Inducible Release of Glial Derived Neurogenic Factors from Novel Neural Scaffolds
Sam Elijah Lien, MSE1, Hannah Hoeun Lee, MD, PhD1, Robert B. Shultz, PhD2 and D. Kacy Cullen, PhD1, 1University of Pennsylvania, Philadelphia, PA, 2Axonova Medical, Philadelphia, PA
Introduction: Nerve scaffolds capable of releasing Glial Derived Neuronal Factors (GDNF) have been proven to be beneficial in accelerating axonal regeneration across nerve defects. However, prolonged release of GDNF can cause axonal entrapment and impair regrowth, known as the candy store effect. Therefore, temporal control over GDNF release is needed. Previous work has demonstrated the efficacy of tissue engineered nerve grafts (TENGs) built from living neurons for preferentially expressing different reporter genes across the scaffold. In this study, we characterized the temporal control of AAV-TENGs fabricated with adeno-associated viral vectors (AAV) with a Tetracycline-on system (TRE).
Methods:
One population of DRG neurons was transduced with AAV to express Tetracycline controlled GDNF (TRE-GDNF) while another population of DRG neurons was transduced with constitutively expressed mCherry (CMV-mCherry)-visible under the red spectrum. The TRE-GDNF DRGs were plated on the base membrane (distal) side, while CMV-mCherry DRGs were plated on the towing membrane (proximal) side. These were stretch grown to 1.5 cm lengths and 0.8 cm width, and then encapsulated into collagen. Three cycles of doxycycline (DOX) stimulation were performed. For each cycle, DOX was added for 3 days, and removed for 4 days. TENGs were cut in halves and separated before the third cycle and conditioned media was collected from each side. ELISA (R&D Systems) was performed.
Results:
We observed mCherry-expression only at the proximal end of the TENGs from DRG cell bodies transduced with AAV-CMV-mCherry. Via ELISA, we also detected significantly higher levels of GDNF from TENG media collected after the second and third cycle of DOX addition, which were expressed only from the distal side. The removal of DOX led to decrease of GDNF levels.
Conclusions:
We built an inducible transgene system to express GDNF on one side of the TENGs, and we characterized the GDNF release profile over 2 separate additions of DX across 2-3 weeks in vitro. These TENGs exhibited higher mCherry expression at the proximal end, while significant levels of GDNF were expressed and released from the distal end after the second addition of DOX. Further investigation of the timing of DOX addition and removal would lead to more precise control over GDNF release levels. Ultimately, a controlled release system capable of delivering neurogenic factors in an on and off manner would allow optimal delivery of GDNF levels throughout treatment, avoiding axonal coil while ensuring accelerated recovery.
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