Human Molecular Genetics, 2011, Vol.

20, Review Issue 1 R28–R41

doi:10.1093/hmg/ddr110
Advance Access published on March 23, 2011

Genetic therapy for the nervous system

William J. Bowers 1, Xandra O. Breakefield 2,3,∗ and Miguel Sena-Esteves 4
1
Department of Neurology, Center for Neural Development and Disease, University of Rochester,
School of Medicine and Dentistry, Rochester, NY 14642, USA, 2Neuroscience Center and Molecular Neurogenetics
Unit, Department of Neurology and 3Center for Molecular Imaging Research, Department of Radiology,
Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA
and 4Department of Neurology, Gene Therapy Center, Interdisciplinary Graduate Program, University of

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Massachusetts Medical School, Worcester, MA 01605, USA

Received February 1, 2011; Revised and Accepted March 11, 2011

Genetic therapy is undergoing a renaissance with expansion of viral and synthetic vectors, use of oligonu-
cleotides (RNA and DNA) and sequence-targeted regulatory molecules, as well as genetically modified
cells, including induced pluripotent stem cells from the patients themselves. Several clinical trials for neuro-
logic syndromes appear quite promising. This review covers genetic strategies to ameliorate neurologic
syndromes of different etiologies, including lysosomal storage diseases, Alzheimer’s disease and other amy-
loidopathies, Parkinson’s disease, spinal muscular atrophy, amyotrophic lateral sclerosis and brain tumors.
This field has been propelled by genetic technologies, including identifying disease genes and disruptive
mutations, design of genomic interacting elements to regulate transcription and splicing of specific precur-
sor mRNAs and use of novel non-coding regulatory RNAs. These versatile new tools for manipulation of
genetic elements provide the ability to tailor the mode of genetic intervention to specific aspects of a disease
state.

INTRODUCTION METHODS OF GENETIC INTERVENTION

Genetic therapy covers a range of methods for modifying the
nervous system, including delivery of genes, sequence-
targeted regulatory molecules, genetically modified cells and
oligonucleotides. In this review, we focus on ways to change
the genetic status of the nervous system, including routes of
access and modes of delivery. Examples are provided of strat-
egies used for a number of diseases, including recessive loss of
function conditions [lysosomal storage diseases and spinal
muscular atrophy (SMA)], dominant toxic mutations [amyo-
trophic lateral sclerosis (ALS)] and conditions of mixed etiol-
ogy [Alzheimer’s disease (AD), Parkinson’s disease (PD) and
brain tumors] (Fig. 1). Neurodegenerative diseases caused by
triplet nucleotide repeats are discussed in an article on
siRNAs in this issue. Many of these strategies have proven
very promising in preclinical studies in mouse and larger
animal models, and a substantial number are now being eval-
uated in clinical trials.
Routes of access
Entrance to the central nervous system (CNS) is limited by the
skeletal structures that enclose it and the blood – brain barrier
(BBB), which plays a central role in regulating the chemical
microenvironment of the CNS by preventing simple diffusion
of most small molecules and proteins from the bloodstream.
The physical barrier properties of the BBB are a result of
the tight junctions between brain microvascular endothelial
cells and low vesicular transport (1; Fig. 2). As a result,
most molecular traffic in and out of the CNS is tightly regu-
lated by specialized transporter systems present on the
luminal and abluminal membranes of the endothelial cells,
with the exception of small solutes such O2 and CO2 gases
and some lipophilic molecules (e.g. ethanol) that can diffuse
freely across cellular membranes. Other components of the
BBB are the basal lamina, astrocyte end-feet that surround

∗
To whom correspondence should be addressed at: Molecular Neurogenetics Unit, Massachusetts General Hospital-East, 13th Street, Building 149,
Charlestown, MA 02129, USA. Tel: +1 6177265728; Fax: +1 6177241537; Email: breakefield@hms.harvard.edu

# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1 R29

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Figure 1. Therapeutic strategies based on genetic etiology. (A) Recessive disease. In the case of a recessively inherited disease where both alleles (or one on the
X chromosome in males) of a gene are mutated, the goal is to replace the defective gene with a functional counterpart or to correct the defect. Typically, a vector
is used to deliver a promoter–cDNA expression cassette which either integrates into the genome (1), e.g. lentivirus vector, or resides as an extrachromosomal
element (4), e.g. AAV vector. Other approaches include attempts to achieve homologous recombination with the resident allele (4) or, if appropriate, to correct
the transcript through transplicing of the precursor mRNA (3). (B) Dominant disease. In the case of dominantly inherited diseases with only one defective neu-
rotoxic gene, a common strategy is to block expression of the mutant message through RNAi (1) which can be delivered either as an shRNA encoded in a vector
or as an oligonucleotide. Another approach is to try to neutralize damage caused by the mutant protein through the use of targeted antibodies (2). (C) Multi-
factorial dysfunction. For many neurodegenerative diseases, there is no one gene that can be targeted to alleviate the condition. Approaches include trying
to supplement the neurotransmitter capacity of ‘sick’ neurons by providing them with enzymes to generate more of their normal neurotransmitter for release
at synapses (1). Alternately, the neurotransmitter profile of interacting neurons can be altered to change their output from excitatory to inhibitory, or vice
versa (2). In another common scenario, cells in the vicinity are genetically modified to produce a growth factor which is supportive for the sick neurons (3).

