September 1, 2017 (Vol. 37, No. 15)
Sophia Ktori Freelance Writer
New Nanotransfection Device Poised to Revolutionize Tissue Engineering
Researchers at the Center for Regenerative Medicine and Cell-Based Therapies at The Ohio State University have developed a portable, thumbnail-sized silicon chip that can, in a fraction of a second, reprogram skin cells so that they transform into just about any other cell type in the body. The noninvasive tissue nanotransfection (TNT) technology has already been used in mice and pigs to prompt skin cells to develop into complete blood vessels that join up with existing vasculature to heal necrotizing skin flaps and to rescue critically injured ischemic legs. In subsequent experiments, TNT directed the transformation of mouse epidermal skin cells into functioning neurons that within just a few weeks could be removed from the skin layer and transplanted into the animals’ brains to reverse the effects of a stroke.
The Ohio State University researchers, led by Chandan Sen, Ph.D., and L. James Lee, Ph.D., describe the TNT technology and report on their mouse revascularization experiments in the August 7, 2017 online issue of Nature Nanotechnology. Daniel Gallego-Perez, Ph.D., Durba Pal, Ph.D., and Subhadip Ghatak, Ph.D., are the three co-first authors of the paper, which is entitled “Topical Tissue Nano-Transfection Mediates Non-Viral Stroma Reprogramming and Rescue.”
Dr. Sen also serves as the editor-in-chief of two journals, Antioxidants & Redox Signaling and Advances in Wound Care, both published by Mary Ann Liebert, Inc.
TNT is a nanoelectroporation technology that fires novel cell reprogramming factor genes directly into epidermal skin cells through temporary channels created in the cells’ outer membranes. The chip is loaded with the requisite reprogramming factors and placed on the skin. A small electrical charge is then passed momentarily through the chip, and this opens up tiny channels in the cell membranes, through which the genes are injected. “Because the electric current is very low due to the very high electric resistance of nanopores, this approach is benign with minimal invasiveness to the transfected cells or tissue,” GEN was told by Dr. Lee, who is professor of chemical and biomolecular engineering with Ohio State's College of Engineering in collaboration with Ohio State's Nanoscale Science and Engineering Center.
Just One Touch
Dr. Sen stressed that TNT requires no laboratory equipment or processing and can be applied easily at point-of-care and out in the field. The whole process occurs in less than a second, just by touching the chip onto the skin surface. “All you need is the chip, the reprogramming factors for the required cell type, and a power source,” he commented to GEN. “We can use TNT to transform skin cells into any type of cell that is required to treat local tissue and organ disease or injury. Alternatively, a patient’s skin can be considered as an ‘agricultural landscape’ for growing and harvesting therapeutic cell types for implantation elsewhere in the body. We have, for example, generated hundreds of thousands of neurons in the skin of mice. It takes just 3-4 weeks for functional neurons to be ready for grafting into the brain.”
Electroporation as a nonviral gene-delivery technique isn’t new, but bulk electroporation techniques have demonstrated limited success, added Dr. Sen, who is director of the Center for Regenerative Medicine and Cell-Based Therapies at The Ohio State University Wexner Medical Center, and also executive director of Ohio State’s Comprehensive Wound Center. “Bulk electroporation renders the entire cell membrane permeable and impacts on the cytoskeleton, which subdues the plasticity of the cell. In contrast, TNT creates a series of tiny channels, which affects just 2% of the cell membrane surface area and doesn't inhibit cell plasticity. Using TNT, we have achieved greater than 98% transfection efficiency and cell transformation.”
In the Nature Nanotechnology paper, the researchers reported two sets of in vivo studies in mice, through which they reprogrammed skin cells to transform into vascular cells, first to prevent necrosis in full-thickness skin flaps, and then in a second set of animals to rescue complete limbs from which the femoral artery had been removed. The legs of untreated control animals quickly became necrotic due to lack of blood flow. In contrast, animals treated using TNT in the lower limb skin grew functional blood vessels within a couple of weeks. By the third week, the affected limbs were well served with new vasculature and healed, without any other form of treatment.
“We have this hardware with arrayed nanochannels that can deliver factors of interest into the body to achieve tissue reprogramming, not just cell reprogramming,” Dr. Sen stressed as he spoke with GEN. “Our technology rescued the legs simply by reprogramming the skin to regenerate blood vessels, without any femoral supply. We didn’t just make vasculogenic cells, we made hundreds of functioning blood vessels. That’s the big distinction here.”
