Download Gene-based treatment of motor neuron diseases

INVITED REVIEW
ABSTRACT: Motor neuron diseases (MND), such as amyotrophic lateral
sclerosis (ALS) and spinal muscular atrophy (SMA), are progressive neurodegenerative diseases that share the common characteristic of upper and/or
lower motor neuron degeneration. Therapeutic strategies for MND are designed to confer neuroprotection, using trophic factors, anti-apoptotic proteins, as well as antioxidants and anti-excitotoxicity agents. Although a large
number of therapeutic clinical trials have been attempted, none has been
shown satisfactory for MND at this time. A variety of strategies have
emerged for motor neuron gene transfer. Application of these approaches
has yielded therapeutic results in cell culture and animal models, including
the SOD1 models of ALS. In this study we describe the gene-based treatment of MND in general, examining the potential viral vector candidates,
gene delivery strategies, and main therapeutic approaches currently attempted. Finally, we discuss future directions and potential strategies for
more effective motor neuron gene delivery and clinical translation.
Muscle Nerve 33: 302–323, 2006
GENE-BASED TREATMENT OF MOTOR
NEURON DISEASES
THAIS FEDERICI, PhD,1 and NICHOLAS M. BOULIS, MD1,2
1
Department of Neuroscience, Cleveland Clinic Foundation, NB2-126A, 9500 Euclid Avenue,
Cleveland, Ohio 44195, USA
2
Center for Neurological Restoration, Cleveland Clinic Foundation, Cleveland, Ohio, USA
Accepted 5 August 2005
Motor neuron diseases (MND) are progressive neurodegenerative disorders with different etiologies
and clinical variability, but a common final event:
loss of upper and/or lower motor neurons. Amyotrophic lateral sclerosis (ALS) and spinal muscular
atrophy (SMA) are the most frequent forms of MND
and therefore the most studied.100,144,184,185 Although a variety of therapeutic trials in ALS and
SMA are ongoing, available treatments remain marAvailable for Category 1 CME credit through the AANEM at www.
aanem.org.
Abbreviations: AAV, adeno-associated virus; ALS, amyotrophic lateral sclerosis; BDNF, brain-derived neurotrophic factor; BHK, baby hamster kidney;
CNS, central nervous system; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin-1; EAAT2, excitatory amino acid transporter 2; EIAV, equine infectious anemia virus; FALS, familial amyotrophic lateral sclerosis; GDNF, glial
cell line– derived neurotrophic factor; GFAP, glial fibrillary acidic protein; GFP,
green fluorescent protein; HIV-1, human immunodeficiency virus type 1; HSV,
herpes simplex virus; IAPs, inhibitors of apoptosis proteins; IGF-1, insulin-like
growth factor 1; MND, motor neuron diseases; NAIP, neuronal apoptosis
inhibitory protein; NT-3, neurotrophin-3; p75NTR, p75 neurotrophin receptor;
pmn, progressive motor neuropathy; PNA, peptide nucleic acid; RabG, rabies
G protein; RNAi, RNA interference; SALS, sporadic amyotrophic lateral sclerosis; siRNA, small interfering RNA; SMA, spinal muscular atrophy; SMN1 and
SMN2, survival motor neuron gene; SOD1, copper–zinc superoxide dismutase; trk, receptor tyrosine kinases; VEGF, vascular endothelial growth
factor; VSV-G, vesicular stomatitis virus G glycoprotein; XIAP, X-linked inhibitors of apoptosis protein
Key words: amyotrophic lateral sclerosis; gene therapy; retrograde axonal
transport; spinal muscular atrophy; viral vectors
Correspondence to: N. M. Boulis; e-mail: [email protected]
© 2005 Wiley Periodicals, Inc.
Published online 14 October 2005 in Wiley InterScience (www.interscience.
wiley.com). DOI 10.1002/mus.20439
302
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ginally effective. A detailed understanding of the
pathogenesis of MND remains elusive, and this gap
continues to impede the elaboration of specific treatment strategies. Nonetheless, based on our limited
insight into the pathogenesis of the MNDs, a variety
of approaches have emerged. Although these are not
likely to be curative, they may provide life-prolonging treatments. Gene-based treatment has several
advantages over alternative paradigms that may deliver these therapies for MND.5,29,167
AMYOTROPHIC LATERAL SCLEROSIS
ALS, also known as Lou Gehrig’s disease or adult
motor neuron disease, was first described by Charcot
in 1869 and is the most common form of MND. ALS
is characterized by a progressive degeneration of
lower and upper motor neurons in the cerebral
cortex, brainstem, and spinal cord. In turn, this neuronal loss causes weakness, spasticity, and muscular
atrophy that may evolve to paralysis. The disease
culminates in death within 2–5 years of onset, generally due to respiratory failure. To date, ALS remains untreatable and all therapeutic trials are
merely palliative.47,100,184,185
ALS is a multifactorial disease, with etiological
heterogeneity and a high variability of clinical presentation. The sporadic form (SALS) is widely pre-
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dominant, including approximately 90% of all cases,
whereas the familial variant (FALS) affects less than
10% of these patients and is usually autosomal-dominant.61,184,185
Close to 20% of familial cases have been found to
have mutations in the copper–zinc (Cu/Zn) superoxide dismutase-1 (SOD1) gene on chromosome
21q.91,163 Over 100 different mutations of the SOD1
gene have already been described and several transgenic animal models for the SOD1 gene mutation
have been created.77,161
No satisfactory treatment is available for ALS at
this time. Patient care focuses on symptomatic treatment and physical therapy. Assisted ventilation and
nutrition can transiently overcome the loss of upper
airway and respiratory muscular control. A large
number of therapeutic trials have been attempted.
Nonetheless, it was not until 1996 that the first drug
approved by the FDA for the treatment of patients
with ALS reached the market. Riluzole is an antiglutamatergic substance that blocks the presynaptic
release of glutamate.103,104 The efficacy of riluzole is
questionable, however, with minimal therapeutic
benefits.177
SPINAL MUSCULAR ATROPHY
SMA is the generic name for a heterogeneous group
of diseases characterized by the degeneration of motor neurons in the anterior horn of the spinal cord
and the brainstem (lower motor neurons), causing
progressive muscle weakness and atrophy. Like ALS,
SMA is invariably fatal. SMA remains one of the most
frequent genetic causes of death in childhood.43,144
The classification of SMA is controversial and
includes several forms, separated according to age of
onset, severity of symptoms, and evolution. Pediatric
SMA can be roughly divided into three forms: type 1,
severe, infantile, also known as Werdnig–Hoffmann
disease; type 2, the intermediate form; and type 3,
mild, juvenile, also called Kugelberg–Welander disease. Adult SMA includes distinct complex syndromes. One example is Kennedy’s syndrome, also
known as progressive spinal and bulbar muscular
atrophy.43,46,90,100
The childhood forms, although phenotypically
different, are recessive autosomal disorders. The majority are caused by homozygous deletion or mutations in the telomeric copy of the survival motor
neuron gene (SMN1) on chromosome 5q, which
codes the functional copy of the SMN protein.
Kennedy’s disease, by contrast, is an X-linked recessive disorder and its molecular basis resides in the
expansion of a trinucleotide (CAG) repeat in the
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androgen receptor gene.66,90,100,170 As with ALS, clinical trials have made no significant impact on SMA
survival.
RESEARCH
Research using animal models that reproduce the
phenotype of MND has provided substantial insight
into the human disease over the past decade.21,58
There are many different mouse models for ALS,
including those with spontaneous mutations, such as
the motor neuron degenerative (mnd) mouse, the
wasted mouse, the wobbler mouse, and the progressive motor neuronopathy (pmn) mouse; and genetically engineered mice, such as the SOD1 transgenic
mouse and the transgenic mouse that overexpresses
mutant neurofilaments. Although all these models
are complementary and very helpful, the complex
etiology of ALS remains unclear and more studies
are required to provide therapies capable of preventing or slowing the disease course.197
In the case of SMA, as SMN is a highly expressed
protein essential for survival, the development of
animal models is extremely difficult. For example,
the attempt to create a complete SMN knockout in
mice was hampered by its embryonic lethality.172 In
fact, the age at onset of disease is a challenge among
all the different mice models of MND. More recently, the creation of several mouse models, including the novel SMN knockout mouse,66,144 has facilitated improved understanding of SMA and the
development of therapeutic strategies.
Currently gene therapy is among the most promising treatments for ALS5,29,59,133 and SMA.13,55,110
This review focuses on the gene-based treatment of
motor neuron diseases in general, surveying the potential viral vector candidates, delivery strategies,
and main therapeutic approaches that have
emerged. Finally, recent progress and potential future strategies toward an effective motor neuron
gene therapy are also discussed, with emphasis on
ALS and SMA.
PATHOGENESIS
Several mechanisms have been postulated to explain
motor neuron death in ALS. However, none of these
is completely satisfactory to elucidate the entire process.58,177,184 Among the proposed mechanisms, glutamatergic oxidation and excitotoxicity have been
explored in the greatest depth. This mechanism postulates that an excess of glutamate outside of the
motor neuron, probably caused by inefficient clearance (e.g., altered metabolism or defective function
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303
of glutamate transporters), leads to an increase in
levels of free intracellular calcium, resulting in a
cascade of damaging processes by the generation of
free radicals.47,177 Conversely, the oxidative stress
that occurs due to mutations in the SOD1 gene can
result in more accumulation of glutamate in the
neural tissue and more excitotoxicity. In addition,
the oxidative lesion damages the membranes of the
neurons and, consequently, blocks cellular functions. The role of glia in this mechanism remains
controversial. Reactive oxygen and reactive nitrogen
species (ROS and RNS) may derive from activated
glia. However, the presence of oxidants can also lead
to inflammatory reactions and activate glial
cells.7,17,82,128
Neurofilament aggregation within the cell body
and proximal axon of motor neurons has also been
proposed as a possible mechanism. As with glial
activation, whether these aggregates are a cause or
effect of the underlying pathophysiological process
is unclear. These deposits can also lead to axonal
transport defects that culminate in neuronal death.
Conversely, they may result from a perturbation of
axonal transport.107,164,177,204
Whether the problem resides in neurons or astrocytes remains an open question. Some evidence
supports the idea that reactive astrocytes cause motor neuron death by a nerve growth factor (NGF)–
mediated mechanism involving nitric oxide and peroxynitrite formation.45 In fact, astrogliosis is a
common finding in ALS patients and ALS transgenic
mice. However, it remains undetermined whether
this is a primary finding or a consequent event upon
the oxidative stress in motor neurons.169 A neuroinflammatory process with the accumulation of reactive microglial cells is also observed in the degenerating areas and may contribute to motor neuron
degeneration.86,218 Finally, a significant body of literature supports the downregulation of the excitatory
amino acid transporter 2 (EAAT2), expressed
mainly in astrocytes, as a cause of motor neuron
excitotoxicity.17,40,73
Apoptosis, triggered by a mitochondrial-dependent pathway or induced by glutamatergic oxidation,
leads to motor neuron death in model systems and
plays an important role in the pathogenesis of
ALS.153 Apoptosis involves the activation of a specific
intracellular cascade that culminates in DNA fragmentation and chromatin clumping. Proteases
known as caspases are a critical precursor to the
cascade that leads to DNA fragmentation. The release of mitochondrial contents can activate this
caspase cascade. The overexpression and functionality of different caspases reported in the mutant
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SOD1, and findings of abnormal mitochondrial morphology are evidence corroborating the role of motor neuron apoptosis in ALS.75,112,123,149,164,203 In the
SOD1 mouse model of ALS, caspase-1 and caspase-3
are sequentially activated.199 Caspase-9 activation, cytoplasmic cytochrome c, and mitochondrial Bax
translocation have also been documented in the
SOD1 mouse spinal cord.75 In addition, expression of
a mutant caspase-1 protein65 as well as the intraventricular injection of caspase inhibitors,112 both capable of inhibiting MN apoptosis, improve the survival
of SOD1 mutant animals.
Finally, autoimmune disease9 and viral infection23 have also been implicated as possible mechanisms for motor neuron degeneration in ALS. In
fact, at present it remains undetermined which process is dominant and whether one of these mechanisms gives rise to the others. The cascade of events
that leads motor neurons to death is complex and
these events are all linked. For this reason, gene
therapy approaches to ALS have, for the most part,
focused on inhibiting the final common pathway of
motor neuron death, apoptosis. The therapeutic
transgenes employed to achieve this end have included intracellular inhibitors of pro-apoptotic cascades and neurotrophic factors. As will be discussed,
attempts have also been made to inhibit motor neuron excitotoxicity and downregulate the expression
of mutant genes in familial forms of ALS.
In contrast to idiopathic ALS, the mechanisms of
SMA appear to have been better elucidated. Like
ALS, a preponderance of data suggest that a programmed neuronal death (apoptosis) plays the major role in this disorder. Indeed, the specific gene
that causes SMA has been identified. Deletion or
mutations in the SMN1 gene leads to motor neuron
apoptosis, forming the pathogenesis of SMA. It was
demonstrated that SMN delays the onset of apoptosis and that the C terminal of hSMN is important for
its cell-death inhibitory function.200 However, the
reasons that motor neuron degeneration occurs selectively in the spinal cord are not yet understood.46,66,90,144,170 More importantly, the developmental time window for the critical insult to the
motor neuron is not clearly understood. Although it
is tempting to believe that SMN1 replacement in
motor neurons will prevent motor neuron degeneration, this approach is doomed to failure if the absence of SMN initiates a delayed deterioration in
utero.129 Similarly, it is not entirely clear that SMN
deficiency in motor neurons is the cause of their
death. Despite these theoretical concerns, SMA gene
therapy focuses on SMN1 replacement in motor neurons as a first-pass approach.
