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HUMAN GENE THERAPY 17:1–9 (January 2006) © Mary Ann Liebert, Inc. Review Lentivirus-Mediated Gene Transfer to the Central Nervous System: Therapeutic and Research Applications LIANG-FONG WONG, LUCY GOODHEAD, CHRISTINE PRAT, KYRIACOS A. MITROPHANOUS, SUSAN M. KINGSMAN, and NICHOLAS D. MAZARAKIS ABSTRACT The management of disorders of the nervous system remains a medical challenge. The key goals are to understand disease mechanisms, to validate therapeutic targets, and to develop new therapeutic strategies. Viral vector-mediated gene transfer can meet these goals and vectors based on lentiviruses have particularly useful features. Lentiviral vectors can deliver 8 kb of sequence, they mediate gene transfer into any neuronal cell type, expression and therapy are sustained, and normal cellular functions in vitro and in vivo are not compromised. After delivery into the nervous system they induce no significant immune responses, there are no unwanted side effects of the vectors per se to date, and manufacturing and safety testing for clinical applications are well advanced. There are now numerous examples of effective long-term treatment of animal models of neurological disorders, such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, motor neuron diseases, lysosomal storage diseases, and spinal injury, using a range of therapeutic genes expressed in lentiviral vectors. Significant issues remain in some areas of neural gene therapy including defining the optimum therapeutic gene(s), increasing the specificity of delivery, regulating expression of potentially toxic genes, and designing clinically relevant strategies. We discuss the applications of lentiviral vectors in therapy and research and highlight the essential features that will ensure their translation to the clinic in the near future. INTRODUCTION therapy. Neural architecture is relevant in designing therapy and in anticipating any side effects. Neurons can have long-range axonal projections and therefore delivery at one site could lead to action at a distant site. The greatest challenge will be to treat disseminated disease, where pathology spreads through the nervous system (e.g., motor neuron disease) or involves large regions of the brain (e.g., Alzheimer’s disease), or where the pathology is in brain and other tissues (e.g., lysosomal storage diseases). It may be essential to express the gene within each affected neuron as with small interfering RNAs (siRNAs) or intrabodies to ablate gene expression, and this will require efficient gene transfer. If therapy is via secreted factors then fewer cells can be transduced, so reducing the delivery challenge, but diffusion or anterograde distribution to other sites may be an issue. Many neural disorders are chronic but not always life threatening, so there must be long-term expression and safety. Vec- T HE ABILITY TO TRANSFER GENES into neurons, using viral vectors, allows therapeutic genes to be delivered to the nervous system and provides new approaches to probe the mechanistic basis of disease and identify new target points for intervention. There are unique opportunities and challenges for gene therapy in the central nervous system (CNS). The potent blood–brain barrier precludes the ready access of systemic molecules as large as a viral vector and so invasive brain surgery may be required. However, knowledge of mammalian neuroanatomy and clinical tools such as magnetic resonance imaging (MRI) allow the precise deposition of viral vectors at the site of interest, so imparting considerable specificity to the treatment. Anatomical isolation results in a degree of “immune privilege,” which may limit the immunological consequences of any Oxford BioMedica (UK), Medawar Centre, Oxford Science Park, Oxford OX4 4GA, UK. 1 2 WONG ET AL. tors that have useful features include those based on herpesviruses, high-capacity adenoviral vectors, adeno-associated viruses (AAVs), and lentiviruses (Davidson and Breakefield, 2003). AAV and lentiviral vectors are attractive because of their simplicity, their excellent safety profile, and their relative ease of production. In many indications both AAV and lentiviral vectors are being assessed but here we focus on lentiviral vectors, which have many important advantages in research and therapy of neurological disorders. LENTIVIRAL VECTORS AND GENE TRANSFER TO THE NERVOUS SYSTEM Transduction of neurons, using lentiviral vectors in vivo in rodent and primate models, has been amply demonstrated (e.g., see Galimi and Verma, 2002; Azzouz et al., 2004a). These vectors are derived from primate (human and simian immunodeficiency viruses [HIV and SIV, respectively]) and nonprimate (e.g., equine, feline, and bovine immunodeficiency viruses [EIAV, FIV, and BIV, respectively]) lentiviruses (reviewed by Delenda, 2004). Relevant vectors for research and