Gene Ther Mol Biol Vol 1, 265-277. March, 1998.
Neurotrophic factor gene therapy for
Martha C. Bohn and Derek L. Choi-Lundberg1
Dept. of Pediatrics, Northwestern Univ. Med. Sch., Chicago, IL 60614
Correspondence: Martha C. Bohn, Ph.D., Medical Research Institute Council Professor, ChildrenÕs Memorial Institute For Education and Research, Northwestern University Medical School, Department of Pediatrics, 2300 ChildrenÕs Plaza, #209, Chicago, IL 60614, Tel: 773-868-8052, Fax: 773-868-8066, E-mail: firstname.lastname@example.org
1 Current address: Department of Neurosciences, Genentech, Inc., DNA Way, South San Francisco, CA 94080, Tel: 415-225-6237
Key words: retrovirus, adenovirus, herpes simplex virus, adenoassociated virus, neurotrophic factors, GDNF, CNTF, BDNF, neurotrophins, adrenal, tyrosine hydroxylase, dopamine, substantia nigra, neurodegeneration, neural transplantation
The delivery of genes to the central nervous system (CNS) whose products protect specific types of nerve cells from dying or stimulate their growth is applicable to a wide range of neurodegenerative diseases and injuries to the nervous system. This review focuses on ParkinsonÕs disease as a prototype for studying adenoviral mediated gene therapy. The dopaminergic (DA) neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF), is a potent DA factor that ameliorates the behavioral and histological consequences of lesioning DA neurons in mice, rats and monkeys following injection of large amounts of recombinant GDNF protein into the brain. Adenoviral mediated gene therapy with GDNF was studied in a rat model of ParkinsonÕs disease in which the DA neurons die slowly following injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), near their terminals. The survival of DA neurons was markedly enhanced in rats injected with Ad GDNF, but not with control vectors (approximately 80% in Ad GDNF treated rats vs 30% in control rats). Moreover, this degree of neuronal protection was observed at levels of biosynthesized GDNF in the nanogram range which persisted in the injection site for at least 7 weeks. These studies suggest that neurotrophic factor gene therapy may be valuable to treat ParkinsonÕs disease in humans. A review of related studies of neurotrophic factor gene therapy suggests that the potential applications of neurotrophic factor gene therapy are extensive, once advances are made in vector delivery, persistence, safety and testing in non-human primate brain.
Gene therapy applied to the nervous system is unique in many ways compared to most tissues. The cellular heterogeneity and complexity of the nervous system present exceptional challenges for directing genes to specific cells that are beyond the blood brain barrier and not accessible by ordinary routes of vector administration. In the brain, most cells are postmitotic, making approaches requiring gene integration into host chromatin not particularly effective. In addition, while gene replacement for hereditary gene defects and delivery of killer genes for brain tumors are similar to approaches taken in other tissues, gene therapies aimed at brain repair address rather unique processes in which neurons fail to develop or degenerate later in life. Neurodegenerative diseases, such as ParkinsonÕs, AlzheimerÕs, and Lou GehrigÕs diseases, have devastating consequences and are incapacitating to the individual, as well as of high cost to society. In addition, there are limited pharmacological means of intervening against the neuronal degenerations that occur in these diseases, as well as those accompanying stroke or trauma, such as in spinal cord and head injuries.
There are interesting parallels in the neurodegenerations that occur in disease and injuries to the nervous system. Regardless of the type of insult and the neuronal cell type involved, most neurodegeneration appears to proceed through apoptosis, an active process requiring new gene expression. This presents a window of opportunity for intervention by delivering genes whose products block or slow the progress of apoptosis. Another approach is to provide genes that replace the neurotransmitter systems that are lost following degeneration of specific classes of neurons. For example, studies in animal models of ParkinsonÕs disease suggest that gene replacement of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine (DA) synthesis, increases DA levels in the brain and in some studies has been shown to improve parkinsonian behavior (Cao et al., 1995; During et al., 1994; Kaplitt et al., 1994; Lundberg et al., 1996; Wolff et al., 1989). While this approach may ameliorate disease symptoms, an approach that prevents neurons from dying or that stimulates regrowth in damaged neurons has the potential of permanently restoring brain function and of reversing or at least slowing the progression of neurodegenerative diseases.
