Principal Investigator University of Pennsylvania, United States
Introduction: The regenerative capacity of current scaffolds to bridge critical-size peripheral nerve defects remains limited. Previous work has demonstrated the efficacy of tissue-engineered nerve grafts (TENGs) built from living neurons and axons for bridging critical length defects in rodent and swine nerves. In this study, TENGs were engineered to express glial-derived neurotrophic factors (GDNF) via Adeno-Associated Viral Vectors (AAV) delivery. TENGs were fabricated from embryonic rat dorsal root ganglia (DRG) and transduced to express reporter or therapeutic genes at opposite ends. Using tetracycline-controlled promoters (TRE), we induced variable GDNF expression across different time points.
Materials and Methods: One population of DRG neurons was transduced with AAV constitutively expressing-mCherry, while another population of DRG neurons was transduced with AAV GDNF. The mCherry-positive DRG were plated on the base membrane (left) side, while GDNF-positive DRG were plated on the towing membrane (right) side. These were stretch grown to 1.5 cm lengths, with 0.8 cm width. TENGs were cultured for 3 weeks. Doxycycline (DOX) was added and removed after 3 days. 4 days after, DOX was added again and the cycle was repeated. Conditioned media was collected, and ELISA (R&D Systems) was performed.
Results, Conclusions, and Discussions: We observed a very bright red expression at one end of the AAV-transduced DRG cell bodies as well as some lower signals along the axon tracts. Via ELISA, we also detected robust expression of GDNF from DRG transduced with AAV-GDNF. In the absence of tetracycline, GDNF expression was low. When tetracycline was administered to TENGs expressing TRE-GDNF, an increased GDNF expression was observed. First, we successfully demonstrated spatial control over transgene expression in the TENG system by fabricating TENGs exhibiting preferential expression of mCherry and GDNF at each end of the graft. These TENGs were grown to both 1.5cm lengths. mCherry transgene expression was higher at one end, while GDNF transgene expression was higher at the other end. Second, we were able to control when GDNF was released from TENGs at varying time points. The next steps include the establishment of more complex temporal control methods in TENGs. Ultimately, the techniques demonstrated and proposed here can be leveraged to build TENGs that provide an inducible GDNF gradient across the graft allowing for accelerated recovery.