all CNS blood vessels and pericytes. Interestingly, pericytes
appear to play a central role in regulating BBB permeability
via their influence over endothelial cells and astrocytes (2).
Not surprisingly, the BBB has proved exceptionally efficient
in excluding the vast majority of gene transfer vehicles
from reaching the CNS via the vasculature. As a result, most
CNS gene therapy approaches have utilized direct infusion
of gene transfer vectors into the brain parenchyma to
target disease-relevant structures. The distribution of viral
vectors in the brain can be improved considerably by
convection-enhanced delivery (CED), a slow pressurized infu-
sion of a larger volume (3,4). Nonetheless, transduction of
CNS cells after intraparenchymal injection of viral vectors
remains mostly limited to the targeted structure.
An alternative route of entry into the CNS is by delivery of
the gene transfer vectors directly into cerebrospinal fluid
(CSF) via either the lateral ventricles (5) or intrathecal space
(6). This approach is exceptionally effective in achieving
widespread transduction in the neonatal mouse brain (5,7),
but the distribution pattern is more restricted in adult
animals (6,8,9). This suggests that there may be additional bar-
riers to distribution of gene transfer vectors from CSF into
adult brains, or that commonly used injection procedures
disrupt normal CSF flow preventing widespread distribution.
Delivery of recombinant lysosomal enzymes into CSF
appears to be effective in providing therapeutic levels of
these enzymes throughout the CNS (10,11). Similarly, CSF
infusion of antisense oligonucleotides (ASOs) directed at
altering the splicing pattern of the SMN2 gene has been dra-
matically effective in a mouse model of SMA (see below).
An ideal route of entry into the CNS to achieve widespread
gene transfer would be through the vasculature. Until recently,
the only exception to the BBB-imposed block to gene therapy
vectors was PEGylated immunoliposomes (PILs) formulated
with monoclonal antibodies specific for receptors, such as
transferrin and insulin receptors which mediate transcytosis
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Figure 2. Routes of gene delivery to the CNS. CED of viral vectors into the brain improves considerably their distribution in target structures and hence transduc-
tion volumes. This technique can yield volumes of transduced cell distribution 3 –3.5-fold larger than the infused volume, which is highly significant for human
applications. Viral vectors or secreted transgene products (growth factors, lysosomal enzymes) can be further distributed from the primary target structure by
axonal transport (top left diagram). Infusion of recombinant proteins or oligonucleotides into the brain ventricular system, or intrathecal space, leads to wide-
spread CNS distribution via CSF flow. An alternative strategy is to use viral vectors to engineer ependymal cells lining the ventricles or choroid plexus cells to
secrete therapeutic proteins into CSF (top right diagram). The BBB with its many constituents has thwarted most gene transfer vehicles from entering the brain
from the vasculature. In recent years, PILs and a new generation of viral vectors (AAV and SV40) have been shown to mediate efficient CNS gene transfer after
i.v. infusion in newborn and adult animals (bottom right diagram).