This chapter will review studies applying gene therapy to prevent neurodegeneration or stimulate regeneration, focusing on animal models of ParkinsonÕs disease in which DA neurons degenerate.
II. Animal models of ParkinsonÕs disease
ParkinsonÕs disease (PD) affects approximately 1% of people over the age of 50, with nearly 500,000 patients in the United States. The cardinal symptoms of PD include bradykinesia or akinesia, rigidity, resting tremor and postural instability. The hallmark of PD is the slow degeneration of DA neurons in the substantia nigra region of the midbrain. Due to this discrete, well-defined lesion and the availability of neurotoxins that selectively kill DA neurons, several well-characterized animal models of ParkinsonÕs disease have been developed (Bankiewicz et al., 1993, review). Transfection of the medial forebrain bundle, which contains nigrostriatal and mesolimbic DA axons, causes a reduction of DA phenotypic markers and cell death in the substantia nigra and ventral tegmental area. Injection of the neurotoxin 6-hydroxydopamine (6-OHDA) into the striatum, medial forebrain bundle or substantia nigra results in a selective loss of DA neurons. 6-OHDA, an analog of DA, is concentrated in DA neurons through uptake by the high affinity DA transporter. 6-OHDA undergoes auto-oxidation, generating hydroxyl radical, hydrogen peroxide and superoxide anion, molecules that damage lipids, proteins and DNA and lead to cell death. Injection of 6-OHDA into the rat striatum produces a progressive loss of DA neurons over several weeks (Przedborski et al., 1995; Sauer and Oertel, 1994), whereas injection into the medial forebrain bundle or substantia nigra produces a rapid loss of DA neurons. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes parkinsonian symptoms in humans, non-human primates and mice following its oxidation to MPP+ (1-methyl-4-phenyl pyridinium) by monamine oxidase type B (MAO-B). MPP+ enters DA neurons by high affinity uptake through the DA transporter and interferes with ATP production by inhibiting complex I of the mitochondrial electron transport chain.
Since a unilateral lesion of the nigrostriatal pathway produces an imbalance in the levels of DA and DA receptors in the target regions of the DA nerve terminals (the striatum), behavioral tests of this asymmetry have been useful for assessing the efficacy of therapeutic approaches. For example, DA stores are depleted and striatal postsynaptic DA receptors are upregulated on the lesioned side. Animals exhibit rotational behavior in response to amphetamine and DA agonists such as apomorphine that can be easily quantified (Ungerstedt, 1971). In addition, unilaterally lesioned animals exhibit deficits in contralateral limb use in several spontaneous behaviors (Olsson et al., 1995; Schallert, 1995). MPTP lesioned non-human primates exhibit symptoms similar to humans with ParkinsonÕs disease, including bradykinesia or akinesia, resting tremor and rigidity.
