In this study, we describe a rationally designed light-inducible RNA-releasing protein (LIRP) capable of inhibiting mRNA translation in the dark while permitting gene expression upon exposure to blue or ambient light. This LIRP-dependent gene switch is compatible with various delivery routes of gene- and cell-based therapy, such as subcutaneous implantation of microencapsulated light-sensitive cells or expression in various light-accessible body sites using single adeno-associated virus (AAV) vectors. To exemplify a gene therapy approach that directly harnesses ambient light as a natural illumination source to induce therapeutic action, we show how intradermal delivery of AAV2 vectors carrying a LIRP-regulated gene switch controlling murine thymic stromal lymphopoietin expression was effective in enabling light-dependent prevention and treatment of diet-induced obesity. To describe another therapeutic scenario, we engineered AAV2 vectors for LIRP-dependent expression of Vascular endothelial growth factor (VEGF) inhibitors for the treatment of retinal neovascular diseases. Upon intravitreal delivery into mice suffering from wet macular degeneration, VEGF inhibitors were constantly produced when animals were exposed to daylight, but therapeutic actions could be flexibly interrupted either by exposure to dark environments or by administration of a selective blue light filter at any point in time. When compared to conventional treatment strategies based on constitutive VEGF inhibition over the course of 3 months, we show that a regulated gene therapy approach through LIRP-dependent optogenetics was advantageous in maintaining a normal retina thickness. This work not only provides a valuable addition to the optogenetic toolbox but also offers a perspective to translate light-dependent gene switches toward therapeutic usage.
Although gene editing offers durable therapeutic potential for genetic disorders such as hypercholesterolemia, widely used adenine base editors (ABEs) face translational obstacles due to potential off-target effects inherent in gene-editing systems. ABE8e has strong clinical potential owing to its high efficiency and rapid editing kinetics, but its genome-wide off-target effects remain poorly characterized. Here, we uncovered substantial genome-wide off-target edits of ABE8e using the GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) assay, including in oncogenes and tumor suppressors, highlighting significant risks for clinical translation. We therefore engineered a next-generation precision base editor, erABE, through electrostatic remodeling of the TadA8e deaminase. erABE reduces ABE8e's genome-wide off-target activity by ~36.5-fold to background levels while retaining an efficiency indistinguishable from ABE8e. In mice, a single low-dose delivery of erABE packaged in lipid nanoparticles achieved robust Pcsk9 knockdown, resulting in a sustained, ~90% reduction in plasma PCSK9 protein and ~60% reduction in low-density lipoprotein cholesterol for at least 6 months. erABE also potently suppresses PCSK9 expression in human liver organoids. These results highlight the safety and durable therapeutic effects of erABE, supporting its translation as a clinical genome-editing platform.
Reproducing the spatiotemporally coordinated emergence of vascular, biliary, and parenchymal compartments of the human liver remains a persistent bottleneck in organoid engineering. We introduce biodegradable poly(lactic-co-glycolic acid) microspheres that display epithelial (E) cadherin and vascular endothelial (VE) cadherin fusion proteins and deliver morphogens, mimicking key developmental signals that guide liver organogenesis. When combined with mesenchymal stem cells (MSCs), the microspheres supply adhesion cues that drive aggregate compaction. E-cadherin engagement induces Yes-associated protein activation, priming MSCs for lineage induction. Complementarily, VE-cadherin and vascular endothelial growth factor jointly establish an endothelial niche that delivers inductive cues for hepatic lineage divergence. The resulting organoids display compartmentalized architecture comprising hepatocytes, cholangiocytes, mesenchymal cells, and endothelial networks and respond to hepatotoxic compounds with clinically relevant phenotypes. Unlike induced pluripotent stem cell-based or multiple cell cocultures, this single-source MSC system achieves self-organization through cadherin-defined adhesion and timed morphogen delivery. These elements define a scalable, cadherin-defined biomaterial platform for liver organoid engineering, enabling translational use in drug evaluation and regenerative medicine.
