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Growth-factor gene therapy for neurodegenerative disorders
Review
Growth-factor gene therapy
Growth-factor gene therapy for neurodegenerative disorders Mark H Tuszynski
Preclinical neuroscience has advanced rapidly over the past two decades. New approaches for treating neurological disease, including gene-based therapies, nervous-system growth factors, stem cells, novel vaccines, and modulation of the immune system, offer the potential to prevent cell loss and degeneration in the brain, rather than attempting to compensate for loss after it has occurred. I will review one of these prospective therapies: growth-factor gene therapy for Alzheimer’s disease, an approach that is currently the subject of a phase I clinical trial. Other disease targets for gene therapy will also be discussed, including Parkinson’s disease, Huntington’s disease, inborn errors of metabolism, and cancer. The progress of gene-therapy clinical trials is aiding the transition to molecular and gene-targeted therapeutic approaches which have the potential to improve dramatically the prognosis of neurological disease. Lancet Neurology 2002; 1: 51–57
Current neurological treatments do not prevent cell loss in disorders such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease. However, remarkable progress in both preclinical and clinical neuroscience has shed light on the mechanisms that contribute to cell loss in these disorders, and has led to the development of therapeutic approaches that might, for the first time, provide sufficient neuroprotection. In addition, research in neurobiology and molecular biology has helped to identify new molecules and delivery systems that could provide the practical means to use molecular and genetic advances clinically. In this review I describe the development of nerve growth factor (NGF) gene therapy for Alzheimer’s disease. Preclinical studies, which lasted for longer than 10 years, confirmed this approach’s efficacy in rodents and primates, and its safety and feasibility in a primate dose-escalation/toxicity study. On the basis of these data, a clinical trial proposal was approved and a phase I safety trial of growth-factor gene therapy in Alzheimer’s disease was initiated. This trial will show whether the delivery of a nervoussystem growth factor, by means of gene therapy, to a vulnerable neuronal population in patients with Alzheimer’s disease can prevent cell loss. Two hypotheses underlie this approach: first, that nervous-system growth factors can prevent the death of brain cells after various insults; and, second, that gene therapy is an effective and simple way of delivering therapeutic substances to the brain.
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Effects of NGF in the adult brain 50 years ago, Levi-Montalcini and Hamburger1 discovered that mouse sarcoma extracts contained a substance that promoted survival of sensory and sympathetic neurons in chick embryos. They called this protein nerve growth factor.2 In the years that followed, the protein structure and DNA sequence of NGF were revealed,3,4 and its role in influencing the fate and patterning of specific neuronal populations in the developing nervous system was described. However, the role of NGF, or any other growth factor, in the adult nervous system was still unknown. The startling discovery that NGF could also influence neuronal survival in the adult nervous system came nearly 40 years after the molecule was first discovered. Initially, several groups reported that NGF was present in the adult brain including the hippocampus.5–8 In 1983, Gnahn and colleagues9 reported that NGF increased functional cholinergic neuronal markers in the basal forebrain. The next year Seiler and Schwab10 reported that NGF was retrogradely transported from the cortex to cholinergic neurons of the nucleus basalis in rats; this finding suggested that NGF was actively utilised by the adult brain. Most importantly, three groups reported nearly simultaneously that injections of NGF protein into the brains of adult rats completely prevented the death of cholinergic neurons in the basal forebrain after axotomy.11–13 Shortly afterwards, another group reported that NGF reversed spontaneous age-related morphological and behavioural decline in rats.14 These findings convincingly established that NGF can prevent neuronal death or agerelated atrophy in the adult brain. The potential relevance of these findings to neuronal loss in Alzheimer’s disease was recognised almost immediately.15,16 Cholinergic neurons in the basal forebrain (which were protected by NGF injections in rats), atrophy and die in the brains of patients with Alzheimer’s disease; this process is thought to contribute to the attention deficits and overall cognitive decline.17,18 Thus, the delivery of NGF into the brains of patients with Alzheimer’s disease could reduce or prevent the cholinergic component of cell loss. However, delivery of NGF into the brain is difficult because the active, beta MHT is at the Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA, and the Veterans Administration Medical Center, San Diego, CA, USA. Correspondence: Dr Mark H Tuszynski, Department of Neurosciences-0626, University of California at San Diego, La Jolla, CA 92093, USA. Tel +1 858 534 8857; fax +1 858 534 5220; email [email protected]
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Review component of the molecule is large and polar, and cannot cross the blood-brain barrier. By use of intracerebroventricular infusions or intermittent injections, NGF was successfully delivered into the CSF of rodents. From there, NGF diffused short distances to reach the cell bodies of cholinergic neurons, where it bound to specific NGF receptors and was actively transported into the cell bodies of cholinergic neurons in the septal nucleus and nucleus basalis.11–13,19 On the basis of these findings, the best method of delivering NGF to patients with Alzheimer’s disease was thought to be intracerebroventricular infusion. Effects of NGF in the adult primate brain
Given the invasive nature of intracerebroventricular infusion in human beings, my group and others sought to establish that this route of administration could prevent cholinergic neuronal degeneration in an animal model more relevant to human beings—the adult monkey.20,21 In 1990, these groups showed that cholinergic neuronal degeneration caused by fornix lesions in the brain of primates could be prevented by intracerebroventricular infusions of rodent NGF (figure 1).20,21 A year later, we and others reported that infusions of recombinant, human NGF also rescued cholinergic neurons in primates.22 Can infusions of NGF be used to treat Alzheimer’s disease?
Considering the extent of the loss of cholinergic neurons in patients with Alzheimer’s disease and the ability of NGF to prevent this loss in rodents and primates, clinical trials were planned.16 However, at about the same time there were several reports describing significant toxic effects associated with intracerebroventricular infusions of NGF. First, Isaacson and colleagues23 reported in 1990 that NGF infusions caused sympathetic axons to sprout and surround the cerebral vasculature, although no adverse physical consequences of this growth response were observed. Subsequently, Williams24 reported that NGF infusions resulted in weight loss in rats due to a reduction in appetite. Finally, Winkler and colleagues25
Figure 1. NGF prevents degeneration of cholinergic neurons in the primate brain. (A) After the cholinergic axonal projections to the hippocampus on the right side of the brain have been lesioned, neurons undergo degeneration as seen by a reduction in immunolabelling for choline acetyltransferase. (B) Normal neuronal labelling is evident on the intact, left side of the brain. Administration of NGF substantially reduces degeneration of cholinergic neurons on the right, lesioned side of the brain.
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reported that intracerebroventricular infusions of NGF in rats induced the migration of Schwann cells into the subpial space surrounding the brainstem and spinal cord, and that the migrating cells eventually formed a dense sheath that was several cell-layers thick. This hypertrophy and division of Schwann cells was reversed when NGF infusion was stopped. These findings were replicated in primates.26 By 1996, it was evident that intracerebroventricular infusions of NGF were impractical as a means of delivering NGF to patients with Alzheimer’s disease. In support of this conclusion, one clinical trial, in which three patients with Alzheimer’s disease were given infusions of murine NGF, had to be stopped because of adverse events including weight loss and pain;27 these adverse events were predicted by animal studies.
