Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex.

Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken ß-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases.

Combining glial cell line-derived neurotrophic factor gene delivery (AdGDNF) with L-arginine decreases contusion size but not behavioral deficits after traumatic brain injury.

Our laboratory has previously demonstrated that viral administration of glial cell line-derived neurotrophic factor (AdGDNF), one week prior to a controlled cortical impact (CCI) over the forelimb sensorimotor cortex of the rat (FL-SMC) is neuroprotective, but does not significantly enhance recovery of sensorimotor function. One possible explanation for this discrepancy is that although protected, neurons may not have been functional due to enduring metabolic deficiencies. Additionally, metabolic events following TBI may interfere with expression of therapeutic proteins administered to the injured brain via gene therapy. The current study focused on enhancing the metabolic function of the brain by increasing cerebral blood flow (CBF) with l-arginine in conjunction with administration of AdGDNF immediately following CCI. An adenoviral vector harboring human GDNF was injected unilaterally into FL-SMC of the rat immediately following a unilateral CCI over the FL-SMC. Within 30min of the CCI and AdGDNF injections, some animals were injected with l-arginine (i.v.). Tests of forelimb function and asymmetry were administered for 4weeks post-injury. Animals were sacrificed and contusion size and GDNF protein expression measured. This study demonstrated that rats treated with AdGDNF and l-arginine post-CCI had a significantly smaller contusion than injured rats who did not receive any treatment, or injured rats treated with either AdGDNF or l-arginine alone. Nevertheless, no amelioration of behavioral deficits was seen. These findings suggest that AdGDNF alone following a CCI was not therapeutic and although combining it with l-arginine decreased contusion size, it did not enhance behavioral recovery.

Distribution and morphology of the catecholaminergic neural elements in the human hypothalamus.

Previous studies have demonstrated that catecholaminergic, tyrosine hydroxylase (TH)-immunoreactive (IR) perikarya and fibers are widely distributed in the human hypothalamus. Since TH is the key and rate-limiting enzyme for catecholaminergic synthesis, these IR neurons may represent dopaminergic, noradrenergic or adrenergic neural elements. However, the distribution and morphology of these neurotransmitter systems in the human hypothalamus is not entirely known. Since the different catecholaminergic systems can be detected by identifying the neurons containing the specific key enzymes of catecholaminergic synthesis, in the present study we mapped the catecholaminergic elements in the human hypothalamus using immunohistochemistry against the catecholaminergic enzymes, TH, dopamine beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). Only a few, PNMT-IR, adrenergic neuronal elements were found mainly in the infundibulum and the periventricular zone. DBH-IR structures were more widely distributed in the human hypothalamus occupying chiefly the infundibulum/infundibular nucleus, periventricular area, supraoptic and paraventricular nuclei. Dopaminergic elements were detected by utilizing double label immunohistochemistry. First, the DBH-IR elements were visualized; then the TH-IR structures, that lack DBH, were detected with a different chromogen. In our study, we conclude that all of the catecholaminergic perikarya and the majority of the catecholaminergic fibers represent dopaminergic neurons in the human hypothalamus. Due to the extremely small number of PNMT-IR, adrenergic structures in the human hypothalamus, the DBH-IR fibers represent almost exclusively noradrenergic neuronal processes. These findings suggest that the juxtapositions between the TH-IR and numerous peptidergic systems revealed by previous reports indicate mostly dopaminergic synapses.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors.

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.

Differentiated dopaminergic MN9D cells only partially recapitulate the electrophysiological properties of midbrain dopaminergic neurons.

The cell line MN9D, a fusion of embryonic ventral mesencephalic and neuroblastoma cells, is extensively used as a model of dopamine (DA) neurons because it expresses tyrosine hydroxylase and synthesizes and releases DA. These cells are also used to test mechanisms and potential therapeutics relevant to the loss of DA neurons in Parkinson's disease. To date, little work has been done to determine whether MN9D cells electrophysiologically resemble mature DA neurons. We examined sodium, calcium and potassium currents in undifferentiated and differentiated MN9D cells, and compared these to those found in acutely dissociated mouse substantia nigra pars compacta DA neurons. It was observed that undifferentiated MN9D cells bore no resemblance to DA neurons. Upon differentiation with butyric acid with or without a prior treatment with glial cell line-derived neurotrophic factor, differentiated MN9D cells produce an electrophysiological profile that more closely resembles substantia nigra pars compacta DA neurons even though the A-type potassium current remains noticeably absent. These observations demonstrate that undifferentiated MN9D cells are not reasonable models of DA neurons. Although differentiated MN9D cells are closer to the mature DA neuronal phenotype, they do not fully mimic DA neurons and are likely to be of questionable value as a model because of their substantive differences, including the lack of the characteristic A-type potassium current. The future use of one or a combination of growth or other factors to differentiate MN9D cells may yield a more useful model system for Parkinson's disease studies in vitro.

Effect of AdGDNF on dopaminergic neurotransmission in the striatum of 6-OHDA-treated rats.