of their ligands across the BBB. These PILs appear to be quite
effective in delivering expression plasmids (with expression
under cell type-specific promoters) and RNAi to normal
brain or brain tumors (12). In a parallel approach, therapeutic
proteins are delivered to the CNS using chimeric recombinant
molecules, e.g. growth factors, single-chain antibodies or lyso-
somal enzymes, fused to receptor-targeting monoclonal anti-
bodies (12) or ligands, such as transferrin (13). Recently,
adeno-associated virus 9 (AAV9) vectors have been found to
enter the CNS of neonatal mice and young cats after intravas-
cular (i.v.) infusion and to transduce large numbers of glia and
motor neurons in the spinal cord (14,15). Transduction of
other neuronal populations in the brain is found mostly in
the hippocampus and Purkinje cells in the cerebellum (14).
SV40 recombinant vectors also appear to mediate efficient
gene transfer to certain regions of the CNS after i.v. infusion
in adult mice combined with intraperitoneal mannitol infusion
(16). Alternative approaches to deliver secretable therapeutic
proteins to the CNS (e.g. lysosomal enzymes, growth
factors, cytokines, tumor killing agents) are to target gene
transfer to brain microcapillary endothelial cells (17), or use
ex vivo genetically modified stem cells [hematopoietic stem
cells (HSCs), neural stem cells, mesenchymal stem cells]
which will themselves, or their progeny in the case of HSCs,
migrate within the brain to regions of injury or tumors
(18 –20). In fact, the whole nervous system can be transduced
with a gene for the lifespan by injecting a lentivirus vector into
the amniotic sac of mouse embryos before the neural groove
has closed (21).
DNA/RNA
The use of non-viral nucleic acid delivery to selectively
modify cellular processes within the brain presents a number
of challenges, including target cell specificity and transient
gene expression duration. Pardridge and colleagues (22)
have developed PILs target to the brain when administered
intravenously (see above). Stachowiak et al. (23) have recently
 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1
reported the use of organically modified silica-based nanopar-
ticles to induce neurogenesis within the subventricular zone of
adult mice via intraventricular delivery of DNA encoding a
recombinant nuclear form of fibroblast growth factor
receptor-1. Electroporation-based nucleic acid transfection
has also been extensively documented in the prenatal and post-
natal rodent brain [reviewed by De Vry (24)], providing the
means to genetically modify significant numbers of neurons
(25,26) within the living brain (27).
Viral vectors
Recombinant viral vector systems remain the most efficient
vehicles to achieve long-term stable gene expression in the
CNS. Over the years, many different viral vector systems
have been investigated for this purpose, including those
derived from herpes simplex virus type 1 (HSV-1), adeno-
virus, AAV, lentiviruses—such as HIV-1, feline immunode-
ficiency virus or equine infectious anemia virus, and more
recently SV40. AAV and lentivirus vectors have emerged
as the vectors of choice for gene transfer to the CNS for
non-oncological applications as they mediate efficient long-
term gene expression with no apparent toxicity. A recent
study has shown that AAV-mediated transgene expression
in the primate brain continues for at least 8 years with no
evidence of neuroinflammation or reactive gliosis (28).
Moreover, several clinical trials have shown that direct infu-
sion of AAV2 vectors into brain parenchyma in humans is
well tolerated (29– 33). The safety profile of direct infusion
of lentivirus vectors into human brain remains to be evalu-
ated. High-capacity adenovirus vectors are attractive
because of their large transgene capacity (30 kb) and
ability to mediate long-term gene expression without
immunological complications (34). These and the large
capacity HSV amplicon vectors (150 kb; 35) may be ideal
choices to transfer large genomic regions necessary to
achieve physiological regulation of gene expression for par-
ticular genes/sets of genes in specific cell populations in the
CNS. Full-length gene copies with intact regulatory elements
(.100 kb) can by delivered in HSV-1 amplicon vectors
(36), verging on the capacity to deliver virtual mini-
chromosomes (37). The main difficulty for high-capacity
adenovirus and HSV-1 amplicon vectors lies in difficulties
in large-scale production. Recombinant SV40 vectors
display some promising properties, namely their apparent
ability to cross the adult mouse BBB after i.v. infusion
and transduce neurons and astrocytes in specific brain
regions and spinal cord (16). Moreover, these vectors can
be administered repeatedly as they do not seem to elicit neu-
tralizing antibodies in rodents. Whether this property is
shared in other mammalian species remains to be deter-
mined. In the neuro-oncology field, recombinant vectors
derived from HSV-1, adenovirus, measles virus and Newcas-
tle virus appear to be the most promising in pre-clinical
models of brain tumors (see below). After initial attempts
to broadly use each viral vector system for any/all CNS
gene transfer applications, now the particular strengths of
each system are being exploited to meet the specific needs
of different applications/diseases.
R31
EXAMPLES OF APPLICATIONS TO SPECIFIC
DISEASES
Lysosomal storage diseases
Lysosomal storage diseases are typically, but not exclu-
sively, childhood diseases resulting from a genetic deficiency
in a lysosomal enzyme involved in a particular metabolic
pathway that results in lysosomal accumulation of its sub-
strate(s). Many of these enzymes can be secreted from
cells genetically engineered to overexpress them and then
taken up by enzyme-deficient cells and correctly targeted
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to lysosomes via mannose-6-phosphate receptors where
they degrade the stored substrate(s). This mechanism,
known as cross-correction (38), is the basis for enzyme
replacement therapy which is now available for a subset of
these diseases without neurological involvement. Unfortu-
nately, the BBB prevents recombinant lysosomal enzymes
infused peripherally from entering the CNS. Alternative
routes of delivery and molecules are being actively investi-
gated (see above). These diseases are particularly well
suited for gene therapy as they are monogenic diseases
with very well established genotype – phenotype correlations,
and the cross-correction mechanism makes it possible to
devise gene delivery strategies to supply essentially the
entire CNS with therapeutic levels of these enzymes. Two
main approaches have shown dramatic therapeutic effects
in animal models of lysosomal storage diseases, namely
intraparenchymal infusion of recombinant viral vectors
(39– 46), and bone marrow transplantation with ex vivo
lentivirus vector modified-autologous HSCs (see article by
Biffi, Cartier and Aubourg in this issue).
The ability of focal viral vector-mediated gene delivery to
supply the CNS with therapeutic levels of lysosomal
enzymes is dependent on widespread distribution based on dif-
fusion (47), axonal transport over long distances (48,49) and
CSF flow in the perivascular space (8). AAV-mediated
genetic modification of highly interconnected structures in
the brain, such as deep cerebellar nuclei (50), ventral tegmen-
tal area (51) or thalamus (4,52), leads to widespread distri-
bution of these enzymes in the CNS. Alternative targets,
such as the external capsule (53) or lateral ventricles (8),
take advantage of either interstitial fluid flow or CSF flow
for distribution of lysosomal enzymes throughout the CNS.
Successful translation of AAV-based (or lentivirus-based)
approaches to humans will likely require targeting one or
more of these structures to achieve therapeutic levels of
these enzymes throughout the CNS. Pre-clinical studies in
large animal models of some of these diseases are highly
encouraging (54,55). Bone marrow transplantation with
lentivirus-modified autologous HSCs has shown exceptional
results in different mouse models of lysosomal storage dis-
eases resulting in correction of pathologic findings throughout
the CNS (18,56,57), as well as peripheral organs (58). This
approach relies on genetically modified HSC-derived cells
(macrophages in the case of CNS) trafficking to the sites of
disease and becoming an in situ source of recombinant
enzyme. Both of these gene therapy approaches are now
being tested in human clinical trials for different lysosomal
storage diseases.
R32 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1

