- Email: [email protected]
Gene therapy in psychiatric disorders: too early, too complex?
68
Gene therapy in psychiatric disorders: too early, too complex? John F Neumaier
Gene transfer strategies are being tested in a variety of animal models for psychiatric disorders. These promise to translate viral-mediated, non-viral and combinatorial techniques for delivery of transgenes into neuroanatomical and cell-type-speci®c therapeutic tools. However, these disorders involve complex functional neurocircuits and developmental aetiologies that may present even greater challenges than other neurological conditions. Addresses University of Washington, Department of Psychiatry and Behavioral Sciences, Box 359911, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104-2499, USA e-mail: [email protected]
Current Opinion in Pharmacology 2003, 3:68±72 This review comes from a themed issue on Neurosciences Edited by Robert S Sloviter and Thomas P Blackburn 1471-4892/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1471-4892(02)00012-7 Abbreviations AON antisense oligonucleotide BDNF brain-derived neurotrophic factor
Introduction
New developments in molecular neurobiology over the past ten years have created great excitement about the possibility of treating neurological and psychiatric illnesses with gene therapy approaches. These promise greater neuroanatomic accuracy of intervention as well as molecular speci®city. Such somatic cell gene therapies can be used to raise or lower the expression of a single target gene and may even be able to change the phenotype of targeted cells by altering the expression of regulatory proteins such as transcription factors; however, enthusiasm for these opportunities must be tempered with the realisation that we do not yet have proof that such manipulations will be more effective than currently available pharmacological strategies. Gene therapies can be thought of as direct remediation of a gene expression abnormality that causes a psychiatric illness, but more likely they would function as molecular manipulations that are akin to drug treatments (which often lead to changes in gene expression less directly). Although gene therapy for a few neurological disorders may be around the corner, the complexity of neuropsyCurrent Opinion in Pharmacology 2003, 3:68±72
chiatric disorders, and our lack of knowledge regarding their fundamental aetiologies, means that the application of gene therapy to mental illnesses is still in an early theoretical stage. This review identi®es the scope of the challenges faced in targeting these illnesses, introduces the gene transfer strategies that are under development and considers how these approaches may be re®ned in the future to ameliorate or even reverse the pathophysiology of illnesses such as schizophrenia, bipolar disorder and depression. The basic object of gene therapy in the brain is to introduce a piece of genetic material (usually DNA) coding for the desired gene sequence into cells of the brain to achieve a therapeutic change in brain function. The critical issue in the development of gene therapies may be the choice of gene transfer technology; however, the optimisation of the mode of delivery, tissue speci®city and degree of gene expression, as well as whether the transgene will be expressed transiently or permanently, are all issues that must be resolved before these approaches can be tested in humans. Progress is being made on each of these issues and the future of gene therapy is bright. In particular, gene therapies have been used in animal models of psychiatric symptoms for two purposes: to test whether certain gene manipulations will cause symptoms and whether gene transfer can be used to prevent or reduce these symptoms, which may ultimately lead to new treatments in humans. Because the serotonergic system plays a fundamental role in regulating behaviours involved in mental illnesses, examples of gene therapy strategies affecting serotonin neurotransmission will be used to illustrate the research and therapeutic potential for these genetic approaches. Altered expression of genes in the brain can be accomplished either by introducing a transgene into existing cells in the brain or by transplanting genetically modi®ed cells into the brain. The latter strategy has been considered for the treatment of Parkinson's disease [1] and is most appropriate if one hopes to replace or augment a critical extracellular signalling molecule, such as a neurotransmitter or a neuropeptide. For example, there is considerable evidence that brain-derived neurotrophic factor (BDNF) is reduced by stress in rodents [2], and is increased by antidepressant treatments in humans [3] and animals [2,4]. However, BDNF cannot be delivered to the brain using currently available delivery strategies, but perhaps it could be increased in speci®c brain regions by transplanting cells that express BDNF. This may be a viable option for treatment-resistant depression. In the future it may even be possible to collect cells from an www.current-opinion.com
Gene therapy in psychiatric disorders: too early, too complex? Neumaier 69
individual, alter the gene expression of these cells in the laboratory, and then reintroduce the cells into the brain where they may be less likely to induce immunological rejection than heterologous cells. Stem cells may also be valuable vehicles for gene therapy. Although transplanting cells in the brain is being vigorously pursued in animal models, it is not discussed further here as it compounds the issue of introducing a transgene with issues regarding the cell biology of transplantation.
