Tissue nano-transfection and Extracellular Vesicle–Based Nanocarriers for Motor Neuron-Like Reprogramming to Support Muscle Protection After Peripheral Nerve Injury

Introduction: : Peripheral nerve injuries (PNI) have an annual incidence of 13 to 23 cases per 100,000 people in the United States, leading to sensory and motor deficits that often result in long-term disability[1]. Without timely reinnervation, skeletal muscle rapidly undergoes atrophy and degradation of motor endplate, underscoring the urgent need for therapies that restore nerve-muscle connectivity to preserve function[2]. While gene and cell therapies offer promise for enhancing regenerative outcomes, effective and targeted delivery remains a significant challenge. Therefore, we propose leveraging Tissue Nano-Transfection (TNT) technology[3], a Silicon-based chip for non-viral delivery of neurogenic factors directly to denervated muscle tissue. This in vivo gene delivery approach reprograms resident cells into motor neuron-like phenotypes, offering localized neurotrophic support and preserving muscle integrity. To further enhance and sustain this therapeutic effect, we combined TNT with engineered extracellular vesicles (EVs), which serve as synergistic carriers of bioactive cargo. This strategy using EV-based nanocarriers, administered locally or systemically, has the potential to amplify the reprogramming process providing protection to the muscle until permanent nerve connections are reestablished.

Materials and

Methods: : In vitro transfection of myoblasts using a neurogenic reprogramming DNA gene cocktail Ascl1/Brn2/Myt1l:ABM + Ngn2/Isl1/Lhx3:NIL (A+N) was performed to induce the conversion into motor-like neurons. An empty vector (sham) was used as control. Reprogramming efficiency was evaluated after 7 and 14 days post-transfection via immunostaining, RNAseq and qRT-PCR. Furthermore, 8-10-week-old C57BL/6 mice (n=8/group) received a sciatic nerve removal as a denervation model, followed by TNT with A+N in the gastrocnemius tissue to promote the conversion of cells into functional motor-neural tissue. Non injured mice, injured mice without repair, and TNT with sham were used as controls. Muscle function assessments (e.g., fibrillations), histology, and RNAseq were evaluated 6 weeks after TNT interventions. EVs isolated during the reprogramming process (Fig.1A) were fully characterized using size and concentration analysis, cryo electron microscopy (Cryo-EM), Western Blot, and qRT-PCR. To evaluate the gene transfer efficiency into myoblasts via EVs, gene expression in the recipient cells were evaluated via qRT-PCR. Most importantly, reprogramming studies using EVs were evaluated using qRT-PCR.

Results, Conclusions, and Discussions:: Our results demonstrate successful transfection of myoblasts with A+N factors at 24 hours, 7-, 14-, and 21-days post-transfection. Reprogramming efficiency analysis showed the expression of motor-like neuron markers (e.g., TUJ1, MAP2, Synapsin I, NKX.6, VcHAT) in the A+N group 14 days post-transfection compared to controls. RNAseq analyses show upregulated genes related to neurogenesis pathways in the A+N group, indicating successful conversion (Fig.1B). Furthermore, histology analysis of muscle tissue displayed higher expression of motor-neuron markers after neurogenic TNT interventions confirmed by qRT-PCR (Fig.1C-D) and a reduction of fibrillations compared to the controls (Fig.1E). RNAseq results demonstrated an enrichment of pathways related to muscle health in the A+N group compared to the control groups, suggesting muscle protection as a result of neurogenic programming. Moreover, full characterization of the isolated EVs displayed proper size distribution and morphology and loading of the A+N factors persistent throughout 14-days (Fig.1F-G). Uptake experiments confirmed the gene transfer to myoblasts via EVs after 24 hours and after 14 days post-EV treatment, expression of motor-like neuron markers were found in the treated groups (Fig.1H). Our study demonstrates the potential of TNT to deliver interventions to denervated muscle tissue to improve outcomes after PNIs. Our results highlight the potential of using EVs to further amplify and propagate the reprogramming signal as a therapeutic potential. Future studies will focus on injecting these loaded EVs in vivo after TNT to improve myoblast conversion to motor-like neurons.

Acknowledgements and/or References (Optional):: [1]. Mushtaq, S., Hina, S., Maqbool, H. et al. Frequency of Peripheral Nerve Injury in Trauma in Emergency Settings. Cureus 13, e14195 (2021).
[2]. Reist, N.E., Werle, M.J. & McMahan, U.J. Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. Neuron 8, 865-868 (1992).
[3]. Gallego-Perez, D., Pal, D., Ghatak, S. et al. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nature Nanotechnology 12, 974-979 (2017).