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The therapeutic potential of gene transfer for the treatment of peripheral neuropathies

Published online by Cambridge University Press:  19 March 2007

James R. Goss
James R. Goss
Affiliation:
Molecular Genetics and Biochemistry, Center for Biotechnology and Bioengineering, University of Pittsburgh, 300 Technology Drive, Rm 208, Pittsburgh, PA 15219, USA. Tel: +1 412 383 9558; Fax: +1 412 383 9760; E-mail: jrgoss@pitt.edu
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Abstract

Peripheral neuropathy is a common medical problem with numerous aetiologies. Unfortunately, for the majority of cases there is no available medical solution for the underlying cause, and the only option is to try to treat the resulting symptoms. Treatment options exist when neuropathy results in positive symptoms such as pain, but there is a significant lack of treatments for negative symptoms such as numbness and weakness. Systemic application of growth factor peptides has shown promise in protecting nerves from neuropathic insults in preclinical animal studies, but translation into human trials has been problematic and disappointing. Significant advancements have been made in the past few years in utilising gene therapy approaches to treat peripheral neuropathy by expressing neuroprotective gene products either systemically or in specific nervous tissues. For example, plasmids expressing vascular endothelial growth factor injected into muscle, or herpes-simplex-virus-based vectors expressing neurotrophin gene products delivered to dorsal root ganglion neurons, have been used to protect peripheral nerve function in animal models of diabetes-associated peripheral neuropathy. Many published studies support the feasibility of this approach, although several questions still need to be addressed as gene therapy to treat peripheral neuropathy moves out of the laboratory and into the clinic.

Information

Type
Research Article
Information
Expert Reviews in Molecular Medicine , Volume 9 , Issue 8 , March 2007, pp. 1 - 20
Copyright
Copyright © Cambridge University Press 2007

Peripheral neuropathy is a general term describing damage to the nerves of the peripheral nervous system (PNS). These include the cranial nerves (except the optic nerve), the nerve roots exiting the spinal cord, the dorsal root ganglia neuron cell bodies, the peripheral nerve trunks and their distal processes, and the nerves constituting the autonomic nervous system. The term peripheral neuropathy also encompasses damage to myelin or to myelin-producing Schwann cells. Peripheral neuropathy is a common neurological problem, with over 100 types described. While the exact prevalence is unknown, it is estimated that approximately 2–8% of the population suffers from some form of peripheral neuropathy (Ref. Reference Hughes1).

The various types of peripheral neuropathies differ in their aetiology, the nerves affected, anatomical location, and progression; furthermore, they can be inherited or acquired. Table 1 summarises the characteristics of several different peripheral neuropathies. Charcot–Marie–Tooth (CMT) disease is an example of an inherited neuropathy. It is the most common inherited disorder of the PNS and leads to either primary peripheral demyelination or primary peripheral axonal pathology (Ref. Reference Warner, Garcia and Lupski2). Neuropathy is a common problem associated with autoimmune demyelinating diseases such as Guillan–Barre syndrome, and with some infectious agents such as human immunodeficiency virus (HIV) where up to 20% of patients may suffer from peripheral neuropathy (Ref. Reference Samson3). Peripheral neuropathy is a major complication in 60–70% of patients with either type 1 or type 2 diabetes mellitus (Refs Reference Dyck4, Reference Dyck5), and can be found in patients with systemic illnesses such as chronic liver disease and kidney failure (Ref. Reference Amato, Dumitru and Dumitru6). Cancer can cause peripheral neuropathy as a result of direct invasion or compression of the nerve or through remote (paraneoplastic) effects. Mechanical compression of nerves, usually at sites where they pass through openings in bone, fibrous tissues or muscle, can cause radiculopathy (damage to the spinal nerve roots, usually due to a herniated or degenerative disc, or bone changes from rheumatoid or osteoarthritis), or entrapment neuropathies such as carpal tunnel syndrome. Peripheral neuropathy is a dose-limiting adverse effect of several common medications, including those used to treat malignancies and HIV (Ref. Reference Peltier and Russell7). Nutritional deficiencies and chronic alcoholism, which leads to vitamin deficiency syndromes, can also result in peripheral neuropathy. The aetiology of peripheral neuropathy in ∼15–20% of patients is not known (Refs Reference Dyck, Oviatt and Lambert8, Reference McLeod9).

Table 1

Classification of some peripheral neuropathiesFootnote a

bPrevalence represents the estimated number of cases of a disorder or disease in the US population – not the number of people suffering from neuropathy.

cPrevalence not known.

Abbreviations: HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; POEMS, ‘polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes’.

Anatomy and pathophysiology of peripheral neuropathies

Various patterns of nerve damage can be distinguished in peripheral neuropathies. The large myelinated axons affected in large-fibre neuropathies include motor neurons, and Aβ sensory neurons responsible for vibration sense, light touch, and proprioception. (Diseases of the motor neuron cell body are classically considered separate from peripheral neuropathies and are not covered in this review.) Small-diameter nerves include thinly myelinated Aδ-fibres, which respond to cold sensation and mechanical nociception and which also have preganglionic autonomic cholinergic functions, and unmyelinated C-fibres, which respond to warmth and a variety of nociceptive stimuli and which have postganglionic autonomic functions. Neuropathies affecting Aδ-fibre and C-fibre neurons are generally referred to as small-fibre neuropathies (Ref. Reference Lauria10).

