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The handle http://hdl.handle.net/1887/43807 holds various files of this Leiden University dissertation
Author: Hoyng, Stefan
Title: Gene therapy and nerve repair
Issue Date: 2016-11-01
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General introduction.
Nerve surgery and gene therapy: a neurobiological and clinical perspective
Stefan A. Hoyng1,2, Martijn R. Tannemaat1, Fred de Winter1, Joost Verhaagen1, Martijn J.A. Malessy1,2
Journal of Hand Surgery (European Volume). 2011 Nov;36(9):735-46.
PMID: 21914696
1. Department of Neuroregeneration, Netherlands Insititute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands
2. Department of Neurosurgery, Leiden University Medical Center, Leiden, the Netherlands
ABSTRACT
Despite major microsurgical improvements the clinical outcome of peripheral nerve sur- gery 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 fol- lowed by a short introduction into 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.
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INTRODUCTION
It is estimated that, on a yearly basis, peripheral nerve injury affects over 67.000 people in the United States alone (Taylor et al. 2008). The leading cause of peripheral nerve injury is trauma to the peripheral extremities in an overrepresented young male popula- tion (Kouyoumdjian 2006). Currently, surgical reconstruction consists mainly of pri- mary repair with direct end-to-end coaptation or the insertion of autologous nerve grafts (Scholz et al. 2009). Despite major microsurgical improvements the clinical outcome is still regarded as suboptimal and it is widely accepted that further research is necessary.
From an historical perspective regeneration research has come a long way; from the observations of Augustus Waller (Waller 1850) to the first successful nerve reconstruc- tions after WW-II (Sunderland 1991). It is however only recently that the collaboration between surgical sciences and the rapidly developing field of molecular neurobiology has intensified. This has led to several novel concepts and the development of a number of innovative techniques (Battiston et al. 2009). Some of the most promising experimen- tal repair strategies include: 1) the application of neurotrophic factors through osmotic pumps, microspheres or gene therapy (Young et al. 2001; Tannemaat et al. 2008b; de Boer R. et al. 2010), 2) the transplantation of Schwann cells and skin cell derived Schwann cell precursors (Walsh and Midha 2009), 3) the use of artificial biodegradable nerve guides (Schmidt and Leach 2003) 4) the use of photodynamic therapy (Rochkind et al.
2009),and 5) brief electrical stimulation of the injured nerve (Gordon et al. 2009).
In this review we will discuss the potential of gene therapy as a strategy to promote peripheral nerve regeneration. We will first briefly discuss the current concepts and limi- tations of surgical nerve repair. We will then summarize the four main factors that limit functional recovery as they have emerged from neurobiological research. Subsequently, we will discuss the potential of gene therapy and its ability to overcome these limitations.
Finally, we consider a number of concrete gene therapeutic strategies that could help the nerve surgeon in the future.
THE CURRENT CLINICAL PRACTICE: WHY IS REGENERATION FOLLOWING SURGICAL REPAIR INCOMPLETE?
The decision to operate a patient with a peripheral nerve injury is usually based on sev- eral factors: a clinical assessment of the severity of the injury, the lack of spontaneous recovery of function and an absence of signs of recovery on electrophysiological record- ings (Sunderland 1990). Unless there is clear evidence of a complete nerve transection, rupture or avulsion (e.g. in open injury due to a traumatic laceration or following a high velocity trauma) surgical nerve reconstruction is performed several months after the initial injury when spontaneous recovery does not occur. Early surgical intervention in severe lesions is preferable, but is not easy to decide upon. This is caused by the limited
possibilities to assess the severity of the injury in closed nerve lesions (Malessy et al.
2009).
The current surgical strategy consists of tension-free coaptation of proximal and dis- tal nerve ends, carefully aligning proximal and distal nerve stumps to ensure maximal functional recovery (Evans et al. 1991). When direct repair is not possible, due to traction at the nerve coaptation site, autologous nerve grafts are applied in order to guide regen- erating axons from the proximal to the distal nerve stump (Isaacs 2010). These grafts are usually derived from the sural nerve, a sensory nerve that can be removed with- out causing major functional impairment. Other donor possibilities are the superficial radial sensory nerve or the anterior branch of the medial antebrachial cutaneous nerve.
