Associate Professor; Department of Biomedical Engineering and Surgery The Ohio State University Columbus, Ohio, United States
Introduction:: Cell and tissue reprogramming have the potential to enable the development of highly promising cell therapies for a wide variety of conditions. Current approaches to cell reprogramming, however, face major practical and translational hurdles, including heavy reliance on viral vectors, and a highly stochastic nature, which often leads to inefficient and/or unpredictable reprogramming outcomes. We developed a novel nanotechnology-based approach that overcomes these barriers by enabling deterministic transfection of reprogramming factors into tissues with single-cell resolution and without the need for viral vectors. Cell and tissue nano-transfection (TNT) promote remarkably fast and efficient direct cellular reprogramming in vivo (Fig. 1). Such platform technology could be applicable to virtually any reprogramming model, and its minimally disruptive and non-viral nature make it an ideal candidate for use in highly complex disease systems, including neurodegenerative conditions and metabolic disorders, among others.
Materials and Methods:: Cell and tissue nano-transfection chips were manufactured from silicon or polymeric track etched membranes using cleanroom-based approaches, as described previously. Scanning electron microscopy was used to ensure the chips had nanochannel sizes ranging between 400-500 nm. These chips were then used to nanotransfect dermal or nerve fibroblasts, in vitro or in vivo, with pro-vasculogenic or pro-adipogenic reprogramming gene cocktails, which were then evaluated (for their therapeutic potential) in different murine models of neurodegenerative conditions, including peripheral nerve injury (PNI), ischemic stroke, and Alzheimer’s disease (AD), as well as models of metabolic dysfunction, including obesity/type 2 diabetes. Fibroblasts nanotransfected with sham genes were used as controls. Different molecular, histological, and functional outcomes studies were conducted in order to assess the extent to which these cells mitigated disease burden in each model.
Results, Conclusions, and Discussions:: In murine models of PNI, TNT-treated fibroblasts promoted nerve revascularization and reduced inflammation, which then led to improved axonal regrowth as well as nerve and muscle function. In murine models of ischemic stroke, nanotransfected fibroblasts improved brain vascularization, perfusion, as well as neuroprotection and neuroregeneration in the motor cortex, which led to improved motor function. In murine models of AD, nanotransfected fibroblasts led to improved brain vascularization, perfusion, and reduced amyloid beta load. This also correlated with improved memory and cognitive function. Finally, in murine models of metabolic dysfunction, TNT-treated dermal fibroblasts and other skin cells were successfully coopted to partially fulfill brown adipogenic functions, from the skin, which led to improved weight control and cardiometabolic function. Altogether, these findings suggest that cell and tissue nanotransfection could potentially be used to effectively drive different reprogramming processes, in vivo, to recover damage or diseased tissue structure and function. Additional studies are also being conducted to evaluate the extent to which cell and tissue-nanotransfection-based approaches can be used to mitigate disease burden in different preclinical models of cancer.