Wyss Institute for Biologically Inspired Engineering

Oral wound healing is a robust process; however, complications from surgery, systemic diseases, and aging can impair healing. While some treatments exist, regenerative therapies to promote mucosal wound healing remain limited. In recent years, there has been a significant rise in FDA-approved cell-based therapies; however, extracellular vesicles represent an emerging cell-free alternative that may mitigate risks associated with cellular therapies, including tumorigenesis and immunogenicity. These lipid-encapsulated nanovesicles can deliver therapeutic cargo, such as proteins, lipids, nucleic acids, or drugs, to the wound site. Extracellular vesicles can be derived from mesenchymal stromal cells, immune cells, bodily fluids, or bacteria, and engineered through genetic modification, preconditioning, or direct cargo loading to enhance therapeutic potency. Furthermore, advanced delivery platforms, including hydrogels, microneedles, and aerosols, allow for sustained and localized EV delivery to the oral wound site. This review examines differences between cutaneous and oral wound healing; factors that impair oral repair; extracellular vesicle sources and engineering strategies; and delivery strategies for developing EV-based therapeutics for oral wound healing.

Abstract Background Early and accurate detection of breast cancer and differentiation from benign breast disease remains a substantial challenge, with about 70% of diagnostic breast biopsies having no malignant findings. Tumor-associated Autoantibodies represent the immune system’s response to a neoplasm and are a promising biomarker group for the early diagnosis of breast cancer by liquid biopsy. Methods In this study, we quantified the IgM and IgG titers to 525 Tumor Associated Antigens in a prospectively-collected cohort of 50 serum samples from donors with benign breast disease and donors with early-stage breast cancer. The considerable number of antibodies analyzed enabled us to account for variations in individual immune profiles through z- score normalization of each donor’s total antibody distribution. Differentially expressed antibodies were identified using Mann-Whitney U tests ( p < 0.05) and fold-change analysis (fold-change > ± 1.2). For each donor, we calculated the total number of “high-titer” antibodies, defined as antibodies with relative concentrations > 3 SD above the cohort mean. Logistic regression classifiers were then built using differentially expressed biomarkers and high-titer antibody counts to distinguish benign breast disease from breast cancer. Results We identified 25 differentially expressed antibodies between the benign and cancer groups. A down-selected panel of eight antibodies demonstrated good performance in a logistic regression classifier to distinguish benign disease from invasive carcinomas (AUC-ROC = 0.83 ± 0.14). High-titer antibody analysis revealed that the benign group had a higher prevalence of donors with elevated IgG immune response, and donors displayed antibody signatures unique to their individual disease pathway. Conclusions This study identifies an eight-antibody panel with promising diagnostic potential to distinguish benign breast disease from early-stage breast cancer. The z-score normalization approach and analysis of individual donors’ high-titer antibody profiles represent a novel approach towards personalized cancer immunology. This study provides encouraging preliminary evidence supporting the promise of tumor-associated autoantibody profiling for distinguishing benign and malignant breast disease, warranting future studies in larger cohorts.

DNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing ΦC31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The method’s ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.

We have explored a simple model for the multicellular interplay between bioelectricity and protein transcription using a top-down perspective that offers new insights complementary to the commonly used bottom-up descriptions. We model the non-excitable cell bioelectrical representation of the external environment, including the neighboring cells, using voltage-gated ion channels and intercellular junctions. The simulations make three predictions: (i) shifts in membrane potential allow transitions between gene expression states, (ii) in the case of different cell potential-gated transcriptions, depolarized cells cannot control the distinct gene expressions as effectively as polarized cells, and (iii) community effects should permit to extend the single-cell control to the multicellular level. Because the spatio-temporal distributions of instructive signaling ions and molecules depend on the local electric potentials, different multicellular potentials correlate with distinct downstream gene expression patterns. A central cell is able to measure the number of neighboring cells and learn their bioelectrical state from the downward-induced membrane potential changes, which suggests that multiscale bioelectricity can operate as a top-down mechanism in development and regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-31696-6.

