Neurosurg Focus 13 (5):Article 5, 2002, Click here to return to Table of Contents

Growth factor gene therapy for Alzheimer disease

MARK H. TUSZYNSKI, M.D., PH.D., HOI SANG U, M.D., JOHN ALKSNE, M.D., ROY A. BAKAY, M.D., MARY MARGARET PAY, R.N., DAVID MERRILL, PH.D., AND LEON J. THAL, M.D. Departments of Neurosciences and Surgery, Division of Neurosurgery, University of California at San Diego, La Jolla; Veterans Administration Medical Center, San Diego, California; and Department of Surgery, Division of Neurosurgery, Rush–Presbyterian Medical Center, Chicago, Illinois

The capacity to prevent neuronal degeneration and death during the course of progressive neurological disorders such as Alzheimer disease (AD) would represent a significant advance in therapy. Nervous system growth factors are families of naturally produced proteins that, in animal models, exhibit extensive potency in preventing neuronal death due to a variety of causes, reversing age-related atrophy of , and ameliorating functional deficits. The main challenge in translating growth factor therapy to the clinic has been delivery of growth factors to the brain in sufficient concentrations to influence neuronal function. One means of achieving growth factor delivery to the central nervous system in a highly targeted, effective manner may be gene therapy. In this article the authors summarize the develop- ment and implementation of gene delivery as a potential means of reducing cell loss in AD.

KEY WORDS • Alzheimer disease • system • gene therapy • nerve growth factor

Alzheimer disease is the most common neurodegenera- the entire and , leads to the loss of tive disorder, afflicting approximately 4 million people in , which in turn is thought to exacerbate sig- the United States. This number will exceed 14 million by nificantly the “cortical” in AD.2 Cholinergic sys- the year 2050. At a current cost of $100 billion per year, tems modulate activity in target hippocampal and cortical AD is an imminent epidemic that threatens to severely regions,16 a mechanism that is thought to be responsible strain the healthcare system unless effective therapies are for the impairment in cognition when these cholinergic developed.32 Alzheimer disease accounts for the majority inputs are lost. Indeed, cholinergic neuronal degeneration of dementia cases, with chief features of memory loss and has been shown to correlate with clinical severity, density dysfunction of at least one other higher cortical function. of amyloid plaques, and the loss of synapses in AD.2,9 In the late stage of the disease, profound loss of memory Thus, therapies targeted at ameliorating or preventing dys- and executive function occurs, rendering individuals inca- function of cholingeric neurons could affect cognitive de- pable of self care. With disease progression, there is pro- cline in AD. To date, the only therapeutic agents approved found degeneration of cortical neurons in frontal, parietal, by the Federal Drug Administration for the treatment of and temporal neocortices but relative sparing of primary AD are the AChE inhibitors, which augment acetylcho- sensory and motor cortices. line levels in the brain.34 Cholinesterase inhibitors, how- Extensive degeneration of subcortical neuronal popula- ever, exert only modest effects on symptoms of AD. It is tions also occurs in AD, including cholingeric, serotoner- gic, and noradrenergic systems. By the midstages of AD, thought that this occurs because the degeneration of the for example, significant degeneration of the subcortical BFC system continues unabated during treatment with BFC system has occurred, a cell population that includes AChE inhibitors, eventually leading to inadequate cholin- the nucleus basalis of Meynert. This degeneration of the ergic compensation. Further, the doses of orally adminis- cholinergic system, which projects extensively throughout tered cholinesterase inhibitors that can be administered are limited because of the development of peripheral side ef- fects. Hence, the therapeutic potential of fully augmenting Abbreviations used in this paper: AChE = acetylcholinester- cholinergic function in AD cannot entirely be tested with ase; AD = Alzheimer disease; BFC = basal forebrain cholinergic; currently available oral cholinesterase medications. CNS = central nervous system; ICV = intracerebroventricular; Novel therapies that target neuronal loss in AD should NGF = nerve growth factor; NTF = neurotophic factor. not only aim to augment neuronal function but ideally

