Objectives/Goals: Our objective in this study was to develop a nanoparticle-based RNA delivery system that reprograms Müller glia toward neuronal fates in human relevant models, enabling localized and transient regeneration without viral vectors. Methods/Study Population: - Results/Anticipated Results: Fabricated nanoparticles encapsulating RNA cargoes were ~110–150 nm in size with polydispersity indices of ~0.2–0.3 and encapsulation efficiencies >95%. Nanoparticles successfully delivered GFP mRNA in both organoid and retinal explant models, showing significant expression of fluorescent reporters in both live cell imaging and in slides prepared using immunohistochemistry. We anticipate that our delivery system will lead to transcription factor expression after delivery of corresponding RNAs with evidence of proliferation and neuronal marker upregulation. We also hypothesize that this approach will enhance neurogenesis in retinal organoids and explants, potentially establishing a platform for non-viral retinal regeneration. Discussion/Significance of Impact: This work demonstrates a clinically translatable, virus-free strategy for retinal repair through targeted RNA delivery. By enabling transient expression of neurogenic factors, nanoparticle-mediated reprogramming presents a scalable and adaptable approach for restoring vision in degenerative retinal diseases.
Objectives/Goals: This collaboration explores magnetic-responsive scaffolds toward modeling Salmonella infection via the small extracellular vesicle (sEV) route. We aim to elucidate the mechanism by which sEVs reflect host–pathogen interactions to inform new Salmonella therapeutics. Methods/Study Population: Human intestinal epithelial cells (INT-407) are being cultured on magnetic-responsive scaffolds under controlled mechanical stimulation and infected with Salmonella Typhimurium 12023 WT. Kinetic assays are assessed via confocal microscopy, flow cytometry, and qPCR. sEV isolation is being performed by NanoPoms immunomagnetic capture and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting for canonical markers (CD9, CD63, and CD81). Additionally, sEVs derived from human THP-1 cells are being used to treat naïve INT-407, in which cytokine responses and proteomic shifts are analyzed. Results/Anticipated Results: We anticipate that INT-407 in 3D magnetic scaffolds will shift the Salmonella infection kinetics compared to the unstimulated controls and traditional 2D culture. We expect the sEVs from infected cells to be enriched in antigenic proteins and canonical sEV markers. Furthermore, sEVs from infected THP-1 cells are anticipated to induce pro-inflammatory cytokine release and proteomic shifts toward intracellular signaling and inflammatory processing in naïve INT-407 following treatment. Taken together, these findings will elucidate the mechanism by which magnetic-responsive scaffolds better recapitulate the gut microenvironment for modeling host–pathogen interactions. Discussion/Significance of Impact: This project’s findings will indicate if 3D magentic scaffolds provide a sound model for Salmonella infection. Analysis of sEVs derived from INT-407 and THP-1 will provide insight into the functional characteristics sEVs have in the gastrointestinal environment upon pathogen insult.
Objectives/Goals: Clinical studies with triple-negative breast cancer (TNBC) are encouraging for immunotherapy combined with chemotherapy. Additional clinical advances may be accelerated by leveraging preclinical TNBC models including syngeneic mammary tumor cell lines. Here, we report two lines exhibit differential immunotherapy responses in vivo . Methods/Study Population: Spontaneous mammary tumors from C57BL/6J MMTV-Cre Trp53fl/+ animals were passaged serially in cell culture and in vivo in the mammary fat pad of fully wildtype animals. The resulting lines, MM001i and MM008i, lost Trp53 and formed 1000 mm3 [https://www.biorxiv.org/content/10.1101/2025.09.18.677171v1#ref-3] tumors in the mammary fat pad within 21–28 days. Tumors from each group were analyzed for their response to αPd-1 and αCtla-4 immunotherapy, infiltrating immune populations, and transcriptional profiles. Results/Anticipated Results: Despite originating from the same genetic background, these lines exhibit differential responses to immunotherapy. For αPd-1 therapy, MM001i is poorly responsive and MM008i is strongly responsive with near-complete tumor regression. In comparison, both MM001i and MM008i respond rapidly to αCtla-4 therapy. Both models express unique tumor antigens as evidenced by immunity to subsequent engraftments. Primary MM008i tumors exhibit greater T-cell infiltration, and CD8-positive T lymphocytes are required for αPd-1 responses. Cancer epithelial cells within MM008i tumors demonstrate a greater abundance of Pd-l1 expressing cells, correlating with observations in human TNBC where PD-L1 expression is a biomarker for αPD-1 response. Discussion/Significance of Impact: These TNBC models are promising for further mechanistic studies and testing future single and combinatorial therapies.
Objectives/Goals: Methicillin-resistant Staphylococcus aureus is a nefarious bacterial pathogen and is classified as a serious threat. MRSA is resistant to most B-lactam antibiotics (penicillins, cephalosporins, etc.). The goal of this study is to identify an antibiotic adjuvant capable of resurrecting B-lactam antibiotics for treatment of MRSA infections. Methods/Study Population: A fluorescence-reporter assay was used to screen a compound library. Minimum-inhibitory concentrations were assessed for the compounds against various MSSA and MRSA strains. A common resistance mechanism to B-lactams by MRSA is by the function of the bla operon. One gene in this operon encodes for a B-lactam sensor/signal transducer protein BlaR, the primary target of this study. Inhibition of BlaR by compound 1 (best potentiator of oxacillin) was studied by nano-differential scanning fluorimetry (nanoDSF), surface plasmon resonance (SPR), scanning electron microscopy (SEM), and in vivo assays. Results/Anticipated Results: We identified 80 compound hits from a 1,974-compound NCI library. Twenty-four compounds showed potentiating ability (2- to 4,096-fold decrease in MIC for oxacillin). Seven compounds exhibited melting temperature shifts by nanoDSF of BlaR, indicating binding. SPR determined compound 1 has a binding affinity of 31 mM to BlaR-SD. SEM images showed disruption in the S. aureuscell wall on exposure to compound 1and oxacillin. S. aureusN315 showed 3-log reduction in bacterial count treated with a mixture of compound 1 and oxacillin. Discussion/Significance of Impact: Compound 1 targets BlaR-SD, which restores S. aureussusceptibility to treatment by oxacillin. There are currently few antibiotics available in the clinic capable of treating MRSA infections. The combination hold promise of a treatment option for MRSA.