Gene therapy for CNS disease The occurrence of intolerable side-effects of intracerebroventricular infusions of growth factors meant that another delivery method was required. The new method had not only to deliver growth factors across the blood-brain barrier, but also to restrict the delivery to precise cellular targets in the brain to avoid adverse events. Gene therapy was an obvious choice because this technique could potentially achieve highly localised and sustained delivery to the brain. Gene therapy is commonly divided into two approaches: ex vivo and in vivo.28 In the former, host cells are genetically modified in vitro (or ex vivo) to produce the desired protein; they are then harvested and put back into the host where they act as biological minipumps for the local delivery of the protein. By contrast, the latter approach involves the injection of viral vectors carrying the gene in question directly into host cells in vivo. When we began studies of gene therapy in the late 1980s, vectors for use ex vivo were generally better developed and more efficient than those for use in vivo, so our initial studies primarily focused on the former. These studies showed that delivery of host cells, modified ex vivo to produce NGF, could prevent the death of cholinergic neurons in the rodent and primate brains; could reverse spontaneous age-related neuronal atrophy in primates; and could be administered to the primate brain safely. On the basis of these findings, we initiated a phase I clinical trial of ex vivo NGF gene therapy in patients with Alzheimer’s disease. Suitable target cells for ex vivo gene therapy must be easily obtainable from an adult animal; be able to survive in vitro; divide at a rate sufficient to allow gene-therapy vectors to enter the host genome and express their gene product; and survive grafting to the host nervous system for extended periods without migrating or forming tumours. Primary (non-immortalised) fibroblasts, Schwann cells, endothelial cells, and, more recently, stem cells all meet these criteria. Immortalised cell lines typically form tumours and are therefore unsuitable for gene therapy in the CNS, unless rendered “conditionally immortal”.29 We have used primary fibroblasts for the studies described below because they meet all the criteria mentioned, and are easily obtainable by skin biopsy. In 1988, a collaborative study resulted in the first successful use of ex vivo gene therapy in the CNS.30 Primary rat fibroblasts were genetically modified to produce and
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Growth-factor gene therapy
secrete human NGF. The researchers lesioned the fimbria fornix in rats to induce degeneration of cholinergic neurons and then grafted the genetically-modified cells into the animals’ septal nuclei. Cholinergic neuronal degeneration did not occur in animals that received NGF-secreting fibroblasts. Chen and Gage31 subsequently showed that grafting NGFsecreting fibroblasts into aged rats improved cognition and ameliorated morphological cholinergic neuronal decline.
NGF gene therapy in primates
Review forebrain cholinergic neurons in monkeys. To assess the functionality of cholinergic neurons, we quantified the number of cells expressing choline acetyltransferase (ChAT)—the enzyme that catalyses acetylcholine synthesis— and low-affinity p75 neurotropin receptor, in both young and old monkeys. Ageing resulted in a 40% reduction in ChAT and p75 labeling in basal forebrain cholinergic neurons.35 However, cholinergic neurons did not die as a result of ageing, shown by the presence of a normal number of cells in the basal forebrain, when brains from older monkeys were compared with those from younger animals on simple Nissl stains. Thus, atrophy and functional decline, but not death, of cholinergic neurons occurs as a normal consequence of ageing in rhesus monkeys. (In Alzheimer’s disease, more extensive cholinergic neuronal atrophy and, eventually, death occur.) Next, autologous NGF-secreting cells or control, ß-galactosidase-producing fibroblasts, were grafted into the cholinergic basal forebrain (nucleus basalis, or intermediate division of Ch4 in the nomenclature of Mesulam) of aged monkeys.36 3 months later, there was a substantial reversal of age-related neuronal atrophy in aged monkeys that received the NGF-secreting grafts. Both the number and size of
To find out whether ex vivo gene therapy with NGF could protect cholinergic neuronal function in primates, we studied two primate models: fornix lesions and ageing. In the first set of experiments, we unilaterally lesioned the fornix of rhesus monkeys and then immediately grafted primary autologous fibroblasts, which were genetically modified to produce and secrete human NGF, into the parenchyma. A month later, the monkeys that received NGFsecreting cell grafts had significantly less cholinergic neuronal degeneration than monkeys that were lesioned but received control grafts of unmodified primary fibroblasts.32 In the control group, only about 25% of medial septal cholinergic neurons were detectable after the fornix was lesioned, whereas in NGF-grafted monkeys 68% of neurons were protected (p<0·05). As we got better at accurately placing the graft, the extent of neuronal protection in monkeys that received NGF-secreting cells was raised to 92%. This degree of neuronal protection was achieved with NGF doses about 500 times lower than the concentrations of NGF protein that had been used in earlier primate studies of intracerebroventricular infusion.20,21 Emerich and colleagues33,34 also reported cholinergic neuronal rescue in monkeys with fornix lesions, using another genetherapy approach that delivered NGF in xenografted, encapsulated cells. In an additional set of experiments in primates, we investigated whether ageing causes spontaneous atrophy of cholinergic neurons in the basal forebrain, and whether such atrophy, if present, could be reversed by ex vivo NGF gene delivery. These studies were initiated in the mid-1990s, at which point the effects of ageing on the primate brain were not well understood. Primates (both human beings and monkeys) experience clear Figure 2. Ex vivo gene delivery of NGF to the brains of aged monkeys significantly ameliorates age-related declines in cognition, but atrophy of cholinergic neurons and reverses the loss of cholinergic innervation to the cortex. (A) the cellular mechanisms underlying Normal density of cortical cholinergic innervation in young monkeys seen by acetylcholinesterase these changes had not been identified. stain. (B) Density of cortical cholinergic innervation in aged monkeys. (C) Density of cortical Using stereological methods for cholinergic innervation in aged monkeys that receive NGF-secreting autologous-fibroblast grafts to the region of cholinergic neuronal cell bodies in the Ch4 region (nucleus basalis). (D) Normalised quantifying neurons, we assessed cholinergic axonal density is significantly decreased in aged monkeys, but increases in aged whether ageing resulted in a change in monkeys treated with NGF gene therapy. Reproduced with permission of the National Academy of the number or function of basal Sciences (Proc Natl Acad Sci USA 2001; 98: 1941–46).
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Review neurons labelled for p75 and ChAT were restored to values that did not differ significantly from those in young monkeys. In addition, age-related declines in cholinergic innervation of the cortex were reversed (figure 2). Dose-escalation/toxicity studies in primates
As discussed above, ex vivo NGF gene delivery prevents cholinergic neuronal degeneration after fornix lesions and ameliorates spontaneous age-related cholinergic neuronal atrophy in primates. Further, NGF diffuses no more than 1–2 mm from genetically modified cells grafted into the parenchyma; this characteristic contrasts with diffusion distances of several centimetres from intracerebroventricular or intraparenchymal sites of protein infusion (Conner and Tuszynski, unpublished observations). Thus, gene delivery could achieve localised delivery of NGF to the brain without the side-effects associated with NGF infusions. Although the short distance of NGF diffusion from implantation sites of genetically modified cells necessitated strict accuracy in graft placement, and required several graft deposits to influence the full rostral to caudal length of cholinergic neurons (five grafts per side of the brain in human and non-human primates), hypothetically NGF could be delivered safely to the brain by gene therapy. To test this possibility, dose escalation/toxicity studies were done in adult monkeys. The animals received injections of autologous NGF-transduced fibroblasts into the nucleus basalis; doses ranged from 5–100 µL (at a concentration of 100 000 cells/µL) per injection site. The duration of gene expression in vivo, NGF concentration in CSF, graft size, weight, and general comfort of the animals were assessed over the next year. At the highest doses, fibroblasts refluxed from injection sites in the cortex, yet no toxic effects were observed at any dose. All the monkeys maintained a stable weight, and there was no evidence of a change in activity or any indication of pain. Grafted cells did not migrate from injection sites and did not form tumours. No abnormal Schwann-cell migration into the subpial space of the brainstem or spinal cord was observed, and NGF was undetectable in spinal-fluid samples 6 months and 1 year after intraparenchymal grafting of NGF-secreting cells. NGF gene expression persisted for at least 1 year shown by NGF ELISA measurement of biopsy samples from grafted sites. After 1 year, NGF concentrations in the parenchyma still exceeded physiological concentrations by five times. Thus, in primates, NGF gene delivery ex vivo prevents cholinergic neuronal degeneration, reverses spontaneous cholinergic atrophy in aged monkeys, and produces sustained gene expression at least 1 year with no evidence of toxic effects.
