Antisense oligonucleotides (ASOs) are a treatment modality for genetic diseases. ASOs bind to ribonucleic acid (RNA) with high specificity, with downstream effects that eventually reduce, restore, or modify protein expression through distinct mechanisms. Conjugating ASOs to targeting ligands has emerged as a promising strategy to improve tissue-specific delivery. Here, we develop streamlined assays that assess the binding kinetics of both the ASO and the targeting ligand using biolayer interferometry (BLI). The utility of this biomolecular sandwich binding assay is demonstrated with phosphorodiamidate morpholino oligomers (PMOs) and peptidenucleic acids (PNAs) conjugated to a peptide or protein ligand. We show that this assay can be used to detect intracellular uptake and predict in vitro efficacy. We believe that the methods developed here can accelerate the development of next-generation ASO therapeutics.

Lipid nanoparticles (LNPs) are the leading nonviral nucleic acid delivery technology, but LNP structure-function data remains fragmented and nonstandardized. Unlike protein engineering which is anchored by the centralized Protein Data Bank, the LNP field lacks a unified repository for systematic analysis. To address this, we develop Lipid Nanoparticle Database (LNPDB) (https://lnpdb.molcube.com), an integrated database and web tool that consolidates structural and functional data for 19,528 LNPs. LNPDB standardizes LNP featurization by encoding lipid composition, experimental methods, and functional results, and generates CHARMM force field files for constituent lipids to enable molecular dynamics simulations. LNPDB also supports future data contributions for continued growth. We examine the utility of LNPDB through two applications: advancing our deep learning model for predicting LNP delivery performance, and simulating bilayer dynamics to identify structural features – bilayer stability and critical packing parameter – that correlate with LNP delivery performance. Altogether, LNPDB provides the digital framework for LNP modeling and data-driven rational design. This study introduces the Lipid Nanoparticle Database (LNPDB) and web tool that consolidates lipid nanoparticle structure-function data. LNPDB facilitates molecular dynamics and deep learning approaches to advance data-driven design for nucleic acid delivery.

Here, we develop a next-generation wireless, battery-free oxygen-generating O2-macrodevice and wearable power transfer platform that can enable long-term immunoprotection and subcutaneous function of therapeutic cells. We demonstrate that this device supports xenogeneic islet transplantation in C57BL/6J mice, evidenced by 90-day diabetes reversal and glucose responsiveness in vivo. We also show partial glycemic control via high-density (>8,000 islets/cm2) human stem-cell-derived islets (SC-islets) without immunosuppression in subcutaneous sites for 90 days. Additionally, we confirmed that the device supports allogenic islet cell survival and 90-day diabetic reversal in rats. Finally, we demonstrate 1-month islet survival in a nonhuman primate without the need for immunosuppression in the subcutaneous space. Collectively, these results indicate that the device supports cell survival and function across multiple transplant models in three species without the need for any immunosuppression or external user intervention. These results represent an important set of advances toward immunosuppression-free, minimally invasive islet transplantation.

Atrial fibrillation and the risk of its lethal complications are propelled by fibrosis, which induces electrical heterogeneity and gives rise to reentry circuits. Atrial TREM2+ macrophages secrete osteopontin (encoded by Spp1), a matricellular signaling protein that engenders fibrosis, inflammation, and atrial fibrillation. Here we developed an antibody-siRNA conjugate (ARC) drug candidate to silence Spp1. The ARC relies on an anti-TREM2 antibody for delivering Spp1-targeted siRNA to a pathogenic macrophage subset that expands in human atrial fibrillation. The ARC preferentially targeted atrial TREM2+ macrophages with limited uptake by other immune or stromal cells of the heart. We observed efficient silencing of the target gene in human myocardium and in mice, where it reduced pro-fibrotic fibroblast activation and atrial fibrosis. Four weeks of systemic ARC treatment suppressed inducible atrial fibrillation in mice exposed to clinically prevalent risk factors. These results suggest that macrophage subset targeting offers a viable immunomodulatory strategy for atrial fibrillation.