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Thus, whereas the molecular mechanisms underlying MND are not entirely clear, all forms of ALS
and SMA have a common outcome: motor neuron
injury and death. We believe that the most effective
treatments will derive from a better understanding
of the possible genetic, toxic, inflammatory, developmental, and infectious etiologies of this process.
However, given the tremendous human cost associated with motor neuron diseases, attempts at employing less specific strategies are not only warranted
but imperative.
GENE THERAPY
Human gene therapy using viral vectors has gained
considerable attention over the past two decades.
Gene therapy is considered one of the most promising approaches for disorders that currently lack effective treatment, including inherited and acquired
diseases. Although its original concept was focused
on the treatment of inherited genetic diseases, the
current status of gene therapy shows cancer as the
major indication in clinical trials (Journal of Gene
Medicine clinical trial site: http://www.wiley.co.uk/
genetherapy/clinical). Gene therapy presents an exciting prospect for treatment of neurodegenerative
disorders, including MND, that also urgently need
an effective treatment.
Viral Vectors. In order to achieve efficient gene delivery, viral vector genomes encode a functional gene
expression cassette. The expression cassette is the
coding region (DNA sequence that contains the
transgene), flanked by the regulatory elements necessary for its transcription (promoter and polyadenylation signal). Promoters drive and can even restrict gene expression to certain cell types, whereas
the polyadenylation (polyA) sequence is involved in
the end of the transcription process. Expression cassettes may be simple, double, or multigenic, for the
expression of one or more genes of interest, according to the required therapy and the cloning capacity
of each vector.
In theory, a viral vector candidate for the treatment of MND should be able to deliver therapeutic
genes to motor neurons, while preserving motor
function. It is also desirable for the vector to be
capable of retrograde transport and to mediate longterm gene expression.167 Retrograde axonal transport provides a direct conduit through which a virus
can move from the periphery into the central nervous system, bypassing the blood– brain barrier. Neurotropic viruses have employed this evolutionary
strategy to avoid the immune system, passing into
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the immunoprivileged compartment of the central
nervous system (CNS). This mechanism can be
equally employed to deliver therapeutic genes to
threatened motor neurons. Vectors derived from
adenovirus, herpes simplex virus (HSV), adeno-associated virus (AAV), and lentivirus are potential candidates for the treatment of neurodegenerative disorders, because they can transduce neurons in vitro
and in vivo.4,52,69,72,142
Delivery of genes, trophic factors, anti-apoptotics,
antioxidants, and other drugs has been used extensively in animal models to prevent or restore neuron
damage. We will discuss the therapeutic strategies,
delivery methods, and candidate genes that are
emerging for a gene-based treatment of MND.154
Adenoviral Vectors. Adenoviral vectors are nonenveloped, double-stranded DNA viral vectors. They
have been one of the most widely used vehicles for
gene delivery. Many studies conducted in the early
1990s characterized the neural tropism of adenoviral
vectors in vitro and in vivo.4,16,42,51,108 Although adenovirus is not known to infect the CNS, work with
adenoviral vectors has demonstrated their capacity
to undergo retrograde axonal transport.36,37,39,131,137
In a therapeutic context, several investigators described the use of adenoviral vectors encoding different neurotrophic factors, by intramuscular injection of these vectors in newborn normal rats, pmn
mutant mice, or SMA mutant mice.19,32,70,78,79,110 In
most reports, investigators used the same recombinant adenoviral vector construction, but different
neurotrophic factor genes driven by the Rous sarcoma virus (RSV) long-terminal repeat (LTR) promoter showing therapeutic effects such as neuroprotection and improved lifespan.
Vectors based on human adenovirus serotypes 2
and 5 continue to show increasing promise as gene
therapy delivery vehicles. However, the host immune
response remains a major safety issue in the context
of adenoviral-mediated human gene therapy, making clinical application of these vectors in the nervous system questionable.117,118 The immune response to adenoviral vectors also plays a critical role
in turning off CNS gene expression mediated by
these vectors.192 Thus, different investigators have
reported varying durations of gene expression by
these vectors in the CNS.
The recent development of new generations of
adenoviral vectors might help to overcome this problem. Helper-dependent adenoviral vectors have
demonstrated reduced toxicity, as well as increased
cloning capacity and prolonged transgene expression, making them viable for use in the future.181
Nonetheless, at present, this vector system has
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proven somewhat cumbersome. These difficulties
are justified if an increased cloning capacity is required by the need for larger therapeutic genes. At
present, most of the therapeutic expression cassettes
that have been developed for application to MND
remain small enough to accommodate in other vector systems. The need for a large cloning capacity for
the delivery of the dystrophin gene in neuromuscular
gene therapy of Duchenne’s muscular dystrophy has
driven the continued development of this class of
vectors.
HSV Vectors. Vectors derived from herpes simplex virus type 1 (HSV-1), a natural neurotropic, enveloped, double-stranded DNA virus, are promising for
gene therapy applications. This virus has the advantages of a large cloning capacity and the potential to
remain in a latent state within neurons. Further, because HSV-1 naturally infects nerve terminals and the
sensory neurons of the dorsal root ganglia, it has
evolved effective mechanisms for retrograde transport.
Vectors derived from HSV-1 have been generated and
demonstrated to infect neurons in vitro and in vivo,
and undergo retrograde transport.62,69,113,121,151 The
tendency to infect sensory neurons lends HSV to application in the treatment of pain and sensory neuropathies. Nonetheless, these vectors are capable of motor
neuron gene delivery.
Together, these characteristics, including the
possibility of insertion of multiple genes, are advantageous for the development of delivery systems for
the treatment of MND.148 Progress has been made to
overcome immunity and transgene expression issues, supporting the use of these vectors as therapeutic candidates for the treatment of MND.143,151 The
ability of the herpes latency promoter to drive gene
expression while the virus remains in a quiescent
state in the neuron has created hope that sustained
gene expression can be achieved in the nervous
system through HSV vectors.155,168 However, at
present, sustained expression has remained difficult
to achieve.
It is also critical to point out that application of
viral vectors to large-scale human therapy will require the production of large quantities of vector.
This so-called “scaleability” problem may undermine
therapies based on elegant vector systems that are
hard to “scale up.” Proponents of HSV vectors point
out that these vectors have been widely employed in
a variety of clinical trials and have proven relatively
easy to produce in large quantities.
AAV Vectors. Adeno-associated viral vectors (AAV)
are based on the adeno-associated virus, a singlestranded DNA, nonpathogenic, defective member of
the Parvoviridae family, genus dependovirus. These vi-
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ruses require a helper virus to infect hosts, which can
be either adenovirus, HSV, or vaccinia virus. Recombinant AAV (rAAV) vectors are capable of transducing
numerous dividing and nondividing cell types. They
promote prolonged in vivo gene expression and are,
for the most part, nonpathogenic in the wild-type,
giving them an attractive safety profile. Despite their
low immunogenicity, some immune response has been
reported against the vector capsid and the transgene.
An intensive effort is currently being made to improve
AAV vector systems in safety, stability, and regulation of
gene expression.217
To date, eight different serotypes have been described, which display different tissue tropisms and
patterns of transduction.50,189 For this reason, the vast
majority of clinical trials ongoing and planned for application to neurodegenerative diseases are based on
AAV vectors. The clinical protocol for Canavan’s disease, using AAV-aspartoacylase (ASPA), was the pioneering approved clinical use of AAV vectors in the
human brain.92 Other examples of approved protocols
include the ongoing phase I clinical trials for Parkinson’s disease, using AAV– hAADC (aromatic-l-amino
acid decarboxylase) and AAV–GAD (glutamic acid decarboxylase), as well as the use of AAV–NGF for Alzheimer’s disease (Journal of Gene Medicine clinical trial
site: http://www.wiley.co.uk/genetherapy/clinical). It
is hoped that all these clinical trials will be able to
confirm the safety of AAV vectors in the human CNS.
Serotype 2 (rAAV2) is the most studied and,
therefore the most used, in in vivo studies and clinical trials. Experiments comparing the transduction
efficiency of distinct serotypes in the neonatal and
adult CNS of rats and mice have shown distinct
patterns of gene expression, depending on the serotype, the promoter, and the injected area of brain.
After intrastriatal injection, for example, whereas
rAAV2 transduces predominantly neurons,18,127 serotypes 1, 4, and 5 show a broader tropism, also transducing astrocytes and ependymal cells.52,201
Furthermore, the ability of these vectors to be
transported retrogradely both in the CNS95 and peripheral nervous system38,39,119 makes them even
more appropriate and attractive candidates for gene
therapy for MND. Nonetheless, in our experience,
rAAV remote delivery has proven less reliable than
HSV and adenoviral vectors.
Lentiviral Vectors. Lentiviral vectors are among
the most promising viral vectors for clinical applications for neurodegenerative diseases. They combine
advantages of midrange cloning capacity with stable
gene expression and an attractive safety profile due
to a minimal inflammatory response to the vectors.
In addition, as discussed in what follows, the mem-
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brane capsule of lentiviral vectors renders alterations
in tropism relatively easy. Lentiviral vectors are based
on the single-stranded RNA (ssRNA) lentiviruses,
which are a subclass of retrovirus. They are able to
transduce both dividing and nondividing cells, including neurons, to integrate transgenes of interest
into their target cells, promoting long-term gene
expression. They can also be retrogradely transported depending on the specific glycoprotein expressed on the envelope.12,126,141,142 Currently, vector systems derived from different lentiviruses are
under development, and a series of improvements
have been made in these vectors, making them both
safer and more efficacious.
The most widely used lentiviral vectors for gene
transfer to the CNS are based on human immunodeficiency virus type 1 (HIV-1). Besides HIV-1, vectors derived from other lentiviruses, such as the nonprimate equine infectious anemia virus (EIAV) and
feline immunodeficiency virus (FIV), represent
other options of gene transfer vector systems to the
CNS.14,152
Lentiviral vectors are usually pseudotyped with
the vesicular stomatitis virus G glycoprotein (VSV-G)
envelope, but the use of alternative envelopes is of
interest and has been explored in the engineering of
new vectors. The lentipseudotyping strategy is discussed in greater detail in a later section focused on
targeted vectors.
The large-scale production of AAV and lentiviral
vectors remains to be established. The production of
these vectors requires stable packaging cell lines.
Moreover, in the case of AAV vectors, the low vector
solubility and aggregation of concentrated vector are
other challenges in production.
MOTOR NEURON GENE DELIVERY
Theoretically, gene transfer to upper and lower motor neurons can be achieved either by direct injection into the brain and spinal cord, remote delivery
using a vector that can be retrogradely transported,
or by ex vivo gene transfer (Fig. 1). These methods
of delivery overcome the challenge that the blood–
brain barrier represents to the passage of therapeutic macromolecules into the CNS. However, the feasibility of these techniques of administration is
hampered by other difficulties.
Direct Injection. All the vectors just described are
capable of neuronal gene delivery in vivo. Direct
injection can reliably deliver high titers of vector into
the targeted region. Further, because remote delivery paradigms depend on axonal transport, they are
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FIGURE 1. Schematic diagram showing methods for gene-based
motor neuron therapies. Therapeutic transgenes can be delivered to upper and lower motor neurons in the brain and spinal
cord by direct injection; remote gene transfer (using a vector that
can be retrogradely transported from muscles and peripheral
nerves), or by ex vivo gene delivery of cells engineered in vitro.
effective for motor neuron gene delivery, but not
glial gene delivery. If the core defect of MND proves
to be glial, remote delivery will be insufficient. The
inherent lack of specificity created by direct injection can be overcome through the modification of
vector tropism or the development of cell-type–specific expression cassettes through specific promoters. Furthermore, a variety of data suggest that practical remote delivery may not be possible in human
MND patients.87,90,107,139,204 First, axonal transport
may be impaired in MND, preventing adequate delivery in symptomatic patients. Second, there are
inherent disadvantages to the remote delivery, as
discussed in greater detail in the next section.
We have demonstrated effective gene transfer
after direct spinal cord injection with three different
vectors, including adenoviral NGF in normal rats,35
AAV2 vectors, and a rabies G (RabG) protein–
pseudotyped lentiviral vector in SOD1 transgenic
mice188 (Fig. 2). Nonetheless, direct injection of the
human spinal cord remains a daunting challenge
with serious potential morbidity. However, taking
into account the grave prognosis of MND patients
and the imperative for practical clinical translation
in the near future, we have begun work to develop
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307
FIGURE 2. Direct gene delivery of an advanced generation vector into the cervical spinal cord of a SOD1 transgenic mouse.
Effective motor neuron gene expression can be achieved in the
cervical spinal cord of mutant mice through intraparenchymal
injection without deleterious behavioral effects, reduced survival,
or substantial inflammation, using an EIAV vector. X-gal staining
showing the transduced area by the EIAV–LacZ vector.
the tools and techniques to make restorative spinal
cord surgery possible.
Remote Delivery. As just discussed, several types of
viral vectors are capable of migrating by retrograde
axonal transport to the cell body of projection neurons, after internalization at the axon terminal36,38
(Fig. 3). This noninvasive remote gene delivery, that
is, from sites outside the CNS (muscle or peripheral
nerves), might be applicable to the future clinical
treatments of a variety of neurodegenerative diseases. In addition, direct intraparenchymal spinal
injection of vectors capable of retrograde axonal
movement provides a strategy that is potentially capable of gene delivery to both upper and lower
motor neurons.
