Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit... Show moreGene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo. Show less
Vliet, A.C. van; Lee, J.; Poel, M. van der; Mason, M.R.J.; Noordermeer, J.N.; Fradkin, L.G.; ... ; Winter, F. de 2021
A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood.... Show moreA human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration. Show less
Eggers, R.; Winter, F. de; Tannemaat, M.R.; Malessy, M.J.A.; Verhaagen, J. 2020
A spinal root avulsion is the most severe proximal peripheral nerve lesion possible. Avulsion of ventral root filaments disconnects spinal motoneurons from their target muscles, resulting in... Show moreA spinal root avulsion is the most severe proximal peripheral nerve lesion possible. Avulsion of ventral root filaments disconnects spinal motoneurons from their target muscles, resulting in complete paralysis. In patients that undergo brachial plexus nerve repair, axonal regeneration is a slow process. It takes months or even years to bridge the distance from the lesion site to the distal targets located in the forearm. Following ventral root avulsion, without additional pharmacological or surgical treatments, progressive death of motoneurons occurs within 2 weeks (Koliatsos et al., 1994). Reimplantation of the avulsed ventral root or peripheral nerve graft can act as a conduit for regenerating axons and increases motoneuron survival (Chai et al., 2000). However, this beneficial effect is transient. Combined with protracted and poor long-distance axonal regeneration, this results in permanent function loss. To overcome motoneuron death and improve functional recovery, several promising intervention strategies are being developed. Here, we focus on GDNF gene-therapy. We first introduce the experimental ventral root avulsion model and discuss its value as a proxy to study clinical neurotmetic nerve lesions. Second, we discuss our recent studies showing that GDNF gene-therapy is a powerful strategy to promote long-term motoneuron survival and improve function when target muscle reinnervation occurs within a critical post-lesion period. Based upon these observations, we discuss the influence of timing of the intervention, and of the duration, concentration and location of GDNF delivery on functional outcome. Finally, we provide a perspective on future research directions to realize functional recovery using gene therapy. Show less
Eggers, R.; Winter, F. de; Smit, L.; Luimens, M.; Muir, E.M.; Bradbury, E.J.; ... ; Verhaagen, J. 2020
Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose... Show moreVentral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45 weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45 weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects. Show less
Eggers, R.; Winter, F. de; Arkenaar, C.; Tannemaat, M.R.; Verhaagen, J. 2019
Root avulsions due to traction to the brachial plexus causes complete and permanent loss of function. Until fairly recent, such lesions were considered impossible to repair. Here we review clinical... Show moreRoot avulsions due to traction to the brachial plexus causes complete and permanent loss of function. Until fairly recent, such lesions were considered impossible to repair. Here we review clinical repair strategies and current progress in experimental ventral root avulsion lesions. The current gold standard in patients with a root avulsion is nerve transfer, whereas reimplantation of the avulsed root into the spinal cord has been performed in a limited number of cases. These neurosurgical repair strategies have significant benefit for the patient but functional recovery remains incomplete. Developing new ways to improve the functional outcome of neurosurgical repair is therefore essential. In the laboratory, the molecular and cellular changes following ventral root avulsion and the efficacy of intervention strategies have been studied at the level of spinal motoneurons, the ventral spinal root and peripheral nerve, and the skeletal muscle. We present an overview of cell-based pharmacological and neurotrophic factor treatment approaches that have been applied in combination with surgical reimplantation. These interventions all demonstrate neuroprotective effects on avulsed motoneurons, often accompanied with various degrees of axonal regeneration. However, effects on survival are usually transient and robust axon regeneration over long distances has as yet not been achieved. Key future areas of research include finding ways to further extend the post-lesion survival period of motoneurons, the identification of neuron-intrinsic factors which can promote persistent and long-distance axon regeneration, and finally prolonging the pro-regenerative state of Schwann cells in the distal nerve. Show less
Hoyng, S.A.; Winter, F. de; Gnavi, S.; Egmond, L. van; Attwell, C.L.; Tannemaat, M.R.; ... ; Malessy, M.J.A. 2015
Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD),... Show moreClinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (MV) and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS) repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS-gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study. Show less
Pascal, D.; Giovannelli, A.; Gnavi, S.; Hoyng, S.A.; Winter, F. de; Morano, M.; ... ; Gambarotta, G. 2014
In dit artikel wil ik op vraag van hoofdredacteur Frank van der Plas graag een analyse maken van de rol die de media vandaag in Vlaanderen vervullen. De analyse zal bestaan uit het belichten van... Show moreIn dit artikel wil ik op vraag van hoofdredacteur Frank van der Plas graag een analyse maken van de rol die de media vandaag in Vlaanderen vervullen. De analyse zal bestaan uit het belichten van verschillende facetten van de media, om zo te komen tot een duidelijke illustratie van haar werkwijze en doel. Onder media w ordt verstaan de hedendaagse ge schreven en audio/visuele massacommunicatiemiddelen, zoals dag- en weekbladen, radio en televisie. Show less