Damage or destruction of an isolated nerve or nerve group is known as mononeuropathy and results in a focal pattern of sensory and/or motor deficits. Mononeuropathies are usually caused by physical trauma or prolonged nerve compression. Some systemic disorders, such as vasculitis, can affect two or more isolated nerve areas (mononeuropathy multiplex), resulting in a multifocal pattern of nerve disability. Usually, systemic disorders result in a generalised symmetric polyneuropathy where several nerves or nerve groups are affected throughout the body. Damage to peripheral nerves occurs at the axon (axonopathy), the cell body (neuronopathy) or the myelin sheath (myelinopathy). Damage to the axon results in degeneration of the axon and often disruption of the myelin sheath as well. Mechanical damage due to trauma causes Wallerian degeneration where the axon and myelin sheath distal from the point of injury degenerate. Metabolic insults (e.g. diabetes) usually cause axonal degeneration from the distal tip, which then proceeds proximally towards the cell body as the disease progresses, a process known as ‘dying-back’. This type of neuropathy is usually length-dependent in that longer nerves, such as those innervating the feet or hands are affected first. Sensory neuron diseases (SNDs) describe a subset of peripheral neuropathies that primarily affect dorsal root ganglion neurons, resulting in non-length-dependent neuropathy, often with degeneration of central projections (Ref. Reference Sghirlanzoni, Pareyson and Lauria11).

Symptoms of peripheral neuropathy correspond to the nerves and/or axons involved and the type of information they convey to the central nervous system (CNS). Large-fibre sensory neuropathies might involve numbness and gait imbalance (sensory ataxia), whereas small-fibre neuropathies often present with paresthesias such as burning pain or tingling or dysesthesias such as allodynia (the sensation of pain from a normally non-noxious stimulus). Distal symmetric neuropathies usually initiate in the toes and/or fingers and progress proximally, resulting in a stocking-glove distribution of symptoms. Impairment of motor function generally presents as weakness and, similarly to sensory deficits, progresses in a distal to proximal manner. Proximal muscle weakness that is not associated with this type of distal to proximal pattern can reflect a radiculopathy. Radiculopathy can also result in painful paresthesia and numbness, usually in a dermatomal distribution. Loss of motor-fibre innervation can lead to muscle wasting; cramps, fasciculations and myalgia are other symptoms. Involvement of autonomic fibres can lead to orthostatic hypotension, decreased sudomotor activity, gastrointestinal dysmotility, urinary bladder dysfunction, impotence and glandular dysfunction.

Symptoms of peripheral neuropathy vary from person to person. Some people experience temporary and/or mild symptoms, whereas other patients suffer extreme pain or paralysis. In acute neuropathies such as Guillan–Barre syndrome, symptoms may manifest within a couple of days and progress rapidly. In chronic disorders such as diabetes, the symptoms might progress slowly over a long period of time. In some patients, chronic peripheral neuropathy reaches a plateau or, rarely, symptoms may wane and wax.

Current treatment options for peripheral neuropathy

Since peripheral axons have the ability to regenerate, endogenous recovery from axonopathies is possible; however, no neuroregenerative treatments are clinically available. Recovery is more problematic in sensory neuronopathies, which can result in degeneration of both the central and peripheral processes. Radiculopathy resulting from nerve root irritation (e.g. herniated disc) can often be resolved with supportive therapies (physical therapy, management of pain), although recovery is poor from severe nerve root trauma resulting in axon transection because of the inability of spinal nerve axons to regenerate properly back into the spinal cord. Acquired myelinopathies that spare the axon are generally sporadic and recovery is often complete, but recovery is poor from acquired myelinopathies that result in axon damage. Likewise, hereditary myelinopathies tend to be progressive with little chance for full recovery.

The treatment of peripheral neuropathy is usually directed at either alleviating the symptoms or treating the underlying disease that causes neuropathy. Unfortunately, there is no currently available treatment for numbness or sensory ataxia – cardinal symptoms of peripheral neuropathy. In neuropathy patients suffering from painful paresthesias, over-the-counter analgesics and/or prescription pain medications are the primary treatment option. Nonsteroidal anti-inflammatory drugs (NSAIDs) suffice for some patients, but most patients do not respond well. Topical analgesics (lidocaine patch, capsaicin cream), anticonvulsants (carbamazepine, phenytoin, pregabalin), antidepressants (tricyclic antidepressants, serotonin noradrenalin reuptake inhibitors), and opioids (morphine) are used to treat pain, but only about half of patients respond to these medications (Ref. Reference Hansson, Lacerenza, Marchettini and Hansson12). Direct injection of nerve-block agents such as lidocaine can also be helpful to relieve pain. Physical and occupational therapy, and or surgery, can be used to alleviate painful symptoms as well as improve mobility and function in neuropathies involving muscle weakness and in patients with compression neuropathies and those susceptible to new nerve injury at pressure points. Prompt diagnosis and treatment of the underlying cause can reduce the severity of peripheral neuropathy or decrease the recovery time. Immune-mediated neuropathies can be treated with immunomodulating agents (e.g. Guillan–Barre syndrome can be treated with intravenous immunoglobulin) and infection-associated neuropathies can be treated with antibiotic or antiviral therapy. Nutritional deficiencies can be treated with changes in diet and dietary supplements.

