Download Lentivirus-Mediated Gene Transfer to the Central Nervous System

HUMAN GENE THERAPY 17:1–9 (January 2006)
© Mary Ann Liebert, Inc.
Review
Lentivirus-Mediated Gene Transfer to the Central Nervous
System: Therapeutic and Research Applications
LIANG-FONG WONG, LUCY GOODHEAD, CHRISTINE PRAT, KYRIACOS A. MITROPHANOUS,
SUSAN M. KINGSMAN, and NICHOLAS D. MAZARAKIS
ABSTRACT
The management of disorders of the nervous system remains a medical challenge. The key goals are to understand disease mechanisms, to validate therapeutic targets, and to develop new therapeutic strategies. Viral
vector-mediated gene transfer can meet these goals and vectors based on lentiviruses have particularly useful
features. Lentiviral vectors can deliver 8 kb of sequence, they mediate gene transfer into any neuronal cell type,
expression and therapy are sustained, and normal cellular functions in vitro and in vivo are not compromised.
After delivery into the nervous system they induce no significant immune responses, there are no unwanted
side effects of the vectors per se to date, and manufacturing and safety testing for clinical applications are well
advanced. There are now numerous examples of effective long-term treatment of animal models of neurological disorders, such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, motor neuron diseases,
lysosomal storage diseases, and spinal injury, using a range of therapeutic genes expressed in lentiviral vectors. Significant issues remain in some areas of neural gene therapy including defining the optimum therapeutic gene(s), increasing the specificity of delivery, regulating expression of potentially toxic genes, and designing clinically relevant strategies. We discuss the applications of lentiviral vectors in therapy and research
and highlight the essential features that will ensure their translation to the clinic in the near future.
INTRODUCTION
therapy. Neural architecture is relevant in designing therapy and
in anticipating any side effects. Neurons can have long-range
axonal projections and therefore delivery at one site could lead
to action at a distant site. The greatest challenge will be to treat
disseminated disease, where pathology spreads through the nervous system (e.g., motor neuron disease) or involves large regions of the brain (e.g., Alzheimer’s disease), or where the
pathology is in brain and other tissues (e.g., lysosomal storage
diseases). It may be essential to express the gene within each
affected neuron as with small interfering RNAs (siRNAs) or intrabodies to ablate gene expression, and this will require efficient gene transfer. If therapy is via secreted factors then fewer
cells can be transduced, so reducing the delivery challenge, but
diffusion or anterograde distribution to other sites may be an
issue.
Many neural disorders are chronic but not always life threatening, so there must be long-term expression and safety. Vec-
T
HE ABILITY TO TRANSFER GENES into neurons, using viral
vectors, allows therapeutic genes to be delivered to the nervous system and provides new approaches to probe the mechanistic basis of disease and identify new target points for intervention.
There are unique opportunities and challenges for gene
therapy in the central nervous system (CNS). The potent
blood–brain barrier precludes the ready access of systemic molecules as large as a viral vector and so invasive brain surgery
may be required. However, knowledge of mammalian neuroanatomy and clinical tools such as magnetic resonance imaging (MRI) allow the precise deposition of viral vectors at the
site of interest, so imparting considerable specificity to the treatment. Anatomical isolation results in a degree of “immune privilege,” which may limit the immunological consequences of any
Oxford BioMedica (UK), Medawar Centre, Oxford Science Park, Oxford OX4 4GA, UK.
1
2
WONG ET AL.
tors that have useful features include those based on herpesviruses, high-capacity adenoviral vectors, adeno-associated
viruses (AAVs), and lentiviruses (Davidson and Breakefield,
2003). AAV and lentiviral vectors are attractive because of their
simplicity, their excellent safety profile, and their relative ease
of production. In many indications both AAV and lentiviral vectors are being assessed but here we focus on lentiviral vectors,
which have many important advantages in research and therapy of neurological disorders.
LENTIVIRAL VECTORS AND GENE
TRANSFER TO THE NERVOUS SYSTEM
Transduction of neurons, using lentiviral vectors in vivo in
rodent and primate models, has been amply demonstrated (e.g.,
see Galimi and Verma, 2002; Azzouz et al., 2004a). These vectors are derived from primate (human and simian immunodeficiency viruses [HIV and SIV, respectively]) and nonprimate
(e.g., equine, feline, and bovine immunodeficiency viruses
[EIAV, FIV, and BIV, respectively]) lentiviruses (reviewed by
Delenda, 2004). Relevant vectors for research and clinical applications are referred to as “minimal self-inactivating (SIN)
vectors.” The entire viral coding regions are removed from the
viral genome and particle proteins and replication enzymes
(Gag and Pol) and an envelope (Env) are provided in trans from
separate expression cassettes. The optimum efficiency is obtained by using a gag-pol gene that is codon optimized and in
general the use of codon-optimized transgenes is desirable (our
unpublished data). The envelope glycoprotein is nonlentiviral;
this is referred to as pseudotyping. The vesicular stomatitis virus
(VSV-G) envelope is widely used and gives vectors broad
species and tissue tropism, but other envelope glycoproteins
with unique features for different applications are available
(Watson et al., 2002; Wong et al., 2004). As there are no residual lentiviral promoters or enhancers remaining in the genome in SIN vectors the transgene is expressed from an internal enhancer-promoter that can be varied according to the
application. The most advanced vectors place less than 1 kb of
viral DNA into the transduced cell genome and express only
the transgene cassette. Useful technical and historical information on lentiviral vector design can be found in Dull et al.
(1998), Kim et al. (1998), Zufferey et al. (1998), Kotsopoulou
et al. (2000), and Rohll et al. (2002).
Gene transfer in the nervous system could be compromised
by innate or adaptive immune responses against virion components or the transgene product. This has certainly limited the
effectiveness of AAV vectors where circulating preexisting antibodies to AAV inhibit gene transfer in the brain (Peden et al.,
2004). However, innate immune responses may not be problematic for lentiviral vectors as minimal acute, asymptomatic
inflammation in animal models has been observed (see Kordower et al., 2000; Mazarakis et al., 2001). Adaptive antivector response were not significant in mediating immune clearance in the CNS transduced with HIV vectors and injection into
the brain did not induce systemic immunity to virions (AbordoAdesida et al., 2005). These data are encouraging for CNS applications, suggesting that there will be no immune suppression
in patients and that repeated treatments with vector in the brain
may not induce adverse immune responses. This important clinical issue requires more work with different transgenes, treat-
ment protocols, and vectors. In particular, scenarios in which
preexisting or posttreatment immune responses could occur, as
with infection with a prevalent virus such as HIV, need to be
explored.
Most studies using lentiviral vectors have used powerful constitutive promoters such as PGK1, CMV, CAG, and EF1 (see
citations in Ramezani et al., 2000). This is pragmatic and rational for nontoxic transgenes with a broad therapeutic window,
if expression in nontarget cells is not an issue and if the potential benefit outweighs the perceived risk in a particular indication. Restricted expression may be desired for some situations, for example, striatal expression of the potent growth
factor glial-derived nerve growth factor (GDNF) led to GDNF
release and aberrant fiber sprouting at downstream striatal targets in animal models of Parkinson’s disease (Georgievska et
al., 2002). These side effects may be limited by confining
GDNF expression to astrocytes by using a glial cell-specific
promoter (hGFAP), leading to more localized secretion (Jakobsson et al., 2003). Regulated gene expression to evaluate temporal aspects or to modulate dosage level will be important in
some applications. Lentiviral vectors are particularly amenable
to developing regulated systems, as their copy number in target cells is reasonably predictable and stable without silencing.
