The nervous system, made up largely of a population of postmitotic nondividing cells, is subject to degenerative conditions that occur with increasing frequency in old age.
Those are recognized as "diseases", as opposed to simply aging, tend to affect selective, defined groups of neurons with specific biochemical and functional characteristics to produce the disease phenotype.
Examples of such focal or restricted neurodegeneration include Parkinson's disease (PD), resulting from degeneration of dopaminergic neurons in the substantia nigra to produce the triad of bradykinesia, rigidity, and tremor, and motor neuron disease (MND), in which degeneration of "lower" motor neurons in the spinal cord and "upper" motor neurons in the brain result in weakness without involvement of sensory or higher cortical functions.
Widespread neuronal degeneration typified by Alzheimer's disease (AD), a condition in which neuronal cell death appears to be related to the accumulation of a toxic protein (amyloid) in the extracellular space in the brain, results in preferential degeneration of cholinergic neurons in the basal forebrain and structures in the hippocampus and parahippocampal gyrus, but many other cell types are also lost.
It is also possible that a nonselective loss of neurons over the decades, leading to a loss of the "safety margin" in brain function, may underlie the gradual deterioration in several aspects of brain function that occur over decades.
While substantial advances have been made in understanding the pathogenesis of these diseases, and treatments that ameliorate the symptoms to some extent have become available (more for the motor symptoms of PD than for the cognitive decline in AD), effective cures are not yet available.
Is there a prospect that gene therapy will be useful in the treatment of these and other conditions afflicting the aging nervous system?
There are several reasons why this approach is particularly applicable to disease of the brain and the nervous system.
The blood-brain barrier limits the penetration of systemically administered macromolecules into brain, and even macromolecules injected directly into the ventricles penetrate only a short distance into brain parenchyma.
In many cases, the regional specialization of brain function dictates that a therapeutic intervention may be best achieved by the local expression of a transgene product such as a neurotrophic or antiapoptotic factor.
In addition, the widespread and redundant use of a limited repertoire of neurotransmitters and receptors in diverse pathways in the nervous system means that the local production of neurotransmitters achieved by therapeutic gene transfer may be used to achieve desired outcomes while avoiding unwanted adverse side effects that would result from activation of the same receptors in other pathways by a systemically administered drug.
Techniques of Gene Transfer
Two different categories of gene transfer are commonly recognized.
In ex vivo applications, cells removed from the body are cultured in a dish, the therapeutic gene inserted into those cells, and after determining that the transgene is expressed appropriately, the cells are transplanted back into the body to achieve the desired therapeutic effect.
In vivo gene therapy refers to those approaches in which the therapeutic gene is inserted directly into the body to transduce cells in their natural location.
Gene delivery in either case is accomplished through the use of "vectors" that may be derived from viruses (viral vectors) or constructed using nonviral elements (nonviral vectors).
The simplest nonviral vectors are plasmids (sometimes referred to as "naked plasmids"), and consist simply of the gene of interest, a promoter element required to drive gene expression, and sequences that allow the propagation of the plasmid in bacteria.
Plasmid DNA can be complexed with any one of a number of lipid formulations to create a particle, "liposome", which may enter cells more efficiently than naked plasmids.
An advantage of plasmids or liposomes as gene transfer vectors is that manufacture is straightforward, and can be easily standardized to produce a pharmaceutical agent for human use.
Unfortunately, naked plasmids have proven to be effective only in transfer of genes into muscle following direct inoculation, and liposome-mediated gene transfer has not proven particularly useful for neuronal cells in vitro or in vivo.
Viral vectors exploit the natural biology of viruses to deliver DNA to the nucleus of the target cell.
Genetic modification is used to reduce or remove viral pathogenicity while leaving the targeting characteristics of the viral vector intact.
Five different viruses have been modified to create the vectors that are used in most of the gene transfer studies reported to date (Table 1).
Retroviral vectors, based principally on the Moloney murine leukemia virus, were the first viral vectors developed.
Retroviral vectors, like the parental virus, must transduce actively dividing cells in order for the cDNA transcribed from the viral RNA to be incorporated into the host cell genome.
The major nervous system application of ex vivo gene therapy involves retroviral transduction of host fibroblasts to produce nerve growth factor to treat AD, and will be considered in some detail below.
Most of the gene transfer applications directed at brain, peripheral nerve, or muscle have employed vectors that do not require host cell division.
