Using Stem Cell and Gene Therapy Technologies to Treat Parkinson's Disease
2010, Vol. 2 No. 03 | pg. 1/1
IN THIS ARTICLE
Introduction to Parkinson’s disease
Parkinson’s disease (PD), a progressive neurodegerative disorder most prevalent in the elderly and for which there is currently no cure, selectively targets nigrostriatal Dopaminergic (DAergic) projection neurons in the substantia nigra pars compacta (SNpc), which generates a consequential loss of Dopamine (DA) in the striatum (ST) (1). Because of the involvement of DA in voluntary movement, the resulting alteration in basal ganglia circuitry culminates in a predominantly motor phenotype of rigidity, rest tremor, bradykinesia, and akinesia, hallmark features of the disease (1, 2).
The sudden onset of Parkinsonism in drug addicts consuming the heroin- analogue MPTP in the 1980’s, led to its discovery as a potential cause of sporadic PD; indeed several chemical agents such as Rotenone have also received considerable attention (2). Although its etiology is still incompletely understood, the role of genetics in the pathogenesis of PD has been hotly debated for decades. The recent discovery of at least five genes (such as a-synuclein and parkin (Park-2))(3) together with animal lesion models (6-OHDA, MPTP, Rotenone) and human post mortem tissue analysis has polarized belief that that the cause of PD may be multivariate (“genetics holds the gun but environment pulls the trigger”) (2). Increasing evidence indicates that mitochondrial dysfunction, oxidative stress, neuroinflammation, and accelerated apoptosis are amongst several mechanisms thought to contribute to the pathogenesis of both sporadic and familial PD . With the presence of increased microglial activation and intracytoplasmic proteinaceous inclusions known as Lewy bodies in the few surviving DA and non-DAergic neurons, the resulting identification of new targets for novel treatment strategies has furthered our knowledge of PD.The “Parkinson’s” phenotype results from an abnormally active subthalamic nucleus (STN) and globus pallidus internus (Gpi), the latter which releases the inhibitory neurotransmitter (NT) GABA, essentially silencing glutamatergic thalamo-cortical neurons projecting to the motor cortex (2). Less appreciated are non-motor symptoms which have now been recognized, and include disturbances of affective function (with depression being the most common) and cognition . These non-motoric features may represent useful clinical benchmarks for initiating early therapeutics prior to the overt motoric symptoms. The significant movement disability caused by PD and its rising prevalence due to an increasing geriatric population worldwide thus warrants an accelerated effort in understanding PD further as well the urgent need for a cure.
The primary goal of drug therapy is to enhance DA and inhibit the concomitant increased cholinergic transmission. Consequently, current treatment options include pharmacotherapy, with the DA precursor L-Dopa being the most effective. In conjunction, DA agonists (Bromocriptine), MAO-B inhibitors (Deprenyl), COMPT inhibitors (Entacapone), GABA agonists (lidocaine) and anti-cholinergics are also prescribed. Surgery, a second more invasive option, is essentially reserved for patients harboring severe symptoms, with stereotactic thalamotomy and pallidotomy, deep brain stimulation (DBS) and STN ablation performed according to the each patient’s disease profile, in an attempt to electrically silent the disinhibited STN (4).
Novel Treatment Strategies
Inevitably however, long term drug intervention and surgery only act to alleviate symptoms and are inadequate in the long run due to resulting side effects (occurrence of dyskinesias upon long term use of L-Dopa for example). Indeed, current options fail to arrest progressive cell death, restore disease inflicted cells to normal or replace lost cells with healthy ones (5). As a result, current focus on PD research has shifted to the development of novel intervention strategies such as Gene Therapy and Stem Cell Therapy, which, based on ongoing research, offer promise for neuroprotection, neurorestoration and cell replacement. Interestingly, the focal (rather than global) pathogenicity of PD warrants it as an ideal candidate for the use of such novel intervention (5, 6).
Gene Therapy (GT) for PD
Gene Therapy involves the use of replication-deficient viral vectors (VV) to deliver therapeutic gene(s) in order to cause a beneficial alteration in the instruction of a cell. The direct injection of the VV constitutes the in- vivo delivery mode, while ex-vivo therapy involves transplantation of genetically modified donor cells into the host. Although the concept was originally conceived in the 1960’s, it was not till 1990 when the first clinical gene therapy trial involving human gene transfer came about (5). Interestingly, early attempts at transducing cell lines (fibroblasts, myoblasts, and glioma) engineered with particular genes were abandoned due to the tumour formation and the development of an immunological response, thus current GT trials involve in -vivo gene therapy (7).