This contrasts with AAV vectors, which have limited cargo capacity and which may produce such high copy numbers that
regulators are potentially diluted out. Regulated systems in evaluation in lentiviral vectors to date use regulator proteins, for
example, derived from bacterial operons such as the Tet repressor or engineered eukaryotic transcription factors (for some
examples of regulation systems that can be applied in lentiviral vectors, see Ye et al., 1999; Reiser et al., 2000; Vigna et
al., 2002; Koponen et al., 2003; Sirin and Park, 2003; Galimi
et al., 2005; Pluta et al., 2005). These systems are now being
refined to ensure that the basal expression is low and that the
induced levels are high enough for the purpose (e.g., Vigna et
al., 2002; Pluta et al., 2005). As all extraneous viral coding regions are assiduously removed from lentiviral vectors it is counterproductive to introduce foreign regulators that may be immunogenic and/or may influence the cell biology. Current work
is therefore aimed at deimmunizing regulator proteins or at developing systems that can be regulated by small-molecule interactions with the proviral DNA or transcripts (Suess et al.,
2003).
Lentiviral vectors might be targeted to specific cell types by
varying the envelope proteins although, as with all vector-targeting attempts to date, this is more likely to result in selectivity rather than absolute specificity. Ross River virus pseudotypes are more efficient on glial cells than neurons (Kang et al.,
2002); lymphocytic choriomeningitis virus (LCMV) pseudotypes preferentially transduce murine neural stem cells and human glioma cell lines (Steffens et al., 2004; Stein et al., 2005);
foamy virus, rabies virus, mokola virus, and amphotropic
murine leukemia virus pseudotypes are enhanced on human
neuroblastoma cell lines (Steffens et al., 2004); and rabies-G
pseudotypes are useful for distal targeting of neurons in vivo
because they are efficiently retrogradely transported (Mazarakis
et al., 2001; Wong et al., 2004).
Gene delivery for neurological disorders often requires invasive surgery and therefore repeat treatments are not desirable;
long-term, stable expression at therapeutic levels is essential.
Lentiviral vectors maintain expression for up to 16 months (e.g.,
LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS
Kordower et al., 2000; Bienemann et al., 2003; Balaggan et al.,
2005) and animals made transgenic with lentiviral vectors maintain expression without silencing (Lois et al., 2002; Pfeifer et
al., 2002; McGrew et al., 2004). Furthermore, lentiviral vector
transduction does not appear to affect the electrophysiological
properties of neurons (Dittgen et al., 2004).
Many types of therapeutic genes are being evaluated including trophic factors and enzymes, as well as intrabodies and
siRNA targeted to defective intracellular proteins and genes.
The versatility of lentiviral vectors allows ex vivo engineering
of stem cells or differentiated cells for transplantation, direct
injection at the target site, and remote delivery and targeting
via retrograde transport. Some impressive efficacy data are
emerging and some of these studies are outlined below.
GENE THERAPY FOR PARKINSON’S DISEASE
Parkinson’s disease (PD) is a progressive neurodegenerative
disorder that arises as a result of the death of neurons in the
substantia nigra region in the brain. Loss of these neurons
causes depletion of dopamine levels in the putamen, leading to
aberrant movement control. The mechanism of selective neuron loss is unknown, although degeneration may be due to
neurotoxicity of accumulated misfolded proteins possibly
induced/exacerbated by an altered redox state of the cells. Familial PD involves mutations in genes encoding proteins that
participate in the ubiquitin–proteasome pathway (e.g., -synuclein, parkin, and ubiquitin C-terminal hydrolase L1), which
may contribute to the formation of cytoplasmic inclusions
termed “Lewy bodies” in neurons, a pathological hallmark of
PD. Early-stage patients can be treated with oral L-3,4-dihydroxyphenylalanine (L-dopa), which crosses the blood–brain
barrier and is metabolized by aromatic-L-amino-acid decarboxylase to produce dopamine. Prolonged depletion of natural
dopamine increases dopamine sensitivity and the ectopic generation of dopamine leads to inappropriate localized concentrations, resulting in a serious side effect of debilitating abnormal movements (dyskinesia). The daily management of PD
becomes difficult as L-dopa dosage requires frequent modulation and patients experience unpredictable periods of immobility and dyskinesia. For some patients surgical interventions such
as deep brain stimulation (DBS) can control tremor or pallidotomy can control dyskinesia (reviewed in Betchen and Kaplitt,
2003). However, there are no curative treatments and patients
cannot be restored to normal movement in a predictable and
stable way.
Late-stage PD is ideal for treatment by gene therapy because
it is a localized disease and the pathology, biochemistry, and
clinical presentations are well characterized. The most conservative approach is to manage the symptoms and one strategy
already in the clinic injects an AAV vector expressing glutamic
acid decarboxylase (GAD) into the subthalamic nucleus to limit
tremor by stimulating the motor-inhibitory -aminobutyric acid
(GABA)-ergic pathway (During et al., 2001). Another gene
therapy strategy is to provide the missing dopamine in a manner designed to minimize side effects and this could displace
oral L-dopa therapy as the standard of care in late-stage patients.
Normally dopamine is produced in the nigral neurons by hydroxylation of tyrosine to L-dopa (via the enzyme tyrosine hydroxylase TH1 and the cofactor tetrahydrobiopterin, the biosyn-
3
thesis of which is rate limited by the enzyme GTP cyclohydrolase I [CH1]) and then L-dopa is decarboxylated to dopamine by aromatic-L-amino-acid decarboxylase (AADC). One
approach in clinical trials uses an AAV vector to deliver just
the AADC, which could augment the background levels of this
enzyme to allow lower doses of L-dopa to be administered and
might reduce the dyskinesia, although ectopic conversion
may still occur with this approach (see http://www.avigen.
com/non_financial_release/2005/2005_Avigen_EarlyData_PD
ClinicalTrial_071805.htm). However, remarkable efficacy has
been observed in animal models when all three genes are coexpressed in the striatum. This was demonstrated in nonhuman
primates with AAV vectors (Muramatsu et al., 2002), but three
different vectors carrying each of the genes were used because
of the size limitation of AAV. The advantage of the higher capacity of lentiviral vectors has been exploited to build a single
vector expressing all three enzymes, TH1, CHI, and AADC, by
using internal ribosome entry sites to create a tricistronic transcript (Azzouz et al., 2002). This tricistronic vector gives a consistent, complete, and long-lasting reversal of the major Parkinsonian clinical and motor symptoms in a chronic rhesus
macaque model of severe PD (Jarraya, B., Azzouz, M., Miskin,
J., Ralph, G.S., Wilkes, F., Walmsley, L., Barber, R.D., Rohll,
J., Drouot, X., Brouillet, E., Conde, F., Kingsman, S.M.,
Hantraye, P., Mitrophanous, K., Mazarakis, N.D., and Palfi, S.,
personal communication). A clinical protocol for gene-based
dopamine replacement in patients who are beginning to fail on
conventional L-dopa therapy is being developed (our unpublished data). The aim would be a single administration of the
tricistronic lentiviral vector bilaterally into the striatum to produce tonic levels of dopamine. This should restore a level of
stable motor function without the dyskinesia resulting from the
fluctuating doses and ectopic sites of production associated with
oral L-dopa therapy and oral L-dopa treatment could be reduced
or eliminated.