Integrin plays a crucial role in human immune system activation and stem cell differentiation through interactions with the extracellular matrix (ECM). Reversible modulation of integrin signaling has illuminated the road toward broad-spectrum disease treatment and tissue regeneration. In this regard, an integrin-like decoy receptor, termed molecularly imprinted polymer (MIP), was synthesized via solid-phase imprinting to target the Arg-Gly-Asp (RGD) sequence, an integrin-binding motif abundantly present in the ECM. The MIP, with temperature-dependent RGD affinity, enabled dynamic intervention in integrin signaling, thereby modulating integrin-RGD-mediated immune response and cell differentiation. The MIP could, on demand, orchestrate macrophage phenotypes to display proinflammatory or anti-inflammatory traits, potentially beneficial at the early stage of tissue injury. Furthermore, stem cell fates could be controlled by MIP, showing potential to prevent hyperplasia caused by excessive differentiation. The proof-of-concept studies demonstrate the feasibility of MIP-based decoy receptors in the dynamic control of integrin signals and cell evolution, showing promise in tissue repair and related diseases.
Chimeric antigen receptor macrophages (CAR-Ms) hold promise for solid tumor immunotherapy, but their efficacy is limited by tumor microenvironment (TME)-driven M2 polarization. Current strategies rely on antigen-dependent activation or in vitro priming, which fail to sustain the M1 phenotype in the immunosuppressive TME. In this research article, we developed a TME-regulated CAR-M (TMER CAR-M) biotechnology by integrating an original M1 phenotype dominance into an active CAR-M (ACT CAR-M) platform. In tumor models, TMER CAR-M remodeled the TME, enhanced CD4+ and CD8+ T cell infiltration, increased the proportion of natural killer cells, reduced the frequency of regulatory T cells (Tregs) and myeloid-derived suppressor cells, effectively reversed the immunosuppressive state, and inhibited tumor growth in vivo. Crucially, human primary armored TMER CAR-M could effectively resist M2 reprogramming and reshape M2 macrophages. In conclusion, an armored TMER CAR-M biotechnology with the original M1 phenotype dominance overcomes TME immunosuppression through two mechanisms, including autonomous M1 maintenance and M2 reprogramming without antigen stimulation. This biotechnology bridges a critical gap in CAR-M therapy for solid tumors, offering a clinically translatable platform for immune-cold malignancies.
Oligoclonal VHH mixtures targeting distinct epitopes offer therapeutic benefits in mitigating complex diseases, including cancer, infectious diseases, and snakebite envenoming. However, current production strategies rely on separate expression and purification of individual VHHs, followed by downstream mixing, resulting in high costs. We present a cocultivation approach for producing an experimental recombinant antivenom using growth-decoupled Escherichia coli strains. Six different cell lines were generated by transforming the same host with different VHH genes. The cell lines displayed high-yield extracellular secretion in microtiter plate and 1-l bioreactor cultivations, and cocultivation at defined inoculation ratios yielded oligoclonal VHH mixtures approaching target compositions. This strategy enables one-pot manufacturing with minimal downstream processing and predictable composition. A techno-economic assessment based on obtained titers and two-step purification indicates reduced per-treatment costs compared with Chinese Hamster Ovary cells produced IgG-based antivenoms. Our findings offer an alternative to conventional oligoclonal VHH production for multivalent therapeutics requiring broad neutralization, including recombinant antivenoms.