III. Neurotrophic factors for dopaminergic neurons
The discovery and characterization of neurotrophic factors that promote the survival, neurite outgrowth and phenotypic differentiation of DA neurons has been an area of intense research in recent years. Early studies showed that glia from different brain regions and glial cell lines produce factors that influence the survival and differentiation of embryonic DA neurons in culture (Engele et al., 1991; Rousselet et al., 1988). Previously identified growth factors, such as fibroblast growth factors 1 and 2, were found to enhance survival of DA neurons in vitro, but their effects were shown to be indirectly mediated through glial cells (Engele and Bohn, 1991). The first neurotrophic factor shown to act directly on DA neurons was brain-derived neurotrophic factor (BDNF) (Hyman et al., 1991). Subsequently, glial cell line-derived neurotrophic factor (GDNF) was purified from the glial cell line, B49, and shown to be a very potent DA factor that enhances survival and neurite outgrowth of embryonic DA neurons in vitro (Lin et al., 1993). A second member of the GDNF family, neurturin, was recently identified and also shown to be a potent DA factor (Kotzbauer et al., 1996). The receptors for GDNF and neurturin are widely expressed in the CNS and periphery in distinct, but overlapping, patterns, and many neuronal populations in addition to DA neurons respond to these factors (Baloh et al., 1997; Buj-Bello et al., 1997; Creedon et al., 1997; Henderson et al., 1994; Klein et al., 1997; Nosrat et al., 1996; Olson, 1997; Williams et al., 1996). Consequently, both GDNF and neurturin are excellent candidates for gene therapy for ParkinsonÕs disease. A summary of growth factors with DA neurotrophic activity is shown in Table I.
Table I: Neurotrophic factors for dopaminergic neurons
GDNF (Lin et al., 1993)
Neurturin (Kotzbauer et al., 1996)
GDF-5 (Krieglstein et al., 1995b)
TGF-b1 (Krieglstein et al., 1995a)
TGF-b2, TGF-b3 (Krieglstein et al., 1995a; Poulsen et al., 1994)
Activin A (Krieglstein et al., 1995a)
BDNF (Hyman et al., 1991)
NT-3 (Hyman et al., 1994)
NT-4/5 (Hyman et al., 1994)
Cardiotrophin-1 (Pennica et al., 1995)
CNTF (Hagg and Varon, 1993; Magal et al., 1993)
Il-1b (Akaneya et al., 1995)
Il-6 (Hama et al., 1991; von Coelln et al., 1995)
Il-7 (von Coelln et al., 1995)
Mitogenic Growth Factors
aFGF, bFGF (Beck et al., 1993; Engele and Bohn, 1991; Ferrari et al., 1989; Otto and Unsicker, 1993)
EGF (Casper et al., 1991)
Insulin (Knusel et al., 1990)
IGF-I (Beck et al., 1993)
IGF-II (Liu and Lauder, 1992)
Midkine (Kikuchi et al., 1993)
x (Othberg et al., 1995)
TGF-a (Alexi and Hefti, 1993)
The identification of factors that affect either survival and/or differentiated properties of specific classes of neurons, such as neurite outgrowth, neurite branching, or neurotransmitter expression, led to the concept of using these factors as therapeutic agents for neurodegenerative diseases as reviewed previously (Arenas, 1996; Eide et al., 1993; Lindsay et al., 1993; Mizuno et al., 1994), see Table II. However, the initial identification of neurotrophic factors has for the most part relied on in vitro assays using cultured embryonic neurons from mouse or rat. Consequently, studies in animals are necessary to determine if damaged or aged neurons respond to these factors in the same manner in vivo.
Table II. Potential applications of neurotrophic factor gene therapy
Enhance cell survival
Prolong life of diseased neurons.
Decrease vulnerability of neurons to damage in the nervous system.
Enhance survival and/or differentiation of grafted neurons or neuronal stem cells.