clinical applications are referred to as “minimal self-inactivating (SIN) vectors.” The entire viral coding regions are removed from the viral genome and particle proteins and replication enzymes (Gag and Pol) and an envelope (Env) are provided in trans from separate expression cassettes. The optimum efficiency is obtained by using a gag-pol gene that is codon optimized and in general the use of codon-optimized transgenes is desirable (our unpublished data). The envelope glycoprotein is nonlentiviral; this is referred to as pseudotyping. The vesicular stomatitis virus (VSV-G) envelope is widely used and gives vectors broad species and tissue tropism, but other envelope glycoproteins with unique features for different applications are available (Watson et al., 2002; Wong et al., 2004). As there are no residual lentiviral promoters or enhancers remaining in the genome in SIN vectors the transgene is expressed from an internal enhancer-promoter that can be varied according to the application. The most advanced vectors place less than 1 kb of viral DNA into the transduced cell genome and express only the transgene cassette. Useful technical and historical information on lentiviral vector design can be found in Dull et al. (1998), Kim et al. (1998), Zufferey et al. (1998), Kotsopoulou et al. (2000), and Rohll et al. (2002). Gene transfer in the nervous system could be compromised by innate or adaptive immune responses against virion components or the transgene product. This has certainly limited the effectiveness of AAV vectors where circulating preexisting antibodies to AAV inhibit gene transfer in the brain (Peden et al., 2004). However, innate immune responses may not be problematic for lentiviral vectors as minimal acute, asymptomatic inflammation in animal models has been observed (see Kordower et al., 2000; Mazarakis et al., 2001). Adaptive antivector response were not significant in mediating immune clearance in the CNS transduced with HIV vectors and injection into the brain did not induce systemic immunity to virions (AbordoAdesida et al., 2005). These data are encouraging for CNS applications, suggesting that there will be no immune suppression in patients and that repeated treatments with vector in the brain may not induce adverse immune responses. This important clinical issue requires more work with different transgenes, treat- ment protocols, and vectors. In particular, scenarios in which preexisting or posttreatment immune responses could occur, as with infection with a prevalent virus such as HIV, need to be explored. Most studies using lentiviral vectors have used powerful constitutive promoters such as PGK1, CMV, CAG, and EF1 (see citations in Ramezani et al., 2000). This is pragmatic and rational for nontoxic transgenes with a broad therapeutic window, if expression in nontarget cells is not an issue and if the potential benefit outweighs the perceived risk in a particular indication. Restricted expression may be desired for some situations, for example, striatal expression of the potent growth factor glial-derived nerve growth factor (GDNF) led to GDNF release and aberrant fiber sprouting at downstream striatal targets in animal models of Parkinson’s disease (Georgievska et al., 2002). These side effects may be limited by confining GDNF expression to astrocytes by using a glial cell-specific promoter (hGFAP), leading to more localized secretion (Jakobsson et al., 2003). Regulated gene expression to evaluate temporal aspects or to modulate dosage level will be important in some applications. Lentiviral vectors are particularly amenable to developing regulated systems, as their copy number in target cells is reasonably predictable and stable without silencing. This contrasts with AAV vectors, which have limited cargo capacity and which may produce such high copy numbers that regulators are potentially diluted out. Regulated systems in evaluation in lentiviral vectors to date use regulator proteins, for example, derived from bacterial operons such as the Tet repressor or engineered eukaryotic transcription factors (for some examples of regulation systems that can be applied in lentiviral vectors, see Ye et al., 1999; Reiser et al., 2000; Vigna et al., 2002; Koponen et al., 2003; Sirin and Park, 2003; Galimi et al., 2005; Pluta et al., 2005). These systems are now being refined to ensure that the basal expression is low and that the induced levels are high enough for the purpose (e.g., Vigna et al., 2002; Pluta et al., 2005). As all extraneous viral coding regions are assiduously removed from lentiviral vectors it is counterproductive to introduce foreign regulators that may be immunogenic and/or may influence the cell biology. Current work is therefore aimed at deimmunizing regulator proteins or at developing systems that can be regulated by small-molecule interactions with the proviral DNA or transcripts (Suess et al., 