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Unauthenticated | Downloaded 09/26/21 11:41 PM UTC M. H. Tuszynski, et al. should also prevent cell loss. Nervous system growth human NGF were reported to prevent degeneration of factors, or NTFs, long known to promote survival and BFC neurons in primates.24,41 function of distinct neuronal populations during nervous Thus, by 1991 substantial evidence derived from rodent system development, are a class of molecules that might and primate models suggested that NGF delivery might achieve these objectives in neurodegenerative disorders. be a logical means of attempting to ameliorate choliner- To date, more than 100 NTFs have been identified in the gic cell loss in AD. Indeed, ICV infusion of NGF was CNS. Most can be grouped into distinct “families” of re- attempted in three AD patients in Sweden.15 Whereas lated growth factors based on common structures and sig- Mini-Mental Status Examination scores (a simple mea- naling mechanisms. The first NTFs to be discovered, and sure of cognitive function) did not improve after NGF the most thoroughly studied to date, is the “classic neu- infusions, transient increases in scores were seen on some rotrophin” family consisting of NGF, brain-derived NTF, episodic memory tests and there was some evidence of –3, and neurotrophin–4/5. Nerve growth fac- improved physiological parameters of brain function, in- tor is the prototypical member of the classic neurotrophin cluding nicotine binding, cortical blood flow, and electro- family, and is a specific survival factor for BFC neurons. encephalographic activity. The trial, however, was dis- It is produced throughout life by neurons in the hippo- continued after the onset of debilitating side effects due campus and neocortex, secreted extracellularly, and then to NGF infusion. In particular, back pain developed in bound by specific receptors on cholinergic terminals patients within 2 to 14 days after the onset of ICV NGF in target regions.12,21,22 It is then retrogradely transported to infusions. Pain was hypothesized to result from NGF dif- the cholinergic cell body. Nerve growth factor also acts fusing throughout the cerebrospinal fluid and reaching locally on the axon terminal via NGF receptor–mediated nociceptive NGF-responsive cell bodies in the dorsal tyrosine kinase signaling pathways. Collectively through horns and dorsal root ganglia. In addition, weight loss these receptor mechanisms, NGF modulates the function occurred, likely due to NGF-mediated activation of satiety of BFC neurons during development and throughout adult centers in the . Notably, the authors of pre- life in a manner that is as yet incompletely understood. vious animal studies reported that whereas ICV NGF in- Notably, levels of NGF protein are diminished in BFC fusions successfully rescued degenerating cholinergic neurons in the brains of patients with AD,31,37 although its neurons, these infusions also induced weight loss,45 sym- production by the cortex and hippocampus is not altered, pathetic axon sprouting around cerebral vasculature,36 and suggesting that deficient NGF transport to cholinergic migration and expansion of Schwann cells into a thick cel- neurons may cause or contribute to the loss of this cell lular layer surrounding the medulla and spinal cord.14,46 population.10 The finding in animal models that NGF de- These findings prohibited the initiation of further clinical livery to the adult brain can ameliorate cholinergic cell trials involving ICV infusions of NGF. Thus, whereas loss and reverse cholinergic atrophy with aging has led to NGF treatment offered substantial potential as a means a focus on NGF delivery as a potential therapy for AD. of reducing cholinergic cell loss in AD, an alternative For reasons to be presented, however, safe NGF delivery method of delivery was required to deliver sufficient NGF requires specific targeting to the BFC system. Gene ther- doses to basal forebrain regions while restricting NGF apy is one means of achieving localized, targeted, and spread to other sensitive neuronal populations of the hypo- restricted delivery of NGF into the brain and is the subject thalamus, brainstem, and spinal cord. of a current Phase I clinical trial in AD.

GENE DELIVERY OF NGF NERVE GROWTH FACTOR EFFECTS IN THE ADULT BRAIN One approach to the specific delivery of NGF to cholin- ergic neurons of the basal forebrain is gene therapy. In Nerve growth factor was serendipitously discovered in general, there are two forms of gene therapy: ex vivo and 1951 by Levi-Montalcini and Hamburger29 in the course in vivo gene therapy. In ex vivo gene therapy, cells are of in vitro experiments in which they examined properties genetically modified in vitro to express a gene of interest, of a sarcoma cell line. Subsequently NGF was discovered and are then implanted into the brain where they in effect to be an essential survival factor for peripheral sensory act as biological micropumps for secretion of the desired and sympathetic neurons during development.28 Nerve protein. In in vivo gene therapy, a gene delivery vector is growth factor was purified and characterized in the early injected into the brain to modify directly host brain cells 1970s,1,6 and by the mid-1980s it was shown to be nor- to increase NGF production.18,44 In early studies investiga- mally produced during adulthood by neurons in both the tors focused on ex vivo gene delivery because the tech- hippocampus and neocortex. This discovery raised the niques for this form of gene delivery were developed first. possibility that NGF continued to play a role in the CNS More recently investigators have focused largely on in into maturity.27 Indeed, Hefti20 reported that injections of vivo gene delivery because more efficient gene delivery NGF into the adult animal CNS prevented the degenera- systems for in vivo gene therapy have been developed. tion and death of BFC neurons after axotomy. In the fol- Ex vivo and in vivo gene therapy have distinct individ- lowing year, NGF was also shown to reverse atrophy of ual advantages and disadvantages. The advantages of in BFC neurons and ameliorate deficits in learning and vivo gene therapy include the following. 1) Simplicity: memory in aged rats.17 These findings were extended to gene delivery is accomplished by the single step of direct the nonhuman primate brain, where NGF infusions were vector injection into the desired target organ, as opposed also shown to rescue lesion-induced cholinergic degen- to the considerable cell processing necessary to perform eration.25,42 Human NGF was cloned,43 and infusions of ex vivo gene therapy. Deactivated adenovirus, adenoasso-