Stimulate Regeneration or Sprouting
Stimulate regeneration of damaged, diseased neurons
Stimulate sprouting in healthy, neighboring neurons
Block inhibitory growth molecules
Enhance neuronal function
Upregulate neurotransmitter expression or metabolism
Affect neuronal activity
In the case of GDNF, there is much in vivo evidence showing that GDNF protects neurons in the adult brain from neurotoxin and axotomy induced degeneration (Arenas et al., 1995; Beck et al., 1995; Choi-Lundberg et al., 1997a,b; Emerich et al., 1996; Gash et al., 1995; Henderson et al., 1994; Houenou et al., 1996; Matheson et al., 1997; Munson and McMahon, 1997; Oppenheim et al., 1995; Sagot et al., 1996; Shults et al., 1996; Tomac et al., 1995; Tseng et al., 1997; Yan et al., 1995). GDNF also may stimulate sprouting or regeneration from damaged DA neurons in the adult brain as in one study of polymer-encapsulated GDNF-secreting BHK cells where DA fibers growing into the capsule were observed (Lindner et al., 1995). However in our studies, we have not observed a GDNF stimulated increase in the density of DA fibers in the striatum (Choi-Lundberg and Bohn, unpublished observations). Similar findings for BDNF have been reported in the 6-OHDA lesioned rat in which BDNF improved DA dependent behavior and increased striatal DA levels in the absence of an effect on the density of DA fibers in the striatum (Altar et al., 1994; Altar et al., 1992; Yoshimoto et al., 1995). Thus, studies of embryonic neurons in culture may not be completely predictive of the effects of neurotrophic factors on neurons in the adult brain. It remains entirely unknown to what extent the effects of neurotrophic factors on neurons in animal models of disease, in which neurons are either physically or chemically damaged, will predict their actions on diseased neurons in the human brain, and only clinical studies will reveal this.
There is abundant evidence supporting an application of GDNF to ParkinsonÕs disease. As discussed above, administration of recombinant GDNF in mouse, rat and non-human primate models of ParkinsonÕs disease protects DA neurons against the effects of at least two neurotoxins acting through different mechanisms and against physical lesions. This had led to clinical trials, newly undertaken by Amgen, which involve repeated intracerebral administration of GDNF. Although the outcome of this trial remains unknown, several issues must be addressed in the development of neurotrophic factor therapies for neurodegenerative diseases. Long-term trophic support for the diseased neurons will be required as neurodegenerative diseases are slowly progressive. Since neurotrophic factors are labile substances that are unable to cross the blood-brain barrier, it will be necessary to develop methods for delivering these substances in a controlled manner to specific types of neurons. These methods need to be minimally invasive, safe and sophisticated enough to avoid general effects on other types of neurons that might lead to intolerable side effects. Repeated injections of recombinant neurotrophic factors into the human brain are unlikely to be practical and are likely to elicit deleterious side-effects over the long term. In this respect, clinical trials in which neurotrophic factors were administered in large amounts in the periphery were stopped due to unanticipated, intolerable side-effects (ALS CNTF Treatment Study Group, 1996; Miller et al., 1996). In addition, one patient with AlzheimerÕs disease experienced the side effects of weight loss, pain and sleep disturbances following intraventricular infusion of NGF (Olson et al., 1994; Olson et al., 1992). Gene therapies for delivering neurotrophic factors to the CNS have the potential to circumvent or even eliminate the drawbacks of recombinant protein therapies. By transferring neurotrophic factor genes to the CNS, there is the potential to produce these substances in a continuous, regulatable manner and even to direct synthesis of these substances only in selected cells through the use of a cell specific promoter. This approach is applicable not only to neurotrophic factor genes, but also to other growth promoting genes and anti-apoptotic genes.
IV. GDNF adenoviruses: Construction and testing
As a first step in studying the potential of neurotrophic factor gene therapy for ParkinsonÕs disease, adenoviral vectors of the serotype Ad5, deleted in E1a and E3, were made containing the DNA sequences for wild type human GDNF preproprotein and a mutant human GDNF with a 12 amino acid deletion (Ad- GDNF and Ad mGDNF, respectively). An Ad vector harboring the §-galactosidase gene with the SV40 large T nuclear localization sequence was also used as an additional control (Ad LacZnl). All vectors used in our studies to date have contained the Rous sarcoma virus (RSV) long terminal repeat promoter (Choi-Lundberg et al, 1997a,b).