Over the past decades several types of biodegradable artificial nerve guides have become commercially available (e.g. Neurolac © (Ascension Orthopedics), Neurotube © (Synovis Life Technologies) and NeuraGen © (Integra LifeSciences)). These guides are consid- ered an acceptable alternative to the autologous nerve graft, but only for small–diameter nerves with gaps up to 10 mm (Weber et al. 2000). Traction lesions of the nerve form a distinct group, as the injury may not lead to a complete rupture. The damaged nerve segment is characterized by the formation of a fibrotic scar (Maggi et al. 2003). These so-called neuromas-in-continuity often contain regenerating axons, but these appear to be particularly prone to abnormal branching and misrouting, thereby impairing func- tional recovery. In these cases, the neuroma is surgically resected and the resulting gap is bridged with autologous nerve grafts (Millesi et al. 1966).
Over the last decades, significant advances have been made in surgical techniques. The use of operating microscopes, the development of fine non-immunogenic suture materi- als and the application of nerve transfers have all had beneficial effects on the outcome of peripheral nerve surgery (Midha 2004). Whether there is a place for end-to-side nerve repair has still to be assessed (Viterbo et al. 2009). Despite these technical refinements, the functional outcome of nerve surgery is generally considered to be disappointing (Scholz et al. 2009).
Recent advances in research on the molecular basis of peripheral nerve regeneration have helped to further determine the bottlenecks that limit functional recovery after nerve surgery. First, it has been shown that a prolonged period of axotomy induces atrophy of motoneurons (Koliatsos et al. 1994; Gu et al. 2005; Eggers et al. 2010). Although this atro- phy is partially reversible, it contributes to the poor functional outcome (Fu and Gordon 1995a; McPhail et al. 2004). Similarly, a long interval between trauma and surgery causes changes in the denervated distal nerve stump, which gradually loses its ability to support regeneration over the course of several months (Fu and Gordon 1995b). The velocity of axonal outgrowth, which is on average 1 to 3 mm/day, is relatively slow (Pan et al. 2003).
Thus, even in the case of swift surgical intervention, neurotrophic factor production in the more distal segments of the nerve will drop significantly before regenerating axons
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make contact with Schwann cells, causing these axons to stall proximally (Sulaiman and Gordon 2000; Hoke et al. 2002; Eggers et al. 2010).
Second, after nerve repair, axotomised axons must cross the coaptation site. This occurs in a random and staggered fashion (McDonald et al. 2006). Axons may not succeed to reach the distal stump or will inevitably end up in the interfascicular perineurial tissue due to misalignment of nerve fascicles. In the case of autologous nerve grafts, two coap- tation sites are needed, duplicating these effects. Furthermore, the size and number of fascicles in a graft differ from those in the proximal and distal nerve stumps. Also, at the microscopical level, endoneurial tubes in grafts derived from sensory nerves may not be ideally suited to accommodate the larger diameter axons of motoneurons (Moradzadeh et al. 2008). Finally, the expression of neurotrophic factors in commonly used sensory nerve grafts is probably not ideal for the regeneration of motor axons (Hoke et al. 2006).
Third, perhaps the largest challenge is to prevent or diminish misrouting of motor and sensory axons. At the coaptation site, regenerating axons randomly enter endoneu- rial tubes that guide them towards an end organ. When motor axons mistakenly enter endoneurial tubes that lead towards sensory end organs, they are “pruned” in a process termed preferential motor reinnervation (Madison et al. 1999). However, the outgrowth of motor axons towards muscles occurs randomly, resulting in the random innervation of (in)appropriate muscles (IJkema-Paassen et al. 2002), or even innervation of two mus- cles by branches from a single motoneuron (de Ruiter et al. 2008). Furthermore, by occu- pying an endoneurial tube, misdirected axons probably physically exclude appropriate axons from finding their targets (Hoke and Brushart 2010).
Finally, by the time axons have been able to regenerate over long distances towards target muscles over the course of several months, these muscles have undergone denervation- induced atrophy. Loss of myofibers can lead up to a 90% loss of their original weight further impeding functional outcome (Eggers et al. 2010).