2003). Lentiviral vectors might be targeted to specific cell types by varying the envelope proteins although, as with all vector-targeting attempts to date, this is more likely to result in selectivity rather than absolute specificity. Ross River virus pseudotypes are more efficient on glial cells than neurons (Kang et al., 2002); lymphocytic choriomeningitis virus (LCMV) pseudotypes preferentially transduce murine neural stem cells and human glioma cell lines (Steffens et al., 2004; Stein et al., 2005); foamy virus, rabies virus, mokola virus, and amphotropic murine leukemia virus pseudotypes are enhanced on human neuroblastoma cell lines (Steffens et al., 2004); and rabies-G pseudotypes are useful for distal targeting of neurons in vivo because they are efficiently retrogradely transported (Mazarakis et al., 2001; Wong et al., 2004). Gene delivery for neurological disorders often requires invasive surgery and therefore repeat treatments are not desirable; long-term, stable expression at therapeutic levels is essential. Lentiviral vectors maintain expression for up to 16 months (e.g., LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS Kordower et al., 2000; Bienemann et al., 2003; Balaggan et al., 2005) and animals made transgenic with lentiviral vectors maintain expression without silencing (Lois et al., 2002; Pfeifer et al., 2002; McGrew et al., 2004). Furthermore, lentiviral vector transduction does not appear to affect the electrophysiological properties of neurons (Dittgen et al., 2004). Many types of therapeutic genes are being evaluated including trophic factors and enzymes, as well as intrabodies and siRNA targeted to defective intracellular proteins and genes. The versatility of lentiviral vectors allows ex vivo engineering of stem cells or differentiated cells for transplantation, direct injection at the target site, and remote delivery and targeting via retrograde transport. Some impressive efficacy data are emerging and some of these studies are outlined below. GENE THERAPY FOR PARKINSON’S DISEASE Parkinson’s disease (PD) is a progressive neurodegenerative disorder that arises as a result of the death of neurons in the substantia nigra region in the brain. Loss of these neurons causes depletion of dopamine levels in the putamen, leading to aberrant movement control. The mechanism of selective neuron loss is unknown, although degeneration may be due to neurotoxicity of accumulated misfolded proteins possibly induced/exacerbated by an altered redox state of the cells. Familial PD involves mutations in genes encoding proteins that participate in the ubiquitin–proteasome pathway (e.g., -synuclein, parkin, and ubiquitin C-terminal hydrolase L1), which may contribute to the formation of cytoplasmic inclusions termed “Lewy bodies” in neurons, a pathological hallmark of PD. Early-stage patients can be treated with oral L-3,4-dihydroxyphenylalanine (L-dopa), which crosses the blood–brain barrier and is metabolized by aromatic-L-amino-acid decarboxylase to produce dopamine. Prolonged depletion of natural dopamine increases dopamine sensitivity and the ectopic generation of dopamine leads to inappropriate localized concentrations, resulting in a serious side effect of debilitating abnormal movements (dyskinesia). The daily management of PD becomes difficult as L-dopa dosage requires frequent modulation and patients experience unpredictable periods of immobility and dyskinesia. For some patients surgical interventions such as deep brain stimulation (DBS) can control tremor or pallidotomy can control dyskinesia (reviewed in Betchen and Kaplitt, 2003). However, there are no curative treatments and patients cannot be restored to normal movement in a predictable and stable way. Late-stage PD is ideal for treatment by gene therapy because it is a localized disease and the pathology, biochemistry, and clinical presentations are well characterized. The most conservative approach is to manage the symptoms and one strategy already in the clinic injects an AAV vector expressing glutamic acid decarboxylase (GAD) into the subthalamic nucleus to limit tremor by stimulating the motor-inhibitory -aminobutyric acid (GABA)-ergic pathway (During et al., 2001). Another gene therapy strategy is to provide the missing dopamine in a manner designed to minimize side effects and this could displace oral L-dopa therapy as the standard of care in late-stage patients. Normally dopamine is produced in the nigral neurons by hydroxylation of tyrosine to L-dopa (via the enzyme tyrosine hydroxylase TH1 and the cofactor tetrahydrobiopterin, the biosyn- 3 thesis of which is rate limited by the enzyme GTP cyclohydrolase I [CH1]) and then L-dopa is decarboxylated to dopamine by aromatic-L-amino-acid decarboxylase (AADC). One approach in clinical trials uses an AAV vector to deliver just the AADC, which could augment the background levels of this enzyme to