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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.
∗
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
all CNS blood vessels and pericytes. Interestingly, pericytes widespread transduction in the neonatal mouse brain (5,7),
appear to play a central role in regulating BBB permeability but the distribution pattern is more restricted in adult
via their influence over endothelial cells and astrocytes (2). animals (6,8,9). This suggests that there may be additional bar-
Not surprisingly, the BBB has proved exceptionally efficient riers to distribution of gene transfer vectors from CSF into
in excluding the vast majority of gene transfer vehicles adult brains, or that commonly used injection procedures
from reaching the CNS via the vasculature. As a result, most disrupt normal CSF flow preventing widespread distribution.
CNS gene therapy approaches have utilized direct infusion Delivery of recombinant lysosomal enzymes into CSF
of gene transfer vectors into the brain parenchyma to appears to be effective in providing therapeutic levels of
target disease-relevant structures. The distribution of viral these enzymes throughout the CNS (10,11). Similarly, CSF
vectors in the brain can be improved considerably by infusion of antisense oligonucleotides (ASOs) directed at
convection-enhanced delivery (CED), a slow pressurized infu- altering the splicing pattern of the SMN2 gene has been dra-
sion of a larger volume (3,4). Nonetheless, transduction of matically effective in a mouse model of SMA (see below).
CNS cells after intraparenchymal injection of viral vectors An ideal route of entry into the CNS to achieve widespread
remains mostly limited to the targeted structure. gene transfer would be through the vasculature. Until recently,
An alternative route of entry into the CNS is by delivery of the only exception to the BBB-imposed block to gene therapy
the gene transfer vectors directly into cerebrospinal fluid vectors was PEGylated immunoliposomes (PILs) formulated
(CSF) via either the lateral ventricles (5) or intrathecal space with monoclonal antibodies specific for receptors, such as
(6). This approach is exceptionally effective in achieving transferrin and insulin receptors which mediate transcytosis
R30 Human Molecular Genetics, 2011, Vol. 20, Review Issue 1
of their ligands across the BBB. These PILs appear to be quite factors, cytokines, tumor killing agents) are to target gene
effective in delivering expression plasmids (with expression transfer to brain microcapillary endothelial cells (17), or use
under cell type-specific promoters) and RNAi to normal ex vivo genetically modified stem cells [hematopoietic stem
brain or brain tumors (12). In a parallel approach, therapeutic cells (HSCs), neural stem cells, mesenchymal stem cells]
proteins are delivered to the CNS using chimeric recombinant which will themselves, or their progeny in the case of HSCs,
molecules, e.g. growth factors, single-chain antibodies or lyso- migrate within the brain to regions of injury or tumors
somal enzymes, fused to receptor-targeting monoclonal anti- (18 –20). In fact, the whole nervous system can be transduced
bodies (12) or ligands, such as transferrin (13). Recently, with a gene for the lifespan by injecting a lentivirus vector into
adeno-associated virus 9 (AAV9) vectors have been found to the amniotic sac of mouse embryos before the neural groove
enter the CNS of neonatal mice and young cats after intravas- has closed (21).
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 DNA/RNA
the hippocampus and Purkinje cells in the cerebellum (14). The use of non-viral nucleic acid delivery to selectively
SV40 recombinant vectors also appear to mediate efficient modify cellular processes within the brain presents a number
gene transfer to certain regions of the CNS after i.v. infusion of challenges, including target cell specificity and transient
in adult mice combined with intraperitoneal mannitol infusion gene expression duration. Pardridge and colleagues (22)
(16). Alternative approaches to deliver secretable therapeutic have developed PILs target to the brain when administered
proteins to the CNS (e.g. lysosomal enzymes, growth intravenously (see above). Stachowiak et al. (23) have recently
Human Molecular Genetics, 2011, Vol. 20, Review Issue 1 R31
reported the use of organically modified silica-based nanopar- EXAMPLES OF APPLICATIONS TO SPECIFIC
ticles to induce neurogenesis within the subventricular zone of DISEASES
adult mice via intraventricular delivery of DNA encoding a
recombinant nuclear form of fibroblast growth factor Lysosomal storage diseases
receptor-1. Electroporation-based nucleic acid transfection
has also been extensively documented in the prenatal and post- Lysosomal storage diseases are typically, but not exclu-
natal rodent brain [reviewed by De Vry (24)], providing the sively, childhood diseases resulting from a genetic deficiency
means to genetically modify significant numbers of neurons in a lysosomal enzyme involved in a particular metabolic
(25,26) within the living brain (27). 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
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:
ubiquitin ligase, parkin (99). However, both the increased axonal transport (114). Also, misfolded SOD1 mutants