A more far-reaching opportunity for gene therapy is to delay symptoms by protecting the substantia nigra neurons. Localized lentivirus-mediated GDNF delivery protects the surviving neurons of the nigrostriatal dopaminergic pathway from
further degeneration in rodent models (Bensadoun et al., 2000;
Deglon et al., 2000; Gill et al., 2003; Azzouz et al., 2004d;
Kirik et al., 2004) and in aged, nonlesioned or young, 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus
monkeys (Kordower et al., 2000). However, there was shutdown of tyrosine hydroxylase in the restored nigral neurons and
aberrant neuronal sprouting (Georgievska et al., 2002, 2004b).
A proposal to evaluate AAV-mediated delivery of a related
growth factor, neurturin (Dass et al., 2005), has also been discussed (NIH Recombinant DNA Advisory Committee [RAC]
Meeting, March 16, 2005; http://www.webconferences.com/
nihoba/16_mar_2005.html). The use of a growth factor as the
therapeutic gene raises many issues concerning potential longterm consequences and highlights the desirability of having
some regulation in the system. Regulated lentiviral vectors may
therefore be critical to advance this strategy into the clinic for
PD and studies are in progress (Georgievska et al., 2004a). In
trials of recombinant GDNF delivered via pumps, early indications of success (Gill et al., 2003) were not reproduced in
larger studies (see http://www.amgen.com/media/media_pr_
detail.jsp?year52005&releaseID5673490) and toxicities have
been seen with this approach (Nutt et al., 2003). It is therefore
4
WONG ET AL.
not yet clear what further efficacy and safety studies will justify growth factor therapy for early-stage PD.
Lentiviral vectors are also being used to create novel animal
models for PD (Lo Bianco et al., 2002; Lauwers et al., 2003)
and to test therapeutic strategies beyond dopamine replacement
and neurotrophic support (Lo Bianco et al., 2002, 2004b). For
example, lentiviral vector-mediated transfer of the A30P mutant -synuclein induces a selective and progressive loss of dopamine neurons associated with the appearance of -synuclein
inclusions. In this model, lentiviral vector-mediated delivery of
parkin to the substantia nigra gave significant neuroprotection,
sparing TH-positive nerve terminals in the striatum and this appeared to be due to an effect on the detoxification of misfolded
proteins (Lo Bianco et al., 2004b). Interestingly, prior injection
of a lentiviral vector expressing GDNF was unable to block this
neurodegeneration, suggesting that once a toxic cascade of protein aggregation has been initiated in a dopaminergic neuron
then growth factors may not have a role to play (Lo Bianco et
al., 2004a).
ALZHEIMER’S DISEASE
In Alzheimer’s disease (AD) there is progressive cognitive
decline and memory loss. The only approved treatments are the
chemical cholinesterase inhibitors to briefly delay the onset of
memory loss. The disease-inducing pathology appears to be the
aggregation of amyloid (A) peptides to form amyloid
plaques, particularly in the hippocampus. Sequential cleavage
of transmembrane amyloid precursor protein (APP) by an aspartyl protease, -secretase (or -site APP-cleaving enzyme
[BACE-1]), and an enzyme complex including -secretase, generates different peptides with varying propensities to form
plaques. Several factors influence the deposition of amyloidogenic peptides. The lipid-binding protein ApoE facilitates the
deposition or clearance of A in the brain, depending on the
isoform, and hence certain alleles of the apoE gene reduce or
increase the risk of developing Alzheimer’s disease. The general transmembrane peptidase neprilysin, and a family of proteins called the presenilins (PS1 and PS1), also play a role in
APP processing. Treatment strategies for AD include using
trophic factors to protect neurons from damage, altering the balance of A accumulation by inhibiting secretases or enhancing
degradative pathways, and clearing away the plaques by providing binding compounds or specific antibodies.
Lentiviral vectors are being used to interrogate transgenic
disease models and to develop new models. Lentivirus-mediated expression of neprilysin in the hippocampus of hAPP transgenic mice led to a 50% reduction in amyloid load and provided neuroprotection against A-induced neurotoxicity (Marr
et al., 2003). Intracerebral administration of lentiviral vectors
expressing ApoE isoforms differentially altered the amyloid
burden in the hippocampus of PDAPP transgenic mice (Dodart
et al., 2005). Lentiviral vector-mediated expression of ApoE2
or neprilysin in vulnerable brain regions in AD is therefore a
rational approach to preventing or treating AD.
The -secretase enzymes are classical targets for small-molecule drugs and the pharmaceutical industry is experienced in
developing aspartyl protease inhibitors. Gene therapy might,
however, have a role if the pharmacokinetics and bioavailabil-
ity of small-molecule drugs are unfavorable for chronic disease
management. One strategy would be to use lentivirus-mediated
siRNA targeted against BACE-1, and this was shown to reduce
amyloid production and behavioral deficits in APP transgenic
mice (Singer et al., 2005). Neurons can also be protected from
neurotoxicity by delivering antiapoptotic genes such as Bcl-xL
(Blomer et al., 1998), although using an oncogene raises safety
concerns. Recombinant nerve growth factor (NGF) delivered
directly or via engrafted fibroblasts retrovirally engineered to
secrete NGF prevented degeneration of the basal forebrain
cholinergic neurons in rat and primate models (reviewed by
Blesch and Tuszynski, 2004). When these strategies were tested
in humans there were side effects after direct delivery of the
protein in a few patients (Eriksdotter Jonhagen et al., 1998),
but the ex vivo approach, using transplanted engineered fibroblasts, was promising in having no adverse effects and there
were some indications of cognitive improvement and increased
metabolic activity in the brain (Tuszynski et al., 2005). Another
NGF clinical trial using an AAV vector is underway in earlystage AD patients (http://www.webconferences.com/nihoba/
16_mar_2005.html). The approval of this trial is a good example of the regulatory balance of potential benefit versus perceived risk in a desperate patient group. Lentiviral vectors can
deliver NGF but the aim would be to deal with symptoms and
causes by including neuroprotective factors and factors that reduce and/or block amyloid formation. Furthermore, lentiviral
vectors can provide access to all affected areas of the brain via
the efficient retrograde transport or the use of cellular delivery
systems (see Disseminated Neurological Disorders, below).
Regulated and neuron-specific promoters would increase the
safety of global distribution by imposing temporal and spatial
restriction of gene expression. For example, Blesch et al. (2005)
used a Tet-regulated lentiviral vector expressing NGF to rescue cholinergic neurons in the fimbria-fornix of rats after traumatic lesion mimicking AD pathology. Neuroprotective strategies will rely to some extent on the ability to reliably diagnose
AD at a sufficiently early stage for neuroprotection to provide
a benefit and the long-term consequences of gene therapy will
require careful evaluation.
HUNTINGTON’S DISEASE
In Huntington’s disease (HD) the inheritance of a dominant
mutated allele of the huntingtin (htt) gene causes a selective
loss of striatal neurons leading to motor and cognitive deficiencies and death after 10 to 15 years. The mutant allele contains expansions of CAG trinucleotide repeats encoding polyglutamine (polyQ). A higher number of CAG repeats correlates
with earlier age of disease onset and there is no treatment.
Regulated lentiviral vectors used to overexpress various mutant htt gene fragments in the brains of rats and primates induced the typical neuronal pathology of Huntington’s disease.