The vectors employed include those that do not integrate into the host cell genome, such as adenovirus (Ad) and herpes simplex virus (HSV)-based vectors, vectors that do integrate into the genome of nondividing cells such as lentiviral vectors (LV), and the adeno-associated virus (AAV)-based vectors, which clearly integrate in a site-specific manner in vitro, but whose behavior in this regard in vivo has not been fully established.
Characteristics of the principal virus vectors used for gene transfer to the nervous system.
* Adenoassociated virus integrates into the host genome in a site-specific manner in vitro. The status of the viral genome after transduction in vivo has not been fully established.
||Particle Diameter (nm)
||Genome Size (kb)
Application of Gene Transfer to the Nervous System
There are 4 different types of neurologic conditions for which preclinical data has been published, suggesting that gene transfer may be used in the: (a) treatment of focal neuronal degeneration, exemplified by Parkinson disease, (b) treatment of global neurologic dysfunction, exemplified by the mucopolysaccharidoses (MPS) and other storage diseases, (c) peripheral nervous system including MND and sensory neuropathies, and (d) use of vectors expressing neurotransmitters to modulate functional neural activity in the treatment of pain (Table 2 ).
Choice of Vectors for Neurologic Applications
|Diffuse brain disease in vivo approach
||Lysosomal storage disease
||Large volume of distribution of small vector particles
|Focal brain disease
||Ad, AAV, LV, HSV
||Local expression following stereotactic inoculation
|Diffuse or focal brain disease ex vivo (cell transplant)
||RV (transformed fibroblasts)
||Dividing cells are tranduced by an integrating vector
|Peripheral nerve disease
||Targeted delivery to DRG neurons from peripheral inoculation
|Motor neuron disease
||Targeted delivery to all lower and upper motor neurons
Parkinson's disease is an attractive target for central nervous system gene therapy.
The pathology in early PD is largely limited to degeneration of dopaminergic neurons projecting from the substantia nigra pars compacta (SNc), a very small brain nucleus, to the caudate putamen.
Second, the neurochemical deficits and the functional consequences of dopaminergic cell loss on local basal ganglia circuitry are well characterized.
Third, while a number of pharmacologic options are available that have substantially improved the survival and quality of life of patients with this disease, no therapies have been developed that slow or reverse the neurodegenerative process, and many patients become refractory to treatment over time.
The biochemical phenotype of the disease is a pathogenic cascade resulting in apoptotic cell death of dopamine (DA) neurons.
It has been used to study the therapeutic possibilities of gene transfer.
Several different approaches have been employed.
Gene transfer to provide the local expression of inhibitors of apoptosis in the SNc prevents both the loss of DA neurons and the development of a PD-like phenotype following the chemical insult.
These peptide inhibitors of apoptosis must be expressed intracellularly in order to block the apoptotic cascade, so that gene transfer is uniquely suited to this approach.
However, the pathogenic trigger for PD in humans is unknown and, while the biochemical and behavioral phenotype of the animal models is faithful to the human disease, the role of apoptosis in dopaminergic cell death in naturally occurring PD is controversial.
In addition, the effects of prolonged expression of antiapoptotic factors in the brain have not been fully explored; some of these proteins are proto-oncogene products, and there might be important issues regarding their safety.
Another approach to prevent cell death in SNc employs the glial cell line-derived neurotrophic factor (GDNF), a peptide that was originally isolated by virtue of its trophic effects on dopaminergic cells in culture.
GDNF gene transfer with any one of a number of different vector systems have been successfully used to affect GDNF gene transfer in experimental models, including LV, Ad, AAV, and HSV.
Several points emerge from these studies, which differ mainly in the details of the experimental paradigms used.
First, robust GDNF expression can be seen after gene transfer into the striatum or substantia nigra, and anterograde transport of GDNF to nerve terminals after transduction of the neuronal soma seems to be a property of GDNF rather than the vector system used.
Second, GDNF appears to provide trophic support preventing degeneration of dopaminergic cells and loss of dopaminergic nerve terminals.
This protection correlates with behavioral measures of nigrostriatal integrity and neurochemical assays examining DA production.
Finally, in many circumstances, the application of GDNF is protective or restorative even after the toxic insult has taken place.
A human trial in which the GDNF peptide was administered intracerebroventricularly failed because of adverse effects from the intracerebroventricular administration, and concern about the penetration of the peptide into brain.