Diabetes is the most common cause of neuropathy in the Western world and tight glycaemic control using a combination of diet and drugs is currently the best treatment option, although this is very difficult to achieve over the lifetime of the disease. Several drug therapies designed to interfere with the pathogenic mechanisms underlying the neuropathy have been tested with unsatisfactory results. For example, animal studies have documented the protective effects of aldose reductase inhibitors on the progression of diabetic neuropathy, but the results of human trials have failed to support the use of this therapy in treatment of the human disease (Refs Reference Greene13, Reference Mayer and Tomlinson14, Reference Engerman, Kern and Larson15, Reference Pfeifer, Schumer and Gelber16). Several researchers have described the use of free-radical scavengers including vitamins A, C and E, probucol, reduced glutathione, and alpha-lipoic acid to treat various diabetic complications (Refs Reference Mekinova17, Reference Karasu18, Reference Cotter19, Reference Bravenboer20, Reference Nagamatsu21) but large-scale clinical studies employing compounds such as vitamin E have proved disappointing, perhaps because it is difficult to achieve large enough doses at the target tissue via systemic delivery (Ref. Reference Wiernsperger22).

Growth factors and peripheral neuropathy

Neurotrophins

Unfortunately, direct treatment of the underlying cause of most peripheral neuropathies is not possible. In such instances, interventions designed to support and protect the affected nerves, such as the use of endogenous growth factor peptides, are an attractive possibility. Neurotrophins (NTFs) are a class of small target-derived trophic factor peptides that were initially identified by their ability to prevent programmed cell death of neurons during development. The most widely studied of the NTFs is nerve growth factor (NGF), which has long been known to be required for survival of sympathetic PNS and sensory CNS neurons during development (Refs Reference Levi-Montalcini and Angeletti23, Reference Levi-Montalcini24). Several NTFs with ∼50% amino acid homology with NGF are known, including brain-derived neurotrophic factor (BDNF) and neurotrophins 3 and 4/5 (NT-3 and NT-4/5). The NTFs bind to two types of postsynaptic receptors: a high-affinity (K d = 10−11 m) tropomyosin-related tyrosine kinase (Trk) receptor and the low-affinity (K d = 10 −9 m) p75NTR receptor (a member of the tumour necrosis factor receptor family). In the adult, NTFs can promote both neuron cell survival and cell death, aid in the preservation and regrowth of axonal networks, and modulate synapses (Refs Reference Kalb25, Reference Verge26, Reference Lindsay27, Reference Rich28). These different functions appear to result from the interactions between the proform and mature form of the peptides, which are both released from target tissues, with the Trk and/or p75 receptors. Retrograde axonal transport of both NGF and NT-3 is decreased in experimentally induced diabetes (Refs Reference Jakobsen and Brimijoin29, Reference Tomlinson, Fernyhough and Diemel30, Reference Fernyhough, Diemel and Tomlinson31, Reference Fernyhough32, Reference Lee33), and there appears to be a decrease in target tissue concentrations of both of these trophic factors (Refs Reference Fernyhough, Diemel and Tomlinson31, Reference Fernyhough32, Reference Hellweg and Hartung34). In small-animal models, systemic administration of NTFs can be used to prevent the progression of sensory polyneuropathy caused by diabetes (Refs Reference Tomlinson, Fernyhough and Diemel30, Reference Unger35, Reference Elias36, Reference Apfel37) or neurotoxic exposure (Refs Reference ter Laak38, Reference Hayakawa39, Reference Helgren40).

The impressive effects of trophic factor therapy in animal models of neuropathy led to speculation that trophic factors might be useful in treating similar conditions in human patients, and resulted in a human trial of recombinant human NGF (rhNGF) to treat diabetic neuropathy. Unfortunately, a multicentre Phase III trial failed to demonstrate any improvement in outcome measurements (Ref. Reference Apfel41). Likewise, no measurable improvement was observed in a clinical trial of rhBDNF in diabetic neuropathy (Ref. Reference Wellmer42). A Phase I clinical trial using NT-3 to treat diabetic neuropathy was apparently halted in 1997 for unknown reasons (Ref. Reference Leinninger, Vincent and Feldman43); however, a Phase I clinical trial utilising NT-3 in patients with Charcot–Marie–Tooth showed some promise (Ref. Reference Sahenk44). The generally disappointing results seen might be due to the inability to achieve adequate local doses of the trophic factors. For example, the preclinical animal studies employed NGF in doses of 3–5 mg/kg, compared with the maximum tolerated dose of 0.1 µg/kg employed in the human trial; higher doses caused hyperalgesia. These peptides have a very short half-life in serum, measured at 7.2 min for NGF and 1.28 min for NT-3 in the rat (Ref. Reference Poduslo and Curran45), and so it is not surprising that intermittent low-dose bolus injections failed to achieve a therapeutic effect.

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a small-molecule trophic factor that was first recognised for its role in angiogenesis. VEGF binds to two separate receptors, although only one (VEGFR-2) activates a signal cascade (Refs Reference Terman46, Reference Waltenberger47). Because of the small contact area between peripheral nerves and the microvascular system surrounding them, peripheral nerves are especially susceptible to hypoxia and ischaemia, and abnormalities in the microvasculature leading to tissue ischaemia have been suggested to cause diabetic neuropathy (Refs Reference Yagihashi48, Reference Boulton and Malik49). Therapeutic angiogenesis has been demonstrated with systemic delivery of VEGF in an ischaemic hind-limb rabbit model (Refs Reference Takeshita50, Reference Bauters51). Apart from its trophic actions on endothelial cells, VEGF also has a direct trophic influence on nervous tissue cells. In cultures of superior cervical and dorsal root ganglia, VEGF induces neurite outgrowth and prolongs survival times of neurons and Schwann cells (Ref. Reference Sondell, Lundborg and Kanje52). It protects cultured neurons against cell death resulting from ischaemia or glutamate toxicity (Refs Reference Jin, Mao and Greenberg53, Reference Jin, Mao and Greenberg54, Reference Matsuzaki55) and has shown similar effects in animal models of cerebral ischaemia and spinal cord injury (Refs Reference Yang56, Reference Widenfalk57). VEGF appears to play an important role in the survival of adult motor neurons (Ref. Reference Oosthuyse58); VEGF deficiencies have been implicated as a possible cause of amyotrophic lateral sclerosis (a motor neuron disease) (Ref. Reference Carmeliet and Storkebaum59), although elevated serum levels have been implicated in POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome (Ref. Reference Scarlato60).