In summary, four main challenges emerge from basic neurobiological research:
1) increasing the velocity of regenerating axons in order to limit the probability of being trapped in an atrophied distal stump, 2) increasing the number of axons successfully crossing surgical coaptation sites, 3) preventing misrouting and 4) preventing atrophy of denervated end-organs.
Gene therapy is a promising approach to help resolve several of these challenges. We will first explain how gene therapy works. Subsequently, the first steps that have been under- taken to deliver genes encoding therapeutic proteins to the injured peripheral nerve will be reviewed including the hurdles that need to be overcome. Finally, we will describe a number of possible future scenarios for the clinical application of gene therapy by the nerve surgeon.
THE BASIC CONCEPT OF VIRAL VECTOR-MEDIATED GENE TRANSFER
Gene therapy in the current context is defined as the introduction of foreign genetic material (DNA or RNA) into living cells in order to positively modify nerve regeneration.
The foreign gene is referred to as the transgene and encodes a promoter and a potentially therapeutic protein. The promoter drives the expression of the therapeutic gene once the transgene is present in its target cell. There are several ways to introduce a transgene into cells, but the use of viral vectors has emerged as the most efficient strategy to achieve this.
Thus, gene therapy is based on the use of a viral vector to deliver a transgene to specific target cells. Delivery of a transgene by a viral vector is termed “transduction” and the cell that is infected by the viral vector is said to be “transduced”. Gene therapy can be performed by directly injecting the vector in the peripheral nerve (in vivo gene therapy) or by transducing cells in culture followed by transplantation of the transduced cells in the injured nerve (ex vivo gene therapy).
The production of a viral vector is performed in a molecular biology laboratory. First, a specific promoter and a therapeutic gene (e.g. a gene encoding a neurotrophic protein) are inserted in a viral vector DNA sequence by standard molecular cloning techniques (Figure 1, step 1 and 2). A part of this DNA or its transcribed RNA is subsequently pack- aged into a viral vector by using helper plasmids. It can be harvested and concentrated to obtain viral vector preparations with high titers (Figure 1, step 3). The viral vector can then be used to transduce cultured cells (in vitro) or can be injected in a living organ- ism (in vivo; e.g. during or after surgery) to transduce cells around the site of injection (Figure 1, step 4). Through viral transduction a transgene is introduced in the target cells and the therapeutic protein for which it codes is produced.
The most important promise of gene therapy lies in the two main advantages it has over the conventional delivery of therapeutic proteins. First, transduced cells will express the selected gene for an extended period of time. Secondly, the expression will be localized and specific: transgene expression is restricted to the cells that are transduced at the site of injection of the viral vector. These advantages are of particular significance for the delivery of neurotrophic factors, since these proteins have a very short half-life and do not penetrate well in neural tissue (Kemp et al. 2007; Tannemaat et al. 2008b).
In order to enhance regeneration in the peripheral nerve, there are three main cellular targets for gene therapy: Schwann cells (Figure 1, step 5), injured neurons and muscle fibers. Schwann cells play an essential role in peripheral nerve regeneration mainly by guiding regenerating axons and supporting continued growth through the secretion of an array of proteins, including neurotrophic factors. Their close proximity to regenerat- ing axons make them very suitable candidates for gene therapy. The goal of overexpress- ing therapeutic genes in Schwann cells is to enhance and perhaps even more importantly,
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sustain the regeneration process over long post-lesion time periods. The second main target is the injured neuron. By targeting the neuron, it may be possible to prevent denervation-induced neuronal atrophy or death. Furthermore, certain genes could acti- vate or sustain a neuron-intrinsic regeneration-associated gene expression programme that stimulates axonal regeneration (Raivich et al. 2004; Mason et al. 2011). The third possible target for gene therapy are muscle fibers: the (over)expression of a myotrophic factor in a denervated muscle could potentially prevent denervation-induced atrophy (Dodd et al. 2009; Senf et al. 2008).