Hydrogels that adhere to biological tissues and resist fibrosis are required to provide both optimal functionality and appropriate stiffness on diverse soft tissues to achieve therapeutic efficacy and biocompatibility. However, their performance is often limited by an intrinsic trade-off between functionality and stiffness. Through the incorporation of polymer brush coatings, we develop a modular hydrogel system to enable independent control of functionality and stiffness. By tailoring coating chemistry, coating thickness, and hydrogel network topology, we obtain consistent bioadhesion (~100 joules per square meter) and fibrosis suppression across the full stiffness range of soft tissues (1 kilopascal to 1 megapascal). Using this approach, we design a hydrogel that can maintain stable adhesion in vivo on a beating mouse heart and a hydrogel with no fibrotic capsule in immunocompetent mice over 40 days. This modular system offers a customizable approach for designing functional implants with tailored mechanical properties.

Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

A strategy to control translation of mRNA vaccines reveals cell type–specific contributions to immunity that may be harnessed to enhance vaccine efficacy.

Nucleic acid nanoparticles (NANPs) fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics. Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars, understanding their cell targeting and uptake mechanisms remains limited. Here, we investigated NANP cellular targeting, uptake, and delivery of small interfering RNAs (siRNAs) to liver and neuronal cell models in vitro. Using a rational design approach, we targeted NANPs to two clinically validated receptors, the asialoglycoprotein receptor (ASGPR) and the low-density lipoprotein receptor (LDLR), respectively, using GalNAc and lipidation. We systematically evaluated how the ligand valency, interligand spacing, linker length, and ligand chemistry affected NANP association with on- and off-target liver cell types, revealing the relative roles of the biomolecular corona, receptor engagement, and endocytosis in these targeting strategies. We found that lipidation enhanced NANP uptake into HepG2 cells, a model cell line for hepatocytes, by promoting apolipoprotein recruitment, LDLR engagement, and clathrin-mediated endocytosis and also increased association with nonparenchymal cells. HepG2 uptake was further improved by conjugating NANPs to lipids with higher valency provided that lipids were adequately displayed away from the surface of NANP edges with more lipophilic lipids yielding greater cell association. We then benchmarked the potential for NANPs to deliver siRNAs to HepG2 cells in comparison with lipid nanoparticle and conjugate technologies and explored lipid functionalization as a strategy for nonhepatic NANP targeting to model neuronal cells. Overall, this study advances the foundational understanding of how clinically relevant targeting ligands mediate NANP interactions with both on- and off-target liver cell types in vitro, offering insights into potential design criteria for nucleic acid therapeutic delivery.

IntroductionMacrophages play a key role in inflammatory diseases. We aimed to identify long noncoding RNAs that regulate pro-inflammatory activation of human macrophages. MethodsWe performed human lncRNA microarray analysis in LPS-stimulated primary human macrophages. We then carried out loss-of-function and gain-of-function experiments, luciferase reporter assays, RNA pulldown, RNA immunoprecipitation, and mouse endotoxemia studies, including humanized mouse models. ResultsWe identified 11 lncRNAs that were significantly increased by LPS. Among them, lnc-FAM164A1 was selected for further study. Silencing of lnc-FAM164A1 by antisense oligonucleotides or siRNA reduced the LPS-induced expression of pro-inflammatory cytokines, including CCL2, IL-6, and TNF-α. In contrast, enforced expression of lnc FAM164A1 enhanced inflammatory responses in human and mouse macrophages. lnc FAM164A1 also promoted NF-κB-related signaling. RNA pulldown and RNA immunoprecipitation identified ACLY as an lnc-FAM164A1-associated protein. ACLY silencing reduced the inflammatory effects induced by lnc-FAM164A1. DiscussionThese findings support that human lnc-FAM164A1 promotes pro-inflammatory activation of macrophages through its interaction with ACLY and NF-κB-related signaling.