Despite the fact that some investigators have described abnormal axonal transport in the SOD1 mutant mouse,139 other groups have achieved successful
delivery using this method.131,157,212 Both routes of
administration, intramuscular and intraneural, have
been used extensively in animal models. Both must
overcome real challenges for human gene therapy,
due to larger muscle volume and the length of human motor neuron axons. Furthermore, the high
binding affinity of some vectors in muscle and the
limited binding affinity at synaptic terminals reduce
neuronal uptake and retrograde axonal transport,
especially of AAV vectors.50,83,124
Progress in vector design, by restricting the binding affinity and enhancing the delivery to target
cells, might optimize these techniques to make them
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more feasible for human application.
Intramuscular Delivery. The feasibility of this
method of gene delivery using viral vectors has previously been described using normal animals. For
example, Kahn and colleagues94 constructed adenoviral vectors encoding genes for various neurotrophic factors and demonstrated axonal transport in
spinal motor neurons after intramuscular injection.
Yamamura and collaborators211 also reported gene
expression in the anterior-horn motor neurons after
intramuscular inoculation with an HSV vector.
Recently, the many successful attempts using animal models of MND have encouraged the use of this
route of delivery. Among others, Wang and colleagues202 showed gene expression and neuroprotective effects in muscle and anterior-horn motor neurons after intramuscular injection of an AAV–GDNF
(glial cell line– derived neurotrophic factor) vector
in a transgenic animal model of ALS. Lesbordes et
al.110 demonstrated that intramuscular injection of
adenoviral vector– cardiotrophin-1 (CT-1) improved
therapeutic effects in a mouse model of SMA. Kaspar
and colleagues96 also achieved retrograde viral delivery and neuroprotective effects by intramuscular injection of AAV vectors encoding insulin-like growth
factor 1 (IGF-1) in a mouse model of ALS. Finally,
Mazarakis and Azzouz used pseudotyped EIAV to
achieve gene expression in the spinal cord, after
FIGURE 3. Schematic representation of peripheral viral vector
uptake. Adenoviral vectors, adeno-associated viral vectors, and
RabG-pseudotyped lentiviral vectors can be retrogradely transported to the cell body of motor neurons (MN) and deliver therapeutic transgenes. In ALS, axonal defects may limit remote
gene delivery. Enhanced uptake and retrograde transport
achieved through vector pseudotyping may be required to overcome these defects.
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FIGURE 4. Adeno-associated viral vector gene expression in the
spinal cord after remote delivery by sciatic nerve injection. Anti–
green fluorescent protein (anti-GFP) immunofluorescence reveals GFP-positive ventral horn motor neurons, after intraneural
injection of rAAV.GFP.
multiple or single intramuscular injections of lentiviral vectors, in different approaches.13,15,157 Attempts have been made in some of these experiments to demonstrate the importance of retrograde
vector delivery for therapy. However, some of the
observed therapeutic effects may result from retrograde transport of the transgene product rather than
the vector itself. Nonetheless, this potential dual
mechanism for therapy represents an advantage for
intramuscular delivery strategies.
As previously alluded to, whereas the intramuscular approach has shown efficacy with different
vector systems in mouse models, it faces a difficult
challenge in translation to humans. The nerves of
humans, particularly those that innervate the distal
extremities and diaphragm, are extremely long.
More importantly, the muscle mass of humans vastly
exceeds that of the mouse, requiring vector diffusion
across long distances to achieve uptake at the neuromuscular junction. To date, there are no data to
support the feasibility of this approach in a largeanimal model.
Intraneural Delivery. In order to avoid the problem of muscle binding and uptake, but more importantly to concentrate vector in a region with many
motor axons, we have experimented with injections
directly into the peripheral nervous system. Although less invasive than spinal cord surgery, this
approach still carries potential morbidity. Furthermore, the majority of viral uptake is likely to occur at
the synaptic terminal rather than the axon itself.
Thus, the precise mechanism whereby vectors enter
axons is unclear.
We previously demonstrated that sciatic nerve
injection resulted in significantly higher adenoviral
gene expression in spinal cord neurons as compared
Motor Neuron Diseases and Gene Therapy
with intramuscular or subcutaneous injection.36
More recently, we confirmed the role of axonal
transport in remote delivery of adenoviral and AAV
vectors, through colchicine inhibition, after intraneural injection.39 Subsequently, we also demonstrated remote delivery of AAV genes to the spinal
cord following peripheral nerve injection38 (Fig. 4).
Finally, we reported EIAV gene expression in cervical spinal cord motor neurons of SOD1 mice after
brachial plexus injection187 (Fig. 5). Together, these
distinct approaches point to the potential of this
methodology for minimally invasive spinal cord motor neuron gene therapy.
In ex vivo gene therapy, cells
are genetically modified and then transplanted into
the region of injury. The use of this strategy has been
explored for the treatment of different neurodegenerative diseases, including MND, by grafting cells
that can secrete neurotrophic factors and thus con-
Ex Vivo Gene Therapy.
FIGURE 5. Remote delivery of a RabG-pseudotyped EIAV vector
in a SOD1 transgenic mouse. (A) Photomicrograph showing
gene expression of RabG.EIAV.LacZ vector. ␤-Galactosidase
expression in cervical spinal cord motor neurons (arrows). (B)
Macroscopic view of the injection site (brachial plexus) and the
remote delivery site (cervical spinal cord).
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fer neuroprotection. This approach can also overcome the difficulties that arise from the systemic
delivery of these molecules.193
Encapsulated baby hamster kidney (BHK) cells
genetically engineered to release hCNTF were implanted in pmn mutant mice, resulting in therapeutic
effects, including an increase in mean survival of the
animals and reduction of motor neuron loss.166 A
phase I study involving six ALS patients also demonstrated the feasibility of the ex vivo gene delivery
technique. Patients were intrathecally implanted
with polymer capsules containing engineered BHK
cells. These capsules were demonstrated to release
CNTF. Consequently, measurable CNTF levels were
detected in the cerebrospinal fluid for at least 17
weeks posttransplantation.3 In another approach,
myoblasts retrovirally transduced with GDNF were
implanted into hindlimb muscles of SOD1 mutant
mice; GDNF gene delivery prevented motoneuron
loss and disease progression.138
Ex vivo gene transfer has also been applied
through spinal cord transplant. Recently, efforts
have been made to integrate the application of stem
cell derivatives and gene transfer through ex vivo
gene transfer. Klein and collaborators98 demonstrated that astrocytes derived from human neural
progenitors could be induced to secrete GDNF after
ex vivo lentiviral vector–mediated GDNF gene delivery. These cells were shown to survive for up to 11
weeks and integrate into the gray and white matter
of the spinal cord in SOD1 mutant rats. GDNFproducing astrocytes resulted in upregulation of
choline acetyltransferase staining, suggesting physiological activity of the trophic factor. However, as
with direct injection for in vivo gene transfer, techniques for direct spinal cord injection must exist to
render this approach feasible for human application.
TARGETED VECTOR
Targeted gene therapy is a strategy that consists in
creating vectors that can achieve restricted cell populations. It aims at increasing the specificity and
safety of gene therapy, avoiding vector diffusion to
unsuitable structures.22 Vectors can be genetically
engineered to ablate their natural tropism and confer new binding specificity. In the context of remote
delivery for MND gene therapy, the goal is to enhance vector uptake at the neuromuscular junction
through increased motor neuron affinity and limiting muscle affinity.
Currently, targeting strategies include modifications of the vector’s capsid or incorporation of tran-
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scriptional elements into viral genomes to restrict
the expression of therapeutic genes to certain target
cells. The use of cell-specific promoters,22,102 as well
as enhancers of genes for motor neuron–specific
expression10,196 are improvements that might also
optimize the targeted gene expression strategy.
Because of their lipid membrane capsule, lentiviral vectors can be easily targeted through the insertion of envelope glycoproteins. These vectors are usually pseudotyped with the
vesicular stomatitis virus G glycoprotein (VSV-G).
VSV-G–pseudotyped lentivectors do not undergo reliable retrograde transport.96 However, the use of
alternative envelopes may allow the targeting of specific cell types. For example, when injected into the
muscle, RabG-pseudotyped lentiviral vectors are retrogradely transported and transduce motor neurons
of lumbar spinal cord, in contrast to VSV-G vectors
that transduce exclusively muscle at the site of injection.126 Another work from the same investigators
described additional lentipseudotyped vectors with
distinct transduction patterns and retrograde transport ability, when injected into the brain or spinal
cord.207
Recently, we reported the neurotropism of novel
HIV-1– based lentiviral vectors pseudotyped with glycoproteins from different members of the Rhabdoviridae family, Lyssavirus genus (rabies virus, European bat Lyssavirus, Lagos bat Lyssavirus, and
Duvenhage). We demonstrated their neuronal gene
transfer in vitro191 and their retrograde transport in
vivo, after sciatic nerve injection (Fig. 6).
Lentipseudotyping.
Because their genomes are almost identical, the serotype-specific differences of
transduction observed with AAV vectors are due to
differences in their capsids and receptors required
for attachment and internalization of these vectors.165 The use of capsids of other serotypes rather
than AAV2 is an alternative that may provide advantages to this vector system.41,156,174
Burger and collaborators41 designed the hybrid
serotypes rAAV2/1, rAAV2/2, and rAAV2/5, and
reported an almost exclusive neurotropism for all
of them after injection into distinct regions of the
CNS, with differences just in distribution. More
recently, Shevtsova and colleagues174 described
the better performance and increased spread of
serotype 5 capsid in the brain compared to the
serotype 2 capsid.
AAV Pseudotyping.
Neurotropic Peptides. Targeted gene delivery can
also be achieved using peptides with selective affinity
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FIGURE 6. LacZ gene expression following intraneural injection of a Lagos-pseudotyped HIV-based lentiviral vector. Dorsal root ganglia
(DRGs) ␤-galactosidase expression revealed by X-gal staining, after remote delivery into the sciatic nerve of a normal adult rat.
for motor neurons, such as the nontoxic tetanus
toxin C-fragment that avidly binds to peripheral neurons.63 Some studies have demonstrated the feasibility and the advantages of this strategy. Nonviral
neuronal gene delivery mediated by the nontoxic
tetanus toxin C-fragment has been demonstrated.99
Neuronal targeting of cardiotrophin-1 has been
achieved through C-fragment coupling.33 Finally, an
SMN:tetanus toxin C-fragment fusion protein was
developed for the targeted delivery of the SMN protein to motor neurons.64 The selective retargeting to
neurons using targeted adenoviral vectors with the
tetanus toxin C-fragment was also achieved after intramuscular injection.171
In addition to chemically modifying the coats of
viral vectors to create novel motor neuron targeting
strategies, protein capsids can be altered through
alterations in the genes encoding the capsid proteins. Alteration in AAV tropism has been achieved
by inserting oligonucleotides into the cap gene of
AAV2 vectors. This modification can be done by
peptide insertion into the VP2208 and VP3 protein.175
We have recently employed phage display to identify
short peptides with selective motor neuron binding
properties60,116 and inserted it into the VP3 capsid
protein of AAV2.53
THERAPEUTIC TRANSGENES
Because the molecular mechanisms of motor neuron diseases remain incompletely understood, the
goals of gene therapy strategies have been focused
on neuroprotection through trophic factors, antiapoptotic proteins, and antioxidants and anti-excitotoxicity genes.5,59 The better understood molecular
Motor Neuron Diseases and Gene Therapy
genetics of SMA have motivated SMN1 gene replacement strategies that have shown promise.13 Similarly,
an understanding of the molecular genetics of the
SOD1 mutant variant of familial ALS has motivated a
selective vector-mediated gene knockdown.134,157,158
Neurotrophins and other trophic
factors are molecules that promote several effects in
neurons such as differentiation, maintenance of
function, synaptic plasticity, survival, and regeneration. Their receptors are the high-affinity members
of the family of receptor tyrosine kinases (trks; trk A,
B, and C) and a low-affinity receptor, the p75 neurotrophin receptor (p75NTR). These receptors are
expressed almost exclusively in the nervous system.
Some neurotrophic factors are able to bind to multiple distinct receptors.54,71,193
Several studies have demonstrated alterations in
neurotrophins and their receptors in ALS57,140 that
may be relevant in the development of therapeutic
strategies. However, the up- or downregulation of
neurotrophic factors does not appear to be a major
cause, but rather a consequence of ALS, contributing to neuronal death. Nonetheless, therapy with
neurotrophins has been extensively explored in
MND models due to their neuroprotective effects.
The use of viral vectors encoding neurotrophic
factors to protect motor neurons from acute death
has been applied exhaustively in a variety of models.
However, different studies using viral vectors to deliver these neurotrophins in vivo have identified limitations in their effectiveness, such as short half-life
of the neurotrophic factors, poor access due the
presence of the blood– brain barrier, insufficient or
Trophic Factors.
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nonspecific delivery, and adverse effects.20,31,193 Even
so, a number of neurotrophic factors have been
demonstrated to promote therapeutic benefits,
prompting the development of clinical trials.
Brain-Derived Neurotrophic Factor. Brain-derived
neurotrophic factor (BDNF), among other neuroprotective properties, can prevent glutamateinduced neurotoxicity, an important mechanism involved in the pathogenesis of ALS.114,176 A phase III
study involving subcutaneous delivery of BDNF
failed to demonstrate clinical benefit in patients with
ALS.8 Likewise, intrathecal administration of BDNF
also failed to show treatment efficacy in ALS patients, as reported in a phase I/II trial.145 Defects in
delivery have been proposed to underlie the gulf
between activity in models and the clinical setting.