This was reversed by switching off gene expression, indicating
that ablating mutant huntingtin is a viable therapeutic strategy
(Regulier et al., 2003). Mutant huntingtin can be knocked out
with an siRNA and this improved behavioral and pathological
abnormalities in HD-N171-82Q transgenic mice overexpressing mutant proteins (Harper et al., 2005). Allele-specific suppression without compromising normal protein function would
LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS
be desirable and this has been achieved in other polyQ disorders (Miller et al., 2003). Finally, trophic factors (ciliary neurotrophic factor [CNTF] and brain-derived neurotrophic factor
[BDNF]) had a protective role when delivered by lentiviral vectors into rat striatal cell culture models of HD (Zala et al., 2005)
and a lentiviral vector expressing CNTF protected against subsequent striatal degeneration induced by quinolinic acid in a rat
model for HD (de Almeida et al., 2001).
The clinical evaluation of any of these strategies will be challenging, given the slow progression of this disease and the limited number of patients. It is vital therefore that the optimum
strategy be well rehearsed in relevant models before entering
the clinic.
SPINAL CORD AND NERVE INJURY
The adult central nervous system cannot undergo repair after traumatic damage but gene transfer strategies are emerging
that appear to overcome this limitation. Adenoviral vectors expressing trophic factors promoted regeneration of injured sensory afferents into the adult rat spinal cord (Zhang et al., 1998;
Romero et al., 2001) and improved hind limb locomotor recovery from spinal cord contusion (Tai et al., 2003). Lentiviral vector-mediated delivery of GDNF protected facial motor
neurons from axotomy-induced cell death in mice (Hottinger et
al., 2000). The transcription factor RAR2 (retinoic acid receptor 2), expressed in an EIAV-based lentiviral vector, promoted neurite outgrowth in adult spinal cord explants (Corcoran et al., 2002) and gave significant protection of sensory and
motor functions in a rat dorsal root ganglion rhizotomy model
(Wong et al., 2005). Lentiviral vectors efficiently transduce
Schwann cells (SCs) and olfactory ensheathing cells (OECs) ex
vivo (Ruitenberg et al., 2002; Barakat et al., 2005) and these
may be useful for producing neurotrophin-secreting SC/OECs
for the promotion of axonal regeneration in spinal cord injury
models. siRNA against axon growth inhibitors (Nogo receptor,
p75NTR, or Rho-A) may also promote CNS axon regeneration
in vivo (Ahmed et al., 2005).
Lentiviral vectors expressing axogenic, neuroprotective, or
regeneration molecules could restore neural function after injury but it is critical to determine how long after injury any of
these strategies would be effective. They may need to be combined with methods for reducing or reversing scar formation or
with methods for bridging or bypassing damaged areas. There
are also significant challenges in defining clinical trials for disorders that are not life threatening, that generally occur in a
younger population, and where the severity and therefore medical need may not be revealed for many months.
DISSEMINATED NEUROLOGICAL DISORDERS
A significant number of neurological disorders (e.g., motor
neuron, Alzheimer’s, and lysosomal storage diseases) are disseminated throughout the brain or spinal cord, which poses a
considerable challenge of access. Lentiviral vectors can cross
the blood–brain barrier after in utero treatment or delivery to
neonates (Kobayashi et al., 2005; Kyrkanides et al., 2005), but
not in adults. Alternative approaches take advantage of specific
5
features of lentiviral vectors such as the property of highly efficient retrograde transport and the ability to transduce hematopoietic stem cells (HSCs) that traffic to the CNS.
Motor neuron diseases are neurodegenerative diseases that
cause progressive paralysis and premature death, and there are
no treatments. The majority of amyotrophic lateral sclerosis
(ALS) cases are sporadic, although there is an inherited form
due to mutations in the superoxide dismutase (SOD1) gene.
Spinal muscular atrophy (SMA) is the second commonest genetic disease affecting children and is due to mutations in the
survival motor neuron (SMN1) gene. In mouse models for ALS
(SOD1 mutant mouse) or SMA (SMN1 knockout mouse)
lentiviral vectors pseudotyped with rabies-G envelope can be
injected into the affected muscle, taken up at nerve terminals,
and expressed in the cognate motor neurons in the spinal cord.
Using this approach, vectors expressing vascular endothelial
growth factor (Azzouz et al., 2004c) or siRNA targeted to a
mutated SOD1 gene (Ralph et al., 2005b) or expressing the normal SMN1 gene (Azzouz et al., 2004b) extended corrected motor defects and extended survival in the relevant models. Remote access to the spinal cord via muscle can provide relatively
simple palliative treatments to spare key muscle groups such as
those that control hand movement, swallowing, and breathing
to enhance the quality of and/or extend life. Alternatively, injections directly into the spinal cord or the brain may allow sufficient spread and access to upper motor neurons. Some success in injecting lentiviral vectors expressing inhibitory siRNA
into the spinal cord of animal models of ALS has been reported
(Raoul et al., 2005).
Lysosomal enzyme deficiency disorders (e.g., Gaucher’s,
Tay-Sachs, Hurler’s, Sly, and metachromatic leukodystrophies)
result in the accumulation of neurotoxic metabolites leading to
various pathologies and symptoms depending on the disease,
including inflammation, demyelination, motor deficit, and death
(Biffi and Naldini, 2005). The aim is to restore normal enzyme
function by providing the wild-type gene and the challenge is
to access all of the affected tissues. If the gene product is diffusible, for example, -glucuronidase (Brooks et al., 2002), or
if enhanced diffusion can be engineered by the addition of peptide transporters (Xia et al., 2001), this can increase distribution throughout the CNS. Alternatively, Biffi et al. (2004) have
used an ex vivo approach to transduce HSCs. They observed a
progressive repopulation of CNS and peripheral nervous system (PNS) macrophage/microglial cells with arylsulfatase Aexpressing cells and obtained significant reversal of the systemic and neurological symptoms in a mouse model for a
metachromatic leukodystrophy. This is an exciting approach
and could be more widely applicable for delivering any secreted
product to the nervous system.
NEUROBIOLOGY RESEARCH APPLICATIONS
OF LENTIVIRAL VECTORS
Lentiviral vectors accelerate the generation of transgenic animals, increasing the range of animal models (Lois et al., 2002;
Pfeifer et al., 2002; McGrew et al., 2004; Whitelaw et al.,
2004). These vectors are particularly useful in identifying genes
that can protect neurons against pathological insults in chronic
settings, as described above, but also in acute settings such as
6
WONG ET AL.
in stroke models (Ralph et al., 2004; Wong et al., 2005). They
can bypass transgenesis altogether by making localized disease
models with vectors expressing mutant proteins or siRNA (reviewed in Kirik and Bjorklund, 2003; Ralph et al., 2005a).
These models can be made conditional by using regulated promoters or by applying Cre–lox recombination systems (Tiscornia et al., 2004; Ventura et al., 2004). An exciting technique
for the future is to combine the power of lentiviral vectors and
siRNA to create gene knockout libraries that will allow the rapid
identification of therapeutic targets. The same lentiviral vector
can transition into the clinic if a gene therapy approach is indicated, thus shortening the preclinical development time.
These research applications of lentiviral vectors are promising
to deliver a major advance in our understanding of the nervous
system in health and disease.
SUMMARY
Lentiviral vectors are powerful tools for research and therapy. Their efficaciousness has been shown in models for major diseases such as Parkinson’s disease and Alzheimer’s disease and for the less prevalent but equally debilitating
diseases such as motor neuron disease and lysosomal storage
diseases. It may even be possible to treat serious spinal cord
injuries with these vectors. All of these diseases or conditions
are chronic and limitations to small-molecule drug therapies
open the way for genetic therapies. Encouraging preclinical
data on efficacy and safety with lentiviral vectors are rapidly
accumulating in many laboratories and progress is being
made in developing all of the relevant analytical, safety testing, and manufacturing methods (e.g., Rohll et al., 2002;
Miskin et al., 2005; Sastry et al., 2005). The regulatory
framework for evaluating viral vectors in the nervous system
is in place and the paradigm of risk-versus-benefit analysis
is being applied rationally for each specific indication
(http://www.fda.gov/cber/gdlns/retrogt1000.pdf,
http://www.emea.eu.int/pdfs/human/bwp/245803en.pdf). A
number of lentiviral vectors are set to enter the clinic and
hold real promise for the treatment and eventually the prevention of these difficult and distressing neurological conditions. Exciting times!
