BRITISH JOURNAL OF 2001), 178, 392^394 EDITORIAL

Gene therapy for neurodegenerative diseases: CURRENT GENE THERAPY STRATEGIES FOR fact or fiction? NEURODEGENERATIVE DISEASES JANET E. CARTER and EDWARD H. SCHUCHMAN To date, gene therapy proper in the CNS has been tested only in experimental animal model systems and has included delivering genes encoding neurotrophic factors to inhibit or sti- mulate regeneration, and the replacement of deficient neurotransmitter proteins or Primary neurodegenerative diseases repre- function in patients with Huntington's dis- metabolic enzymes. sent debilitating and progressive condi- ease and Parkinson's disease, respectively. In an animal model of Parkinson's tions for which no cure is currently The first of these strategies gene therapy) disease, for example, in vivoinvivo andand ex vivoexvivo available and for which therapeutic inter- is discussed further here. approaches to replace dopamine have ventions remain limited to palliative and included direct stereotactic intracerebral symptomatic treatment. Advances in gene injection of viral vectors expressing the transfer technology now present the realis- GETGETTING TING GENES INTO tyrosine hydroxylase gene to striatal neu- tic prospect of delivering therapeutic genes CELLS ^ VECTOR-MEDIATED rons, or transplanting cells fibroblasts or to the brain for neuroprotection, restora- GENE DELIVERY )expressing tyrosine hydrox- tion of neuronal function or replacement ylase Horrelou et aletal, 1994: Ridet et aletal,, of deficient proteins as treatments for To date, two approaches to gene delivery 1999). Co-expression of several enzymes these diseases. Thus, although central ner- into somatic cells exist: ex vivoexvivo andand in vivoinvivo.. on the biosynthetic pathway to dopamine vous system CNS)gene therapy presents a Ex vivoExvivo approaches require vector- e.g.e.g. LL-aromatic amino acid decarboxylase number of unique challenges and clinical mediated gene transfer into cells before and tyrosine hydroxylase)from one viral applications remain in their infancy, the transplantation into the target, whereas in vector or coincident expression of the two realisation of this goal is approaching at the context of the CNS in vivoinvivo refers torefersto enzymes from two separate vectors, deliv- an ever-increasing rate. directdirect in situinsitu intracerebral gene delivery. ered at the same time, has also been used to The vectors of choice for both methods of maximise dopamine production. In a related gene transfer are viruses modified to impair strategy, tyrosine hydroxylase has been THERAPEUTIC STRATEGIES ^ their replication after entering cells i.e. expressed virally together with guanosine- OVERVIEW OF APPROACHES replication-replication-deficient).deficient). Five such viral vector 55'' -triphosphate GTP)cyclohydolase I, the systems are currently available, including enzyme necessary for generation of the Broadly speaking, four categories of thera- retrovirus, adenovirus, adeno-associated tetrahydrobiopterin cofactor for tyrosine peutic strategy have become available as a virus, herpes simplex virus and lentivirus, hydroxylase Mandel et aletal, 1998). Al- result of recent advances in genetics and together with a range of artificially con- though interpretation must remain cau- cell biology. The first is gene therapy structed vectors or chimeras that are tious because of the limitations of proper, which describes attempts to deliver hybrids of the above. Parkinsonian animal models, improve- `healthy' genes to a target organ such as All currently available viral vector ments in behaviour and increased dopa- the