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Unauthenticated | Downloaded 09/26/21 11:41 PM UTC Growth factor gene therapy for Alzheimer disease ciated virus, herpes virus, and lentivirus have been used In rodent and then primate experiments, ex vivo NGF successfully to deliver genes of interest in experimental gene delivery has been shown to prevent cholinergic animal models.3,44 2) Minimal invasiveness: injections of neuronal degeneration after lesions in the adult brain. in vivo vectors deliver several microliters of vector par- Cholinergic neurons were induced to degenerate by tran- ticles in an injection fluid solution, a procedure that is secting their projections from the septal nucleus to the hip- simple and safe. 3) Repeatability: the same location can pocampus. After transections of cholinergic axonal pro- be injected more than once using in vivo gene delivery jections to the hippocampus, 60 to 90% of cholinergic approaches. There are also, however, relative potential neurons undergo atrophy and eventually die. Ex vivo NGF disadvantages of in vivo gene therapy including the fol- gene delivery, however, was shown to rescue most cholin- lowing. 1) Nonspecificity of target cell infection: many ergic neurons in both rodent and primate brains.13,26,35,40 different cell types can be infected when in vivo vectors In a second set of experiments, the ability of ex vivo are injected in the CNS, including neurons, glia, and vas- NGF gene therapy to prevent age-related neuronal atrophy cular cells. 2) Toxicity: some in vivo vectors are toxic to was studied. Aged rats and, in separate experiments, aged host cells (for example, herpes virus and rabies virus) and monkeys received grafts of NGF-secreting fibroblasts to elicit immune responses (such as adenovirus).23 Lentiviral another component of the BFC referred to as the basal and adenoassociated viral vector systems have not shown nucleus of Meynert, or Ch4 region. In aged rats, ex vivo adverse effects, and newer-generation herpes virus and NGF gene therapy reversed age-related cholinergic neu- “gutless” adenoviruses without deleterious properties are ronal atrophy and improved spatial memory function.7 being developed. Subsequent experiments in aged primates also demon- The relative advantages of ex vivo gene delivery in- strated that ex vivo gene therapy reversed spontaneous clude the following. 1) It has the ability to target selec- atrophy of BFC neurons,38 and reversed cholinergic termi- tively specific cell types for production of the gene of nal degeneration in widespread cortical areas.8 Indeed, as interest before engrafting of cells into the host brain. 2) a normal function of aging in primates there is atrophy Immunocompatability: host cells are obtained via biopsy (but not death) of 40% of BFC neurons, and ex vivo NGF sampling, grown in vitro, genetically modified, and then gene delivery restored the number of healthy cholinergic implanted into the same host. Thus, no foreign cells are neurons to values within 7% of those observed in young introduced, eliminating any need for immunosuppression. monkeys.38 Aging in the primate brain is also associated 3) Safety: because infectious virus particles are not made with an approximate 25% reduction in cholinergic axon by genetically modified host cells in vitro, there is little density in the cortex; ex vivo NGF delivery completely risk of inadvertently introducing wild-type virus into a restored cholinergic axon density in the aged monkey host with ex vivo gene therapy, and little risk of recombi- brain to values observed in young monkeys. Thus ex vivo nation of the vector with wild-type viruses that may exist NGF delivery effectively prevented and reversed cholin- in the host body. The potential disadvantages of ex vivo ergic neuronal degeneration in the animal brain and did gene delivery include the following. 1) To be maintained so without eliciting adverse effects. Further, genetically and genetically modified in vitro, host cells must be ca- modified fibroblasts grafted into the brain sustained NGF pable of dividing, thus certain postmitotic cell popula- gene expression for at least 1 year in primates, the longest tions such as neurons cannot be targets of transduction time point that we examined. Effects of ex vivo NGF gene for ex vivo gene therapy. Current ex vivo gene therapy therapy on cognitive decline with aging in primates are approaches target primary fibroblasts, stem cells, tumor being examined. cells, Schwann cells, or endothelial cells. 2) Invasiveness: Cholinergic neuronal degeneration is an integral com- grafting of cells is an intrinsically more invasive process ponent of generalized cell loss in the brains of individuals than injection of suspensions of in vivo gene therapy vec- with AD, and loss of cholinergic function correlates tors. 3) Although tumor formation has not been observed strongly in AD with dementia severity and synapse with more than 200 grafts of primary fibroblasts into the loss.2,9,39 Indeed, to date in the only effective treatments primate CNS, delivery of dividing cells bears the risk of for AD cholinergic systems are targeted.11,30 The studies tumor formation. Tumors have been observed when graft- summarized in the preceding paragraphs indicate that ex ing immortalized cell lines; however, more recent derived vivo gene therapy offers the potential, for the first time, to conditionally immortalized cell lines do not form tumors prevent ongoing cholinergic neuronal degeneration in AD. when grafted. Before proceeding with clinical trials, however, we sought to determine whether ex vivo NGF gene therapy was safe. Thus, in a dose-escalation design, adult rhesus monkeys NERVE GROWTH FACTOR GENE THERAPY received steadily increasing volumes of autologous, NGF- FOR AD secreting fibroblasts. Over a 20-fold dose range, no ad- Ex vivo gene delivery techniques were better refined verse effects of ex vivo NGF gene delivery were observed than in vivo gene therapy vectors at the time that we began in primates. NGF gene therapy experiments in animal models several Based on this extensive set of efficacy and safety data years ago. Thus, our current clinical trial of ex vivo NGF in primates, we proposed a Phase I clinical safety trial of gene therapy for AD is based on an ex vivo gene delivery ex vivo NGF gene delivery in humans, which involved a approach, established after several years of efficacy and dose-escalation design in eight patients with AD. Serial safety studies in rodents and nonhuman primates. In vivo safety parameters are being monitored in the individuals gene therapy, however, will likely become commonly per- in this trial, as are neuropsychological measures. Overall, formed in the near future in clinical trials of gene delivery there are two objectives of this clinical program of ex vivo for a variety of nervous system diseases. NGF gene therapy in AD after the Phase I safety testing

