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  • Genetic therapy for the nervous system

    William J. Bowers1, Xandra O. Breakefield2,3, and Miguel Sena-Esteves4

    1Department 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

    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 modifiedcells, 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 neurologicsyndromes of different etiologies, including lysosomal storage diseases, Alzheimers disease and other amy-loidopathies, Parkinsons disease, spinal muscular atrophy, amyotrophic lateral sclerosis and brain tumors.This field has been propelled by genetic technologies, including identifying disease genes and disruptivemutations, 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 ofgenetic elements provide the ability to tailor the mode of genetic intervention to specific aspects of a diseasestate.

    INTRODUCTION

    Genetic therapy covers a range of methods for modifying thenervous system, including delivery of genes, sequence-targeted regulatory molecules, genetically modified cells andoligonucleotides. In this review, we focus on ways to changethe genetic status of the nervous system, including routes ofaccess and modes of delivery. Examples are provided of strat-egies used for a number of diseases, including recessive loss offunction conditions [lysosomal storage diseases and spinalmuscular atrophy (SMA)], dominant toxic mutations [amyo-trophic lateral sclerosis (ALS)] and conditions of mixed etiol-ogy [Alzheimers disease (AD), Parkinsons disease (PD) andbrain tumors] (Fig. 1). Neurodegenerative diseases caused bytriplet nucleotide repeats are discussed in an article onsiRNAs in this issue. Many of these strategies have provenvery promising in preclinical studies in mouse and largeranimal models, and a substantial number are now being eval-uated in clinical trials.

    METHODS OF GENETIC INTERVENTION

    Routes of access

    Entrance to the central nervous system (CNS) is limited by theskeletal structures that enclose it and the bloodbrain barrier(BBB), which plays a central role in regulating the chemicalmicroenvironment of the CNS by preventing simple diffusionof most small molecules and proteins from the bloodstream.The physical barrier properties of the BBB are a result ofthe tight junctions between brain microvascular endothelialcells 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 theluminal and abluminal membranes of the endothelial cells,with the exception of small solutes such O2 and CO2 gasesand some lipophilic molecules (e.g. ethanol) that can diffusefreely across cellular membranes. Other components of theBBB 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: [email protected]

    # The Author 2011. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

    Human Molecular Genetics, 2011, Vol. 20, Review Issue 1 R28R41doi:10.1093/hmg/ddr110Advance Access published on March 23, 2011

  • all CNS blood vessels and pericytes. Interestingly, pericytesappear to play a central role in regulating BBB permeabilityvia their influence over endothelial cells and astrocytes (2).Not surprisingly, the BBB has proved exceptionally efficientin excluding the vast majority of gene transfer vehiclesfrom reaching the CNS via the vasculature. As a result, mostCNS gene therapy approaches have utilized direct infusionof gene transfer vectors into the brain parenchyma totarget disease-relevant structures. The distribution of viralvectors in the brain can be improved considerably byconvection-enhanced delivery (CED), a slow pressurized infu-sion of a larger volume (3,4). Nonetheless, transduction ofCNS cells after intraparenchymal injection of viral vectorsremains 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 adultanimals (6,8,9). This suggests that there may be additional bar-riers to distribution of gene transfer vectors from CSF intoadult brains, or that commonly used injection proceduresdisrupt normal CSF flow preventing widespread distribution.Delivery of recombinant lysosomal enzymes into CSFappears to be effective in providing therapeutic levels ofthese enzymes throughout the CNS (10,11). Similarly, CSFinfusion of antisense oligonucleotides (ASOs) directed ataltering 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 therapyvectors was PEGylated immunoliposomes (PILs) formulatedwith monoclonal antibodies specific for receptors, such astransferrin and insulin receptors which mediate transcytosis

    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 theX 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 vectoris used to deliver a promotercDNA expression cassette which either integrates into the genome (1), e.g. lentivirus vector, or resides as an extrachromosomalelement (4), e.g. AAV vector. Other approaches include attempts to achieve homologous recombination with the resident allele (4) or, if appropriate, to correctthe 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 vectoror 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 tryingto supplement the neurotransmitter capacity of sick neurons by providing them with enzymes to generate more of their normal neurotransmitter for releaseat synapses (1). Alternately, the neurotransmitter profile of interacting neurons can be altered to change their output from excitatory to inhibitory, or viceversa (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).

