Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, -6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.
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.
Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.
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.
Extracellular vesicles (EVs) have emerged as a promising carrier system for the delivery of therapeutic payloads in multiple disease models, including cancer. However, effective targeting of EVs to cancerous tissue remains a challenge. Here, it is shown that nonviral transfection of myeloid-derived suppressor cells (MDSCs) can be leveraged to drive targeted release of engineered EVs that can modulate transfer and overexpression of therapeutic anticancer genes in tumor cells and tissue. MDSCs are immature immune cells that exhibit enhanced tropism toward tumor tissue and play a role in modulating tumor progression. Current MDSC research has been mostly focused on mitigating immunosuppression in the tumor niche; however, the tumor homing abilities of these cells present untapped potential to deliver EV therapeutics directly to cancerous tissue. In vivo and ex vivo studies with murine models of breast cancer show that nonviral transfection of MDSCs does not hinder their ability to home to cancerous tissue. Moreover, transfected MDSCs can release engineered EVs and mediate antitumoral responses via paracrine signaling, including decreased invasion/metastatic activity and increased apoptosis/necrosis. Altogether, these findings indicate that MDSCs can be a powerful tool for the deployment of EV-based therapeutics to tumor tissue.
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.
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.
Functionalized Extracellular Vesicles In article number 2100805 by Natalia Higuita‐Castro and co‐workers, designer extracellular vesicles (EVs) are manufactured via non‐viral engineering of donor cells through electroporation. Designer EVs are packed with plasmid DNA, mRNA, and protein content. Transfecting donor cells with expression plasmids for membrane proteins can be used to tailor the surface decoration of the EVs. Pro‐neurogenic designer EVs drive neurogenesis and improve targeting of neuronal tissue, in vitro and in vivo.
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