Viral transduction of primary Schwann cells using a Cre‐lox system to regulate GDNF expression

Abstract

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.

Transgenic SCs expressing GDNF‐IRES‐DsRed impair nerve regeneration within acellular nerve allografts

“…SC cultures were prepared using previously described methods (Kaewkhaw et al, ; Morrissey et al, ; Wu‐Fienberg et al, ). Briefly, the sciatic nerve was harvested from adult male Lewis rats (Charles River Laboratories, Wilmington, MA) using aseptic technique.…”

“…SC transduction was performed similar to previous methods (Wu‐Fienberg et al, ). The day prior to transduction, normal SCs were seeded at 1 × 10 4 cells/well on pLL‐coated 24‐well plates and on the day of transduction, the number of cells in each well was determined by microscopy.…”

Finely Tuned Temporal and Spatial Delivery of GDNF Promotes Enhanced Nerve Regeneration in a Long Nerve Defect Model

“…The GDNF Tet-on and tdTomato (in vivo control vector) vectors were made using similar established protocols. 30 Briefly, DsRed cDNA sequence was amplified and inserted into FCIV-GDNF (containing GDNF-IRES-Venus, from Milbrandt Lab at Washington University) resulting in FCIV-GDNF-IRES-DsRed. The GDNF-IRES-DsRed fragment was excised and inserted into pEN-TRE-DsRed-PL (derived from pEN_TRmiRc2, ATCC Catalog No.…”

An engineered biocompatible drug delivery system enhances nerve regeneration after delayed repair

“…Current management of delayed nerve repair mainly focuses on restoring nerve continuity through surgical interventions. However, due to unsatisfactory clinical outcomes, a great body of research has been merging on improving immediate and delayed nerve regeneration via recruitment of biological pathways by means such as electrical stimulation or delivery of exogenous factors to the surgical site via mini‐osmotic pumps or via lentiviral factor delivery . This work focuses on development of a fibrin gel based drug delivery system (DDS) containing drug loaded poly(lactic‐ co ‐glycolic acid) (PLGA) microspheres (MS).…”