A phase I clinical trial of ex vivo NGF gene delivery in Alzheimer’s disease The efficacy and safety data discussed previously suggest that gene therapy offers a practical and safe method of testing the hypothesis that NGF reduces cholinergic neuronal degeneration in patients with Alzheimer’s disease. NGF could be beneficial in Alzheimer’s disease either by reducing the extent of cholinergic neuronal loss, or by augmenting neuronal function by directly stimulating cholinergic
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transmission. However, an important question remains: is the mechanism of cholinergic neuronal degeneration sensitive to NGF gene delivery? The mechanism of neuronal death in Alzheimer’s disease is unknown. There are no extensive amyloid deposits in the cholinergic basal forebrain of patients with Alzheimer’s disease, although cholinergic neurons show substantial neurofibrillary degeneration. Also, whereas NGF production is not reduced in the cortex of patients with Alzheimer’s disease, NGF concentrations in the cholinergic basal forebrain are much lower than in age-matched controls.37,38 Thus, there may be defects in NGF retrograde transport in Alzheimer’s disease that account for low concentrations of NGF in basal forebrain neurons and result in cholinergic neuronal degeneration. Delivery of NGF to the cholinergic cell bodies by ex vivo gene therapy could bypass this transport defect, thereby sustaining cholinergic neurons. Furthermore, even if a primary NGF deficiency does not contribute to cholinergic neuronal loss in Alzheimer’s disease, NGF gene delivery could be beneficial by augmentation of cholinergic transmission. Since gene delivery of NGF in the non-human primate brain was safe and effective, we designed a phase I safety trial of NGF gene delivery in human beings with early Alzheimer’s disease. We planned to graft primary autologous fibroblasts that were modified to express human NGF into the nucleus basalis of patients with Alzheimer’s disease, with doses well within the range used in monkeys (figure 3); the smallest dose that we used in the clinical trial had been shown to be effective in our studies with monkeys. As of March 15, 2002, eight individuals have been enrolled in the phase I study, and will receive cell doses that vary over an approximate 5-fold range paralleling doses used in studies with monkeys. A new patient is entered into the study every 3 months, to aid monitoring of adverse events. People with early-stage “probable Alzheimer’s disease” by research criteria are eligible for the trial. We chose early-stage patients for this trial to ensure that they are truly able to understand the implications and risks of their participation in the study and are capable of giving informed consent. The patient’s primary caregiver is also asked to give consent to ensure that trial participation is in the patient’s best interests. Also, to be effective, NGF needs to be delivered while cholinergic neurons are still present in the brain, and this condition is met only in the early and middle stages of Alzheimer’s disease. Potential risks of participation in the study include pain, weight loss, or Schwann-cell migration into the subpial space of the medulla and spinal cord. These adverse effects could occur if NGF diffuses from its site of injection and reaches significant concentrations in the CSF. Other risks include haemorrhage, tumour formation from grafted cells, or cell migration from intended injection sites. However, these adverse effects have not been observed among more than 200 monkeys with NGF-secreting cell implants. These adverse events would be difficult to treat in human beings, but given the lack of effective therapies for Alzheimer’s disease, the relentlessly progressive disease course, and the extensive data supporting the safety of NGF gene delivery in primates, the clinical trial has gone ahead.
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The first patient was implanted with NGF-producing autologous fibroblasts in April, 2001, and the second patient was treated in August, 2001. The study will be complete after all participants have received cell implants and have been studied for 1 year. Follow-up will include monitoring for safety and change in neuropsychological function, and patients will undergo functional magnetic resonance imaging. The trial will address two questions. First, can ex vivo gene therapy be safely accomplished in human beings? And second, are growth factors effective for preventing neuronal degeneration in Alzheimer’s disease?
Growth-factor gene therapy for other disorders To date, more than 50 nervous-system growth factors have been identified.39 Growth-factor proteins offer the potential to reduce or prevent neuronal degeneration in several neurodegenerative or traumatic disorders, including Parkinson’s disease,40 Huntington’s disease,41 amyotrophic lateral sclerosis,42 and spinal-cord injury.43,44 Several clinical trials of growth factors for treating patients with various neurological diseases have been done (table),45–54 all with infusions of growth-factor protein, but none have proved
Clinical trials of growth factors for neurological disease Ref Disease 45–47 Amyotrophic lateral sclerosis
Growth factor CNTF, BDNF, CNTF + BDNF, GDNF, IGF-1
48
Spinal muscular atrophy
BDNF
49
Alzheimer’s disease
NGF
50–53 Peripheral neuropathy
NGF, BDNF, NT-3
54
FGF-2
Stroke
CNTF=ciliary neurotropic factor; BDNF=brain-derived neurotropic factor; GDNF=glial cell line-derived neurotropic factor; IGF-1=insulin-like growth factor 1; NGF= nerve growth factor; NT-3=neurotropin 3; FGF-2=fibroblast growth factor 2.
effective. However, none of these clinical trials are likely to have used delivery methods that could provide therapeutic quantities of growth factors to their neuronal targets. In the past, growth factors were either administered peripherally— with the blood-brain barrier limiting their concentration in the CNS—or they were given by intracerebroventricular infusion and so significant concentrations could not diffuse beyond the ependymal lining. Thus, our trial which uses gene therapy to deliver growth factors to the brain may offer the first opportunity to find out whether these proteins will ultimately be useful for treating neurological disease.
Figure 3. Schematic diagram of the gene therapy protocol. A gene therapy "vector" is constructed from a retrovirus (upper left). The long terminal repeat (LTR) region of the vector contains a promoter region that continually expresses the gene of interest (transgene). A second promoter derived from simian virus (SV) expresses a gene for neomycin resistance (neo) that allows us to select cells in vitro that have successfully incorporated the vector DNA. A packaging cell line contains genes that, when exposed to the vector DNA, package it into viral particles (middle left). These are then secreted from the cell and infect dividing cells (including fibroblasts) with the vector DNA. The fibroblasts are obtained from skin biopsies from the patients to be grafted (centre bottom). Before injection into the patient, the genetically modified fibroblasts are characterised for expression of the protein of interest (upper right). Those flasks of fibroblasts that produce the most of the desired protein are cultured further (middle right). When enough cells are available, they are injected into the host to deliver the gene for human nerve growth factor. Reproduced with permission of Armin Blesch, University of California, San Diego, USA.
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Review Parkinson’s disease is another adult-onset neurodegenerative disorder that may be a suitable candidate for growth-factor gene therapy. Kordower and colleagues40 reported that delivery of the growth factor glial-cell-linederived neurotropic factor (GDNF) prevented the loss of neurons in the substantia nigra and improved functional recovery in rhesus monkeys with experimentally induced Parkinson’s disease. GDNF gene delivery also improved neuronal function in rhesus monkeys with spontaneous, agerelated atrophy of nigral neurons.40 Kordower and colleagues used in vivo gene delivery techniques with lentiviral (HIVbased) vectors. These researchers have established that GDNF gene delivery is effective in two monkey models of Parkinson’s disease, and clinical trials are now likely after additional safety and toxicity studies are completed. Early fears that HIV-based gene therapy held the potential for spontaneous recombination with wild-type virus have now been mitigated; modifications of vectors that eliminate many of the wild-type genomic sequences make the probability of recombination with wild-type HIV extremely remote. Emerich and colleagues41 reported that ex vivo delivery of the CNS growth factor ciliary neurotropic factor (CNTF) improved striatal-cell pathology and functional deficits in a monkey model of Huntington’s disease. In separate studies, we and others have reported that growth-factor gene delivery augmented axonal regeneration and achieved partial functional recovery in rats after experimental spinal-cord injury.43,44 Thus, the principle of growth-factor gene delivery to prevent neuronal degeneration, stimulate neuronal function, or promote axonal growth, may eventually be tested in several neurological disorders.