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  • of their ligands across the BBB. These PILs appear to be quiteeffective in delivering expression plasmids (with expressionunder cell type-specific promoters) and RNAi to normalbrain or brain tumors (12). In a parallel approach, therapeuticproteins are delivered to the CNS using chimeric recombinantmolecules, 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 toenter the CNS of neonatal mice and young cats after intravas-cular (i.v.) infusion and to transduce large numbers of glia andmotor neurons in the spinal cord (14,15). Transduction ofother neuronal populations in the brain is found mostly inthe hippocampus and Purkinje cells in the cerebellum (14).SV40 recombinant vectors also appear to mediate efficientgene transfer to certain regions of the CNS after i.v. infusionin adult mice combined with intraperitoneal mannitol infusion(16). Alternative approaches to deliver secretable therapeuticproteins to the CNS (e.g. lysosomal enzymes, growth

    factors, cytokines, tumor killing agents) are to target genetransfer to brain microcapillary endothelial cells (17), or useex vivo genetically modified stem cells [hematopoietic stemcells (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(1820). In fact, the whole nervous system can be transducedwith a gene for the lifespan by injecting a lentivirus vector intothe amniotic sac of mouse embryos before the neural groovehas closed (21).

    DNA/RNA

    The use of non-viral nucleic acid delivery to selectivelymodify cellular processes within the brain presents a numberof challenges, including target cell specificity and transientgene expression duration. Pardridge and colleagues (22)have developed PILs target to the brain when administeredintravenously (see above). Stachowiak et al. (23) have recently

    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 33.5-fold larger than the infused volume, which is highly significant for humanapplications. Viral vectors or secreted transgene products (growth factors, lysosomal enzymes) can be further distributed from the primary target structure byaxonal 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 tosecrete therapeutic proteins into CSF (top right diagram). The BBB with its many constituents has thwarted most gene transfer vehicles from entering the brainfrom 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 afteri.v. infusion in newborn and adult animals (bottom right diagram).

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  • reported the use of organically modified silica-based nanopar-ticles to induce neurogenesis within the subventricular zone ofadult mice via intraventricular delivery of DNA encoding arecombinant nuclear form of fibroblast growth factorreceptor-1. Electroporation-based nucleic acid transfectionhas also been extensively documented in the prenatal and post-natal rodent brain [reviewed by De Vry (24)], providing themeans to genetically modify significant numbers of neurons(25,26) within the living brain (27).

    Viral vectors

    Recombinant viral vector systems remain the most efficientvehicles to achieve long-term stable gene expression in theCNS. Over the years, many different viral vector systemshave been investigated for this purpose, including thosederived from herpes simplex virus type 1 (HSV-1), adeno-virus, AAV, lentivirusessuch as HIV-1, feline immunode-ficiency virus or equine infectious anemia virus, and morerecently SV40. AAV and lentivirus vectors have emergedas the vectors of choice for gene transfer to the CNS fornon-oncological applications as they mediate efficient long-term gene expression with no apparent toxicity. A recentstudy has shown that AAV-mediated transgene expressionin the primate brain continues for at least 8 years with noevidence of neuroinflammation or reactive gliosis (28).Moreover, several clinical trials have shown that direct infu-sion of AAV2 vectors into brain parenchyma in humans iswell tolerated (2933). The safety profile of direct infusionof lentivirus vectors into human brain remains to be evalu-ated. High-capacity adenovirus vectors are attractivebecause of their large transgene capacity (30 kb) andability to mediate long-term gene expression withoutimmunological complications (34). These and the largecapacity HSV amplicon vectors (150 kb; 35) may be idealchoices to transfer large genomic regions necessary toachieve physiological regulation of gene expression for par-ticular genes/sets of genes in specific cell populations in theCNS. 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-capacityadenovirus and HSV-1 amplicon vectors lies in difficultiesin large-scale production. Recombinant SV40 vectorsdisplay some promising properties, namely their apparentability to cross the adult mouse BBB after i.v. infusionand transduce neurons and astrocytes in specific brainregions and spinal cord (16). Moreover, these vectors canbe administered repeatedly as they do not seem to elicit neu-tralizing antibodies in rodents. Whether this property isshared in other mammalian species remains to be deter-mined. In the neuro-oncology field, recombinant vectorsderived from HSV-1, adenovirus, measles virus and Newcas-tle virus appear to be the most promising in pre-clinicalmodels of brain tumors (see below). After initial attemptsto broadly use each viral vector system for any/all CNSgene transfer applications, now the particular strengths ofeach system are being exploited to meet the specific needsof different applications/diseases.