VIRAL VECTORS: EFFECTIVE GENE DELIVERY VEHICLES
An extensive review of all viral vectors that have been used for gene therapy for the peripheral nerve has recently been published elsewhere (Mason et al. 2011). To target the peripheral nervous system, adeno-associated viral (AAV) vectors (Kaplitt et al. 1994) and lentiviral (LV) vectors (Naldini et al. 1996) have emerged as the most promising
Figure 1: Schematic overview of the concept of gene therapy. DNA encoding a therapeutic gene (1) is inserted into a transfer vector (2). This transfer vector is used to create viral vector particles (3). Following purification and concentration these vector particles can be injected in the tissue of interest; in this case the ulnar nerve.
(4). Inside the nerve, viral vector particles enter the Schwann cells and deliver their genetic material, including the therapeutic gene (5), to the nucleus of the Schwann cells that subsequently start to produce the desired therapeutic protein (green dots).
vectors. LV is a retrovirus that uses a reverse transcriptase and an integrase to insert a specific transgene into the genome of the host cell. AAV is a naturally occurring parvo- virus with a small genome of which multiple naturally occurring variants or serotypes exist. AAV vectors deliver their genome to the nucleus, where it is either integrated in the host cells genome or is stably maintained in the nucleus. Both vectors do not have the capacity to self-replicate (the capacity to create new viral particles by an infected cell has been lost), evoke little or no immune response and are capable of transducing both dividing and non-dividing cells (Naldini et al. 1996; Kay et al. 2001; Glorioso and Fink 2009). An important difference between AAV and LV vectors is their capacity to trans- duce different cell types. LV is capable of transducing a wide variety of cells, and is gener- ally considered a “promiscuous” viral vector. In the context of the peripheral nerve, LV has been shown to consistently and durably transduce Schwann cells in rodents (Hen- driks et al. 2007; Eggers et al. 2008; Tannemaat et al. 2008b) although in cultured human nerve segments, LV-vectors directed transgene expression is mostly limited to fibroblasts (Tannemaat et al. 2007). The transduction profile of AAV depends on its serotype. Sev- eral AAV serotypes, in particular AAV1 and AAV5 are very effective gene delivery vehi- cles to target sensory neurons (Mason et al. 2010). AAV6, on the other hand, is capable of transducing skeletal muscles (Blankinship et al. 2004). A recent study reported that AAV8 directs transgene expression in Schwann cells after injection in the sciatic nerve (Homs et al. 2011), although the number of transduced Schwann cells appears to be low and transgene expression levels are modest. The discovery of a large number of AAV serotypes, and the engineering of at least 12 of these serotypes in AAV vectors with dif- ferent cellular transduction properties, makes AAV one of the most flexible and promis- ing viral vector systems to date (Kwon and Schaffer 2008; Zincarelli et al. 2008; Mason et al. 2010).
Another difference between LV and AAV is that LV can deliver a much larger transgene to the host cell (Uchida et al. 1998; Kumar et al. 2001). This allows for the simultane- ous expression of multiple genes and offers the advantage of using large tissue-specific promoters to target transgene expression to specific cell types [e.g. the S100β promoter which is selectively active in Schwann Cells in the peripheral nervous system (Zuo et al.
2004)]. The use of a cell type specific promoter will normally result in transgene expres- sion in those cells where this promoter is naturally active. Transgene expression in other cell types that are transduced will normally not occur, thereby increasing the cellular specificity. Furthermore, LV vectors integrate their genetic material into the host cell which ensures prolonged transgene expression even in proliferating cells (Lundberg et al. 2008).
Recent research on gene therapy for peripheral nerve lesions in rodents has focused on the delivery of neurotrophic factors to regenerating axons in denervated nerve stumps (Haastert and Grothe 2007; Tannemaat et al. 2008b) and to injured spinal and facial motor neurons (Baumgartner and Shine 1998; Watabe et al. 2000; Blits et al. 2004;
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Nakajima et al. 2007). A variety of factors, including nerve growth factor (NGF) (Goins et al. 1999), glial cell line-derived neurotrophic factor (GDNF) (Araki et al. 2006), neuro trophin-3 (NT-3) (Chattopadhyay et al. 2002), ciliary neurotrophic factor (CNTF) (Homs et al. 2011) and fibroblast growth factor-2 (Haastert et al. 2006) have success- fully been overexpressed and have shown promising results. Transgene production has been shown for up to 4 months implying that long-term expression can be achieved (Hendriks et al. 2007). Lentiviral vector-mediated overexpression of GDNF in Schwann cells of reimplanted ventral roots did prevent avulsion-induced atrophy of spinal cord motoneurons and enhanced regrowth of motoraxons into the re-implanted ventral root.