Insulin-like growth factors

The insulin-like growth factors IGF1 and IGF2, which are trophic factors with sequence homology to pro-insulin and bind to the type 1 IGF receptor, are essential to normal nervous system development (Refs Reference Daughaday and Rotwein61, Reference Rotwein62) and can promote both sensory and motor nerve regeneration (Refs Reference Hansson63, Reference Kanje64, Reference Svenningsen and Kanje65, Reference Pu66, Reference Vergani67). The IGFs also have mitogenic effects on Schwann cells, and reduce demyelination associated with autoimmune encephalopathy (Refs Reference McMorris68, Reference Sondell, Fex-Svenningsen and Kanje69, Reference Yao70). Serum IGF1 levels are reduced in adult patients with insulin-dependent or non-insulin-dependent diabetes (Ref. Reference Tan and Baxter71), and IGF1 mRNA levels are reduced in the spinal cord and nerves of streptozotocin diabetic rats (Refs Reference Wuarin, Guertin and Ishii72, Reference Ishii, Glazner and Pu73). Systemic administration of IGF1 and IGF2 to diabetic animals can prevent and reverse sensory neuropathy (Refs Reference Froesch, Bianda and Hussain74, Reference Ishii and Lupien75, Reference Zhuang76) and IGF1 can reverse neuroaxonal dystrophy in autonomic ganglia of diabetic rats (Ref. Reference Schmidt77). In addition, IGF1 administration can prevent neuropathy in vincristine-induced toxicity (Ref. Reference Contreras78).

Other growth factors

Ciliary neurotrophic factor (CNTF), glial-cell-line-derived neurotrophic factor (GDNF) and erythropoietin (EPO) are additional growth factors with roles in the development and support of peripheral nerves. CNTF belongs to a family of cytokine-like growth factors called neurokines that bind to receptor complexes that share a common structure. CNTF is primarily found in adult glial cells. It lacks the signal peptide typical of secreted proteins including other classical growth factors, and might act only in response to injury or other stresses (Ref. Reference Ito79). In agreement with this idea, the levels of CNTF expression are higher in the adult than in the developing embryo. In addition, mice lacking CNTF gene expression develop normally, and show progressive atrophy and motor neuron loss only as adults (Ref. Reference Masu80). CNTF promotes the survival of parasympathetic ganglion neurons and sympathetic and sensory neurons (Ref. Reference Ip81), but its major role appears to be as a target-derived trophic factor for damaged motor neurons (Refs Reference Li82, Reference Sendtner83). In instances of diabetic neuropathy in rodents and various peripheral neuropathies in humans, CNTF levels actually decrease whereas the receptor levels increase (Refs Reference Ito79, Reference Calcutt84). Interest in CNTF has been primarily as a potential therapy for motor neuron disease; however, a Phase I/II clinical trial utilising CNTF to treat amyotrophic lateral sclerosis demonstrated no efficacy and was halted because of adverse effects (Ref. 85).

GDNF, a member of the transforming growth factor β superfamily, was first recognised for its trophic support of dopaminergic neurons in the CNS (Ref. Reference Lin86). It binds to a receptor complex formed by a tyrosine kinase protein (Ret) and a glycosylphosphatidylinositol-linked α subunit (GDNFRα) (Ref. Reference Treanor87). In the PNS, GDNF acts as a trophic factor for autonomic, sensory and motor neurons (Refs Reference Buj-Bello88, Reference Oppenheim89). GDNF plays an important role in the development of sensory and autonomic neurons; during embryogenesis, a subset of small sensory dorsal root ganglia neurons (nonpeptidergic) switch from requiring NGF to requiring GDNF (Ref. Reference Molliver90), and mice deficient for Ret or GDNFRα fail to develop an enteric nervous system (Refs Reference Schuchardt91, Reference Tomac92). In response to peripheral nerve damage, GDNF expression increases in Schwann cells and the damaged nerve, and decreased GDNF is associated with impaired nerve regeneration (Refs Reference Hammarberg93, Reference Hoke, Cheng and Zochodne94, Reference Hoke95). In adult animals, systemic GDNF has been shown to rescue the nonpeptidergic dorsal root ganglia neurons following axotomy (Ref. Reference Bennett96) and promote myelination in normally unmyelinated nerve fibres (Ref. Reference Hoke97). In diabetic animals, intrathecal GDNF treatment also rescues the nonpeptidergic sensory neurons, and diabetic mice that overexpress GDNF show no enteric neuropathy compared with normal mice (Ref. Reference Anitha98).

