Martijn R. Tannemaat

Differential effects of lentiviral vector-mediated overexpression of nerve growth factor and glial cell line-derived neurotrophic factor on regenerating sensory and motor axons in the transected peripheral nerve

Even after reconstructive surgery, major functional impairments remain in the majority of patients with peripheral nerve injuries. The application of novel emerging therapeutic strategies, such as lentiviral (LV) vectors, may help to stimulate peripheral nerve regeneration at a molecular level. In the experiments described here, we examined the effect of LV vector‐mediated overexpression of nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF) on regeneration of the rat peripheral nerve in a transection/repair model in vivo. We showed that LV vectors can be used to locally elevate levels of NGF and GDNF in the injured rat peripheral nerve and this has profound and differential effects on regenerating sensory and motor neurons. For sensory neurons, increased levels of NGF and GDNF do not affect the number of regenerated neurons 1 cm distal to a lesion at 4 weeks post‐lesion but do cause changes in the expression of markers for different populations of nociceptive neurons. These changes are accompanied by significant alterations in the recovery of nociceptive function. For motoneurons, overexpression of GDNF causes trapping of regenerating axons, impairing both long‐distance axonal outgrowth and reinnervation of target muscles, whereas NGF has no effect on these parameters. These observations show the feasibility of combining surgical repair of the transected nerve with the application of viral vectors. Furthermore, they show a difference between the regenerative responses of motor and sensory neurons to locally increased levels of NGF and GDNF.

Neuroregenerative effects of lentiviral vector-mediated GDNF expression in reimplanted ventral roots

Traumatic avulsion of spinal nerve roots causes complete paralysis of the affected limb. Reimplantation of avulsed roots results in only limited functional recovery in humans, specifically of distal targets. Therefore, root avulsion causes serious and permanent disability. Here, we show in a rat model that lentiviral vector-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) in reimplanted nerve roots completely prevents motoneuron atrophy after ventral root avulsion and stimulates regeneration of axons into reimplanted roots. However, over the course of 16 weeks neuroma-like structures are formed in the reimplanted roots, and regenerating axons are trapped at sites with high levels of GDNF expression. A high local concentration of GDNF therefore impairs long distance regeneration. These observations show the feasibility of combining neurosurgical repair of avulsed roots with gene-therapeutic approaches. Our data also point to the importance of developing viral vectors that allow regulated expression of neurotrophic factors.

Gene therapy for the peripheral nervous system: a strategy to repair the injured nerve.

Peripheral nerve injury in humans often leads to incomplete functional recovery. In this review we discuss the potential for gene therapy to be used as a strategy alongside surgical repair techniques for the study of peripheral nerve regeneration in rodent models and with a view to its eventual use for the promotion of successful regeneration in the clinic. Gene therapy vectors based on herpes simplex virus, adenovirus, lentivirus and adeno-associated virus have been developed to deliver genes to the neurons of the peripheral nervous system, i.e. primary sensory neurons in the dorsal root ganglia and primary motor neurons. Adenoviral and lentiviral vectors have also been used to transduce Schwann cells and fibroblasts in the injured nerve. We present an overview of these vectors, their application so far in the peripheral nervous system, their potential as vectors for enhancing peripheral nerve repair, and the successful interventions that have been demonstrated in animal models. We also discuss some of the limitations of current vectors and how they may be overcome. While the technology for gene delivery is approaching a state of readiness for clinical translation, the current range of therapeutic genes for the repair of the traumatically injured peripheral nerve is mostly limited to neurotrophic factors delivered to neurons, Schwann cells or possibly the target organs. Finally, therefore, we consider what type of therapeutic transgene may be desirable to enhance nerve regeneration in the future.

Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation, Schwann Cell Maturation and Myelination

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.

Human neuroma contains increased levels of semaphorin 3A, which surrounds nerve fibers and reduces neurite extension in vitro

Neuroma formation after peripheral nerve injury is detrimental to functional recovery and is therefore a significant clinical problem. The molecular basis for this phenomenon is not fully understood. Here, we show that the expression of the chemorepulsive protein semaphorin 3A (sema3A), but not semaphorin 3F, is increased in human neuroma tissue that has formed in severe obstetric brachial plexus lesions. Sema3A is produced by fibroblasts in the epineurial space and appears to be secreted into the extracellular matrix. It surrounds fascicles, minifascicles, or single axons, suggesting a role in fasciculation and inhibition of neurite outgrowth. Lentiviral vector-mediated knock-down of Neuropilin 1, the receptor for sema3A, leads to increased neurite outgrowth of F11 cells cultured on neuroma tissue, but not of F11 cells cultured on normal nerve tissue. These findings demonstrate the putative inhibitory role of sema3A in human neuroma tissue. Our observations are the first demonstration of the expression of sema3A in human neural scar tissue and support a role for this protein in the inhibition of axonal regeneration in injured human peripheral nerves. These findings contribute to the understanding of the outgrowth inhibitory properties of neuroma tissue.

