Poster Z2 - Engineering Targeted Therapeutic Exosomes for Peripheral Nerve Regeneration

Introduction: Peripheral neuropathies can cause lifelong disability due to the difficulty of restoring damaged peripheral nerve tissue The current treatment using autologous nerve grafts has limitations, such as limited availability of donor nerve, graft sizes, and low recovery efficiency. Administering therapeutics to peripheral nerve tissue is difficult due to the peripheral neuroanatomy and constraints posed by the blood-nerve barrier (BNB). This study aims to engineer an exosome (Exo) delivery system using erythrocyte-derived Exos targeted to peripheral nerve tissue. One approach involves attaching targeting proteins such as tetanus toxin-C fragment (TTC) from the clostridium neurotoxin family to the Exo surface via a bio-orthogonal click chemistry. Another approach is to label peptide by exploiting the glycan metabolic pathway in erythrocytes using a sugar precursor that can be incorporated onto the Exo surface for click chemistry modifications.

Materials and

Methods: We isolated Exos from patient red blood cells (RBCs) using chemical and metabolic labelling methods (Fig.1A). In the chemical approach, we modified TTC with a PEG5000-azide linkage and crosslinked it with DBCO-Sulfo-NHS to create triazole linkages. In the metabolic method, we co-incubated RBCs with Ac4ManNAz for 72h and isolated ManNAz-integrated exosomes, which we reacted with DBCO-functionalized Cy7 dye and DBCO-TET1 peptide via azide-alkyne cycloaddition click chemistry (Fig. 1B). The targeted Exo system's uptake and specificity were also evaluated in neuronal cell lines and animal models (Fig. 1C).

Results, Conclusions, and Discussions: The TTC-modified Exo system was evaluated in vitro Neuro-2A cell lines, and indicate efficient accumulation along the soma and axonal circumference in comparison to unmodified Exos (Fig. 1D). Our investigations in mouse model reveal accumulation of TTC-Exos in isolated sciatic nerve tissues after 24h and 72h (Fig. 1E). Immunostaining of isolated sciatic nerve tissue sections demonstrated that TTC-Exos were successfully transported into sciatic nerve after 72 h of intramuscular administration (Fig. 1F). This indicates the crucial role played by the TTC label on Exo delivery system facilitating the retrograde axonal transport mechanism.
We have developed a targeted delivery system for peripheral nerve regeneration using a biorthogonal click chemistry-based approach. This TTC-targeted Exo system shows improved uptake and transport to peripheral nerve tissues. We have also utilized metabolic labelling to label sialic glycoproteins on cell surfaces with azide groups, enabling functionalization via a broad range of bioorthogonal reactions with targeting peptides and tags. In summary, we have developed a versatile, next-generation, biocompatible and stable targeted therapeutic Exo-delivery system.

Acknowledgements (Optional): UN Collaboration Initiative Grant, LB606 Nebraska Stem Cell Grant, NIH R21NS131938 and R21AR078439, Great Plains IDeA-CTR Early Career Investigator Program (ECIP) Award