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Unauthenticated | Downloaded 09/26/21 11:41 PM UTC M. H. Tuszynski, et al. has been completed: 1) to determine whether NGF will tetracycline-regulated NGF vectors showed very sparse significantly reduce the rate of cognitive decline in AD axonal growth. Thus, we were able to control completely and 2) to determine whether NGF will augment the ac- the biological effects of NGF gene expression in the brain tivity and function of remaining cholinergic neurons in by using a regulatable vector system. the brains of individuals with AD. If the Phase I trial is More studies are required to allow investigation of successful, subsequent Phase II and III trials are planned. whether long-term regulation of gene expression can be This clinical program will eventually address two key maintained as well as to improve the available regulatable questions that cannot be answered in animal models. First, systems. The studies conducted to date, however, provide will NGF prevent cholinergic loss in AD, given the basis for the future application of controlled gene that the precise cause of cell loss in the AD brain is un- delivery in the CNS to enhance the safety and effective- known? Second, will preventing or delaying degeneration ness of neurological gene therapy. of the cholinergic system in AD be sufficient to ameliorate cognitive decline? OTHER GENE DELIVERY STUDIES REGULATING GENE EXPRESSION As noted previously, multiple neuronal populations de- generate in AD, and NGF therapy for basal cholinergic An important need in gene therapy applications is the neurons may or may not be sufficient to reduce progres- capacity to regulate gene expression. The ability to in- sion of the disorder. In future approaches to AD investi- crease the level of gene expression with progression of gators may target other neuronal populations with other the disease or, conversely, to downregulate gene expres- growth factors. The concept of growth factor gene de- sion in the event of adverse effects is a highly desirable livery may also be relevant to the treatment of other feature of gene therapy approaches. Thus, exogenous con- neurodegenerative disorders including Parkinson disease, trol over the expression of transferred genes might im- Huntington disease, and amyotrophic lateral scleorsis. prove the safety and efficacy of gene therapy. Several different systems have been developed to con- trol the expression of genes in viral vectors, including 19 tetracycline regulatable systems. In the originally de- References scribed vector system, administration of tetracycline (or its analog, doxycycline) reduces expression of the gene 1. 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