Neurodegenerative Diseases Amenable To Gene Therapy

The Parkinson's-Reversing Breakthrough

What is Parkinsons Disease

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The following discussion illustrates the most common forms of neurodegenerative diseases (summarized in Table 2) and potential gene therapy approaches that have been employed based on current understandings of disease mechanisms.

A. Parkinson's Disease

Parkinson's disease (PD) is the second most common chronic neurodegenerative disease of humans. In 1995, the incidence ofPD was estimated to be between 1:100 to 1:500 individuals (17,18). This incidence translates to approximately 1% of the population over the age of 65 (19-21). The disorder, initially described in 6 patients by Dr. James Parkinson, is typified clinically by symptoms including bradykinesia, resting tremor, rigidity, and gait abnormalities, followed by postural instability, dementia, and autonomic dysfunction. Pathologically, PD patients experience specific degeneration of dopami-nergic neurons in the substantia nigra pars compacta as well as dopaminergic ventral tegmental area (VTA) neurons and noradrenergic neurons of the locus coeruleus. Furthermore, neuronal loss has been reported in other brain areas such as the cerebral cortex, anterior thalamus, hypothalamus, amygdala, and basal forebrain. In addition to neuronal loss, accumulation of proteinaceous cytoplasmic inclusions called Lewy bodies is a neuropathological hallmark of PD. The exact role of Lewy bodies in PD is unclear, but other neurodegenerative disorders also exhibit intracellular and extracellular protein aggregates. Understanding the molecular mechanisms underlying protein aggregates in neurodegenerative diseases may assist to illuminate a common target for gene therapy.

Although more than 180 years have passed since Parkinson's first description, the disease etiology is still largely unknown. Because the evolution of successful gene therapeutic strategies will rely heavily on a detailed understanding of the molecular and cellular processes governing the clinical presentations of PD, exploring the pathophysiology of PD is crucial. The following section summarizes possible mechanisms of PD and how methods in gene delivery and expression might be applied to interdict the pathogenic pathway(s) of this neuro-degenerative disorder.

1. Mechanisms of Disease

PD exists as both a sporadic and familial disorder. Although the exact etiology of PD is unknown, the common pathway of both sporadic and familial PD is a loss of dopamine (DA) neurons. Importantly, this decline in DA neuron number below a critical threshold produces early symptomatic PD [reviewed in (22-24)]. Given that environmental factors such as pesticides, herbicides, and industrial chemicals have been

Table 2 Neurological Disorders Amenable to Gene Therapy

Disease

Age onset

Disease mechanism

Basis

Parkinson's

50-70s

Loss pigmented dopamine neurons in midbrain

Genetic (rare): a-synuclein, C-terminal ubiquitin hydrolase; parkin Environmental: pesticides, fungicides, neurotoxicants

Alzheimer's

50-80s

Aß accumulation; tau hyperphosphorylation; synapse loss

Genetic (infrequent): APP, PS

(susceptibility): ApoE4 Environmental: Viral infection/diet?

Lysosomal storage

Infancy

Enzyme deficiency leading to lysosomal protein accumulation and eventual peripheral and CNS degeneration

Genetic: lysosomal enzyme/transport gene deletion/mutation

Huntington's

20-50s

Loss of striatal medium spiny neurons

Genetic: Polyglutamine expansion in huntingtin gene locus

Stroke

Varies

Hypoxic insult leads to necrotic and apoptotic cell death

Potential genetic and environmental susceptibility interaction

Epilepsy

Childhood

Aberrant hypersynchronization of local or global neuronal networks

Unknown: potential early lifetime subclinical event

Motor neuron disorders

40-60s

Loss of spinal motor neurons

Genetic: missense mutations (ALS); gene deletions/aberrant splicing (SMA)

