Improved Functional Outcomes Following Peripheral Nerve Injury Mediated by Direct Cell Reprogramming via Tissue Nano-Transfection

Introduction: Peripheral nerve injuries (PNIs) can have long-lasting consequences, including sensory/motor deficits, pain, and muscle loss, leading to significant morbidity and disability. Although gene and cell therapies have shown potential to improve outcomes following PNIs, there is a paucity of technologies to facilitate their translation and approaches to ameliorate advancing degeneration. Here we are proposing the use of a novel technology, Tissue Nano-Transfection (TNT), that can deliver multi-pronged gene/cell therapies to nerve/muscle tissue. This technology sought to improve the rate of peripheral nerve regeneration in mangled or graftable nerve tissue or denervated muscle (Figure 1). Our goal is to induce controlled vasculogenic and neurogenic reprogramming at the injury site, promoting healing by establishing new vasculature to support axonal growth and temporarily reinnervating muscles with newly reprogrammed neurons.

Materials and

Methods: Fibroblasts were transfected in vitro using a vasculogenic reprogramming DNA gene cocktail ETV2/FLI1/FOXC2: EFF, varying the stoichiometry of the factors to induce conversion into endothelial cells. Similarly, transfection of myoblasts was carried out in vitro with a neurogenic reprogramming cocktail ASCL1/BRN2/MYT1L: ABM to induce the conversion into neurons. An empty vector pCMV6 (Sham) served as the control. To test the reprogramming outcomes, cells were subjected to RNAseq, immunocytochemistry, and qRT-PCR. Additionally, TNT was used to deliver DNA into solid tissues to promote the conversion of fibroblasts into vascular tissue or functional neural tissue as previously demonstrated. 8-10-week-old C57BL/6 mice received a sciatic nerve crush injury only (control), crush followed by TNT with EFF (varying the stoichiometry of the factors), and TNT with the empty (sham) vehicle (n=10/group). Histological assessments and RT-qPCR were used to evaluate the response of the injured tissue to specific TNT regimens evaluated at 7-14 days post-intervention. Muscle function assessments (e.g., compound muscle action potential, CMAP), were evaluated after TNT with ABM to the denervated muscle tissue after 28 days. Lastly, using a more complex model, 8-10-week-old C57BL/6 mice were subjected to TNT (EFF or Sham) followed by cellular or acellular nerve allograft (CNAs) transplantation and functional outcomes (e.g., grip, nerve functionality, and neuromuscular injury) were assessed over a 13-week post-surgery period. Graft-only and non-injured mice were included as controls.

Results, Conclusions, and Discussions: Our findings demonstrate that fibroblasts were successfully transfected with the EFF factors after 24 hours and 7 days by gene expression analysis (Figure 2A). Moreover, we found that doubling the amount of FOXC2 (1:1:2) led to significantly increased endothelial conversion compared to the conventional 1:1:1 EFF ratio (Figure 2B). Additionally, we display that myoblasts were effectively transfected with ABM factors at 24 hours, 7-, 14-, and 21 days post-transfection (Figure 2C). Expression of neuronal markers (e.g., TUJ1, MAP2, Synapsin I) was found in the ABM group compared to controls via qRT-PCR and immunostaining (Figure 2D). Vasculogenic TNT interventions in crushed nerve tissue led to overexpression of the EFF factors after 24 hours (Figure 3A-B) and show a significant increase in vascularity via RT-qPCR and immunostaining (e.g., expression of vascular markers VEGF-D and CD31, respectively), and newly formed axons (e.g., neurofilament) compared to Sham after 7 and 14 days (Figure 3C-E). Similarly, it was found that doubling the amount of FOXC2 (1:1:2) resulted in greater increased vascularity compared to the conventional 1:1:1 EFF ratio. Furthermore, 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 (data not shown). Finally, RNAseq data from the sciatic nerve tissue after TNT show upregulated genes related to angiogenesis pathways in the EFF group compared to controls (Figure 4A-B). Moreover, EFF-treated grafts show greater functional recovery (e.g., grip) after 8-13 weeks post-surgery, compared to control groups (Figure 4C). Our study demonstrates the potential of using this TNT technology to deliver interventions to damaged nerve and muscle tissue to improve outcomes after PNIs. We hypothesized that localized induction of vasculogenesis and neurogenic signaling at the injury site could facilitate healing to support axonal growth.

Acknowledgements (Optional): This work is funded by the Department of Defense (DOD).