Direct instillation of GDNF into brain parenchyma is being studied, but gene transfer may offer an alternative approach.
Inhibition of overactive neurons of the subthalamic nucleus (STN) by stereotactic ablation or deep brain stimulation has been shown to ameliorate motor signs in late-stage PD.
A gene transfer strategy based on this approach has recently been reported.
Transduction of STN neurons with glutamic acid decarboxylase (GAD), the rate-limiting enzyme for synthesis of the inhibitory neurotransmitter gamma-amino butyric acid (GABA) using an AAV vector resulted in synthesis and activity-dependent release of GABA from STN nerve terminals.
Microelectrode studies in control animals showed that stimulation of the STN resulted in excitation of the majority of substantia nigra pars reticulata (SNr) neurons from which recordings were obtained, consistent with the known glutamatergic neurochemical phenotype of STN neurons.
Gene Therapy for Diffuse Brain Disease
Gene transfer has also been applied to the treatment of diseases that affect the central nervous system globally, such as AD.
In this case, the aim of gene transfer is a diffuse distribution of a corrective gene product throughout the nervous system.
One such approach utilizes ex vivo gene transfer to release nerve growth factor (NGF).
Animal studies have demonstrated that NGF released from fibroblasts, transduced with a retroviral vector to express NGF, and transplanted into brain can protect the cholinergic phenotype of axotomized basal forebrain neurons.
Loss of cholinergic activity in cells in this nucleus is characteristic of AD, although the disease process involves many other cell types in widespread areas of the brain.
Other studies demonstrated that grafts of NGF-expressing fibroblasts into the brains of aging primates increased cholinergic expression in the basal forebrain and resulted in improved behavioral measures of cognitive performance in the animals.
Like the use of GDNF in PD, NGF cannot be effectively administered as a peptide by intracerebroventricular injection, but it may be possible that continuous production and release of NGF from the grafts might provide a beneficial effect.
A human study is now under way to test the safety and feasibility of this ex vivo approach in patients with AD.
An alternative approach to treating AD is to attempt to prevent the accumulation of amyloid protein in the brain, for example, by producing an enzyme such as neprilysin that would degrade amyloid peptides in vivo.
The principle of using gene transfer to affect release of an enzyme diffusely through the brain has, to date, been explored principally in studies of lysosomal storage diseases such as MPS.
For example, injection of a recombinant Ad vector expressing beta-glucuronidase directly into the lateral ventricles of mutant mice increased the beta-glucuronidase activity in crude brain homogenates to 30% of heterozygote activity.
Histochemical demonstration of beta-glucuronidase activity in the brain revealed that the enzymatic activity was found principally in ependymal cells and choroids plexus.
An Ad vector expressing aspartylglucosaminidase (AGA) injected intraventricularly into the brain mice with aspartylglucosaminuria resulted in AGA expression in the ependymal cells lining the ventricles and diffusion of AGA into the neighboring neurons.
One month after administration of the wild-type Ad-AGA, a total correction of lysosomal storage in the liver and a partial correction in brain tissue surrounding the ventricles were observed.
One advantage of using gene transfer to express an enzyme is that the secreted enzyme may disseminate along the neuraxis, resulting in widespread reversal of the hallmark pathology, and suggesting that a limited number of appropriately spaced sites of gene transfer may provide overlapping spheres of enzyme diffusion to cover a large volume of brain tissue.
Diseases of Peripheral Nervous System, Polyneuropathy and Mind
Development of treatments for diseases of the peripheral nervous system involves a number of challenges that are distinct from those confronting the development of treatments for CNS disease, the most obvious one of which is the physical distribution of affected sites, but the underlying rationale for the use of gene therapy is similar to the one described above; local and/or focal production of short-lived potent peptide factors may be used to achieve therapeutic effects that cannot be achieved by the systemic delivery of those factors.
A good example is found in the approach to sensory polyneuropathy, degeneration of peripheral sensory nerve fibers, a common neurologic condition for which no current therapies exist.
Studies with recombinant peptides have demonstrated that any one of a number of neurotrophic factors, including NGF, neurotrophin-3 (NT-3), insulin-like growth factor, or vascular endothelial growth factor (VEGF) can prevent the degeneration of peripheral sensory axons in many different models of polyneuropathy.