Genetic modification of human sural nerve segments by a lentiviral vector encoding nerve growth factor.

OBJECTIVEAutologous nerve grafts are used to treat severe peripheral nerve injury, but recovery of nerve function after grafting is rarely complete. Exogenous application of neurotrophic factors may enhance regeneration, but thus far the application of neurotrophic factors has been hampered by fast degradation after local application and unwanted side effects after systemic application. These problems may be overcome with the use of lentiviral (LV) vectors that direct sustained local transgene expression in cells. METHODSHuman sural nerve segments were either submerged in or injected with LV vectors encoding green fluorescent protein and cultured in vitro. Production of nerve growth factor (NGF) by nerve segments after injection of LV-NGF was quantified. The effect of NGF produced by LV-transduced fibroblasts derived from human sural nerve segments was assessed on neurite outgrowth in vitro. RESULTSThe injection of vector into nerve segments is a more effective way to deliver the vector than submersion of the nerve in vector-containing medium, leading to large numbers of transduced fibroblasts over a significant extent inside the nerve. The injection of LV-NGF leads to a gradual increase of NGF production, reaching a plateau after 4 days. LV-NGF-transduced human fibroblasts promote neurite outgrowth in vitro. CONCLUSIONWe have developed a method to transduce cells in human sural nerve segments with LV vector. This approach holds promise as a powerful novel adjuvant therapy for peripheral nerve surgery and can be performed without changing the routine practice of nerve grafting.

Nerve surgery and gene therapy: a neurobiological and clinical perspective

Despite major microsurgical improvements the clinical outcome of peripheral nerve surgery is still regarded as suboptimal. Over the past decade several innovative techniques have been developed to extend the armamentarium of the nerve surgeon. This review evaluates the potential of gene therapy in the context of peripheral nerve repair. First the main challenges impeding peripheral nerve regeneration are presented. This is followed by a short introduction to gene therapy and an overview of its most important advantages over the classical delivery of therapeutic proteins. Next, this review focuses on the most promising viral vectors capable of targeting the peripheral nervous system and their first application in animal models. In addition, the challenges of translating these experimental results to the clinic, the limitations of current vectors and the further developments needed, are discussed. Finally, four strategies are presented on how gene therapy could help patients that have to undergo reconstructive nerve surgery in the future.

From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve.

Reconstructive surgery of the peripheral nerve has undergone major technical improvements over the last decades, leading to a significant improvement in the clinical outcome of surgery. Nonetheless, functional recovery remains suboptimal in the majority of patients after nerve repair surgery. In this review, we first discuss the molecular mechanisms involved in peripheral nerve injury and regeneration, with a special emphasis on the role of neurotrophic factors. We then identify five major challenges that currently exist in the clinical practice of nerve repair and their molecular basis. The first challenge is the slow rate of axonal outgrowth after peripheral nerve repair. The second problem is that of scar formation at the site of nerve injury, which is detrimental to functional recovery. As a third issue, we discuss the difficulty in assessing the degree of injury in closed traction lesions without total loss of continuity of the involved nerve elements. The fourth challenge is the problem of misrouting of regenerating axons. As a fifth and final issue we discuss the potential drawbacks of using sensory nerve grafts to support the regeneration of motoneurons. For all these challenges, solutions are likely to emerge from (a) a better understanding of their molecular basis and (b) the ability to influence these processes at a molecular level, possibly with the aid of viral vectors. We discuss how lentiviral vectors have been applied in the peripheral nerve to express neurotrophic factors and summarize both the advantages and drawbacks of this approach. Finally, we discuss how lentiviral vectors can be used to provide new, molecular neurobiology-based, approaches to address the clinical challenges described above.

Complement inhibition accelerates regeneration in a model of peripheral nerve injury.

Complement (C) activation is a crucial event in peripheral nerve degeneration but its effect on the subsequent regeneration is unknown. Here we show that genetic deficiency of the sixth C component, C6, accelerates axonal regeneration and recovery in a rat model of sciatic nerve injury. Foot-flick test and Sciatic Function Index monitored up to 5 weeks post-injury showed a significant improvement of sensory and motor function in the C6 deficient animals compared to wildtypes. Retrograde tracing experiments showed a significantly higher number of regenerated neurons at 1 week post-injury in C6 deficient rats than wildtypes. Pathology showed improved nerve regeneration in tibials of C6 deficient animals compared to wildtypes. Reconstitution with purified human C6 protein re-established the wildtype phenotype whereas pharmacological inhibition of C activation with soluble C receptor 1 (sCR1) facilitated recovery and improved pathology similarly to C6 deficient animals. We suggest that a destructive C-mediated event during nerve degeneration hampers the subsequent regenerative process. These findings provide a rationale for the testing of anti-complement agents in human nerve injury.