ACKNOWLEDGMENTS
The authors are grateful to Mimoun Azzouz and Alan Kingsman for scientific discussions and critical comments.
REFERENCES
ABORDO-ADESIDA, E., FOLLENZI, A., BARCIA, C., SCIASCIA,
S., CASTRO, M.G., NALDINI, L., and LOWENSTEIN, P.R. (2005).
Stability of lentiviral vector-mediated transgene expression in the
brain in the presence of systemic antivector immune responses. Hum.
Gene Ther. 16, 741–751.
AHMED, Z., DENT, R.G., SUGGATE, E.L., BARRETT, L.B.,
SEABRIGHT, R.J., BERRY, M., and LOGAN, A. (2005). Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite out-
growth on CNS myelin by siRNA-mediated knockdown of NgR,
p75NTR and Rho-A. Mol. Cell Neurosci. 28, 509–523.
AZZOUZ, M., MARTIN-RENDON, E., BARBER, R.D., MITROPHANOUS, K.A., CARTER, E.E., ROHLL, J.B., KINGSMAN,
S.M., KINGSMAN, A.J., and MAZARAKIS, N.D. (2002). Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic
L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson’s
disease. J. Neurosci. 22, 10302–10312.
AZZOUZ, M., KINGSMAN, S.M., and MAZARAKIS, N.D. (2004a).
Lentiviral vectors for treating and modeling human CNS disorders.
J. Gene Med. 6, 951–962.
AZZOUZ, M., LE, T., RALPH, G.S., WALMSLEY, L., MONANI,
U.R., LEE, D.C., WILKES, F., MITROPHANOUS, K.A., KINGSMAN, S.M., BURGHES, A.H., and MAZARAKIS, N.D. (2004b).
Lentivector-mediated SMN replacement in a mouse model of spinal
muscular atrophy. J. Clin. Invest. 114, 1726–1731.
AZZOUZ, M., RALPH, G.S., STORKEBAUM, E., WALMSLEY,
L.E., MITROPHANOUS, K.A., KINGSMAN, S.M., CARMELIET,
P., and MAZARAKIS, N.D. (2004c). VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS
model. Nature 429, 413–417.
AZZOUZ, M., RALPH, S., WONG, L.F., DAY, D., ASKHAM, Z., BARBER, R.D., MITROPHANOUS, K.A., KINGSMAN, S.M., and
MAZARAKIS, N.D. (2004d). Neuroprotection in a rat Parkinson model
by GDNF gene therapy using EIAV vector. Neuroreport 15, 985–990.
BALAGGAN, K.S., BINLEY, K., ESAPA, M., IQBALL, S., NAYLOR, S., ASKHAM, Z., KAN, O., TSCHERNUTTER, M., BAINBRIDGE, J., and ALI, R. (2005). Stable and efficient intraocular
gene transfer using pseudotyped EIAV lentiviral vectors. J. Gene
Med. Nov 21; (Epub ahead of print).
BARAKAT, D.J., GAGLANI, S.M., NERAVETLA, S.R., SANCHEZ,
A.R., ANDRADE, C.M., PRESSMAN, Y., PUZIS, R., GARG, M.S.,
BUNGE, M.B., and PEARSE, D.D. (2005). Survival, integration,
and axon growth support of glia transplanted into the chronically
contused spinal cord. Cell Transplant. 14, 225–240.
BENSADOUN, J.C., DEGLON, N., TSENG, J.L., RIDET, J.L., ZURN,
A.D., and AEBISCHER, P. (2000). Lentiviral vectors as a gene delivery system in the mouse midbrain: Cellular and behavioral improvements in a 6-OHDA model of Parkinson’s disease using GDNF.
Exp. Neurol. 164, 15–24.
BETCHEN, S.A., and KAPLITT, M. (2003). Future and current surgical therapies in Parkinson’s disease. Curr. Opin. Neurol. 16, 487–493.
BIENEMANN, A.S., MARTIN-RENDON, E., COSGRAVE, A.S.,
GLOVER, C.P., WONG, L.F., KINGSMAN, S.M., MITROPHANOUS, K.A., MAZARAKIS, N.D., and UNEY, J.B. (2003).
Long-term replacement of a mutated nonfunctional CNS gene: Reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin. Mol. Ther. 7, 588–596.
BIFFI, A., and NALDINI, L. (2005). Gene therapy of storage disorders
by retroviral and lentiviral vectors. Hum. Gene Ther. 16,1133–1142.
BIFFI, A., DE PALMA, M., QUATTRINI, A., DEL CARRO, U.,
AMADIO, S., VISIGALLI, I., SESSA, M., FASANO, S., BRAMBILLA, R., MARCHESINI, S., BORDIGNON, C., and NALDINI,
L. (2004). Correction of metachromatic leukodystrophy in the mouse
model by transplantation of genetically modified hematopoietic stem
cells. J. Clin. Invest. 113, 1118–1129.
BLESCH, A., and TUSZYNSKI, M.H. (2004). Gene therapy and cell
transplantation for Alzheimer’s disease and spinal cord injury. Yonsei Med. J. 45(Suppl.), 28–31.
BLESCH, A., CONNER, J., PFEIFER, A., GASMI, M., RAMIREZ,
A., BRITTON, W., ALFA, R., VERMA, I., and TUSZYNSKI, M.H.
(2005). Regulated lentiviral NGF gene transfer controls rescue of
medial septal cholinergic neurons. Mol. Ther. 11, 916–925.
BLOMER, U., KAFRI, T., RANDOLPH-MOORE, L., VERMA, I.M.,
LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS
and GAGE, F.H. (1998). Bcl-xL protects adult septal cholinergic neurons from axotomized cell death. Proc. Natl. Acad. Sci. U.S.A. 95,
2603–2608.
BROOKS, A.I., STEIN, C.S., HUGHES, S.M., HETH, J., MCCRAY,
P.M., Jr., SAUTER, S.L., JOHNSTON, J.C., CORY-SLECHTA,
D.A., FEDEROFF, H.J., and DAVIDSON, B.L. (2002). Functional
correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors. Proc. Natl. Acad. Sci. U.S.A. 99,
6216–6221.
CORCORAN, J., SO, P.L., BARBER, R.D., VINCENT, K.J.,
MAZARAKIS, N.D., MITROPHANOUS, K.A., KINGSMAN,
S.M., and MADEN, M. (2002). Retinoic acid receptor 2 and neurite outgrowth in the adult mouse spinal cord in vitro. J. Cell Sci.
115, 3779–3786.
DASS, B., KLADIS, T., CHU, Y., and KORDOWER, J.H. (2005). RET
expression does not change with age in the substantia nigra pars compacta of rhesus monkeys. Neurobiol. Aging Jun 9; (Epub ahead of
print).
DAVIDSON, B.L., and BREAKEFIELD, X.O. (2003). Viral vectors for
gene delivery to the nervous system. Nat. Rev. Neurosci. 4, 353–364.
