Engineered Vasculogenic Extracellular Vesicles Drive Nonviral Direct Conversions of Human Dermal Fibroblasts into Induced Endothelial Cells and Improve Wound Closure.

Vasculogenic cell therapies have emerged as a powerful tool to increase vascularization and promote tissue repair/regeneration. Current approaches to cell therapies, however, rely mostly on progenitor cells, which pose significant risks (e.g., uncontrolled differentiation, tumorigenesis, and genetic/epigenetic abnormalities). Moreover, reprogramming methodologies used to generate induced endothelial cells (iECs) from induced pluripotent stem cells rely heavily on viral vectors, which pose additional translational limitations. This work describes the development of engineered human extracellular vesicles (EVs) capable of driving reprogramming-based vasculogenic therapies without the need for progenitor cells and/or viral vectors. The EVs were derived from primary human dermal fibroblasts (HDFs), and were engineered to pack transcription factor genes/transcripts of ETV2, FLI1, and FOXC2 (EFF). Our results indicate that in addition of EFF, the engineered EVs were also loaded with transcripts of angiogenic factors (e.g., VEGF-A, VEGF-KDR, FGF2). In vitro and in vivo studies indicate that such EVs effectively transfected HDFs and drove direct conversions towards iECs within 7-14 days. Finally, wound healing studies in mice indicate that engineered EVs lead to improved wound closure and vascularity. Altogether, our results show the potential of engineered human vasculogenic EVs to drive direct reprogramming processes of somatic cells towards iECs, and facilitate tissue repair/regeneration.

ICAM-1-decorated extracellular vesicles loaded with miR-146a and Glut1 drive immunomodulation and hinder tumor progression in a murine model of breast cancer.

Tumor-associated immune cells play a crucial role in cancer progression. Myeloid-derived suppressor cells (MDSCs), for example, are immature innate immune cells that infiltrate the tumor to exert immunosuppressive activity and protect cancer cells from the host's immune system and/or cancer-specific immunotherapies. While tumor-associated immune cells have emerged as a promising therapeutic target, efforts to counter immunosuppression within the tumor niche have been hampered by the lack of approaches that selectively target the immune cell compartment of the tumor, to effectively eliminate "tumor-protecting" immune cells and/or drive an "anti-tumor" phenotype. Here we report on a novel nanotechnology-based approach to target tumor-associated immune cells and promote "anti-tumor" responses in a murine model of breast cancer. Engineered extracellular vesicles (EVs) decorated with ICAM-1 ligands and loaded with miR-146a and Glut1, were biosynthesized (in vitro or in vivo) and administered to tumor-bearing mice once a week for up to 5 weeks. The impact of this treatment modality on the immune cell compartment and tumor progression was evaluated via RT-qPCR, flow cytometry, and histology. Our results indicate that weekly administration of the engineered EVs (i.e., ICAM-1-decorated and loaded with miR-146a and Glut1) hampered tumor progression compared to ICAM-1-decorated EVs with no cargo. Flow cytometry analyses of the tumors indicated a shift in the phenotype of the immune cell population toward a more pro-inflammatory state, which appeared to have facilitated the infiltration of tumor-targeting T cells, and was associated with a reduction in tumor size and decreased metastatic burden. Altogether, our results indicate that ICAM-1-decorated EVs could be a powerful platform nanotechnology for the deployment of immune cell-targeting therapies to solid tumors.