EPO is a glycoprotein originally discovered as a result of its involvement in red blood cell production. Several studies have shown it to possess neuroprotective activities. Administration of EPO protects CNS neurons from apoptosis via receptor-mediated signalling through both the phophoinositide-3-kinase–Akt and nuclear factor κB pathways (Ref. Reference Digicaylioglu and Lipton99). It enhances recovery from ischaemia and trauma and reduces the injury associated with experimental autoimmune encephalomyelitis (Refs Reference Gorio100, Reference Siren101, Reference Erbayraktar102); in addition, some encouraging results have been found in a clinical trial utilising EPO in patients with acute stroke (Ref. Reference Ehrenreich103). In the PNS, Schwann cells appear to be the predominant source of EPO expression, whereas nerves express the EPO receptor; both increase following nerve injury (Ref. Reference Campana and Myers104). Systemic EPO administration inhibits apoptosis of dorsal root ganglia neurons following peripheral nerve injury (Refs Reference Campana and Myers105, Reference Campana106). Evidence suggests that EPO is effective in preventing dying-back neuropathies. For example, EPO prevents sensory nerve axon degeneration in an in vitro model of HIV neuropathy (Ref. Reference Keswani, Leitz and Hoke107), and systemic EPO treatment protects both sensory and motor nerve damage in acrylamide-treated rats (Refs Reference Keswani108, Reference Hoke and Keswani109). Systemic EPO or its carbamylated derivative partially preserved sensory nerve conduction deficits in cisplatin-intoxicated rats (Ref. Reference Bianchi110). It also has been shown to attenuate nerve conduction deficits, improve behavioural changes and preserve cutaneous innervation in streptozotocin diabetic rats (Ref. Reference Bianchi111).

Gene therapy approaches for treating peripheral neuropathy

The failure of the systemic rhNGF clinical trial highlights the difficulty of applying the findings from preclinical animal studies to human diseases. One alternative to systemic treatment is to utilise gene therapy in an attempt to either target the affected nerves directly or enable continuous production of the therapeutic product (Fig. 1).

Figure 1

Possible routes of entry for gene therapy for peripheral neuropathies. Local or systemic production of a potentially therapeutic gene product can be achieved by nonviral- or viral-based delivery at various sites. The principal aims of injections at each site, in terms of local or systemic action of the gene product, are indicated. (a) Direct injection into the spinal cord will usually result in local production of the gene product, although release into the cerebral spinal fluid is possible. Injection into the spinal cord utilising some viral-based systems capable of retrograde transport may also result in infection of dorsal root ganglion (DRG) neurons. (b) Direct injection into DRG neurons will result in local production of the gene product. If the gene product is released by transduced neurons it may enter both the spinal cord and the periphery. (c) Injection into adipose tissue can result in release of the gene product into the circulation, thus providing an avenue for systemic treatment. (d) Direct injection into nerves usually results in transduction of neuron cell bodies in the DRG and expression of the gene product. It may be more specific to a particular nerve or nerve bundle than injection into DRG. Localised expression of the gene product only at the site of the injection is possible, especially if the glial cells are transduced. (e) Injection into muscle will result in either systemic release or broad local release (e.g. a leg or arm). This has been the preferred route for nonviral-based systems using plasmids. (f) Viral-based vectors capable of retrograde transport can be subcutaneously injected into a specific dermatome to infect specific DRG. This method is much less invasive than direct injection into DRG or nerves but the results are similar. This has been the preferred route of entry for vectors based on herpes simplex virus.

Vector-mediated gene therapy

Gene therapy (or gene transfer) refers to the introduction and expression of potentially therapeutic DNA or RNA sequences in tissues and/or cells. One major advantage of gene therapy compared with traditional pharmacological approaches is the ability to target specific regions and/or cells in the neuraxis. As a result, the effective dose of a therapeutic protein could be substantially less than the effective dose of a traditional pharmaceutical, thus decreasing adverse effects associated with systemic drug treatment. In addition, gene therapy offers the possibility of long-term expression of therapeutic proteins in specific cells. This is an especially attractive benefit for chronic conditions such as peripheral neuropathy.

Two basic delivery systems have been devised to introduce exogenous genes into cells: viral-based delivery and nonviral-based delivery. Nonviral methods include the use of plasmids to express a desired gene, or antisense oligonucleotides directed against specific gene targets. The use of plasmids and antisense oligonucleotides for gene therapy offers several advantages including specificity, ease of construction, and minimal immunogenicity. A major problem with this approach concerns delivery – that is, how to transfer the DNA from the outside of a cell into its nucleus. Although several delivery systems have been developed to help alleviate this problem including encapsulation into an artificial lipid complex (liposome) (Ref. Reference Luo and Saltzman112), efficient gene transfer often requires microgram to milligram quantities of plasmid DNA.

Viral-based vector gene therapy takes advantage of the natural ability of viruses to infect cells and have their genes expressed by the host cells. Viral-based vector systems can express transgenes for a prolonged period of time and some can express multiple genes at once or exceptionally large gene constructs. They can be used to target specific cell types by altering their viral coat proteins and some can be peripherally delivered and travel to the CNS via retrograde axonal transport. There are several recombinant viral vector systems that are in use today in human gene therapy clinical trials, including retroviruses (35%), adenovirus (AdV) (28.3%), adeno-associated virus (AAV) (3%) and herpes simplex virus (HSV) (1%) (Refs Reference Kay, Glorioso and Naldini113, Reference Davidson and Breakefield114, Reference Lotze and Kost115). Each of these vector systems has advantages and disadvantages with regard to stability, toxicity, immunogenicity, payload capacity, production, safety, and transduction efficiency (Table 2). Vectors that persist in the host are best suited for long-term gene transfer and gene replacement therapies. These vectors include those that integrate their genomes into the host, such as retroviruses, lentiviruses and AAVs and those that persist as episomal molecules in a nonintegrated state like AdV and HSV.

