel-mediated combinatory treatment with lipid nanoparticles and IL-2 remodels the immunosuppressive tumor microenvironment and promotes tumor regression in renal carcinoma animal models. In summary, our findings establish that the proinflammatory phenotype of CAR-Ms can be modulated by a synthetic IL-2 receptor, benefiting the antitumor immunotherapy of CAR-Ms with broad application in other solid malignancies. Jing et al., perform local injections of lipid nanoparticles carrying synthetic IL-2 and circRNAs encoding IL-2R–TLR4 chimeric receptors and anti-CA9 CARs to target renal tumor macrophages, thereby promoting their antitumorigenic potential in situ.

Epi-immunotherapy appears promising for hepatocellular carcinoma (HCC) treatment, but immunosuppressive macrophages limit the capacity of epigenetic regulation to activate T cell-mediated tumoricidal immunity. Here we report an epi-immune nanosatellite (stEiNS) that co-delivers siRNA targeting the YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) alongside the histone deacetylase IIa inhibitor TMP195, enabling epigenetic reprogramming of HCC tumor cells and M2 macrophages to enhance the immunotherapeutic response. stEiNS assembles size-mismatched nanoparticles via dynamic locks in a satellite-like structure, enabling deep tissue penetration. Knockdown of YTHDF1 by stEiNS in HCC cells, along with stEiNS-driven antitumor macrophage phenotype induction, intensifies macrophages-cytotoxic T lymphocytes interactions with tumor cells. stEiNS suppresses TNF/NF-κB signaling in tumor cells to inhibit CCL2-driven recruitment of myeloid-derived suppressor cells while activating the IFNγ/STAT1 pathway in M2-phenotype macrophages to promote their polarization toward an M1 phenotype. Collectively, these effects trigger robust tumoricidal immunity, leading to efficient tumor eradication, as validated in patient-derived tumor organoids, orthotopic HCC models, and recurrence models. In summary, we establish a dual-targeting stEiNS with promising epi-immunotherapeutic potential against advanced HCC and diverse malignancies.

Nanobodies, one-tenth the size of conventional antibodies, have gained attention as therapeutic agents for autoimmune diseases, cancer, and viral infections. However, traditional methods for nanobody discovery are often time-consuming and labor-intensive. In this study, we present a computational design framework that integrates deep generative modeling with epitope profiling. We first developed a generative adversarial network (GAN)-based model named AiCDR, which incorporates two external discriminators to enhance its ability to distinguish native CDR3 sequences from random sequences and peptides. This design enables the generator to produce CDR3 sequences with natural-like properties. Approximately 10,000 CDR3 sequences were generated and grafted onto a humanized scaffold. After structural prediction, we obtained a library of about 5200 high-confidence nanobody models. Using this structure-based library, we conducted epitope profiling across six representative protein targets. The nanobody-enriched epitopes showed strong overlap with known functional regions, suggesting potential biological activity. As a case study, we selected ten nanobodies designed to target the SARS-CoV-2 Omicron RBD. Two of these showed detectable neutralization activity in vitro. Overall, our results demonstrate that computational design and structure-based profiling offer an efficient strategy for early-stage therapeutic nanobody discovery.

Kirsten rat sarcoma virus (KRAS ) is a common oncogene in human cancers. Approximately 40% of the patients diagnosed with colorectal cancer (CRC) have KRAS mutations that exhibit strong resistance to targeted molecular therapy and EGFR antibody treatment. In this study, we present photocatalytic silica nanoparticles (A6-FS/BiVO 4 DMSNs) for targeted therapy of KRAS mutant CRC with the induction of cascadic ferroptosis events. Dendritic mesoporous silica nanoparticles (DMSNs) were impregnated with photocatalytic BiVO 4 , loaded with ferroptotic agents (benzoyl ferrocene: B and sorafenib: S), and encoded with CD44-targeting A6 peptides. For the targeting design, we observed CD44 overexpression in KRAS mutant CRC cells using CPTAC data analysis. Upon laser irradiation, A6-FS/ BiVO 4 DMSNs generate electron-hole pairs (e -/h + ), which produce hydroxyl radical (OH ) and superoxide anions (O 2 - ). Laser irradiation simultaneously initiates the dissociation of iron (Fe 2+ ) from benzoyl ferrocene and the release of sorafenib. This cascade induces ferroptosis in KRAS mutant CRC cells, especially under conditional inhibition of redox-regulating proteins (cystine/glutamate antiporter and glutathione peroxidase 4), and significantly inhibits tumor growth in a KRAS mutant CRC xenograft animal model.

Cancer-associated fibroblasts (CAFs) are key components of the pancreatic adenocarcinoma (PAAD) tumor microenvironment (TME), where they promote tumor progression and metastasis through immunosuppressive functions. Although significant progress has been made in understanding the crosstalk between cancer cells and CAFs, many underlying mechanisms remain unclear. Recent studies have highlighted the importance of calcium signaling in enhancing interactions between tumor cells and the surrounding stroma, with the S100 family of proteins serving as important regulators. While the roles of some S100 proteins have been extensively studied, others, such as S100A13, remain less well understood. Bioinformatic analysis was employed to predict the pathogenic potential of CAFs and S100A13. Stable S100A13 knockdown CAFs were generated using a short hairpin RNA system. Cellular viability and apoptosis rates were evaluated through CCK-8 and flow cytometry tests, respectively. Additionally, the wound healing and migration assays were conducted to assess the invasive and metastatic capabilities. Transcriptome analysis was conducted to identify differential gene expression and associated signaling pathways in PAAD cells derived from an indirect culture system. Furthermore, the protumoral role of S100A13 in PAAD was further verified using both 3D bioprinting and cell line-based xenograft tumor models. In this study, we identified a strong association between S100A13, a calcium-binding protein, and CAFs in PAAD. Gene expression analysis revealed that S100A13 was highly expressed in CAFs and correlated with poor prognosis. Knockdown of S100A13 in CAFs reduced the metastatic potential of PAAD cells. In addition, S100A13 depletion impaired cell motility and calcium signaling pathways within the TME. Furthermore, silencing S100A13 in CAFs markedly slowed PAAD progression in both tumor spheroids and Balb/c nude mice. Together, our findings underscore the critical role of CAFs-derived S100A13 in PAAD progression and suggest that targeting S100A13 may offer a promising therapeutic strategy for PAAD.