Growth-factor gene therapy
Search strategy and selection criteria Data for this review were identified by searches of Medline, PubMed, and references from relevant articles; many articles were also identified through searches of the extensive files of the author. The search terms “gene therapy”, “neurodegeneration”, “growth factors”, “clinical trials”, “NGF”, “BDNF”, “NT-3”, “NT-4/5”, “IGF-1”, “FGF”, and “GDNF” were used. Abstracts and reports from meetings were included only when they related directly to previously published work. Only papers published in English were reviewed.
necessary, would substantially improve the safety profile of gene therapy.71 One of these promising vector systems has a tetracycline-sensitive element incorporated into the construct.71 We recently reported that the biological effects of ex vivo NGF gene therapy in animals could be completely eliminated by use of this tetracycline-sensitive system in animals.72 Another issue that affects the potential effectiveness of gene therapy is the extent and duration of therapeutic gene expression. In the past, many of the therapeutic genes used in these studies were expressed for a few weeks or less, which limited their effectiveness. However, more recent vectors can maintain gene expression in vivo for months and possibly years. These improvements are a result of the use of novel promoters, expression enhancers, and post-transcriptional stabilisers of mRNA. Furthermore, modern vectors, unlike earlier versions, express few, if any, wild-type viral genes and therefore elicit a negligible inflammatory response. Thus, the new generation of adeno-associated virus, lentivirus, and “gutless” adenovirus vectors hold new promise for sustainable gene expression.
Other gene therapy trials This review focuses on the use of gene therapy to deliver growth factors to the nervous system, but gene therapy also has potential for treating several other diseases of the nervous system. For example, inborn errors of metabolism could be treated by delivery of the deficient gene, and gliomas might be more effectively treated by delivery of cytotoxic genes such as thymidine kinase.55,56 Indeed, several clinical trials of gene therapy for intracranial glioma are underway57,58 and a clinical trial of gene therapy for Canavan’s disease, a metabolic disorder affecting the nervous system, has been initiated.59 Positive findings for the use of gene therapy in animal models of other nervous-system disorders, including several metabolic defects,60–67 muscular dystrophy,68 epilepsy,69 and stroke,70 may lead to novel approaches for the treatment of these diseases.
The future of gene therapy The ultimate usefulness of growth-factor gene delivery for neurological disease will depend on the findings of careful, objective, and appropriately controlled clinical trials. In addition, improvements in design and safety of gene-therapy vectors will contribute to the advancement of gene therapybased approaches to the clinic. One shortcoming of current gene-therapy technology is that the amount of gene product delivered cannot be controlled. The development of vector systems in which the expression of a gene can be regulated, and turned off if
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Conclusion Growth-factor gene therapy makes use of two recent advances and may generate effective therapies for diseases of the nervous system that are currently untreatable. First, many types of growth factors now known to exist in the CNS have the potential to prevent neuronal loss and augment neuronal function in various cell systems. Second, advances in molecular biology have led to better gene-therapy vector systems that can sustain and potentially regulate gene expression in vivo. Taken together, these developments may help achieve what has not been possible before: to target delivery of neuroprotective substances in the brain and maintain expression over time. Clinical trials in the coming years will show whether the vast potential of these approaches for treating human disease will be realised. Conflict of interest
The author is: a founding scientist of Ceregene, Inc, which is developing gene therapy for neurological disease; a member of the scientific advisory board of Acorda Therapeutics, a company developing therapies for spinal-cord injury; and a member of the scientific advisory board of the Reeve Paralysis Foundation, the Heumann Foundation, the Paralysis Project, and the Canadian and American Spinal Research Organization. Role of the funding source
MHT’s research is funded by grants from National Institute for Aging (AG10435), National Institute of Neurological Disorders and Stroke (NS42291), Veterans Affairs, the Alzheimer’s Association, the National Paralysis Foundation, and the Hollfelder Foundation. None of these organisations had a role in the writing of this review.
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recombinant human nerve growth factor in primate brain following intracerebroventricular infusion. Exp Neurol 1996; 140: 151–60. Eriksdotter Jonhagen M, Nordberg A, Amberla K, et al. Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer’s disease. Dement Geriatr Cogn Disord 1998; 9: 246–57. Blesch A, Grill RJ, Tuszynski MH. Neurotrophin gene therapy in models of CNS trauma and neurodegeneration. Prog Brain Res 1998; 117: 473–84. Brustle O, McKay RD. Neuronal progenitors as tools for cell replacement in the nervous system. Curr Opin Neurobiol 1996; 6: 688–95. Rosenberg MB, Friedmann T, Robertson RC, et al. Grafting genetically modified cells to the damaged brain: restorative effects of NGF expression. Science 1988; 242: 1575–78. Chen KS, Gage FH. Somatic gene transfer of NGF to the aged brain: behavioral and morphological amelioration. J Neurosci 1995; 15: 2819–25. Tuszynski MH, Roberts J, Senut MC, U H-S, Gage FH. Gene therapy in the adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. Gene Ther 1996; 3: 305–14. Emerich DW, Winn S, Harper J, Hammang JP, Baetge EE, Kordower JH. Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: rescue and sprouting of degenerating cholinergic basal forebrain neurons. J Comp Neurol 1994; 349: 148–64. Kordower JH, Winn SR, Liu Y-T, et al. The aged monkey basal forebrain: rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor. Proc Natl Acad Sci USA 1994; 91: 10898–902. Smith DE, Roberts J, Gage FH, Tuszynski MH. Ageassociated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy. Proc Natl Acad Sci USA 1999; 96: 10893–98. Mesulam MM, Mufson EJ, Levey AI, Wainer BH. Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey. J Comp Neurol 1983; 214: 170–197. Mufson EJ, Conner JM, Kordower JH. Nerve growth factor in Alzheimer’s disease: defective retrograde transport to nucleus basalis. Neuroreport 1995; 6: 1063–66. Scott SA, Mufson EJ, Weingartner JA, Skau KA, Crutcher KA. Nerve growth factor in Alzheimer’s disease: increased levels throughout the brain coupled with declines in nucleus basalis. J Neurosci 1995; 15: 6213–21. Tuszynski MH. Neurotrophic factors. In: Tuszynski MH,Kordower JH, eds. CNS regeneration: basic science and clinical advances. San Diego: Academic Press, 1999: 109–58. Kordower JH, Emborg ME, Bloch J, et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 2000; 290: 767–73. Emerich DF, Winn SR, Hantraye PM, et al. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington’s disease. Nature 1997; 386: 395–99. Yan Q, Matheson C, Lopez OT. In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. Nature 1995; 373: 341–44. Grill R, Murai K, Blesch A, Gage FH, Tuszynski MH. Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci 1997; 17: 5560–72. Liu Y, Kim D, Himes BT, et al. Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J Neurosci 1999; 19: 4370–87. ALS CNTF Treatment Study Group. A double-blind placebo-controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis. Neurology 1996; 46: 1244–49. The BDNF Study Group. A controlled trial of recombinant methionyl human BDNF in ALS. Neurology 1999; 52: 1427–33. Lai EC, Felice KJ, Festoff BW, et al. Effect of recombinant human insulin-like growth factor-I on progression of ALS. A placebo-controlled study. Neurology 1997; 49: 1621–30. Miller RG. Carrell-Krusen Symposium invited lecture. Clinical trials in motor neuron diseases. J Child Neurol 1999; 14: 173–79. Olson L, Nordberg A, von Holst H, et al. Nerve growth factor affects 11C-nicotine binding, blood flow, EEG, and verbal episodic memory in an Alzheimer’s patient.