allow lower doses of L-dopa to be administered and might reduce the dyskinesia, although ectopic conversion may still occur with this approach (see http://www.avigen. com/non_financial_release/2005/2005_Avigen_EarlyData_PD ClinicalTrial_071805.htm). However, remarkable efficacy has been observed in animal models when all three genes are coexpressed in the striatum. This was demonstrated in nonhuman primates with AAV vectors (Muramatsu et al., 2002), but three different vectors carrying each of the genes were used because of the size limitation of AAV. The advantage of the higher capacity of lentiviral vectors has been exploited to build a single vector expressing all three enzymes, TH1, CHI, and AADC, by using internal ribosome entry sites to create a tricistronic transcript (Azzouz et al., 2002). This tricistronic vector gives a consistent, complete, and long-lasting reversal of the major Parkinsonian clinical and motor symptoms in a chronic rhesus macaque model of severe PD (Jarraya, B., Azzouz, M., Miskin, J., Ralph, G.S., Wilkes, F., Walmsley, L., Barber, R.D., Rohll, J., Drouot, X., Brouillet, E., Conde, F., Kingsman, S.M., Hantraye, P., Mitrophanous, K., Mazarakis, N.D., and Palfi, S., personal communication). A clinical protocol for gene-based dopamine replacement in patients who are beginning to fail on conventional L-dopa therapy is being developed (our unpublished data). The aim would be a single administration of the tricistronic lentiviral vector bilaterally into the striatum to produce tonic levels of dopamine. This should restore a level of stable motor function without the dyskinesia resulting from the fluctuating doses and ectopic sites of production associated with oral L-dopa therapy and oral L-dopa treatment could be reduced or eliminated. A more far-reaching opportunity for gene therapy is to delay symptoms by protecting the substantia nigra neurons. Localized lentivirus-mediated GDNF delivery protects the surviving neurons of the nigrostriatal dopaminergic pathway from further degeneration in rodent models (Bensadoun et al., 2000; Deglon et al., 2000; Gill et al., 2003; Azzouz et al., 2004d; Kirik et al., 2004) and in aged, nonlesioned or young, 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys (Kordower et al., 2000). However, there was shutdown of tyrosine hydroxylase in the restored nigral neurons and aberrant neuronal sprouting (Georgievska et al., 2002, 2004b). A proposal to evaluate AAV-mediated delivery of a related growth factor, neurturin (Dass et al., 2005), has also been discussed (NIH Recombinant DNA Advisory Committee [RAC] Meeting, March 16, 2005; http://www.webconferences.com/ nihoba/16_mar_2005.html). The use of a growth factor as the therapeutic gene raises many issues concerning potential longterm consequences and highlights the desirability of having some regulation in the system. Regulated lentiviral vectors may therefore be critical to advance this strategy into the clinic for PD and studies are in progress (Georgievska et al., 2004a). In trials of recombinant GDNF delivered via pumps, early indications of success (Gill et al., 2003) were not reproduced in larger studies (see http://www.amgen.com/media/media_pr_ detail.jsp?year52005&releaseID5673490) and toxicities have been seen with this approach (Nutt et al., 2003). It is therefore 4 WONG ET AL. not yet clear what further efficacy and safety studies will justify growth factor therapy for early-stage PD. Lentiviral vectors are also being used to create novel animal models for PD (Lo Bianco et al., 2002; Lauwers et al., 2003) and to test therapeutic strategies beyond dopamine replacement and neurotrophic support (Lo Bianco et al., 2002, 2004b). For example, lentiviral vector-mediated transfer of the A30P mutant -synuclein induces a selective and progressive loss of dopamine neurons associated with the appearance of -synuclein inclusions. In this model, lentiviral vector-mediated delivery of parkin to the substantia nigra gave significant neuroprotection, sparing TH-positive nerve terminals in the striatum and this appeared to be due to an effect on the detoxification of misfolded proteins (Lo Bianco et al., 2004b). Interestingly, prior injection of a lentiviral vector expressing GDNF was unable to block this neurodegeneration, suggesting that once a toxic cascade of protein aggregation has been initiated in a dopaminergic neuron then growth factors may not have a role to play (Lo Bianco et al., 2004a). ALZHEIMER’S DISEASE In Alzheimer’s disease (AD) there is progressive cognitive decline and memory loss. The only approved treatments are the chemical cholinesterase inhibitors to briefly delay the onset of memory loss. The disease-inducing pathology appears to be the aggregation of amyloid (A) peptides to form amyloid plaques, particularly in the hippocampus. Sequential cleavage of transmembrane amyloid precursor protein (APP) by