We have previously observed that the delivery of an adenoviral vector encoding for glial cell line-derived neurotrophic factor (AdGDNF) into the substantia nigra (SN) 7 days after intrastriatal administration of 6-hydroxydopamine (6-OHDA) protects dopamine (DA)-dependent behaviors, tyrosine hydroxylase immunoreactive (TH+) cells in SN, and amphetamine-induced c-fos induction in striatum. In the present study, we sought to determine if the behavioral protection observed in 6-OHDA-treated rats receiving AdGDNF was associated with an increase in DA availability in the striatum as measured by microdialysis. Rats received intrastriatal 6-OHDA (16 microg/2.8 microl) or vehicle followed 7 days later by intranigral AdGDNF (3.2x10(7) pfu/2 microl), AdLacZ (3.2 x 10(7) pfu/2 microl), or phosphate buffered saline (PBS). Three weeks later, microdialysis samples were collected from the same striatal region under basal conditions, following KCl (100 mM) or amphetamine (250 microM) administered via the striatal microdialysis probe, or amphetamine administered systemically (6.8 mg/kg i.p). Animals given 6-OHDA followed by either PBS or AdLacZ showed a decrease in basal extracellular striatal DA levels to 24% of control. In contrast, basal extracellular DA in 6-OHDA-lesioned rats with a nigral injection of AdGDNF was almost 3-fold higher than 6-OHDA-vehicle treated animals, 65% of control DA levels. Moreover, although KCl and amphetamine produced no increase in striatal DA release in 6-OHDA-treated rats that subsequently were given either PBS or AdLacZ, these manipulations increased DA levels significantly in 6-OHDA-treated rats later given AdGDNF. Thus, DA neurotransmission within the striatum of 6-OHDA treated rats appears to be enhanced by increased expression of GDNF in the nigra.

Quantitative analyses of GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the nigrostriatal system reveal bilateral compensatory changes following unilateral 6-OHDA lesions in the rat.

Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.

Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Quantitative analysis of transgene protein, mRNA, and vector DNA following injection of an adenoviral vector harboring glial cell line-derived neurotrophic factor into the primate caudate nucleus.

Gene therapy for neurodegenerative diseases relies on stable expression of a vector-mediated transgene in the human central nervous system (CNS). In nonhuman primate CNS, transgene expression has been primarily assessed using descriptive histological methods. Here, we quantified the expression of a human glial cell line-derived neurotrophic factor (hGDNF) transgene using an ELISA specific for hGDNF protein and real-time quantitative RT-PCR and PCR for hGDNF mRNA and vector DNA, respectively. Transgene expression was assessed 1 week after injection of an E1-, E3-deleted adenovirus harboring hGDNF into the caudate nucleus of St. Kitts green monkey. We found that 57-147 million and 116-771 million copies of hGDNF mRNA and vector DNA, respectively, were present per 10,000 copies of the beta-actin gene. In the same sites, 40-152 pg of hGDNF protein per milligram of tissue was measured. Comparisons of these measures among monkeys demonstrated variable vector DNA and protein levels, but consistent mRNA levels at one-third of the level of vector DNA. This suggests that local responses to the vector play a role in the level of transgene expression and that high levels of vector DNA do not necessarily predict a high level of transgene protein. However, the results of this study do show that neuroprotective levels of GDNF transgene expression can be achieved following injection of an adenoviral vector into nonhuman primate caudate. Moreover, these assays provide quantitative methods for evaluating and comparing viral vectors in primate CNS.

Gene transfer for neuroprotection in animal models of Parkinson's disease and amyotrophic lateral sclerosis.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MN) and dopaminergic (DA) neurons, neurons which selectively die in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). GDNF gene delivery has been studied in rodent models of ALS and PD. In a mouse model of ALS, implantation of myoblasts retrovirally transduced with GDNF into hindlimb muscles at 6 weeks of age, i.e. prior to the onset of disease symptoms, increased the number of large MNs that maintained projections to treated muscles at 18 weeks of age. GDNF-treated mice also performed better on tests of motor function and had a delayed onset of disease. In a progressive degeneration rat model of PD, effects of in vivo GDNF gene therapy using an adenoviral vector (AdGDNF) were studied in young and aged rats. AdGDNF protected DA neurons against the neurotoxin, 6-hydroxydopamine (6-OHDA), and was effective whether injected either before or after 6-OHDA damage had commenced. However, if AdGDNF was injected prior to 6-OHDA, it was most effective in protecting against DA-dependent changes in the brain when injected near the terminals of the DA neurons. In contrast, if 6-OHDA damage had already commenced, AdGDNF was most effective if injected near the DA soma. These studies suggest that GDNF gene delivery into specific sites in the CNS or into muscle where MNs have access to secreted GDNF may slow the progression of PD and ALS, respectively. Neurotrophic factor gene therapy offers novel interventions not only for PD and ALS, but also other neurodegenerative diseases and injuries to the nervous system.

Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections.

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) as a defensive molecule for neurodegenerative disease: a tribute to the studies of antonia vernadakis on neuronal-glial interactions.