The plethora of knowledge gained from molecular and cell biology has resulted in the establishment of various replication deficient VV such as Retroviruses (RV), adenoviruses (Ad), Adeno-associated viruses (AAV), herpes simplex viruses (HV), and pox viruses. However AAVs constitute the majority of vectors used for gene therapy trials in humans. This small sized, 4.7 kb single stranded DNA virus has enabled the development of targeted, cell specific GT, with the isolation of over 40 serotypes from animals till date. Indeed, its ability to transduce both diving and non-diving cells to generate long term gene expression, coupled with its non-pathogenicity, makes this vector ideal for PD therapy.
Current GT strategies act to biochemically augment DA via the transfer of DA biosynthetic genes such as Tyrosine Hydroxylase (TH) and Aromatic L-amino acid decarboxylase (AADC), into the ST and associated basal ganglia nuclei. Because catalysis via TH is the rate determining step in DA biosynthesis, attempts to deliver TH cDNA have met with promise, with the intraST transduction of AdV or HSV in 6-OHDA lesioned rats shown to decrease ipsilesional rotation bias (8). However technical and safety issues regarding expression of TH only (and not DA) as well as ensuing side effects has meant that the larger genome size of a lentiviral system has been paid particular attention to. The delivery of TH, AADC and GTP-cyclohyrolase 1 (GCH), the latter a rate determining step for the biosynthesis of BH4 (a cofactor for TH biosynthesis), into the ST of a 6-OHDA rat, resulted in sustained expression of DA as well as each enzyme, with an improved behavioural phenotype as well (8).
Current Status of Gene Therapy
Each of the 3 gene therapy clinical trials currently being conducted for PD use AAV vectors. Earlier pre-clinical success with animal models (with the transduction of striatal cells found to enhance conservation of endogenous L-dopa and improve motor phenotype of 6-OHDA challenged rodents, as well as a non-human primate model depicting long term motor improvement with reduced L-dopa -induced side effects) have resulted in L-AADC reaching phase 1 clinical trials. The underlying strategy which this trial utilizes is that “AAV2-hAADC” delivery, administered in conjunction with L-dopa, will result in improved effects of L-dopa. This symptomatic approach therefore has limitations as it only seeks to enhance the brain’s capacity to process L-dopa more efficiently. The dose-response approach used by the investigators has some advantages however. Interim results from a small cohort (5) of subjects are now being reported (8, 9).
Glutamic acid decarboxylase (GAD) GT has now reached clinical trial with approved success thus far (10). The human trial stems from pre-clinical data in rodents, whereby Luo and colleagues utilised an inhibitory approach, based on the pharmacotherapeutic effects of Muscimol (a GABA agonist) in suppressing the overactive STN. Upon delivery of AAV-GAD, the biosynthetic gene for GABA biosynthesis, a phenotypic shift was produced in the STN. There was a significant increase in inhibitory neurons both in the SN and ST as characterised by electrophysiology but also an increase in GABA release relative glutamate. In addition, GAD 65 rats showed marked motor improvement in behavioural tests compared to GAD 67 and control rats. Concurrent with rodent data, a MPTP model using non-human primates also generated similar results (10, 11).
The GAD GT trial examines safety, tolerability and potential efficacy of GAD therapy into the STN of PD patients (12). 12 patients were administered with AAD-GAD unilaterally followed by clinical assessment upto 12 months post gene therapy. Upon follow up, all patients depict statistically significant improvements in clinical ratings, as depicted by scales of activities of daily living (ADL), neuropsychological testing, and PET imaging. No adverse events related to gene therapy have been reported as yet. Significant improvements in motor UPDRS scores ipsilesional were seen 3 months after gene therapy and persisted up to 12 months (12).
The final clinical trial underway for PD tackles the problem using neuroprotective approach. Glial-cell-line-derevied neurotrophic factor (GDNF) is a glycosylated protein (13). The rationale for the use of GDNF as a neurotrophic factor for PD became apparent with the demonstration by Lin and colleagues that GDNF acted to promote the survival of midbrain DA neurons (13). Since then numerous studies have examined the protective effects of GDNF, with positive results: GDNF facilitates neurotransmission in intact adult DA neurons by increasing DA turnover and regulating expression of TH (14, 15). Sustained delivery, essential for efficacy due to the progressive nature of PD, has been obtained by direct intracerebral administration using rAAV or lentiviruses in intact and lesioned rodents. In addition Kirik and colleagues have also demonstrated that it induces regenration and sprouting in injured DA neurons.