    EXAMPLES OF APPLICATIONS TO SPECIFIC

    DISEASES

    Lysosomal storage diseases

    Lysosomal storage diseases are typically, but not exclu-sively, childhood diseases resulting from a genetic deficiencyin a lysosomal enzyme involved in a particular metabolicpathway that results in lysosomal accumulation of its sub-strate(s). Many of these enzymes can be secreted fromcells genetically engineered to overexpress them and thentaken up by enzyme-deficient cells and correctly targetedto lysosomes via mannose-6-phosphate receptors wherethey degrade the stored substrate(s). This mechanism,known as cross-correction (38), is the basis for enzymereplacement therapy which is now available for a subset ofthese diseases without neurological involvement. Unfortu-nately, the BBB prevents recombinant lysosomal enzymesinfused peripherally from entering the CNS. Alternativeroutes of delivery and molecules are being actively investi-gated (see above). These diseases are particularly wellsuited for gene therapy as they are monogenic diseaseswith very well established genotypephenotype correlations,and the cross-correction mechanism makes it possible todevise gene delivery strategies to supply essentially theentire CNS with therapeutic levels of these enzymes. Twomain approaches have shown dramatic therapeutic effectsin animal models of lysosomal storage diseases, namelyintraparenchymal infusion of recombinant viral vectors(3946), and bone marrow transplantation with ex vivolentivirus vector modified-autologous HSCs (see article byBiffi, Cartier and Aubourg in this issue).The ability of focal viral vector-mediated gene delivery to

    supply the CNS with therapeutic levels of lysosomalenzymes is dependent on widespread distribution based on dif-fusion (47), axonal transport over long distances (48,49) andCSF flow in the perivascular space (8). AAV-mediatedgenetic modification of highly interconnected structures inthe 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 flowfor distribution of lysosomal enzymes throughout the CNS.Successful translation of AAV-based (or lentivirus-based)approaches to humans will likely require targeting one ormore of these structures to achieve therapeutic levels ofthese enzymes throughout the CNS. Pre-clinical studies inlarge animal models of some of these diseases are highlyencouraging (54,55). Bone marrow transplantation withlentivirus-modified autologous HSCs has shown exceptionalresults in different mouse models of lysosomal storage dis-eases resulting in correction of pathologic findings throughoutthe CNS (18,56,57), as well as peripheral organs (58). Thisapproach relies on genetically modified HSC-derived cells(macrophages in the case of CNS) trafficking to the sites ofdisease and becoming an in situ source of recombinantenzyme. Both of these gene therapy approaches are nowbeing tested in human clinical trials for different lysosomalstorage diseases.

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  • Alzheimers disease and other amyloidopathies

    AD is a neurodegenerative disorder characterized by severememory loss and cognitive impairment with no availablecure. Neuropathological correlates include extracellularamyloid-beta peptide deposition, intracellular neurofibrillarytangle formation, decreased synaptic integrity and neuronalloss. The basal forebrain cholinergic complex is significantlyaffected by AD-related neurodegeneration (5963). Toaugment cholinergic function, Tuszynski and colleagues(6468) delivered nerve growth factor (NGF), the prototypicalneurotrophin with demonstrated neuroprotective properties,using retrovirus vector-transduced fibroblast grafts anddemonstrated restoration and survival of cholinergic neuronsin lesioned rodents and aged non-human primates. Theseinitial studies set the stage for the first phase I clinical trialof ex vivo NGF gene therapy (ClinicalTrials.gov Identifier:NCT00017940). The rate of cognitive decline was slowed,and no apparent detrimental effects were observed arisingfrom NGF expression 22 months post-engraftment (69).More recently, Ceregene has conducted phase I and II clinicaltrials using an AAV vector that expresses NGF (Clinical-Trials.gov Identifiers: NCT00087789 and NCT00876863,respectively). While the phase I trial demonstrated that stereo-tactic infusions of an NGF-expressing AAV vector is well tol-erated, it is too early to know whether this gene therapy-basedstrategy will significantly impact the course and symptomol-ogy of the disease, given the phase II trial is currently ongoing.During the past several years, promising efforts have

