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In Vivo Transgene Expression in Rat Sciatic Nerve Injury Model with AAV-Transduced Tissue Engineered Nerve Grafts
Viviana Alpizar Vargas, B.S.
1,2, Franco A. Laimo, BS
1, Chung-Hsun Lin, B.S.
1,3, Zarina S. Ali, MD
1, D. Kacy Cullen, PhD
1; Hannah Hoeun Lee, MD, PhD
4(1)University of Pennsylvania, Philadelphia, PA, (2)CMC VA Medical Center, Center for Neurotrauma, Neurodegeneration & Restoration, philadelphia, PA, (3)CMC VA Medical Center, Center for Neurotrauma, Neurodegeneration & Restoration, Philadelphia, PA, (4)CMC VA Medical Center, Philadelphia, PA
Introduction: We developed a tissue engineered nerve graft (TENG) transduced with adeno-associated virus (AAV) to generate a novel nerve scaffold. This study characterizes in vivo survival and transgene expression of AAV-TENGs out to 6 weeks in a rat sciatic nerve injury model.
Materials and MethodsTENGs were fabricated from dorsal root ganglia (DRG) isolated from embryonic day 16 Lewis rats. The DRG were transduced with AAV encoding for a reporter transgene, firefly luciferase, under constitutively active cytomegalovirus (CMV) promoter. The DRG were then cultured in custom mechanobioreactors and axonal tracts were "stretch-grown" to 1-cm. The stretched AAV-TENGs were encapsulated in collagen, transferred into a nerve guidance tube, and transplanted to bridge a 1-cm sciatic nerve defect in 8-week-old Lewis rats. The luciferase transgene expression was imaged using IVISŪ bioluminescence at various time points until the endpoint of 6 weeks. Prior to all imaging sessions, luciferin substrate was injected into the intraperitoneal space of the animal. Nerve graft and muscle were harvested for histology at the study endpoint.
ResultsLuciferase signal was successfully detected in all rats with the 1 cm sciatic nerve repaired with AAV-TENGs, out to the terminal timepoint of 6 weeks (Figure 1). Histological analysis showed TENG cell survival, luciferase positive axons, Schwann cell migration, and endothelial cell migration.
ConclusionsIn vivo longitudinal assessment of survival and transgene expression of AAV-transduced tissue engineered nerve grafts was demonstrated. Prior to this study, TENG survival could only be observed histologically after the terminal time point. This study presents promise for a novel method for
in vivo assessment of tissue engineered implants following peripheral nerve repair. Future analyses of bioluminescence signal and cell count will elucidate a quantifiable relationship between signal strength and implant survival. Future work will include
in vivo longitudinal assessment of spatial and temporal control of transgene expression in AAV-TENG constructs.
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