Alzheimer’s disease and other amyloidopathies the substantia nigra innervating the striatum in the basal
ganglia (85). Although environmental toxins and aging are
AD is a neurodegenerative disorder characterized by severe
risk factors, genetic susceptibility is critical with 16 gene
memory loss and cognitive impairment with no available
loci implicated in 5% of cases, and multifactorial hereditary
cure. Neuropathological correlates include extracellular
risk in many others (85,86). These risk factors point to oxi-
amyloid-beta peptide deposition, intracellular neurofibrillary
dative stress, mitochondrial dysfunction and reduced ability
tangle formation, decreased synaptic integrity and neuronal
to degrade abnormal proteins as etiologic factors. Although
loss. The basal forebrain cholinergic complex is significantly
L-dopa has been used for decades to relieve motor symptoms
affected by AD-related neurodegeneration (59– 63). To
of PD, it does not prevent degeneration and eventually ceases
augment cholinergic function, Tuszynski and colleagues
to be effective.
(64– 68) delivered nerve growth factor (NGF), the prototypical
New gene/cell therapy strategies have been explored exper-
neurotrophin with demonstrated neuroprotective properties,
imentally with some translated into clinical trials, including:

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using retrovirus vector-transduced fibroblast grafts and
delivery or upregulation of neurotrophic factors, generation
demonstrated restoration and survival of cholinergic neurons
of endogenous dopamine and alterations in neuronal circuitry,
in lesioned rodents and aged non-human primates. These
as well as implantation of supportive cells and dopaminergic
initial studies set the stage for the first phase I clinical trial
neurons. Gene delivery has typically been carried out using
of ex vivo NGF gene therapy (ClinicalTrials.gov Identifier:
AAV vectors with CED injection into the striatum. Vectors
NCT00017940). The rate of cognitive decline was slowed,
have delivered both glial-derived neurotrophic factor
and no apparent detrimental effects were observed arising
(GDNF; 87) and its analogue, neurturin (88), which serve as
from NGF expression 22 months post-engraftment (69).
trophic factors for dopaminergic neurons. Since abnormally
More recently, Ceregene has conducted phase I and II clinical
high GDNF can have toxic effects, efforts have gone into reg-
trials using an AAV vector that expresses NGF (Clinical-
ulating its expression through a tetracycline analogue system
Trials.gov Identifiers: NCT00087789 and NCT00876863,
(89) and by using highly specific zinc finger transcription
respectively). While the phase I trial demonstrated that stereo-
factors to upregulate expression of the endogenous gene,
tactic infusions of an NGF-expressing AAV vector is well tol-
with the latter imposing a physiologically controlled upper
erated, it is too early to know whether this gene therapy-based
limit (90).
strategy will significantly impact the course and symptomol-
In terms of modulating neurotransmitter pathways, all
ogy of the disease, given the phase II trial is currently ongoing.
three biosynthetic enzymes for dopamine have been included
During the past several years, promising efforts have
in the same lentivirus vector, including tyrosine hydroxylase
focused on reducing the levels of neurotoxic Ab peptide
(TH) and aromatic amino acid decarboxylase (AADC), as
species within the brain through removal of pathogenic Ab
well as GTP cyclohydrolase for synthesis of the biopterin
peptides or halting the proteolytic release of the amyloido-
co-factor for TH, with the assumption that dopamine, as a
genic form of Ab arising from pathogenic amyloid precursor
paracrine neurotransmitter, can be made by any cell in the
protein (APP) processing. Such preclinical vector-based thera-
striatum (91). However, serotonergic or GABAergic
pies include active and passive vaccines directed against
neurons can also produce dopamine as a false transmitter
specific epitopes of pathogenic Ab peptides (70– 75),
leading to dyskinesia (92). AAV-mediated delivery of
expression of Ab proteases (75– 78), delivery of small inhibi-
AADC alone has the advantage of making production of
tory RNAs (RNAi) designed to specifically suppress the
dopamine dependent on L-dopa intake, with ongoing phase
expression of APP processing enzymes (79 –82) and delivery
I trials indicating continued expression of AADC for at
of a cholesterol degrading enzyme into the brain (83). Each
least 2 years in the human brain (93). Investigators have
experimental approach has exhibited strong preclinical effi-
also tried to block hyperactive neurotransmission in PD
cacy in rodent models of AD, hence increasing enthusiasm
brains using an AAV vector to deliver the enzyme glutamic
for eventual translation of one or more of these strategies to
acid decarboxylase for synthesis of the inhibitory neurotrans-
clinical testing. The common challenge shared by gene thera-