DE ALMEIDA, L.P., ZALA, D., AEBISCHER, P., and DEGLON, N.
(2001). Neuroprotective effect of a CNTF-expressing lentiviral vector in the quinolinic acid rat model of Huntington’s disease. Neurobiol. Dis. 8, 433–446.
DEGLON, N., TSENG, J.L., BENSADOUN, J.C., ZURN, A.D., ARSENIJEVIC, Y., PEREIRA DE ALMEIDA, L., ZUFFEREY, R.,
TRONO, D., and AEBISCHER, P. (2000). Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene
transfer system in Parkinson’s disease. Hum. Gene Ther. 11,
179–190.
DELENDA, C. (2004). Lentiviral vectors: Optimization of packaging,
transduction and gene expression. J. Gene Med. 6(Suppl. 1),
S125–S138.
DITTGEN, T., NIMMERJAHN, A., KOMAI, S., LICZNERSKI, P.,
WATERS, J., MARGRIE, T.W., HELMCHEN, F., DENK, W.,
BRECHT, M., and OSTEN, P. (2004). Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl. Acad. Sci. U.S.A. 101,
18206–18211.
DODART, J.C., MARR, R.A., KOISTINAHO, M., GREGERSEN,
B.M., MALKANI, S., VERMA, I.M., and PAUL, S.M. (2005). Gene
delivery of human apolipoprotein E alters brain A burden in a mouse
model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A. 102,
1211–1216.
DULL, T., ZUFFEREY, R., KELLY, M., MANDEL, R.J., NGUYEN,
M., TRONO, D., and NALDINI, L. (1998). A third-generation
lentivirus vector with a conditional packaging system. J. Virol. 72,
8463–8471.
DURING, M.J., KAPLITT, M.G., STERN, M.B., and EIDELBERG,
D. (2001). Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation. Hum. Gene
Ther. 12, 1589–1591.
ERIKSDOTTER JONHAGEN, M., NORDBERG, A., AMBERLA, K.,
BACKMAN, L., EBENDAL, T., MEYERSON, B., OLSON, L.,
SEIGER, SHIGETA, M., THEODORSSON, E., VIITANEN, M.,
WINBLAD, B., and WAHLUND, L.O. (1998). Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer’s disease. Dement. Geriatr. Cogn. Disord. 9, 246–257.
GALIMI, F., and VERMA, I.M. (2002). Opportunities for the use of
lentiviral vectors in human gene therapy. Curr. Top. Microbiol. Immunol. 261, 245–254.
GALIMI, F., SAEZ, E., GALL, J., HOONG, N., CHO, G., EVANS,
R.M., and VERMA, I.M. (2005). Development of ecdysone-regulated lentiviral vectors. Mol. Ther. 11, 142–148.
GEORGIEVSKA, B., KIRIK, D., and BJORKLUND, A. (2002). Aber-
7
rant sprouting and downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer. Exp. Neurol. 177, 461–474.
GEORGIEVSKA, B., JAKOBSSON, J., PERSSON, E., ERICSON, C.,
KIRIK, D., and LUNDBERG, C. (2004a). Regulated delivery of glial
cell line-derived neurotrophic factor into rat striatum, using a tetracycline-dependent lentiviral vector. Hum. Gene Ther. 15, 934–944.
GEORGIEVSKA, B., KIRIK, D., and BJORKLUND, A. (2004b).
Overexpression of glial cell line-derived neurotrophic factor using a
lentiviral vector induces time- and dose-dependent downregulation
of tyrosine hydroxylase in the intact nigrostriatal dopamine system.
J. Neurosci. 24, 6437–6445.
GILL, S.S., PATEL, N.K., HOTTON, G.R., O’SULLIVAN, K., MCCARTER, R., BUNNAGE, M., BROOKS, D.J., SVENDSEN, C.N.,
and HEYWOOD, P. (2003). Direct brain infusion of glial cell linederived neurotrophic factor in Parkinson disease. Nat. Med. 9,
589–595.
HARPER, S.Q., STABER, P.D., HE, X., ELIASON, S.L., MARTINS,
I.H., MAO, Q., YANG, L., KOTIN, R.M., PAULSON, H.L., and
DAVIDSON, B.L. (2005). RNA interference improves motor and
neuropathological abnormalities in a Huntington’s disease mouse
model. Proc. Natl. Acad. Sci. U.S.A. 102, 5820–5825.
HOTTINGER, A.F., AZZOUZ, M., DEGLON, N., AEBISCHER, P.,
and ZURN, A.D. (2000). Complete and long-term rescue of lesioned
adult motoneurons by lentiviral-mediated expression of glial cell linederived neurotrophic factor in the facial nucleus. J. Neurosci. 20,
5587–5593.
JAKOBSSON, J., ERICSON, C., JANSSON, M., BJORK, E., and
LUNDBERG, C. (2003). Targeted transgene expression in rat brain
using lentiviral vectors. J. Neurosci. Res. 73, 876–885.
KANG, Y., STEIN, C.S., HETH, J.A., SINN, P.L., PENISTEN, A.K.,
STABER, P.D., RATLIFF, K.L., SHEN, H., BARKER, C.K., MARTINS, I., SHARKEY, C.M., SANDERS, D.A., MCCRAY, P.B., Jr.,
and DAVIDSON, B.L. (2002). In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River virus glycoproteins. J. Virol. 76, 9378–9388.
KIM, V.N., MITROPHANOUS, K., KINGSMAN, S.M., and KINGSMAN, A.J. (1998). Minimal requirement for a lentivirus vector based
on human immunodeficiency virus type 1. J. Virol. 72, 811–816.
KIRIK, D., and BJORKLUND, A. (2003). Modeling CNS neurodegeneration by overexpression of disease-causing proteins using viral
vectors. Trends Neurosci. 26, 386–392.
KIRIK, D., GEORGIEVSKA, B., and BJORKLUND, A. (2004). Localized striatal delivery of GDNF as a treatment for Parkinson disease. Nat. Neurosci. 7, 105–110.
KOBAYASHI, H., CARBONARO, D., PEPPER, K., PETERSEN, D.,
GE, S., JACKSON, H., SHIMADA, H., MOATS, R., and KOHN,
D.B. (2005). Neonatal gene therapy of MPS I mice by intravenous
injection of a lentiviral vector. Mol. Ther. 11, 776–789.
KOPONEN, J.K., KANKKONEN, H., KANNASTO, J., WIRTH, T.,
HILLEN, W., BUJARD, H., and YLA-HERTTUALA, S. (2003).
Doxycycline-regulated lentiviral vector system with a novel reverse
transactivator rtTA2S-M2 shows a tight control of gene expression
in vitro and in vivo. Gene Ther. 10, 459–466.
KORDOWER, J.H., EMBORG, M.E., BLOCH, J., MA, S.Y., CHU,
Y., LEVENTHAL, L., MCBRIDE, J., CHEN, E.Y., PALFI, S.,
ROITBERG, B.Z., BROWN, W.D., HOLDEN, J.E., PYZALSKI, R.,
TAYLOR, M.D., CARVEY, P., LING, Z., TRONO, D.,
HANTRAYE, P., DEGLON, N., and AEBISCHER, P. (2000). Neurodegeneration prevented by lentiviral vector delivery of GDNF in
primate models of Parkinson’s disease. Science 290, 767–773.
KOTSOPOULOU, E., KIM, V.N., KINGSMAN, A.J., KINGSMAN,
S.M., and MITROPHANOUS, K.A. (2000). A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits
a codon-optimized HIV-1 gag-pol gene. J. Virol. 74, 4839–4852.