brain. This category includes the spe- systems have inherent limitations but it is mine synthesis have been reported. cialised field of stem cell transplantation, important to note that innovations in Neuroprotective paradigms for Parkin- which exploits the inherent biology of vector design and purification strategies son's disease, in vivoinvivo andand ex vivoexvivo, have,have organ-specific stem cells to provide a have helped to overcome some of these included delivering genes encoding trophic source of genetically engineered replace- disadvantages. For example, `gutless' adeno- factors such as glial-derived neurotrophic ment cells for the delivery of therapeutic viral vectors, stripped of all structural viral factor and brain-derived neurotrophic fac- genes to localised and widespread regions genes and disguising pathogenic viruses in tor and anti-apoptotic genes such as the of the CNS. The second set of strategies the `coats' of non-virulent strains, help to proto-oncogene Bcl-2 Bilang-Bleuel et aletal,, aims to reduce expression of mutated genes minimise toxicity. Manipulation of specific 1997; Choi-Lundberg et aletal, 1997). En- and includes the use of antisense oligo- gene sequences i.e. promoters, enhancers) couraging results demonstrate successful nucleotides and ribozymes. The third has been used to confine transgene expres- rescue of degenerating striatal approach is to use positional cloning sion to specific cell types in the brain and and improved behavioural function in strategies to identify mutated genes in- to produce longer-term expression for a animal models. volved in disease, and then to make use comprehensive review of viral vectors, see Gene therapy in animal models for of the identified gene variants for new Hermans & Verhaagen, 1998). Huntington's disease has similarly concen- drug discovery. Finally, transplantation of A number of non-viral gene transfer trated upon protective ex vivoexvivo approaches, non-genetically engineered primary em- methods also have been developed for gene transplanting cells secreting nerve growth bryonic neural tissue has been used in clin- therapy, but at the present time the ineffi- factor NGF)or human ciliary neuro- ical trials, with varying degrees of success, ciency of these methods precludes clinical trophic factor for a review, see Kordower to improve cognitive function and motor application. et aletal, 1999).,1999).

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Experimental animal models with JANET E.CARTER, MRCPsych, Department of and Section of Old Age Psychiatry,Institute chemically induced lesions of cholinergic of Psychiatry,King's College, London,UK and and Department Department of of Human Human Genetics, Genetics, Mount Mount Sinai Sinai School School of of Medicine, Medicine, basal forebrain neurons have been used to New York; EDWARD H. SCHUCHMAN, PhD,Department of Human Genetics, Mount Sinai School mimic the behavioural and neurodegenera- of Medicine, New York tive changes of Alzheimer's disease. In exex vivovivo approaches, implantation of fibroblasts, Correspondence: Dr Janet Carter,Department of Neuroscience and Old Age Psychiatry,Institute genetically manipulated to produce NGF,NGF, of Psychiatry,De Crespigny Park,Park,London London SE5 8AF,UK.Tel: 020 7848 0707; Fax: 020 7708 0017; e-mail: janet.carter@@iop.kcl.ac.uk into the basal forebrain before lesioning prevented degeneration of cholinergic neu- :First received 26 April 2000, finalfinalrevision revision 27 27 October October 2000, 2000, accepted accepted11December 11December 2000) 2000) rons in an adult primate model Tuszynski et aletal, 1996).,1996).

