Tissue Nanotransfection Strategies for the Treatment of Diabetic Neuropathy and Volumetric Muscle Loss

Gene delivery is one approach being used in the new field of regenerative medicine, with the goal of modifying cells, tissues, or organs of the body to restore function. Gene delivery can be used to correct dysfunctional genes but gene cocktails can also cause expression of genes with a broad impact even to the point of converting cells to a different cell type. This dissertation aims to use tissue nanotransfection (TNT) of reprogramming factors in different pathological settings in vivo in order to restore function.Tissue nanotransfection (TNT) uses principles of nanoelectroporation to transfect exposed tissue. In this procedure, a plasmid solution is separated from the tissue by a nanoporous device and an electric current is passed between the two to transfect the tissue. In Chapter 2, we tested a new nanofabricated device for its ability to transfect skin. We performed TNT on both porcine and murine skin and found differences in penetration depending on the voltage. Voltage appeared to have a biphasic response on plasmid penetration with a threshold voltage separating the two. This threshold voltage was higher in the porcine skin than the murine. In mice, skin underwent TNT at two different locations. Both locations had successful overexpression of the transfected genes, however the difference in overexpression was significantly different between the two locations. Previous studies using TNT with plasmids Ascl1, Brn2, and Myt1l (ABM) in skin shown conversion of skin cells into neuron-like cells that survived for several weeks. In Chapter 3, we investigated how the skin environment is affected by TNT with ABM and its potential use as a therapy in diabetic neuropathy. First, mouse embryonic fibroblasts were transfected in vitro using ABM. These fibroblasts converted into neuron-like cells and were found to secrete NGF. Using TNT with ABM in murine skin, we were able to convert skin cells into neurons and saw increase in NGF in this skin after 4 weeks. Through immunofluoresnce, we observed that much of this increase in NGF was localized to the epidermis. We then performed TNT with ABM in a type 2 diabetic mouse model that developed diabetic neuropathy. ABM transfected diabetic mice had increased NGF levels 4 and 9 weeks after TNT. Additionally these mice had a higher density of PGP9.5 (pan-neuronal marker) fibers in the skin, showing that this therapy may be useful for neuroprotection.In volumetric muscle loss (VML), a large mass of muscle is removed which gets filled with fibrotic tissue. In addition muscle function is drastically reduced. In Chapter 4, we used TNT with myogenic transcription factor MyoD as a therapy for restoring function in VML. First we transfected mouse embryonic fibroblasts in vitro with MyoD and saw conversion to myogenic cells expressing muscle contractile elements. Next we performed TNT with MyoD in murine skin to see if it was capable of causing myogenic conversion in vivo. After 10 days, we saw upregulation of myogenic genes in the skin as well as cells expressing myosin heavy chain in the dermis, where skeletal muscle is not usually seen. Lastly we used TNT on a rat tibialis anterior model of VML. We performed TNT on the fibrotic scare and surrounding musculature 1 week after injury once cells had already infiltrated the defect and hematoma and acute inflammation had subsided. Three weeks after TNT, rats that were transfected with MyoD encoded plasmids had increased muscle function in the form of maximal dorsiflexion torque production when compared to the mock TNT control group. Rats that underwent TNT with MyoD also had increase muscle mass, however no differences were seen in the amount of fibrosis between the two groups after Masson’s trichrome staining.