mitter GABA into the subthalamic nucleus (94). Cell thera-
peutic approaches for AD which require intraparenchymal
pies have included a number of cell types, such as human
delivery relates to sufficiency of brain tissue coverage.
fetal mesencephalic tissue, which includes precursors of
Given AD impacts a number of human brain sub-regions
dopaminergic and serotonergic neurons, with the current
that are integral to learning and memory, viral vector strategies
focus on human dopaminergic neuroblasts generated from
must include the means to monitor vector infusion in real time
stem cells, in particular induced pluripotent stem (iPS)
to widely and safely disseminate vector particles within
cells derived from adult tissue (95).
afflicted networks. Recent advances in real-time monitoring
of CED-mediated AAV vector infusions within the non-human Strangely, few of these therapies address the etiology of the
primate brain indicate that this delivery hurdle can be disease based on genetic insights, and in fact most animal
overcome (84). models of PD employ lesioning of dopaminergic neuronal
connections, with the exception of transgenic mice overex-
pressing alpha-synuclein and recently developed models of
other PD genetic syndromes (96). Since three copies of the
Parkinson’s disease alpha-synuclein gene alone can cause PD (97) and upregulated
PD is a common progressive neurodegenerative disease. While alpha-synuclein inhibits neurotransmitter release (98), a
many regions of the brain are affected, the primary motor logical approach would be to decrease alpha-synuclein syn-
symptoms are caused by the loss of dopaminergic neurons in thesis with RNAi (84) or increase its degradation with the
 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1
ubiquitin ligase, parkin (99). However, both the increased
and decreased levels alpha-synuclein can cause neurodegen-
eration (84).
Spinal muscular atrophy
SMA is an autosomal recessive disease caused by the loss of
function of the ‘survival of motor neuron’ gene, SMN1,
which leads to degeneration of motor neurons and infant mor-
tality. One approach to gene therapy would be to replace the
missing gene in multiple motor neurons, which may now be
possible with i.v. administration of AAV9 vectors which
were able to deliver the SMN1 cDNA to the spinal cord in a
mouse model of SMA with marked correction of motor func-
tion and a dramatic increase in survival (100,101). However,
scaling delivery from mice to humans remains a huge chal-
lenge. Other strategies have taken advantage of the presence
of a second copy of this gene, SMN2, coding for an identical
amino acid sequence in the human genome (102). The
SMN2 gene is defective due to a point mutation in an intron
which interferes with correct splicing, such that a truncated,
non-functional protein is produced. In one therapeutic
modality, an ASO homologous to the last translated exon in
SMN2 is used to guide an exonic splicing enhancer sequence
to the region as a binding platform for pre-mRNA splicing
factors (103). This strategy gives increased SMN2 expression
when infused into the lateral cerebral ventricles in an SMA
mouse model (104,105). In another modality, transplicing is
utilized in which the ASO binds to the defective intronic
sequence and carries a splicing domain and an intact final
exon from SMN1, with injection into the cerebral ventricles
also extending survival in a mouse model of SMA (106).
Amyotrophic lateral sclerosis
ALS is characterized by progressive muscle weakness result-
ing from the loss of motor neurons in the brain and spinal
cord. Mutations in the SOD1 gene encoding cytosolic Cu/Zn
superoxide dismutase were the first to be linked to familial
forms of ALS (fALS), 10% of all cases. In recent years,
there has been a dramatic increase in the number of genes
shown to be associated with fALS, such as ANG encoding
angiogenin (107), TARDP encoding transactive response
(TAR) DNA-binding protein TDP-43 (108), FUS encoding
fused in sarcoma protein (also known as TLS for translocated
in liposarcoma) (109,110) and, more recently, optineurin
(111). Several other genes have been associated with rare
forms of fALS (reviewed in 112). The disease mechanism(s)
remains elusive. However, the fact that many fALS cases
are autosomal dominant suggests that disease-associated
mutations generate protein species with toxic functions
instead of simple loss of function. An emerging picture in
the field is that some of the fALS-associated proteins may
also be involved in sporadic ALS (sALS) cases. As an
example, recent work has shown that oxidized wild-type
SOD1 shares a conformational epitope with fALS-associated
mutant SOD1 (113), and can be found in spinal cord motor
neurons of a subset of sALS patients (114). Moreover, oxi-
dized wild-type SOD1 and mutant SOD1 share toxic proper-
ties to neurons by inhibition of kinesin-dependent fast
R33
axonal transport (114). Also, misfolded SOD1 mutants
have been shown to directly bind and inhibit mitochondrial