Unfortunately, these axons were trapped in the ventral root in the areas of high GDNF expression near the original injection site and as a result long distance axon regeneration was impaired (Eggers et al. 2008; Tannemaat et al. 2008a). This phenomenon has been dubbed the “candy store” effect. In a rodent model, selective viral overexpression of NGF in the sensory sapheneous branch resulted in increased correct sensory reinnervation after injury (Hu et al. 2010). From the results of this study one might cautiously conclude that the viral vector mediated overexpression of neurotrophic factors may also become an effective strategy to address the misrouting of regenerating axons.
In summary, these first attempts to apply gene therapy in a number of rodent models of peripheral nerve injury paradigms show that viral vectors have been successfully applied to 1) counteract the atrophy of spinal motor neurons following ventral root avul- sion, 2) mediate long-term overexpression of neurotrophic factors by Schwann cells in the injured nerve and 3) address the issue of misrouting of regenerating sensory axons.
Regrettably, long term and high-level expression of GDNF in injured peripheral nerve stumps is causing lingering of axons near areas of high concentrations of GDNF. In order to enhance functional recovery in the clinical setting of peripheral nerve surgery, further refinements in the application of gene therapy are necessary. The recent developments and advances in viral vector technology which address this issue will be discussed in the following sections.
ADVANCES IN VIRAL VECTORS TECHNOLOGY
Long lasting transgene expression is one of the advantages of gene therapy. For a number of progressive neurodegenerative diseases a continuous supply of the therapeutic pro- tein throughout the life of the patient is required. Phase I and II clinical trials based on AAV vectors with non-regulated, persistent transgene expression have recently been completed for Parkinson’s disease (Kaplitt et al. 2007; Marks, Jr. et al. 2008). However, for applications such as peripheral nerve repair, it is desirable, if not essential that transgene expression can be regulated. This is important because unwanted side effects follow- ing continual application of several neurotrophic factors have been documented. The systemic administration of CNTF (1996; Lambert et al. 2001) and BDNF (Pelleymounter
et al. 1995; Cao et al. 2009) leads to substantial weight loss and anorexia. Treatments with NGF cause local thermal and mechanical hyperalgesia (Shu and Mendell 1999).
Moreover, as described above we observed trapping of axons at sites of persistent LV- vector-mediated overexpression of a neurotrophic factor (Eggers et al 2008). An ideal viral vector would therefore direct transgene expression as long as the therapeutic pro- tein is needed, that is as long as the therapeutic protein promotes axon regeneration.
Regulatable transgene expression has been obtained by using the reverse tetracycline responsive transactivator (rtTA). This protein binds to an element in the promoter that controls the expression of the transgene only upon the administration of tetracycline (Gossen and Bujard 1992; Gossen et al. 1995). This system has successfully been applied in rodents and in non-human primates (Stieger et al. 2009). By giving doxycycline, an ana- logue of tetracycline, orally or through intravenous injection the expression of numerous transgenes has successfully been regulated. These include several neurotrophic factors such as NGF, GDNF, CNTF and BDNF (Stieger et al. 2009). Unfortunately, immune reactions against the rtTA protein preclude the use of this system in human subjects. The rtTA protein is a fusion protein created from the tetracycline repressor protein derived from the E. coli bacteria and the VP16 transactivator domain from the Herpes simplex virus (HSV) making it considerably immunogenic. In peripheral tissues, including mus- cle, retina and liver, a cellular and humoral immune reaction destroys rtTA expressing cells (Favre et al. 2002; Latta-Mahieu et al. 2002; Chenuaud et al. 2004). To overcome the immune response, a immune-inert version of the rtTA protein has been developed recently (Zaldumbide et al. 2010). We are currently investigating whether reliable reg- ulatable transgene expression in the peripheral nerve can be attained with this novel immune-inert version of rtTA.