Microalgae are robust microorganisms with versatile metabolic functions, including oxygen generation, making them crucial components in wastewater treatment plants, food production, or extraterrestrial life-support systems. Immobilizing microalgae in hydrogels could eliminate costly separation steps required in free-floating cultivation. Volumetric bioprinting (VBP), enabling rapid fabrication of large, complex constructs from cell-laden hydrogels, presents a promising solution. This study investigates the possibilities and limitations of VBP with microalgae in poly(ethylene glycol) diacrylate-cellulose nanofibrils (PEGDA-CNF) hydrogels. Despite chlorophyll-induced interference, the printing of stable 3D structures is achievable in high-resolution up to certain cell densities. During cultivation, printed microalgae exhibited exceptionally high viability and cell density compared to alternative bioprinting methods, and their photosynthetic efficiency remained high even in the nonphysiological PEGDA-CNF environment, as confirmed by pulse-amplitude modulated fluorometry and oxygen measurements. This work demonstrates the feasibility of integrating microalgae into VBP, enabling scalable production of photosynthetically active geometries and significantly simplifying their implementation in cascade-based processes.
The growing recognition of the skin microbiome in regulating the host's metabolic and immune functions has spurred the development of in vitro platforms designed to recapitulate their intricate interactions, aiding research into both skin-microbiome and microbe-microbe interactions within their distinct niches. Despite these efforts, challenges remain in dissecting skin-microbiome interactions, especially due to the absence of a standardized platform for the long-term coculture of bacterial and mammalian cells. In this review, we highlight the key components in modeling an in vitro skin ecosystem and discuss the therapeutic potential of skin commensals, including recent advances and applications for engineered live biotherapeutics targeting skin diseases, to underscore the translational value of in vitro skin-microbiome interaction studies.
Antimicrobial resistance in pathogenic mycobacteria remains a critical challenge due to poor drug penetration through their complex cell wall, which necessitates prolonged multidrug regimens. Mycobacteriophages encode a lytic machinery that can disrupt this barrier. In this research article, we describe a modular mycolysin platform combining phage enzymes Lysin A and Lysin B with outer membrane-permeabilizing peptides and protein transduction domains using VersaTile shuffling technology. Screening the chimeric libraries against Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG), followed by the evaluation of selected mycolysin hits, identified potent candidates with minimum inhibitory concentration values as low as 1.28 μg/ml against M. bovis BCG and up to 75 μg/ml against pathogenic nontuberculous mycobacterium Mycobacterium avium. The three most potent mycolysins showed intracellular efficacy, serum stability, noncytotoxicity, in vivo proof-of-concept efficacy in rat wound and pulmonary infection models, and synergy with rifampicin treatment. This biotechnology framework illustrates the promise of translating phage enzymes into next-generation antimycobacterial therapies.
Burn injuries cause substantial morbidity, mortality, and long-term functional and aesthetic deficits. Autologous skin grafting, the gold standard for wound coverage, is lifesaving but limited by donor-site availability and can lead to scarring and contracture, highlighting the need for bioengineered skin substitutes that better restore native skin function. Stem cell-based skin substitutes have emerged as promising candidates, yet their application in burn care remains limited, warranting a review of potential gaps and opportunities. This review summarizes recent innovations in stem cell-based tissue-engineered skin substitutes for burn care, highlighting biofabrication platforms including 3D bioprinting, handheld devices, hydrogels, spray-on-skin systems, electrospinning, freeze-drying, stem cell sheets, molding, and gas foaming and discusses the key challenges and opportunities for burn-specific clinical translation.
Extrahepatic islet transplantation offers a clinically attractive alternative to intrahepatic delivery, but its success has been constrained by inadequate vascularization and lack of a supportive microenvironment. To address this, we engineered vascularized endocrine constructs (VECs) by coencapsulating pancreatic islets with human blood outgrowth endothelial cells in a good manufacturing practice-compliant amniotic membrane hydrogel (Amniogel). Amniogel provides extracellular matrix-bound prosurvival cues that enhance islet viability and function and promote endothelial self-assembly into vascular networks-improving β-cell gap junction coupling and restoring dynamic glucose-responsive insulin secretion. After subcutaneous implantation in diabetic mice, VECs anastomosed with host vessels and reestablished normoglycemia, outperforming nonvascularized controls. Furthermore, Amniogel impeded chemokine-driven cytotoxic T cell migration and delayed β-cell killing in vitro in a concentration-dependent manner. This integrative strategy, combining a scalable biological scaffold with vascularization and intrinsic barrier properties that may limit early immune cell infiltration, offers a clinically relevant path toward durable β-cell replacement therapies in type 1 diabetes.
