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

Abstract

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. EVs are derived from primary human dermal fibroblasts (HDFs), and are engineered to pack transcription factor genes/transcripts of ETV2, FLI1, and FOXC2 (EFF). In addition to EFF, the engineered EVs are 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 drive direct conversions towards iECs within 714 days. Finally, wound healing studies in mice indicate that engineered EVs lead to improved wound closure and vascularity. Altogether, these 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

“…Engineered EVs were derived from primary mouse embryonic fibroblast (MEF) cultures co-transfected with expression plasmids for miR-146a, Glut1, and ICAM-1, following methods previously published by us and others. [28][29][30][55][56][57] Control EVs were obtained by transfecting cells with sham/ empty plasmids (Fig. 2A).…”

“…27 Previous studies have shown the production of engineered EVs, after the manipulation of donor cells, decorated with ligands for targeted delivery of specific molecular cargo for different applications. 28–30 Additionally, EVs circumvent many of the practical and translational barriers that are characteristic of other delivery systems by offering flexibility in cargo size, unlike viral vectors that have inherent capsid size restrictions, 29 and improved transfection efficiency and low toxicity compared to synthetic carriers. 25 However, EVs derived from progenitor/stem cell populations still face several challenges limiting their clinical translation, such as an increased risk of tumorgenicity, immunogenicity, and limited cell sources.…”

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

“…[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.…”

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype

“…Furthermore, recent work from our group has demonstrated successful reprogramming of degenerate human NP cells toward a healthy pro‐anabolic anti‐catabolic phenotype mediated by the developmental transcription factors Brachyury or FOXF1 . Thus, we propose applying this concept to human AF cells using MKX or SCX to promote a healthy pro‐anabolic phenotype in diseased AF cells 43–45 …”

“…Thus, we propose applying this concept to human AF cells using MKX or SCX to promote a healthy pro-anabolic phenotype in diseased AF cells. [43][44][45] Therefore, the overall goal of this proof of concept study was to assess the therapeutic effect of nonviral delivery of MKX or SCX to diseased human AF cells, to promote a healthy pro-anabolic cell phenotype. We hypothesized that transfection of MKX or SCX can increase phenotypic marker expression and collagen protein content, as well as decrease catabolic enzyme expression and secretion of inflammatory cytokines, leading to a healthier IVD.…”