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J Neural Transm 1992; 4: 79–95. 50 Apfel SC, Kessler JA, Adornato BT, Litchy WJ, Sanders C, Rask CA. Recombinant human nerve growth factor in the treatment of diabetic polyneuropathy. Neurology 1998; 51: 695–702. 51 Bensa S, Hadden RD, Hahn A, Hughes RA, Willison HJ. Randomized controlled trial of brain-derived neurotrophic factor in Guillain-Barre syndrome: a pilot study. Eur J Neurol 2000; 7: 423–26. 52 Chaudhry V, Giuliani M, Petty BG, et al. Tolerability of recombinant-methionyl human neurotrophin-3 (rmetHuNT3) in healthy subjects. Muscle Nerve 2000; 23: 189–92. 53 Wellmer A, Misra VP, Sharief MK, Kopelman PG, Anand P. A double-blind placebo-controlled clinical trial of recombinant human brain-derived neurotrophic factor (rhBDNF) in diabetic polyneuropathy. J Peripher Nerv Syst 2001; 6: 204–10. 54 Ay H, Ay I, Koroshetz WJ, Finklestein SP. Potential usefulness of basic fibroblast growth factor as a treatment for stroke. Cerebrovasc Dis 1999; 9: 131–35. 55 Short MP, Choi BC, Lee JK, Malick A, Breakefield XO, Martuza RL. Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line. J Neurosci Res 1990; 27: 427–39. 56 Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vectorproducer cells for treatment of experimental brain tumors. Science 1992; 256: 1550–52. 57 Nanda D, Driesse MJ, Sillevis Smitt PA. Clinical trials of adenoviral-mediated suicide gene therapy of malignant gliomas. Prog Brain Res 2001; 132: 699–710. 58 Alavi JB, Eck SL. Gene therapy for high grade gliomas. Expert Opin Biol Ther 2001; 1: 239–52. 59 Leone P, Janson CG, Bilaniuk L, et al. Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann Neurol 2000; 48: 27–38. 60 Li T, Davidson BL. Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer. Proc Natl Acad Sci USA 1995; 92:7700–04. 61 Learish R, Ohashi T, Robbins PA, et al. Retroviral gene transfer and sustained expression of human arylsulfatase A. Gene Ther 1996; 3: 343–49. 62 Lacorazza HD, Flax JD, Snyder EY, Jendoubi M. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nat Med 1996; 2: 424–29. 63 Hahn CN, del Pilar Martin M, Zhou XY, Mann LW, d’Azzo A. Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter. Proc Natl Acad Sci USA 1998; 95: 14880–85. 64 Stein CS, Ghodsi A, Derksen T, Davidson BL. Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice. J Virol 1999; 73: 3424–29. 65 Daly TM, Vogler C, Levy B, Haskins ME, Sands MS. Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease. Proc Natl Acad Sci USA 1999; 96: 2296–300. 66 Takenaka T, Murray GJ, Qin G, et al. Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells. Proc Natl Acad Sci USA 2000; 97: 7515–20. 67 Consiglio A, Quattrini A, Martino S, et al. In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. Nat Med 2001; 7: 310–16. 68 Allamand V, Donahue KM, Straub V, Davisson RL, Davidson BL, Campbell KP. Early adenovirus-mediated gene transfer effectively prevents muscular dystrophy in alpha-sarcoglycan-deficient mice. Gene Ther 2000; 7: 1385–91. 69 O’Connor WM, Davidson BL, Kaplitt MG, et al. Adenovirus vector-mediated gene transfer into human epileptogenic brain slices: prospects for gene therapy in epilepsy. Exp Neurol 1997; 148: 167–78. 70 Betz AL, Yang GY, Davidson BL. Attenuation of stroke size in rats using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist in brain. J Cereb Blood Flow Metab 1995; 15: 547–51. 71 Gossen M, Bonin AL, Freundlieb S, Bujard H. Inducible gene expression systems for higher eukaryotic cells. Curr Opin Biotechnol 1994; 5: 516–20. 72 Blesch A, Conner JM, Tuszynski MH. Modulation of neuronal survival and axonal growth in vivo by tetracycline-regulated neurotrophin expression. Gene Ther 2001; 8: 954–60.
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Growth-factor gene therapy
Growth-factor gene therapy for neurodegenerative disorders Mark H Tuszynski
Preclinical neuroscience has advanced rapidly over the past two decades. New approaches for treating neurological disease, including gene-based therapies, nervous-system growth factors, stem cells, novel vaccines, and modulation of the immune system, offer the potential to prevent cell loss and degeneration in the brain, rather than attempting to compensate for loss after it has occurred. I will review one of these prospective therapies: growth-factor gene therapy for Alzheimer’s disease, an approach that is currently the subject of a phase I clinical trial. Other disease targets for gene therapy will also be discussed, including Parkinson’s disease, Huntington’s disease, inborn errors of metabolism, and cancer. The progress of gene-therapy clinical trials is aiding the transition to molecular and gene-targeted therapeutic approaches which have the potential to improve dramatically the prognosis of neurological disease. Lancet Neurology 2002; 1: 51–57
Current neurological treatments do not prevent cell loss in disorders such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease. However, remarkable progress in both preclinical and clinical neuroscience has shed light on the mechanisms that contribute to cell loss in these disorders, and has led to the development of therapeutic approaches that might, for the first time, provide sufficient neuroprotection. In addition, research in neurobiology and molecular biology has helped to identify new molecules and delivery systems that could provide the practical means to use molecular and genetic advances clinically. In this review I describe the development of nerve growth factor (NGF) gene therapy for Alzheimer’s disease. Preclinical studies, which lasted for longer than 10 years, confirmed this approach’s efficacy in rodents and primates, and its safety and feasibility in a primate dose-escalation/toxicity study. On the basis of these data, a clinical trial proposal was approved and a phase I safety trial of growth-factor gene therapy in Alzheimer’s disease was initiated. This trial will show whether the delivery of a nervoussystem growth factor, by means of gene therapy, to a vulnerable neuronal population in patients with Alzheimer’s disease can prevent cell loss. Two hypotheses underlie this approach: first, that nervous-system growth factors can prevent the death of brain cells after various insults; and, second, that gene therapy is an effective and simple way of delivering therapeutic substances to the brain.
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Effects of NGF in the adult brain 50 years ago, Levi-Montalcini and Hamburger1 discovered that mouse sarcoma extracts contained a substance that promoted survival of sensory and sympathetic neurons in chick embryos. They called this protein nerve growth factor.2 In the years that followed, the protein structure and DNA sequence of NGF were revealed,3,4 and its role in influencing the fate and patterning of specific neuronal populations in the developing nervous system was described. However, the role of NGF, or any other growth factor, in the adult nervous system was still unknown. The startling discovery that NGF could also influence neuronal survival in the adult nervous system came nearly 40 years after the molecule was first discovered. Initially, several groups reported that NGF was present in the adult brain including the hippocampus.5–8 In 1983, Gnahn and colleagues9 reported that NGF increased functional cholinergic neuronal markers in the basal forebrain. The next year Seiler and Schwab10 reported that NGF was retrogradely transported from the cortex to cholinergic neurons of the nucleus basalis in rats; this finding suggested that NGF was actively utilised by the adult brain. Most importantly, three groups reported nearly simultaneously that injections of NGF protein into the brains of adult rats completely prevented the death of cholinergic neurons in the basal forebrain after axotomy.11–13 Shortly afterwards, another group reported that NGF reversed spontaneous age-related morphological and behavioural decline in rats.14 These findings convincingly established that NGF can prevent neuronal death or agerelated atrophy in the adult brain. The potential relevance of these findings to neuronal loss in Alzheimer’s disease was recognised almost immediately.15,16 Cholinergic neurons in the basal forebrain (which were protected by NGF injections in rats), atrophy and die in the brains of patients with Alzheimer’s disease; this process is thought to contribute to the attention deficits and overall cognitive decline.17,18 Thus, the delivery of NGF into the brains of patients with Alzheimer’s disease could reduce or prevent the cholinergic component of cell loss. However, delivery of NGF into the brain is difficult because the active, beta MHT is at the Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA, and the Veterans Administration Medical Center, San Diego, CA, USA. Correspondence: Dr Mark H Tuszynski, Department of Neurosciences-0626, University of California at San Diego, La Jolla, CA 92093, USA. Tel +1 858 534 8857; fax +1 858 534 5220; email [email protected]
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Review component of the molecule is large and polar, and cannot cross the blood-brain barrier. By use of intracerebroventricular infusions or intermittent injections, NGF was successfully delivered into the CSF of rodents. From there, NGF diffused short distances to reach the cell bodies of cholinergic neurons, where it bound to specific NGF receptors and was actively transported into the cell bodies of cholinergic neurons in the septal nucleus and nucleus basalis.11–13,19 On the basis of these findings, the best method of delivering NGF to patients with Alzheimer’s disease was thought to be intracerebroventricular infusion. Effects of NGF in the adult primate brain
Given the invasive nature of intracerebroventricular infusion in human beings, my group and others sought to establish that this route of administration could prevent cholinergic neuronal degeneration in an animal model more relevant to human beings—the adult monkey.20,21 In 1990, these groups showed that cholinergic neuronal degeneration caused by fornix lesions in the brain of primates could be prevented by intracerebroventricular infusions of rodent NGF (figure 1).20,21 A year later, we and others reported that infusions of recombinant, human NGF also rescued cholinergic neurons in primates.22 Can infusions of NGF be used to treat Alzheimer’s disease?