an aspartyl protease, -secretase (or -site APP-cleaving enzyme [BACE-1]), and an enzyme complex including -secretase, generates different peptides with varying propensities to form plaques. Several factors influence the deposition of amyloidogenic peptides. The lipid-binding protein ApoE facilitates the deposition or clearance of A in the brain, depending on the isoform, and hence certain alleles of the apoE gene reduce or increase the risk of developing Alzheimer’s disease. The general transmembrane peptidase neprilysin, and a family of proteins called the presenilins (PS1 and PS1), also play a role in APP processing. Treatment strategies for AD include using trophic factors to protect neurons from damage, altering the balance of A accumulation by inhibiting secretases or enhancing degradative pathways, and clearing away the plaques by providing binding compounds or specific antibodies. Lentiviral vectors are being used to interrogate transgenic disease models and to develop new models. Lentivirus-mediated expression of neprilysin in the hippocampus of hAPP transgenic mice led to a 50% reduction in amyloid load and provided neuroprotection against A-induced neurotoxicity (Marr et al., 2003). Intracerebral administration of lentiviral vectors expressing ApoE isoforms differentially altered the amyloid burden in the hippocampus of PDAPP transgenic mice (Dodart et al., 2005). Lentiviral vector-mediated expression of ApoE2 or neprilysin in vulnerable brain regions in AD is therefore a rational approach to preventing or treating AD. The -secretase enzymes are classical targets for small-molecule drugs and the pharmaceutical industry is experienced in developing aspartyl protease inhibitors. Gene therapy might, however, have a role if the pharmacokinetics and bioavailabil- ity of small-molecule drugs are unfavorable for chronic disease management. One strategy would be to use lentivirus-mediated siRNA targeted against BACE-1, and this was shown to reduce amyloid production and behavioral deficits in APP transgenic mice (Singer et al., 2005). Neurons can also be protected from neurotoxicity by delivering antiapoptotic genes such as Bcl-xL (Blomer et al., 1998), although using an oncogene raises safety concerns. Recombinant nerve growth factor (NGF) delivered directly or via engrafted fibroblasts retrovirally engineered to secrete NGF prevented degeneration of the basal forebrain cholinergic neurons in rat and primate models (reviewed by Blesch and Tuszynski, 2004). When these strategies were tested in humans there were side effects after direct delivery of the protein in a few patients (Eriksdotter Jonhagen et al., 1998), but the ex vivo approach, using transplanted engineered fibroblasts, was promising in having no adverse effects and there were some indications of cognitive improvement and increased metabolic activity in the brain (Tuszynski et al., 2005). Another NGF clinical trial using an AAV vector is underway in earlystage AD patients (http://www.webconferences.com/nihoba/ 16_mar_2005.html). The approval of this trial is a good example of the regulatory balance of potential benefit versus perceived risk in a desperate patient group. Lentiviral vectors can deliver NGF but the aim would be to deal with symptoms and causes by including neuroprotective factors and factors that reduce and/or block amyloid formation. Furthermore, lentiviral vectors can provide access to all affected areas of the brain via the efficient retrograde transport or the use of cellular delivery systems (see Disseminated Neurological Disorders, below). Regulated and neuron-specific promoters would increase the safety of global distribution by imposing temporal and spatial restriction of gene expression. For example, Blesch et al. (2005) used a Tet-regulated lentiviral vector expressing NGF to rescue cholinergic neurons in the fimbria-fornix of rats after traumatic lesion mimicking AD pathology. Neuroprotective strategies will rely to some extent on the ability to reliably diagnose AD at a sufficiently early stage for neuroprotection to provide a benefit and the long-term consequences of gene therapy will require careful evaluation. HUNTINGTON’S DISEASE In Huntington’s disease (HD) the inheritance of a dominant mutated allele of the huntingtin (htt) gene causes a selective loss of striatal neurons leading to motor and cognitive deficiencies and death after 10 to 15 years. The mutant allele contains expansions of CAG trinucleotide repeats encoding polyglutamine (polyQ). A higher number of CAG repeats correlates with earlier age of disease onset and there is no treatment. Regulated lentiviral vectors used to overexpress various mutant htt gene fragments in the brains of rats and primates induced the typical neuronal pathology of Huntington’s disease. This was reversed by switching off gene expression, indicating that ablating mutant huntingtin is a viable