Conflicting evidence pertaining to the precise level of GDNF delivery for substantial neuroprotection however required the need for a dose-response study (5). Using both immunohistochemical and behavioural paradigms it was established that the regulation of GDNF was pertinent to its use and that a low but continious level of GDNF delivery was sufficient to provide optimal fucntonal outcome. In contrast, high GDNF levels were a matter of safety concern as the use of a GDNF high vector produced both high and non-specific increases in TH immunoreactivity and DA metabolites (6). In recent years, the repertoire of growth factors has grown to include related ligands such as Persephin (PSPN) and Artemin (ATN), and Neurturin (NTN). GDNF and NTN however offer the most promise. Kordower and colleagues have recently depicted using rAAV encoding NTN (CERE 120), the neuroprotective, restorative and enhanced activity of nigrostriatal DA neurons in the MPTP lesioned and aged monkeys. The phase 1 trial currently ongoing concludes that CERE-120 is relatively safe and well-tolerated in patients; with mean decrease in UPDRS scores of 35 % 12 months post gene therapy (4).
Recent evidence points to apoptotic-like cell death as a key cell death mechanism in PD . As a result numerous studies have delivered anti-apoptotic elements in the hope to dampen programmed cell death of DA neurons. In rats, Adv delivery of neuronal apoptosis inhibitor protein (NAIP) resulted in protection of the degeneration and loss of function of nigrostriatal neurons induced by the 6-OHDA insult. Likewise, AAV transduction of the anti-apoptotic factor (apaf-1) prevented DAergic cell death in the mouse model (16). The Bcl-2 family of macromolecules known to mediate cell death have also been manipulated in a study whereby nigrostriatal neurons from transgenic mice overexpressing this mediator were resistant to MPTP and 6-OHDA neurotoxicity. Several studies now suggest that the concomitant administration of anti-apoptotic molecules with GDNF may offer greater viability to DA survival and protection. However a major drawback of the anti-apoptotic approach is the potential for adverse effects from the risk of oncogenesis; for this reason a transient rather than long term expression may be more suited (6, 15).
Emerging gene therapy strategies have considered the delivery of a normal copy of a mutated recessive gene to target familial PD. Evidence for this stems from a study demonstrating the restoration of Park7 expression of Park7-null mice via AAV as well as a biochemical and behavioral resistance to the 6-OHDA insult (6). Anti-oxidant administration via AAV delivery of human glutathione peroxidase 1 gene (GPX1) has also been established to resist 6-OHDA induced neurotoxicity of nigrostriatal DA neurons in the rat. Given the inflammatory changes inherent to PD pathology, development of anti-inflammatory gene therapeutics is an important avenue to consider in PD treatment (16, 17).
The treatment for PD in humans however requires targeted delivery of genes and proteins. This may be accomplished by viral vectors with known tropisms and cell-specific promoters. Neuronal, microglial and astrocytic specific expression has been accomplished utilizing cell-specific promoters, such as the neuron specific neuron-specific enolase (NSE) promoter (18).
The onset of symptoms in PD occurs when approximately 80% of neurons have been lost (2). Thus, despite extensive advances, PD gene therapy will be most successful when delivered at specific time points of pathogenesis such as the pre-symptomatic phase. Gene therapy has not only provided us with potential therapeutic intervention strategies but ongoing gene therapy research allows for the identification of new targets, so that we can better understand the currently un-established pathophysiology of PD (19) (18).
Cell Replacement Therapy for PD
Cell-based replacement therapy for PD made its debut in the late 1980’s (20). The study led by Bjorklund and colleagues saw the first in vivo transplantation of pre-dissociated cells in-vitro, grafted unilaterally into the ST of 6-OHDA compromised rats (21) (22). Using immunofluorescence and established behavioral paradigms such as amphetamine-induced rotations, survival and fiber outgrowth of DA neurons, and phenotypic behavior of transplanted rats was analyzed, respectively. Taken together, the results were strongly suggestive of the idea that implanted DA neurons were capable of restoring DAergic neurotransmission in the denervated striatum (21) (22). Indeed, since the late 1980s several hundred PD patients have since received grafts of DAergic human embryonic neural tissue, with the grafted tissue shown to survive and ameliorate typical motor symptoms. Despite these early successes however, the poor survival rate, on average about 2–5% of implanted cells, instigated the search for alternative resources of cells (23). Initial clinical studies of transplantation in PD have also employed adrenal medulla (AM) tissue as donor tissue. The rationale for this was based on animal models, which showed AM grafts of DA cells capable of surviving transplantation as well functional recovery (24).