voltage-dependent anion channel (VDAC1) leading to mito-
chondrial dysfunction (115). Taken together, these results
suggest that conformational changes in SOD1 (and possibly
also other fALS-associated proteins), caused by mutations
associated with some forms of fALS, and other as yet undeter-
mined factors, may be the basis for fALS and sALS. Gene
therapy approaches for ALS have centered on delivery of
growth factors such as IGF-1 (116– 118) and vascular endo-
thelial growth factor (116,119), and RNAi-based silencing of
mutant SOD1 alleles (120 – 123) in transgenic ALS mouse
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models. The most commonly used ALS mouse model carries
a human SOD1 G93A transgene expressed at high levels
with a mean survival of 129 days. To date, many of the
gene therapy studies have demonstrated a significant increase
in median survival, but ultimately all treated mice have suc-
cumbed to disease progression. The reasons for the apparent
failure of those interventions can be multifactorial, but an
interesting aspect to consider is the fact that silencing
mutant SOD1 expression in motor neurons delays disease
onset but not progression, while silencing it in microglia
does the opposite (124). This suggests that other CNS cell
types contribute significantly to the disease phenotype. Devel-
opment of new mouse models using mutant alleles from other
ALS genes will enhance our understanding of common disease
pathways and development of effective gene therapies for this
disease. The recent findings on the toxicity of misfolded SOD1
proteins (normal or mutant) strongly suggest that either immu-
nization (125), or passive immunization with conformation-
specific antibodies (126) may also be viable approaches to
develop effective therapies for ALS.
Brain tumors
Malignant glioma tumors (glioblastoma; GBM) are the most
common adult brain tumor and are virtually untreatable,
with most patients dying within 2 years of diagnosis in spite
of neurosurgery, radiation and chemotherapy. These tumors
are very invasive in the brain and genetically heterogeneous
with a cancer stem cell population that defies all current che-
motherapies (127). Some benign tumors of the nervous
system, e.g. vestibular schwannomas, regress in response to
anti-angiogenic therapy (128), while for GBM tumors this
treatment can enhance invasiveness without suppressing pro-
liferation (129).
Treatment of malignant brain tumors presents a major thera-
peutic challenge requiring multicombinatorial approaches,
with gene/cell therapy showing promise as adjunct therapies.
Experimental therapies in brain tumor models have focused
on direct elimination of tumor cells, including a ‘bystander’
effect to confer selective toxicity to non-transduced tumor
cells in the vicinity and on targeting invasive tumor cells.
The basic strategy is to remove the bulk of the tumor mass,
and during that intervention to inject virus vectors or cells
into the resected region to kill remaining tumor cells over an
expanded radius. Concepts employed in this arsenal include:
(i) Prodrug activation (or suicide genes). In this case, viral
vectors or cells are used to express enzymes, such as viral thy-
midine kinase (130), bacterial cytosine deaminase/uracil
R34 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1
phosphoribosyltransferase (131) or mammalian cytochrome
P450/carboxyesterase (132) within the brain to activate pro-
drugs (ganciclovir, 5-fluorocytosine, cyclophosphamide/irino-
tecan, respectively) that can pass the BBB and be converted
into active chemotherapeutic agents within the tumor. (ii)
Viral oncolysis. In this approach mutant forms of viruses, typi-
cally HSV-1 and adenovirus, are used which replicate selec-
tively in tumor versus normal brain based on cancer-related
mutations or their increased proliferation rate (133). These
oncolytic vectors can also be armed with therapeutic genes.
An increasing number of different viruses are being tested in
this context, including measles virus, reovirus, Newcastle
disease virus (134), polio virus (135) and vaccinia virus
(136). Although promising, this field is still wrestling with
immune responses to the virus and an unfavorable tumor
microenvironment which restrict virus spread within the
tumor (134). (iii) Cellular delivery. Several studies have
shown that some normal cells introduced into the brain, such
as neural stem cells (137) and mesenchymal stem cells (138)
migrate towards tumor foci. These cells can deliver agents
which are selectively toxic to tumor cells, e.g. antibodies
against tumor antigens (139,140), oncolytic virus vectors
(141,142), apoptotic factors (143) and anti-angiogenic proteins
(144), as well as prodrug activating enzymes. (iv) Immu-
notherapy to target tumor antigens. Several strategies have
been used to increase recognition of tumor antigens and
empower the immune system. These include vaccination
with a common and unique GBM antigen, EGFRvIII (145)