identified as potential risk factors for PD and genetic mutations have also been identified, it is likely that either alone or in combination, these triggers will produce a clinical syndrome similar to PD [reviewed in (24)]. Figure 2 outlines a common pathway for PD and suggests several targets for gene therapy without necessarily interdicting the initiating mechanism. In this ''common pathway'' model for PD, multiple triggering mechanisms such as genetic, toxicant, and environmental trigger, plus genetic vulnerability, converge on a shared common pathway to cell death. The first step along this pathway may encompass presynaptic injury and dysfunction followed by cellular compensation and metabolic stress. This preclinical injury and damage would result in DA deficiency and cell death. Gene therapy treatment opportunities would include (1) targeting specific triggering mechanisms, (2) targeting shared early pathways prior to presynaptic dopami-nergic dysfunction, (3) targeting shared later pathways when dysfunction occurs, and (4) restoring DA biosynthesis in the denervated striatum. As stated above, PD is likely the result of a combination of environmental, toxin, and genetic triggering factors. Because of this, it is difficult to review the impact of each individually, but we attempt to briefly outline the potential impact of these factors on the etiology of PD and summarize their convergence on the pathophysiology of this disease.

2. Environmental Factors

Several findings support an etiologic role for exogenous factors in PD. The earliest observation was that a synthetic by product of meperidine production produced a syndrome similar to PD. 1,2,3,6-Methyl-phenyl-tetrahydropyridine (MPTP) treatment of mice and monkeys has become a common method to achieve dopaminergic neuronal loss and an animal ''model'' of PD. The toxic compound MPTP is converted in glia to the pyridinium ion (MPP + ) by monoaminoxidase type B (MAO-B) and subsequently taken up by dopamine neurons via the dopamine transporter. MPP + is then actively transported to the mitochondria where it inhibits complex I, interfering with mitochondrial respiration and resulting in increased production of the superoxide anion. (25).

Recent reports lend biological plausibility to pesticide exposure as both an alternative model of nigrostriatal dopami-nergic degeneration and a risk factor for PD (26-28). Greena-myre's group reported that chronic, systemic treatment of rats with the lipophilic pesticide, rotenone, resulted in highly selective nigrostriatal dopaminergic degeneration (27,29). This degeneration is associated with PD-like behavioral changes, including hypokinesia and rigidity. Neuropathologically, these animals accumulate fibrillar cytoplasmic inclusions that contain both ubiquitin and a-synuclein. This treatment represents an important alternate PD model because these inclusions resemble Lewy bodies, a hallmark of PD. Of equal importance is the suggestion from this model that mitochondrial dysfunction in dopaminergic neurons, in particular, complex I inhibition, plays an important role in the pathophysiology of Lewy body formation and some cases of idiopathic PD.

Figure 2 Schematic depicting the common pathway model for PD and points along this pathway where gene-based therapy may be applied. (From Ref. 252, © 2001 Elsevier Science B.V.)

Other groups have implicated a combination of herbicide and fungicide exposure as a potent risk factor for PD (26,28). Mice treated with a combination of paraquat and maneb demonstrated a sustained decrease in motor activity and reduced tyrosine hydroxylase immununoreactivity in the dorsal striatum. These effects were greater in combination than either paraquat or maneb alone, suggesting that multiple compound exposure alone, or in concert with genetic vulnerability, may trigger a cascade of events leading to PD.

3. Genetic Factors

Several genetic factors have recently been identified to play a central role in the etiology of familial PD. In 1996, genetic linkage strategies were applied to a large Italian family with early onset PD (17,30). The susceptibility gene was identified on the long arm of chromosome 4q21-q23 and positional cloning identified a missense mutation in the a-synuclein gene at position 53 (A53T) (31). Another mutation in a-synuclein, A30P, was identified in a German family (32). Although mutant a-synuclein is responsible for a small number of PD cases, it has vaulted to the forefront of PD research since wild-type a-synuclein has been identified as a key component of Lewy bodies. In fact, a-synuclein staining is now widely used as a neuropathological criterion for PD (33-38).