Viral-mediated gene transfer
Currently, viral-mediated gene transfer is the most commonly utilised strategy for achieving gene transfer in the brains of experimental animals leading to altered behaviour. Several different viral vectors have been tested, modi®ed and optimised: each of these has advantages and disadvantages. These approaches have been reviewed recently [5,6]. The most commonly used vectors include replication de®cient herpes simplex virus, adenovirus, adeno-associated virus and several retroviruses. In each case, a desired transgene substitutes for a portion of the viral genome such that transgene will be expressed in infected brain cells. The vector lacks the viral structural genes necessary to induce subsequent cycles of infection. We have used a herpes simplex virus based `amplicon' system to overexpress 5-HT1B autoreceptors into neurons in the dorsal raphe nucleus (a major locus of serotonergic cell bodies that projects widely to limbic forebrain). This vector preferentially infects neurons and leads to ef®cient, but transient, gene expression by infected neurons with lower non-speci®c toxicity than many other viral vectors [5,7]. Direct injection of the vector by a stereotaxic method targets a speci®c neuronal type and the behaviours associated with these cells. Using a vector that expresses both epitope tagged 5-HT1B receptors and green ¯uorescent protein from separate transcription units [8], we increased 5-HT1B terminal autoreceptor expression in this anatomically focused manner, leading to altered stress reactivity and anxiety-related behaviours. By contrast, injection of the same vector into nucleus accumbens sensitised rats to cocaine-induced behaviours [9], showing that the same gene transfer manipulation in different neurocircuits can have different behavioural consequences. One important disadvantage of this technique is that the promoters used may drive gene expression in a non-speci®c manner, and are easier to use for compact subcortical nuclei than large or laminarly organised regions. The greater the cellular heterogeneity in a targeted region, the more valuable a neuron-type-speci®c gene therapy will be in minimising unintended side effects. It is possible to use either viral or synthetic promoters to control transcription of the transgene. Synthetic promoters derived from genes with discrete patterns of expression in the brain, such as that associated with dopaminecontaining neurons, may be valuable ways to produce www.current-opinion.com
expression only in the desired groups of cells [10]. For example, the tyrosine hydroxylase promoter should only be activated in dopaminergic cells, which expresses the necessary transacting factors to drive expression from this promoter. Although the techniques associated with these viruses continue to be improved, non-speci®c toxicity and inadequate cellular speci®city can be signi®cant problems, even though the viruses can be injected directly into a discrete brain region using stereotaxic procedures. It is likely that these complications can be reduced through further re®nements and the ef®cient expression associated with viral vectors is likely to make them attractive delivery systems in the future.
Non-viral gene transfer
Non-viral transfection of genes into cells grown in culture has been a popular research strategy for years and can be accomplished using a variety of simple methods. Some of these have also been attempted in brain [11]; for example, DNA can be transferred into brain cells using commercially available lipid carriers (lipofection) or polymers (such as polyethylenimine). Fabre et al. [12] used polyethylenimine to either increase or decrease serotonin transporter expression in dorsal raphe nucleus cells using a full length copy of, or partial length, antisense sequence from the serotonin transporter. They demonstrated changes in transporter density by radioligand binding and altered neurochemical and circadian behavioural activities. Although they achieved regional precision by injecting the DNA directly into the dorsal raphe nucleus, it is likely that both neurons and glial cells were transfected, and both serotonergic and non-serotonergic neurons expressed the transgenic serotonin transporter. This was an important achievement, nevertheless, because such non-viral approaches could easily be adapted to genetic material that might not be appropriate for expression with viral-based strategies, such as antisense oligonucleotides (discussed below).