In contrast, foetal replacement utilises pluripotent neurons from the aborted foetus (25). In their study Svendsen and colleagues transplanted human fetal expanded neural precursor cells (ENPs) into the striatum of rats lesioned with 6-OHDA (4). Despite ENPs surviving implantation and differentiating into neurons and glia, immunohistochemical evidence for TH immunoreactivity depicted functionally active dopaminergic neurones at low and variable numbers, with the extent of DAergic differentiation also small (4).
Stem Cell Technology
Stem cells therapy has won wider acclaim in recent years. Stem cells are undifferentiated progenitor cells which depict pluripotency - the potential to develop into any cell type in the body (25). For PD, this cell type is ideally a SNpc projection neuron which uses DA as its N .
Embryonic stem cells (ESC) from the embryonic blastocyst are most versatile in the body (25). The production of neurons positive for TH was first demonstrated in vitro from mouse ES cells by Lee and colleagues (26). Recently this has been extended to in vivo studies in rodent models of PD, where transplanted murine ES cells give rise to DAergic neurons, which in turn generate a gradual but sustained decrease in amphetamine-induced rotation after grafting (26). This behavioural recovery has been accompanied by functional scans of PET and fMRI, suggesting dopamine-mediated hemodynamic changes in the striatum and associated brain circuitry. The pioneering demonstration that ES cells could differentiate into mature and functional DA neurons that are able to integrate appropriately within basal ganglia circuitry to give stable functional recovery was nullified by the discovery that 20% of rats developed teratomas at the transplant site within 9 weeks of implantation. Ensuing technical challenges and ethical considerations however has meant the search for alternative sources of cells (26).
Adult neural stem cells - Neuronal replacement strategies from endogenous neural precursors in the adult brain
Adult neural stem cells (NSC) are multipotent cells which have the ability to differentiate into each of three neural lineages – glial (astrocytes), neural (neurons) and oligodendrocytes(27) . Today we know that there are two discrete regions - the Subgranular Zone (SGZ) of the dentate gyrus and the Subventricular Zone (SVZ) of the lateral ventricle, where the production of migrating neuroblasts occurs(27). Fortunately, the migration of new born cells is not limited to these areas. The discovery of neurogenesis in the adult human hippocampus in 1998 (28) has offered a tantalizing possibility to medical science: that endogenous progenitor cells might be manipulated to provide an endogenous neuronal replacement therapy for brain diseases such as PD (29) (30). Given the neurogenic property of the SVZ, it is essential to examine to what extent it responds to in a changing environment. However a fundamental question is whether neuronal replacement from endogenous precursors occurs in the brain in response to a toxic insult, and whether or not new cells migrate to the site of injury to replace those cells which have died. The pioneering study in 2002 led by Arvidsson and colleagues (31) was the first to comprehensively show that new neurons have the capability to replace cells lost at the site of an insult.
A number of studies based on animal and post mortem human tissue have now established that, far from being dormant tissues, the neurogenic regions of the brain respond to neurodegerative disease in a way which makes them potential targets for therapeutic intervention (32) (33-35).
A recent study led by Aponso and colleagues provides first quantitative evidence for an alteration in the production of endogenous cell proliferation in the SVZ, SN and midbrain (with a failure of neurogenesis) as a result of a partial progressive 6-OHDA lesion (36). Results are suggestive of endogenous neural progenitor cells in the striatum and the SN can provide potential repair for PD, but require alteration or modulation of environmental cues to suppress astrogenesis in the striatum and enhance or direct neurogenesis or DAergic differentiation to replace the degenerating neurons in the SN (36). However, despite the establishment that proliferation takes place (33), the appropriate and adequate Migration, Differentiation and integration of DA neurons is essential for a consequent change and improvement in motor phenotype. Indeed, autologous transplantation of adult neural stem cells has been attempted clinically in at least one patient with PD, with mixed results (37).
The stagnant success of pharmacotherapy and surgical methods in treating PD has instigated the search for novel treatment options, two of which are discussed here. Gene therapy is a relatively new field. Apart from increasing DA, it has been used to deliver anti-apoptotic agents, grown factors and recessive genes in an attempt to provide neurorestoration and neuroprotection. There exist 3 clinical trials for PD date, all of which have seen some success in impeding PD and achieving behavioural motor improvements in patients. Novel cell replacement strategies transplant DA into the depleted ST. Knowledge about the integration of DAergic neurons, more specifically the synaptic and neurotrophic mechanisms controlling their involvement in the basal ganglia circuitry is essential for clear relevance in human trials. Stem cell technology however has gained more focus recently, with ESC and NSC being used. The stimulation of endogenous neural stem cells in particular holds great potential. Novel strategies thus hold great potential for the modification of the natural course of PD, particularly with respect to protecting healthy cells and preventing loss of persisting ones.
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