Two other genetic mutations related to proteasome function have been identified in familial PD. One mutation in the parkin gene was discovered in an early onset juvenile autosomal recessive form of parkinsonism (AR-JP) that presents with mild symptoms, slow progression, and the absence of Lewy bodies (39-41). Homozygous deletions and point mutations in parkin also account for the majority of autosomal recessive inherited PD (42-48). A second mutation in the ubiquitin carboxy-terminal-hydrolase-L1 (UCH-L1) gene has been identified in a German family with PD (49). Both Parkin and UCH-L1 are involved in the regulation of the ubiquitin-proteasome pathway that suggests dysregulation of protein processing in the pathophysiology of PD (21,50,51).

4. Potential Gene-based Therapies for Parkinson's Disease

As outlined in Fig. 1 and discussed above, toxicant and environmental triggers combined with genetic vulnerability are likely to converge on a common pathway toward cell death in PD. These common downstream events that culminate in neuronal cell death are all possible targets for gene therapy in the treatment of PD. Teaming the appropriate gene with the most powerful vector system will make these treatments a clinical reality for PD-afflicted individuals. The following sections summarize the present state of the art for the application of gene transfer technologies to treatment of PD. a. Ad Vectors and PD. Adenovirus-based therapies for PD have been tested extensively in rodent models. A firstgeneration Ad vector encoding the TH gene, when introduced into the striatum of 6-hydroxydopamine (6-OHDA) lesioned rats, led to a reduction in amphetamine-induced rotational behavior (52). Vector-directed TH gene expression was observed for 1 to 2 weeks following gene transfer, but was ac companied by a vigorous inflammatory response, gliosis, and local tissue damage. Ad vectors expressing the TGF-p family member, glial cell line-derived neurotrophic factor (GDNF), have also been tested in 6-OHDA-lesioned rats where protection of the dopaminergic phenotype from chemical-induced damage is observed for up to 6 weeks postlesion (53). This protection was equivalent in scope if the vector was delivered to the striatum or the SN prior to the 6-OHDA lesion (54,55). GDNF delivery via an Ad vector was shown to exhibit differential effects in the lesioned aged rat as opposed to younger rats. Connor and colleagues demonstrated that Ad-mediated GDNF delivery was only protective in aged rats when the virus was delivered to the striatum, whereas the dopaminergic system of young 6-OHDA-lesioned rats could be protected by delivery to either the SN or striatum (56). This group hypothesized, based proposals by Zigmond et al., that compensatory changes occur in the CNS of the aged rat that likely increases its sensitivity to Parkinsonian lesioning (57).

b. AAV Vectors and PD. Recombinant AAV vectors exhibit great promise in the arena of PD gene therapy. For example, Kaplitt and others injected AAV vectors expressing either p-galactosidase or human tyrosine hydroxylase (hTH) into the brains of 6-OHDA-lesioned rats and demonstrated long-term transgene expression (3 months) and functional recovery (58). Mandel and colleagues demonstrated longer-term transgene expression (at least 1 year) in the rat striatum (59). In this series of experiments, AAV vectors were constructed to express either hTH or human GTP-cyclohydrolase I (GTPCHI) in the 6-OHDA-lesioned rat striatum. Elevated levels of L-dihydroxyphenlaline (L-DOPA) were observed in animals receiving both vectors, but disappointingly, no reduction in apo-morphine-induced rotational behavior was apparent in these animals. CED-mediated delivery of AAV expressing the aromatic amino acid decarboxylase (AADC) gene to the striata of MPTP-lesioned monkeys resulted in L-DOPA-regulated dopamine production and release in these parkinsonian animals (11). An illustration of an experiment from this study is shown in Fig. 3. Another study using AAV vectors to deliver hTH and AADC genes to the brains of MPTP-treated monkeys showed that AAV vectors could direct long-term transgene expression devoid of significant toxicity (60). Recently, Shen and colleagues have used a triple AAV vector administration approach to combat PD (1). These vectors encode for TH, AADC, and GTPCHI, and were shown in combination in rats to enhance levels of tetrahydrobiopterin (BH4) and dopamine production, as well as to improve apomorphine-induced rotational behavior for up to 1 year. Other studies have also demonstrated extended expression in the CNS using AAV vectors (61-63).