Antisense strategies
The expression level of endogenous genes can be reduced by using antisense oligonucleotides (AONs); this has been referred to as gene `knockdown' [13,14]. The premise of this technique is that the AON hybridises with a speci®c sequence within the mRNA, and this heteroduplex leads to enzymatic degradation of the target mRNA [15]. Reduction in the amount of targeted mRNA usually leads to about 50% reduction in the associated protein in most studies. One advantage of the knockdown approach is that, if designed correctly, the AON will only have effects in cells that express the target gene, so that there may be a more selective effect than can be achieved with overexpression of the same protein or with pharmacological antagonists. However, there are likely to be subtle differences between preventing receptors from being synthesised and blocking them with an antagonist drug, which may have variable degrees of inverse Current Opinion in Pharmacology 2003, 3:68±72
70 Neurosciences
agonism, `¯utter' block, etc. Many different chemistries for AONs are being explored [13,16]. They differ in their sensitivity to degradation before speci®c hybridisation. This is important as it reduces the effectiveness of the AON, requiring higher doses to achieve knockdown and greater likelihood of non-speci®c toxicity. Morpholino AONs may be a promising method for inducing gene knockdown [17], but these uncharged oligonucleotides may be more dif®cult to introduce into cell cytoplasm. The delivery of AONs has also not been optimised, although lipofection and polyethylenimine methods have been used. AONs are generally injected into brain stereotaxically in animal models, but intravenous delivery may become feasible if the blood brain barrier can be ef®ciently penetrated [18]. The pharmacological similarity among several of the serotonin receptors has hampered the development of highly selective ligands, but the selectivity based on gene sequence circumvents this problem. AONs have been used extensively to alter serotonergic function in animals by targeting 5-HT2A receptors [19], 5-HT3 receptors [20] and monoamine oxidase B [21]. The 5-HT6 receptor has been studied with this technique by several groups, even though it was discovered recently and is poorly understood. These studies have shown AON-mediated reduction in 5-HT6 receptor-binding sites in brain with associated changes in motor, cognitive, stress and anxiety behaviours [22±24,25,26]. Indeed, these studies have played an important role in de®ning the repertoire of behaviours that 5-HT6 receptors mediate. Lastly, a new and exciting method of small interfering RNA technology may offer even more ef®cient degradation of target mRNAs than AONs [27,28], but has yet to be successfully demonstrated in a range of mammalian cells and has not been reported in vivo. Intracellular delivery of DNA has also been achieved in vitro through ballistic [29] and electroporation [30] strategies. Electroporation involves the application of brief pulses of electricity, transiently inducing micropores in the plasma membrane, thus allowing exogenous molecules (such as DNA plasmids or oligonucleotides) to enter the cell. This technique has also recently been used in developing brain to express transgenes [31] and may, ultimately, be useful in adult brain as well [32±34]. Although electroporation in vivo might involve surgical procedures, it may avoid toxicity associated with the infection and transfection techniques discussed above. The preceding section discussed several strategies for modulating gene expression. Although each of these approaches has the potential for making very speci®c modi®cations in target neurons, some newer transgenic strategies may offer additional lessons. Once a gene construct is delivered, it may be possible to modulate expression levels further by using conditional expression with vectors controlled by tetracycline, ecdysone or other Current Opinion in Pharmacology 2003, 3:68±72
administered drugs [35]. This approach can be used to turn gene expression either on or off, and might allow ®ne tuning of gene expression. Most gene therapies proposed to date have attempted to change the expression of endogenously expressed genes; however, it may be possible to design novel receptors designed to cause a desired biological effect under the control of an administered drug that otherwise has no human binding site. For example, the binding site of a receptor, enzyme or transcription factor can be modi®ed by site-directed mutagenesis such that it is activated by a novel agent exclusively; the activity can then be stimulated only when the agent is administered orally. Although complicated, these types of combinatorial therapies may increase the precision of gene therapies. Gene therapies hold great promises for the treatment of severe mental disorders; however, signi®cant barriers still exist. First of all, we must identify gene targets that are very likely to lead to substantial therapeutic results when modulated. Currently, there is no single target that we can be certain will assure adequate results in any mental illness. Indeed, mental illnesses are characterised by highly individual responses to currently available treatments. This contrasts with some neurological disorders, such as Parkinson's disease, where we can be reasonably certain that regionally targeted increased expression of tyrosine hydroxylase will increase dopamine release thereby relieving symptoms. De®ciency syndromes (like Parkinson's disease) are likely to be easier to treat than conditions that require optimisation of function in widely distributed networks. Although parkinsonism may be relieved by gene therapy in a small proportion of targeted neurons, this may be insuf®cient for psychiatric disorders. Finally, the neuropathology of mental illnesses, especially schizophrenia [36] and mood disorders [37], may involve the interaction of genes and environment very early in brain development. The hope for suitable genetic treatments for psychiatric disorders in adults may be unrealistic in some cases; however, gene manipulations in animal models may be one of the most powerful means for clarifying the cause of these disorders now, and may allow much more selective interventions in human brain function in the future.