Considering the extent of the loss of cholinergic neurons in patients with Alzheimer’s disease and the ability of NGF to prevent this loss in rodents and primates, clinical trials were planned.16 However, at about the same time there were several reports describing significant toxic effects associated with intracerebroventricular infusions of NGF. First, Isaacson and colleagues23 reported in 1990 that NGF infusions caused sympathetic axons to sprout and surround the cerebral vasculature, although no adverse physical consequences of this growth response were observed. Subsequently, Williams24 reported that NGF infusions resulted in weight loss in rats due to a reduction in appetite. Finally, Winkler and colleagues25
Figure 1. NGF prevents degeneration of cholinergic neurons in the primate brain. (A) After the cholinergic axonal projections to the hippocampus on the right side of the brain have been lesioned, neurons undergo degeneration as seen by a reduction in immunolabelling for choline acetyltransferase. (B) Normal neuronal labelling is evident on the intact, left side of the brain. Administration of NGF substantially reduces degeneration of cholinergic neurons on the right, lesioned side of the brain.
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reported that intracerebroventricular infusions of NGF in rats induced the migration of Schwann cells into the subpial space surrounding the brainstem and spinal cord, and that the migrating cells eventually formed a dense sheath that was several cell-layers thick. This hypertrophy and division of Schwann cells was reversed when NGF infusion was stopped. These findings were replicated in primates.26 By 1996, it was evident that intracerebroventricular infusions of NGF were impractical as a means of delivering NGF to patients with Alzheimer’s disease. In support of this conclusion, one clinical trial, in which three patients with Alzheimer’s disease were given infusions of murine NGF, had to be stopped because of adverse events including weight loss and pain;27 these adverse events were predicted by animal studies.
Gene therapy for CNS disease The occurrence of intolerable side-effects of intracerebroventricular infusions of growth factors meant that another delivery method was required. The new method had not only to deliver growth factors across the blood-brain barrier, but also to restrict the delivery to precise cellular targets in the brain to avoid adverse events. Gene therapy was an obvious choice because this technique could potentially achieve highly localised and sustained delivery to the brain. Gene therapy is commonly divided into two approaches: ex vivo and in vivo.28 In the former, host cells are genetically modified in vitro (or ex vivo) to produce the desired protein; they are then harvested and put back into the host where they act as biological minipumps for the local delivery of the protein. By contrast, the latter approach involves the injection of viral vectors carrying the gene in question directly into host cells in vivo. When we began studies of gene therapy in the late 1980s, vectors for use ex vivo were generally better developed and more efficient than those for use in vivo, so our initial studies primarily focused on the former. These studies showed that delivery of host cells, modified ex vivo to produce NGF, could prevent the death of cholinergic neurons in the rodent and primate brains; could reverse spontaneous age-related neuronal atrophy in primates; and could be administered to the primate brain safely. On the basis of these findings, we initiated a phase I clinical trial of ex vivo NGF gene therapy in patients with Alzheimer’s disease. Suitable target cells for ex vivo gene therapy must be easily obtainable from an adult animal; be able to survive in vitro; divide at a rate sufficient to allow gene-therapy vectors to enter the host genome and express their gene product; and survive grafting to the host nervous system for extended periods without migrating or forming tumours. Primary (non-immortalised) fibroblasts, Schwann cells, endothelial cells, and, more recently, stem cells all meet these criteria. Immortalised cell lines typically form tumours and are therefore unsuitable for gene therapy in the CNS, unless rendered “conditionally immortal”.29 We have used primary fibroblasts for the studies described below because they meet all the criteria mentioned, and are easily obtainable by skin biopsy. In 1988, a collaborative study resulted in the first successful use of ex vivo gene therapy in the CNS.30 Primary rat fibroblasts were genetically modified to produce and
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Growth-factor gene therapy
secrete human NGF. The researchers lesioned the fimbria fornix in rats to induce degeneration of cholinergic neurons and then grafted the genetically-modified cells into the animals’ septal nuclei. Cholinergic neuronal degeneration did not occur in animals that received NGF-secreting fibroblasts. Chen and Gage31 subsequently showed that grafting NGFsecreting fibroblasts into aged rats improved cognition and ameliorated morphological cholinergic neuronal decline.
NGF gene therapy in primates
Review forebrain cholinergic neurons in monkeys. To assess the functionality of cholinergic neurons, we quantified the number of cells expressing choline acetyltransferase (ChAT)—the enzyme that catalyses acetylcholine synthesis— and low-affinity p75 neurotropin receptor, in both young and old monkeys. Ageing resulted in a 40% reduction in ChAT and p75 labeling in basal forebrain cholinergic neurons.35 However, cholinergic neurons did not die as a result of ageing, shown by the presence of a normal number of cells in the basal forebrain, when brains from older monkeys were compared with those from younger animals on simple Nissl stains. Thus, atrophy and functional decline, but not death, of cholinergic neurons occurs as a normal consequence of ageing in rhesus monkeys. (In Alzheimer’s disease, more extensive cholinergic neuronal atrophy and, eventually, death occur.) Next, autologous NGF-secreting cells or control, ß-galactosidase-producing fibroblasts, were grafted into the cholinergic basal forebrain (nucleus basalis, or intermediate division of Ch4 in the nomenclature of Mesulam) of aged monkeys.36 3 months later, there was a substantial reversal of age-related neuronal atrophy in aged monkeys that received the NGF-secreting grafts. Both the number and size of
To find out whether ex vivo gene therapy with NGF could protect cholinergic neuronal function in primates, we studied two primate models: fornix lesions and ageing. In the first set of experiments, we unilaterally lesioned the fornix of rhesus monkeys and then immediately grafted primary autologous fibroblasts, which were genetically modified to produce and secrete human NGF, into the parenchyma. A month later, the monkeys that received NGFsecreting cell grafts had significantly less cholinergic neuronal degeneration than monkeys that were lesioned but received control grafts of unmodified primary fibroblasts.32 In the control group, only about 25% of medial septal cholinergic neurons were detectable after the fornix was lesioned, whereas in NGF-grafted monkeys 68% of neurons were protected (p<0·05). As we got better at accurately placing the graft, the extent of neuronal protection in monkeys that received NGF-secreting cells was raised to 92%. This degree of neuronal protection was achieved with NGF doses about 500 times lower than the concentrations of NGF protein that had been used in earlier primate studies of intracerebroventricular infusion.20,21 Emerich and colleagues33,34 also reported cholinergic neuronal rescue in monkeys with fornix lesions, using another genetherapy approach that delivered NGF in xenografted, encapsulated cells. In an additional set of experiments in primates, we investigated whether ageing causes spontaneous atrophy of cholinergic neurons in the basal forebrain, and whether such atrophy, if present, could be reversed by ex vivo NGF gene delivery. These studies were initiated in the mid-1990s, at which point the effects of ageing on the primate brain were not well understood. Primates (both human beings and monkeys) experience clear Figure 2. Ex vivo gene delivery of NGF to the brains of aged monkeys significantly ameliorates age-related declines in cognition, but atrophy of cholinergic neurons and reverses the loss of cholinergic innervation to the cortex. (A) the cellular mechanisms underlying Normal density of cortical cholinergic innervation in young monkeys seen by acetylcholinesterase these changes had not been identified. stain. (B) Density of cortical cholinergic innervation in aged monkeys. (C) Density of cortical Using stereological methods for cholinergic innervation in aged monkeys that receive NGF-secreting autologous-fibroblast grafts to the region of cholinergic neuronal cell bodies in the Ch4 region (nucleus basalis). (D) Normalised quantifying neurons, we assessed cholinergic axonal density is significantly decreased in aged monkeys, but increases in aged whether ageing resulted in a change in monkeys treated with NGF gene therapy. Reproduced with permission of the National Academy of the number or function of basal Sciences (Proc Natl Acad Sci USA 2001; 98: 1941–46).