Engineered Extracellular Vesicles Derived from Dermal Fibroblasts Attenuate Inflammation in a Murine Model of Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) represents a significant burden to the healthcare system, with ≈200 000 cases diagnosed annually in the USA. ARDS patients suffer from severe refractory hypoxemia, alveolar-capillary barrier dysfunction, impaired surfactant function, and abnormal upregulation of inflammatory pathways that lead to intensive care unit admission, prolonged hospitalization, and increased disability-adjusted life years. Currently, there is no cure or FDA-approved therapy for ARDS. This work describes the implementation of engineered extracellular vesicle (eEV)-based nanocarriers for targeted nonviral delivery of anti-inflammatory payloads to the inflamed/injured lung. The results show the ability of surfactant protein A (SPA)-functionalized IL-4- and IL-10-loaded eEVs to promote intrapulmonary retention and reduce inflammation, both in vitro and in vivo. Significant attenuation is observed in tissue damage, proinflammatory cytokine secretion, macrophage activation, influx of protein-rich fluid, and neutrophil infiltration into the alveolar space as early as 6 h post-eEVs treatment. Additionally, metabolomics analyses show that eEV treatment causes significant changes in the metabolic profile of inflamed lungs, driving the secretion of key anti-inflammatory metabolites. Altogether, these results establish the potential of eEVs derived from dermal fibroblasts to reduce inflammation, tissue damage, and the prevalence/progression of injury during ARDS via nonviral delivery of anti-inflammatory genes/transcripts.

Engineered extracellular vesicles from human skin cells induce pro-ß-cell conversions in pancreatic ductal cells.

Direct nuclear reprogramming has the potential to enable the development of ß cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite their promise, current approaches to ß cell-directed reprogramming rely heavily on the use of viral vectors. Here we explored the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) as novel non-viral carriers of endocrine cell-patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone-expressing cells. Electrotransfection of HDFs with expression plasmids for Pdx1, Ngn3, and MafA (PNM) led to the release of EVs loaded with PNM at the gene, mRNA, and protein level. Exposing PDC cultures to PNM-loaded EVs led to successful transfection and increased PNM expression in PDCs, which ultimately resulted in endocrine cell-directed conversions based on the expression of insulin/c-peptide, glucagon, and glucose transporter 2 (Glut2). These findings were further corroborated in vivo in a mouse model following intraductal injection of PNM- vs sham-loaded EVs. Collectively these findings suggest that dermal fibroblast-derived EVs could potentially serve as a powerful platform technology for the development and deployment of non-viral reprogramming-based cell therapies for insulin-dependent diabetes.

Designer Extracellular Vesicles Modulate Pro-Neuronal Cell Responses and Improve Intracranial Retention.

Gene/oligonucleotide therapies have emerged as a promising strategy for the treatment of different neurological conditions. However, current methodologies for the delivery of neurogenic/neurotrophic cargo to brain and nerve tissue are fraught with caveats, including reliance on viral vectors, potential toxicity, and immune/inflammatory responses. Moreover, delivery to the central nervous system is further compounded by the low permeability of the blood brain barrier. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for neurogenic/neurotrophic therapies, overcoming many of the limitations mentioned above. However, the manufacturing processes used for therapeutic EVs remain poorly understood. Here, we conducted a detailed study of the manufacturing process of neurogenic EVs by characterizing the nature of cargo and surface decoration, as well as the transfer dynamics across donor cells, EVs, and recipient cells. Neurogenic EVs loaded with Ascl1, Brn2, and Myt1l (ABM) are found to show enhanced neuron-specific tropism, modulate electrophysiological activity in neuronal cultures, and drive pro-neurogenic conversions/reprogramming. Moreover, murine studies demonstrate that surface decoration with glutamate receptors appears to mediate enhanced EV delivery to the brain. Altogether, the results indicate that ABM-loaded designer EVs can be a promising platform nanotechnology to drive pro-neuronal responses, and that surface functionalization with glutamate receptors can facilitate the deployment of EVs to the brain.

Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke.

Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.

Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.

OBJECTIVES: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influence the integrity and quality of proteins and nucleic acids. METHODS: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection; after 1, 2, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas nucleic acid integrity values determined DNA and RNA degradation. RESULTS: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. CONCLUSIONS: Adding PIs immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.

Nanochannel-Based Poration Drives Benign and Effective Nonviral Gene Delivery to Peripheral Nerve Tissue.

While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage-dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe-spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT-based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT-treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming-based cell therapies for nerve repair/regeneration.