Conclusions
Gene therapies hold great promise for increasing the neuroanatomical speci®city of treatments for mental illnesses while reducing unnecessary or dangerous side effects. A range of viral, non-viral and combinatorial strategies are rapidly becoming available for animal models of psychiatric disorders and may eventually become available for humans. Perhaps the most exciting next steps will involve the use of tissue-speci®c promoters for transgenes and expression regulated by administered drugs; however, these strategies must be based on greater understanding of the causes of mental illnesses. www.current-opinion.com
Gene therapy in psychiatric disorders: too early, too complex? Neumaier 71
Acknowledgements
The author's work was supported by the National Institute of Mental Health (MH63303).
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Ishida A, Yasuzumi F: Approach to ex vivo gene therapy in the treatment of Parkinson's disease. Brain Dev 2000, 22(Suppl 1): S143-S147.
17. Corey DR, Abrams JM: Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol 2001, 2:REVIEWS1015. 18. Pardridge WM: Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov 2002, 1:131-139. 19. Scalzitti JM, Berg KA, Kratowicz SA, Hensler JG: Regulation of serotonin2A receptor expression by an antisense oligodeoxynucleotide. J Neurochem 1998, 71:1457-1463. 20. Paul D, Yao D, Zhu P, Minor LD, Garcia MM: 5-hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach. J Pharmacol Exp Ther 2001, 298:674-678. 21. Falk EM, Cook VJ, Nichols DE, Sprague JE: An antisense oligonucleotide targeted at MAO-B attenuates rat striatal serotonergic neurotoxicity induced by MDMA. Pharmacol Biochem Behav 2002, 72:617-622.
2.
Nibuya M, Morinobu S, Duman RS: Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J Neurosci 1995, 15:7539-7547.
3.
Chen B, Dowlatshahi D, MacQueen GM, Wang JF, Young LT: Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiatry 2001, 50:260-265.
4.
Russo-Neustadt A, Beard RC, Cotman CW: Exercise, antidepressant medications, and enhanced brain derived neurotrophic factor expression. Neuropsychopharmacology 1999, 21:679-682.
23. Hamon M, Doucet E, Lefevre K, Miquel MC, Lanfumey L, Insausti R, Frechilla D, Del Rio J, Verge D: Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 1999, 21:68S-76S.
5.
Carlezon WA Jr, Nestler EJ, Neve RL: Herpes simplex virus-mediated gene transfer as a tool for neuropsychiatric research. Crit Rev Neurobiol 2000, 14:47-67.
24. Sleight AJ, Monsma FJ Jr, Borroni E, Austin RH, Bourson A: Effects of altered 5-HT6 expression in the rat: functional studies using antisense oligonucleotides. Behav Brain Res 1996, 73:245-248.
6.
Neve RL: Overview of gene delivery into cells using HSV-1-based vectors. In Current Protocols in Neuroscience. Edited by Hall ZW. John Wiley and Sons; 1999.
7.
Neve RL: Generation of high-titer defective HSV-1 vectors. In Current Protocols in Neuroscience. Edited by Hall ZW. John Wiley and Sons; 1999.
8.
Clark MS, Sexton TJ, McClain M, Root DC, Kohen R, Neumaier JF: Overexpression of 5-HT1B receptor in dorsal raphe nucleus using herpes simplex virus gene transfer increases anxiety behavior after inescapable stress. J Neurosci 2002, 22:4550-4562.
25. Woolley ML, Bentley JC, Sleight AJ, Marsden CA, Fone KC: A role for 5-ht6 receptors in retention of spatial learning in the Morris water maze. Neuropharmacology 2001, 41:210-219. This paper describes the comparison of AON knockdown of central 5-HT6 receptors and systemic administration of a 5-HT6 antagonist. This study is especially interesting because it suggests that reduced 5-HT6 function enhances cholinergic activity with resulting improvement in learning behaviour. The comparison of AON knockdown and pharmacological blockade reduces the likelihood of non-speci®c mechanisms accounting for their behavioural observations.
9.
Neumaier JF, Vincow E, Arvanitogiannis A, Wise RA, Carlezon WA: Elevated expression of 5-HT1B receptors in nucleus accumbens efferents sensitizes animals to cocaine. J Neurosci 2002, in press.