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Review neurons labelled for p75 and ChAT were restored to values that did not differ significantly from those in young monkeys. In addition, age-related declines in cholinergic innervation of the cortex were reversed (figure 2). Dose-escalation/toxicity studies in primates
As discussed above, ex vivo NGF gene delivery prevents cholinergic neuronal degeneration after fornix lesions and ameliorates spontaneous age-related cholinergic neuronal atrophy in primates. Further, NGF diffuses no more than 1–2 mm from genetically modified cells grafted into the parenchyma; this characteristic contrasts with diffusion distances of several centimetres from intracerebroventricular or intraparenchymal sites of protein infusion (Conner and Tuszynski, unpublished observations). Thus, gene delivery could achieve localised delivery of NGF to the brain without the side-effects associated with NGF infusions. Although the short distance of NGF diffusion from implantation sites of genetically modified cells necessitated strict accuracy in graft placement, and required several graft deposits to influence the full rostral to caudal length of cholinergic neurons (five grafts per side of the brain in human and non-human primates), hypothetically NGF could be delivered safely to the brain by gene therapy. To test this possibility, dose escalation/toxicity studies were done in adult monkeys. The animals received injections of autologous NGF-transduced fibroblasts into the nucleus basalis; doses ranged from 5–100 µL (at a concentration of 100 000 cells/µL) per injection site. The duration of gene expression in vivo, NGF concentration in CSF, graft size, weight, and general comfort of the animals were assessed over the next year. At the highest doses, fibroblasts refluxed from injection sites in the cortex, yet no toxic effects were observed at any dose. All the monkeys maintained a stable weight, and there was no evidence of a change in activity or any indication of pain. Grafted cells did not migrate from injection sites and did not form tumours. No abnormal Schwann-cell migration into the subpial space of the brainstem or spinal cord was observed, and NGF was undetectable in spinal-fluid samples 6 months and 1 year after intraparenchymal grafting of NGF-secreting cells. NGF gene expression persisted for at least 1 year shown by NGF ELISA measurement of biopsy samples from grafted sites. After 1 year, NGF concentrations in the parenchyma still exceeded physiological concentrations by five times. Thus, in primates, NGF gene delivery ex vivo prevents cholinergic neuronal degeneration, reverses spontaneous cholinergic atrophy in aged monkeys, and produces sustained gene expression at least 1 year with no evidence of toxic effects.
A phase I clinical trial of ex vivo NGF gene delivery in Alzheimer’s disease The efficacy and safety data discussed previously suggest that gene therapy offers a practical and safe method of testing the hypothesis that NGF reduces cholinergic neuronal degeneration in patients with Alzheimer’s disease. NGF could be beneficial in Alzheimer’s disease either by reducing the extent of cholinergic neuronal loss, or by augmenting neuronal function by directly stimulating cholinergic
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transmission. However, an important question remains: is the mechanism of cholinergic neuronal degeneration sensitive to NGF gene delivery? The mechanism of neuronal death in Alzheimer’s disease is unknown. There are no extensive amyloid deposits in the cholinergic basal forebrain of patients with Alzheimer’s disease, although cholinergic neurons show substantial neurofibrillary degeneration. Also, whereas NGF production is not reduced in the cortex of patients with Alzheimer’s disease, NGF concentrations in the cholinergic basal forebrain are much lower than in age-matched controls.37,38 Thus, there may be defects in NGF retrograde transport in Alzheimer’s disease that account for low concentrations of NGF in basal forebrain neurons and result in cholinergic neuronal degeneration. Delivery of NGF to the cholinergic cell bodies by ex vivo gene therapy could bypass this transport defect, thereby sustaining cholinergic neurons. Furthermore, even if a primary NGF deficiency does not contribute to cholinergic neuronal loss in Alzheimer’s disease, NGF gene delivery could be beneficial by augmentation of cholinergic transmission. Since gene delivery of NGF in the non-human primate brain was safe and effective, we designed a phase I safety trial of NGF gene delivery in human beings with early Alzheimer’s disease. We planned to graft primary autologous fibroblasts that were modified to express human NGF into the nucleus basalis of patients with Alzheimer’s disease, with doses well within the range used in monkeys (figure 3); the smallest dose that we used in the clinical trial had been shown to be effective in our studies with monkeys. As of March 15, 2002, eight individuals have been enrolled in the phase I study, and will receive cell doses that vary over an approximate 5-fold range paralleling doses used in studies with monkeys. A new patient is entered into the study every 3 months, to aid monitoring of adverse events. People with early-stage “probable Alzheimer’s disease” by research criteria are eligible for the trial. We chose early-stage patients for this trial to ensure that they are truly able to understand the implications and risks of their participation in the study and are capable of giving informed consent. The patient’s primary caregiver is also asked to give consent to ensure that trial participation is in the patient’s best interests. Also, to be effective, NGF needs to be delivered while cholinergic neurons are still present in the brain, and this condition is met only in the early and middle stages of Alzheimer’s disease. Potential risks of participation in the study include pain, weight loss, or Schwann-cell migration into the subpial space of the medulla and spinal cord. These adverse effects could occur if NGF diffuses from its site of injection and reaches significant concentrations in the CSF. Other risks include haemorrhage, tumour formation from grafted cells, or cell migration from intended injection sites. However, these adverse effects have not been observed among more than 200 monkeys with NGF-secreting cell implants. These adverse events would be difficult to treat in human beings, but given the lack of effective therapies for Alzheimer’s disease, the relentlessly progressive disease course, and the extensive data supporting the safety of NGF gene delivery in primates, the clinical trial has gone ahead.
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Review
Growth-factor gene therapy
The first patient was implanted with NGF-producing autologous fibroblasts in April, 2001, and the second patient was treated in August, 2001. The study will be complete after all participants have received cell implants and have been studied for 1 year. Follow-up will include monitoring for safety and change in neuropsychological function, and patients will undergo functional magnetic resonance imaging. The trial will address two questions. First, can ex vivo gene therapy be safely accomplished in human beings? And second, are growth factors effective for preventing neuronal degeneration in Alzheimer’s disease?