A paper by During and colleagues (64) described the use of AAV vectors to express the glutamic acid decarboxylase (GAD) gene GAD65 in the setting of experimental PD to phenoconvert excitatory neurons (glutamate neurotransmitter releasing) to inhibitory neurons (GABA neurotransmitter-re-leasing). The rationale behind this novel approach relates to the altered neurotransmission that occurs within the basal gan

Figure 3 Convection-enhanced delivery of an AAV vector expressing the aromatic L-amino acid decarboxylase (AADC) gene in Parkinsonian nonhuman primates. Rhesus monkeys received the dopaminergic toxin MPTP to establish a stable parkinsonian state. A combined unilateral intracarotid artery and intravenous MPTP injection protocol was performed to produce a nearly complete dopaminergic lesion on the side of the carotid artery infusion (ipsilateral side) and a partial lesion on the other side (contralateral side). Once this was achieved, the animals received ipsilateral infusions of either AAV-AADC or the p-galactosidase control vector AAV-LacZ via convection-enhanced delivery into the striatum. Positron emission tomography studies of uptake of the AADC tracer 6-[18F]fluoro-L-rn-tyrosine (FMT) in the striatum are shown for AAV-LacZ (upper segments of A and B) and AAV-AADC-injected monkeys (upper segments of C and D) pre- and postviral injection. FMT uptake was negligible in the ipsilateral striatum in all monkeys prior to AAV administration. After virus injection, AAV-LacZ-treated monkeys showed little change in ipsilateral FMT uptake, whereas AAV-AADC-treated animals demonstrated a dramatic increase in ipsilateral FMT uptake. After sacrifice, immunohistochemical analyses for tyrosine hydroxylase (TH-IR) and AADC (AADC-IR) expression were performed on the striatum (row 1 of each panel) and substantia nigra (row 2 of each panel). TH- and AADC-IR were markedly reduced in the ipsilateral striatum of AAV-LacZ-injected monkeys, whereas only AADC-IR was restored on the ipsilateral striatum of AAV-AADC-treated animals. In data not shown, AAV-AADC monkeys exhibited a marked enhancement in dopamine production when the animals were given L-DOPA, indicating that the AADC transgene product was bioactive. (From Ref. 11, © 2000 Elsevier Science B.V.) See the color insert for a color version of this figure.

Figure 3 Convection-enhanced delivery of an AAV vector expressing the aromatic L-amino acid decarboxylase (AADC) gene in Parkinsonian nonhuman primates. Rhesus monkeys received the dopaminergic toxin MPTP to establish a stable parkinsonian state. A combined unilateral intracarotid artery and intravenous MPTP injection protocol was performed to produce a nearly complete dopaminergic lesion on the side of the carotid artery infusion (ipsilateral side) and a partial lesion on the other side (contralateral side). Once this was achieved, the animals received ipsilateral infusions of either AAV-AADC or the p-galactosidase control vector AAV-LacZ via convection-enhanced delivery into the striatum. Positron emission tomography studies of uptake of the AADC tracer 6-[18F]fluoro-L-rn-tyrosine (FMT) in the striatum are shown for AAV-LacZ (upper segments of A and B) and AAV-AADC-injected monkeys (upper segments of C and D) pre- and postviral injection. FMT uptake was negligible in the ipsilateral striatum in all monkeys prior to AAV administration. After virus injection, AAV-LacZ-treated monkeys showed little change in ipsilateral FMT uptake, whereas AAV-AADC-treated animals demonstrated a dramatic increase in ipsilateral FMT uptake. After sacrifice, immunohistochemical analyses for tyrosine hydroxylase (TH-IR) and AADC (AADC-IR) expression were performed on the striatum (row 1 of each panel) and substantia nigra (row 2 of each panel). TH- and AADC-IR were markedly reduced in the ipsilateral striatum of AAV-LacZ-injected monkeys, whereas only AADC-IR was restored on the ipsilateral striatum of AAV-AADC-treated animals. In data not shown, AAV-AADC monkeys exhibited a marked enhancement in dopamine production when the animals were given L-DOPA, indicating that the AADC transgene product was bioactive. (From Ref. 11, © 2000 Elsevier Science B.V.) See the color insert for a color version of this figure.

