Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting the peripheral nervous system, characterized by the development of benign and malignant tumors, including neurofibromas. NF1 is primarily caused by the loss of function of neurofibromin, a tumor suppressor protein predominantly expressed in Schwann cells, where it negatively regulates the RAS/MAPK signaling pathway. Current treatment options for NF1 are limited to surgical tumor removal and pharmacological interventions, which often yield suboptimal outcomes. In this project, we propose a novel approach utilizing engineered extracellular vesicles (EVs) as a targeted delivery system for Schwann cells. Our current results demonstrate the feasibility of producing Schwann cell-targeting, NF1-loaded EVs and confirm the effectiveness of targeting ligands in mediating specific delivery both in vitro and in vivo. We are further optimizing the engineered EV formulation and advancing our therapeutic approach by employing tissue nanotransfection (TNT) to induce the production of Schwann cell-targeting designer EVs within the epidermis of NF1 model mice.
Lower back pain is a leading cause of disability, with intervertebral disc degeneration (IVDD) contributing through inflammation, neurovascularization, and loss of matrix production. Current treatments are invasive and fail to address IVDD-associated mechanisms. Engineered extracellular vesicles (eEVs) containing developmental transcription factors (TFs) offer a promising therapeutic alternative. This study characterizes eEVs loaded with Mohawk (MKX) and Scleraxis (SCX) for annulus fibrosus (AF) cells and assesses their effects on degenerate human AF cells seeded on polycaprolactone (PCL) nanofiber scaffolds mimicking healthy (Organized) and degenerative (Disorganized) architectures. eEVs were successfully loaded and delivered to AF cells, with RNA sequencing revealing 290 differentially expressed genes between Disorganized vs. Organized controls, including reductions in CCL8 (inflammation) and increases in COL5A3, ELF3, and MMP1 (matrix remodeling). Synergy-eEVs in Organized fibers induced 366 differentially expressed genes, including upregulation of SEMA3A, SEMA3D, and TAC1, suggesting modulation of neurovascularization and pain pathways. In Disorganized fibers, 257 genes were differentially expressed, with downregulation of ACAN, SMAD1, and VCAM1 (osteoarthritis and NP-like pathways). Comparing Synergy-eEVs in Disorganized vs. Organized fibers, 307 genes showed differential expression, with decreases in IFNA, IFNB, IFNG, and ACAN and increases in COL5A3, VTN, MMP14, and A2M, suggesting a shift toward matrix remodeling and reduced inflammatory signaling. These findings demonstrate that eEVs effectively deliver TF cargo to AF cells, with therapeutic effects influenced by matrix architecture. This study highlights the importance of substrate relevance in screening and the use of eEVs in therapuetic approaches for IVDD.
NFκB is a stress-response transcription factor that is held in the cytoplasm in an inhibited state until an extracellular stress signal activates the IκB kinase which phosphorylates the disordered N-terminal domain of the inhibitor, IκBα. Subsequent ubiquitylation and proteasome-mediated degradation results in release of NFκB which translocates to the nucleus and activates transcription of hundreds of genes. NFκB signaling is oscillatory with a period of ~1 hr indicating that the nuclear NFκB is rapidly returned to the cytoplasm. We showed that newly synthesized IκBα enters the nucleus and removes NFκB from transcription sites in a molecular stripping (facilitated dissociation) process. Single-molecule FRET reveals the disordered regions of IκBα that fold and unfold in the native state and fold upon binding to NFκB facilitating dissociation. Single-molecule FRET also reveals that DNA-bound NFκB is dynamic transiently leaving an opening for IκBα-mediated dissociation. The mechanisms of each step of this process and how disordered linkers and domains are involved will be discussed.
Last update: 3/31/2025, Ralf Bundschuh