Localized Immunosuppression in Nerve Allografts via Tissue Nano-Transfection of FasL to Improve Graft Survival and Regeneration

Introduction: : Peripheral nerve injuries (PNIs) cause neuropathy, muscle atrophy, and functional loss. While autografts are the gold standard, cellular nerve allografts (CNAs) offer accessible, regenerative options but face challenges from immune rejection. Systemic immunosuppressants are effective but impose life-threatening side effects such as tumorigenicity and complicated infections. Localized immunosuppression provides a safer, targeted approach by limiting immune modulation to the graft site. Fas ligand (FasL) is a potent immunosuppressive protein that induces apoptosis in infiltrating T cells via Fas–FasL interaction, selectively reducing alloreactive responses and preventing graft rejection. Local gene delivery enables targeted immunomodulation at the graft site while minimizing systemic toxicity. Therefore, we proposed using Tissue Nano-Transfection (TNT), a non-viral, nanochannel-based electroporation method, which enables safe and efficient gene delivery with minimal cellular damage. We hypothesize that one-time localized delivery of FasL via TNT will provide a localized immunosuppressive environment by reducing T-cell infiltration in CNAs through Fas-mediated apoptosis and increasing graft survival and integration.

Materials and

Methods: : In vitro transfection of fibroblasts was carried out using an expression plasmid DNA encoding for FasL. Transfection with an empty vector (sham) served as the control. These cells were collected 24 hours post-transfection, and FasL gene expression was quantified using RT-qPCR. Immunocytochemistry (ICC) was performed to confirm the presence of FasL on the cell membrane. Immunocytochemistry (ICC) staining was done with DAPI to locate the cell nucleus and FasL to show the ligand around the cell membrane. These in vitro studies confirmed the increased expression of the FasL gene along with membrane localization before in vivo experiments were conducted. Sciatic nerves were collected from Balb/C donor mice and subjected to TNT with plasmid encoding for FasL to promote their immunoprotection. Following TNT, the nerves were dissected and placed subcutaneously in the right leg of C57Bl/6 recipient mice (Fig. 1-A). Histological analysis of FasL expression in the donor nerves was assessed 24 hours and 3 days post-implantation. To evaluate immune cell infiltration, CD4 and CD8 T cell markers were evaluated in nerves collected at 7 and 14 days post-implantation via immunostaining. Healthy sciatic nerve grafts with no intervention and grafts transfected with sham served as controls. One-way ANOVA was used for statistical analysis.

Results, Conclusions, and Discussions::

Results: Our findings demonstrate successful transfection of fibroblasts, showing a >15-fold increase in FasL gene expression compared to sham-treated cells at 24 hours post-transfection (p < 0.0001, Fig. 1-B). Moreover, immunostaining confirmed FasL protein expression and its localization on the cell surface, validating the effective transgene delivery and membrane targeting in FasL-transfected cells (Figure 1-C). Quantitative imaging showed sustained FasL expression at 24 and 3 days post-TNT (p < 0.0001) compared to sham (Figure 1-D). Most importantly, histological analyses of nerve grafts demonstrated the ability of TNT with FasL to significantly reduce CD4+ and CD8+ T cell infiltration compared to control groups at both 7 and 14 days post-implantation (p ⪅ 0.0001, Figure 1-E).

Conclusion: Our study demonstrates the potential of using TNT to deliver FasL to nerve tissue, leading to sustained gene expression and providing a localized immunosuppressive environment to improve graft survival and functional integration in peripheral nerve repair. Ongoing studies are looking at nerve reconstruction surgeries with FasL-TNT treated grafts and functional outcomes.

Acknowledgements and/or References (Optional)::