To determine whether these vectors conferred to cells the ability to synthesize the transgene, and in the case of GDNF to secrete bioactive protein, several assay systems were used. PC12 cells, a pheochromocytoma clonal cell line, were used to test infectivity and degree of cytotoxicity. Counts of viable cells four days after infection with vector stocks with different titers and total particles showed that the degree of cytotoxicity correlated with particle ratio rather than infectious titer (Figure 1; Choi-Lundberg, 1997a,b). Consequently, in vivo studies (discussed below) were performed with the same titer of vector stocks with similar particle ratios in order to control for both the degree of cytotoxicity and the multiplicity of infection in experimental groups. Since medium conditioned by PC12 cells does not contain neurotrophic activity for DA neurons (Engele et al., 1991), PC12 cells were also used to determine whether bioactive GDNF was secreted following infection with the Ad GDNF vector. Media from PC12 cells infected with Ad GDNF or Ad mGDNF at 10 pfu/cell were collected and tested for presence of DA neurotrophic factor activity. Cultures of dissociated embryonic mesencephalon containing DA neurons were used as the assay system.
As shown in Figure 2, the survival of DA neurons as assessed by counting neurons positive for tyrosine hydroxylase (TH), the rate limiting enzyme in DA synthesis, was significantly increased in cultures grown in the presence of medium conditioned by Ad GDNF infected PC12 cells, but not by control medium (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997b). A further demonstration of the bioactivity conferred by the vectors was shown by directly infecting cultures of embryonic mesencephalon at 1 or 10 pfu/cell . The survival of DA neurons also was significantly increased only by the Ad GDNF vector (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997b); Figure 2.
Figure 1. Cytotoxicity of Ad vectors in vitro correlates more closely with total particle number per cells (A) than with infectious plaque forming units per cell (B).
PC12 cells were infected for two hours with 100 to 100,000 particles per cell of Ad vectors with particle ratios 20, 65 or 190, or mock infected. Four days later, cells were trypsinized, centrifuged, resuspended and counted in a hemacytometer. Cell counts were plotted versus total particle number per cell (A) and infectious plaque forming units (pfu) per cell (B).
Prior to testing Ad vectors in rodents models of ParkinsonÕs disease in vivo, it was deemed important to optimize in vivo injection paradigms to obtain a high level of transgene expression that persisted for at least two months, but which resulted in minimal tissue damage. Fischer 344 rats were injected in the striatum with 4 x 105, 4 x 106, or 4 x 107 pfu of Ad LacZnl containing approxi-
Figure 2. Bioactive GDNF is produced in vitro after infection with Ad GDNF.
The number of TH immunoreactive (TH-IR) neurons was counted in E14.5 cultures of dissociated rat ventral mesencephalon maintained on 50% PC12 CM or directly infected with Ad GDNF or Ad mGDNF at 10 pfu/cell, or mock-infected (mean ± SEM, n=6). Ad GDNF increased survival versus Ad mGDNF and no virus in both groups (analysis of variance (ANOVA), F=10.49, P<0.001; symbol indicates TukeyÕs post hoc pairwise comparisons at a family error rate of 0.02). Reprinted with permission from Choi-Lundberg et al., 1997b. Copyright 1997 American Association for the Advancement of Science.
mately 1 x 107, 1 x 108, or 1 x 109 total particles, respectively. All vector stocks had a particle ratio between 20 and 26. Rats were sacrificed at 4, 30 or 60 days and the number of blue nuclei in the injection site counted. At 4 days little tissue damage was noted around the needle tract in rats injected with the 2 lower titers, whereas significant tissue damage extending up to 0.5mm from the site of injection was observed at the highest titer. At 30 and 60 days, many blue cells persisted in rats injected with the two highest titers; however, macrophages were present in the needle tracts of rats injected with each titer.