Table 2

Comparison of different viral-based vector systems with potential for gene therapy

Abbreviations: AAV, adeno-associated virus; AdV, adenovirus; HIV, human immunodeficiency virus; HSV, herpes simplex virus.

Table 3 summarises several published studies that have utilised plasmid- and viral-mediated gene therapy to treat peripheral neuropathies.

Table 3

Gene therapy studies involving peripheral neuropathy

aTime point refers to latest time point examined in the study and is not an indication of the maximal length of effectiveness.

Abbreviations: AAV, adeno-associated virus; AdV, adenovirus; BCL2, B-cell CLL/lymphoma 2; EIAV, equine infectious anaemia virus; eNOS, endothelial nitric oxide synthase; FGF2, fibroblast growth factor 2; GDNF, glial-cell-line-derived neurotrophic factor; HGF, hepatocyte growth factor; HSV, herpes simplex virus; NGF, nerve growth factor; NT-3, neurotrophin 3; PS, prostacyclin synthase; RARβ2, retinoic acid receptor β2; VEGF, vascular endothelial growth factor.

Gene therapy studies involving diabetic neuropathy

VEGF-expressing plasmids

Many reports have used plasmids as a vehicle to deliver and overexpress potential therapeutic genes in order to treat diabetic neuropathy. Muscle tissue has been the site of preference for the delivery and expression of plasmid transgenes, resulting in a constitutive systemic production of the therapeutic substance. Critical limb ischaemia with peripheral neuropathy is a frequent complication in diabetes. Intramuscular gene transfer of a plasmid encoding VEGF in a rabbit model of hind-limb ischaemia significantly improved compound muscle action potentials (CMAPs) and sensory nerve action potentials (SNAPs) and protected against ischaemia-induced myelin wasting and axonal loss, whether applied at the time of ischaemic insult or 10 days after (Ref. Reference Schratzberger116). Similar findings have been reported in diabetic rats and rabbits. Intramuscular injection of VEGF-expressing plasmids 12 or 26 weeks after the induction of diabetes in rats or rabbits normalised motor nerve conduction velocities (MNCVs), sensory nerve conduction velocities (SNCVs), and reversed thermal hypoalgesia within 4 weeks (Ref. Reference Schratzberger117). Delivery of a VEGF plasmid to muscle by electroporation was shown to improve mechanical hypoalgesia in diabetic mice (Ref. Reference Murakami118). Comparison of VEGF with netrin-1 and netrin-4, two axonal guidance factors that also have potent neovascularisation capabilities, found that intramuscular injection of plasmids encoding any of the three genes reversed declines in MNCVs and SNCVs in the db/db diabetic mouse (Ref. Reference Wilson119). Recently, two reports have demonstrated positive effects using hepatocyte growth factor (HGF), another peptide with angiogenic and neurotrophic functions similar to VEGF. Cotransfection of muscle tissue with plasmids encoding HGF and prostacyclin synthase (a vasodilator) improved MNCVs and SNCVs in the streptozotocin diabetic mouse (Ref. Reference Koike120), and liposome-mediated delivery of HGF DNA to muscle improved MNCVs, CMAPs and tactile allodynia in the streptozotocin diabetic rat (Ref. Reference Kato121).

The promising findings regarding plasmid-derived VEGF overexpression led to an open-label, dose-escalating clinical trial in human patients with chronic ischaemic neuropathy. Patients were injected with a total of 3000–9000 µg of plasmid encoding human VEGF into the muscles below the knee multiple times over a 4-week time course and were followed for 6 months. Symptom score, sensory examination score, and total examination score showed significant improvement in more than half of the treated limbs; in addition, there was improvement in CMAPs and SNAPs (Ref. Reference Simovic122). A Phase I/II clinical trial to treat diabetic neuropathy is currently under way; results of this study should be available within the next year (Ref. Reference Isner, Ropper and Hirst123). Sangamo Bioscience recently completed a Phase I clinical trial utilising a plasmid encoding a zinc-finger DNA-binding protein transcription factor designed to upregulate the VEGFA gene; improvements in pain, numbness and neurological symptoms were observed in about 50% of the patients (http://www.Sangamo.com).

Neurotrophin-expressing viral vectors

Viral-based vectors can, like plasmids, be used to achieve systemic production of transgene products. Recombinant AdV encoding NT-3 injected at multiple sites into forelimb muscles of streptozotocin- or acrylamide-treated rats partially prevented decreases in MNCVs of the sciatic nerve and SNCVs of the caudal nerve (Ref. Reference Pradat124). Systemic release of NGF can be obtained following injection of an HSV-based vector into adipose tissue, preventing the normal diabetes-related reductions in SNAPs in streptozotocin-treated mice (Ref. Reference Goss125).

An alternative to systemic production is to transfer therapeutic genes directly into affected nerves. Direct injection of an NGF-expressing HSV vector into the sciatic nerve perineural sheath protected against the development of thermal hypoalgesia in the Leprdb progressive diabetes mouse model (Ref. Reference Walwyn126). Vectors based on HSV can also be used to infect nerves by peripheral inoculation. As part of the virus's natural life cycle, it possesses the ability to spread from cells of the periphery such as epithelial or mucosal cells to the sensory nerves that innervate the site of primary infection, by retrograde axonal transport. By inoculating HSV vectors in specific dermatomes, one can target the neurons in which one wants to express the therapeutic product. Transduction of dorsal root ganglia following subcutaneous inoculation into the foot with HSV vectors expressing NGF protected against the decrease in SNAPs in streptozotocin diabetic mice (Ref. Reference Goss125); it also preserved autonomic function as measured by pilocarpine-induced sweating and prevented the development of thermal hypoalgesia, probably by reducing the diabetes-induced loss of cutaneous innervation (Ref. Reference Chattopadhyay127).