glia during PD. In this disease, dopaminergic neuron projections from the substantia nigra pars compacta to the striatum degenerate. The observed reduction in dopamine release in the striatum results in reduced GABA neurotransmission, also called ''disinhibition,'' in the subthalamic nucleus (STN). This state, coupled with direct disinhibition within the striatum itself, results in increased glutamate neurotransmission and thus excessive excitation of the substantia nigra pars reticulata (SNr) and the globus pallidum internal segment (GPi). Delivery of AAV expressing GAD65 to the STN led to increased GABA release in the SNr, shifted SNr single-unit recording responses from primarily excitatory to inhibitory, and if delivered prior to 6-OHDA lesioning protected rats from the neurotoxin. These results, although intriguing, require a comprehensive evaluation of long-term effects that neuronal phenoconversion may have on basal ganglia circuitry. c. Lentivirus Vectors and PD. Lentivirus-based therapies for PD have been tested extensively in rodent and nonhuman primate models. Injection of a self-inactivating (SIN) lentivirus vector expressing GDNF has been shown to be protective in the 6-OHDA rat and MPTP nonhuman primate models of PD and in nonlesioned aged rhesus monkeys (65-67). An example of one of these studies is shown in Fig.

4. Long-term striatal overexpression of this potent factor by a lentiviral vector in nonlesioned rats has recently been shown to markedly down-regulate TH expression (68). The mechanism responsible for GDNF-mediated repression of TH expression is presently not understood, but the observation supplicates caution in implementation of gene transfer approaches clinically that involve long-term, uncontrolled GDNF expression.

Lentiviral vectors have been shown preclinically to be effective with other approaches to treat PD. The dopamine bio-synthetic pathway, which is compromised in PD, may require the reintroduction of multiple components to restore physiological levels of dopamine. Due to the moderate size capacity of lentiviral vectors, multicistronic versions of the vector platform have been developed. Azzouz and colleagues recently created a lentiviral vector that coexpresses aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohy-drolase I (69). Delivery of this vector to the striata of 6-OHDA-lesioned rats led to stable dopamine biosynthesis and functional improvement for up to 5 months posttreatment. d. HSV Vectors and PD. Dependent the transgene size capacity of a given gene transfer vector, the use of cellular promoters to direct transgene expression appears to be another

Figure 4 Delivery of a lentiviral vector expressing the glial cell line-derived neurotrophic factor (GDNF) gene in Parkinsonian nonhuman primates. Rhesus monkeys received the dopaminergic toxin MPTP unilaterally (right side) to establish a parkinsonian state. One week later, the animals received ipsilateral infusions of lentiviral vector expressing glial cell line-derived neurotrophic factor (lenti-GDNF) or one expressing the reporter protein p-galactosidase (lenti-pGal). Three months following treatment, the animals were sacrificed and immunohistochemistry was performed. A and B depict low-power, dark-field photomicrographs through the right striatum of TH-immunostained sections of MPTP-treated monkeys treated with lenti-pGal (A) or lenti-GDNF (B). There appeared to be a comprehensive diminution of TH immunoreactivity in the caudate and putamen of lenti-pGal-treated animals, whereas a nearly normal level of TH immunoreactivity was seen in the animals receiving lenti-GDNF. Low-power (C and D) and medium-power (E and F) photomicrographs are shown of a TH-immunostained section through the substantia nigra of animals treated with lenti-pGal (C and E) and lenti-GDNF (D and F). Note the loss of TH-immunoreactive neurons in the lenti-pGal-treated animals on the side of the MPTP infusion. TH-immunoreactive sprouting fibers, as well as an above normal number of TH-positive nigral perikarya, are observed in lenti-GDNF-treated animals on the side of the MPTP injection. G and H depict low-power, bright-field photomicrographs of a TH-immunostained section from a lenti-GDNF-treated monkey. Note the normal TH-immunoreactive fiber density through the globus pallidus on the intact side that was not treated with lenti-GDNF (G). In contrast, an enhanced network of TH-immunoreac-tive fibers is seen on the side treated with both MPTP and lenti-GDNF. Scale bar in (G) represents the following magnifications: A, B, C, and D at 3500 ^m; E, F, G, and H at 1150 ^m. (From Ref. 66, © 2000 AAAS.) See the color insert for a color version of this figure.