Establishing efficient cell factories involves a continuous process of trial and error due to metabolic complexity. This complexity makes predicting effective engineering targets a challenging task. Therefore, successful previous designs are vital for future cell factory development. In this study, we developed a method using large language models to extract metabolic engineering strategies from research articles. We created a database containing over 29 006 metabolic engineering entries, 1210 products, and 751 organisms. Using this database, we trained a deep learning model to predict engineering targets for cell factories. Our model outperformed traditional algorithms, demonstrated strong generalization to unseen products and multigene combinations, and was experimentally validated with geraniol overproduction in yeast, leading to the identification of several novel targets. Our study provides a valuable dataset, a chatbot, and an engineering target prediction model for the metabolic engineering field and exemplifies an efficient method for leveraging existing knowledge for future predictions.
Systems engineering has transformed chemical manufacturing, but bioprocessing has lagged in adopting comprehensive approaches. This review explores strategies that successfully engineer integrated upstream and downstream bioprocesses. Our analysis reveals a critical gap: bioprocess subsystems are typically optimized in isolation ('subsystems optimization'), which limits the overall performance. We identify four key leverage points for systems engineering: engineering product accessibility to eliminate cell lysis, modifying strains to remove contaminants, adapting products for simplified purification, and enhancing strain tolerance for improved separation. While these integrative approaches substantially improve process consolidation, our findings show that there remains a significant misalignment between academic research and industrial needs (failing commercially relevant metrics). Embracing a holistic systems perspective is essential for future bioprocesses to have a transformative impact.
The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.
Marine microalgae are frequently promoted as sustainable biofuel feedstocks because of their halotolerance, high photosynthetic efficiency, and limited land requirements, yet commercial deployment remains elusive. This gap is primarily systemic rather than biological, reflecting the fragmented development of strain engineering, harvesting, conversion, and sustainability assessment. This review reframes marine microalgae as circular biofactories and advances a system-centric paradigm for integrated biorefineries. We synthesise recent advances in metabolic and genetic engineering, low-energy harvesting, and thermochemical and biochemical conversion, highlighting how cross-stage interdependencies dominate overall performance. We further discuss how artificial intelligence, digital twins, nutrient recycling, carbon utilisation, and high-value coproducts enable predictive optimisation and techno-economic viability. This perspective provides a road map for translating marine microalgal biofuels from laboratory promise to industrial relevance.
Extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSC-EVs) hold great therapeutic promise, yet challenges in scalable production and the impact of 3D cellular architecture on EV content and function continue to hinder clinical translation. Here, we demonstrate a perfusion-based stirred-tank bioreactor (BR) system to scale up hiPSC-EV production, eliminating labor-intensive static cultures while preserving physiologically relevant cellular characteristics. The system was successfully scaled from 0.2 l BRs to 2 l BRs, yielding clinically relevant EV doses (1011 EVs per 2 l BR). Notably, only EVs secreted under BR conditions elicited significant proangiogenic effects in vitro, promoting endothelial cell survival, proliferation, migration, and sprouting. Small RNA and proteomic profiling revealed enrichment of proangiogenic miRNA and proteins regulating endothelial function in BR-derived EVs, indicating a shift driven by the culture environment. These findings underscore the role of BR dynamics in shaping EV properties and supporting the scalable production of therapeutically potent hiPSC-EVs.
