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Engineering Primary Schwann Cells using Lentiviruses to Control GDNF Expression
Yuewei Wu-Fienberg, BS, Laura Marquardt, BS, Piyaraj Newton, BS, Philip J. Johnson, PhD, Susan E. Mackinnon, MD, Shelly E. Sakiyama-Elbert, PhD, Amy M. Moore, MD, Matthew D. Wood, PhD.
Washington University in St. Louis, St. Louis, MO, USA.
PURPOSE: Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to increase axonal regeneration. Recent studies found that prolonged excess GDNF production from transgenic cells caused sequestering of motor axons at the site of GDNF expression and prevented improved distal nerve regeneration, a phenomenon called the “candy-store effect.” In the present study, we attempted to capitalize on the beneficial effects of GDNF delivery and limit the “candy store effect” by engineering transgenic Schwann cells (SCs) in which constitutive GDNF overexpression can be downregulated using a tetracycline-inducible Cre/lox excision mechanism. The purpose of this study was to create transgenic SCs that could provide temporally-controlled, local GDNF delivery to be used in peripheral nerve injuries to enhance axonal regeneration.
METHODS: Lewis rat SCs were transduced with 2 distinct lentiviral vectors: a GDNF FUIV vector in which full-length rat GDNF cDNA is flanked by two loxP sites under the control of an ubiquitin promoter, and a Cre vector based on the pSLIK platform in which Cre recombinase is expressed in a Tet-On fashion. Cells were transduced using our 2 vectors at a multiplicity of infection (MOI) of 20 for each vector. Successful transduction was assessed by visualization of a red fluorescent protein reporter. GDNF expression was quantified by enzyme-linked immunosorbent assay (ELISA) of cell medium samples collected daily and normalized to corresponding daily cell counts. Cre recombinase expression was visualized qualitatively using immunohistochemistry. Biological activity of the GDNF produced by engineered SCs was measured by neurite extension assays.
RESULTS: Engineered SCs allowing temporally-controlled GDNF expression were produced by lentiviral transduction of 2 unique viral vectors. These SCs expressed and secreted significantly more GDNF as compared to untransduced controls, as measured by ELISA. Further, the SCs produced biologically active GDNF as demonstrated by neurons extending neurites. To demonstrate control of GDNF expression, engineered SCs were exposed to doxycycline (a tetracycline analog) to excise the GDNF transgene. The addition of doxycycline to SC culture medium demonstrated a marked increase in Cre recombinase staining as visualized by immunohistochemistry and produced significantly lower levels of GDNF on ELISA in comparison to engineered SCs that were not exposed to doxycycline.
CONCLUSION: We have engineered SCs that constitutively overexpress GDNF, and in which expression can be controlled, i.e.“turned off,” by exposure to tetracycline-family antibiotics using a tetracycline-inducible Cre/lox excision mechanism.
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