therapeutic strategy (Regulier et al., 2003). Mutant huntingtin can be knocked out with an siRNA and this improved behavioral and pathological abnormalities in HD-N171-82Q transgenic mice overexpressing mutant proteins (Harper et al., 2005). Allele-specific suppression without compromising normal protein function would LENTIVIRUS-MEDIATED GENE TRANSFER TO THE CNS be desirable and this has been achieved in other polyQ disorders (Miller et al., 2003). Finally, trophic factors (ciliary neurotrophic factor [CNTF] and brain-derived neurotrophic factor [BDNF]) had a protective role when delivered by lentiviral vectors into rat striatal cell culture models of HD (Zala et al., 2005) and a lentiviral vector expressing CNTF protected against subsequent striatal degeneration induced by quinolinic acid in a rat model for HD (de Almeida et al., 2001). The clinical evaluation of any of these strategies will be challenging, given the slow progression of this disease and the limited number of patients. It is vital therefore that the optimum strategy be well rehearsed in relevant models before entering the clinic. SPINAL CORD AND NERVE INJURY The adult central nervous system cannot undergo repair after traumatic damage but gene transfer strategies are emerging that appear to overcome this limitation. Adenoviral vectors expressing trophic factors promoted regeneration of injured sensory afferents into the adult rat spinal cord (Zhang et al., 1998; Romero et al., 2001) and improved hind limb locomotor recovery from spinal cord contusion (Tai et al., 2003). Lentiviral vector-mediated delivery of GDNF protected facial motor neurons from axotomy-induced cell death in mice (Hottinger et al., 2000). The transcription factor RAR2 (retinoic acid receptor 2), expressed in an EIAV-based lentiviral vector, promoted neurite outgrowth in adult spinal cord explants (Corcoran et al., 2002) and gave significant protection of sensory and motor functions in a rat dorsal root ganglion rhizotomy model (Wong et al., 2005). Lentiviral vectors efficiently transduce Schwann cells (SCs) and olfactory ensheathing cells (OECs) ex vivo (Ruitenberg et al., 2002; Barakat et al., 2005) and these may be useful for producing neurotrophin-secreting SC/OECs for the promotion of axonal regeneration in spinal cord injury models. siRNA against axon growth inhibitors (Nogo receptor, p75NTR, or Rho-A) may also promote CNS axon regeneration in vivo (Ahmed et al., 2005). Lentiviral vectors expressing axogenic, neuroprotective, or regeneration molecules could restore neural function after injury but it is critical to determine how long after injury any of these strategies would be effective. They may need to be combined with methods for reducing or reversing scar formation or with methods for bridging or bypassing damaged areas. There are also significant challenges in defining clinical trials for disorders that are not life threatening, that generally occur in a younger population, and where the severity and therefore medical need may not be revealed for many months. DISSEMINATED NEUROLOGICAL DISORDERS A significant number of neurological disorders (e.g., motor neuron, Alzheimer’s, and lysosomal storage diseases) are disseminated throughout the brain or spinal cord, which poses a considerable challenge of access. Lentiviral vectors can cross the blood–brain barrier after in utero treatment or delivery to neonates (Kobayashi et al., 2005; Kyrkanides et al., 2005), but not in adults. Alternative approaches take advantage of specific 5 features of lentiviral vectors such as the property of highly efficient retrograde transport and the ability to transduce hematopoietic stem cells (HSCs) that traffic to the CNS. Motor neuron diseases are neurodegenerative diseases that cause progressive paralysis and premature death, and there are no treatments. The majority of amyotrophic lateral sclerosis (ALS) cases are sporadic, although there is an inherited form due to mutations in the superoxide dismutase (SOD1) gene. Spinal muscular atrophy (SMA) is the second commonest genetic disease affecting children and is due to mutations in the survival motor neuron (SMN1) gene. In mouse models for ALS (SOD1 mutant mouse) or SMA (SMN1 knockout mouse) lentiviral vectors pseudotyped with rabies-G envelope can be injected into the affected muscle, taken up at nerve terminals, and expressed in the cognate motor neurons in the spinal cord. Using this approach, vectors expressing vascular endothelial growth factor (Azzouz et al., 2004c) or siRNA targeted to a mutated SOD1 gene (Ralph et al., 2005b) or expressing the normal SMN1 gene (Azzouz et al., 2004b) extended corrected motor defects and extended survival in the relevant models. Remote access to the spinal cord via muscle can provide relatively simple palliative treatments to spare key muscle groups such as those that control hand movement, swallowing, and breathing