and decreased levels alpha-synuclein can cause neurodegen- have been shown to directly bind and inhibit mitochondrial
eration (84). voltage-dependent anion channel (VDAC1) leading to mito-
chondrial dysfunction (115). Taken together, these results
suggest that conformational changes in SOD1 (and possibly
Spinal muscular atrophy also other fALS-associated proteins), caused by mutations
SMA is an autosomal recessive disease caused by the loss of associated with some forms of fALS, and other as yet undeter-
function of the ‘survival of motor neuron’ gene, SMN1, mined factors, may be the basis for fALS and sALS. Gene
which leads to degeneration of motor neurons and infant mor- therapy approaches for ALS have centered on delivery of
tality. One approach to gene therapy would be to replace the growth factors such as IGF-1 (116– 118) and vascular endo-
missing gene in multiple motor neurons, which may now be thelial growth factor (116,119), and RNAi-based silencing of
possible with i.v. administration of AAV9 vectors which mutant SOD1 alleles (120 – 123) in transgenic ALS mouse
phosphoribosyltransferase (131) or mammalian cytochrome using single-stranded DNA (73 bp; 152). In addition, there is
P450/carboxyesterase (132) within the brain to activate pro- the potential to deliver human artificial mini-chromosomes
drugs (ganciclovir, 5-fluorocytosine, cyclophosphamide/irino- into the cell nucleus for gene-deficiency syndromes
tecan, respectively) that can pass the BBB and be converted (153,154). These strategies include control of transplicing,
into active chemotherapeutic agents within the tumor. (ii) for example in SMA therapies (see above). Naturally occur-
Viral oncolysis. In this approach mutant forms of viruses, typi- ring and synthetic transposable elements, including the Sleep-
cally HSV-1 and adenovirus, are used which replicate selec- ing Beauty transposon, have shown promise in stable
tively in tumor versus normal brain based on cancer-related integration and gene expression in cells, including neurons
mutations or their increased proliferation rate (133). These in vivo, and are discussed in more detail elsewhere in this
oncolytic vectors can also be armed with therapeutic genes. issue. All these more ‘advanced’ manipulations of the
An increasing number of different viruses are being tested in genome will depend on large vector capacity and more effi-
this context, including measles virus, reovirus, Newcastle cient delivery to the nervous system. A new arena will be
cells that are part of the BBB; (iii) molecular evolution of carrying genomes with mammalian expression cassettes
AAV vectors has been propelled by using new AAV capsid flanked by AAV2 inverted terminal repeats (169). These
libraries developed by DNA shuffling of existing AAV AAVP vectors appear to be quite effective at targeting brain
capsid genes (162– 164). This approach has been used to gen- tumor vasculature (170).
erate new AAV vectors with improved transduction properties The recent developments in engineering new CNS-targeted
for particular targets, such as lung epithelium (165), myocar- vectors give great optimism that within the next decade we
dium (166) and more recently to a particular subset of will finally achieve the long-standing goal of global gene
neurons in the piriform cortex in the brain following delivery to the post-natal CNS via the vasculature. This will
seizure-induced temporary disruption of the BBB (167). This undoubtedly revolutionize neuroscience research, and mark a
selection is performed in live animals, and one of the highly new era in neurology with the genetic tools existing to
significant findings has been newly identified chimeric AAV develop effective therapies for many conditions that remain
capsids with dramatically reduced transduction of liver upon untreatable today. But before then, we already have excep-
systemic infusion. The work by Kumar et al. (168) where a tional tools at hand to change the outcome of many neurologi-
rabies virus glycoprotein-derived peptide was used to target cal diseases using viral vectors for focal gene delivery to
an artificial siRNA to the CNS suggests that the distinction particular structures in the CNS. Most work thus far has
between artificial/synthetic and viral vectors is likely to relied on the use of strong constitutive promoters, but in
become increasingly blurred as both sides borrow from each many instances it may be necessary to regulate gene
other to achieve the ultimate goal. AAV-phage (AAVP) expression. This has been accomplished to a large extent by
vectors are another example of this melding of components incorporating into the gene transfer vectors a combination
with target peptide-displaying M13-derived phage capsids 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 Conflict of Interest statement. None declared.
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 FUNDING
drug-regulated viral vector systems have yet to be tested in Support for X.O.B. comes from NIH/NCI CA069246 and NIH/
the CNS of patients. NINDS NS242791, for W.J.B. from NIH R01-AG026328 and
for M.S.-E. from NIH/NINDS R01NS066310, U01NS064096
and NIH/NICHHD R01HD060576.
IPs and stem cells
Neuronal cell death is associated with most of the major dis-
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