Gene therapy studies involving other neuropathies

Several toxic chemicals as well as many common medications can cause peripheral neuropathy. In the case of antineoplastic agents such as paclitaxel, cisplatin and thalidomide, the neuropathy is dose-dependent and dose-limiting; patients often cannot tolerate the doses necessary to treat the underlying cancer. Gene therapy could represent a potential prophylactic treatment allowing for greater dosing of chemotherapeutic drugs. Intramuscular injection of either an AdV vector or plasmid expressing NT-3 has been shown to improve the distal latency of caudal nerve SNAPs in cisplatin-treated mice (Ref. Reference Pradat128). Similarly, intramuscular injection of a VEGF-expressing plasmid preserves and reverses cisplatin-associated reductions in MNCVs and SNCVs in the rat (Ref. Reference Kirchmair129). Pyridoxine (vitamin B6) in high doses causes a pure large-fibre sensory neuropathy characterised by loss of proprioception. Transduction of dorsal root ganglia by subcutaneous inoculation of HSV vectors expressing NGF or NT-3 protected against pyridoxine-induced reductions in SNAPs, SNCVs and H-wave amplitudes (a pure sensory-mediated reflex arc). Furthermore, HSV-mediated neurotrophin treatment also protected against loss of nerve fibres in the sciatic nerve and improved the ability of the rats to cross a narrow beam (Refs Reference Chattopadhyay130, Reference Chattopadhyay131, Reference Chattopadhyay132). In a rat model of cisplatin-induced neuropathy, these vectors preserved SNAPs and H-wave amplitudes following a 6-week course of cisplatin treatment. Behavioural function, as analysed by the ability of rats to walk along a beam and stay on a rotarod, was unaffected in the HSV–neurotrophin-treated, cisplatin-intoxicated, rats. Nerve anatomy was also preserved, as measured by the amount of cutaneous innervation in the skin and central innervation in the dorsal horn of the spinal cord (Ref. Reference Chattopadhyay133).

A few studies have examined the use of gene therapy to treat other types of peripheral neuropathies. Charcot–Marie–Tooth type 1B disease is modelled in mice with homozygous deletions of the gene encoding P0, a myelinating Schwann cell protein. An AdV vector containing the rat P0 gene restored P0 expression in abnormal Schwann cells in these mice; however, no measurements to assess function were performed (Ref. Reference Guenard134). Hereditary spastic paraplegia, a disorder characterised by progressive loss of peripheral motor axons, is primarily a motor neuron disease, although complex forms can include peripheral neuropathy. It can be modelled in mice deficient for the inner mitochondrial membrane protease paraplegin. AAV-mediated gene transfer of the paraplegin gene via intramuscular injection halted the progression of axonopathy, reduced mitochondrial abnormalities in sciatic nerve and improved rotarod-based motor behaviour, although sensory abnormalities were not measured (Ref. Reference Pirozzi135).

Damaged peripheral nerves can regenerate and recover, but complete recovery from severely damaged spinal nerves is almost never observed. Injury to dorsal spinal nerves results in sensory deficits and incomplete regeneration of central projections; injury to ventral roots results in motor neuron cell loss and motor function. AdV vectors expressing NGF or fibroblast growth factor 2 (FGF2) directly injected into the spinal cord induced regeneration of central projections following a dorsal root nerve crush, which was correlated with the functional recovery of thermal nociception (Ref. Reference Romero136). HSV vectors expressing either the anti-apoptotic peptide BCL2 or GDNF directly injected into the ventral horn of the spinal cord protected against motor neuron cell death following ventral root avulsion. In addition, motor function, as measured by MNCVs and gait analysis, was significantly improved, especially in animals receiving both vectors (Refs Reference Yamada137, Reference Natsume138). Recently a study utilising a rabies pseudotyped lentivirus (equine infectious anaemia virus) vector expressing retinoic acid receptor β2, reported regeneration of dorsal root ganglia axons through the dorsal root entry zone following a dorsal root nerve crush, and improved sensorimotor function. In this study, the nerve crush was performed 3 weeks after the vector was directly injected into the spinal cord (Ref. Reference Wong139).

Summary and conclusions

Peripheral neuropathy is a major medical complication of a wide variety of diseases. The only medical options currently available are to either treat the underlying disease, which in many chronic illnesses is not possible, or to treat the symptoms. One promising option is the use of gene therapy to limit the extent of nerve damage. While the past several years have seen significant advances in the utilisation and refinement of gene therapy approaches to peripheral neuropathy in preclinical animal experiments, of the over 750 human gene-transfer protocols reviewed by the Recombinant DNA Advisory Committee, only the two VEGF studies cited above are intended to treat peripheral neuropathy (http://www4.od.nih.gov/oba/rac/PROTOCOL.pdf). Given the magnitude of the problem, the number of clinical trials is expected to grow substantially within the next several years.