    focused on reducing the levels of neurotoxic Ab peptidespecies within the brain through removal of pathogenic Abpeptides or halting the proteolytic release of the amyloido-genic form of Ab arising from pathogenic amyloid precursorprotein (APP) processing. Such preclinical vector-based thera-pies include active and passive vaccines directed againstspecific epitopes of pathogenic Ab peptides (7075),expression of Ab proteases (7578), delivery of small inhibi-tory RNAs (RNAi) designed to specifically suppress theexpression of APP processing enzymes (7982) and deliveryof a cholesterol degrading enzyme into the brain (83). Eachexperimental approach has exhibited strong preclinical effi-cacy in rodent models of AD, hence increasing enthusiasmfor eventual translation of one or more of these strategies toclinical testing. The common challenge shared by gene thera-peutic approaches for AD which require intraparenchymaldelivery relates to sufficiency of brain tissue coverage.Given AD impacts a number of human brain sub-regionsthat are integral to learning and memory, viral vector strategiesmust include the means to monitor vector infusion in real timeto widely and safely disseminate vector particles withinafflicted networks. Recent advances in real-time monitoringof CED-mediated AAV vector infusions within the non-humanprimate brain indicate that this delivery hurdle can beovercome (84).

    Parkinsons disease

    PD is a common progressive neurodegenerative disease. Whilemany regions of the brain are affected, the primary motorsymptoms are caused by the loss of dopaminergic neurons in

    the substantia nigra innervating the striatum in the basalganglia (85). Although environmental toxins and aging arerisk factors, genetic susceptibility is critical with 16 geneloci implicated in 5% of cases, and multifactorial hereditaryrisk in many others (85,86). These risk factors point to oxi-dative stress, mitochondrial dysfunction and reduced abilityto degrade abnormal proteins as etiologic factors. AlthoughL-dopa has been used for decades to relieve motor symptomsof PD, it does not prevent degeneration and eventually ceasesto be effective.New gene/cell therapy strategies have been explored exper-

    imentally with some translated into clinical trials, including:delivery or upregulation of neurotrophic factors, generationof endogenous dopamine and alterations in neuronal circuitry,as well as implantation of supportive cells and dopaminergicneurons. Gene delivery has typically been carried out usingAAV vectors with CED injection into the striatum. Vectorshave delivered both glial-derived neurotrophic factor(GDNF; 87) and its analogue, neurturin (88), which serve astrophic factors for dopaminergic neurons. Since abnormallyhigh GDNF can have toxic effects, efforts have gone into reg-ulating its expression through a tetracycline analogue system(89) and by using highly specific zinc finger transcriptionfactors to upregulate expression of the endogenous gene,with the latter imposing a physiologically controlled upperlimit (90).In terms of modulating neurotransmitter pathways, all

    three biosynthetic enzymes for dopamine have been includedin the same lentivirus vector, including tyrosine hydroxylase(TH) and aromatic amino acid decarboxylase (AADC), aswell as GTP cyclohydrolase for synthesis of the biopterinco-factor for TH, with the assumption that dopamine, as aparacrine neurotransmitter, can be made by any cell in thestriatum (91). However, serotonergic or GABAergicneurons can also produce dopamine as a false transmitterleading to dyskinesia (92). AAV-mediated delivery ofAADC alone has the advantage of making production ofdopamine dependent on L-dopa intake, with ongoing phaseI trials indicating continued expression of AADC for atleast 2 years in the human brain (93). Investigators havealso tried to block hyperactive neurotransmission in PDbrains using an AAV vector to deliver the enzyme glutamicacid decarboxylase for synthesis of the inhibitory neurotrans-mitter GABA into the subthalamic nucleus (94). Cell thera-pies have included a number of cell types, such as humanfetal mesencephalic tissue, which includes precursors ofdopaminergic and serotonergic neurons, with the currentfocus on human dopaminergic neuroblasts generated fromstem cells, in particular induced pluripotent stem (iPS)cells derived from adult tissue (95).Strangely, few of these therapies address the etiology of the

    disease based on genetic insights, and in fact most animalmodels of PD employ lesioning of dopaminergic neuronalconnections, with the exception of transgenic mice overex-pressing alpha-synuclein and recently developed models ofother PD genetic syndromes (96). Since three copies of thealpha-synuclein gene alone can cause PD (97) and upregulatedalpha-synuclein inhibits neurotransmitter release (98), alogical approach would be to decrease alpha-synuclein syn-thesis with RNAi (84) or increase its degradation with the

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  • ubiquitin ligase, parkin (99). However, both the increasedand decreased levels alpha-synuclein can cause neurodegen-eration (84).

