GDNF Gene Yherapy with a Novel Immune-Evasive Gene Switch Promotes Regeneration of Ventral Roots
Ruben Eggers, BSc1; Fred De Winter, PhD2; S. a. Hoyng, Ph.D1; Martijn JA Malessy, MD, PhD3; Martijn Tannemaat, MD, PhD4; Joost Verhaagen, PhD2; (1)Neuroregeneration, Netherlands Institute for Neurosciences, Amsterdam, Netherlands, (2)Netherlands Institute for Neuroscience, Amsterdam, Netherlands, (3)Department of Neurosurgery, Leiden University Medical Center, Leiden, Netherlands, (4)University of Leiden, Leiden, Netherlands
Introduction. In patients lesions of the brachial plexus causes permanent loss of function. After experimental lumbar ventral root avulsion, reimplantation promotes motoneuron survival and a limited degree of axon regeneration. Prolonged, uncontrolled lentiviral vector-mediated expression of glial cell-line derived neurotrophic factor (GDNF) in reimplanted ventral roots enhanced motoneuron survival and axonal outgrowth into the root. Persistent, uncontrolled GDNF expression results in trapping of axons and a failure to reinnervate the hind paw. The goal of the current study was to overcome these adverse effects by exerting temporal control over the expression of GDNF by using a novel immune-evasive gene switch which can be turned on by the antibiotic doxycycline.
Materials and Methods. High titer (~1010 gc/ml) lentiviral vectors were produced in HEK cells and applied to L3-L5 ventral roots of adult Wistar rats as previously reported. GDNF expression was regulated in the transduced reimplanted ventral roots using doxycycline-supplemented food for 4 (on/off group, n=14) or 24 weeks (on group, n=14) post-surgery.
Results. A 5 fold increased expression of GDNF for either 4 wk followed by a decline to baseline expression levels, or in persistent expression of GDNF for 12 and 24 wk was detected using a GDNF Elisa. Motoneuron survival was increased in all GDNF treated groups irrespective of the doxycycline treatment period. At the reimplantation site, GDNF induced robust regrowth of regenerating motor fibers into the root. Persistent GDNF expression resulted in coiled fiber growth. The ventral roots exposed to persistent GDNF expression are enlarged in comparison to GFP controls. In contrast, in the 4 wk GDNF group, axons in the ventral root display a longitudinally organized growth pattern and the roots are less enlarged. Biweekly compound muscle action potentials (CMAP) measurements revealed that 4 wk GDNF expression led to an earlier recovery of CMAPs and a significant increase in amplitude compared to animals with persistent GDNF expression and GFP controls. Further histological analysis of axon regeneration revealed a two fold increase in the number of axons in the sciatic nerve at 9 cm from the reimplantation site.
Conclusion. Timed GDNF expression using our novel stealth gene switch in a long distance regeneration model results in enhanced motoneuron survival and reduces local trapping of regenerating motor axons at sites of high GDNF expression. The observed improved CMAP values indicate that timed GDNF expression results in a potentiation of long distance regeneration and muscle innervation.
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