and co-treatment with oncolytic HSV vectors and cyclopho-
sphamide to temporarily suppress the immune curtailment of
virus spread, and inclusion of immune enhancing cytokines
(146). In addition, T cells have been modified to express chi-
meric receptors targeting antigens, such as the IL13 receptor
which is abundant on GBM tumors (147). (v) Zone of resist-
ance. Other efforts have tried to increase the resistance of
the brain to tumor invasion, including the use of AAV
vectors to infect normal brain cells so they release interferon-
beta which depresses tumor growth (148).
FUTURE INITIATIVES
New genetic manipulation methods
A number of new genetic strategies are being explored to
manipulate the endogenous cell genome in order to regulate
expression or correct mutated gene transcripts. In one
modality, zinc finger proteins (ZFPs) which recognize a typi-
cally 18 bp site in the promoter region of a gene are linked to a
transcriptional activator (149) to selectively turn on a gene, as
in the case of SMN2 (above; 90). This approach can also be
used to stimulate gene correction by linking the ZFP to a
nuclease causing double-stranded breaks in the DNA in com-
bination with a correct extrachromosomal DNA sequence such
that homologous recombination is stimulated (149). Another
potent tool that can be linked to ZFPs are meganucleases
which recognize .12 bp sequences and have been shown to
inactivate HIV sequences (150). Other methods to facilitate
gene correction have included triplex-forming oligonucleo-
tides which can be used to modify sequence or inhibit gene
expression (151). Gene correction has also been achieved
using single-stranded DNA (73 bp; 152). In addition, there is
the potential to deliver human artificial mini-chromosomes
into the cell nucleus for gene-deficiency syndromes
(153,154). These strategies include control of transplicing,
for example in SMA therapies (see above). Naturally occur-
ring and synthetic transposable elements, including the Sleep-
ing Beauty transposon, have shown promise in stable
integration and gene expression in cells, including neurons
in vivo, and are discussed in more detail elsewhere in this
issue. All these more ‘advanced’ manipulations of the
genome will depend on large vector capacity and more effi-
cient delivery to the nervous system. A new arena will be
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modulation of miRNA levels which have such an important
role in development and cancer, including decreasing levels
of oncogenic miRNAs and increasing levels of tumor suppres-
sor miRNAs (155). Epigenetic modulation of the genome is an
expanding arena, with for example histone deacetylase inhibi-
tors being used to slow down degeneration in mouse models of
Huntington’s disease (156).
Improved vectors
The new breed of viral vectors that can reach the CNS after
peripheral administration (14,16) also transduce peripheral
organs at high efficiency, namely the liver. This potential
limitation could be simply addressed with the use of
CNS-specific promoters such as the synapsin-1, neuron-
specific enolase or glial fibrillary acidic protein promoters.
However, some of these promoters are relatively weak and
some are rather large, thus reducing considerably the trans-
gene capacity of the vectors. An alternative approach is to
incorporate into the transgene expression cassette targets for
miRNAs expressed at high levels in peripheral organs but
absent or expressed at low levels in the CNS to prevent vector-
mediated gene expression outside of the CNS (157, see
below). This approach appears to be quite universal to
de-target vector-mediated expression from specific cell
lineages (57,158,159). Another approach to addressing the
undesired targeting of peripheral organs consists in engineer-
ing the viral vector particles themselves. Lentivirus vectors
can be pseudotyped with envelope proteins derived from
other viruses, and the vesicular stomatitis virus glycoprotein
is the most commonly used due to its pantropism. Previous
studies have shown that incorporation of the rabies glyco-
protein allows lentivirus vectors administered peripherally to
reach the CNS via retrograde axonal transport (119,160). Non-
integrating lentivirus vectors can also be used effectively in
the neural cells, thus reducing the risk of oncogenic insertion
events (161).
Development of CNS-targeted AAV vectors has followed
three different routes: (i) in recent years hundreds of new
AAV capsids have been identified from humans and quite a
few other species, and systematic screening for new
CNS-targeting properties may yet yield new AAV pseudo-
types more powerful than AAV9; (ii) chimeric AAV2
capsids carrying CNS-targeting peptides identified from in
vivo phage display experiments can increase delivery. This
approach had met with limited success until recently Chen
et al. (17) showed that it can be used to develop AAV
vectors highly specific for brain microcapillary endothelial
 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1 R35