    Spinal muscular atrophy

    SMA is an autosomal recessive disease caused by the loss offunction 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 themissing gene in multiple motor neurons, which may now bepossible with i.v. administration of AAV9 vectors whichwere able to deliver the SMN1 cDNA to the spinal cord in amouse 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 presenceof a second copy of this gene, SMN2, coding for an identicalamino acid sequence in the human genome (102). TheSMN2 gene is defective due to a point mutation in an intronwhich interferes with correct splicing, such that a truncated,non-functional protein is produced. In one therapeuticmodality, an ASO homologous to the last translated exon inSMN2 is used to guide an exonic splicing enhancer sequenceto the region as a binding platform for pre-mRNA splicingfactors (103). This strategy gives increased SMN2 expressionwhen infused into the lateral cerebral ventricles in an SMAmouse model (104,105). In another modality, transplicing isutilized in which the ASO binds to the defective intronicsequence and carries a splicing domain and an intact finalexon from SMN1, with injection into the cerebral ventriclesalso 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 spinalcord. Mutations in the SOD1 gene encoding cytosolic Cu/Znsuperoxide dismutase were the first to be linked to familialforms of ALS (fALS), 10% of all cases. In recent years,there has been a dramatic increase in the number of genesshown to be associated with fALS, such as ANG encodingangiogenin (107), TARDP encoding transactive response(TAR) DNA-binding protein TDP-43 (108), FUS encodingfused in sarcoma protein (also known as TLS for translocatedin liposarcoma) (109,110) and, more recently, optineurin(111). Several other genes have been associated with rareforms of fALS (reviewed in 112). The disease mechanism(s)remains elusive. However, the fact that many fALS casesare autosomal dominant suggests that disease-associatedmutations generate protein species with toxic functionsinstead of simple loss of function. An emerging picture inthe field is that some of the fALS-associated proteins mayalso be involved in sporadic ALS (sALS) cases. As anexample, recent work has shown that oxidized wild-typeSOD1 shares a conformational epitope with fALS-associatedmutant SOD1 (113), and can be found in spinal cord motorneurons 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

    axonal transport (114). Also, misfolded SOD1 mutantshave been shown to directly bind and inhibit mitochondrialvoltage-dependent anion channel (VDAC1) leading to mito-chondrial dysfunction (115). Taken together, these resultssuggest that conformational changes in SOD1 (and possiblyalso other fALS-associated proteins), caused by mutationsassociated with some forms of fALS, and other as yet undeter-mined factors, may be the basis for fALS and sALS. Genetherapy approaches for ALS have centered on delivery ofgrowth factors such as IGF-1 (116118) and vascular endo-thelial growth factor (116,119), and RNAi-based silencing ofmutant SOD1 alleles (120123) in transgenic ALS mousemodels. The most commonly used ALS mouse model carriesa human SOD1 G93A transgene expressed at high levelswith a mean survival of 129 days. To date, many of thegene therapy studies have demonstrated a significant increasein median survival, but ultimately all treated mice have suc-cumbed to disease progression. The reasons for the apparentfailure of those interventions can be multifactorial, but aninteresting aspect to consider is the fact that silencingmutant SOD1 expression in motor neurons delays diseaseonset but not progression, while silencing it in microgliadoes the opposite (124). This suggests that other CNS celltypes contribute significantly to the disease phenotype. Devel-opment of new mouse models using mutant alleles from otherALS genes will enhance our understanding of common diseasepathways and development of effective gene therapies for thisdisease. The recent findings on the toxicity of misfolded SOD1proteins (normal or mutant) strongly suggest that either immu-nization (125), or passive immunization with conformation-specific antibodies (126) may also be viable approaches todevelop effective therapies for ALS.