Cell count data (Figure 3; Choi-Lundberg, 1997) revealed that a high level of persistent gene expression could be obtained with titers in the range of 4 x 106 to 4 x 107 pfu with infected cells extending 0.6 mm or greater from the injection site. Notably, there were no significant
differences in the numbers of blue nuclei at 4, 30 or 60 days at any of the titers, suggesting transgene expression from Ad vectors is stable in the Fischer 344 rat brain. Retrograde transport of either the Ad LacZnl vector or §-
Figure 3. Expression of §-galactosidase transgene protein in the striatum at 4, 30 and 60 days after injection of 4 x 105, 4 x 106 , or 4 x 107 pfu.
Tissue sections of striatum at 160 µm intervals were stained with X-gal histochemistry and the number of blue nuclei in the striatum counted.
galactosidase protein also occurred since numerous DA neurons in the substantia nigra with blue nuclei were observed. However, these labeled neurons were observed only in rats injected with the highest titer of vector (Choi-Lundberg, 1997).
V. Ad GDNF gene therapy directed to DA cell bodies
Injection of microgram amounts of recombinant GDNF protein into the striatum or substantia nigra has been shown to rescue DA neurons from cell death following chemical lesions from 6-hydroxydopamine (6-OHDA) and MPTP, as well as that caused by physical lesions (Beck et al., 1995; Bowenkamp et al., 1995; Gash et al., 1995; Gash et al., 1996; Kearns and Gash, 1995; Shults et al., 1996; Tomac et al., 1995). To determine whether low levels of GDNF biologically synthesized near the DA neurons would also protect against cell death, Ad GDNF, Ad-mGDNF, or Ad LacZnl were injected stereotaxically immediately above the substantia nigra in the ventral midbrain. In the same surgery as the vector injection, a subpopulation of DA neurons that projected to a specific site in the striatum were labeled with the retrograde tracer fluorogold (FG). This labeling permitted a quantitative assessment of the degree of cell survival without relying on any DA neuronal phenotypic marker.
This was considered important since previous studies in which rats were injected with GDNF following a 6-OHDA lesion suggested that neurons that had supposedly degenerated had actually lost TH-immunoreactivity and seemed to ŅreappearÓ after GDNF administration (Bowenkamp et al., 1996; Hoffer et al., 1994; Kearns and Gash, 1995). Although our cell counts were made of FG labeled cells, we also noted that virtually all of the FG labeled cells were also positive for TH prior to lesioning (unpublished observations). One week following the vector and FG injections, the subpopulation of DA neurons on one side of the brain was lesioned by injection of 6-OHDA into the striatum in the vicinity of their terminals. The DA neurons on the other side of the brain were labeled with FG, but remained unlesioned and untreated as an internal control in each animal. Seven weeks after vector injection, rats were assessed for the degree of DA cell survival and the persistence of transgene expression.
DA neurons undergo a slow, progressive degeneration following injection of 6-OHDA near their terminals so that by 2 months, approximately 75% of the neurons have died (Sauer and Oertel, 1994). This degeneration was almost completely prevented by the Ad GDNF vector. As shown in Figure 4, Ad-GDNF protected an average of about 80% of the FG labeled DA neurons with two of the rats displaying nearly complete protection (Choi-Lundberg et al., 1997b). In contrast, only an average of about 30% of the neurons remained in the 3 control groups. In lesioned rats that received no treatment or injection of Ad LacZnl or Ad mGDNF, there were few large FG labeled DA neurons remaining and many small FG labeled cells. Many of the small cells had the characteristic morphology of microglia, which presumably had phagocytosed dying DA neurons, or alternatively were shrunken DA neurons in the process of dying. These observations clearly demonstrated that increasing levels of synthesis of a neurotrophic factor in the brain can have remarkable effects on protecting neurons from chemically induced cell death. It remains to be determined whether this approach will also protect diseased neurons from cell death.