selective cholinergic lesioning Martinez- adult bone marrow demonstrate the ability STEM CELL Serrano & Bjorklund, 1998). to differentiate into astrocytic and neuronal TRANSPLANTATION ^ Significantly, homogeneous clonal phenotypes neuroectodermal origin)when SPECIALISED EX VIVOEXVIVO GENE populations of genetically immortalised transplanted into the non-haematopoietic THERAPY human embryonic NSCs have been shown environment of the rodent brain ± so-called recently to be capable of differentiation inter-germ layer differentiation Kopen et aletal,, Perhaps one of the most remarkable ad- into mature neuronal cell types on trans- 1999; Pittenger et aletal, 1999). Because mesen- vances for the field of CNS gene therapy plantation into adult rat striatum. This sug- chymal stem cells can be isolated readily has been the identification and isolation of gests their potential as vehicles for from a patient's own bone marrow, their neural stem cells NSCs)from embryonic molecular therapies for neurodegenerative use in transplantation would circumvent or foetal neural tissue. On transplantation conditions Rubio et aletal, 2000).,2000). the problem of graft versus host disease. into developing or adult mouse brain, Despite the promise of brain-derived It can be seen that this radical transfor- human embryonic NSCs can migrate over NSCs, there remain reservations about mation in the concepts of stem cell biology distance, integrate into the surrounding their use in transplantation to the mature advances the prospect of achieving the ulti- microenvironment and demonstrate multi- CNS. It may be that the normal adult mate goal for molecular medicine in the potency i.e. an ability to differentiate into brain lacks the appropriate signals to re- CNS: an easily isolated source of multi- regionally appropriate neuronal cells); for alise their proliferative potential or that potent adult human stem cells with high a review, see Scheffler et aletal 1999). It is specific neuropathological conditions may proliferative capacity that can be lineage- these inherent biological features that, on alter the balance of required environmen- directed into different types of neurons for transplantation, allow for the generation tal signals. One answer may lie in exploit- the treatment of neurodegeneration. of brain regions where integrated donor- ing the multipotency of stem cells by derived cells can replace lost cells, provide promoting lineage commitment in vitroinvitro support to degenerating cells or, if genetic- from a founder stem cell population prior ally modified to do so, be used to target to transplantation to the damaged brain, CONCLUSION genes.genes. to provide a limitless source of mature neuronal cells for transplantation and Advances in gene transfer technologies genetic manipulation. now provide an array of versatile tools IMMORTALISED STEM CELLS for gene delivery to localised or wide- ALTERNATIVE STEM CELLS spread areas of the brain. However, the Neural stem cells also can be rendered `im- FORFOR EX VIVOCNS GENE optimism generated by results in animal mortal' by the addition of growth factors or THERAPY models must be tempered because many by genetic manipulation, thereby promot- improvements in safety, efficacy, stability ing continuous cell division in vitroinvitro that isthatis The ongoing issues of limited accessibility and regulation of gene transfer are re- `conditional' on the presence or absence of and ethical concern over the use of foetal quired before clinically effective CNS gene manipulatable factors such as temperature. tissues have continued to drive the search therapy can be accomplished for human Using this approach, immortalised NSCs for alternative populations of stem cells patients. A cautionary note, too, is from rodents, genetically modified in addi- for neural transplantation. Stem cells de- sounded by gene therapy attempts in tiontionbyby viral vectors to express disease- rived from adult tissues such as the bone organs with greater accessibility than the specific deficient enzymes or neurotrophic marrow and skin may have potential in brain e.g. liver, bone marrow), because factors, have been used as biological `mini- this respect. A body of evidence undoubt- these have, so far, met with disappointing pumps' to correct the widespread neuro- edly now supports the extraordinary con- outcomes. Despite such reservations, the pathology in animal models of lysosomal clusion that adult stem cells extracted successful application of these gene ther- storage disease, mucopolysaccharidosis VII from their usual niche can be diverted apy strategies in treating CNS disorders Sly syndrome)and Tay±Sachs disease from their normal fates and redirected to in animal models already has demon- LacorazzaLacorazza et aletal, 1998). A similar approach atypical lineages determined by their new strated their enormous potential. Col- has been employed to reconstitute partially microenvironment Fuchs & Segre, 2000). laborative efforts between clinicians and the spatial memory deficits in rodent mod- Remarkably, mesenchymal stem cells basic science researchers continue to make els of Alzheimer's disease generated by mesodermal origin)isolated from human CNS gene therapy an achievable goal.