    Brain tumors

    Malignant glioma tumors (glioblastoma; GBM) are the mostcommon adult brain tumor and are virtually untreatable,with most patients dying within 2 years of diagnosis in spiteof neurosurgery, radiation and chemotherapy. These tumorsare very invasive in the brain and genetically heterogeneouswith a cancer stem cell population that defies all current che-motherapies (127). Some benign tumors of the nervoussystem, e.g. vestibular schwannomas, regress in response toanti-angiogenic therapy (128), while for GBM tumors thistreatment 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 focusedon direct elimination of tumor cells, including a bystandereffect to confer selective toxicity to non-transduced tumorcells 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 cellsinto the resected region to kill remaining tumor cells over anexpanded radius. Concepts employed in this arsenal include:(i) Prodrug activation (or suicide genes). In this case, viralvectors or cells are used to express enzymes, such as viral thy-midine kinase (130), bacterial cytosine deaminase/uracil

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  • phosphoribosyltransferase (131) or mammalian cytochromeP450/carboxyesterase (132) within the brain to activate pro-drugs (ganciclovir, 5-fluorocytosine, cyclophosphamide/irino-tecan, respectively) that can pass the BBB and be convertedinto 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-relatedmutations or their increased proliferation rate (133). Theseoncolytic vectors can also be armed with therapeutic genes.An increasing number of different viruses are being tested inthis context, including measles virus, reovirus, Newcastledisease virus (134), polio virus (135) and vaccinia virus(136). Although promising, this field is still wrestling withimmune responses to the virus and an unfavorable tumormicroenvironment which restrict virus spread within thetumor (134). (iii) Cellular delivery. Several studies haveshown that some normal cells introduced into the brain, suchas neural stem cells (137) and mesenchymal stem cells (138)migrate towards tumor foci. These cells can deliver agentswhich are selectively toxic to tumor cells, e.g. antibodiesagainst 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 havebeen used to increase recognition of tumor antigens andempower the immune system. These include vaccinationwith a common and unique GBM antigen, EGFRvIII (145)and co-treatment with oncolytic HSV vectors and cyclopho-sphamide to temporarily suppress the immune curtailment ofvirus 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 receptorwhich is abundant on GBM tumors (147). (v) Zone of resist-ance. Other efforts have tried to increase the resistance ofthe brain to tumor invasion, including the use of AAVvectors 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 tomanipulate the endogenous cell genome in order to regulateexpression or correct mutated gene transcripts. In onemodality, zinc finger proteins (ZFPs) which recognize a typi-cally 18 bp site in the promoter region of a gene are linked to atranscriptional activator (149) to selectively turn on a gene, asin the case of SMN2 (above; 90). This approach can also beused to stimulate gene correction by linking the ZFP to anuclease causing double-stranded breaks in the DNA in com-bination with a correct extrachromosomal DNA sequence suchthat homologous recombination is stimulated (149). Anotherpotent tool that can be linked to ZFPs are meganucleaseswhich recognize .12 bp sequences and have been shown toinactivate HIV sequences (150). Other methods to facilitategene correction have included triplex-forming oligonucleo-tides which can be used to modify sequence or inhibit geneexpression (151). Gene correction has also been achieved

    using single-stranded DNA (73 bp; 152). In addition, there isthe potential to deliver human artificial mini-chromosomesinto 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 stableintegration and gene expression in cells, including neuronsin vivo, and are discussed in more detail elsewhere in thisissue. All these more advanced manipulations of thegenome will depend on large vector capacity and more effi-cient delivery to the nervous system. A new arena will bemodulation of miRNA levels which have such an importantrole in development and cancer, including decreasing levelsof oncogenic miRNAs and increasing levels of tumor suppres-sor miRNAs (155). Epigenetic modulation of the genome is anexpanding arena, with for example histone deacetylase inhibi-tors being used to slow down degeneration in mouse models ofHuntingtons disease (156).

    Improved vectors

    The new breed of viral vectors that can reach the CNS afterperipheral administration (14,16) also transduce peripheralorgans at high efficiency, namely the liver. This potentiallimitation could be simply addressed with the use ofCNS-specific promoters such as the synapsin-1, neuron-specific enolase or glial fibrillary acidic protein promoters.However, some of these promoters are relatively weak andsome are rather large, thus reducing considerably the trans-gene capacity of the vectors. An alternative approach is toincorporate into the transgene expression cassette targets formiRNAs expressed at high levels in peripheral organs butabsent or expressed at low levels in the CNS to prevent vector-mediated gene expression outside of the CNS (157, seebelow). This approach appears to be quite universal tode-target vector-mediated expression from specific celllineages (57,158,159). Another approach to addressing theundesired targeting of peripheral organs consists in engineer-ing the viral vector particles themselves. Lentivirus vectorscan be pseudotyped with envelope proteins derived fromother viruses, and the vesicular stomatitis virus glycoproteinis the most commonly used due to its pantropism. Previousstudies have shown that incorporation of the rabies glyco-protein allows lentivirus vectors administered peripherally toreach the CNS via retrograde axonal transport (119,160). Non-integrating lentivirus vectors can also be used effectively inthe neural cells, thus reducing the risk of oncogenic insertionevents (161).Development of CNS-targeted AAV vectors has followed

