Using Stem Cell and Gene Therapy Technologies to Treat Parkinson's Disease

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).