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ACKNOWLEDGEMENTS Horrelou, P.,Vigne, E., Castel, M., et aletal 191994) 94) DirectDirect Martinez-Serrano, A. & Bjorklund, A. 1998) Ex vivoExvivo intracerebral gene transfer of an adenoviral vector nerve growth factor gene transfer to to the the basal basal forebrain forebrain expressing tyrosine hydroxylase hydroxylase in in a a rat rat model model of of inpresymptomatic middle-aged rats preventspreventsthe the J.E.C. is a Medical Research Council Clinician Scien- Scien- Parkinson's disease. Neuroreport,, 66, 49^53.,49^53. development of cholinergic atrophy and tist Fellow. This article was supported inpart by cognitive impairment during ageing. Proceedings of the grants from the National Institutes of Health, includ- Kopen, G. C., Prockop, D. J. & Phinney, D. G. 1999) National Academy of Sciences of the United States of ing research grants ROI HD28607 and POI Marrow stromal cells migrate throughout forebrainand America,, 95,1858^1863. HD28822.HD28822. cerebellum and differentiate into astrocytes after injection into into neonatal neonatal mouse mouse brains. brains.Proceedings of the Pittenger, M. F., Mackay, A. M., Beck, S. C., et aletal National Academy of Sciences of the United States of 1999)1999) Multilineage potential of of adult adult mesenchymal stem stem REFERENCES AmericaAmerica,, 9696,10711^10716. cells. Science,, 284,143^147.,143^147. Ridet, J. L.,Corti,O., Pencalet, P., et aletal 191999) 9 9) Tow a rdrdTow Kordower, J., Isacson, O. & Emerich, D. 1999) Bilang-Bleuel, A., Revah, F., Colin, P., et aletal 19 9 7) autologousautologous ex vivoexvivo gene therapy for the central nervous Intrastriatal injection of an adenoviral vector expressing Cellular delivery of trophic factors for the treatment system with humanadult astrocytes. Human Gene glial-cell-line derived neurotrophic factor prevents of Huntington's disease. Experiments in ,, 159159,, TherapyTherapy,, 20, 271^280.,271^280. dopaminergic neuron degeneration and behavioural 4^20.4^20. Rubio, F. J., Bueno, C.,Villa, A., et aletal 2000) impairment in a rat model of Parkinson's disease. Lacorazza, H. F., Flax, J. D., Snyder, E.Y., et aletal 19 9 8) Genetically perpetuated human neural stem cells engraft Proceedings of the National Academy of Sciences of the Expressionof human bb-hexosaminidase aa-subunit gene gene and differentiate into the adult mammalian brain. United States of America,, 9494, 8818^8823. :the gene defect of Tay^Sachs disease) in mouse brains Molecular and Cell Neuroscience,, 1616,1^13.,1^13. Choi-Lundberg, D. L., Lin, Q., Chang,Y., et aletal 19 9 7) uponuponthe the engraftmentof engraftment of transduced transduced progenitor progenitor cells. cells. Scheffler,B.,Horn,M.,Blumcke,L.,Scheffler, B., Horn, M., Blumcke, L., et aletal 1999)1999) Dopaminergic neurones protected from degeneration Natural Medicine,, 22, 424^429. Marrow-mindedness: a perspective on neuropoiesis. by GDNF gene therapy. Science,1997, 255, 838^841.,838^841. Trends in Neuroscience,, 2222, 348^357. Mandel, R., Rendahl, K. G., Spratt, S. K., et aletal 19 9 8) Fuchs, E. & Segre, J. A. 2000) Stem cells: a new lease CharacterisationofCharacterisation of intrastriatal intrastriatal recombinantadeno- recombinant adeno- Tuszynski, M. H., Roberts, J., Senut, M. C., et aletal onlife. CellCell,, 100,143^155.,143^155. associated virus-mediated gene transfer of human 19 9 6) Gene therapy in the adult primate brain: Hermans,W.T. J. M. C. & Verhaagen, J. 1998) ViralViral tyrosine hydroxylase and human GTP-cyclohydrolase I intraparenchymal grafts of cells genetically modified to vectors, tools for gene transfer in the . ina rat model of Parkinson's disease. Journal ofofJournal produce nerve growth factor prevent cholinergic Progress in Neurobiology,, 5555, 399^434. Neuroscience,, 1212, 4271^4284. neuronal degeneration. Gene Therapy,, 44,,304^314. 304^314.

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