8
KYRKANIDES, S., MILLER, J.H., BROUXHON, S.M.,
OLSCHOWKA, J.A., and FEDEROFF, H.J. (2005). -Hexosaminidase lentiviral vectors: Transfer into the CNS via systemic
administration. Brain Res. Mol. Brain Res. 133, 286–298.
LAUWERS, E., DEBYSER, Z., VAN DORPE, J., DE STROOPER,
B., NUTTIN, B., and BAEKELANDT, V. (2003). Neuropathology
and neurodegeneration in rodent brain induced by lentiviral vectormediated overexpression of -synuclein. Brain Pathol. 13, 364–372.
LO BIANCO, C., RIDET, J.L., SCHNEIDER, B.L., DEGLON, N., and
AEBISCHER, P. (2002). -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A. 99, 10813–10818.
LO BIANCO, C., DEGLON, N., PRALONG, W., and AEBISCHER,
P. (2004a). Lentiviral nigral delivery of GDNF does not prevent neurodegeneration in a genetic rat model of Parkinson’s disease. Neurobiol. Dis. 17, 283–289.
LO BIANCO, C., SCHNEIDER, B.L., BAUER, M., SAJADI, A.,
BRICE, A., IWATSUBO, T., and AEBISCHER, P. (2004b). Lentiviral vector delivery of parkin prevents dopaminergic degeneration in
an -synuclein rat model of Parkinson’s disease. Proc. Natl. Acad.
Sci. U.S.A. 101, 17510–17515.
LOIS, C., HONG, E.J., PEASE, S., BROWN, E.J., and BALTIMORE,
D. (2002). Germline transmission and tissue-specific expression of
transgenes delivered by lentiviral vectors. Science 295, 868–872.
MARR, R.A., ROCKENSTEIN, E., MUKHERJEE, A., KINDY, M.S.,
HERSH, L.B., GAGE, F.H., VERMA, I.M., and MASLIAH, E.
(2003). Neprilysin gene transfer reduces human amyloid pathology
in transgenic mice. J. Neurosci. 23, 1992–1996.
MAZARAKIS, N.D., AZZOUZ, M., ROHLL, J.B., ELLARD, F.M.,
WILKES, F.J., OLSEN, A.L., CARTER, E.E., BARBER, R.D., BABAN, D.F., KINGSMAN, S.M., KINGSMAN, A.J., O’MALLEY,
K., and MITROPHANOUS, K.A. (2001). Rabies virus glycoprotein
pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum.
Mol. Genet. 10, 2109–2121.
MCGREW, M.J., SHERMAN, A., ELLARD, F.M., LILLICO, S.G.,
GILHOOLEY, H.J., KINGSMAN, A.J., MITROPHANOUS, K.A.,
and SANG, H. (2004). Efficient production of germline transgenic
chickens using lentiviral vectors. EMBO Rep. 5, 728–733.
MILLER, V.M., XIA, H., MARRS, G.L., GOUVION, C.M., LEE, G.,
DAVIDSON, B.L., and PAULSON, H.L. (2003). Allele-specific silencing of dominant disease genes. Proc. Natl. Acad. Sci. U.S.A. 100,
7195–7200.
MISKIN, J., CHIPCHASE, D., ROHLL, J., BEARD, G., WARDELL,
T.M., ANGELL, D., ROEHL, H., JOLLY, D., KINGSMAN, S.M.,
and MITROPHANOUS, K. (2005). A replication competent
lentivirus (RCL) assay for equine infectious anemia virus (EIAV)based lentiviral vectors. Gene Ther. Oct 6; (Epub ahead of print).
MURAMATSU, S., FUJIMOTO, K., IKEGUCHI, K., SHIZUMA, N.,
KAWASAKI, K., ONO, F., SHEN, Y., WANG, L., MIZUKAMI,
H., KUME, A., MATSUMURA, M., NAGATSU, I., URANO, F.,
ICHINOSE, H., NAGATSU, T., TERAO, K., NAKANO, I., and
OZAWA, K. (2002). Behavioral recovery in a primate model of
Parkinson’s disease by triple transduction of striatal cells with adenoassociated viral vectors expressing dopamine-synthesizing enzymes.
Hum. Gene Ther. 13, 345–354.
NUTT, J.G., BURCHIEL, K.J., COMELLA, C.L., JANKOVIC, J.,
LANG, A.E., LAWS, E.R., Jr., LOZANO, A.M., PENN, R.D.,
SIMPSON, R.K., Jr., STACY, M., and WOOTEN, G.F. (2003). Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD. Neurology 60, 69–73.
PEDEN, C.S., BURGER, C., MUZYCZKA, N., and MANDEL, R.J.
(2004). Circulating anti-wild-type adeno-associated virus type 2
(AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated,
but not rAAV5-mediated, gene transfer in the brain. J. Virol. 78,
6344–6359.
WONG ET AL.
PFEIFER, A., IKAWA, M., DAYN, Y., and VERMA, I.M. (2002).
Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc.
Natl. Acad. Sci. U.S.A. 99, 2140–2145.
PLUTA, K., LUCE, M.J., BAO, L., AGHA-MOHAMMADI, S., and
REISER, J. (2005). Tight control of transgene expression by
lentivirus vectors containing second-generation tetracycline-responsive promoters. J. Gene Med. 7, 803–817.
RALPH, G.S., PARHAM, S., LEE, S.R., BEARD, G.L., CRAIGON,
M.H., WARD, N., WHITE, J.R., BARBER, R.D., RAYNER, W.,
KINGSMAN, S.M., MUNDY, C.R., MAZARAKIS, N.D., and
KRIGE, D. (2004). Identification of potential stroke targets by
lentiviral vector mediated overexpression of HIF-1 and HIF-2 in
a primary neuronal model of hypoxia. J. Cereb. Blood Flow Metab.
24, 245–258.
RALPH, G.S., MAZARAKIS, N.D., and AZZOUZ, M. (2005a). Therapeutic gene silencing in neurological disorders, using interfering
RNA. J. Mol. Med. 83, 413–419.
RALPH, G.S., RADCLIFFE, P.A., DAY, D.M., CARTHY, J.M., LEROUX, M.A., LEE, D.C., WONG, L.F., BILSLAND, L.G., GREENSMITH, L., KINGSMAN, S.M., MITROPHANOUS, K.A.,
MAZARAKIS, N.D., and AZZOUZ, M. (2005b). Silencing mutant
SOD1 using RNAi protects against neurodegeneration and extends
survival in an ALS model. Nat. Med. 11, 429–433.
RAMEZANI, A., HAWLEY, T.S., and HAWLEY, R.G. (2000).
Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol. Ther. 2, 458–469.
RAOUL, C., ABBAS-TERKI, T., BENSADOUN, J.C., GUILLOT, S.,
HAASE, G., SZULC, J., HENDERSON, C.E., and AEBISCHER, P.
(2005). Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of
ALS. Nat. Med. 11, 423–428.
REGULIER, E., TROTTIER, Y., PERRIN, V., AEBISCHER, P., and
DEGLON, N. (2003). Early and reversible neuropathology induced
by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum. Hum. Mol. Genet. 12, 2827–2836.
REISER, J., LAI, Z., ZHANG, X.Y., and BRADY, R.O. (2000). Development of multigene and regulated lentivirus vectors. J. Virol. 74,
10589–10599.
