(M-490) Tissue Nano-Transfection drives improved outcomes following peripheral nerve injury

Introduction:: Even though peripheral nerve injuries (PNIs) have a reported rate incidence of just 3%, there are significant associated morbidity and disability outcomes that can lead to severe and irreversible long-term consequences (e.g., sensory and motor deficits, muscle loss, weakness, and pain) that limit the functional recovery of these patients^1,2. Causes of this might include that the treatment and diagnosis of the nerve injury are often delayed, the slow rate of nerve regeneration, and that the standard of care has fallen short in terms of providing optimum results. Therefore, while there have been significant breakthroughs in the fields of gene and cell therapies for PNIs, there is still a paucity of technologies to enable its translation and a need for strategies that can significantly curve progressive degeneration until surgical reconstruction can be performed. Here we describe the use of a novel technology, Tissue Nano-Transfection (TNT), which could potentially be used to controllably induce vasculogenic reprogramming at the injury site, protecting denervated muscle tissue, and imparting immunoprivileged properties to nerve tissue to support grafting procedures by delivering multi-pronged gene and cell therapies to PNIs.

Materials and Methods:: TNT utilizes Si nanochannels to deliver plasmid DNA into solid tissues in a controlled, benign, and efficient manner, without the need for viral vectors. TNT devices were fabricated using standard Si manufacturing techniques using a combination of lithography and deep reactive ion etching. Our published studies demonstrate that a one-time TNT intervention can promote the conversion of adult mouse skin cells into vascular tissue or fully functional neural tissue^3,4. Three intervention modes were tested, including (1) the delivery of vasculogenic reprogramming gene cocktail of Etv2, Foxc2, and Fli1 (EFF), varying the stoichiometry of the factors to determine the optimal formulation, to injured nerve tissue, (2) neurogenic reprogramming gene cocktail of Ascl1, Brn2, and Myt1l to denervated muscle tissue, and (3) immunoprotective interventions by delivering FasL to cellular nerve allografts (CNAs). TNT delivery of empty plasmids (sham) served as a control in all cases. All these experiments were conducted on mice (n=5). Laser speckle imaging (LSI), muscle function, histological assessments, and RT-qPCR were collectively used to evaluate the response of the injured tissue to specific TNT intervention regimens.

Results, Conclusions, and Discussions:: Vasculogenic TNT interventions in crushed nerve tissue of 8-10-week-old C57BL/6 mice led to an increase in nerve tissue perfusion as measured by LSI, vascularity via immunostaining and RT-qPCR (e.g., expression of the vascular markers CD31 and VEGF-D, respectively), and newly formed axons (e.g., neurofilament, NF) compared to the sham group and evaluated 7 and 14 days post-intervention. Additional experiments aimed to optimize the molar ratio of EFF showed that by doubling the amount of either Etv2, Fli1, or Foxc2 the perfusion and vascularity were improved compared to the conventional 1:1:1 EFF ratio. CMAP measurements also showed improved outcomes in response to vasculogenic TNT interventions after nerve injury. Neurogenic TNT interventions, on the other hand, showed that denervated muscle exhibited reduced fibrillation and improved neuromuscular junction morphology compared to TNT interventions with sham plasmids. Such results likely stem from the localized induction of neurotrophic signaling in the denervated muscle, which presumably led to improved muscle health. Finally, immunoprotective TNT interventions on CNAs showed that overexpression of FasL correlated with reduced infiltration of cytotoxic T cells post-implantation and improved graft survival.
Our results show the feasibility of using TNT to deliver various types of interventions to mangled nerve and muscle tissue, as well as graftable nerve tissue, which could potentially lead to improved treatment strategies for PNIs. The introduction of new vasculature through vasculogenic TNT is likely working via localized induction of new endothelial cells through reprogramming processes, that can facilitate healing by supporting axonal growth. Neurogenic TNT is likely working by localized induction of neurons via reprogramming processes, which seemingly maintain neurotrophic signaling essential for preserving health after denervation. Finally, immunoprotective TNT is likely working through FasL-Fas receptor interactions, which lead to T cell apoptosis and reduced graft rejection. While these experiments have been mainly conducted in mouse models of crushed nerve injury, current studies are focused on evaluating the applicability of TNT in the repair of segmental nerve defects, in conjunction with nerve grafting procedures. Currently, no model uses reprogramming strategies via non-viral nanotechnology platforms to treat PNIs. Therefore, this proposed work is fundamentally innovative, with a near-term beneficial impact.


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References (Optional): : 1 Yegiyants, S., Dayicioglu, D., Kardashian, G. & Panthaki, Z. J. "Traumatic peripheral nerve injury: a wartime review." [In eng] (Jul 2010) J Craniofac Surg 21, no. 4: 998-1001.

2 Mushtaq, S. et al. "Frequency of Peripheral Nerve Injury in Trauma in Emergency Settings." [In eng] (Mar 30 2021) Cureus 13, no. 3: e14195.

3 Gallego-Perez, D. et al. "Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue." [In eng] (Oct 2017) Nat Nanotechnol 12, no. 10: 974-979.

4 Moore, J. T. et al. "Nanochannel-Based Poration Drives Benign and Effective Nonviral Gene Delivery to Peripheral Nerve Tissue." (2020) Advanced Biosystems 4, no. 11: 2000157.