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

Abstract

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.

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

“…istics and display tropism for particular cells or tissues, [78,79] they can also be modified with ligands (e.g., integrins, protein ligands, peptides, nucleic acids) to facilitate the targeting/recognition of specific receptors for preferential binding and payload delivery, avoiding major off-target effects. [43,[80][81][82] Type II pneumocytes constitute 60% of alveolar epithelial cells and make up 10-15% of all cells present in the lungs. [83] The relevance of this is that alveolar epithelial cells are the first line of cells that secrete proinflammatory mediators (e.g., cytokines, chemokines) in response to pathogenic/harmful stimuli, leading and orchestrating the recruitment of innate and adaptative immune cells to the site of injury and/or infection.…”

“…EV Isolation and Characterization: eEVs were isolated from culture media 24 h after transfection of the donor cells, as previously reported. [43,46,47] The culture media was centrifuged at 2000g for 30 min at 4 °C to remove dead cells and debris. After centrifugation, Total exosome isolation reagent (Thermo Fisher Scientific, 44-783-59) was added to the supernatant containing the cell-free culture media following manufacturer instructions.…”

“…[41,42] We have previously shown that donor cells can be engineered to stimulate the release of engineered (eEVs) packed with specific molecular cargo for diverse therapeutic applications and that eEVs can be functionalized with ligands of interest to achieve targeted delivery to cells and tissues. [43][44][45][46][47][48][49] Here we report on the implementation of eEVs derived from dermal fibroblasts, loaded with genes, mRNA transcripts, and protein content of anti-inflammatory cytokines interleukin-4 and -10 (IL-4 and IL-10), to dampen lung injury and inflammation, and decorated with Surfactant Protein A (SPA) to promote preferential retention by cell compartments in the injured lung. SPA is an abundant glycoprotein component present in the lung surfactant, which is implicated in the reduction of the surface tension at the pulmonary air-liquid interphase, prevention of alveolar collapse, and is also considered a major inflammatory immunomodulator.…”

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

“…[7][8][9][10] Additionally, our lab and others have shown their significant potential to serve as efficient nanocarriers for an extensive array of therapeutic applications, including regenerative medicine and cancer. [7,[11][12][13][14][15][16][17][18][19][20] Ischemic disorders and tissue injury often result in deficient vascularization, which can lead to infections, inflammation, and impaired tissue regeneration (e.g., non-healing ulcers, partial necrosis). [21,22] Cell therapies, involving the use of vascular progenitor cells or induced endothelial cells (iECs), have emerged as a powerful approach to increase neovascularization and promote tissue repair and regeneration under multiple conditions, including myocardial ischemia, limb ischemia, and stroke, among others.…”

“…[ 7–10 ] Additionally, our lab and others have shown their significant potential to serve as efficient nanocarriers for an extensive array of therapeutic applications, including regenerative medicine and cancer. [ 7,11–20 ]…”