Growth-factor gene therapy for other disorders To date, more than 50 nervous-system growth factors have been identified.39 Growth-factor proteins offer the potential to reduce or prevent neuronal degeneration in several neurodegenerative or traumatic disorders, including Parkinson’s disease,40 Huntington’s disease,41 amyotrophic lateral sclerosis,42 and spinal-cord injury.43,44 Several clinical trials of growth factors for treating patients with various neurological diseases have been done (table),45–54 all with infusions of growth-factor protein, but none have proved
Clinical trials of growth factors for neurological disease Ref Disease 45–47 Amyotrophic lateral sclerosis
Growth factor CNTF, BDNF, CNTF + BDNF, GDNF, IGF-1
48
Spinal muscular atrophy
BDNF
49
Alzheimer’s disease
NGF
50–53 Peripheral neuropathy
NGF, BDNF, NT-3
54
FGF-2
Stroke
CNTF=ciliary neurotropic factor; BDNF=brain-derived neurotropic factor; GDNF=glial cell line-derived neurotropic factor; IGF-1=insulin-like growth factor 1; NGF= nerve growth factor; NT-3=neurotropin 3; FGF-2=fibroblast growth factor 2.
effective. However, none of these clinical trials are likely to have used delivery methods that could provide therapeutic quantities of growth factors to their neuronal targets. In the past, growth factors were either administered peripherally— with the blood-brain barrier limiting their concentration in the CNS—or they were given by intracerebroventricular infusion and so significant concentrations could not diffuse beyond the ependymal lining. Thus, our trial which uses gene therapy to deliver growth factors to the brain may offer the first opportunity to find out whether these proteins will ultimately be useful for treating neurological disease.
Figure 3. Schematic diagram of the gene therapy protocol. A gene therapy "vector" is constructed from a retrovirus (upper left). The long terminal repeat (LTR) region of the vector contains a promoter region that continually expresses the gene of interest (transgene). A second promoter derived from simian virus (SV) expresses a gene for neomycin resistance (neo) that allows us to select cells in vitro that have successfully incorporated the vector DNA. A packaging cell line contains genes that, when exposed to the vector DNA, package it into viral particles (middle left). These are then secreted from the cell and infect dividing cells (including fibroblasts) with the vector DNA. The fibroblasts are obtained from skin biopsies from the patients to be grafted (centre bottom). Before injection into the patient, the genetically modified fibroblasts are characterised for expression of the protein of interest (upper right). Those flasks of fibroblasts that produce the most of the desired protein are cultured further (middle right). When enough cells are available, they are injected into the host to deliver the gene for human nerve growth factor. Reproduced with permission of Armin Blesch, University of California, San Diego, USA.
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Review Parkinson’s disease is another adult-onset neurodegenerative disorder that may be a suitable candidate for growth-factor gene therapy. Kordower and colleagues40 reported that delivery of the growth factor glial-cell-linederived neurotropic factor (GDNF) prevented the loss of neurons in the substantia nigra and improved functional recovery in rhesus monkeys with experimentally induced Parkinson’s disease. GDNF gene delivery also improved neuronal function in rhesus monkeys with spontaneous, agerelated atrophy of nigral neurons.40 Kordower and colleagues used in vivo gene delivery techniques with lentiviral (HIVbased) vectors. These researchers have established that GDNF gene delivery is effective in two monkey models of Parkinson’s disease, and clinical trials are now likely after additional safety and toxicity studies are completed. Early fears that HIV-based gene therapy held the potential for spontaneous recombination with wild-type virus have now been mitigated; modifications of vectors that eliminate many of the wild-type genomic sequences make the probability of recombination with wild-type HIV extremely remote. Emerich and colleagues41 reported that ex vivo delivery of the CNS growth factor ciliary neurotropic factor (CNTF) improved striatal-cell pathology and functional deficits in a monkey model of Huntington’s disease. In separate studies, we and others have reported that growth-factor gene delivery augmented axonal regeneration and achieved partial functional recovery in rats after experimental spinal-cord injury.43,44 Thus, the principle of growth-factor gene delivery to prevent neuronal degeneration, stimulate neuronal function, or promote axonal growth, may eventually be tested in several neurological disorders.
Growth-factor gene therapy
Search strategy and selection criteria Data for this review were identified by searches of Medline, PubMed, and references from relevant articles; many articles were also identified through searches of the extensive files of the author. The search terms “gene therapy”, “neurodegeneration”, “growth factors”, “clinical trials”, “NGF”, “BDNF”, “NT-3”, “NT-4/5”, “IGF-1”, “FGF”, and “GDNF” were used. Abstracts and reports from meetings were included only when they related directly to previously published work. Only papers published in English were reviewed.
necessary, would substantially improve the safety profile of gene therapy.71 One of these promising vector systems has a tetracycline-sensitive element incorporated into the construct.71 We recently reported that the biological effects of ex vivo NGF gene therapy in animals could be completely eliminated by use of this tetracycline-sensitive system in animals.72 Another issue that affects the potential effectiveness of gene therapy is the extent and duration of therapeutic gene expression. In the past, many of the therapeutic genes used in these studies were expressed for a few weeks or less, which limited their effectiveness. However, more recent vectors can maintain gene expression in vivo for months and possibly years. These improvements are a result of the use of novel promoters, expression enhancers, and post-transcriptional stabilisers of mRNA. Furthermore, modern vectors, unlike earlier versions, express few, if any, wild-type viral genes and therefore elicit a negligible inflammatory response. Thus, the new generation of adeno-associated virus, lentivirus, and “gutless” adenovirus vectors hold new promise for sustainable gene expression.
Other gene therapy trials This review focuses on the use of gene therapy to deliver growth factors to the nervous system, but gene therapy also has potential for treating several other diseases of the nervous system. For example, inborn errors of metabolism could be treated by delivery of the deficient gene, and gliomas might be more effectively treated by delivery of cytotoxic genes such as thymidine kinase.55,56 Indeed, several clinical trials of gene therapy for intracranial glioma are underway57,58 and a clinical trial of gene therapy for Canavan’s disease, a metabolic disorder affecting the nervous system, has been initiated.59 Positive findings for the use of gene therapy in animal models of other nervous-system disorders, including several metabolic defects,60–67 muscular dystrophy,68 epilepsy,69 and stroke,70 may lead to novel approaches for the treatment of these diseases.
The future of gene therapy The ultimate usefulness of growth-factor gene delivery for neurological disease will depend on the findings of careful, objective, and appropriately controlled clinical trials. In addition, improvements in design and safety of gene-therapy vectors will contribute to the advancement of gene therapybased approaches to the clinic. One shortcoming of current gene-therapy technology is that the amount of gene product delivered cannot be controlled. The development of vector systems in which the expression of a gene can be regulated, and turned off if
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Conclusion Growth-factor gene therapy makes use of two recent advances and may generate effective therapies for diseases of the nervous system that are currently untreatable. First, many types of growth factors now known to exist in the CNS have the potential to prevent neuronal loss and augment neuronal function in various cell systems. Second, advances in molecular biology have led to better gene-therapy vector systems that can sustain and potentially regulate gene expression in vivo. Taken together, these developments may help achieve what has not been possible before: to target delivery of neuroprotective substances in the brain and maintain expression over time. Clinical trials in the coming years will show whether the vast potential of these approaches for treating human disease will be realised. Conflict of interest
The author is: a founding scientist of Ceregene, Inc, which is developing gene therapy for neurological disease; a member of the scientific advisory board of Acorda Therapeutics, a company developing therapies for spinal-cord injury; and a member of the scientific advisory board of the Reeve Paralysis Foundation, the Heumann Foundation, the Paralysis Project, and the Canadian and American Spinal Research Organization. Role of the funding source
MHT’s research is funded by grants from National Institute for Aging (AG10435), National Institute of Neurological Disorders and Stroke (NS42291), Veterans Affairs, the Alzheimer’s Association, the National Paralysis Foundation, and the Hollfelder Foundation. None of these organisations had a role in the writing of this review.
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Review
Growth-factor gene therapy
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