Viral gene transfer has been applied in an attempt to
enhance delivery. Pretreatment with adenoviral vector–BDNF prevented the death of axotomized facial
motoneurons in newborn rats.70 BDNF has not been
applied using more practical, advanced generation
vector systems.
Cardiotrophin-1. Cardiotrophin-1 (CT-1) is a
muscle-derived cytokine that has been shown to prevent or delay motor axonal degeneration in different
mouse models of MND, including pmn,32 wobbler,136
SOD1,34 and SMA mutant mice (carrying a deletion
of Smn exon 7).110 Most of these studies explored
the use of adenoviral vectors encoding the CT-1
gene, after intramuscular injection, and demonstrated neuroprotection. Indeed, in the SMA approach, the investigators also pointed out the neuroprotective role of CT-1 during the postnatal
period, in addition to its previously reported role
during the embryonic period.147 As discussed earlier,
because neurotrophic factors can themselves be
taken up and delivered through retrograde axonal
transport, intramuscular vector injection may result
in gene- or gene product–mediated effects on motor
neurons.
Ciliary Neurotrophic Factor. Ciliary neurotrophic
factor (CNTF), a member of the same family of
CT-1, promotes motor neuron survival in vitro and
in vivo like other neurotrophic factors.97,120,166 CNTF
has also been shown to effectively reduce motor
neuron degeneration in animal models such as pmn
mice.173
The systemic delivery of recombinant human
CNTF has failed to demonstrate efficacy. A placebocontrolled trial in 570 patients with ALS showed no
beneficial effects, but rather side effects at all tested
doses.132 However, a phase I study using an ex vivo
gene delivery approach demonstrated measurable
levels of CNTF without deleterious side effects. In
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this study, encapsulated BHK cells were modified to
secrete hCNTF and implanted into the lumbar intrathecal space of six ALS patients.3
Vector systems to deliver CNTF, such as adenoviral and lentiviral vectors, have been shown to be
successful in achieving neuroprotection. In the adenoviral study, the investigators compared different
routes of administration of adenoviral vector–CNTF
in pmn mutant mice. While intramuscular and intravenous injections resulted in transduction of skeletal
muscle fibers and hepatocytes, increased lifespan,
and reduction in motor axonal degeneration, the
intracerebroventricular injection resulted in transgene expression restricted to ependymal cells and
no neuroprotective effects.80 In the other study, tetracycline-regulated lentiviral vectors were used to
investigate the dose-dependent neuroprotective effect of CNTF. Using a quinolinic acid (QA) rat
model of Huntington’s disease, the researchers
showed that, in QA-lesioned rats, gene-controlled
delivery of CNTF was associated with reduction of
the motor deficit.160
Recently, conflicting data have emerged from
studies on endogenous CNTF in clinical MND. Some
postmortem investigations and CNTF measurements
in the cervical enlargement of the spinal cord have
attributed motor neuron loss to decreased CNTF
expression in the spinal cord of ALS patients.6,109,146
By contrast, it has been demonstrated that CNTF
serum levels are increased in patients with ALS.89
Moreover, gene expression of CNTF is upregulated
in degenerating spinal motor neurons from autopsied patients with SALS.93 Therefore, although
CNTF might have potential therapeutic utility in
ALS and other neurodegenerative diseases, its role
in the pathogenesis of ALS remains unclear.
Glial Cell Line–Derived Neurotrophic Factor. Glial
cell line– derived neurotrophic factor (GDNF) was
discovered over a decade ago and is currently considered to be one of the most potent neuroprotective molecules.26,28,85,219 Consequently, viral vector–
mediated delivery of GDNF has been applied to the
CNS in a variety of animal models of neurodegenerative diseases, including ALS.
Many different groups have reported the efficacy of
GDNF gene delivery using the SOD1(G93A) transgenic
mouse model of ALS. Intramuscular injections of adenoviral vector–GDNF1,122 and AAV–GDNF96,119,202 at
different ages before the disease onset, conferred neuroprotective effects, delayed disease onset, and prolonged survival of animals (Fig. 7). Also, as reported
previously, the ex vivo gene delivery of GDNF using
myoblasts retrovirally transduced and implanted into
hindlimb muscles, prevented motor neuron degener-
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FIGURE 7. Survival analysis of G93A SOD1 transgenic mice after different treatments. Kaplan–Meier graph demonstrates increase in
lifespan of SOD1 mice, after intramuscular injection of Ad–GDNF (red), AAV–IGF-1 (green), EIAV–VEGF (blue), or EIAV-mediating
expression of RNAi targeted to the human SOD1 gene (orange). Untreated SOD1 animals survive an average of 135 days (black). 1:
Acsadi et al.1; 2: Kaspar et al.96; 3: Azzouz et al.15; 4: Ralph et al.157
ation and delayed the progression of disease in ALS
mice.138 However, although direct injection of lentiviral GDNF into the spinal cord promoted long-term
expression in motor neurons, it did not prevent motor
neuron loss and muscle denervation in transgenic
mice.76
Zhao and collaborators219 used the glial fibrillatory acidic protein (GFAP)–specific promoter to express GDNF in astrocytes of transgenic mice, demonstrating its effects on motor neuron survival. They
achieved restricted gene expression predominantly
in astrocytes throughout the brain and spinal cord.
The overexpression of GDNF prevented programmed cell death during development and protected facial motoneurons following axotomy.
For a detailed description of GDNF experimental
models the reader is referred to recent articles by
Bohn and colleagues.27,28
Insulin-Like Growth Factor 1. The neuroprotective role of insulin-like growth factor 1 (IGF-1) was
reported over a decade ago.111 IGF-1 has the distinct
advantage of direct trophic effects on both muscle
and motor neurons. Because diffuse muscle atrophy
Motor Neuron Diseases and Gene Therapy
affects MND patients, this dual effect has generated
significant enthusiasm for the application of IGF-1.
However, to date, studies addressing the efficacy of
this trophic factor in the treatment of MND have had
conflicting results.24,133,135
Recent placebo-controlled studies showed recombinant human IGF-1 to be a modestly effective
drug treatment for ALS. Although one investigation,
a European trial, found no satisfactory clinical effects,30 a North American trial showed slowing of
disease progression in patients treated with recombinant IGF-1.105 Other clinical trials are currently in
progress.
Kaspar and colleagues96 reported efficient remote AAV-mediated IGF-1 gene delivery followed
intramuscular injections, promoting prolonged survival and delaying disease progression in SOD1 mutant animals (Fig. 7). In an in vitro model of ALS,
experiments suggested that motor neurons expressing the IGF-1 gene may be capable of protecting
bystander neurons through secretion of the factor.198 We have also constructed RabG.EIAV vectors
for the delivery of the IGF-1 gene. This vector has
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proven to induce motor neuron axon elongation
and neuroprotection in vitro.190 In addition, this
vector sporadically prolonged survival in the SOD1/
G93A model after intramuscular remote injections at
relatively low titers.
Nerve Growth Factor. Nerve growth factor
(NGF) was one of the first members of the neurotrophic factor family to be described.54,193 Although
NGF levels were decreased postmortem in cerebral
motor cortex and the dorsal spinal cord of ALS
patients, levels in the lateral column of spinal cord
were increased.6 This paradoxical observation has
been attributed to deleterious reactive astrocyte production.150
In early experiments, we demonstrated the feasibility of adenoviral-mediated NGF gene delivery and
expression in the spinal cord for application to spinal cord injury.35 Despite NGF’s neuroprotective activity, it has not been successfully applied for ALS
treatment. In fact, the suggestion that motor neuron
death is exacerbated by reactive astrocyte NGF secretion has prompted the proposal that NGF gene silencing, rather than delivery, should be applied in
ALS.194
Neurotrophin-3. Like NGF and BDNF, neurotrophin-3 (NT-3) is a member of the neurotrophin
family. The intramuscular administration of an adenoviral vector coding for NT-3 in pmn mutant mice
resulted in beneficial effects, including increased
survival, reduction of motor axon loss, improvement
in neuromuscular function, and efficient reinnervation of muscle fibers.79 However, since these initial
experiments, NT-3 has not been explored extensively for motor neuron protection. It is important to
consider that NT-3 utilizes the trk C receptor, which
is not abundantly expressed in either normal or ALS
spinal cord motor neurons.57
Vascular Endothelial Growth Factor. As implied
by its name, vascular endothelial growth factor
(VEGF) was originally described in the context of
angiogenesis. However, recent observations have
demonstrated that this trophic factor has potent direct neuroprotective properties.15,48,74,106,182,183
Because of its mixed vascular and neurotrophic
effects, VEGF can exercise direct and indirect protective effects. VEGF can act directly on motor neurons as a neurotrophic factor and also regulate
blood supply to these cells.182 Some studies revealed
that reduced levels of VEGF can predispose mice
and humans to ALS, suggesting a role for VEGF in
the pathogenesis of ALS.48 This hypothesis was corroborated by the observation that VEGF␦/␦– knock-in
mice developed degeneration of adult motor neurons and symptoms similar to those of ALS.106
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Two different study approaches pointed out its
neuroprotective effect and its application in ALS
treatment. The first study demonstrated that direct
intracerebroventricular delivery of recombinant
VEGF was able to delay disease onset, improve motor
performance, and prolong survival in two rat models
of ALS. Moreover, the investigators showed that
rVEGF was anterogradely transported in peripheral
axons, protecting cervical motoneurons and preserving neuromuscular junctions.183 More recently, Azzouz and colleagues15 pointed out its neuroprotective effect and its application in ALS treatment.
Intramuscular injection of RabG-pseudotyped EIAV–
VEGF resulted in retrograde transport and motor
neuron delivery of VEGF, promoting prolonged survival in an ALS mouse model (Fig. 7).
Summary. Although therapeutic application of
protein trophic factors has yielded disappointing results in clinical trials, gene delivery may overcome
the barrier of sustained and targeted delivery. The
combination of two or more factors may also improve the chances of success with this strategy.24,78,97
Apoptosis plays an important role in neuronal cell death and is considered to
be one of the main possible causes of MND.153,170,200
The apoptotic mechanism is driven by a family of
proteases (caspases) and controlled by a family of
proteins (Bcl-2), including Bcl-xL, which regulates
the cell death repressor activity in the apoptotic
pathway. The downregulation of Bcl-2 has been demonstrated in ALS123 and SMA.180 Because anti-apoptotic proteins function in the intracellular space,
gene transfer provides the advantage of circumventing the need for uptake across the neuronal membrane.
Bcl-2 and Bcl-xL. The Bcl-2 family includes both
anti-apoptotic and pro-apoptotic members. Bcl-2
family proteins regulate apoptosis by the mitochondrial pathway. Bcl-xL is a member of the Bcl-2 family
of proteins and is the dominant inhibitor of apoptosis in cell death repressor activity.2,159 The anti-apoptotic activity of both molecules, Bcl-2 and Bcl-xL, has
been achieved in vitro and in vivo, making their
therapeutic use an attractive tool to confer neuroprotection.
Adenoviral vector–Bcl-xL125 and AAV–Bcl-xL67
gene delivery have been shown to prevent apoptosis
in vitro. These results were obtained in normal primary rat neuronal cultures,125 as well as in cultured
motor neurons (Fig. 8) and in a glutamate-induced
apoptosis neuroblastoma cell model.67
Adenoviral vector–Bcl-2 and AAV–Bcl-2 have also
been applied to achieve neuroprotective effects in
Anti-Apoptotic Proteins.
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FIGURE 8. Cultured motor neurons transduced by an AAV–Bcl-xL.GFP vector. (A) E15 primary rat motor neurons in vitro (Phase). (B)
GFP expression, after transduction with an AAV–Bcl-xL.GFP vector.
animal models of ALS. Yamashita and co-workers212
used intramuscular injection of adenoviral vector–
Bcl-2 into the tongue of mutant SOD1 transgenic
mice to induce Bcl-2 expression in motor neurons of
the hypoglossal nuclei by retrograde transport of the
vector. They reported that Bcl-2 expression was able
to promote motor neuron survival213 by regulating
caspase-1 activation and retarding the release of the
pro-apoptotic protein cytochrome c into the cytosol.214 Using the same SOD1 transgenic mice, Azzouz and collaborators11 also demonstrated Bcl-2 expression in motor neurons, as well as increased
motor neuron survival, after intraspinal injection of
an rAAV–Bcl-2 vector. Furthermore, the investigators reported a delay in the onset of disease, without
increase in lifespan.
X-Linked Inhibitor of Apoptosis Protein/Neuronal Apoptosis Inhibitory Protein. The inhibitors of apoptosis proteins (IAPs) regulate both the upstream “initiator” caspase (caspase-9) as well as downstream
“effector” caspases (caspase-3 and -7). Seven members of the IAP family have been identified. They are
the neuronal apoptosis inhibitory protein (NAIP),
X-linked inhibitor of apoptosis protein (XIAP),
c-IAP1 (HIAP-1), c-IAP2 (HIAP-2), survivin, livin,
and Ts-IAP.115 Transfer of the NAIP and XIAP genes
has proven to have a therapeutic effect in a variety of
neurodegenerative disease animal models. In the
four-vessel occlusion model, adenoviral vector–
mediated delivery of IAPs has been shown to prevent
CA-1 neuronal death209 and associated behavioral
deficit.210 Vector-mediated XIAP delivery was able
to protect axotomy-induced apoptosis in retinal ganglion.101 It was also reported that NAIP gene delivery
reduced 6-hydroxydopamine–induced apoptosis in
the rat substantia nigra.49
Motor Neuron Diseases and Gene Therapy
IAPs are considered stronger and, thus, are potential candidates for anti-apoptotic treatment strategies, as we showed in an in vitro model of ALS.
