AAHS - Persistent in vivo spatial control of AAV-transgene using tissue engineered nerve grafts in a 1.5-cm rat sciatic nerve defect model

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Persistent in vivo spatial control of AAV-transgene using tissue engineered nerve grafts in a 1.5-cm rat sciatic nerve defect model
Viviana Alpizar Vargas, B.S.¹; Franco A. Laimo, BS¹; Sam Elijah Lien, MSE¹; Mykhailo Tatarchuk, MD, PhD¹; Robert B. Shultz, PhD²; Kritika Katiyar, PhD²; D. Kacy Cullen, PhD¹; Hannah Hoeun Lee, MD, PhD¹
¹University of Pennsylvania, Philadelphia, PA; ²Axonova Medical, Philadelphia, PA

Introduction

Spatial control of neurogenic growth factors in vivo can increase the regenerative capacity of a neural scaffold in a segmental nerve defect. We previously presented in vitro data on tissue engineered nerve grafts (TENGs) that spatially express glial derived neurotrophic factor (GDNF) in the distal segment of the graft using adeno-associated virus serotype-2 (AAV-2). This study characterizes release profiles of AAV-TENGs with spatial control of transgene expression in vivo with the aim to improve the regenerative capacity of the scaffolds to bridge a segmental nerve defect.

Materials and methods

TENGs were fabricated from dorsal root ganglia (DRG) isolated from embryonic day 16 rats. For the reporter gene AAV-TENGs, the DRG population at the proximal end was transduced with AAV-CMV-GFP, while the distal population was transduced with AAV-CMV-mCherry. For the growth factor release AAV-TENGs, the DRG population intended for the proximal end of the TENG was transduced with AAV-CMV-GFP and the distal population with AAV-CMV-GDNF. They were then cultured in custom mechanobioreactors and axonal tracts were "stretch-grown� to 1.5cm. Once at desired length, AAV-TENGs were encapsulated in a collagen mixture mimicking the extracellular matrix, transferred into a nerve guidance tube, and transplanted to bridge in a 1.5 cm sciatic nerve defect in adult Sprague-Dawley rats. Nerve and muscle were harvested for histology at the study endpoint of 24 weeks.

Results

Spatial distribution of transgene expression was observed in both proximal and distal ends of the AAV-TENGs. Our study showed spatial differences in GFP and MCherry expression in our control AAV-TENGs after 24 weeks. Intensity signal of GFP in the proximal end was almost two-fold higher than the GFP signal in the distal end. Similarly, mCherry signal was higher in the distal end of the TENG compared to the proximal end.

Conclusions

Spatial control of transgene expression may aid in more suitable scaffolds for long-segmental nerve defects. This presents promise for persistent spatial control of growth factors up to 24 weeks in vivo. Ongoing analyses of release of GDNF expression profile in vivo may corroborate findings of the reporter transgenes. Future work will include in vivo temporal control of growth factor release once spatial control in confirmed.

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