To determine the amount and stability of GDNF transgene expression in the brain following injection of Ad GDNF, a time course of vector expression was undertaken. A comparison was made of levels of transgene protein, transgene mRNA and vector DNA for both GDNF and LacZnl at 1, 4 and 7 weeks after injection of vector into brain. The levels of human GDNF protein at 1, 4 and 7 weeks in the ventral midbrain were found to be 13, 5 and 2 nanograms, respectively, while those of §-
Figure 4. In vivo gene therapy with Ad GDNF prevents progressive degeneration of fluorogold-prelabeled DA neurons.
Six weeks following intrastriatal 6-OHDA, many large, FG positive cells (DA neurons, arrows) were observed in the SN on the unlesioned side (A) and on the lesioned side of a rat treated with Ad GDNF (B). In contrast, fewer large FG+ cells, but many small, secondarily labeled FG+ cells (microglia and other non-neuronal cells, arrowheads) were noted in rats treated with Ad mGDNF (C) or untreated (D). Scale bars in A-D are 100 µm and 50 µm for the insets. Reprinted with permission from Choi-Lundberg et al., 1997b. Copyright 1997 American Association for the Advancement of Science.
galactosidase protein were 57, 20 and 17 nanograms (Choi-Lundberg, 1997b; Choi-Lundberg et al., 1997). Although the levels of transgene declined over time, nanogram levels of GDNF are high with respect to the Kd for components of the GDNF receptor system that are in the picomolar range (Durbec et al., 1996; Jing et al., 1996; Trupp et al., 1996). The levels of transgene mRNAs also declined. In contrast, the levels of vector DNA did not decline between 1 and 7 weeks, suggesting that host responses to the vectors repressed transgene expression, but did not result in the loss of infected cells (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997b). This observation also suggests that the use of different promoters, e.g. cellular promoters, may increase the stability of transgene expression from adenoviral vectors.
The observation that neurotrophic factor gene therapy near the cell bodies of the DA neurons provided neuroprotection against the effects of 6-OHDA raises several issues. One issue is whether GDNF gene therapy directed to the terminals of DA neurons will also offer neuroprotection. This question is important since a clinical application of this therapy would require injection into striatum, a brain region relatively accessible and safe compared to injection into the midbrain. Another issue is whether the GDNF gene therapy results in a functional improvement. Studies addressing these questions were undertaken in the same progressive degeneration rat model of ParkinsonÕs disease as above, but in which vectors were injected into the striatum rather than into the mesencephalon. Preliminary results from these studies show that Ad-GDNF injected near the terminals of DA neurons also protected against 6-OHDA induced cell death (Choi-Lundberg, 1997a; Choi-Lundberg et al., 1997). Furthermore, DA dependent behaviors were also improved in the Ad GDNF treated rats (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997a).
VI. Other directions for neurotrophic factor based gene therapies.
The idea of using vectors to deliver genes that may slow or even reverse the progression of neurodegenerative diseases is exciting as there are presently no cures for diseases such as ParkinsonÕs, HuntingtonÕs, AlzheimerÕs or Lou GehrigÕs. However, this research area is in its infancy and new vectors need to be developed that are safe and offer stable gene expression in the CNS, most specifically in the primate CNS. Vectors that target genes to specific cell types in the nervous system, as well as those that contain promoters that can be regulated by peripheral drug administration, may offer distinct advantages over those presently in use.