promising strategy for development of a gene-based therapy for PD because these promoters tend to yield longer-term, cell-specific expression. This has been demonstrated using HSV amplicons equipped with either the preproenkephalin or 9-kb tyrosine hydroxylase (TH) promoter in vivo. Kaplitt and colleagues showed extended expression duration and striatal cell specificity using a version of the preproenkephalin promoter inserted into an HSV amplicon (70). Using 9-kb rat TH promoter to drive expression of the p-galactosidase (lacZ) reporter gene in the mouse striatum, Jin et al. observed expression of lacZ in TH-positive dopaminergic neurons in the substantia nigra due to retrograde transport of amplicon virions (71). In one of the few published PD-related studies using amplicons as the chosen gene therapy vector, During and colleagues treated 6-OHDA-lesioned rats with an HSV amplicon expressing human TH (72). Behavioral and biochemical recovery was maintained for 1 year following vector introduction.

e. Nonviral Vectors and PD. The use of nonviral means of DNA delivery for treatment of PD is appealing due to the inherent lack of immunogenic and/or cytotoxic viral gene products in the system. Nonviral delivery of genes includes the following means of transfer: ''naked'' DNA, DNA encapsulated within cationic lipids or polycationic polymers, or DNA attached to positively charged metal particles and introduced via particle bombardment (3,73-75). Until recently, successful implementation of this gene transfer modality has largely been impeded by the low transfection efficiencies observed in neurons. In vivo nonviral means of DNA transfer were initially demonstrated to occur in muscle cells with surprising efficiency (76). DNA transfer using polycationic lipid formulations to glia and neurons has been also demonstrated, albeit at low efficiencies (10,73). Martinez-Fong and colleagues recently described their use of a neurotensin-SPDP-poly-L-lysine conjugate that was competent to bind and transfer plasmid DNA to neurotensin receptor-expressing cell lines (N1E-115 and HT-29) (77). Because the high-affinity neuro-tensin receptor is expressed by a subset of neurons of the nigrostriatal and mesolimbic dopaminergic systems, this non-viral gene delivery modality could prove useful in the treatment of PD. In Addition, the 25-kD cationic polymer polye-thylenimine (PEI) was shown by Abdallah et al. to mediate transfer of a luciferase-expressing plasmid to neurons and glia of adult mice (78).

Pardridge and colleagues have developed an interesting nonviral gene delivery modality that has been recently tested in the 6-OHDA rat model of PD (79). A TH-expressing plasmid, encapsulated in PEGylated immunoliposomes (PIL) that were targeted to the brain with a rat transferrin receptor-specific monoclonal antibody, was administered intravenously. The plasmid was shown to effectively cross the blood-brain barrier and lead to transient normalization of TH levels within the striatum of 6-OHDA-lesioned rats. If issues regarding transgene expression silencing can be resolved and strict targeting to the striatum can be achieved, this approach may be one of the more promising applications of nonviral gene transfer-based therapies for PD.

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