to enhance the quality of and/or extend life. Alternatively, injections directly into the spinal cord or the brain may allow sufficient spread and access to upper motor neurons. Some success in injecting lentiviral vectors expressing inhibitory siRNA into the spinal cord of animal models of ALS has been reported (Raoul et al., 2005). Lysosomal enzyme deficiency disorders (e.g., Gaucher’s, Tay-Sachs, Hurler’s, Sly, and metachromatic leukodystrophies) result in the accumulation of neurotoxic metabolites leading to various pathologies and symptoms depending on the disease, including inflammation, demyelination, motor deficit, and death (Biffi and Naldini, 2005). The aim is to restore normal enzyme function by providing the wild-type gene and the challenge is to access all of the affected tissues. If the gene product is diffusible, for example, -glucuronidase (Brooks et al., 2002), or if enhanced diffusion can be engineered by the addition of peptide transporters (Xia et al., 2001), this can increase distribution throughout the CNS. Alternatively, Biffi et al. (2004) have used an ex vivo approach to transduce HSCs. They observed a progressive repopulation of CNS and peripheral nervous system (PNS) macrophage/microglial cells with arylsulfatase Aexpressing cells and obtained significant reversal of the systemic and neurological symptoms in a mouse model for a metachromatic leukodystrophy. This is an exciting approach and could be more widely applicable for delivering any secreted product to the nervous system. NEUROBIOLOGY RESEARCH APPLICATIONS OF LENTIVIRAL VECTORS Lentiviral vectors accelerate the generation of transgenic animals, increasing the range of animal models (Lois et al., 2002; Pfeifer et al., 2002; McGrew et al., 2004; Whitelaw et al., 2004). These vectors are particularly useful in identifying genes that can protect neurons against pathological insults in chronic settings, as described above, but also in acute settings such as 6 WONG ET AL. in stroke models (Ralph et al., 2004; Wong et al., 2005). They can bypass transgenesis altogether by making localized disease models with vectors expressing mutant proteins or siRNA (reviewed in Kirik and Bjorklund, 2003; Ralph et al., 2005a). These models can be made conditional by using regulated promoters or by applying Cre–lox recombination systems (Tiscornia et al., 2004; Ventura et al., 2004). An exciting technique for the future is to combine the power of lentiviral vectors and siRNA to create gene knockout libraries that will allow the rapid identification of therapeutic targets. The same lentiviral vector can transition into the clinic if a gene therapy approach is indicated, thus shortening the preclinical development time. These research applications of lentiviral vectors are promising to deliver a major advance in our understanding of the nervous system in health and disease. SUMMARY Lentiviral vectors are powerful tools for research and therapy. Their efficaciousness has been shown in models for major diseases such as Parkinson’s disease and Alzheimer’s disease and for the less prevalent but equally debilitating diseases such as motor neuron disease and lysosomal storage diseases. It may even be possible to treat serious spinal cord injuries with these vectors. All of these diseases or conditions are chronic and limitations to small-molecule drug therapies open the way for genetic therapies. Encouraging preclinical data on efficacy and safety with lentiviral vectors are rapidly accumulating in many laboratories and progress is being made in developing all of the relevant analytical, safety testing, and manufacturing methods (e.g., Rohll et al., 2002; Miskin et al., 2005; Sastry et al., 2005). The regulatory framework for evaluating viral vectors in the nervous system is in place and the paradigm of risk-versus-benefit analysis is being applied rationally for each specific indication (http://www.fda.gov/cber/gdlns/retrogt1000.pdf, http://www.emea.eu.int/pdfs/human/bwp/245803en.pdf). A number of lentiviral vectors are set to enter the clinic and hold real promise for the treatment and eventually the prevention of these difficult and distressing neurological conditions. Exciting times! ACKNOWLEDGMENTS The authors are grateful to Mimoun Azzouz and Alan Kingsman for scientific discussions and critical comments. REFERENCES ABORDO-ADESIDA, E., FOLLENZI, A., BARCIA, C., SCIASCIA, S., CASTRO, M.G., NALDINI, L., and LOWENSTEIN, P.R. (2005). Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses. Hum. Gene Ther. 16, 741–751. 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Address reprint requests to: Dr. Liang-Fong Wong Oxford BioMedica (UK) Ltd. Medawar Centre Robert Robinson Avenue The Oxford Science Park Oxford OX4 4GA, UK E-mail: [email protected] Received for publication October 3, 2005; accepted after revision November 11, 2005. Published online: December 15, 2005.