Research stemming from interests in neuronal-glial interactions has led to the identification of a number of novel trophic factors, such as the dopaminergic neurotrophic factor glial cell line-derived neurotrophic factor (GDNF). Delivery of the GDNF gene to rat models of Parkinson's disease suggests a potential clinical use of GDNF gene therapy for humans with this disease. This review article briefly summarizes the history of GDNF and the effects of GDNF gene delivery prior to or after a lesion of the rat nigrostriatal system.

GDNF is trophic for mouse motoneurons that express a mutant superoxide dismutase (SOD-1) gene.

BACKGROUND AND METHODS: An in vitro system of motoneurons was established from mice carrying a transgene for a human superoxide dismutase-1 (SOD-1) with a gly93ala mutation that has been linked to familial amyotrophic lateral sclerosis (FALS). These cultures were characterized and used to compare the effects of glial cell line-derived neurotrophic factor (GDNF) on motoneurons expressing the mutant gene with those on normal motoneurons. RESULTS: Recombinant human GDNF (100 ng/ml) significantly promoted the survival of a subpopulation of choline acetyltransferase (ChAT)-immunoreactive motoneurons that were also immunoreactive for the homeoprotein islet-1 in cultures from both wild type and mutant SOD-1 mice. However, GDNF did not increase the total number of ChAT-immunoreactive neurons in cultures from either wild type or transgenic mice. A distinct subpopulation of islet-1-immunoreactive motoneurons characterized by a soma 3 1/2 times larger and a ten-fold increase in neurite length was observed exclusively in GDNF-treated cultures. In cultures from mutant SOD-1 mice, there were 3 1/2 times as many motoneurons of this subpopulation as in wild type cultures at 6 days in vitro. In addition, this subpopulation of neurons survived for 10 days in vitro, the longest time point studied, in culture from mutant SOD-1 mice, but not in cultures from wild type mice. This subpopulation was also present at 6 days in vitro in cultures from mutant SOD-1 mice that received GDNF at 3 days in vitro instead of at the time of plating, suggesting that GDNF promotes the differentiation of these neurons. CONCLUSION: Our observations suggest that the expression of a mutant SOD-1 gene, as occurs in familial ALS, does not compromise the trophic effects of GDNF on motoneuron survival, but may affect the development of motoneurons.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis.

Effects of ex vivo GDNF gene delivery on the degeneration of motoneurons were studied in the G1H transgenic mouse model of familial ALS carrying a human superoxide dismutase (SOD1) with a Gly93Ala mutation (Gurney et al., 1994). Retroviral vectors were made to produce human GDNF or E. coli beta-galactosidase (beta-Gal) by transient transfection of the Phoenix cell line and used to infect primary mouse myoblasts. In 6-week-old G1H mice, 50,000 myoblasts per muscle were injected bilaterally into two hindlimb muscles. Untreated G1H and wild-type mice served as additional controls. At 17 weeks of age, 1 week before sacrifice, these muscles were injected with fluorogold (FG) to retrogradely label spinal motoneurons that maintained axonal projections to the muscles. There were significantly more large FG-labeled alpha motoneurons at 18 weeks in GDNF-treated G1H mice than in untreated and beta-Gal-treated G1H mice. A morphometric study of motoneuron size distribution showed that GDNF shifted the size distribution of motoneurons toward larger cells compared with control G1H mice, although the average size and number of large motoneurons in GDNF-treated mice were less than that in wild-type mice. GDNF also prolonged the onset of disease, delayed the deterioration of performance in tests of motor behavior, and slowed muscle atrophy. Quantitative, real-time RT-PCR and PCR showed persistence of transgene mRNA and DNA in muscle for up to 12 weeks postgrafting. These observations demonstrate that ex vivo GDNF gene therapy in a mouse model of FALS promotes the survival of functional motoneurons, suggesting that a similar approach might delay the progression of neurodegeneration in ALS.

Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

A commentary on glial cell line-derived neurotrophic factor (GDNF). From a glial secreted molecule to gene therapy.

Glial cell line-derived neurotrophic factor (GDNF) was identified as a consequence of the hypothesis that glia secrete factors that influence growth and differentiation of specific classes of neurons. Glia are a likely source of additional neurotrophic factors; however, this strategy has not been applied extensively. The discovery of GDNF in 1993 led to an abundance of studies that within only a few years qualified GDNF as a bona fide neurotrophic factor. Of particular interest are studies demonstrating the effectiveness of GDNF protein in ameliorating neurodegeneration in animal models of Parkinson's disease and amyotrophic lateral sclerosis (ALS). It remains to be determined whether GDNF will be an effective therapy in humans with these diseases. However, since these diseases are slowly progressive and the CNS relatively inaccessible, the delivery of GDNF as a therapeutic molecule to the CNS in a chronic manner is problematic. Studies addressing this problem are applying viral vector mediated transfer of the GDNF gene to the CNS in order to deliver biosynthesized GDNF to a specific location in a chronic manner. Recent studies suggest that these GDNF gene therapy approaches are effective in rat models of Parkinson's disease. These studies are reviewed in the context of what developments will be needed in order to apply GDNF gene therapy to the clinic.