Using a glutamate-induced model of excitotoxicity,
we demonstrated that the reduction in cell death was
associated with the neuroprotective effects of the
adenoviral vector–XIAP.GFP vector present in the
primary cultured motor neurons.68
Excitatory Amino Acid Transporter 2. Excitotoxicity and glutamate-induced apoptosis has been considered a major mechanism in the
pathogenesis of ALS.154,177,185 Increased concentrations of glutamate in the cerebrospinal fluid of ALS
patients, decreased levels in the glial glutamate transporter EAAT2 in the spinal cord and motor cortex of
ALS patients, as well as reports of loss of function of
EAAT2 in transgenic SOD1 models, provide substantial
support for the involvement of this mechanism.84,88
Engineered cells overexpressing EAAT2 have
been used to deliver EAAT2 gene in vitro and thus
confer neuroprotection of motor neurons against
glutamate toxicity.206 More recently, AAV and lentiviral vector systems have been developed to deliver
EAAT2 gene in vitro and in vivo.81,205 Both studies
showed that EAAT2 can be delivered to motor neurons, but have not yet demonstrated a neuroprotective effect in ALS mouse models.
In fact, because EAAT2 is naturally expressed in
astrocytes, targeted delivery is an approach that may
be considered in this case. A gene transfer protocol
utilizing rAAV vector to drive expression of EAAT2
by the GFAP-specific promoter has been proposed.
The GFAP promoter is particularly active in the reactive astrocytes present in the spinal cord of ALS
patients (Paola Leone, personal communication).
Anti-Excitotoxicity.
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OTHER STRATEGIES
Gene Silencing.
RNA Interference. Traditionally,
gene transfer has provided a means for correction of
genetic mutations and the restoration of genes or
proteins that they encode. However, RNA interference (RNAi) is emerging as an exciting therapeutic
approach for the treatment of several genetic disorders, selectively silencing the expression of mutant,
but not wild-type, genes.44 RNAi is an evolutionary
conserved process by which double-stranded RNA
degrades messenger RNA where there is homology,
silencing posttranscriptional gene expression.44,216
The potential of RNAi as a therapy for ALS was
demonstrated by selective inhibition of the mutant
SOD1 form, using small interfering RNA (siRNA)
sequences with this specificity.56,215
Improvements on the “vectorization” of siRNA
and the targeted delivery of viral vectors will make
this technology more practical for application to
neurodegenerative diseases. Two very recent approaches using lentiviral vectors, which can integrate
the host genome and thus provide long-term gene
expression, showed success in silencing the SOD1
gene in SOD1 models of ALS.157,158 Furthermore, the
investigators demonstrated a significant increase in
the lifespan of SOD1 mutant animals157 (Fig. 7). A
third study also showed the benefit of siRNA therapy
for ALS, after retrograde transport of an AAV vector
to the spinal cord following intramuscular injection.134
A distinct alternative for silencing gene expression is the antisense peptide nucleic acid (PNA).
Systemic intraperitoneal administration of this antisense PNA to SOD1 mutant mice conferred significant reduction in p75 neurotrophin receptor gene
expression.17,194 Because this approach would be expected to damp neurotrophic activity, it further supports the idea that NGF may contribute to motor
neuron degeneration.
Gene Replacement. The direct replacement of defective genes is ideal to treat SMA by increasing the
expression of functional SMN protein from the
SMN2 gene. This approach has been attempted recently using a lentiviral vector expressing human
SMN. The ability of this system to restore SMN protein levels in both SMA type 1 fibroblasts and in SMA
mice, has been demonstrated after multiple intramuscular injections of a RabG–EIAV vector.13
Stem Cell Therapy. Ex vivo gene therapy, employing stem cell derivatives genetically engineered to enhance neurotrophic factors, has already been discussed. In addition, several reports
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have suggested that stem cell therapy, in which
degenerated neurons are replaced in order to promote recovery in the areas of motor neurons loss,
is also a good strategy for the treatment of neurodegenerative diseases, such as Parkinson’s and
Huntington’s diseases and, more recently, ALS
and SMA. A recent study has demonstrated the
functionality of motor neurons derived from embryonic stem cells, supporting the idea of cell
replacement.130 However, many challenges limit
the success of the stem cell therapy. Besides clinical safety and ethical issues concerning the use of
embryonic stem cells, the ability of these cells to
migrate, survive, and become functional in the
environment where they will be grafted has to be
considered.178,179 In addition, stem cell– derived
neurons must integrate into a functioning network
forming afferent and efferent synapses. In order to
reproduce the neuromuscular junction, the axons
of these transplanted cells must cross the white
matter of the ventral spinal cord. This white matter contains molecules that suppress axonal
growth. Further, these axons can be misdirected
into the white matter tracks. Thus, strategies are
required that can guide these axons into the ventral horn. Viral gene transfer has been used to
guide axons in the corpus callosum and spinal
cord.162 An alternative approach is grafting of
adult stem cells, in order to help “sick” neurons to
recover.186
TOWARD THE CLINIC
Neurodegenerative disorders are progressive debilitating diseases that usually have a dire outcome, due
to the loss of neural tissue. There remain no effective
treatments for this group of disorders. Gene therapy
employing neuroprotective approaches is an exciting strategy that may prevent neuronal death. Ex
vivo growth factor gene therapies involving CNTF
gene delivery for Huntington’s disease,25 as well as
NGF for Alzheimer’s disease,195 are examples of
growth factor phase I clinical trials that have been
recently performed for neurodegenerative disorders.
The present review has emphasized different
therapeutic strategies focusing on neuroprotection
for MND. A preponderance of evidence supports the
feasibility of this approach in animal models, especially in SOD1 models of ALS. However, these models
reproduce only the FALS, which affects 10% or less
of ALS patients and, among these, only about 20%
have the mutation in the SOD1 gene. Moreover,
none of these strategies has been tested in humans.
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Practical barriers to gene-based treatment in humans include the duration of gene expression, the
distribution of gene expression (targeting of gene
delivery and broad distribution), and the safety of
the system (random insertion into human genome,
insertion into germ cell lines and inflammatory injury to tissue). The appropriate routes for delivery
and control of gene expression remain as significant
obstacles. We have begun delivery experiments in
large-animal models to investigate the limits of direct
and remote injections. The availability of sufficient
resources to “scale-up” vectors for human application on a large scale will similarly limit application to
MND.
FUTURE DIRECTIONS
The development of practical gene-based therapy
for MND requires a better understanding of the
pathophysiology of these diseases. For example, the
reasons for this selective motor neuron vulnerability
may hold important keys to novel neuroprotective
strategies. Targeting the early steps of these diseases
may be the key to preventing motor neuron damage,
instead of correcting damage that has already occurred. Toward this end, better diagnostic aids may
be required. Microarray technology may provide direct information on the genes leading to neurodegeneration.93 Unlocking the specific mechanisms of
MND may provide novel therapeutic target genes.
Finally, the co-delivery of therapeutic agents may
provide synergistic or additive effects.24,59,78,97 Regardless of these improvements, variations of the
strategies discussed are likely to be subjected to clinical trials over the next decade.
The authors thank the Amyotrophic Lateral Sclerosis Association
(ALSA) and the National Institute of Neurological Diseases and
Stroke (KO8 NS43305) for support.
REFERENCES
1. Acsadi G, Anguelov RA, Yang H, Toth G, Thomas R, Jani A,
et al. Increased survival and function of SOD1 mice after
glial cell– derived neurotrophic factor gene therapy. Hum
Gene Ther 2002;13:1047–1059.
2. Adams J, Cory S. The Bcl-2 protein family: arbiters of cell
survival. Science 1998;281:1322–1326.
3. Aebischer P, Schluep M, Deglon N, Joseph JM, Hirt L, Heyd
B, et al. Intrathecal delivery of CNTF using encapsulated
genetically modified xenogeneic cells in amyotrophic lateral
sclerosis patients. Nat Med 1996;2:696 – 699.
4. Akli S, Caillaud C, Vigne E, Stratford-Perricaudet LD, Poenaru L, Perricaudet M, et al. Transfer of a foreign gene into
the brain using adenovirus vectors. Nat Genet 1993;3:224 –
228.
5. Alisky JM, Davidson BL. Gene therapy for amyotrophic lateral sclerosis and other motor neuron diseases. Hum Gene
Ther 2000;11:2315–2329.
Motor Neuron Diseases and Gene Therapy
6. Anand P, Parrett A, Martin J, Zeman S, Foley P, Swash M, et
al. Regional changes of ciliary neurotrophic factor and nerve
growth factor levels in post mortem spinal cord and cerebral
cortex from patients with motor disease. Nat Med 1995;1:
168 –172.
7. Anneser JM, Chahli C, Ince PG, Borasio GD, Shaw PJ. Glial
proliferation and metabotropic glutamate receptor expression in amyotrophic lateral sclerosis. J Neuropathol Exp
Neurol 2004;63:831– 840.
8. Anonymous. A controlled trial of recombinant methionyl
human BDNF in ALS: the BDNF Study Group (Phase III).
Neurology 1999;52:1427–1433.
9. Appel SH, Smith RG, Engelhardt JI, Stefani E. Evidence for
autoimmunity in amyotrophic lateral sclerosis. J Neurol Sci
1993;118:169 –174.
10. Arber S, Han B, Mendelsohn M, Smith M, Jessell TM, Sockanathan S. Requirement for the homeobox gene Hb9 in
the consolidation of motor neuron identity. Neuron 1999;
23:659 – 674.
11. Azzouz M, Hottinger A, Paterna JC, Zurn AD, Aebischer P,
Bueler H. Increased motoneuron survival and improved
neuromuscular function in transgenic ALS mice after intraspinal injection of an adeno-associated virus encoding
Bcl-2. Hum Mol Genet 2000; 9:803– 811.
12. Azzouz M, Kingsman SM, Mazarakis ND. Lentiviral vectors
for treating and modeling human CNS disorders. J Gene
Med 2004;6:951–962.
13. Azzouz M, Le T, Ralph GS, Walmsley L, Monani UR, Lee DC,
et al. Lentivector-mediated SMN replacement in a mouse
model of spinal muscular atrophy. J Clin Invest 2004;114:
1726 –1731.
14. Azzouz M, Mazarakis N. Non-primate EIAV-based lentiviral
vectors as gene delivery system for motor neuron diseases.
Curr Gene Ther 2004;4:277–286.
15. Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous KA, Kingsman SM, et al. VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse
ALS model. Nature 2004;429:413– 417.
16. Bajocchi G, Feldman SH, Crystal RG, Mastrangeli A. Direct
in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors. Nat
Genet 1993;3:229 –234.
17. Barbeito LH, Pehar M, Cassina P, Vargas MR, Peluffo H,
Viera L, et al. A role for astrocytes in motor neuron loss in
amyotrophic lateral sclerosis. Brain Res Brain Res Rev 2004;
47:263–274.
18. Bartlett JS, Samulski RJ, McCown TJ. Selective and rapid
uptake of adeno-associated virus type 2 in brain. Hum Gene
Ther 1998;9:1181–1186.
19. Baumgartner BJ, Shine HD. Targeted transduction of CNS
neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduced population. J Neurosci 1997;17:6504 – 6511.
20. Begley DJ. Delivery of therapeutic agents to the central
nervous system: the problems and the possibilities. Pharmacol Ther 2004;104:29 – 45.
21. Bendotti C, Carri MT. Lessons from models of SOD1-linked
familial ALS. Trends Mol Med 2004;10:393– 400.
22. Benitez JA, Segovia J. Gene therapy targeting in the central
nervous system. Curr Gene Ther 2003;3:127–145.
23. Berger MM, Kopp N, Vital C, Redl B, Aymard M, Lina B.
Detection and cellular localization of enterovirus RNA sequences in spinal cord of patients with ALS. Neurology
2000;54:20 –25.
24. Bilak MM, Corse AM, Kuncl RW. Additivity and potentiation
of IGF-I and GDNF in the complete rescue of postnatal
motor neurons. Amyotroph Lateral Scler Other Motor Neuron Disord 2001;2:83–91.
25. Bloch J, Bachoud-Levi AC, Deglon N, Lefaucheur JP, Winkel
L, Palfi S, et al. Neuroprotective gene therapy for Huntington’s disease, using polymer-encapsulated cells engineered
MUSCLE & NERVE
March 2006
317
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
318
to secrete human ciliary neurotrophic factor: results of a
phase I study. Hum Gene Ther 2004;15:968 –975.
Bohn MC. A commentary on glial cell line– derived neurotrophic factor (GDNF). From a glial secreted molecule to
gene therapy. Biochem Pharmacol 1999;57:135–142.
Bohn MC, Connor B, Kozlowski DA, Mohajeri MH. Gene
transfer for neuroprotection in animal models of Parkinson’s disease and amyotrophic lateral sclerosis. Novartis
Found Symp 2000;231:70 – 89.
Bohn MC. Motoneurons crave glial cell line– derived neurotrophic factor. Exp Neurol 2004;190:263–275.
Boillee S, Cleveland DW. Gene therapy for ALS delivers.
Trends Neurosci 2004;27:235–238.
Borasio GD, Robberecht W, Leigh PN, Emile J, Guiloff RJ,
Jerusalem F, et al. A placebo-controlled trial of insulin-like
growth factor-I in amyotrophic lateral sclerosis. European
ALS/IGF-I Study Group. Neurology 1998;51:583–586.
Borasio GD, Sloan R, Pongratz DE. Breaking the news in
amyotrophic lateral sclerosis. J Neurol Sci 1998;160(suppl
1):S127–133.