Bioelectronic implants for brain stimulation are used to treat brain disorders but require invasive surgery. To provide a noninvasive alternative, we report nonsurgical implants consisting of immune cell-electronics hybrids, an approach we call Circulatronics. The devices can be delivered intravenously and traffic autonomously to regions of inflammation in the brain, where they implant and enable neuromodulation, circumventing the need for surgery. To achieve suitable electronics, we designed and built subcellular-sized, wireless, photovoltaic electronic devices that harvest optical energy with high power conversion efficiency. In mice, we demonstrate nonsurgical implantation in an inflamed brain region, as an example of therapeutic target for several neural diseases, by employing monocytes as cells, covalently attaching them to the subcellular-sized, wireless, photovoltaic electronic devices and administering the resulting hybrids intravenously. We also demonstrate neural stimulation with 30-µm precision around the inflamed region. Thus, by fusing electronic functionality with the biological transport and targeting capabilities of living cells, this technology can form the foundation for autonomously implanting bioelectronics.

Ribonuclease A (RNase A) has long been known to spontaneously refold to its native conformation and recover catalytic activity following denaturation. Yet, the molecular heterogeneity of its folding pathway has remained unclear due to ensemble averaging in traditional bulk measurements. Here, we use single-molecule enzymology (SME) to monitor the catalytic recovery of urea-denatured RNase A at the single-molecule level. We directly observe that individual RNase A molecules exhibit either fast or slow refolding kinetics, indicating distinct renaturation pathways that implicate slow prolyl peptide bond isomerization. These findings establish the utility of SME in revealing hidden heterogeneity in protein folding pathways.

Herpes simplex viruses are prevalent human pathogens that cause significant morbidity and mortality in neonates and adults. They undergo acute infection in the mucosae and spread to peripheral neurons including sensory and autonomic neurons, where they establish lifelong latent infection. Reactivation from latency leads to recurrent infection and disease, including central nervous system infection that can be life-threatening. Experimental studies of HSV latency have mostly been conducted in animals, which may differ from the human situation. In this study, we establish a system for differentiation of human-inducible pluripotent stem cells into sensory neurons and for latent infection and reactivation by herpes simplex virus 1. This system will enable studies of the mechanism of HSV latent infection in human sensory neurons and therapeutic approaches to curtail it.

Meiotic failure is a major cause of infertility, but the lack of an in vitro model of human meiosis is a barrier to understanding its mechanism. Here, we establish a method to initiate meiosis directly from male or female human-induced pluripotent stem cells (iPSCs). DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (antiapoptotic BCL2 and promeiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis over a 15-day protocol. Our protocol bypasses the primordial germ cell stage and directly generates cells expressing genes similar to meiotic oogonia, including oogonia markers, all synaptonemal complex components, and meiotic recombination machinery. DNMT1 inhibition rapidly erases DNA methylation, including at imprinting control regions and promoters of meiotic genes. Microscopy shows key aspects of meiosis, including chromosome axis formation and synapsis in live human cells. Our model of human meiosis provides opportunities for studying this critical reproductive process under chemically defined conditions in vitro.

Surfactant protein-B (SP-B) deficiency is a lethal neonatal respiratory disease with few therapeutic options. Gene therapy using adeno-associated viruses (AAVs) to deliver human SFTPB cDNA (AAV-hSPB) can improve survival in a mouse model of SP-B deficiency. However, the effect of this gene therapy wanes. Gene therapy efficacy could be prolonged if AAV vectors could be redosed, but readministering vectors is hindered by an immune response that includes TLR9 (Toll-like receptor 9) recognition of unmethylated CpG DNA motifs in the AAV genome. One strategy to mitigate TLR9 recognition of AAV is to incorporate decoy nucleotide sequences within the AAV genome. In this work we examined if an AAV containing these TLR9 inhibitory oligonucleotide sequences (AAV-hSPBTLR9i) could mitigate the immune response sufficiently to redose AAV in the lungs and prolong the survival of SP-B-deficient mice. Indeed, AAV-hSPBTLR9i could be redosed multiple times, which significantly improved survival in our mouse model. This was partially a result of long-term increased SFTPB RNA and SP-B protein expression. Conversely, redosing AAV-hSPB resulted in the rapid death of SP-B-deficient mice after the second AAV dose. TLR9 inhibition enabled readministration by avoiding the broad stimulation of genes belonging to multiple pathways in the host immune and inflammatory responses, including components of the IFN pathways. Thus, redosing of AAV vectors in the lungs using TLR9 inhibitory sequences is a promising strategy for prolonging gene therapy efficacy, with a proof of concept for AAV readministration in a clinically relevant mouse model of SP-B deficiency.