ROHLL, J.B., MITROPHANOUS, K.A., MARTIN-RENDON, E., ELLARD, F.M., RADCLIFFE, P.A., MAZARAKIS, N.D., and KINGSMAN, S.M. (2002). Design, production, safety, evaluation, and clinical applications of nonprimate lentiviral vectors. Methods Enzymol.
346, 466–500.
ROMERO, M.I., RANGAPPA, N., GARRY, M.G., and SMITH, G.M.
(2001). Functional regeneration of chronically injured sensory afferents into adult spinal cord after neurotrophin gene therapy. J. Neurosci. 21, 8408–8416.
RUITENBERG, M.J., PLANT, G.W., CHRISTENSEN, C.L., BLITS,
B., NICLOU, S.P., HARVEY, A.R., BOER, G.J., and VERHAAGEN, J. (2002). Viral vector-mediated gene expression in olfactory
ensheathing glia implants in the lesioned rat spinal cord. Gene Ther.
9, 135–146.
SASTRY, L., XU, Y., DUFFY, L., KOOP, S., JASTI, A., ROEHL, H.,
JOLLY, D., and CORNETTA, K. (2005). Product-enhanced reverse
transcriptase assay for replication-competent retrovirus and lentivirus
detection. Hum. Gene Ther. 16, 1227–1236.
SINGER, O., MARR, R.A., ROCKENSTEIN, E., CREWS, L.,
COUFAL, N.G., GAGE, F.H., VERMA, I.M., and MASLIAH, E.
(2005). Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat. Neurosci. 8,
1343–1349.
SIRIN, O., and PARK, F. (2003). Regulating gene expression using
self-inactivating lentiviral vectors containing the mifepristone-inducible system. Gene 323, 67–77.
STEFFENS, S., TEBBETS, J., KRAMM, C.M., LINDEMANN, D.,
9
LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS
FLAKE, A., and SENA-ESTEVES, M. (2004). Transduction of human glial and neuronal tumor cells with different lentivirus vector
pseudotypes. J. Neurooncol. 70, 281–288.
STEIN, C.S., MARTINS, I., and DAVIDSON, B.L. (2005). The lymphocytic choriomeningitis virus envelope glycoprotein targets
lentiviral gene transfer vector to neural progenitors in the murine
brain. Mol. Ther. 11, 382–389.
SUESS, B., HANSON, S., BERENS, C., FINK, B., SCHROEDER, R.,
and HILLEN, W. (2003). Conditional gene expression by controlling translation with tetracycline-binding aptamers. Nucleic Acids
Res. 31, 1853–1858.
TAI, M.H., CHENG, H., WU, J.P., LIU, Y.L., LIN, P.R., KUO, J.S.,
TSENG, C.J., and TZENG, S.F. (2003). Gene transfer of glial cell
line-derived neurotrophic factor promotes functional recovery following spinal cord contusion. Exp. Neurol. 183, 508–515.
TISCORNIA, G., TERGAONKAR, V., GALIMI, F., and VERMA,
I.M. (2004). CRE recombinase-inducible RNA interference mediated
by lentiviral vectors. Proc. Natl. Acad. Sci. U.S.A. 101, 7347–7351.
TUSZYNSKI, M.H., THAL, L., PAY, M., SALMON, D.P., U, H.S.,
BAKAY, R., PATEL, P., BLESCH, A., VAHLSING, H.L., HO, G.,
TONG, G., POTKIN, S.G., FALLON, J., HANSEN, L., MUFSON,
E.J., KORDOWER, J.H., GALL, C., and CONNER, J. (2005). A
phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat. Med. 11, 551–555.
VENTURA, A., MEISSNER, A., DILLON, C.P., MCMANUS, M.,
SHARP, P.A., VAN PARIJS, L., JAENISCH, R., and JACKS, T.
(2004). Cre–lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. U.S.A. 101, 10380–10385.
VIGNA, E., CAVALIERI, S., AILLES, L., GEUNA, M., LOEW, R.,
BUJARD, H., and NALDINI, L. (2002). Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol. Ther. 5, 252–261.
WATSON, D.J., KOBINGER, G.P., PASSINI, M.A., WILSON, J.M.,
and WOLFE, J.H. (2002). Targeted transduction patterns in the
mouse brain by lentivirus vectors pseudotyped with VSV, Ebola,
Mokola, LCMV, or MuLV envelope proteins. Mol. Ther. 5, 528–537.
WHITELAW, C.B., RADCLIFFE, P.A., RITCHIE, W.A., CARLISLE,
A., ELLARD, F.M., PENA, R.N., ROWE, J., CLARK, A.J., KING,
T.J., and MITROPHANOUS, K.A. (2004). Efficient generation of
transgenic pigs using equine infectious anaemia virus (EIAV) derived vector. FEBS Lett. 571, 233–236.
WONG, L.F., AZZOUZ, M., WALMSLEY, L.E., ASKHAM, Z.,
WILKES, F.J., MITROPHANOUS, K.A., KINGSMAN, S.M., and
MAZARAKIS, N.D. (2004). Transduction patterns of pseudotyped
lentiviral vectors in the nervous system. Mol. Ther. 9, 101–111.
WONG, L.F., RALPH, G.S., WALMSLEY, L.E., BIENEMANN, A.S.,
PARHAM, S., KINGSMAN, S.M., UNEY, J.B., and MAZARAKIS,
N.D. (2005). Lentiviral-mediated delivery of Bcl-2 or GDNF protects against excitotoxicity in the rat hippocampus. Mol. Ther. 11,
89–95.
WONG, L.F., YIP, P.K., BATTAGLIA, A., GRIST, J., CORCORAN,
J., MADEN, M., AZZOUZ, M., KINGSMAN, S.M., KINGSMAN,
A.J., MAZARAKIS, N.D., and MCMAHON, S.B. (2005). Retinoic
acid receptor 2 promotes functional regeneration across the dorsal
root entry zone. Nat. Neurosci. (in press).
XIA, H., MAO, Q., and DAVIDSON, B.L. (2001). The HIV Tat protein transduction domain improves the biodistribution of -glucuronidase expressed from recombinant viral vectors. Nat. Biotechnol. 19, 640–644.
YE, X., RIVERA, V.M., ZOLTICK, P., CERASOLI, F., Jr.,
SCHNELL, M.A., GAO, G., HUGHES, J.V., GILMAN, M., and
WILSON, J.M. (1999). Regulated delivery of therapeutic proteins
after in vivo somatic cell gene transfer. Science 283, 88–91.
ZALA, D., BENCHOUA, A., BROUILLET, E., PERRIN, V., GAILLARD, M.C., ZURN, A.D., AEBISCHER, P., and DEGLON, N.
(2005). Progressive and selective striatal degeneration in primary
neuronal cultures using lentiviral vector coding for a mutant huntingtin fragment. Neurobiol. Dis. 20, 785–798.
ZHANG, Y., DIJKHUIZEN, P.A., ANDERSON, P.N., LIEBERMAN,
A.R., and VERHAAGEN, J. (1998). NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the
spinal cord of adult rats. J. Neurosci. Res. 54, 554–562.
ZUFFEREY, R., DULL, T., MANDEL, R.J., BUKOVSKY, A.,
QUIROZ, D., NALDINI, L., and TRONO, D. (1998). Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.
J. Virol. 72, 9873–9880.
Address reprint requests to:
Dr. Liang-Fong Wong
Oxford BioMedica (UK) Ltd.
Medawar Centre
Robert Robinson Avenue
The Oxford Science Park
Oxford OX4 4GA, UK
E-mail: [email protected]
Received for publication October 3, 2005; accepted after revision November 11, 2005.
Published online: December 15, 2005.