Immunoisolation devices containing therapeutic protein-secreting cells offer potential for long-term therapy without immune suppression. However, scar tissue formation driven by the foreign body response (FBR) hinders nutritional exchange and ultimately leads to graft failure. We previously showed that inhibiting the colony-stimulating factor-1 receptor (CSF1R) pathway in monocytes and macrophages can block the FBR to implanted materials. Here, we demonstrate that coencapsulation of slow-releasing CSF1R inhibitor (GW2580) crystals with human stem cell-derived β cells (SC-β) in alginate spheres enables stable glycemic control for 1 year in immune-competent diabetic C57BL/6 mice. In nonhuman primates (NHPs), GW2580 crystals similarly protected viable, glucose-responsive allogeneic β cells for 1 month without systemic immune suppression. In contrast, the same xenogeneic human SC-β cell formulation that functioned long-term in mice elicited extensive sphere overgrowth and graft failure in NHPs. Serum cytokine profiling and transcriptomic analysis of omental biopsies at day 30 revealed pronounced adaptive immune activation in xenogeneic recipients, including enrichment of CD4+ T cells, CD19+ B cells, and antigen-presenting cell programs marked by elevated MHC class II expression. Chemokines CCL17, CCL22, and CXCL13 were among the most highly up-regulated transcripts, mirroring responses observed previously with profibrotic alginate formulations without cells. These findings underscore the issues associated with xenogeneic cell sources in higher-order species yet indicate that targeting innate immune pathways with localized CSF1R inhibition may be sufficient to enable function of encapsulated allogeneic cell therapies.

Hi1a is a venom peptide isolated from the Australian funnel-web spider Hadronyche infensa, containing an intricate "double knot" tertiary structure, and is currently under investigation for the treatment of ischemic stroke. The recombinant expression and chemical synthesis of Hi1a remain a significant challenge due to laborious protocols, delaying progress in translation to the clinic. We describe the first single-shot chemical synthesis of Hi1a (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (<4.5 h total synthesis time) to milligram quantities of linear Hi1a (>10 mg). This work highlights the utility of AFPS as a technology that enables chemical production of biomolecules challenging to obtain by recombinant methods.

Membranes that prevent cellular infiltration are essential components of immune-isolation devices. Stringent pore size control is critical to robustly exclude immune cells while preserving the efficient transport of nutrients and therapeutic molecules. Conventional membrane fabrication methods, however, struggle to balance these two properties. For instance, phase separation and fiber spinning generate highly tortuous pores that restrict diffusion, while random pore overlaps due to ion track etching result in oversized defects that compromise immune isolation. Here, we leverage scalable laser-writing tools to achieve spatially controlled open-through and dense pores with uniformly distributed submicrometer sizes for rapid transport and robust immune cell exclusion. Given the optical limits of laser aligners when fabricating submicrometer features, we tuned substrate reflectivity and exposure parameters to overcome these resolution limits. Self-standing and flexible membranes were achieved via thickness-tunable grid layers supporting membrane mechanical integrity. We report pore sizes as small as ca. 600 nm (below the resolution limits of the laser exposure systems) and over 20% open area at the exposure times of ∼3 min/cm2. Compared with conventional methods, this approach minimizes pore tortuosity, narrows the pore distribution, and improves the open pore density. Enhanced pore control is highlighted by macrophage infiltration studies, which indicate that these membranes reliably exclude macrophages at nominal pore sizes greater than those previously achievable. This approach thus provides a scalable pathway toward next-generation immunoisolation membranes for enhanced implantable therapeutic devices.

Self-driving laboratories that integrate robotic production with artificial intelligence have the potential to accelerate innovation in biotechnology. Because self-driving labs can be complex and not universally applicable, it is useful to consider their suitable use cases for successful integration into discovery workflows. Here, we review strategies for assessing the suitability of self-driving labs for biochemical design problems.