We present a sustainable microbial platform utilizing gut bacteria and a plant-based medium for stereoselective neurosteroid biosynthesis. Through bioinformatics- and structural biology-guided screening of over 3000 bacterial isolates, we identified three gut strains exhibiting distinct stereospecificities: Holdemania filiformis produces isopregnanolone, Clostridium innocuum generates epipregnanolone, and Hungatella effluvii synthesizes pregnanolone. Following heterologous expression in Escherichia coli, we characterized two H. filiformis proteins: steroid 5α-reductase (Hp5αR) and 3β-hydroxysteroid dehydrogenase/reductase (Hf4205). We developed molasses-okara medium (MOM), a plant-derived composite medium combining sugarcane molasses with enzymatically hydrolyzed okara devoid of animal-derived components. In multigram batch whole-cell biotransformation trials using MOM, we achieved over 95% progesterone conversion into target neurosteroid isomers. The inherent stereoselectivity of these whole-cell biotransformations bypasses downstream chiral chromatographic separation, enabling pharmaceutical-grade product recovery through a simple open-column purification. Compared with peptone-yeast extract-glucose media, MOM reduced production costs by 90% and carbon footprint by 95% in whole-cell biotransformation, demonstrating sustainable bioeconomy principles in pharmaceutical biotechnology.
Prime editing (PE) and base editing (BE) are potent gene-editing techniques that avoid introducing exogenous DNA donors and double-strand breaks, but their large molecular size hinders efficient delivery and widespread application. Here, we engineer and evolve compact obligate mobile element-guided activity (OMEGA)-based PE and BE systems, termed OMEGA-PE and OMEGA-adenine base editor, which achieve superior editing efficiency and versatility compared with existing PE and BE tools in Escherichia coli. Notably, OMEGA-PE enables rapid, programmable targeted mutagenesis with outstanding specificity and efficiency compared with current tools. Moreover, it exhibits excellent compatibility with several high-throughput screening platforms and can be used to enhance the efficiency of cofactor regeneration and protein translation. These groundbreaking advancements offer significant potential for gene editing, directed evolution, metabolic engineering, and protein expression optimization. Combining compact size with high performance, our OMEGA-based systems address key bottlenecks in current tools, offering new possibilities for basic research and industrial biotechnology.
Rapid yet high-information-content monitoring of cellular metabolism during fermentation is highly desirable for process optimization. While single-cell Raman spectroscopy can rapidly distinguish intracellular biopolymer classes, finer-resolution discrimination of monomeric units remains challenging. In this study, we demonstrated that the ramanome can classify polyhydroxyalkanoate (PHA)-producing Halomonas bluephagenesis cells that synthesize either polyhydroxybutyrate or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 99.75% accuracy and simultaneously quantify total intracellular PHA and its constituent monomeric units (3-hydroxybutyrate and 4-hydroxybutyrate) at the single-cell level (median absolute deviation <3.8%). Such information enabled timely, data-driven selection of the optimal harvest time and revealed precursor competition between nucleic acid and PHA biosynthesis. Moreover, when monitoring PHA production in an industrial-scale 5000 l-fermenter, our method achieved a 12-min turnaround time, representing a more than100-fold acceleration over gas chromatography. Furthermore, tests on protein production by Saccharomyces cerevisiae and on lipid synthesis in Rhodococcus opacus supported its versatility. Thus, ramanomics is a valuable approach for process control and strain evaluation in biomanufacturing.
Corneal diseases remain a leading cause of blindness, and corneal transplantation is the primary treatment for severe cases. However, the persistent shortage of donor corneas significantly limits this approach. Artificial corneas with engineered biomaterials designed to mimic the structure and function of native corneal tissue offer a promising alternative, and significant progress has been made, including FDA approval and clinical trials. Among emerging technologies, 3D printing enables personalized customization of implants tailored to patient needs, anatomy, and pathology. This method also enables precise control over the biomaterial composition and integration of living cells within the implant, enhancing post-transplantation regeneration. In this review, we examine the advancements in 3D-printed, proregenerative artificial corneas, focusing on innovative biofabrication and the challenges that remain in ensuring implant quality.