10. Song S, Wang Y, Bak SY, Lang P, Ullrey D, Neve RL, O'Malley KL, Geller AI: An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-speci®c expression in the midbrain. J Neurochem 1997, 68:1792-1803. 11. Li S, Ma Z: Nonviral gene therapy. Curr Gene Ther 2001, 1:201-226. An excellent review of the technical issues regarding the development of non-viral gene transfer strategies in vivo. 12. Fabre V, Boutrel B, Hanoun N, Lanfumey L, Fattaccini CM, Demeneix B, Adrien J, Hamon M, Martres MP: Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer. J Neurosci 2000, 20:5065-5075. 13. Bennett CF, Cowsert LM: Application of antisense oligonucleotides for gene functionalization and target validation. Curr Opin Mol Ther 1999, 1:359-371. 14. Robinson ES, Nutt DJ, Jackson HC, Hudson AL: Antisense oligonucleotides in psychopharmacology and behaviour: promises and pitfalls. J Psychopharmacol 1997, 11:259-269. 15. Zamaratski E, Pradeepkumar PI, Chattopadhyaya J: A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H. J Biochem Biophys Methods 2001, 48:189-208. 16. Mickle®eld J: Backbone modi®cation of nucleic acids: synthesis, structure and therapeutic applications. Curr Med Chem 2001, 8:1157-1179. www.current-opinion.com
22. Bourson A, Borroni E, Austin RH, Monsma FJ Jr, Sleight AJ: Determination of the role of the 5-ht6 receptor in the rat brain: a study using antisense oligonucleotides. J Pharmacol Exp Ther 1995, 274:173-180.
26. Yoshioka M, Matsumoto M, Togashi H, Mori K, Saito H: Central distribution and function of 5-HT6 receptor subtype in the rat brain. Life Sci 1998, 62:1473-1477. 27. Hammond SM, Caudy AA, Hannon GJ: Post-transcriptional gene silencingby double-stranded RNA. NatRevGenet 2001,2:110-119. 28. Hannon GJ: RNA interference. Nature 2002, 418:244-251. This review introduces the reader to a new technique for gene silencing that may become a fundamental new way to alter gene expression in vivo. 29. Biewenga JE, Destree OH, Schrama LH: Plasmid-mediated gene transfer in neurons using the biolistics technique. J Neurosci Methods 1997, 71:67-75. 30. Schoenwolf GC: Cutting, pasting and painting: experimental embryology and neural development. Nat Rev Neurosci 2001, 2:763-771. 31. Saito T, Nakatsuji N: Ef®cient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 2001, 240:237-246. This paper carefully documents the conditions to achieve gene transfer in vivo using electroporation with embryos. The authors injected green ¯uorescent protein vector intracranially and performed square wave electroporation using extracranial electrodes. They carefully documented energy parameters, green ¯uorescent protein expression and toxicity. 32. Kondoh T, Motooka Y, Bhattacharjee AK, Kokunai T, Saito N, Tamaki N: In vivo gene transfer into the periventricular region by electroporation. Neurol Med Chir (Tokyo) 2000, 40:618-622. 33. Mo X, Yokoyama A, Oshitari T, Negishi H, Dezawa M, Mizota A, Adachi-Usami E: Rescue of axotomized retinal ganglion cells by BDNF gene electroporation in adult rats. Invest Ophthalmol Vis Sci 2002, 43:2401-2405. 34. Aberg MA, Ryttsen F, Hellgren G, Lindell K, Rosengren LE, MacLennan AJ, Carlsson B, Orwar O, Eriksson PS: Selective Current Opinion in Pharmacology 2003, 3:68±72
72 Neurosciences
introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis. Mol Cell Neurosci 2001, 17:426-443. 35. Yamamoto A, Hen R, Dauer WT: The ons and offs of inducible transgenic technology: a review. Neurobiol Dis 2001, 8:923-932.
Current Opinion in Pharmacology 2003, 3:68±72
36. Sawa A, Snyder SH: Schizophrenia: diverse approaches to a complex disease. Science 2002, 296:692-695. 37. Ressler KJ, Nemeroff CB: Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders. Depress Anxiety 2000, 12(Suppl 1):2-19.