Both ex vivo and in vivo gene therapy studies employing neurotrophic factors have generated promising results in animal models of disease and aging. Other in vivo gene therapy studies in rat models of ParkinsonÕs disease using adenoviral and adeno-associated viral vectors harboring GDNF have demonstrated protective effects similar to those observed in our studies (Bilang-Bleuel et al., 1997; Horellou et al., 1997; Mandel et al., 1997). Injection of an Ad vector harboring nerve growth factor (NGF) into aged rats increased the size of cholinergic neurons (Castel-Barthe et al., 1996). Another study using herpes simplex virus harboring NGF prevented the decline in TH activity in sympathetic neurons that occurs after axotomy (Federoff et al., 1992). In a rat model of stroke, injection of an adenoviral vector harboring a cDNA for interleukin receptor antagonist decreased the amount of tissue damage in the area of focal ischemia (Betz et al., 1995). Adenoviral vectors for ciliary neurotrophic factor (CNTF) and neurotrophin 3 (NT-3) improved indices of motoneuron function and increased life span of pmn mice, a mouse strain characterized by motoneuron nerve terminal and axonal degeneration (Haase et al., 1997). Motoneuron cell death following facial nerve lesions in newborn rats was reduced by muscular injection of Ad vectors harboring BDNF or GDNF (Gimenez y Ribotta et al., 1997). Retinal degeneration is also a potential target of neurotrophic factor gene therapy. In this respect, injection of an Ad vector harboring ciliary neurotrophic factor (CNTF) intravitreally in rd transgenic mice rescued photoreceptors from apoptosis (Cayouette and Gravel, 1997). Although not yet studied in vivo, transduction of explant cultures of rat spiral ganglia with an HSV amplicon vector harboring BDNF elicited neuritic outgrowth, suggesting that neurotrophic factor gene therapy may also be applicable to deafness (Geschwind et al., 1996).
Ex vivo gene therapy approaches with neurotrophic factors have used a variety of cell types, including fibroblasts, myoblasts, astrocytes, stem cells and a variety of cell types encapsulated in polymers. Ex vivo neurotrophic factor gene therapies have shown protective effects or behavioral improvement in animals whose cholinergic, dopaminergic or striatal systems have been damaged; however, these studies are too numerous to be reviewed here (for reviews, see (Choi-Lundberg and Bohn, 1997; Gage et al., 1987; Jinnah et al., 1993). In the case of Lou GehrigÕs disease, ex vivo neurotrophic factor gene therapy has progressed to the clinical trial stage where encapsulated cells secreting CNTF are being implanted intrathecally (Aebischer et al., 1996a, b). In animal models of spinal cord injury, implanted fibroblasts secreting NGF or BDNF increased sprouting from sensory, motor and noradrenergic fibers and reduced the area of spinal tissue injury and locomotor deficits (Kim et al., 1996; Tuszynski et al., 1996). In aged rats, implantation of GDNF-secreting fibrobasts increased locomotor activity and bar pressing, as well as increased the density of TH staining in the striatum suggesting an effect of GDNF on DA neurons in aged rats (Emerich et al., 1997). An interesting recent study showed that implantation of mouse stem cells responsive to the proliferative effects of epidermal growth factor (EGF) and generated from mice in which NGF is under control of the astrocyte specific promoter, glial fibrillary acidic protein (GFAP), not only protected striatal neurons from excitotoxin-induced cell death, but also resulted in the integration of grafted cells as astrocytes into host tissue (Kordower et al., 1997). The grafting of neuronal precursors carrying transgenes, including neurotrophic factor genes, in which such cells integrate into host tissue is beginning to be explored (Martinez-Serrano et al., 1995; Snyder and Fisher, 1996; Snyder et al., 1995), and has significant potential for both cell replacement and gene therapies.
In conclusion, the merging of gene therapy with recent discoveries in the field of developmental neurobiology, such as novel neurotrophic factors, molecules involved in neuronal death and differentation, and techniques for generating neuronal precursor cells, presents exciting, relatively unexplored horizons for developing therapies for neurodegenerative diseases and trauma to the nervous system. Yet, advances in vectors that will elicit safe, stable gene expression in the human brain are pivotal to bringing these potentials to practical use. As a first step, it is crucial that new generation vectors be developed that elicit persistent transgene expression in specific cell types and that these be demonstrated to be effective and safe in the non-human primate CNS.
This review was supported by NIH grant NS31957 and the Medical Research Institute Council of ChildrenÕs Memorial Hospital. The secretarial assistance of Ms. Gabrielle Pearlman is appreciated.
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