Before gene therapy can become an accepted medical option to treat peripheral neuropathy, several issues will need to be considered (Ref. Reference Mata, Chattopadhyay and Fink140). First are issues with proper application and dosing. Some neuropathies, such as symmetric distal neuropathies, might require systemic treatment, while other types, such as mononeuropathies, might be better resolved with targeted treatment strategies. In instances of systemic treatment, enough of the gene product needs to be produced to rescue endangered nerves without producing an overabundance, resulting in adverse side effects as observed in the rhNGF clinical trial. The issue of re-dosing needs to be addressed as well. Peripheral neuropathy is generally a chronic condition, which might require constant expression of a therapeutic transgene to achieve relief. A couple of the preclinical animal studies reviewed in Table 3 demonstrated effectiveness at time points of 6 months or longer, and the report by Simovic and colleagues showed that a single inoculum of a plasmid expressing VEGF resulted in positive clinical signs 6 months later in human patients with critical limb ischaemia (Ref. Reference Simovic122). Still, these time points are short term when compared with the potential need for life-long therapy. This may require re-application of a gene therapy vector and none of the studies reviewed examined the effect of re-dosing. The initial administration of some viral-based vectors such as AdV or AAV can cause the production of neutralising antibodies, thus increasing the likelihood that re-dosing will be ineffective or even dangerous. Our group, using rodent models of acute and chronic pain, has demonstrated that analgesia can be restored following re-administration of HSV-based vectors after the initial dose has lost its effectiveness, suggesting that neutralising antibodies might not be a concern for these types of vectors (Refs Reference Goss141, Reference Hao142).

Another issue is the necessity of regulatable gene expression systems. So far, none of the preclinical animal studies has utilised a system capable of shutting off transgene expression. This might be a vital safety feature when one considers that, once applied, many gene-transfer vehicles could persist in cells for the life of the patient. Regulatable systems have been used in other gene therapy applications and will no doubt be tested in animal models of peripheral neuropathy in the near future.

A practical concern is one of cost. The costs for growing, isolating and purifying gene therapy vectors are significantly different and more expensive than traditional pharmaceuticals. A comparison of cost structures for the various viral-based systems can be interpreted from Table 2: vectors with low production capabilities and low transduction efficiency (e.g. AAV) would expect to have a higher cost per dose, whereas high production and high transduction efficiency vectors (e.g. HSV) would expect to have a lower cost per dose. Still, it may be wrong to think of gene therapy in terms of traditional drug therapy with regard to cost; the future cost to treat severe combined immunodeficiency disease with retroviral gene therapy was recently estimated to be $30 000–$50 000 per patient after commercialisation of the product – more in line with the cost of major surgery, not a pharmaceutical drug (Ref. Reference Judson143).

One area of research that needs considerably more focus is the use of gene therapy to treat inherited peripheral neuropathies; only the study by Guenard et al. (Ref. Reference Guenard134) addressed this issue. More than 30 genetic causes of inherited peripheral neuropathies have been identified, each of which could be potential targets for gene therapy (Ref. Reference Shy144). One possible reason for the lack of studies could be the paucity of accessible animal models. Whereas peripheral neuropathy secondary to diabetes or chemical toxicity can be easily modelled in any laboratory, animals carrying genetic mutations need to be created and/or bred – tasks that many laboratories are not equipped to handle. Spontaneous or genetically engineered rodent models for Charcot–Marie–Tooth type 1A, type 1B and type 1X, Dejerine–Sottas disease, and hereditary neuropathy with liability to pressure palsies do exist, allowing for additional gene therapy studies in the future (Ref. Reference Martini145).

As our understanding of the physiology and biochemistry of peripheral neuropathy continues to grow, there is no doubt that additional molecular targets and neuroprotective substances will be discovered that will be amenable to alterations by gene therapy. The next decade will be an exciting and productive one for this area of research.

Acknowledgements and funding

A hearty thank you is owed to the peer reviewers, whose thoughtful suggestions greatly enhanced this review. J.R.G. is supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK074026) and the Juvenile Diabetes Research Foundation (18-2006-1074).

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Further reading, resources and contacts

The Neuropathy Association site provides general information and support services for patients suffering from peripheral neuropathy:

Poncelet, A.N. (1998) An algorithm for the evaluation of peripheral neuropathy. Am Fam Physician 57, 755-764 This is a useful reference in diagnosing and categorising various peripheral neuropathies.Google Scholar
Shy, M.E. (2006) Therapeutic strategies for the inherited neuropathies. Neuromolecular Med 8, 255-278. This excellent review discusses potential molecular targets for treating inherited neuropathies, including the use of gene therapy.Google Scholar
Davidson, B.L and Breakefield, X.O. (2003) Viral vectors for gene delivery to the nervous system. Nat Rev Neurosci 4, 353-364.Google Scholar
http://www.neuropathy.org/site/PageServer Google Scholar
http://www.asgt.org/Google Scholar
Poncelet, A.N. (1998) An algorithm for the evaluation of peripheral neuropathy. Am Fam Physician 57, 755-764 This is a useful reference in diagnosing and categorising various peripheral neuropathies.Google Scholar
Shy, M.E. (2006) Therapeutic strategies for the inherited neuropathies. Neuromolecular Med 8, 255-278. This excellent review discusses potential molecular targets for treating inherited neuropathies, including the use of gene therapy.Google Scholar
Davidson, B.L and Breakefield, X.O. (2003) Viral vectors for gene delivery to the nervous system. Nat Rev Neurosci 4, 353-364.Google Scholar
http://www.neuropathy.org/site/PageServer Google Scholar
http://www.asgt.org/Google Scholar