The range of potential applications is huge, he maintains. As well as demonstrating that TNT can generate blood vessels and functional neurons from skin cells, the researchers have also transformed mouse skin cells into insulin-producing cells that can sense glucose in the animals’ blood and secrete insulin in response.
Not Just for Surface Skin
“There are many potential opportunities for skin-based transfection or reprogramming,” Dr. Lee continued. “One is a DNA vaccine and another is neuron regeneration for diabetic patients, for example. It may also be applicable for hair regrowth in some situations.” TNT could, in addition, feasibly be used with other tissues, he suggested. “We have successfully demonstrated TNT on exposed muscle tissue and fat tissue (to reprogram white fat cells to brown fat cells). It should be applicable to any surface tissues other than skin (for example, eye, ear) and surgically exposed tissues (such as bone repair or during organ surgery), as long as the transfection vectors are available.”
Dr. Sen pointed out that while transfection per se is limited to epidermal cells in the upper layer of the skin, the effects of transfection propagate down to the dermis. As the authors write in the published Nature Nanotechnology paper, “Our findings showed that TNT can not only be used for topical delivery of reprogramming factors, but that it can also orchestrate a coordinated response that results in reprogramming stimuli propagation (that is, epidermis to dermis) beyond the initial transfection boundary (the epidermis)… .”
“We are quite surprised that the transfection is able to propagate into the deeper layers of skin tissue cells,” Dr. Lee admitted to GEN. “Our current results show that the transfected surface cells are able to release functional biomolecules, including mRNA and proteins, some in secreted extracellular vesicles, to relay the transfection to other cells in the tissue.”
The detailed mechanism by which this happens will require further investigation, he stated. “Compared with existing physical in vivo tissue transfection techniques, such as bulk electroporation and gene gun, our TNT approach is much more benign with minimal tissue damage. I don't think damaged tissue or dead cells caused by transfection is the reason for the observed transfection/reprogramming propagation in tissue… . We still don’t know how deep the propagation can go in a tissue or organ.”
The TNT technology relies on two unique components, Dr. Sen continued. “First, the design of cargo that may be plasmid, DNA, or even RNA to induce plasticity. The ability to use RNA for such purposes minimizes the risk for genomic integration. Second, a 3D high-throughput nanoelectroporation (NEP) chip generated using cleanroom-based micro-/nanofabrication techniques.” The nanoelectroporation technology was originally developed six years ago by Dr. Lee’s team. In vivo application of the platform was realized when Dr. Lee’s lab responded to interest from Dr. Sen to progress to tissue reprogramming in vivo.
microRNA, siRNA and CRISPR Editing
Dr. Lee’s earlier research had also demonstrated the potential to use nanoelectroporation/TNT to deliver other nucleic acid cargos, and this is a key area for ongoing research, he told GEN. “In our in vitro cell transfection research, we have demonstrated that 2D and 3D NEP biochips can also deliver microRNA (miRNA) and small interfering RNA (siRNA) cargos to cancer cells causing cell death by oncogene silencing and knock-off. We are working on CRISPR/Cas9 gene editing now. The same technique can be applied by TNT to tissue in vivo. We now need to investigate its efficacy.”
Dr. Sen projects that, with expedited FDA approval, initial TNT clinical trials could start within a year for serious limb ischemia applications. A smal NIH grant is separately now funding early work in the field of neuropathy, and the team is also collaborating with the Walter Reed Army Medical Center for potential field-based applications of TNT in rescuing injured extremities and peripheral nerve injury.
“We have a novel 3D TNT chip design optimized for clinical applications,” Dr. Sen noted. “Further work will probably involve collaboration with industrial partners.” Work is ongoing to develop cost-effective fabrication techniques using optimum biocompatible materials. “Miniature TNT chips are also required for delicate tissue (e.g., eye, ear) transfection,” Dr. Lee pointed out.
The researchers are currently in the early stages of a potential agreement with Taiwan’s electronics giant Foxconn, and additional licensees for clinical applications of TNT are anticipated, Dr. Sen stated. “The IP has been secured, and we want different groups to take this technology and develop it for widespread applications.”