But these potent short-lived peptides cannot be administered to patients in the same doses that are effective in the animal models because of unwanted adverse systemic effects.
Selective transduction of dorsal root ganglion (DRG) neurons to express a neurotrophic factor may be used to produce a local (autocrine or paracrine) protective effect while avoiding systemic side effects.
In this regard, HSV-based vectors are particularly well suited because of the natural tropism of the wild-type virus that affords efficient uptake into DRG neurons from peripheral inoculation of the vector.
Using transduction of DRG neurons by peripheral inoculation of an HSV vector, the protective effect against the development of neuropathy have been demonstrated.
Selective large fiber nerve degeneration caused by overdose of pyridoxine can be prevented by subcutaneous inoculation of an HSV-based vector containing the coding sequence for NT-3, measured by the amplitude and conduction velocity of the evoked sensory response, as well as preservation of H-wave amplitude.
Treated animals show preservation of a population of large myelinated fibers that otherwise degenerate in this condition, and the preservation of electrophysiologic and histologic parameters is reflected in behavioral testing of treated animals.
Inoculation of an HSV-based vector expressing NGF under the control of the human cytomegalovirus promoter prior to the start of pyridoxine intoxication provides a similar protective effect.
Injection of an replication-incompetent HSV vector expressing NGF under the control of the human cytomegalovirus promoter 2 weeks after the induction of diabetes (by injection of streptozotocin) prevents the development of neuropathy measured by reduction in evoked sensory nerve amplitude, and also increases expression of neuropeptides in the DRG.
A protective effect has also been observed by transfer of VEGF using a plasmid injected into muscle in models of ischemic and diabetic neuropathy, although one must assume that the protective effect in those models results from circulating levels of VEGF achieved by muscle transduction and thus may not avoid the potential for systemic side effects.
MND is a serious and fatal affliction without currently effective treatment.
Like polyneuropathies, administration of trophic factors appears to slow the progression of the disease in rodent models, but a human trial of ciliary neuronotrophic factor in MND had to be abandoned because of the cytokine-like side effects of the systemically administered trophic factor.
An AAV-based vector expressing GDNF has been demonstrated to protect a motor neuron-like cell line from apoptotic cell death in vitro.
After intramuscular injection of the NT-3 adenoviral vector, mice (a model of MND) showed a 50% increase in life span, reduced loss of motor axons, and improved neuromuscular function as assessed by electromyography.
This approach relies on systemic release from injected muscle, and thus may not avoid the problems of systemic administration.
Achieving adequate systemic levels from muscle transduction in larger animals may prove difficult.
To date, no vectors have been created from viruses that would naturally target motor neurons in a manner similar to the targeting of DRG neurons by HSV-based vectors, and efforts to construct vectors that would target to motor neurons have been unsuccessful to date.
Gene Transfer for Treatment of Pain
In a manner analogous to the correction of PD by using gene transfer to achieve focal neurotransmitter release (transduction with a TH vector to produce DA, transduction with a GAD-expressing vector to produce GABA), several studies have demonstrated that gene transfer may be used to provide an analgesic effect in the treatment of pain.
Why should one contemplate gene therapy for a problem such as pain?
Opiate drugs are exceptionally potent analgesic agents, but the action of these drugs on central and peripheral opioid receptors resulting in nausea, sedation, respiratory suppression, and constipation or urinary retention respectively limit the dose that may be used.
Continued use of opiate drugs in chronic pain leads to tolerance, and addiction is also a problem.
Local production and release of analgesic substances by gene transfer might offer the possibility of sustained analgesic effect while avoiding the side effects.
In the 30 years since it was first proposed, there has been enormous technical progress in the field of gene therapy.
Vectors have been constructed that are essentially apathogenic and that can target gene delivery to specific cell types.
The unvarnished optimism that characterized the field in the early 1990s has given way to a more restrained recognition that gene transfer will not be used to cure all disease but may play an important role in our therapeutic armamentarium alongside and often in combination with other therapies.
For diseases that commonly afflict the aging nervous system, it seems likely that gene transfer will be used to target the delivery of therapeutic proteins of peptides to specific regions of the nervous system to achieve therapeutic effects to treat conditions that are not principally genetic in origin.
Human trials for the treatment of AD have begun, and other trials targeting PD and intractable pain are poised to begin.
The results of these studies will serve as an important milestone in the development of this field.