Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally-controlled GDNF source to promote axon growth.

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 enzymelinked 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.