A second potential strategy to allow for controllable transgene expression would be to use an “injury-induced” promoter. An ideal injury-induced promoter would drive the expression of the therapeutic transgene in response to nerve injury and would be shut off after regeneration is completed. The promoter of the glial fibrillary acidic protein (GFAP) would be a suitable candidate since GFAP expression is induced in Schwann cells in the peripheral nerve after injury (Jessen et al. 1990; Cheng and Zochodne 2002;
Xu et al. 2008; Wang et al. 2010). Gene expression was selectively driven in Mueller glia cells following the injection of an AAV vector that contained the GFAP promoter in front of a reporter gene (Aartsen et al. 2010). This proof of principle study in the retina may be translatable to the injured peripheral nerve where an injury-induced promoter could be used to drive neurotrophic factor expression.
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FROM EXPERIMENT TO CLINICAL APPLICATION: THE CHALLENGE OF TRANSLATION
The translation of the results obtained with gene therapy from animal models to humans form an additional challenge. Experiments on human nerve surplus tissue either removed during elective surgery, from nerve grafting surgery or from cadaveric obduction may be helpful to establish the efficacy of different viral delivery vehicles in vitro before con- ducting larger scale in vivo experiments. A first study in this direction was performed by injecting a lentiviral vector encoding either for a reporter gene or NGF into segments of cultured human sural nerve (Tannemaat et al. 2007). This experiment resulted in the successful transduction of human perineural and epineural fibroblasts (Figure 2).
Interestingly this finding contrasted with observations in rodents where most of the transduced cells were Schwann cells (Hendriks et al. 2007; Tannemaat et al. 2008a). The reason for this difference between rodents and humans is not known. It could be due to the high density of myelin in the human nerve, which may form a physical barrier for the vector particles to reach the Schwann cells. It is also not known whether it is crucial to target the Schwann cells or whether transducing fibroblasts surrounding the lesion may be equally sufficient. Fortunately transduced fibroblasts from the human nerve segments were still able to secrete biologically active NGF. Further research is required to confi- dently establish the functionality of other types of viral vectors, including AAV vectors, in the human peripheral nervous system.
At this moment, several phase I and II clinical trials with AAV vectors in the human cen- tral nervous system are in progress or have recently ended and so far, not a single major adverse biosafety event that could be attributed to the vector has been reported (Snyder et al. 2010). AAV possesses, ever since it was first applied to the central nervous system (Kaplitt et al. 1994), an excellent safety record.
Unfortunately for LV a major biosafety issue needs to be resolved before clinical appli- cation can be considered. Insertional mutagenesis is caused by the random integra- tion of transgenes in the genome of transduced cells which can theoretically lead to the (in) activation of genes and can cause cancer. An early gene therapeutic trial for X-linked severe combined immunodeficiency with a different (µ-)retroviral (µRV) vector caused leukemogenesis in 2 out of 10 patients (Hacein-Bey-Abina et al. 2003). This raised serious concerns about retroviral integration and insertional mutagenesis. Fortunately several major differences between LV and µRV exist and insertional mutagenesis for LV has still not been reported (Montini et al. 2006; Beard et al. 2007; Hargrove et al. 2008). In a recent clinical trial involving LV in the treatment of X-linked adrenoleukodystrophy, fear of insertional mutagenesis led to extensive repeated deep-sequencing analysis of blood samples of all patients, but not a single instance of oncogenesis was found (Cartier et al.
2009). It is however widely accepted that further developments are warranted before LV vectors can be considered safe for large clinical trials.
Other important improvements in the biosafety of LV and AAV have recently been made through the reduction of contaminants from viral stocks. These are mainly composed of residuals from culture components and other cellular debris following production and are thought to contribute to the inflammatory response following vector administration.
The use of novel purification and filtration methods has allowed the production of large- scale, clean and highly concentrated LV and AAV stocks (Kutner et al. 2009; Ayuso et al.