Bordet T, Schmalbruch H, Pettmann B, Hagege A, Castelnau-Ptakhine L, Kahn A, et al. Adenoviral cardiotrophin-1
gene transfer protects pmn mice from progressive motor
neuronopathy. J Clin Invest 1999;104:1077–1085.
Bordet T, Castelnau-Ptakhine L, Fauchereau F, Friocourt G,
Kahn A, Haase G. Neuronal targeting of cardiotrophin-1 by
coupling with tetanus toxin C fragment. Mol Cell Neurosci
2001;17:842– 854.
Bordet T, Lesbordes JC, Rouhani S, Castelnau-Ptakhine L,
Schmalbruch H, Haase G, et al. Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular
degeneration in transgenic ALS mice. Hum Mol Genet 2001;
10:1925–1933.
Boulis NM, Bhatia V, Brindle TI, Holman HT, Krauss DJ,
Blaivas M, et al. Adenoviral nerve growth factor and betagalactosidase transfer to spinal cord: a behavioral and histological analysis. J Neurosurg Spine 1999;90:99 –108.
Boulis NM, Turner DE, Dice JA, Bhatia V, Feldman EL.
Characterization of adenoviral gene expression in spinal
cord after remote vector delivery. Neurosurgery 1999;45:
131–137.
Boulis NM, Turner DE, Imperiale MJ, Feldman EL. Neuronal survival following remote adenovirus gene delivery.
J Neurosurg Spine 2002;96:212–219.
Boulis NM, Noordmans AJ, Song DK, Imperiale MJ, Rubin A,
Leone P, et al. Adeno-associated viral vector gene expression
in the adult rat spinal cord following remote vector delivery.
Neurobiol Dis 2003;14:535–541.
Boulis NM, Willmarth NE, Song DK, Feldman EL, Imperiale
MJ. Intraneural colchicine inhibition of adenoviral and adeno-associated viral vector remote spinal cord gene delivery.
Neurosurgery 2003;52:381–387.
Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA,
Copeland NG, et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive
disease with SOD1-containing inclusions. Neuron 1997;18:
327–338.
Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P,
Zolotukhin S, et al. Recombinant AAV viral vectors
pseudotyped with viral capsids from serotypes 1, 2, and 5
display differential efficiency and cell tropism after delivery
to different regions of the central nervous system. Mol Ther
2004;10:302–317.
Caillaud C, Akli S, Vigne E, Koulakoff A, Perricaudet M,
Poenaru L, et al. Adenoviral vector as a gene delivery system
into cultured rat neuronal and glial cells. Eur J Neurosci
1993;5:1287–1291.
Camu W. Spinal muscular atrophies in the adult. Rev Neurol
(Paris) 2004;160:225–228.
Caplen NJ. Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference.
Gene Ther 2004;11:1241–1248.
Motor Neuron Diseases and Gene Therapy
45. Cassina P, Peluffo H, Pehar M, Martinez-Palma L, Ressia A,
Beckman JS, et al. Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J Neurosci Res 2002;67:21–29.
46. Cifuentes-Diaz C, Frugier T, Melki J. Spinal muscular atrophy. Semin Pediatr Neurol 2002;9:145–150.
47. Cleveland DW, Rothstein JD. From Charcot to Lou Gehrig:
deciphering selective motor neuron death in ALS. Nat Rev
Neurosci 2001;2:806 – 819.
48. Cleveland JL. A new piece of the ALS puzzle. Nat Genet
2003;34:357–358.
49. Crocker S, Wigle N, Liston P, Thompson C, Lee C, Xu D, et
al. NAIP protects the nigrostriatal dopamine pathway in an
intrastriatal 6-OHDA rat model of Parkinson’s disease. Eur
J Neurosci 2001;14:391– 400.
50. Daly TM. Overview of adeno-associated viral vectors. Methods Mol Biol 2004;246:157–165.
51. Davidson B, Allen E, Kozarsky K, Wilson J, Roessler B. A
model system for in vivo gene transfer into the CNS using an
adenoviral vector. Nat Genet 1993;3:219 –223.
52. Davidson BL, Stein CS, Heth JA, Martins I, Kotin RM, Derksen TA, et al. Recombinant adeno-associated virus type 2, 4,
and 5 vectors: transduction of variant cell types and regions
in the mammalian central nervous system. Proc Natl Acad
Sci USA 2000;97:3428 –3432.
53. Davis A, Stachler M, Shi W, Liu J, Boulis N, Bartlett J.
Manipulating AAV2 tropism for enhanced delivery of AAV
vectors to the spinal cord. Mol Ther 2005;11(suppl):S46.
54. Dawbarn D, Allen SJ. Neurotrophins and neurodegeneration. Neuropathol Appl Neurobiol 2003;29:211–230.
55. DiDonato CJ, Parks RJ, Kothary R. Development of a gene
therapy strategy for the restoration of survival motor neuron
protein expression: implications for spinal muscular atrophy
therapy. Hum Gene Ther 2003;14:179 –188.
56. Ding H, Schwarz DS, Keene A, el Affar B, Fenton L, Xia X,
et al. Selective silencing by RNAi of a dominant allele that
causes amyotrophic lateral sclerosis. Aging Cell 2003;2:209 –
217.
57. Duberley RM, Johnson IP, Anand P, Leigh PN, Cairns
NJ. Neurotrophin-3-like immunoreactivity and Trk C expression in human spinal motoneurones in amyotrophic lateral
sclerosis. J Neurol Sci 1997;148:33– 40.
58. Dupuis L, Muller A, Meininger V, Loeffler JP. Molecular
mechanisms of amyotrophic lateral sclerosis: recent contributions from studies in animal models. Rev Neurol (Paris)
2004;160:35– 43.
59. Eisen A, Weber M. Treatment of amyotrophic lateral sclerosis. Drugs Aging 1999;14:173–196.
60. Federici T, Liu J, Teng Q, Garrity-Moses M, Yang J, Boulis N.
In vivo spinal cord uptake and neuronal binding properties
of Tet1: a potential means for enhanced spinal cord AAV
delivery. Mol Ther 2005;11(suppl):S332.
61. Figlewicz DA, Orrell RW. The genetics of motor neuron
diseases. Amyotroph Lateral Scler Other Motor Neuron Disord 2003;4:225–231.
62. Fink DJ, Sternberg LR, Weber PC, Mata M, Goins WF, Glorioso JC. In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer. Hum
Gene Ther 1992;3:11–19.
63. Francis JW, Bastia E, Matthews CC, Parks DA, Schwarzschild
MA, Brown RH Jr, et al. Tetanus toxin fragment C as a vector
to enhance delivery of proteins to the CNS. Brain Res 2004;
1011:7–13.
64. Francis JW, Figueiredo D, van der Spek JC, Ayala LM, Kim
YS, Remington MP, et al. A survival motor neuron:tetanus
toxin fragment C fusion protein for the targeted delivery of
SMN protein to neurons. Brain Res 2004;995:84 –96.
65. Friedlander R, Brown R Jr, Gagliardini V, Wang J, Yuan
J. Inhibition of ICE slows ALS in mice. Nature 1997;388:31.
66. Frugier T, Nicole S, Cifuentes-Diaz C, Melki J. The molecular
bases of spinal muscular atrophy. Curr Opin Genet Dev
2002;12:294 –298.
MUSCLE & NERVE
March 2006
67. Garrity-Moses M, Teng Q, Tanase D, Liu J, Bhat M, Boulis N.
AAVCMV.Bcl-xLGFP gene delivery to SHSY-5Y neuroblastoma cells as a neuroprotective agent in glutamate-induced
apoptosis. Mol Ther 2003;7(suppl):S96.
68. Garrity-Moses M, Teng Q, Liu J, Tanase D, Rezai A, Boulis N.
Adenoviral XIAP expression protects motor neurons in an in
vivo model of ALS. Soc Neurosci Abstr 2004;312–310.
69. Geller AI, Freese A. Infection of cultured central nervous
system neurons with a defective herpes simplex virus 1 vector
results in stable expression of Escherichia coli beta-galactosidase. Proc Natl Acad Sci USA 1990;87:1149 –1153.
70. Gimenez y Ribotta M, Revah F, Pradier L, Loquet I, Mallet J,
Privat A. Prevention of motoneuron death by adenovirusmediated neurotrophic factors. J Neurosci Res 1997;48:281–
285.
71. Glass DJ, Yancopoulos GD. The neurotrophins and their
receptors. Trends Cell Biol 1993;3:262–268.
72. Glorioso JC, Mata M, Fink DJ. Therapeutic gene transfer to
the nervous system using viral vectors. J Neurovirol 2003;9:
165–172.
73. Gong YH, Parsadanian AS, Andreeva A, Snider WD, Elliott
JL. Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause
motoneuron degeneration. J Neurosci 2000;20:660 – 665.
74. Greenberg DA, Jin K. VEGF and ALS: the luckiest growth
factor? Trends Mol Med 2004;10:1–3.
75. Guegan C, Vila M, Rosoklija G, Hays AP, Przedborski S.
Recruitment of the mitochondrial-dependent apoptotic
pathway in amyotrophic lateral sclerosis. J Neurosci 2001;21:
6569 – 6576.
76. Guillot S, Azzouz M, Deglon N, Zurn A, Aebischer P. Local
GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in
SOD1(G93A) transgenic mice. Neurobiol Dis 2004;16:139 –
149.
77. Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY,
Alexander DD, et al. Motor neuron degeneration in mice
that express a human Cu,Zn superoxide dismutase mutation. Science 1994;264:1772–1775.
78. Haase G, Kennel P, Pettmann B, Vigne E, Akli S, Revah F, et
al. Gene therapy of murine motor neuron disease using
adenoviral vectors for neurotrophic factors. Nat Med 1997;
3:429 – 436.
79. Haase G, Pettmann B, Vigne E, Castelnau-Ptakhine L,
Schmalbruch H, Kahn A. Adenovirus-mediated transfer of
the neurotrophin-3 gene into skeletal muscle of pmn mice:
therapeutic effects and mechanisms of action. J Neurol Sci
1998;160(suppl 1):S97–105.
80. Haase G, Pettmann B, Bordet T, Villa P, Vigne E, Schmalbruch H, et al. Therapeutic benefit of ciliary neurotrophic
factor in progressive motor neuronopathy depends on the
route of delivery. Ann Neurol 1999;45:296 –304.
81. Haenggeli C, Kaspar B, Lee H, Gage F, Rothstein J. Therapeutic viral vector delivery of excitatory amino acid transporters in a mouse model of ALS. Amyotroph Lateral Scler
Other Motor Neuron Disord 2004;5:55.
82. Hall ED, Oostveen JA, Gurney ME. Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS. Glia 1998;23:
249 –256.
83. Hauck B, Xiao W. Characterization of tissue tropism determinants of adeno-associated virus type 1. J Virol 2003;77:
2768 –2774.
84. Heath PR, Shaw PJ. Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve 2002;26:438 – 458.
85. Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, et al. GDNF: a potent survival factor
for motoneurons present in peripheral nerve and muscle.
Science 1994;266:1062–1064.
86. Henkel JS, Engelhardt JI, Siklos L, Simpson EP, Kim SH, Pan
T, et al. Presence of dendritic cells, MCP-1, and activated
Motor Neuron Diseases and Gene Therapy
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
microglia/macrophages in amyotrophic lateral sclerosis spinal cord tissue. Ann Neurol 2004;55:221–235.
Holzbaur EL. Motor neurons rely on motor proteins. Trends
Cell Biol 2004;14:233–240.
Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, et
al. Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic
lateral sclerosis (ALS). Proc Natl Acad Sci USA 2002;99:
1604 –1609.
Ilzecka J. Increased serum CNTF level in patients with amyotrophic lateral sclerosis. Eur Cytokine Netw 2003;14:192–
194.
Jablonka S, Sendtner M. Molecular and cellular basis of
spinal muscular atrophy. Amyotroph Lateral Scler Other
Motor Neuron Disord 2003;4:144 –149.
Jafari-Schluep HF, Khoris J, Mayeux-Portas V, Hand C, Rouleau G, Camu W. Superoxide dismutase 1 gene abnormalities in familial amyotrophic lateral sclerosis: phenotype/
genotype correlations. The French experience and review of
the literature. Rev Neurol (Paris) 2004;160:44 –50.
Janson C, McPhee S, Bilaniuk L, Haselgrove J, Testaiuti M,
Freese A, et al. Clinical protocol. Gene therapy of Canavan
disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum Gene Ther
2002;13:1391–1412.
Jiang YM, Yamamoto M, Kobayashi Y, Yoshihara T, Liang Y,
Terao S, et al. Gene expression profile of spinal motor
neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol 2005;57:236 –251.
Kahn A, Haase G, Akli S, Guidotti JE. Gene therapy of
neurological diseases. C R Seances Soc Biol Fil 1996;190:9 –
11.
Kaspar BK, Erickson D, Schaffer D, Hinh L, Gage FH, Peterson DA. Targeted retrograde gene delivery for neuronal
protection. Mol Ther 2002;5:50 –56.
Kaspar BK, Llado J, Sherkat N, Rothstein JD, Gage FH.
Retrograde viral delivery of IGF-1 prolongs survival in a
mouse ALS model. Science 2003;301:839 – 842.
Kato AC, Lindsay RM. Overlapping and additive effects of
neurotrophins and CNTF on cultured human spinal cord
neurons. Exp Neurol 1994;130:196 –201.
Klein SM, Behrstock S, McHugh J, Hoffmann K, Wallace K,
Suzuki M, et al. GDNF delivery using human neural progenitor cells in a rat model of ALS. Hum Gene Ther 2005;16:
509 –521.