www.current-opinion.com
Gene therapy in psychiatric disorders: too early, too complex? John F Neumaier
Gene transfer strategies are being tested in a variety of animal models for psychiatric disorders. These promise to translate viral-mediated, non-viral and combinatorial techniques for delivery of transgenes into neuroanatomical and cell-type-speci®c therapeutic tools. However, these disorders involve complex functional neurocircuits and developmental aetiologies that may present even greater challenges than other neurological conditions. Addresses University of Washington, Department of Psychiatry and Behavioral Sciences, Box 359911, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104-2499, USA e-mail: [email protected]
Current Opinion in Pharmacology 2003, 3:68±72 This review comes from a themed issue on Neurosciences Edited by Robert S Sloviter and Thomas P Blackburn 1471-4892/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1471-4892(02)00012-7 Abbreviations AON antisense oligonucleotide BDNF brain-derived neurotrophic factor
Introduction
New developments in molecular neurobiology over the past ten years have created great excitement about the possibility of treating neurological and psychiatric illnesses with gene therapy approaches. These promise greater neuroanatomic accuracy of intervention as well as molecular speci®city. Such somatic cell gene therapies can be used to raise or lower the expression of a single target gene and may even be able to change the phenotype of targeted cells by altering the expression of regulatory proteins such as transcription factors; however, enthusiasm for these opportunities must be tempered with the realisation that we do not yet have proof that such manipulations will be more effective than currently available pharmacological strategies. Gene therapies can be thought of as direct remediation of a gene expression abnormality that causes a psychiatric illness, but more likely they would function as molecular manipulations that are akin to drug treatments (which often lead to changes in gene expression less directly). Although gene therapy for a few neurological disorders may be around the corner, the complexity of neuropsyCurrent Opinion in Pharmacology 2003, 3:68±72
chiatric disorders, and our lack of knowledge regarding their fundamental aetiologies, means that the application of gene therapy to mental illnesses is still in an early theoretical stage. This review identi®es the scope of the challenges faced in targeting these illnesses, introduces the gene transfer strategies that are under development and considers how these approaches may be re®ned in the future to ameliorate or even reverse the pathophysiology of illnesses such as schizophrenia, bipolar disorder and depression. The basic object of gene therapy in the brain is to introduce a piece of genetic material (usually DNA) coding for the desired gene sequence into cells of the brain to achieve a therapeutic change in brain function. The critical issue in the development of gene therapies may be the choice of gene transfer technology; however, the optimisation of the mode of delivery, tissue speci®city and degree of gene expression, as well as whether the transgene will be expressed transiently or permanently, are all issues that must be resolved before these approaches can be tested in humans. Progress is being made on each of these issues and the future of gene therapy is bright. In particular, gene therapies have been used in animal models of psychiatric symptoms for two purposes: to test whether certain gene manipulations will cause symptoms and whether gene transfer can be used to prevent or reduce these symptoms, which may ultimately lead to new treatments in humans. Because the serotonergic system plays a fundamental role in regulating behaviours involved in mental illnesses, examples of gene therapy strategies affecting serotonin neurotransmission will be used to illustrate the research and therapeutic potential for these genetic approaches. Altered expression of genes in the brain can be accomplished either by introducing a transgene into existing cells in the brain or by transplanting genetically modi®ed cells into the brain. The latter strategy has been considered for the treatment of Parkinson's disease [1] and is most appropriate if one hopes to replace or augment a critical extracellular signalling molecule, such as a neurotransmitter or a neuropeptide. For example, there is considerable evidence that brain-derived neurotrophic factor (BDNF) is reduced by stress in rodents [2], and is increased by antidepressant treatments in humans [3] and animals [2,4]. However, BDNF cannot be delivered to the brain using currently available delivery strategies, but perhaps it could be increased in speci®c brain regions by transplanting cells that express BDNF. This may be a viable option for treatment-resistant depression. In the future it may even be possible to collect cells from an www.current-opinion.com
Gene therapy in psychiatric disorders: too early, too complex? Neumaier 69
individual, alter the gene expression of these cells in the laboratory, and then reintroduce the cells into the brain where they may be less likely to induce immunological rejection than heterologous cells. Stem cells may also be valuable vehicles for gene therapy. Although transplanting cells in the brain is being vigorously pursued in animal models, it is not discussed further here as it compounds the issue of introducing a transgene with issues regarding the cell biology of transplantation.