The intersection of biotechnology and materials science has driven medical and scientific innovation for decades and is poised to make similar transformative impacts over the next 50 years. Advanced drug delivery systems, including nanoparticles and larger delivery material platforms, are enhancing therapeutic precision, while tissue engineering and regenerative medicine are laying the groundwork for bioprinting complex organs, offering new possibilities for transplantation and repair. Nanotechnology and biomedical devices are reshaping diagnostics and therapeutics, enabling real-time monitoring essential for personalized health care. Additionally, emerging fields such as space biotechnology and machine learning-driven biomaterials design hold potential for cutting-edge discoveries. This article examines the historical trajectory, current state-of-the-art applications, and bold future directions of biotechnology in materials science, emphasizing its impact on human health and its untapped potential yet to be explored.

Lipid nanoparticle (LNP) components can impact the safety and immunogenicity of mRNA vaccines. Here we examine the mechanisms contributing to the performance of mRNA-LNP vaccines by exploring the impact of nucleoside modifications and LNP components on translational efficiency, innate immune activation, and immunogenicity. Our data reveals several molecular and immunological parameters affected by nucleoside modification including a synergistic effect of the mRNA and ionizable lipid composition on the immune activation triggered by the mRNA-LNP formulation. Our results indicate changes in the LNP composition, independent from whether the mRNA is modified or unmodified, caused differential expression of genes associated with innate and antiviral immunity. We believe these findings offer valuable insights into mRNA vaccine function and offer strategies for enhancing vaccine efficacy and reducing the reactogenicity of next generation mRNA vaccines.

Background— Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. The BolA Family Member 3 (BOLA3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. Methods— In vitro assessment of BOLA3 regulation and gain and loss of function assays were performed in human pulmonary artery endothelial cells (PAECs) using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was utilized for lung endothelial-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. Results— In cultured hypoxic PAECs as well as lung from human Group 1 and 3 PH patients as well as multiple rodent models of PH, endothelial BOLA3 expression was down-regulated, which involved HIF-2 α -dependent transcriptional repression via HDAC-mediated histone deacetylation. In vitro gain and loss of function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency down-regulated the glycine cleavage system protein H (GCSH), thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction, while decreasing angiogenic potential. In vivo, pharmacologic knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histologic and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. Conclusions— BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic re-programming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, iron-sulfur biogenesis, and glycine biology, for diagnostic and therapeutic development. provide crucial support for the of central dysregulation of Fe-S integrity a biogenesis

Bioelectronic devices hold transformative potential for healthcare diagnostics and therapeutics. Yet, traditional electronic implants often require invasive surgeries and are mechanically incompatible with biological tissues. Injectable hydrogel bioelectronics offer a minimally invasive alternative that interfaces with soft tissue seamlessly. A major challenge is the low conductivity of bioelectronic systems, stemming from poor dispersibility of conductive additives in hydrogel mixtures. We address this issue by engineering doping conditions with hydrophilic biomacromolecules, enhancing the dispersibility of conductive polymers in aqueous systems. This approach achieves a 5-fold increase in dispersibility and a 20-fold boost in conductivity compared to conventional methods. The resulting conductive polymers are molecularly and in vivo degradable, making them suitable for transient bioelectronics applications. These additives are compatible with various hydrogel systems, such as alginate, forming ionically cross-linkable conductive inks for 3D-printed wearable electronics toward high-performance physiological monitoring. Furthermore, integrating conductive fillers with gelatin-based bioadhesive hydrogels substantially enhances conductivity for injectable sealants, achieving 250% greater sensitivity in pH sensing for chronic wound monitoring. Our findings indicate that hydrophilic dopants effectively tailor conducting polymers for hydrogel fillers, enhancing their biodegradability and expanding applications in transient implantable biomonitoring.