Knight A, Carvajal J, Schneider H, Coutelle C, Chamberlain
S, Fairweather N. Non-viral neuronal gene delivery mediated
by the HC fragment of tetanus toxin. Eur J Biochem 1999;
259:762–769.
Krivickas LS. Amyotrophic lateral sclerosis and other motor
neuron diseases. Phys Med Rehabil Clin N Am 2003;14:327–
345.
Kugler S, Straten G, Kreppel F, Isenmann S, Liston P, Bahr
M. The X-linked inhibitor of apoptosis (XIAP) prevents cell
death in axotomized CNS neurons in vivo. Cell Death Differ
2000;7:815– 824.
Kugler S, Lingor P, Scholl U, Zolotukhin S, Bahr M. Differential transgene expression in brain cells in vivo and in vitro
from AAV-2 vectors with small transcriptional control units.
Virology 2003;311:89 –95.
Lacomblez L, Bensimon G, Leigh PN, Guillet P, Meininger
V. Dose-ranging study of riluzole in amyotrophic lateral
sclerosis. Amyotrophic Lateral Sclerosis/Riluzole Study
Group II. Lancet 1996;347:1425–1431.
Lacomblez L, Bensimon G, Leigh PN, Guillet P, Powe L,
Durrleman S, et al. A confirmatory dose-ranging study of
riluzole in ALS. ALS/Riluzole Study Group II. Neurology
1996;47(suppl):S242–250.
Lai EC, Felice KJ, Festoff BW, Gawel MJ, Gelinas DF, Kratz R,
et al. Effect of recombinant human insulin-like growth factor-I on progression of ALS. A placebo-controlled study. The
MUSCLE & NERVE
March 2006
319
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
320
North America ALS/IGF-I Study Group. Neurology 1997;49:
1621–1630.
Lambrechts D, Storkebaum E, Carmeliet P. VEGF: necessary
to prevent motoneuron degeneration, sufficient to treat
ALS? Trends Mol Med 2004;10:275–282.
LaMonte BH, Wallace KE, Holloway BA, Shelly SS, Ascano J,
Tokito M, et al. Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron 2002;34:715–727.
Le Gal La Salle G, Robert JJ, Berrard S, Ridoux V, StratfordPerricaudet LD, Perricaudet M, et al. An adenovirus vector
for gene transfer into neurons and glia in the brain. Science
1993;259:988 –990.
Lee DA, Gross L, Wittrock DA, Windebank AJ. Localization
and expression of ciliary neurotrophic factor (CNTF) in
postmortem sciatic nerve from patients with motor neuron
disease and diabetic neuropathy. J Neuropathol Exp Neurol
1996;55:915–923.
Lesbordes JC, Cifuentes-Diaz C, Miroglio A, Joshi V, Bordet
T, Kahn A, et al. Therapeutic benefits of cardiotrophin-1
gene transfer in a mouse model of spinal muscular atrophy.
Hum Mol Genet 2003;12:1233–1239.
Lewis ME, Neff NT, Contreras PC, Stong DB, Oppenheim
RW, Grebow PE, et al. Insulin-like growth factor-I: potential
for treatment of motor neuronal disorders. Exp Neurol
1993;124:73– 88.
Li M, Ona VO, Guegan C, Chen M, Jackson-Lewis V, Andrews LJ, et al. Functional role of caspase-1 and caspase-3 in
an ALS transgenic mouse model. Science 2000;288:335–339.
Lilley CE, Groutsi F, Han Z, Palmer JA, Anderson PN, Latchman DS, et al. Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to
neurons in culture and widespread gene delivery to the
central nervous system in vivo. J Virol 2001;75:4343– 4356.
Lindholm D, Dechant G, Heisenberg CP, Thoenen H. Brainderived neurotrophic factor is a survival factor for cultured
rat cerebellar granule neurons and protects them against
glutamate-induced neurotoxicity. Eur J Neurosci 1993;5:
1455–1464.
Liston P, Fong W, Korneluk R. The inhibitors of apoptosis:
there is more to life than Bcl2. Oncogene 2003;22:8568 –
8580.
Liu JK, Teng Q, Garrity-Moses M, Federici T, Tanase D,
Imperiale MJ, et al. A novel peptide defined through phage
display for therapeutic protein and vector neuronal targeting. Neurobiol Dis 2005;19:407– 418.
Lowenstein PR. Immunology of viral-vector-mediated gene
transfer into the brain: an evolutionary and developmental
perspective. Trends Immunol 2002;23:23–30.
Lowenstein PR, Castro MG. Inflammation and adaptive immune responses to adenoviral vectors injected into the
brain: peculiarities, mechanisms, and consequences. Gene
Ther 2003;10:946 –954.
Lu YY, Wang LJ, Muramatsu S, Ikeguchi K, Fujimoto K,
Okada T, et al. Intramuscular injection of AAV-GDNF results
in sustained expression of transgenic GDNF, and its delivery
to spinal motoneurons by retrograde transport. Neurosci
Res 2003;45:33– 40.
Magal E, Burnham P, Varon S. Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: survival and
expression of choline acetyltransferase and low-affinity nerve
growth factor receptors. Brain Res Dev Brain Res 1991;63:
141–150.
Maguire-Zeiss KA, Bowers WJ, Federoff HJ. HSV vector–
mediated gene delivery to the central nervous system. Curr
Opin Mol Ther 2001;3:482– 490.
Manabe Y, Nagano I, Gazi MS, Murakami T, Shiote M, Shoji
M, et al. Adenovirus-mediated gene transfer of glial cell
line– derived neurotrophic factor prevents motor neuron
loss of transgenic model mice for amyotrophic lateral sclerosis. Apoptosis 2002;7:329 –334.
Motor Neuron Diseases and Gene Therapy
123. Martin LJ. Neuronal death in amyotrophic lateral sclerosis is
apoptosis: possible contribution of a programmed cell death
mechanism. J Neuropathol Exp Neurol 1999;58:459 – 471.
124. Martinov VN, Sefland I, Walaas SI, Lomo T, Nja A, Hoover F.
Targeting functional subtypes of spinal motoneurons and
skeletal muscle fibers in vivo by intramuscular injection of
adenoviral and adeno-associated viral vectors. Anat Embryol
(Berl) 2002;205:215–221.
125. Matsuoka N, Yukawa H, Ishii K, Hamada H, Akimoto M,
Hashimoto N, et al. Adenovirus-mediated gene transfer of
Bcl-xL prevents cell death in primary neuronal culture of the
rat. Neurosci Lett 1999;270:177–180.
126. Mazarakis ND, Azzouz M, Rohll JB, Ellard FM, Wilkes FJ,
Olsen AL, et al. Rabies virus glycoprotein pseudotyping of
lentiviral vectors enables retrograde axonal transport and
access to the nervous system after peripheral delivery. Hum
Mol Genet 2001;10:2109 –2121.
127. McCown TJ, Xiao X, Li J, Breese GR, Samulski RJ. Differential and persistent expression patterns of CNS gene transfer
by an adeno-associated virus (AAV) vector. Brain Res 1996;
713:99 –107.
128. McGeer PL, McGeer EG. Glial cell reactions in neurodegenerative diseases: pathophysiology and therapeutic interventions. Alzheimer Dis Assoc Disord 1998;12(suppl 2):S1– 6.
129. McWhorter ML, Monani UR, Burghes AH, Beattie CE.
Knockdown of the survival motor neuron (Smn) protein in
zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol 2003;162:919 –931.
130. Miles GB, Yohn DC, Wichterle H, Jessell TM, Rafuse VF,
Brownstone RM. Functional properties of motoneurons derived from mouse embryonic stem cells. J Neurosci 2004;24:
7848 –7858.
131. Millecamps S, Mallet J, Barkats M. Adenoviral retrograde
gene transfer in motoneurons is greatly enhanced by prior
intramuscular inoculation with botulinum toxin. Human
Gene Therapy 2002;13:225–232.
132. Miller RG, Petajan JH, Bryan WW, Armon C, Barohn RJ,
Goodpasture JC, et al. A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF) factor in
amyotrophic lateral sclerosis. rhCNTF ALS Study Group.
Ann Neurol 1996;39:256 –260.
133. Miller TM, Cleveland DW. Has gene therapy for ALS arrived? Nat Med 2003;9:1256 –1257.
134. Miller TM, Kaspar BK, Kops GJ, Yamanaka K, Christian LJ,
Gage FH, et al. Virus-delivered small RNA silencing sustains
strength in amyotrophic lateral sclerosis. Ann Neurol 2005;
57:773–776.
135. Mitchell JD, Wokke JH, Borasio GD. Recombinant human
insulin-like growth factor I (rhIGF-I) for amyotrophic lateral
sclerosis/motor neuron disease. Cochrane Database Syst Rev
2002:CD002064.
136. Mitsumoto H, Klinkosz B, Pioro EP, Tsuzaka K, Ishiyama T,
O’Leary RM, et al. Effects of cardiotrophin-1 (CT-1) in a
mouse motor neuron disease. Muscle Nerve 2001;24:769 –
777.
137. Miwa H, Shibata M, Okado H, Hirano S. Tracing axons in
the peripheral nerve using lacZ gene recombinant adenovirus and its application to regeneration of the peripheral
nerve. J Neuropathol Exp Neurol 2001;60:671– 675.
138. Mohajeri M, Figlewicz D, Bohn M. Intramuscular grafts of
myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic
lateral sclerosis. Hum Gene Ther 1999;10:1853–1866.
139. Murakami T, Nagano I, Hayashi T, Manabe Y, Shoji M,
Setoguchi Y, et al. Impaired retrograde axonal transport of
adenovirus-mediated E. coli LacZ gene in the mice carrying
mutant SOD1 gene. Neurosci Lett 2001;308:149 –152.
140. Mutoh T, Sobue G, Hamano T, Kuriyama M, Hirayama M,
Yamamoto M, et al. Decreased phosphorylation levels of
TrkB neurotrophin receptor in the spinal cords from pa-
MUSCLE & NERVE
March 2006
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
tients with amyotrophic lateral sclerosis. Neurochem Res
2000;25:239 –245.
Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a
lentiviral vector. Proc Natl Acad Sci USA 1996;93:11382–
11388.
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH,
et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263–
267.
Natsume A, Mata M, Wolfe D, Oligino T, Goss J, Huang S, et
al. Bcl-2 and GDNF delivered by HSV-mediated gene transfer after spinal root avulsion provide a synergistic effect.
J Neurotrauma 2002;19:61– 68.
Nicole S, Diaz CC, Frugier T, Melki J. Spinal muscular
atrophy: recent advances and future prospects. Muscle
Nerve 2002;26:4 –13.
Ochs G, Penn RD, York M, Giess R, Beck M, Tonn J, et al. A
phase I/II trial of recombinant methionyl human brain
derived neurotrophic factor administered by intrathecal infusion to patients with amyotrophic lateral sclerosis.
Amyotroph Lateral Scler Other Motor Neuron Disord 2000;
1:201–206.
Ono S, Imai T, Igarashi A, Shimizu N, Nakagawa H, Hu J.
Decrease in the ciliary neurotrophic factor of the spinal cord
in amyotrophic lateral sclerosis. Eur Neurol 1999;42:163–
168.
Oppenheim RW, Wiese S, Prevette D, Armanini M, Wang S,
Houenou LJ, et al. Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons. J Neurosci 2001;21:1283–1291.
Palmer JA, Branston RH, Lilley CE, Robinson MJ, Groutsi F,
Smith J, et al. Development and optimization of herpes
simplex virus vectors for multiple long-term gene delivery to
the peripheral nervous system. J Virol 2000;74:5604 –5618.
Pasinelli P, Borchelt DR, Houseweart MK, Cleveland DW,
Brown RH Jr. Caspase-1 is activated in neural cells and tissue
with amyotrophic lateral sclerosis-associated mutations in
copper–zinc superoxide dismutase. Proc Natl Acad Sci USA
1998;95:15763–15768.
Pehar M, Cassina P, Vargas MR, Castellanos R, Viera L,
Beckman JS, et al. Astrocytic production of nerve growth
factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J Neurochem 2004;89:464 – 473.
Perez MC, Hunt SP, Coffin RS, Palmer JA. Comparative
analysis of genomic HSV vectors for gene delivery to motor
neurons following peripheral inoculation in vivo. Gene Ther
2004;11:1023–1032.
Poeschla EM. Non-primate lentiviral vectors. Curr Opin Mol
Ther 2003;5:529 –540.
Przedborski S. Programmed cell death in amyotrophic lateral sclerosis: a mechanism of pathogenic and therapeutic
importance. Neurologist 2004;10:1–7.
Przedborski S. Molecular targets for neuroprotection.
Amyotroph Lateral Scler Other Motor Neuron Disord 2004;
5(suppl 1):14 –18.
Puskovic V, Wolfe D, Goss J, Huang S, Mata M, Glorioso JC,
et al. Prolonged biologically active transgene expression
driven by HSV LAP2 in brain in vivo. Mol Ther 2004;10:67–
75.
Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X,
et al. Cross-packaging of a single adeno-associated virus
(AAV) type 2 vector genome into multiple AAV serotypes
enables transduction with broad specificity. J Virol 2002;76:
791– 801.
Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA,
Lee DC, et al. Silencing mutant SOD1 using RNAi protects
against neurodegeneration and extends survival in an ALS
model. Nat Med 2005;11:429 – 433.
Raoul C, Abbas-Terki T, Bensadoun JC, Guillot S, Haase G,
Szulc J, et al. Lentiviral-mediated silencing of SOD1 through
Motor Neuron Diseases and Gene Therapy
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
RNA interference retards disease onset and progression in a
mouse model of ALS. Nat Med 2005;11:423– 428.