Viral-mediated gene transfer
Currently, viral-mediated gene transfer is the most commonly utilised strategy for achieving gene transfer in the brains of experimental animals leading to altered behaviour. Several different viral vectors have been tested, modi®ed and optimised: each of these has advantages and disadvantages. These approaches have been reviewed recently [5,6]. The most commonly used vectors include replication de®cient herpes simplex virus, adenovirus, adeno-associated virus and several retroviruses. In each case, a desired transgene substitutes for a portion of the viral genome such that transgene will be expressed in infected brain cells. The vector lacks the viral structural genes necessary to induce subsequent cycles of infection. We have used a herpes simplex virus based `amplicon' system to overexpress 5-HT1B autoreceptors into neurons in the dorsal raphe nucleus (a major locus of serotonergic cell bodies that projects widely to limbic forebrain). This vector preferentially infects neurons and leads to ef®cient, but transient, gene expression by infected neurons with lower non-speci®c toxicity than many other viral vectors [5,7]. Direct injection of the vector by a stereotaxic method targets a speci®c neuronal type and the behaviours associated with these cells. Using a vector that expresses both epitope tagged 5-HT1B receptors and green ¯uorescent protein from separate transcription units [8], we increased 5-HT1B terminal autoreceptor expression in this anatomically focused manner, leading to altered stress reactivity and anxiety-related behaviours. By contrast, injection of the same vector into nucleus accumbens sensitised rats to cocaine-induced behaviours [9], showing that the same gene transfer manipulation in different neurocircuits can have different behavioural consequences. One important disadvantage of this technique is that the promoters used may drive gene expression in a non-speci®c manner, and are easier to use for compact subcortical nuclei than large or laminarly organised regions. The greater the cellular heterogeneity in a targeted region, the more valuable a neuron-type-speci®c gene therapy will be in minimising unintended side effects. It is possible to use either viral or synthetic promoters to control transcription of the transgene. Synthetic promoters derived from genes with discrete patterns of expression in the brain, such as that associated with dopaminecontaining neurons, may be valuable ways to produce www.current-opinion.com
expression only in the desired groups of cells [10]. For example, the tyrosine hydroxylase promoter should only be activated in dopaminergic cells, which expresses the necessary transacting factors to drive expression from this promoter. Although the techniques associated with these viruses continue to be improved, non-speci®c toxicity and inadequate cellular speci®city can be signi®cant problems, even though the viruses can be injected directly into a discrete brain region using stereotaxic procedures. It is likely that these complications can be reduced through further re®nements and the ef®cient expression associated with viral vectors is likely to make them attractive delivery systems in the future.
Non-viral gene transfer
Non-viral transfection of genes into cells grown in culture has been a popular research strategy for years and can be accomplished using a variety of simple methods. Some of these have also been attempted in brain [11]; for example, DNA can be transferred into brain cells using commercially available lipid carriers (lipofection) or polymers (such as polyethylenimine). Fabre et al. [12] used polyethylenimine to either increase or decrease serotonin transporter expression in dorsal raphe nucleus cells using a full length copy of, or partial length, antisense sequence from the serotonin transporter. They demonstrated changes in transporter density by radioligand binding and altered neurochemical and circadian behavioural activities. Although they achieved regional precision by injecting the DNA directly into the dorsal raphe nucleus, it is likely that both neurons and glial cells were transfected, and both serotonergic and non-serotonergic neurons expressed the transgenic serotonin transporter. This was an important achievement, nevertheless, because such non-viral approaches could easily be adapted to genetic material that might not be appropriate for expression with viral-based strategies, such as antisense oligonucleotides (discussed below).
Antisense strategies
The expression level of endogenous genes can be reduced by using antisense oligonucleotides (AONs); this has been referred to as gene `knockdown' [13,14]. The premise of this technique is that the AON hybridises with a speci®c sequence within the mRNA, and this heteroduplex leads to enzymatic degradation of the target mRNA [15]. Reduction in the amount of targeted mRNA usually leads to about 50% reduction in the associated protein in most studies. One advantage of the knockdown approach is that, if designed correctly, the AON will only have effects in cells that express the target gene, so that there may be a more selective effect than can be achieved with overexpression of the same protein or with pharmacological antagonists. However, there are likely to be subtle differences between preventing receptors from being synthesised and blocking them with an antagonist drug, which may have variable degrees of inverse Current Opinion in Pharmacology 2003, 3:68±72
70 Neurosciences