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Figure 3. Schematic representation of existing cellular therapies, including iPS cells, which require vector-based strategies to generate neuroprecursor cells and
neurons to ultimately treat neurodegenerative diseases. A variety of cellular therapies have been devised to repair and replace degenerated neuronal networks
within the CNS. These strategies utilize multiple sources of neurons and neuroprecursor cells, including fetal brain, pre-implantation embryos, mesenchymal
stem cells and iPS cells. The generation of iPS cells requires the use of viral vectors to deliver multiple genes key for the molecular reprogramming of
patient fibroblasts. These iPS cells can be differentiated into neurons or neuroprecursor cells that are subsequently transplanted into the diseased brain.

cells that are part of the BBB; (iii) molecular evolution of
AAV vectors has been propelled by using new AAV capsid
libraries developed by DNA shuffling of existing AAV
capsid genes (162– 164). This approach has been used to gen-
erate new AAV vectors with improved transduction properties
for particular targets, such as lung epithelium (165), myocar-
dium (166) and more recently to a particular subset of
neurons in the piriform cortex in the brain following
seizure-induced temporary disruption of the BBB (167). This
selection is performed in live animals, and one of the highly
significant findings has been newly identified chimeric AAV
capsids with dramatically reduced transduction of liver upon
systemic infusion. The work by Kumar et al. (168) where a
rabies virus glycoprotein-derived peptide was used to target
an artificial siRNA to the CNS suggests that the distinction
between artificial/synthetic and viral vectors is likely to
become increasingly blurred as both sides borrow from each
other to achieve the ultimate goal. AAV-phage (AAVP)
vectors are another example of this melding of components
with target peptide-displaying M13-derived phage capsids
carrying genomes with mammalian expression cassettes
flanked by AAV2 inverted terminal repeats (169). These
AAVP vectors appear to be quite effective at targeting brain
tumor vasculature (170).
The recent developments in engineering new CNS-targeted
vectors give great optimism that within the next decade we
will finally achieve the long-standing goal of global gene
delivery to the post-natal CNS via the vasculature. This will
undoubtedly revolutionize neuroscience research, and mark a
new era in neurology with the genetic tools existing to
develop effective therapies for many conditions that remain
untreatable today. But before then, we already have excep-
tional tools at hand to change the outcome of many neurologi-
cal diseases using viral vectors for focal gene delivery to
particular structures in the CNS. Most work thus far has
relied on the use of strong constitutive promoters, but in
many instances it may be necessary to regulate gene
expression. This has been accomplished to a large extent by
incorporating into the gene transfer vectors a combination
of drug-responsive transcription factors and respective
R36 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1
promoters, thus being able to regulate transgene expression in
the CNS by peripheral administration of a particular drug.
The most commonly used and investigated systems for
this purpose are the tetracycline-responsive (89,171– 173)
and rapamycin-responsive systems (174,175). These
drug-regulated viral vector systems have yet to be tested in
the CNS of patients.
IPs and stem cells
Neuronal cell death is associated with most of the major dis-
eases that afflict the CNS. Stem cell therapeutics holds
promise for replacing degenerating or ablated neurons to ulti-
mately restore neuronal network integrity. While embryonic
stem cell-based therapy has been approved for clinical trial
testing in patients with spinal cord lesions (176), the ethical
implications and funding policy inconsistency associated
with the use of stem cells isolated from human embryos for
CNS therapy have led to the rapid development of new cell
sources, including iPS cells (Fig. 3). iPS cells require the con-
trolled ‘reprogramming’ of adult somatic cells through careful
introduction of key molecular cues and re-derivation of viable
clones that lack tumorigenic potential (177,178). Viral vectors,
including those derived from retrovirus and lentivirus, have
proved useful for efficient delivery of genes encoding these
molecular cues (Oct3/4, Sox, Klf, Myc, Nanog and LIN28)
to adult human fibroblasts (179 – 181). The challenges facing
the clinical implementation of iPS cells for neurodegenerative
diseases relate to achieving an optimal balancing of repro-
gramming factors [reviewed by Lewitzky and Yamanaka
(182)], lowering risk of integrating vector-mediated gene dis-
ruption (183,184) and prevention of teratoma formation,
which is an inherent risk of iPS cell derivation (185,186).
State-of-the-art summary
In the past 20 years, genetic therapy has moved from a dream
to a near medical reality for some diseases, with marked ben-
eficial effects on immune function in X-linked SCID cases
(187) and restoration of some vision in Leber’s optic
atrophy (188). There have been many hurdles along the way,
including toxicity of vectors, oncogenic insertions into the
genome, immune rejections and difficulties in scaling up
from mouse models to humans. The burgeoning successes
have depended in large part on genetics, including identifying
disease genes and types of mutations, design of
genome-interacting elements, manipulation of splicing events
and novel genetic elements, such as microRNAs and regulat-
ory non-coding RNAs. Critical factors lie in tailoring the
mode and content of the genetic vehicle to the specifics of
the disease, and effectively utilizing the broad range of target-
ing modalities and vector types available.
ACKNOWLEDGEMENTS
We thank Ms Suzanne McDavitt for skilled editorial assist-
ance, and Ms Emily Mills at Millstone Design (http
://www.millstone-design.com) for preparation of figures.
Conflict of Interest statement. None declared.
FUNDING
Support for X.O.B. comes from NIH/NCI CA069246 and NIH/
NINDS NS242791, for W.J.B. from NIH R01-AG026328 and
for M.S.-E. from NIH/NINDS R01NS066310, U01NS064096
and NIH/NICHHD R01HD060576.
Downloaded from https://academic.oup.com/hmg/article-abstract/20/R1/R28/631150 by guest on 15 March 2019
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