2010). Future improvements will almost certainly further reduce residual contaminants of the production process.
HOW CAN GENE THERAPY BECOME PART OF THE ARMAMENTARIUM OF THE NERVE SURGEON?
At the beginning of this article we defined the challenges that exist to improve the func- tional outcome following surgical nerve repair. These include the necessity to prevent the atrophy and eventual death of axotomized motor neurons, to increase the number and the velocity of regenerating axons, limit the misrouting of motor and sensory axons and prevent muscle atrophy. Figure 3 illustrates how gene therapy could help to overcome some of these challenges in patients that have to undergo reconstructive nerve surgery.
First, short-term overexpression of a neurotrophic factor distal from a coaptation site may increase the speed and number of regenerating axons that successfully cross the
Figure 2: Ex-vivo gene transfer in a human sural nerve segment. The photomicrograph shows a longitudinal section of a human sural nerve segment 3 days after the injection of a lentiviral vector encoding Green Fluorescent Protein (GFP). Transduced cells express GFP and are therefore green. A sural nerve as shown here is commonly used in human reconstructive nerve surgery. This experiment demonstrates that it is possible to genetically modify human nerve segments, without interfering with the current clinical practice of neurosurgical nerve reconstruction. Scale bar: 1 mm.
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Figure 3: Illustration of four possible future applications of gene therapy to enhance the results of peripheral nerve repair.
A: Short-term overexpression of a neurotrophic factor may be able to increase the speed and number of regenerating axons crossing the nerve coaptation site.
B: Regeneration across autologous nerve transplants is poor: temporary over-expression of neurotrophic factors in the graft may promote the number of axons that enter the graft and could enhance the speed with which axons grow across the graft into the distal nerve stump.
C: Some neurotrophic factors appear to have differential effects on motoneurons and sensory neurons. Short term expression of a motoneuron-specific factor in a selected nerve branch may specifically attract regenerating motoneurons towards that nerve branch. This would facilitate the reinnervation of the denervated muscle, whereas sensory axons are not affected. Alternatively, short term expression of a sensory-specific factor could be useful, depending on the clinical situation.
D: Following proximal nerve injuries, target muscle atrophy is a major problem. This could possibly be prevented by injecting a viral vector encoding a gene that reduces denervation-induced atrophy of the target muscle fibers.
nerve coaptation site (Figure 3A). Second, regeneration across autologous nerve trans- plants is poor. The temporary over-expression of a neurotrophic factor in the graft may promote the number of axons that enter the graft and may in addition enhance the speed by which axons grow across the graft into the distal nerve stump (Figure 3B). Alterna- tively, a second injection of a viral vector distal from the graft may facilitate the growth of axons towards the distal nerve stump. Thirdly, certain neurotrophic factors appear to have a differential neurotropic influence on motoneurons and sensory neurons. Short term expression of a motoneuron-specific factor in a selected nerve branch may spe- cifically attract regenerating motor axons towards that nerve branch (Figure 3C). This
would facilitate the reinnervation of the denervated muscle, whereas sensory axons are not affected. Alternatively, depending on the clinical situation, short term expression of a sensory-specific factor could be useful. Finally, following proximal nerve injuries, target muscle atrophy is a major problem. This could possibly be prevented by injecting a vector encoding a myotrophic gene that reduces denervation-induced atrophy of the denervated muscle fibers (Figure 3D).
These clinical scenarios will only become reality if future viral vectors meet the biosafety requirements previously discussed, including the ability to create safe immune-inert regulatable vectors that efficiently transduce the human peripheral nerve and do not cause potentially harmful insertional mutations.
CONCLUSION
In the next decade the expanding field of viral vector technology, in combination with new insights and lessons from multiple ongoing clinical trials for hematological, neuro- degenerative, pulmonary, and retinal diseases will most likely provide us with efficient, regulatable, safe and cell-specific viral vectors to target the injured human peripheral nervous system. Continued collaboration between nerve surgeons and neurobiologists is essential to implement gene therapy in the field of nerve surgery. It will be fascinating to witness this evolution of the surgical sciences in the not too distant future.
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