Reed J. Bcl-2 family proteins. Oncogene 1998;17:3225–3236.
Regulier E, Pereira de Almeida L, Sommer B, Aebischer P,
Deglon N. Dose-dependent neuroprotective effect of ciliary
neurotrophic factor delivered via tetracycline-regulated lentiviral vectors in the quinolinic acid rat model of Huntington’s disease. Hum Gene Ther 2002;13:1981–1990.
Ripps ME, Huntley GW, Hof PR, Morrison JH, Gordon JW.
Transgenic mice expressing an altered murine superoxide
dismutase gene provide an animal model of amyotrophic
lateral sclerosis. Proc Natl Acad Sci USA 1995;92:689 – 693.
Romero M, Rangappa N, Garry M, Smith G. Functional
regeneration of chronically injured sensory afferents into
adult spinal cord after neurotrophin gene therapy. J Neurosci 2001;21:8408 – 8416.
Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P,
Hentati A, et al. Mutations in Cu/Zn superoxide dismutase
gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362:59 – 62.
Rowland LP. Six important themes in amyotrophic lateral
sclerosis (ALS) research, 1999. J Neurol Sci 2000;180:2– 6.
Rutledge EA, Halbert CL, Russell DW. Infectious clones and
vectors derived from adeno-associated virus (AAV) serotypes
other than AAV type 2. J Virol 1998;72:309 –319.
Sagot Y, Tan SA, Baetge E, Schmalbruch H, Kato AC, Aebischer P. Polymer encapsulated cell lines genetically engineered to release ciliary neurotrophic factor can slow down
progressive motor neuronopathy in the mouse. Eur J Neurosci 1995;7:1313–1322.
Sahenk Z, Seharaseyon J, Mendell JR, Burghes AH. Gene
delivery to spinal motor neurons. Brain Res 1993;606:126 –
129.
Scarpini CG, May J, Lachmann RH, Preston CM, Dunnett
SB, Torres EM, et al. Latency associated promoter transgene
expression in the central nervous system after stereotaxic
delivery of replication-defective HSV-1– based vectors. Gene
Ther 2001;8:1057–1071.
Schiffer D, Fiano V. Astrogliosis in ALS: possible interpretations according to pathogenetic hypotheses. Amyotroph Lateral Scler Other Motor Neuron Disord 2004;5:22–25.
Schmalbruch H, Haase G. Spinal muscular atrophy: present
state. Brain Pathol 2001;11:231–247.
Schneider H, Groves M, Muhle C, Reynolds PN, Knight A,
Themis M, et al. Retargeting of adenoviral vectors to neurons using the Hc fragment of tetanus toxin. Gene Ther
2000;7:1584 –1592.
Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith
AG, et al. Inactivation of the survival motor neuron gene, a
candidate gene for human spinal muscular atrophy, leads to
massive cell death in early mouse embryos. Proc Natl Acad
Sci USA 1997;94:9920 –9925.
Sendtner M, Schmalbruch H, Stockli KA, Carroll P,
Kreutzberg GW, Thoenen H. Ciliary neurotrophic factor
prevents degeneration of motor neurons in mouse mutant
progressive motor neuronopathy. Nature 1992;358:502–504.
Shevtsova Z, Malik JM, Michel U, Bahr M, Kugler S. Promoters and serotypes: targeting of adeno-associated virus vectors
for gene transfer in the rat central nervous system in vitro
and in vivo. Exp Physiol 2005;90:53–59.
Shi W, Bartlett J. RGD inclusion in VP3 provides adenoassociated virus type 2 (AAV2)-based vectors with a heparan
sulfate–independent cell entry mechanism. Mol Ther 2003;
7:515–525.
Shimohama S, Tamura Y, Akaike A, Tsukahara T, Ohara O,
Watanabe S, et al. Brain-derived neurotrophic factor pretreatment exerts a partially protective effect against glutamate-induced neurotoxicity in cultured rat cortical neurons.
Neurosci Lett 1993;164:55–58.
Silani V, Braga M, Cardin V, Scarlato G. The pathogenesis of
ALS: implications for treatment strategies. Neurol Neurochir Pol 2001;35:25–39.
MUSCLE & NERVE
March 2006
321
178. Silani V, Leigh N. Stem therapy for ALS: hope and reality.
Amyotroph Lateral Scler Other Motor Neuron Disord 2003;
4:8 –10.
179. Silani V, Cova L, Corbo M, Ciammola A, Polli E. Stem-cell
therapy for amyotrophic lateral sclerosis. Lancet 2004;364:
200 –202.
180. Soler-Botija C, Ferrer I, Alvarez JL, Baiget M, Tizzano EF.
Downregulation of Bcl-2 proteins in type I spinal muscular
atrophy motor neurons during fetal development. J Neuropathol Exp Neurol 2003;62:420 – 426.
181. St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene Ther 2003;10:1135–1141.
182. Storkebaum E, Lambrechts D, Carmeliet P. VEGF: once
regarded as a specific angiogenic factor, now implicated in
neuroprotection. Bioessays 2004;26:943–954.
183. Storkebaum E, Lambrechts D, Dewerchin M, Moreno-Murciano MP, Appelmans S, Oh H, et al. Treatment of motoneuron degeneration by intracerebroventricular delivery of
VEGF in a rat model of ALS. Nat Neurosci 2005;8:85–92.
184. Strong M, Rosenfeld J. Amyotrophic lateral sclerosis: a review of current concepts. Amyotroph Lateral Scler Other
Motor Neuron Disord 2003;4:136 –143.
185. Strong MJ. The basic aspects of therapeutics in amyotrophic
lateral sclerosis. Pharmacol Ther 2003;98:379 – 414.
186. Svendsen CN, Langston JW. Stem cells for Parkinson disease
and ALS: replacement or protection? Nat Med 2004;10:224 –
225.
187. Tanase D, Garrity-Moses M, Liu J, Teng Q, Mazarakis N,
Boulis N. Techniques for remote cervical spinal cord delivery of neurotherapeutic RabG-EIAV encoded genes. Mol
Ther 2003;7(suppl):S97.
188. Tanase K, Teng Q, Krishnaney AA, Liu JK, Garrity-Moses
ME, Boulis NM. Cervical spinal cord delivery of a rabies G
protein pseudotyped lentiviral vector in the SOD-1 transgenic mouse. J Neurosurg Spine 2004;1:128 –136.
189. Tenenbaum L, Chtarto A, Lehtonen E, Velu T, Brotchi J,
Levivier M. Recombinant AAV-mediated gene delivery to the
central nervous system. J Gene Med 2004;6(suppl 1):S212–
222.
190. Teng Q, Garrity-Moses M, Liu J, Tanase D, Mazarakis N,
Walmsely L, et al. EIAV-IGF-I gene transfer to motor
neurons enhances axonal length in vitro. Mol Ther 2004;
9(suppl):S19.
191. Teng Q, Garrity-Moses M, Federici T, Yang J, Kutner R,
Tordo N, et al. Neuronal gene transfer with HIV-1– based
lentiviral vectors pseudotyped with Lyssavirus glycoproteins.
Mol Ther 2005;11(suppl):S187.
192. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR. Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected
intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases. Proc Natl Acad Sci
USA 2000;97:7482–7487.
193. Thorne RG, Frey WH II. Delivery of neurotrophic factors to
the central nervous system: pharmacokinetic considerations.
Clin Pharmacokinet 2001;40:907–946.
194. Turner BJ, Cheah IK, Macfarlane KJ, Lopes EC, Petratos S,
Langford SJ, et al. Antisense peptide nucleic acid–mediated
knockdown of the p75 neurotrophin receptor delays motor
neuron disease in mutant SOD1 transgenic mice. J Neurochem 2003;87:752–763.
195. Tuszynski MH, Thal L, Pay M, Salmon DP, Bakay R, et al. A
phase 1 clinical trial of nerve growth factor gene therapy for
Alzheimer disease. Nat Med 2005;11:551–555.
196. Uemura O, Okada Y, Ando H, Guedj M, Higashijima S,
Shimazaki T, et al. Comparative functional genomics revealed conservation and diversification of three enhancers
of the isl1 gene for motor and sensory neuron-specific expression. Dev Biol 2005;278:587– 606.
322
Motor Neuron Diseases and Gene Therapy
197. Veldink JH, Van den Berg LH, Wokke JH. The future of
motor neuron disease: the challenge is in the genes. J Neurol 2004;251:491–500.
198. Vincent AM, Mobley BC, Hiller A, Feldman EL. IGF-I prevents glutamate-induced motor neuron programmed cell
death. Neurobiol Dis 2004;16:407– 416.
199. Vukosavic S, Stefanis L, Jackson-Lewis V, Guegan C, Romero
N, Chen C, et al. Delaying caspase activation by Bcl-2: a clue
to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci 2000;20:9119 –9125.
200. Vyas S, Bechade C, Riveau B, Downward J, Triller A. Involvement of survival motor neuron (SMN) protein in cell death.
Hum Mol Genet 2002;11:2751–2764.
201. Wang C, Wang CM, Clark KR, Sferra TJ. Recombinant AAV
serotype 1 transduction efficiency and tropism in the murine
brain. Gene Ther 2003;10:1528 –1534.
202. Wang LJ, Lu YY, Muramatsu S, Ikeguchi K, Fujimoto K,
Okada T, et al. Neuroprotective effects of glial cell line–
derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis. J Neurosci 2002;22:6920 – 6928.
203. Wengenack TM, Holasek SS, Montano CM, Gregor D, Curran GL, Poduslo JF. Activation of programmed cell death
markers in ventral horn motor neurons during early presymptomatic stages of amyotrophic lateral sclerosis in a
transgenic mouse model. Brain Res 2004;1027:73– 86.
204. Williamson TL, Cleveland DW. Slowing of axonal transport
is a very early event in the toxicity of ALS-linked SOD1
mutants to motor neurons. Nat Neurosci 1999;2:50 –56.
205. Wisman L, Coenen S, Van Wingerden W, Hol E, Verhaagen
J, Van Muiswinkel F, et al. Lentiviral vectors coding for
EAAT2 to protect motoneurons from glutamate toxicity. Soc
Neurosci Abstr 2003;96.96.
206. Wisman LA, van Muiswinkel FL, de Graan PN, Hol EM, Bar
PR. Cells over-expressing EAAT2 protect motoneurons from
excitotoxic death in vitro. Neuroreport 2003;14:1967–1970.
207. Wong LF, Azzouz M, Walmsley LE, Askham Z, Wilkes FJ,
Mitrophanous KA, et al. Transduction patterns of
pseudotyped lentiviral vectors in the nervous system. Mol
Ther 2004;9:101–111.
208. Wu P, Xiao W, Conlon T, Hughes J, Agbandje-McKenna M,
Ferkol T, et al. Mutational analysis of the adeno-associated
virus type 2 (AAV2) capsid gene and construction of AAV2
vectors with altered tropism. J Virol 2000;74:8635– 8647.
209. Xu D, Crocker S, Doucet J, St-Jean M, Tamai K, Hakim A, et
al. Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus. Nat Med 1997;3:997–
1004.
210. Xu D, Bureau Y, McIntyre DC, Nicholson DW, Liston P, Zhu
Y, et al. Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis
protein overexpression in the rat hippocampus. J Neurosci
1999;19:5026 –5033.
211. Yamamura J, Kageyama S, Uwano T, Kurokawa M, Imakita
M, Shiraki K. Long-term gene expression in the anterior
horn motor neurons after intramuscular inoculation of a live
herpes simplex virus vector. Gene Ther 2000;7:934 –941.
212. Yamashita S, Mita S, Arima T, Maeda Y, Kimura E, Nishida Y,
et al. Bcl-2 expression by retrograde transport of adenoviral
vectors with Cre–loxP recombination system in motor neurons of mutant SOD1 transgenic mice. Gene Ther 2001;8:
977–986.
213. Yamashita S, Mita S, Kato S, Okado H, Ohama E, Uchino M.
Effect on motor neuron survival in mutant SOD1 (G93A)
transgenic mice by Bcl-2 expression using retrograde axonal
transport of adenoviral vectors. Neurosci Lett 2002;328:289 –
293.
214. Yamashita S, Mita S, Kato S, Okado H, Ohama E, Uchino M.
Bcl-2 expression using retrograde transport of adenoviral
vectors inhibits cytochrome c release and caspase-1 activation in motor neurons of mutant superoxide dismutase 1
(G93A) transgenic mice. Neurosci Lett 2003;350:17–20.
MUSCLE & NERVE
March 2006
215. Yokota T, Miyagishi M, Hino T, Matsumura R, Tasinato A,
Urushitani M, et al. siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS,
compared with ribozyme and DNA enzyme. Biochem Biophys Res Commun 2004;314:283–291.
216. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002;99:6047– 6052.
217. Zaiss AK, Muruve DA. Immune responses to adeno-associated virus vectors. Curr Gene Ther 2005;5:323–331.
Motor Neuron Diseases and Gene Therapy
218. Zhao W, Xie W, Le W, Beers DR, He Y, Henkel JS, et al.
Activated microglia initiate motor neuron injury by a nitric
oxide and glutamate-mediated mechanism. J Neuropathol
Exp Neurol 2004;63:964 –977.
219. Zhao Z, Alam S, Oppenheim RW, Prevette DM, Evenson
A, Parsadanian A. Overexpression of glial cell line– derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their
long-term survival following axotomy. Exp Neurol 2004;
190:356 –372.
MUSCLE & NERVE
March 2006
323