agonism, `¯utter' block, etc. Many different chemistries for AONs are being explored [13,16]. They differ in their sensitivity to degradation before speci®c hybridisation. This is important as it reduces the effectiveness of the AON, requiring higher doses to achieve knockdown and greater likelihood of non-speci®c toxicity. Morpholino AONs may be a promising method for inducing gene knockdown [17], but these uncharged oligonucleotides may be more dif®cult to introduce into cell cytoplasm. The delivery of AONs has also not been optimised, although lipofection and polyethylenimine methods have been used. AONs are generally injected into brain stereotaxically in animal models, but intravenous delivery may become feasible if the blood brain barrier can be ef®ciently penetrated [18]. The pharmacological similarity among several of the serotonin receptors has hampered the development of highly selective ligands, but the selectivity based on gene sequence circumvents this problem. AONs have been used extensively to alter serotonergic function in animals by targeting 5-HT2A receptors [19], 5-HT3 receptors [20] and monoamine oxidase B [21]. The 5-HT6 receptor has been studied with this technique by several groups, even though it was discovered recently and is poorly understood. These studies have shown AON-mediated reduction in 5-HT6 receptor-binding sites in brain with associated changes in motor, cognitive, stress and anxiety behaviours [22±24,25,26]. Indeed, these studies have played an important role in de®ning the repertoire of behaviours that 5-HT6 receptors mediate. Lastly, a new and exciting method of small interfering RNA technology may offer even more ef®cient degradation of target mRNAs than AONs [27,28], but has yet to be successfully demonstrated in a range of mammalian cells and has not been reported in vivo. Intracellular delivery of DNA has also been achieved in vitro through ballistic [29] and electroporation [30] strategies. Electroporation involves the application of brief pulses of electricity, transiently inducing micropores in the plasma membrane, thus allowing exogenous molecules (such as DNA plasmids or oligonucleotides) to enter the cell. This technique has also recently been used in developing brain to express transgenes [31] and may, ultimately, be useful in adult brain as well [32±34]. Although electroporation in vivo might involve surgical procedures, it may avoid toxicity associated with the infection and transfection techniques discussed above. The preceding section discussed several strategies for modulating gene expression. Although each of these approaches has the potential for making very speci®c modi®cations in target neurons, some newer transgenic strategies may offer additional lessons. Once a gene construct is delivered, it may be possible to modulate expression levels further by using conditional expression with vectors controlled by tetracycline, ecdysone or other Current Opinion in Pharmacology 2003, 3:68±72
administered drugs [35]. This approach can be used to turn gene expression either on or off, and might allow ®ne tuning of gene expression. Most gene therapies proposed to date have attempted to change the expression of endogenously expressed genes; however, it may be possible to design novel receptors designed to cause a desired biological effect under the control of an administered drug that otherwise has no human binding site. For example, the binding site of a receptor, enzyme or transcription factor can be modi®ed by site-directed mutagenesis such that it is activated by a novel agent exclusively; the activity can then be stimulated only when the agent is administered orally. Although complicated, these types of combinatorial therapies may increase the precision of gene therapies. Gene therapies hold great promises for the treatment of severe mental disorders; however, signi®cant barriers still exist. First of all, we must identify gene targets that are very likely to lead to substantial therapeutic results when modulated. Currently, there is no single target that we can be certain will assure adequate results in any mental illness. Indeed, mental illnesses are characterised by highly individual responses to currently available treatments. This contrasts with some neurological disorders, such as Parkinson's disease, where we can be reasonably certain that regionally targeted increased expression of tyrosine hydroxylase will increase dopamine release thereby relieving symptoms. De®ciency syndromes (like Parkinson's disease) are likely to be easier to treat than conditions that require optimisation of function in widely distributed networks. Although parkinsonism may be relieved by gene therapy in a small proportion of targeted neurons, this may be insuf®cient for psychiatric disorders. Finally, the neuropathology of mental illnesses, especially schizophrenia [36] and mood disorders [37], may involve the interaction of genes and environment very early in brain development. The hope for suitable genetic treatments for psychiatric disorders in adults may be unrealistic in some cases; however, gene manipulations in animal models may be one of the most powerful means for clarifying the cause of these disorders now, and may allow much more selective interventions in human brain function in the future.
Conclusions
Gene therapies hold great promise for increasing the neuroanatomical speci®city of treatments for mental illnesses while reducing unnecessary or dangerous side effects. A range of viral, non-viral and combinatorial strategies are rapidly becoming available for animal models of psychiatric disorders and may eventually become available for humans. Perhaps the most exciting next steps will involve the use of tissue-speci®c promoters for transgenes and expression regulated by administered drugs; however, these strategies must be based on greater understanding of the causes of mental illnesses. www.current-opinion.com
Gene therapy in psychiatric disorders: too early, too complex? Neumaier 71
Acknowledgements
The author's work was supported by the National Institute of Mental Health (MH63303).
References and recommended reading
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