    three different routes: (i) in recent years hundreds of newAAV capsids have been identified from humans and quite afew other species, and systematic screening for newCNS-targeting properties may yet yield new AAV pseudo-types more powerful than AAV9; (ii) chimeric AAV2capsids carrying CNS-targeting peptides identified from invivo phage display experiments can increase delivery. Thisapproach had met with limited success until recently Chenet al. (17) showed that it can be used to develop AAVvectors highly specific for brain microcapillary endothelial

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  • cells that are part of the BBB; (iii) molecular evolution ofAAV vectors has been propelled by using new AAV capsidlibraries developed by DNA shuffling of existing AAVcapsid genes (162164). This approach has been used to gen-erate new AAV vectors with improved transduction propertiesfor particular targets, such as lung epithelium (165), myocar-dium (166) and more recently to a particular subset ofneurons in the piriform cortex in the brain followingseizure-induced temporary disruption of the BBB (167). Thisselection is performed in live animals, and one of the highlysignificant findings has been newly identified chimeric AAVcapsids with dramatically reduced transduction of liver uponsystemic infusion. The work by Kumar et al. (168) where arabies virus glycoprotein-derived peptide was used to targetan artificial siRNA to the CNS suggests that the distinctionbetween artificial/synthetic and viral vectors is likely tobecome increasingly blurred as both sides borrow from eachother to achieve the ultimate goal. AAV-phage (AAVP)vectors are another example of this melding of componentswith target peptide-displaying M13-derived phage capsids

    carrying genomes with mammalian expression cassettesflanked by AAV2 inverted terminal repeats (169). TheseAAVP vectors appear to be quite effective at targeting braintumor vasculature (170).The recent developments in engineering new CNS-targeted

    vectors give great optimism that within the next decade wewill finally achieve the long-standing goal of global genedelivery to the post-natal CNS via the vasculature. This willundoubtedly revolutionize neuroscience research, and mark anew era in neurology with the genetic tools existing todevelop effective therapies for many conditions that remainuntreatable 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 toparticular structures in the CNS. Most work thus far hasrelied on the use of strong constitutive promoters, but inmany instances it may be necessary to regulate geneexpression. This has been accomplished to a large extent byincorporating into the gene transfer vectors a combinationof drug-responsive transcription factors and respective

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

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  • promoters, thus being able to regulate transgene expression inthe CNS by peripheral administration of a particular drug.The most commonly used and investigated systems forthis purpose are the tetracycline-responsive (89,171173)and rapamycin-responsive systems (174,175). Thesedrug-regulated viral vector systems have yet to be tested inthe 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 holdspromise for replacing degenerating or ablated neurons to ulti-mately restore neuronal network integrity. While embryonicstem cell-based therapy has been approved for clinical trialtesting in patients with spinal cord lesions (176), the ethicalimplications and funding policy inconsistency associatedwith the use of stem cells isolated from human embryos forCNS therapy have led to the rapid development of new cellsources, including iPS cells (Fig. 3). iPS cells require the con-trolled reprogramming of adult somatic cells through carefulintroduction of key molecular cues and re-derivation of viableclones that lack tumorigenic potential (177,178). Viral vectors,including those derived from retrovirus and lentivirus, haveproved useful for efficient delivery of genes encoding thesemolecular cues (Oct3/4, Sox, Klf, Myc, Nanog and LIN28)to adult human fibroblasts (179181). The challenges facingthe clinical implementation of iPS cells for neurodegenerativediseases 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 dreamto 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 Lebers opticatrophy (188). There have been many hurdles along the way,including toxicity of vectors, oncogenic insertions into thegenome, immune rejections and difficulties in scaling upfrom mouse models to humans. The burgeoning successeshave depended in large part on genetics, including identifyingdisease genes and types of mutations, design ofgenome-interacting elements, manipulation of splicing eventsand novel genetic elements, such as microRNAs and regulat-ory non-coding RNAs. Critical factors lie in tailoring themode and content of the genetic vehicle to the specifics ofthe 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 andfor M.S.-E. from NIH/NINDS R01NS066310, U01NS064096and NIH/NICHHD R01HD060576.

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